Pig Lymphocytes Research Articles

Identification of Potential miRNA-mRNA Regulatory Network Associated with Regulating Immunity and Metabolism in Pigs Induced by ASFV Infection

African swine fever (ASF) is a devastating infectious disease in domestic pigs caused by African swine fever virus (ASFV) with a mortality rate of about 100%. However, the understanding of the interaction between ASFV and host is still not clear. In this study, the expression differences and functional analysis of microRNA (miRNA) in porcine peripheral blood lymphocytes of ASFV infected pigs and healthy pigs were compared based on Illumina high-throughput sequencing, then the GO and KEGG signal pathways were analyzed. The miRNA related to immunity and inflammation were screened, and the regulatory network of miRNA-mRNA was drawn. A total of 70 differentially expressed miRNAs were found (p ≤ 0.05). Of these, 45 were upregulated and 25 were downregulated in ASFV-infected pigs vs. healthy pigs. A total of 8179 mRNA genes targeted by these 70 differentially expressed miRNA were predicted, of which 1447 mRNA genes were targeted by ssc-miR-2320-5p. Five differentially expressed miRNA were validated by RT-qPCR, which were consistent with the RNA-Seq results. The GO analysis revealed that a total of 30 gene functions were significantly enriched, including 7 molecular functions (MF), 13 cellular components (CC), and 10 biological processes (BP). The KEGG enrichment analysis revealed that the differentially expressed genes were significantly enriched in pathways related to immunity, inflammation, and various metabolic processes, in which a total of two downregulated miRNAs after infection and eight upregulated miRNAs related to immunity and inflammation were screened in ASFV-infected pigs vs. healthy pigs. The network of miRNA-mRNA showed that the mRNA target genes were strongly regulated by ssc-miR-214, ssc-miR-199b-3p, and ssc-miR-199a-3p. The mRNA target genes were enriched into the MAPK signaling pathway, Toll-like receptor signaling pathway, TNF signaling pathway, and IL-17 signaling pathway by using a KEGG enrichment analysis. Therefore, ASFV could regulate immunity and metabolism-related pathways in infected pigs by inducing differential expression of miRNAs. These results provided a new basis for further elucidating the interactions between ASFV and the host as well as the immunity regulation mechanisms of ASFV, which will be conducive to better controlling ASF.

APPLICATION OF VERMIGHUMUS IN FEEDING PIGS

The article analyzes a new method of fattening young pigs by introducing a biologically active additive into the diet - vermighumus, which is a product of pig manure processing by California worms. In addition to the standard diet, the youngsters of the research groups consumed vermighumus in a dose of 130, 180, 230 g per head per day. The young of the control group consumed standard compound feed. It was established that the application of vermighumus into the diet of young pigs improved their fattening indicators. In particular, the youngsters of the second and third experimental groups exceeded the control analogues in terms of live weight at the end of fattening by 3.73 and 5.53%, respectively. Youngsters of the second and third experimental groups also had higher growth energy by 4.80 and 7.40%, respectively, and better feed payment (by 0.43 and 071 kg). In terms of half-carcass length, young animals of the experimental group 3 did not significantly exceed the counterparts of the control group. In the 2nd and 3rd experimental groups, the fat thickness at the level of the 6th-7th thoracic vertebra was smaller (by 5.85-7.50%). They prevailed over the control analogues in the area of the "muscle eye" (by 2.83-5.45%). The weight of the rear third half of the carcass and the content of meat in the carcass was the highest in young animals of the 3 experimental group (by 7.85% and 4.91%, respectively), and the fat content in it was lower than in the control group (by 14.52%). The smallest fat thickness at the level of the 6-7 thoracic vertebra, as well as the content of fat in the carcass, was in young animals of the third experimental group. Among the indicators of the physical and chemical properties of the muscle tissue of young pigs of the experimental groups, no statistically significant differences were found. However, there is a certain trend towards a decrease in such indicators as the level of acidity, tenderness and losses during cooking, and an increase in the moisture-holding capacity and intensity of coloring in the young of the control group. The tasting evaluation revealed minor differences among the experimental animals. The relative number of lymphocytes (% of the total number of leukocytes) in pigs treated with vermighumus was higher (by 4.44-6.66% Р<0.05) than in counterparts fed with conventional compound feed, which indicates an improvement in resistance animalsEnrichment of the ration of young pigs with vermihumus as a biologically active additive contributed to the improvement of fattening and meat qualities and ensures obtaining additional basic products in the amount of UAH 320.38-354.34 per head.

