Pig Leukocytes Research Articles

Long‐term dietary supplementation of organic selenium modulates gene expression profiles in leukocytes of adult pigs

Seventy-two pigs at 34.4 kg body weight (BW) were allotted to two treatments with six replicates/treatment and six pigs/pen: the CON (negative control, no added selenium (Se)) and the OS (0.36 mg/kg added selenium from selenium-enriched yeast). Pigs were fed until 130 kg BW. The CON diet contained 0.18 mg/kg indigenous Se whereas the OS diet contained 0.54 mg/kg Se. Blood samples were collected at 130 kg BW and further processed for microarray analysis, prepared with 885 genes related to immune function of pigs. Among those, 28 genes related to improved immune status and innate immunity were up-regulated (P < 0.05) in leukocytes from Se-fed pigs and those include major histocompatibility class I (> 1.66), arginase I (> 1.27), integrin beta-1-subunit (> 1.20), toll like receptor 2 (> 1.12) and double-stranded RNA-dependent protein kinase. However, 24 genes including tissue factor (< 4.70), serum amyloid A-2 protein (< 3.11) and p27Kip1 (< 1.42) were down-regulated (P < 0.05) in leukocytes from Se-fed pigs. Expression of four selected genes was validated using quantitative PCR (qPCR) showing significant correlation between mircroarray analysis and qPCR analysis. This study indicates that a long- term dietary supplementation (0.3%) of organic Se improves the expression of genes that are related to enhanced immunity of pigs.

Membrane Disruption by Antimicrobial Peptide Protegrin-1 is Tuned by Incorporation of Cholesterol and Phosphoethanolamine Lipids

Antimicrobial peptides (AMPs) are a class of small (less than 100 amino acid residues) host defense peptides that induce selective membrane lytic activity against microbial pathogens. To understand the mechanism of membrane disruption by AMPs, we investigated, via atomic force microscopy, topological changes induced by protegrin-1 (PG-1), an 18-residue, cationic, β-sheet AMP isolated from pig leukocytes, in supported phospholipid bilayers (SPBs). Lipid mixtures of dioleoylphosphatidylserine (DOPS), dioleoylphosphatidylcholine (DOPC), and cholesterol were used to mimic eukaryotic cell membranes while bacterial cell membranes were emulated by substitution of cholesterol for dioleoylphosphatidylethanolamine (DOPE). We have previously shown that AMP disruption of zwitterionic dimyristoylphosphatidylcholine (DMPC) SPBs induce concentration dependent structural transformations that progress from fingerlike instabilities at bilayer edges, to the formation of sievelike nanoporous structures, and finally to a network of wormlike micellar structures. The observed transformations suggest that the peptides act to lower the interfacial energy of the bilayer in a manner similar to detergents. Detergent solubilization of membranes encompass processes such as pore formation, blebbing, budding, and vesiculation that share common saddle-splay (“negative Gaussian”) curved topologies. We pose that membranes rich in negative curvature lipids such as those with phosphoethanolamine (PE) headgroups enhance the efficacy of AMP disruption while those membranes containing cholesterol retard disruption. Results have shown that cholesterol incorporation shifts the disruption susceptibility of a bilayer to a higher peptide dosage regime while an opposite effect is observed in the presence of PE. The observed trend sheds light on a lingering debate as to how nature has evolved AMPs to discriminate between host and pathogen.

Mannan oligosaccharide modulates gene expression profile in pigs experimentally infected with porcine reproductive and respiratory syndrome virus12

