Sheep Peripheral Blood Mononuclear Cells Research Articles

Screening for immune biomarkers associated with infection or protection against Ehrlichia ruminantium by RNA-sequencing analysis

Heartwater is one of the most economically important tick-borne fatal diseases of livestock. The disease is caused by the bacteria Ehrlichia ruminantium transmitted by Amblyomma ticks. Although there is evidence that interferon-gamma controls E. ruminantium growth and that cellular immune responses are protective, an effective recombinant vaccine for this disease is lacking. Analyses of markers associated with infection as well as protection will lead to a better understanding of the E. ruminantium immune response and corresponding pathways induced in sheep peripheral blood mononuclear cells (PBMC) will assist in development of such a vaccine. In this study, Biomarkers of infection (BMI) were identified as uniquely expressed genes during primary infection and biomarkers of protection (BMP) associated with immune to heartwater were identified post challenge. Sheep were experimentally infected and challenged with E. ruminantium infected ticks. The immune phenotypic and transcriptome profile of their PBMC were compared to their own naïve PBMC collected before infection. The study revealed 305 differentially expressed genes (DEGs) as BMI, of these 17 were upregulated at all three time-points investigated. These DEGs, form part of the bacterial invasion of epithelial cells Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway, and others detected from day 1 post infection and are considered predictive markers for early heartwater infection in ruminants. Similarly, a total of 332 DEGs were identified as BMP, of these 100 were upregulated and 75 were downregulated at all three time-points investigated. However, at D1PC most DEGs were downregulated (n = 1312) that correlated with a reduction in the % CD4 and CD8 T cells detected with flow cytometry. KEGG pathway analyses showed complete down regulation of T cell specific pathways possibly due to homing of immune cells to the site of infection after acquired immunity developed. At D4PC, expression levels of most of these downregulated genes increased and by D6PC they were upregulated. This indicates that the sampling time-point for biomarker analyses is important when results for acquired immune responses are inferred. This data identified DEGs that could be considered as biomarkers of protective immunity that can be used for identification of vaccine antigens and provides a strong foundation to further development of heartwater recombinant vaccines.

Green extraction of bioactive compounds from wine lees and their bio-responses on immune modulation using in vitro sheep model

The objective of this study was to apply microwave-assisted extraction using green solvents starting from 3 different wine (white, rosé, and red) lees and to test their bio-response on sheep peripheral blood mononuclear cells proliferation, Bax/Bcl-2 ratio, and cytokines secretion. Wine lees (WL) of local organic farming from white wine, produced with Trebbiano cultivar, rosé and red wine, made with Nero di Troia cultivar, were collected. The WL were subjected to microwave-assisted extraction using 2 green solvents (water and ethanol) in 3 different combinations (water; water/ethanol 1:1 vol/vol; ethanol) with a dry matter-to-solvent ratio of 1:40 (wt/vol) at 4 temperature levels: 50, 100, 150, and 200°C. Sodium carbonate Na2CO3 (2 mmol/g of dry weight of lees) was used for increasing the polyphenol extraction yield. A total number of 6 extracts, 2 for each kind of WL investigated, according to their total phenolic content and in vitro antioxidant capacity, were selected to be tested on sheep peripheral blood mononuclear cells, as an animal model. All the WL extracts demonstrated a strong antiproliferative action. On the contrary, the cytokines' profile was mainly dependent on the different winemaking derived WL and the extraction solvent combination procedures. Red WL extract obtained by a combination of water/Na2CO3 and tested at 0.8 mg/mL, resulted in an increase of both IL-6 secretion and Bax/Bcl-2 ratio. Data from the present study demonstrated that WL extracts derived from different winemaking and solvent extraction could have a bimodal action on control of inflammatory mediated damage and highlighted the importance for further studies aimed at applying the biorefinery process on by-products to increase their economic value and exploit new derived bioactive compound.

Identification of Different Extracellular Vesicles in the Hydatid Fluid of Echinococcus granulosus and Immunomodulatory Effects of 110 K EVs on Sheep PBMCs.