Intestinal single-cell atlas reveals novel lymphocytes in pigs with similarities to human cells.

Lymphocytes can heavily influence intestinal health, but resolving intestinal lymphocyte function is challenging as the intestine contains a vastly heterogeneous mixture of cells. Pigs are an advantageous biomedical model, but deeper understanding of intestinal lymphocytes is warranted to improve model utility. Twenty-six cell types were identified in the porcine ileum by single-cell RNA sequencing and further compared with cells in human and murine ileum. Though general consensus of cell subsets across species was revealed, some porcine-specific lymphocyte subsets were identified. Differential tissue dissection and in situ analyses conferred spatial context, revealing similar locations of lymphocyte subsets in Peyer's patches and epithelium in pig-to-human comparisons. Like humans, activated and effector lymphocytes were abundant in the ileum but not periphery of pigs, suggesting tissue-specific and/or activation-associated gene expression. Gene signatures for peripheral and ileal innate lymphoid cells newly discovered in pigs were defined and highlighted similarities to human innate lymphoid cells. Overall, we reveal novel lymphocyte subsets in pigs and highlight utility of pigs for intestinal research applications.

ELISpot Assay for the Detection of ASFV-Specific Interferon-Gamma (IFN-γ)-Producing Cells.

The enzyme-linked immunospot (ELISpot) assay is a technique of unparalleled sensitivity to determine the frequency of antigen-specific immune cells secreting an immunomodulatory mediator upon recall antigen stimulation, making it a valuable tool in vaccine research. Typically done in multi-well microplate format, it also allows a high-throughput analysis of numerous immune cell samples, e.g., from different donor subjects, especially with the help of automated plate readers and specialized software that currently exist in most laboratories. IFN-γ is a hallmark cytokine secreted especially by T-cell subsets in recall response to pathogens, and consequently the IFN-γ ELISpot assay is one of the most widely used. The cellular arm of the immune response is known to be fundamental in protection against virulent ASFV, and therefore this assay is frequently employed in ASFV vaccine research to evaluate the results from immunization experiments.The technique involves the use of plates with wells that have a membrane for base with a strong binding capacity for amino acids that thus can be densely coated with an antibody for IFN-γ. Upon adding cells and specific antigen or other control stimuli, responding cells will release IFN-γ that is captured by the antibody in close proximity and revealed using a second antibody (sandwich method) through either chromogenic or fluorescent methods, leading to the detection of a "spot" on the membrane for each positive cell. Here we detail our protocol to detect the frequency of ASFV antigen-specific IFN-γ-producing cells in immunized pig lymphocytes and give an example of a typical result using the technique.

Morphological and Functional Activity Dynamics of Blood Lymphocytes in Large White Breed Pigs in Postnatal Ontogenesis and during Pregnancy

The purpose of the study was to research the activity of nucleolar organizers of blood lymphocytes in large white breed pigs in postnatal ontogenesis and during pregnancy. The prepared blood smears were stained with silver nitrate (AgNO3) and additionally stained with azur and eosin. By measuring the parameters of the nucleus and nucleolar organizers’ activity (AgNORs), the changes in the morphological and functional state of the protein synthesizing apparatus of blood lymphocytes in large white breed pigs in postnatal ontogenesis were evaluated. It was found that the area of lymphocyte nuclei in newborn piglets was 83.72±3.37 mm2. By the age of three months, the area of the lymphocyte nuclei of pigs significantly increased by 13.41% (p⩽0.05) and amounted to 96.69±2.57 mm2, at the age of six months this indicator was 15.78% more than at the age of three months (114.80±3.69), by the age of nine months the number of AgNORs significantly (p⩽0.05) decreased by 11.32% compared to six-month-old animals (101.80±2.97). From 1 to 4 units of AgNORs are detected in the lymphocyte nuclei. By the age of three months, the total area of nucleolar organizers’ zones decreased by 29.30% (p⩽0.05), and by the age of twelve months increased by 16.72% (p⩽0.05) compared to the previous age period. A slight increase in the area of the lymphocyte nucleus was observed in pigs after delivery by 9.15% (p⩽0.05). After delivery, the number of AgNORs in pigs significantly increased by 11.26% (p⩽0.05). During pregnancy, from 1 to 3 AgNORs were detected in pigs with a total area of 4.29±0.27 to 4.33±0.30 microns. Based on the conducted research, it can be concluded that the functional state of lymphocytes undergoes obvious changes in postnatal ontogenesis and during pregnancy, this can be seen in alterations in the area of their nuclei, the number and total area of AgNORs.