This study characterized gene expression in peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage fluid (BALF) cells from control- or mannan oligosaccharide (MOS)-fed pigs with or without porcine reproductive and respiratory syndrome virus (PRRSV) at d 7 postinfection (PI). Weaned pigs (3 wk old) fed 0 or 0.2% MOS (Bio-Mos) diets were intranasally inoculated with PRRSV or a sterile medium at 5 wk of age. Total RNA (3 pigs/treatment) was extracted from cells. Double-stranded cDNA was amplified, labeled, and further hybridized to the Affymetrix GeneChip Porcine Genome Array consisting of 23,937 probe sets representing 20,201 genes. Microarray data were analyzed in R using packages from the Bioconductor project. Differential gene expression was tested by fitting a mixed linear model equivalent to a 2 × 2 factorial ANOVA using the limma package. Dietary MOS and PRRSV changed the expression of thousands of probe sets in PBMC and BALF cells (P < 0.05). The MOS × PRRSV interaction altered the expression of more nonimmune probe sets in PBMC (977 up, 1,128 down) than in BALF cells (117 up, 78 down). The MOS × PRRSV interaction (P < 0.05) for immune probe sets in PBMC affected genes encoding key inflammatory mediators. In uninfected pigs, gene expression of IL-1α, IL-6, myeloid differentiation factor 88, Toll-like receptor (TLR) 4, major histocompatibility complex (MHC) II, and dead box polypeptide 58 increased in PBMC of MOS-fed pigs (P < 0.05). This suggests that MOS enhances disease resistance in pigs and supports the fact that MOS induced a rapid increase in leukocytes at d 3 and 7 PI. Within infected pigs, however, MOS reduced the expression of IL-1β, IL-6, IL-8, macrophage inflammatory protein (MIP)-1α, MIP-1β, monocyte chemotactic protein (MCP)-1, and TLR4 genes in PBMC (P < 0.05). This finding may explain why fever was ameliorated in infected pigs fed MOS by d 7 PI. The expression of IL-1β, IL-6, MIP-1β, MCP-1, and TLR4 genes was confirmed by quantitative real-time reverse-transcription PCR. In BALF cells of infected pigs, MOS reduced the gene expression of TLR4, MHCII, and molecules associated with the complement system, but increased the gene expression of MHCI. In short, MOS regulated the expression of nonimmune and immune genes in pig leukocytes, perhaps providing benefits by enhancing the immune responses of the pigs to an infection, while preventing overstimulation of the immune system.

Investigations of the possible glycosylation of monoamine oxidase B from pig leucocytes.

Monoamine oxidase B (MAO B) from pig liver has been reported to be a sialoglycoprotein. However, when that enzyme from pig lymphocytes and granulocytes was separated by polyacrylamide gel electrophoresis after labelling with the specific irreversible inhibitor [3H]pargyline, staining with 1-ethyl-2-[3-(1-ethyl-naphtho [1,2d] thiazolin-2-ylidene)-2-methylpropenyl] naphtho [1,2d] thiazolium bromide ("Stains-all") failed to detect the presence of sialic acid residues. Treatment of the enzyme in disrupted lymphocytes and granulocytes, or in mitochondrial fractions prepared from them, with neuraminidase resulted in a decrease in MAO activity. However, after the enzyme was rendered soluble by treatment with octylglucoside, treatment with neuraminidase had no effect on the activity. These results indicate that sialic acid residues are not an intrinsic component of MAO B, although associated material containing such groups appears to affect the activity of the membrane-bound enzyme. The activities of membrane-bound preparations of MAO B from pig lymphocytes and granulocytes were unaffected by treatment with trypsin or beta-chymotrypsin. After the preparations had been rendered soluble by treatment with octylglucoside there was a decrease in the activity on treatment with beta-chymotrypsin, but trypsin treatment had no effect. Thus solubilization resulted in residues sensitive to cleavage by the former enzyme becoming accessible to it. Tryptic and chymotryptic peptides separated from the sodium dodecyl sulphate denatured enzymes by polyacrylamide gel electrophoresis revealed no differences between MAO B prepared from lymphocytes and granulocytes.

Characterizing a Detergent-Like Commonality Among Antimicrobial Peptides with Structural and Mechanistic Differences

Largely distributed among living organisms, antimicrobial peptides are a class of small (<100 amino acid residues) host defense peptides that induce selective membrane lytic activity against microbial pathogens. The permeabilizing behavior of these diverse peptides has been commonly applied to the formation of pores, categorized as the barrel-stave, toroidal, or carpet type. With the continuing discovery of new peptide species, many are uncharacterized and the exact mechanism is unknown. Through the use of atomic force microscopy, we have previously shown that the disruption of mica supported lipid bilayer patches by protegrin-1, an 18-residue, cationic, beta sheet antimicrobial peptide isolated from pig leukocytes, is concentration dependent. The intercalation of antimicrobial peptide into the bilayer results in structures beyond that of pore formation including the observance of worm-like micelles. This suggests that antimicrobial peptide acts to lower the interfacial energy of the bilayer in a way similar to detergents. We pose that antimicrobial peptides act universally within a detergent-like mechanism in which pore formation is a small part of a much more complex phase diagram that spans peptide-lipid aggregates beyond that of lamellar bilayers. Antimicrobial peptides with structural and mechanistic differences have been studied and current results with magainin-1 and aurein 1.1 for example exhibit a mechanistic commonality. Future aspirations of antimicrobial peptides as new sources of antibiotics may be realized as a universal mechanism can streamline the synthesis of de novo designed peptides.