Echinococcosis, mainly caused by Echinococcus granulosus, is one of the 17 neglected tropical diseases. Extracellular vesicles (EVs) play an essential role in the host–parasite interplay. However, the EVs in the hydatid fluid (HF) of E. granulosus are not fully characterized. Herein, three different types of HF EVs, designated as 2 K, 10 K, and 110 K EVs based on the centrifugal force used, were morphologically identified. A total of 97, 80, and 581 proteins were identified in 2 K, 10 K, and 110 K EVs, respectively, 39 of which were commonly shared. Moreover, 11, 8, and 25 miRNAs were detected, respectively, and all of the 7 selected miRNAs were validated by qPCR to be significantly lower abundant than that in protoscoleces. It was further deemed that 110 K EVs were internalized by sheep peripheral blood mononuclear cells (PBMCs) in a time-dependent manner and thus induced interleukin (IL)-10, tumor necrosis factor-α (TNF-α), and IRF5 were significantly upregulated and IL-1β, IL-17, and CD14 were significantly downregulated (p < 0.05). These data demonstrate the physical discrepancy of three HF EVs and an immunomodulatory effect of 110 K EVs on sheep PMBCs, suggesting a role in immune responses during E. granulosus infection.

Novel circRNA discovery in sheep shows evidence of high backsplice junction conservation

Circular RNAs (circRNAs) are covalently closed circular non-coding RNAs. Due to their structure, circRNAs are more stable and have longer half-lives than linear RNAs making them good candidates for disease biomarkers. Despite the scientific relevance of these molecules, the study of circRNAs in non-model organisms is still in its infancy. Here, we analyse total RNA-seq data to identify circRNAs in sheep from peripheral blood mononuclear cells (PBMCs) and parietal lobe cortex. Out of 2510 and 3403 circRNAs detected in parietal lobe cortex and in PBMCs, a total of 1379 novel circRNAs were discovered. Remarkably, around 63% of all detected circRNAs were found to be completely homologous to a circRNA annotated in human. Functional enrichment analysis was conducted for both tissues based on GO terms and KEGG pathways. The enriched terms suggest an important role of circRNAs from encephalon in synaptic functions and the involvement of circRNAs from PBMCs in basic immune system functions. In addition to this, we investigated the role of circRNAs in repetitive vaccination experiments via differential expression analysis and did not detect any significant relationship. At last, our results support both the miRNA sponge and the miRNA shuttle functions of CDR1-AS in sheep brain. To our knowledge, this is the first study on circRNA annotation in sheep PBMCs or parietal lobe cortex samples.

Development and evaluation of a real-time PCR panel for the detection of 20 immune markers in cattle and sheep

The establishment of a panel of immune markers is of paramount importance to understand the different transcription patterns of infectious diseases in livestock. The array of commercially available immunological assays for cattle and sheep is currently limited, due to the lack of antibodies for these species. Even though SYBR Green based real time quantitative PCR (qPCR) is the most commonly used method to study cytokine transcription in ruminants, a lack of standardization impairs its implementation in the study of different ruminant diseases. In order to obtain reliable qPCR results, several variables need to be considered: choice of reference genes for optimal normalization, variation of annealing temperature among primer sets, and assay specificity and sensitivity.In this study, we developed and validated a panel of immune markers in bovine and ovine samples using SYBR Green based qPCR in a cost-effective way with multiple primer sets optimised to amplify at a common thermal cycling temperature. Twenty primer sets were designed to quantify immune markers (IL-1b, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, TNF-α, IFN-γ, IFN-α, Ki-67, NFkB-65, TLR-3, TLR-4, TLR-8 and Rig-1) in ovine and bovine templates. For optimal normalization and selection of suitable reference genes, primer sets that measure the transcription of five reference genes were also included in the panel. The amplification efficiency, linearity and specificity was validated for all target genes. Optimal amplification conditions were achieved in both ovine and bovine samples for all gene targets, with the exception of Ki67. Relative quantification studies were performed on ovine and bovine mRNA obtained from sheep peripheral blood mononuclear cells (PBMCs) stimulated with three different treatments (PMA/Ionomycin, Concanavalin A (Con A) and pokeweed mitogen (PWM)). Pokeweed and ConA efficiently induced gene transcription of most of the targeted genes, while PMA/Ionomycin showed a weaker induction. Finally, we further assessed usability of our panel by running it on bovine monocyte derived dendritic cells (MoDCs) stimulated with different vaccines. Results confirmed the induction of a specific pro-inflammatory gene transcription pattern by rabies vaccine, which resembles the one occurring during viral infection.Altogether, we validated the efficiency and usability of an extended real-time PCR panel that gives the possibility to rapidly measure a broad spectrum of ovine and bovine immune markers by using a single set of reagents and protocol thus representing a valid and cost-effective tool for research purposes.