Chronic Hyperglycemia Drives Functional Impairment of Lymphocytes in Diabetic INSC94Y Transgenic Pigs.

People with diabetes mellitus have an increased risk for infections, however, there is still a critical gap in precise knowledge about altered immune mechanisms in this disease. Since diabetic INSC94Y transgenic pigs exhibit elevated blood glucose and a stable diabetic phenotype soon after birth, they provide a favorable model to explore functional alterations of immune cells in an early stage of diabetes mellitus in vivo. Hence, we investigated peripheral blood mononuclear cells (PBMC) of these diabetic pigs compared to non-diabetic wild-type littermates. We found a 5-fold decreased proliferative response of T cells in INSC94Y tg pigs to polyclonal T cell mitogen phytohemagglutinin (PHA). Using label-free LC-MS/MS, a total of 3,487 proteins were quantified, and distinct changes in protein abundances in CD4+ T cells of early-stage diabetic pigs were detectable. Additionally, we found significant increases in mitochondrial oxygen consumption rate (OCR) and higher basal glycolytic activity in PBMC of diabetic INSC94Y tg pigs, indicating an altered metabolic immune cell phenotype. Thus, our study provides new insights into molecular mechanisms of dysregulated immune cells triggered by permanent hyperglycemia.

Effects of deoxynivalenol on mitochondrial dynamics and autophagy in pig spleen lymphocytes

Deoxynivalenol (DON) is a type of cytotoxic mycotoxin that targets the mitochondria in cells. However, effects of DON on mitochondrial dynamics have not yet been reported. In this study, we investigated the effects of DON on mitochondrial dynamics and mitochondrial autophagy. Spleen lymphocytes of primary pigs were treated with different concentrations of DON. 24 h later, the cells were collected and the indexes were measured as follow: contents of ROS and MDA; activities of SDH and SOD; total antioxidant capacity; expression of MFN1, MFN2 and OPA1; the mRNA expression of the genes encoding mitochondrial autophagy proteins LC3 and P62, as well as mitochondrial respiratory chain complex activities. The results showed that, compared with the control group, the contents of ROS and MDA in the DON groups increased, while the activities of T-AOC, SDH and SOD decreased in a dose-dependent manner. With increasing DON concentrations, the expression levels of MFN1/2, OPA1 and mitochondrial respiratory chain complex activities decreased, while LC3, P62 increased. We suspect that DON causes oxidative damage, which in turn leads to down-regulation of MFN1, MFN2, OPA1 and up-regulation of LC3 and P62 mRNA, thereby inhibiting mitochondrial fusion and promoting mitochondrial autophagy.

Impacts of dietary microalgae (Schizochytrium JB5) on growth performance, blood profiles, apparent total tract digestibility, and ileal nutrient digestibility in weaning pigs.