Oral Vaccine Formulations Stimulate Mucosal and Systemic Antibody Responses against Staphylococcal Enterotoxin B in a Piglet Model

Despite the potential for its use as an agent of biowarfare or bioterrorism, no approved vaccine against staphylococcal enterotoxin B (SEB) exists. Nontoxic, mutant forms of SEB have been developed; however, it has been difficult to determine the efficacy of such subunit vaccine candidates due to the lack of superantigen activity of native SEB in rodents and due to the limitations of primate models. Since pigs respond to SEB in a manner similar to that of human subjects, we utilized this relevant animal model to investigate the safety and immunogenicity of a triple mutant of SEB carrying the amino acid changes L45R, Y89A, and Y94A. This recombinant mutant SEB (rmSEB) did not possess superantigen activity in pig lymphocyte cultures. Furthermore, rmSEB was unable to compete with native SEB for binding to pig leukocytes. These in vitro studies suggested that rmSEB could be a safe subunit vaccine. To test this possibility, piglets immunized orally with rmSEB formulations experienced no significant decrease in food consumption and no weight loss during the vaccination regimen. Oral vaccination with 1-mg doses of rmSEB on days 0, 7, 14, and 24 resulted in serum IgG and fecal IgA levels by day 36 that cross-reacted with native SEB. Surprisingly, the inclusion of cholera toxin adjuvant in vaccine formulations containing rmSEB did not result in increased antibody responses compared to formulations using the immunogen alone. Taken together, these studies provide additional evidence for the potential use of nontoxic forms of SEB as vaccines.

Porcine CD18 mediatesActinobacillus pleuropneumoniaeApxIII species-specific toxicity

Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, produces Apx toxins that are recognized as major virulence factors. Recently, we showed that ApxIIIA-cytotoxic activity specifically targets Sus scrofa leukocytes. Since both LtxA from Aggregatibacter actinomycetemcomitans (aggressive periodontitis in humans) and LktA from Mannheimia haemolytica (pneumonia in ruminants) share this characteristic, respectively towards human and ruminant leukocytes, and because both use the CD18 subunit to interact with their respective LFA-1, we hypothesized that ApxIIIA was likely to bind porcine CD18 to exercise its deleterious effects on pig leukocytes. A β 2−integrin-deficient ApxIIIA-resistant human erythroleukemic cell line was transfected either with homologous or heterologous CD11a/CD18 heterodimers using a set of plasmids coding for human (ApxIIIA-resistant), bovine (-resistant) and porcine (-susceptible) CD11a and CD18 subunits. Cell preparations that switched from ApxIIIA-resistance to -susceptibility were then sought to identify the LFA-1 subunit involved. The results showed that the ApxIIIA-resistant recipient cell line was rendered susceptible only if the CD18 partner within the LFA-1 heterodimer was that of the pig. It is concluded that porcine CD18 is necessary to mediate A. pleuropneumoniae ApxIIIA toxin-induced leukolysis.

Genomic expression profiling of peripheral blood leukocytes of pigs infected with highly virulent classical swine fever virus strain Shimen

Classical swine fever (CSF), caused by a virus of the same name (CSFV), is a highly contagious swine pyrexic disease featuring extensive haemorrhagic lesions and leukopenia, but little is known about the molecular mechanisms of its pathogenesis. To gain insight into the interaction between the virus and host cells, microarray analyses were performed to detect alterations in genomic expression of pig peripheral blood leukocytes (PBLs) following CSFV infection. Three healthy pigs were inoculated with a lethal dose of highly virulent CSFV strain Shimen. PBLs were isolated at the onset of typical clinical signs and total RNA was subjected to microarray analyses with Affymetrix Porcine Genome Array GeneChips. Of all 20,201 pig genes arrayed in the chip, 1745 showed altered expression (up- or downregulation) after infection. These were classified into eight functional groups, relating to cell proliferation (3.6%), immune response (2.1%), apoptosis (1.4%), kinase activity (1.4%), signal transduction (1.4%), transcription (0.7%), receptor activity (0.7%) and cytokines/chemokines (0.4%). The remaining 88.3% of genes had unknown functions. Alterations in genomic expression were confirmed by real-time RT-PCR of selected cellular genes and Western blotting of annexin 2, a cellular protein relating to virus infection. The observed expression changes of numerous genes involved in immune and inflammatory responses and in the apoptosis process indicate that CSFV has developed sophisticated mechanisms to cause leukopenia in infected pigs. These data provide a basis for exploring the molecular pathogenesis of CSFV infection through an understanding of the interaction between viral and cellular components.