Transcriptional profiling of innate immune responses in sheep PBMCs induced by Haemonchus contortus soluble extracts

BackgroundPattern recognition receptors (PRRs) can recognize pathogen-associated molecular patterns and activate downstream signalling pathways, resulting in modulation of host immunity against pathogens. Here, we investigated whether PRR-mediated recognition is involved in host immune responses to the blood-feeding nematode Haemonchus contortus.MethodsDuring blood-feeding, H. contortus secretes immune-modulating antigens into host blood. Therefore, we stimulated sheep peripheral blood mononuclear cells (PBMCs) with H. contortus soluble extract (HcAg) and performed transcriptional profiling.ResultsHcAg upregulated two genetically linked CLRs (CLEC2L and KLRG2), two NLRs attenuating inflammation (NLRP12 and NLRC3) and one G protein-coupled receptor with potent anti-inflammatory effects (HCAR2). Furthermore, several Th2-related transcription factors (ATF3, IRF4, BCL3 and NFATC) were also upregulated, which may confer anti-inflammatory type 2 immune responses to HcAg.ConclusionsTogether, our preliminary studies provide new insights into how the host innate immune system controls type 2 immunity to H. contortus. Further work will be needed to identify H. contortus products recognized by the host innate immune system and determine the Th2 polarization ability of these putative PRR ligands.

Extracts from Microalga Chlorella sorokiniana Exert an Anti-Proliferative Effect and Modulate Cytokines in Sheep Peripheral Blood Mononuclear Cells.

Simple SummaryIn the last several years, the animal production sector has been very concerned about the overuse of antibiotics and the concomitant increase in antimicrobial resistance. The objective of this paper was the evaluation of the immune properties of different extracts isolated from Chlorella sorokiniana applied to sheep peripheral blood mononuclear cells (PBMCs). In particular, the effects of the unsaponified fraction (UP), the acetylated unsaponified fraction (AUP), and the total lipid fraction (TL) at two concentrations (0.4 and 0.8 mg/mL) on the proliferative response of sheep cells and their cytokine secretion were tested. All the extracts showed an inhibitory effect on cell proliferation, particularly in the presence of the UP fraction at a 0.4 mg/mL concentration. As regards cytokine production, the UP extract tested at a 0.8 mg/mL concentration increased interleukin(IL)-10 production, whereas the TL fraction at 0.4 mg/mL showed a cytokine profile characterized by an increase in both IL-10 secretion and the anti-inflammatory cytokine IL-6, and, to a lesser extent, IL-1β secretions, which are proinflammatory cytokines. These results supported the hypothesis that extracts from Chlorella sorokiniana could be considered useful ingredients to be integrated into animal feed with the aim to control immune responses during inflammation and minimize the use of antibiotics.The objective of this experiment was to study the effects of the unsaponified fraction (UP), the acetylated unsaponified fraction (AUP), and the total lipid fraction (TL) extracted and purified from Chlorella sorokiniana (CS) on the proliferation and cytokine profile of sheep peripheral blood mononuclear cells (PBMCs). Cells were cultured with 0.4 mg/mL and 0.8 mg/mL concentrations of each extract (UP, AUP, and TL fractions) and activated with 5 μg/mL concanavalin A (ConA) and 1 μg/mL lipopolysaccharide (LPS) at 37 °C for 24 h. PBMCs cultured with ConA and LPS represented the stimulated cells (SC), and PBMCs without ConA and LPS represented the unstimulated cells (USC). Cell-free supernatants were collected to determine IL-10, IL-1β, and IL-6 secretions; on cells, measurement of proliferation was performed. All the extracts tested significantly decreased the cell proliferation; in particular, the UP fraction at 0.4 mg/mL showed the lowest proliferative response. Furthermore, at 0.8 mg/mL, the UP fraction enhanced IL-10 secretion. On the contrary, the TL fraction at 0.4 mg/mL induced an increase in IL-10, IL-6, and, to a lesser extent, IL-1β secretions by cells. The AUP fraction did not change cytokine secretion. The results demonstrated that CS extracts could be useful ingredients in animal feed in order to minimize the use of antibiotics by modulating cell proliferation and cytokine response.