Microalgae can serve as an alternative to animal feed additives or supplements in the diet of weaning pigs, to increase their potential and performance. Two experiments were conducted with three dietary treatments: CON (basal diet), MA1 (basal diet + 0.5% microalgae), and MA2 (basal diet + 1.0% microalgae). In Experiment 1, 150 crossbred weaning pigs were used in a 5-week experiment. In Experiment 2, 15 cannulated barrows were used. The objective of this study was to determine the effects of dietary supplementation of Schizochytrium microalgae on growth performance, blood profiles, apparent total tract digestibility, and ileal nutrient digestibility in weaning pigs. In Experiment 1, pigs fed with MA2 had higher growth (P < 0.05) than pigs fed with MA1 from day 8 to day 21 but the experimental result showed no significant effects (P > 0.05) on growth performance, white blood cell, total cholesterol, and triglyceride among the treatments. However, lymphocytes (%) of pigs fed with MA1 and MA2 diets were significantly higher (P < 0.05) than those of pigs fed with a CON diet at day 35. In Experiment 2, the apparent total tract digestibility and ileal nutrient digestibility of dry matter and nitrogen were higher (P < 0.05) in pigs fed with MA1 and MA2 than in those fed with a CON diet. Dietary microalgae supplementation had more dramatic and beneficial effects on nutrient digestibility than a CON diet. It also increased lymphocyte concentration but had no significant effect on growth performance in weaning pigs. © 2019 Society of Chemical Industry.

Chloramphenicol-Induced Alterations in the Liver and Small Intestine Epithelium in Pigs

Abstract An effect of the exposure to chloramphenicol (CAP) at doses used therapeutically was studied in pigs at the age of slaughter. Pigs were treated with CAP intramuscularly (20 mg/kg b.w. two times every 24 hours). Histomorphometrical and immunohistochemical analyses of small intestine and liver were done. CAP increased the thickness of myenteron and submucosa, and the length of villi; decreased the depth of crypts in the duodenum and jejunum. CAP influenced the Auerbach plexus. A decrease in cell proliferation, an increase in the number of apoptotic cells and T lymphocytes in the CAP-treated pigs were observed. CAP induces hepatotoxicity, neurotoxicity and disturbed intestinal epithelium. It can be concluded that short exposure of pigs to CAP at doses used therapeutically results in disturbed digestion and absorption process in the intestine.

Improved Cytotoxic T Lymphocyte Responses to Vaccination with Porcine Reproductive and Respiratory Syndrome Virus in 4-1BB Transgenic Pigs.

Vaccination is the most reliable measure to prevent infectious diseases in domestic animals. Development of novel vaccines demands extensive studies with new technologies, such as using novel adjuvants and immunomodulatory molecules. The co-stimulatory molecule 4-1BB provides a key signal that directs the fate of T cells during activation, and thus is important to their function in immune protection. To determine whether host immune responses to viral infection could be promoted by enhancing 4-1BB co-stimulation, in this study, we produced transgenic pig clones expressing an extra copy of the 4-1BB gene by clustered regularly interspaced short palindromic repeats/CRISPR-associated gene 9-mediated homologous recombination at the Rosa26 locus. The immune responses of transgenic pigs to porcine reproductive and respiratory syndrome virus (PRRSV) vaccine were determined on day 14. We show that peripheral blood lymphocytes of transgenic pigs expressed around twice the level of 4-1BB mRNA than those of control pigs. We also found IL-2, TNF-α, and granzyme B mRNA levels as well as PRRSV-specific IFN-γ response were significantly upregulated in 4-1BB transgenic pigs, leading to more efficient cytotoxic T lymphocyte (CTL) killing, whereas the expressions of IL-4, IL-17, and Foxp3 were not affected. These results indicate that higher levels of 4-1BB expression involve in promoting Th1 differentiation and enhancing specific CTL responses to PRRSV, and provide a novel approach to increase the efficacy of current vaccines to control the infectious diseases.

The 15N and 46R Residues of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein Enhance Regulatory T Lymphocytes Proliferation