Ultrafiltered pig leukocyte extract (IMUNOR®) decreases nitric oxide formation and hematopoiesis-stimulating cytokine production in lipopolysaccharide-stimulated RAW 264.7 macrophages

A low-molecular-weight (< 12 kDa) ultrafiltered pig leukocyte extract, IMUNOR®, was tested in experiments in vitro on non-stimulated and lipopolysaccharide (LPS)-stimulated murine RAW 264.7 macrophages in order to assess modulation of nitric oxide (NO) production (measured indirectly as the concentration of nitrites), hematopoiesis-stimulating activity of the supernatant of the macrophage cells (ascertained by counting cell colonies growing from progenitor cells for granulocytes and macrophages (GM-CFC) in vitro), and the release of hematopoiesis-stimulating cytokines. No hematopoiesis-stimulating activity and cytokine or NO production were found in the supernatant of non-stimulated macrophages. It was found that IMUNOR® does not influence this status. Supernatant of LPS-stimulated macrophages was characterized by hematopoiesis-stimulating activity, as well as by the presence of nitrites, interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). A key role in the hematopoiesis-stimulating activity of the supernatant of LPS-stimulated macrophages could be ascribed to G-CSF since the formation of the colonies could be abrogated nearly completely by monoclonal antibodies against G-CSF. IMUNOR® was found to suppress all the mentioned manifestations of the LPS-activated macrophages. When considering these results together with those from our previous in vivo study revealing stimulatory effects of IMUNOR® on radiation-suppressed hematopoiesis, a hypothesis may be formulated which postulates a homeostatic role of IMUNOR®, consisting in stimulation of impaired immune and hematopoietic systems but also in cutting back the production of proinflammatory mediators in cases of overstimulation which threats with undesirable consequences.

The role of G-CSF and IL-6 in the granulopoiesis-stimulating activity of murine blood serum induced by perorally administered ultrafiltered pig leukocyte extract, IMUNOR®

IMUNOR®, a low-molecular weight (< 12 kD) ultrafiltered pig leukocyte extract, has been previously found to have significant stimulatory effects on murine hematopoiesis supressed by ionizing radiation or cytotoxic drugs. This communication shows data on the mechanisms of these effects. Using ELISA assay, significantly increased levels of granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6) were observed. On the contrary, no detectable levels of granulocyte-macrophage colony-stimulating factor (GM-CFC) and interleukin-3 (IL-3) have been found in blood serum of IMUNOR®-treated mice. Incubation of the serum from IMUNOR®-treated mice with antibodies against G-CSF caused abrogation of the ability of the sera to stimulate in vitro growth of colonies originating from granulocyte-macrophage progenitor cells (GM-CFC). In contrast, incubation of the serum with antibodies against IL-6 did not change its colony-stimulating activity. It may be inferred from these findings that G-CSF is probably the main cytokine responsible for the granulopoiesis-stimulating effects of IMUNOR®. When the serum from IMUNOR®-treated mice with G-CSF inactivated by anti-G-CSF antibodies (but with elevated IL-6) was added to cultures of bone marrow cells together with a suboptimum concentration of IL-3, a significant increase in the numbers of GM-CFC colonies was found. Moreover, conjoint inactivation of G-CSF and IL-6 significantly decreased the numbers of GM-CFC colonies in comparison with those observed when only G-CSF was inactivated. This observation strongly suggests that though IMUNOR®-induced IL-6 is not able to induce the growth of GM-CFC colonies alone, it is able to potentiate the hematopoiesis-stimulating effect of IL-3. These findings represent a new knowledge concerning the hematopoiesis-stimulating action of IMUNOR®, a promising immunomodulatory agent.