Ehrlichia ruminantium antigens and peptides induce cytotoxic T cell responses in vitro

Since CD8+ T cells play an important role in resistance to infection with heartwater, effective vaccines against this disease will likely require identification of antigens that contain CD8+ T cell epitopes responsible for cytotoxic T lymphocyte (CTL) responses. With the use of the fluorescent antigen-transfected target cell (FATT)-CTL assay, IFN-γ ELISPOT and flow cytometry, peptides that induce CTL, proliferation of CD8 + T cells and IFN-γ production were identified as possible target antigens for vaccine development. Of particular relevance was the finding that different peptides from different antigens were able to elicit varied cytotoxic activities by immune peripheral blood mononuclear cells (PBMC) from heartwater immune tick-infected sheep. Several peptides derived from Erum0660, Erum2330, Erum2540, Erum2580 and Erum5000 induced CTL in immune sheep PBMC. Peptide Erum2540-6 was the only peptide that induced significant CTL, CD8+CD45RO+ and CD8+IFN-γ+ by PBMC from all three sheep, and Erum2540 and p2540-20 induced the highest % CTL response in all three outbred sheep. These results suggest that these epitopes may be of major importance in heartwater recombinant vaccine development.

Innate immune transcriptomic evaluation of PBMC isolated from sheep after infection with E. ruminantium Welgevonden strain

Heartwater is a tick-borne non-infectious fatal disease of wild and domestic ruminants caused by the bacterium Ehrlichia ruminantium, transmitted by Amblyomma ticks. Although there is evidence that interferon-gamma (IFN-γ) controls E. ruminantium growth and that cellular immune responses could be protective, an effective recombinant vaccine for this disease is lacking. An overall analysis of which immune pathways are up- or down-regulated in sheep peripheral blood mononuclear cells is expected to lead to a better understanding of the global immune response of sheep to E. ruminantium infection. Therefore, a systems biology oriented approach following the infection with E. ruminantium was investigated from peripheral blood mononuclear cells to aid recombinant vaccine development. In this study, heartwater naïve sheep were infected and challenged by allowing E. ruminantium infected ticks to feed on them. After primary infection, all the animals were treated with antibiotic during the resulting febrile response. Blood was collected daily for E. ruminantium detection by qPCR (pCS20 assay). The pCS20 assay only detected the pathogen in the blood one day prior to and during the febrile stage of infection confirming infection of the sheep. IFN-γ real-time PCR indicated that this cytokine was expressed at specific time points: post infection, during the febrile stage of the disease and after challenge. These were used as a guide to select samples for transcriptome sequencing. This paper focuses on transcripts that are associated with innate activating pathways that were identified to be up- and down-regulated after primary infection and the subsequent challenge. These included the CD14 monocyte marker, toll-like receptor (TLR), nod-like receptor, chemokine, cytosolic and cytokine–cytokine interaction receptor pathways. In particular, TLR4, TLR9 and CD14 were activated together with DNA detection pathways, suggesting that vaccine formulations may be improved if CpG motifs and lipopolysaccharides are included. This data indicates that innate immune activation, perhaps by using adjuvants, should be an important component for consideration during future heartwater recombinant vaccine development.

Phytosterols from Dunaliella tertiolecta Reduce Cell Proliferation in Sheep Fed Flaxseed during Post Partum.