Porcine reproductive and respiratory syndrome virus (PRRSV) negatively modulates host immune responses, resulting in persistent infection and immunosuppression. PRRSV infection increases the number of PRRSV-specific regulatory T lymphocytes (Tregs) in infected pigs. However, the target antigens for Tregs proliferation in PRRSV infection have not been fully understood. In this study, we demonstrated that the highly pathogenic PRRSV (HP-PRRSV) induced more CD4+CD25+Foxp3+ Tregs than classical PRRSV (C-PRRSV) strain. Of the recombinant GP5, M and N proteins of HP-PRRSV expressed in baculovirus expression systems, only N protein induced Tregs proliferation. The Tregs assays showed that three amino-acid regions, 15–21, 42–48 and 88–94, in N protein played an important role in induction of Tregs proliferation with synthetic peptides covering the whole length of N protein. By using reverse genetic methods, it was firstly found that the 15N and 46R residues in PRRSV N protein were critical for induction of Tregs proliferation. The phenotype of induced Tregs closely resembled that of transforming-growth-factor-β-secreting T helper 3 Tregs in swine. These data should be useful for understanding the mechanism of immunity to PRRSV and development of infection control strategies in the future.

Effect of therapeutic doses of enrofloxacin on circulating lymphocyte subpopulations in pigs

Abstract Twenty pigs of similar genetics (PIC) were used. Pigs were randomly divided into two groups: experimental (ENRO, n = 10) and control (C, n = 10). From day 0 to day 4, pigs from ENRO group received enrofloxacin at the recommended therapeutic dose. Pigs from C group received PBS instead of enrofloxacin. Blood samples were collected on days 0 (before antibiotic administration), 2, 4 (during antibiotic therapy), 9, and 13 of the study (after enrofloxacin administration). Haematological examination and flow cytometry were used to establish the relative and absolute counts of various leukocyte subsets. Lymphocyte subpopulations were measured by fluorochrom-labelled antibodies according to following definitions: CD3+ (T cells), CD21+ (B cells), CD4+CD8- (helper T cells, Th), CD4-CD8+ (cytolytic T cells, CLT), CD4+CD8+ (cytolytic and memory T cells). The present study revealed the modulating effect of enrofloxacin on the composition of circulating lymphocytes in pigs. Concentration and percentage of CD8+ cells decreased significantly after treatment with enrofloxacin and as a result the absolute CD4/CD8 ratio increased significantly as compared to control group (P &lt; 0.05).These findings should prompt further studies on the practical significance of the results obtained in terms of clinical implications. In view of the results, it cannot be excluded that enrofloxacin may also have immunomodulatory effects on host response to infection.

Generation of recombination activating gene-1-deficient neonatal piglets: a model of T and B cell deficient severe combined immune deficiency.

Although severe combined immune deficiency (SCID) is a very important research model for mice and SCID mice are widely used, there are only few reports describing the SCID pig models. Therefore, additional research in this area is needed. In this study, we describe the generation of Recombination activating gene-1 (Rag-1)-deficient neonatal piglets in Duroc breed using somatic cell nuclear transfer (SCNT) with gene targeting and analysis using fluorescence-activated cell sorting (FACS) and histology. We constructed porcine Rag-1 gene targeting vectors for the Exon 2 region and obtained heterozygous/homozygous Rag-1 knockout cell colonies using SCNT. We generated two Rag-1-deficient neonatal piglets and compared them with wild-type neonatal piglets. FACS analysis showed that Rag-1 disruption causes a lack of Immunoglobulin M-positive B cells and CD3-positive T cells in peripheral blood mononuclear cells. Consistent with FACS analysis, histological analysis revealed structural defects and an absence of mature lymphocytes in the spleen, mesenteric lymph node (MLNs), and thymus in Rag-1-deficient piglets. These results confirm that Rag-1 is necessary for the generation of lymphocytes in pigs, and Rag-1-deficient piglets exhibit a T and B cell deficient SCID (T-B-SCID) phenotype similar to that of rodents and humans. The T-B-SCID pigs with Rag-1 deficiency generated in this study could be a suitably versatile model for laboratory, translational, and biomedical research, including the development of a humanized model and assessment of pluripotent stem cells.

Illegitimate recombination between T cell receptor genes in humans and pigs (Sus scrofa domestica).