Mycobacterium bovis BCG vaccination modulates TNF- α production after pulmonary challenge with virulent Mycobacterium tuberculosis in guinea pigs

Tumor necrosis factor-alpha (TNF-alpha) plays critical and opposing roles in the pathogenesis of tuberculosis (TB). We examined the effects of Mycobacterium bovis BCG vaccination on TNF-alpha production in three distinct guinea pig leukocyte populations before and after pulmonary infection with M. tuberculosis H37Rv. Following BCG vaccination alone, and following challenge, bronchoalveolar lavage cells (BALC), resident peritoneal cells (PC), and splenocytes (SPC) were stimulated with purified protein derivative (PPD). Before virulent challenge, BCG vaccination clearly enhanced the ability of BALC, PC and SPC to produce TNF-alpha in response to PPD stimulation ex vivo. Following challenge, the TNF-alpha production of all three leukocyte populations from BCG-vaccinated animals remained relatively constant at pre-challenged levels. In sharp contrast, 5 weeks post-challenge, all three leukocyte populations from unvaccinated animals produced very high amounts of TNF-alpha in response to PPD. Three weeks post-challenge, SPC from one of the unvaccinated animals produced higher levels of TNF-alpha but the others produced lower levels of TNF-alpha than BCG-vaccinated animals. As expected, BCG vaccination reduced the levels of virulent mycobacteria in both the lungs and spleens. Thus, BCG vaccination allows guinea pigs to modulate TNF-alpha levels in conjunction with a reduction in bacillary loads in their tissues.

Anti-hemostatic Effects of a Serpin from the Saliva of the Tick Ixodes ricinus

Serpins (serine protease inhibitors) are a large family of structurally related proteins found in a wide variety of organisms, including hematophagous arthropods. Protein analyses revealed that Iris, previously described as an immunomodulator secreted in the tick saliva, is related to the leukocyte elastase inhibitor and possesses serpin motifs, including the reactive center loop (RCL), which is involved in the interaction between serpins and serine proteases. Only serine proteases were inhibited by purified recombinant Iris (rIris), whereas mutants L339A and A332P were found devoid of any protease inhibitory activity. The highest Ka was observed with human leukocyte-elastase, suggesting that elastase-like proteases are the natural targets of Iris. In addition, mutation M340R completely changed both Iris substrate specificity and affinity. This likely identified Met-340 as amino acid P1 in the RCL. The effects of rIris and its mutants were also tested on primary hemostasis, blood clotting, and fibrinolysis. rIris increased platelet adhesion, the contact phase-activated pathway of coagulation, and fibrinolysis times in a dose-dependent manner, whereas rIris mutant L339A affected only platelet adhesion. Taken together, these results indicate that Iris disrupts coagulation and fibrinolysis via the anti-proteolytic RCL domain. One or more other domains could be responsible for primary hemostasis inhibition. To our knowledge, this is the first ectoparasite serpin that interferes with both hemostasis and the immune response.

Novel two-parameter flow cytometry (MIL4/SSC followed by MIL4/CT7) allows for identification of five fractions of guinea pig leukocytes in peripheral blood and lymphoid organs

Though the guinea pig has been an extremely useful animal model for a variety of diseases, the tools necessary to undertake a full-scale immunological analysis of the guinea pig have been lacking. For instance, traditional two-parameter forward/side scatter (FSC/SSC) flow cytometry, though effective in human and other animal models, is unable to adequately identify the distinct fractions of guinea pig peripheral blood leukocytes (PBL). We introduce here a new flow cytometric technique (MIL4/SSC followed by MIL4/CT7) which redresses this lack by identifying and characterizing five distinct fractions of PBL: neutrophils, lymphocytes, monocytes, eosinophils plus basophils, and the novel MIL4 −SSC largeCT7 high population. The MIL4 −SSC largeCT7 high cells possess cytoplasmic inclusion bodies of variable size that were positive for periodic acid Schiff (PAS). Their cell surface stained positive for the helper/inducer lymphocyte markers, T cell markers, CD45, Thy-1, asialo GM1 and FcR, but negative for B cell markers, such as membrane-type IgM, CD8 and MHC class II. The novel flow cytometric technique also allowed us to establish that the five leukocyte fractions were found in PBL, splenocytes, thymocytes and lymph node cells. Cells which were positive for inclusion bodies comprised 16.6% of splenocytes, 9.9% of PBL and 4.3% of liver cells, but were comparatively rare in lymph node cells, thymocytes, and BM cells. The novel flow cytometric technique introduced here will allow a better understanding of the response of each type of guinea pig leukocyte and thereby shed light on the diseases with which they are associated.