The post partum period is characterized by immunosuppression and increased disease susceptibility. Both phytosterols from microalga Dunaniella tertiolecta and dietary supplementation with n-3 polyunsaturated fatty acids (PUFA) influence cell proliferation and cytokine release during inflammation. The objective of this paper was the evaluation of the effects of physterols, extracted and purified from D. tertiolecta, on the in vitro immune responses of ewes supplemented with flaxseed during post partum. Twenty Comisana parturient ewes were divided in two balanced groups, and supplemented with flaxseed (FS, 250 g/day) or fed with a conventional diet (CON). Blood samples (15 mL) were collected for five weeks, starting from lambing, in order to isolate peripheral blood mononuclear cells (PBMC). Stimulated PBMC were treated with a total sterols fraction from D. tertiolecta (TS), a mix of ergosterol and 7-dehydroporiferasterol (purified extract, PE), and a mix of acetylated ergosterol and 7-dehydroporiferasterol (acetylated purified extract, AcPE), extracted and purified from D. tertiolecta at two concentrations (0.4 and 0.8 mg/mL). Results of the experiment demonstrated that n-3 PUFA from flaxseed induced an anti-inflammatory cytokine profile, with an increase of both IL-10, IL-6 and a decrease of IL-1β. TS, PE, and AcPE purified from D. tertiolecta showed an anti-proliferative effect on sheep PBMC regardless their chemical composition and concentration.

Peripheral blood mononuclear cell proliferation and cytokine production in sheep as affected by cortisol level and duration of stress

A large number of studies recognize glucocorticoids (Gc) as suppressors of inflammation; Gc exert an important role in coordinating the magnitude and duration of host immune responses. In the present in vitro investigation, we tested incremental levels of cortisol to verify the immunosuppressive or immunopermissive role of cortisol in sheep peripheral blood mononuclear cells (PBMC) after acute and chronic stress. Phytohemagglutinin (PHA)-stimulated PBMC were cultured for 24h and 96h at 37°C with 5% of CO2 and varying cortisol levels: 10 ng/mL (baseline), 100 ng/mL (physiological poststressor), and 1,000 ng/mL [hyperactivated hypothalamic-pituitary-adrenal (HPA) axis]. The cell-free supernatants were collected for determination of IL-6, IL-1β, and IL-10 by ELISA, and the bromodeoxyuridine assay was performed on cells. Physiological cortisol concentration negatively affected the levels of IL-6 secreted by PBMC, resulting in increased cell proliferation after acute stress (24h of incubation). However, physiological cortisol concentration exhibited a reduction in cell proliferation induced by increased levels of IL-6 secreted by PBMC during chronic stress (96h of incubation). The cortisol concentration representing a hyperactivated HPA axis led to a reduction in cell proliferation after acute stress, which was probably induced by the elevated IL-10 production. Our results demonstrate that in sheep the effect of Gc on the immune system was related to the magnitude and the duration of stress. In particular, cortisol levels higher than physiological concentrations suppressed cell proliferation soon after acute stress. Instead, the physiological poststressor concentration of cortisol affected the immune responses in a bidirectional manner depending on the duration of the stressor.

Potential of Tissue Engineered Blood Vessel as Model to Study Effect of Flow and Wall Thickness on Cellular Communication

In physiology, blood vessel function is maintained mainly through nitric oxide (NO)-mediated cross-talk between endothelial cells (ECs) and smooth muscle cells (SMCs), which is compromised in pathology. Lack of an appropriate in vitro model hampers the study of vascular disease progression mechanisms. This study attempted to use tissue engineered blood vessel (TEBV) as a model system to understand the effect of wall thickness and shear stress on EC to SMC communication. Differentiated ECs and SMCs obtained by in vitro culture of sheep peripheral blood mononuclear cells (PBMNCs) were seeded on biodegradable €-polycaprolactone (PCL) conduits of different wall thicknesses and exposed to shear stress in a two-channel bioreactor to construct functional TEBV. Phenotypes of ECs and SMCs were studied in terms of nitric oxide synthase (eNOS) and basic calponin expressions respectively, using real time polymerase chain reaction. Endothelial to SMC cross-talk under the influence of wall thickness and shear stress was interrelated to NO and cyclic GMP (cGMP) production. Shear stress accelerates, but wall thickness has no influence on endothelial NO production. Increased release of NO in response to shear stress resulted in augmented cGMP production, but only when the wall thickness was lower. Both wall thickness and shear stress affect cGMP production and smooth muscle contractile phenotype. From this study, it is also suggested that TEBV may be a suitable model to study various risk factors on vessel integrity. Keywords: Blood vessel wall thickness, cGMP synthesis in SMC, endothelial nitric oxide synthetase, functional tissue engineering, nitric oxide synthesis, shear stress, tissue engineered blood vessel.