T cell receptor (TCR) genes (TRA/TRD, TRB and TRG) reside in three regions on human chromosomes (14q11.2, 7q34 and 7p14, respectively) and pig chromosomes (7q15.3-q21, 18q11.3-q12 and 9q21-22, respectively). During the maturation of T cells, TCR genes are rearranged by site-specific recombination. Occasionally, interlocus recombination of different TCR genes takes place, resulting in chromosome rearrangements. It has been suggested that the absolute number of these "innocent" trans-rearrangements correlates with the risk of lymphoma. The aims of this work were to assess the frequencies of rearrangements with breakpoints in TCR genes in domestic pig lymphocytes and to compare these with the frequencies of corresponding rearrangements in human lymphocytes by using fluorescence in situ hybridization with chromosome painting probes. We show that frequencies of trans-rearrangements involving TRA/TRD locus in pigs are significantly higher than the frequency of translocations with breakpoints in TRB and TRG genes in pigs and the frequencies of corresponding trans-rearrangements involving TRA/TRD locus in humans. Complex structure of the pig TRA/TRD locus with high number of potential V(D)J rearrangements compared to the human locus may account for the observed differences. Furthermore, we demonstrated that trans-rearrangements involving pig TRA/TRD locus occur at lower frequencies in γδ T cells than in αβ T lymphocytes. The decrease of the frequencies in γδ T cells is probably caused by the absence of TRA recombination during maturation of this T cell lineage. High numbers of innocent trans-rearrangements in pigs may indicate a higher risk of T-cell lymphoma than in humans.

Development of a neutralizing mouse-pig chimeric antibody with therapeutic potential againstHaemophilus parasuisinPichia pastoris

Haemophilus parasuis is one of the most important bacterial diseases of pigs worldwide. The lack of a vaccine against a broad spectrum of strains and the limitation of antimicrobial susceptibility hamper the control of disease. In this study, we cloned the constant regions of gamma heavy chains and kappa light chain of pig lymphocytes in frame with the variable regions of heavy and light chains of mouse monoclonal antibody 1D8, which reacts with all 15 serotypes of H.parasuis and has neutralizing activity. The constructed mouse-pig chimeric antibody was expressed in Pichia pastoris. Results demonstrated that the expressed chimeric antibody inhibited the growth of H.parasuis in vitro. Furthermore, the experiments in mice showed that chimeric antibody increased survival rate of the mice compared with that of the control group (P<0.05). Importantly, the chimeric antibody partially protected piglets against H.parasuis infection according to the clinical lesion scores and PCR results of H.parasuis in the tissues from piglets of the chimeric antibody-inoculated group and the PBS group. In summary, our results demonstrated that the mouse-pig chimeric antibody could be a therapeutic candidate to prevent the H.parasuis infection and control the prevalence of disease.

Preliminary investigations on the single and combined cytotoxic effect of T-2 and HT-2 toxin measured by Methyl Thiazol Tetrazolium (MTT) cytotoxicity test using pig lymphocytes

Fusariotoxins are frequently existing contaminants in cereal and other plant products. High relationship was found between the levels of the trichothecene T-2 and HT-2 toxin detected in contaminated commodities. Many in vitro studies investigated the cytotoxicity of T-2 and HT-2, but the interaction between them has not been thoroughly studied yet. The aim of the study was to examine the dose dependent cytotoxic effect of T-2 and HT-2 toxins, single and in combination, using pig lymphocytes in the methyl thiazol tetrazolium (MTT ) assay. The mycotoxins were added at various concentrations, i.e. 0.5, 0.1, 0.05, 0.01 and 0.001 μM of T-2; 1.0, 0.5, 0.2., 0.1 and 0.05 μM of HT-2; and both mycotoxins combined. Two exposure times (6 and 24 h) were tested. Both T-2 and HT-2 toxins exerted a dose dependent effect. After 6 h incubation, the increase in concentration of T-2 from 0.001 to 0.5 μM and HT-2 for 0.05 to 1.0 μM resulted in lower cell viability by 22 and 17%, respectively. After 24 h cell viability was significantly lower compared to values obtained at 6 h, except 0.5 μM T-2 and 0.05, 1.0 μM HT-2, respectively. Measured cell viability for combinations of T-2 and HT-2 was higher compared to the calculated expected values. The two toxins caused lower cell survival when applied together than in single administration after 6 h incubation. After 24 h incubation this tendency was not consistent.