Comparative analysis of innate immune response following in vitro stimulation of sheep and goat peripheral blood mononuclear cells with bluetongue virus – serotype 23

Bluetongue is an infectious disease caused by bluetongue virus (BTV), which affects sheep, goat, cattle and certain wild ruminants. However severe clinical signs are usually seen with significant mortality in sheep than cattle and goat. To date, comparative studies on innate immune responses of sheep and goat infected with BTV is lacking. In this study, we compared the innate immune response of sheep and goat by infecting the peripheral blood mononuclear cells (PBMCs) with BTV serotype 23. In our study, we observed that sheep PBMCs supports higher virus replication than goat PBMCs. To delineate the role of innate immune response in differential viral replication observed in this study, we examined TLR3 (Receptor for dsRNA virus) mRNA expression and cytokine profiles (IL-1β, Il-6, IL-8, Il-10, IL-12p40, TNF-α, IFN-γ and IFN-α) following Poly I:C (TLR3 ligand) stimulation and BTV 23 infection. In our present study, sheep PBMCs had significantly higher TLR3 mRNA levels, TLR3 specific ligand (Poly I:C) stimulation resulted in increased levels of IFN-γ at transcriptional and translational levels along with IL-8 and IL-10 at transcriptional levels. Whereas, the levels of TNF-α was higher in goat PBMCs at transcriptional levels. BTV infected sheep PBMCs expressed significantly higher levels of IFN-γ at transcriptional and translational levels along with IL-6, IL-8 and IL-10 at transcriptional levels. Whereas the expression levels of TNF-α and IFN-α at transcriptional and translational levels were significantly high in goat PBMCs. To examine the potential factor for consistent increase in the expression of TNF-α, we sequenced the promoter region of TNF-α and identified a total of five single nucleotide polymorphisms (SNP) and one indel in goat TNF-α promoter region. Luciferase assay for transcriptional activity of the promoter showed that goat TNF-α has significantly enhanced transcriptional activity in comparison with sheep TNF-α promoter. Altogether, our data suggests that the expression levels of TNF-α and IFN-α and/or IL-10 plays crucial role in replication of BTV 23.

The measurement of three cytokine transcripts in naïve and sensitized ovine peripheral blood mononuclear cells following in vitro stimulation with bluetongue virus serotype-23

Bluetongue (BT), a serious disease of sheep and some wild ruminants, is caused by bluetongue virus (BTV), a member of the family, Reoviridae. The current research thrust for controlling BT is on development of efficient vaccines, necessitating clear understanding of ovine immunology. At present, comparative studies on cytokine gene expression profiles of naïve and BTV-sensitized peripheral blood mononuclear cells (PBMC) in sheep have not been clearly understood. In the present study, PBMC from naïve and BEI-inactivated-saponin-adjuvanted BTV-1 vaccinated sheep were stimulated in vitro with heterologous BTV-23. At various intervals, RT-qPCR was carried out to estimate cytokine (interferon-gamma, interleukin-12 and interleukin-2) mRNA expressions that are linked to cell-mediated immunity. The results showed that PBMC cytokine profiles were relatively increased both temporally and quantitatively in immunized sheep PBMC compared to naïve ones, suggesting that BTV-1 vaccination may prime immune system that can cross-react with BTV-23 antigens.

Co-stimulation with TLR7/8 and TLR9 agonists induce down-regulation of innate immune responses in sheep blood mononuclear and B cells

Toll-like receptors (TLRs) play an important role in the activation of innate and adaptive immune responses. Stimulation with multiple TLR agonists may result in synergistic, complimentary or inhibitory effects on innate immune responses. In this study, we investigated the effects of co-stimulation of sheep peripheral blood mononuclear cells (PBMC) and B cells with agonists for TLR3, 4, 7/8 and 9. Sheep PBMC stimulated with either CpG (TLR9 agonist) or RNA oligoribonucleotides ([ORNs], TLR7/8 agonist) exhibited significant IL-12 production, but only CpG induced IFNalpha, IgM and proliferative responses. In contrast, poly(I:C) (TLR3 agonist) and LPS (TLR4 agonist) did not induce any of these responses. Interestingly, we observed that co-stimulation of PBMC with CpG+ORN or CpG+imiquimod (another TLR7/8 agonist) resulted in significant reduction in CpG-induced IFNalpha production, B cell proliferation and IgM responses. Pre-incubation of cells with CpG prior to exposure of the cells to imiquimod resulted in similar inhibitory responses indicating that the down-regulatory mechanisms are not associated with competition for cellular uptake or for receptors of the two agonists. Sheep B cells constitutively expressed TLR7, TLR8 and TLR9 mRNA transcripts, suggesting a possible role of TLR cross-talk in the down-regulatory mechanisms. Down-regulation of responses by co-stimulation with closely related TLRs may be a regulatory mechanism by which the host prevents overstimulation of innate immune responses.

Differential Transcription of Ovine Herpesvirus 2 Genes in Lymphocytes from Reservoir and Susceptible Species

Ovine herpesvirus 2 (OvHV-2) is a lymphotropic gammaherpesvirus that asymptomatically infects most sheep, but causes malignant catarrhal fever in cattle, bison, pigs and deer. There is no permissive cell culture system but OvHV-2-infected T lymphocytes can be cultured from diseased animals. We showed that the OvHV-2 genome was in a circular conformation in sheep peripheral blood mononuclear cells and that the latency-associated ORF73 was transcribed, while expression of the productive cycle genes ORF9 (DNA polymerase) and ORF50 (R-transactivator) was barely detectable, suggestive of latency. Doxorubicin treatment of these cells induced the appearance of linear viral DNA and transcription of productive cycle genes along with several viral unique genes. In contrast, cultured T cells from diseased cattle and rabbits contained a mixture of circular and linear genome configurations indicative of a mixture of latently- and productively-infected cells. Most of the OvHV-2 unique genes were transcribed in these cells but ORF50 expression was only seen after doxorubicin treatment indicating a 'leaky' latent pattern of gene expression. 5-azacytidine treatment increased the proportion of circular DNA and inhibited the expression of most of the OvHV-2 unique genes except Ov2.5 (vIL-10) and Ov4.5 (Bcl-2 homologue) in the cattle cell line. These studies provide key insights into the differences in OvHV-2 gene expression in cells from reservoir and susceptible species and, for the first time, an in vitro system for studying the latent and productive phases of the OvHV-2 virus life cycle.

Detection of the μ-opioid receptor in sheep peripheral blood mono-nuclear cells

Detection of the μ-opioid receptor in sheep peripheral blood mono-nuclear cells

Molecular cloning and mRNA tissue-expression of two isoforms of the ovine costimulatory molecule CD80 (B7-1)