Varicella zoster virus (VZV) infects and establishes latency in enteric neurons.

Case reports have linked varicella-zoster virus (VZV) to gastrointestinal disorders, including severe abdominal pain preceding fatal varicella and acute colonic pseudoobstruction (Ogilvie's syndrome). Because we had previously detected DNA and transcripts encoding latency-associated VZV gene products in the human gut, we sought to determine whether latent VZV is present in the human enteric nervous system (ENS) and, if so, to identify the cells in which it is located and its route to the bowel. Neither DNA, nor transcripts encoding VZV gene products, could be detected in resected gut from any of seven control children (<1year old) who had not received the varicella vaccine or experienced varicella; however, VZV DNA and transcripts were each found to be present in resected bowel from 6/6 of children with a past history of varicella and in that of 6/7 of children who received the varicella vaccine. Both wild-type (WT) and vaccine-type (vOka) VZV thus establish latent infection in human gut. To determine routes by which VZV might gain access to the bowel, we injected guinea pigs with human or guinea pig lymphocytes expressing green fluorescent protein (GFP) under the control of the VZV ORF66 gene (VZV(OKA66.GFP)). GFP-expressing enteric neurons were found throughout the bowel within 2days and continued to be present for greater than 6weeks. DNA encoding VZV gene products also appeared in enteric and dorsal root ganglion (DRG) neurons following intradermal administration of WT-VZV and in enteric neurons after intradermal injection of VZV(OKA66.GFP); moreover, a small number of guinea pig DRG neurons were found to project both to the skin and the intraperitoneal viscera. Viremia, in which lymphocytes carry VZV, or axonal transport from DRG neurons infected through their epidermal projections are thus each potential routes that enable VZV to gain access to the ENS.

The expression of CD25, CD11b, SWC1, SWC7, MHC-II, and family of CD45 molecules can be used to characterize different stages of γδ T lymphocytes in pigs

The expression of selected molecules was chosen to study porcine γδ lymphocytes and their CD2/CD8 subsets in different lymphoid organs in vivo and in vitro. Results indicate that many γδ T cells can constitutively express CD25 and MHC-II and that the frequency of γδ T cells positive for CD25, CD11b, SWC1 and SWC7 can be increased by stimulation. A diversified TCRδ repertoire was found inside CD25+, CD11b+, SWC1− and CD45RA− cells. Ontogenetic studies revealed various age and/or colonization dependency for expression of all studied molecules except of SWC7. Findings generally indicate that CD25 represent an activation molecule that probably marks a functionally distinct subsets, expression of CD11b is perhaps connected to early functions of naive γδ T cells in the periphery, SWC1 is lineage specific marker, SWC7 may represent an activation molecule with intrinsic or transient expression, and the expression of CD45RA/RC most likely defines naive and terminally differentiated cells.

Induction, regulation and physiological role of IL-17 secreting helper T-cells isolated from PBMC, thymus, and lung lymphocytes of young pigs

Interleukin-17 (IL-17) producing cells, referred to as Th17, have recently emerged as a third subset of the T helper (Th) cell family. Studies in mice have demonstrated that Th17 cells and their associated cytokines are involved in several autoimmune diseases and host defense against infection. Murine Th17 cells differentiate from naïve CD4 + T-cells in the presence of TGFβ and IL-6, however, there are contradicting reports as to the role of TGFβ in the differentiation of human Th17 cells and very little is known about these cells in other animals. We report here the presence of IL-17 secreting lymphocytes in the lung and peripheral blood of pigs. The cDNA of porcine IL-17 gene was cloned and sequenced from activated lung lymphocytes and PBMC from piglets. A 17 kDa recombinant protein was expressed and purified both under denaturing and native conditions from E. coli BL21 cells. Furthermore, we demonstrate that TGFβ in the presence of IL-6 and/or IL-1β induces in vitro differentiation of Th17 cells from naïve porcine CD4 + thymocytes.

Immuno-modulating effect of Astragalus polysaccharides on the immune reactions of peripheral blood lymphocytes in pigs

Immuno-modulating effect of Astragalus polysaccharides on the immune reactions of peripheral blood lymphocytes in pigs