Using RT-PCR and rapid amplification of cDNA ends (RACE), we cloned two putative alternatively spliced transcripts of the sheep CD80 (B7-1) molecule that encode both transmembrane (TM) and secreted (s) forms of CD80 protein. Comparison of the amino acid sequence of the TM form of ovine CD80 with the sequence of cattle, swine and human CD80 indicated that the deduced protein had a higher degree of similarity to cattle (87% of amino acid identity) than to pig (68%) and human sequence (53% of homology). In tissues, RT-PCR using primers for the TM and the sCD80 transcripts indicated that the expression of both CD80 transcripts was almost exclusively expressed in the hematolymphoid system, with the exception of the uterus. The sCD80 transcript was expressed in peripheral blood mononuclear cells (PBMC), uterus and lymph node, whereas the TM-CD80 transcript was very weakly detected only in PBMC cells. Our result indicates that mRNA transcripts encoding both membrane-bound and secreted CD80 proteins are expressed in sheep like in other animals. However, in contrast with the CD80 molecules from other species, the secreted form of sheep CD80 seems to be the predominant form expressed in the ovine PBMC and other tissues, suggesting that the TM-CD80 represents a rare transcript in this species. Interestingly, the expression of both forms of the CD80 molecule was not affected by treatment of sheep PBMC with Concanavalin A (ConA), as detected by RT-PCR. This is the first report describing the identification of a B7 costimulatory transcript in sheep.

Effects of endotoxin exposure on cationic amino acid transporter function in ovine peripheral blood mononuclear cells.

Rodent models of sepsis differ from clinical human disease in that humans make substantially less whole-body nitric oxide and have different cellular responses to endotoxin. Sheep, when exposed to endotoxin, behave in a manner more similar to humans. Many studies of rodent peripheral blood mononuclear cells (PBMCs) exposed to endotoxin demonstrate increased cationic amino acid transporter function (particularly through the y+ transporter) to supply arginine substrate to upregulated nitric oxide synthase. Whether this is true in sheep is not known. We have studied cationic amino acid transport in sheep PBMCs stimulated with endotoxin, using labelled lysine. PBMCs stimulated both in vitro and in vivo show an initial reduction in total and y+ lysine transport (after 1-2 h exposure to endotoxin): a previously undescribed effect of endotoxin. In in vitro activated cells, the reduction in y+ transport was prevented by the lipoxygenase inhibitor, nordihydroguaretic acid (NDGA), and the phospholipase inhibitor 4-bromophenacyl bromide (4-BPAB), but not cyclohexamide or a number of other inhibitors of intracellular second-messenger pathways. In contrast after 14 h incubation, the expected increase in total and y+ lysine transport was seen. The increase in y+ transport could be prevented by cyclohexamide, dexamethasone, ibuprofen, the protein kinase C inhibitor sphingosine, NDGA and 4-BPAB. These results suggest that in response to endotoxin exposure there is an initial decrease in y+ activity mediated by a lipoxygenase product, followed by a substantial increase in y+ activity mediated by the products of either cyclo-oxygenase or lipoxygenase. Cyclo-oxygenase and/or lipoxygenase inhibition might be useful in reducing arginine transport, and hence nitric oxide production, in these cells.

Modulation of sheep lymphocyte responses by Fasciola hepatica excretory–secretory products

The effect of Fasciola hepatica excretory–secretory products (FhESPs) on mitogen-induced proliferation of sheep peripheral blood mononuclear cells (PBMCs) and PBMC subsets (CD2 +, CD4 +, CD8 +, γδTCR + or CD21 + cells) were studied. PBMCs were incubated with Concanavalin A (ConA) or phytohemagglutinin (PHA) at optimal (1 μg per well) or suboptimal (0.25 μg per well) doses and with FhESPs at several doses (1.25–20 μg per well). PBMC subsets were incubated with ConA at a suboptimal dose and with FhESPs at 5 μg per well. These cells were incubated with or without monocytes (CD14 + cell). FhESPs slightly increased the proliferation of PBMCs stimulated with optimal doses of PHA. FhESPs (10 and 20 μg per well) inhibited the PBMCs stimulated with optimal doses of ConA. FhESP dose-dependent inhibition was observed on PBMCs stimulated with suboptimal doses of ConA. CD21 + lymphocytes (B lymphocytes), CD14 + cells (monocytes) and γδTCR + cells were not stimulated by ConA. T lymphocyte subsets (CD2 +, CD4 + or CD8 + cells) proliferation was decreased by FhESPs at 5 μg per well. FhESPs inhibits the ConA-induced stimulation of sheep PBMCs and sheep T lymphocyte subsets. Further studies should be done to investigate the mechanism of this FhESP immunomodulatory effect.