Peroral IMUNOR®, A Low-Molecular-Weight Immunomodulator Prepared from Disintegrated and Ultrafiltered Leukocytes, Enhances Recovery from Myelosuppression Induced by Cisplatin or 5-Fluorouracil

A single dose of IMUNOR®, a low-molecular-weight immunodulator prepared from disintegrated and ultrafiltered pig leukocytes, was found to enhance recovery of murine pool of hemopoietic progenitor cells for granulocytes and macrophages damaged by a single injection of cytotoxic drugs 5-fluorouracil or cisplatin. The best results were obtained after the treatment with IMUNOR® on days 3 or 4 after the injection of 5-fluorouracil or cisplatin. These results together with previous findings obtained in our laboratory suggest that IMUNOR® has the potential to become a part of treatment schemes in oncological practice aimed at alleviation of myelosuppression evoked by cytotoxic anti-tumor therapy.

Ultrafiltered Pig Leukocyte Extract (UPLE, IMUNOR®) Potentiates Hematopoiesis–Stimulating Effects of G-CSF In Vitro and Improves the Outcome of Treatment of Hematopoietic Radiation Damage in Mice with G-CSF

Ultrafiltered pig leukocyte extract (UPLE, Imunor®), a heterogeneous mixture of low molecular weight (<10 kD) substances released from disintegrated pig leukocytes was tested from the point of view of its hematopoiesis-modulating activities using experiments in vitro and in vivo. Attention was focused especially on evaluation of the contingent ability of UPLE to potentiate the hematopoiesis-stimulating effects of recobinant human granulocyte colony-stimulating factor (G-CSF). Experiments in vitro revealed the capability of sera from mice administered UPLE perorally (p.o.) to stimulate proliferation of progenitor cells for granulocytes and macrophages (GM-CFC) in cultures of normal bone marrow cells. In addition, UPLE, as well as sera from mice given UPLE, added to the cultures in combination with G-CSF enhanced the numbers of GM-CFC significantly over those induced by sera after administration of either of the preparations alone. In in vivo experiments, UPLE was found to increase the counts of GM-CFC per femur and femoral bone marrow cellularity in sublethally irradiated mice when administered p.o. after irradiation in combination with G-CSF in comparison with the effects of G-CSF alone. These results indicate the possibility of using UPLE, a commercially available preparation, for treatment of hematopoietic suppression of various etiology.

The normalization of guinea pig leukocyte fractions and lymphocyte subsets in blood and lymphoid tissues using a flow cytometric procedure.

Many hematological and immunological parameters remain unclear in the study of the guinea pig. In this study, we established the mean values of blood counts, the percentage of leukocyte fractions and lymphocyte subsets in blood and various lymphoid tissues of the guinea pig with a flow cytometric procedure using MIL4/SSC. The mean counts of WBC and RBC in the blood were lower, and MCV and MCH were higher than those of other rodents, resembling those of humans. Furthermore, the mean percentages of blood lymphocytes were smaller and that of granulocyte was larger than those of other rodents, resembling those of humans. We further established a flow cytometric procedure for lymphocyte subsets and clarified the mean percentages of T- and B-cells, CD4(+)-, CD8(+)- and MHC Class II(+)- T-cells, and CD4(-)CD8 (-) T-cells. The latter were morphologically larger in cell size and cytoplasm than CD4(+)- plus CD8(+) T-cells, and this subset had a significantly higher percentage in newborn animals. Furthermore, the appearance of the MHC Class II(+) T-cell subset was suggested to be a marker of hyper-activation of T-cells in BCG-immunized animals. Thus, both the novel flow cytometric procedure for leukocyte fractions and lymphocyte subsets, and the established normal values will be useful tools in studying guinea pigs as models of various diseases and biological phenomena.

DNA fragmentation in leukocytes following subacute low-dose nerve agent exposure.

The objective of the present study was to determine levels of DNA fragmentation in blood leukocytes from guinea pigs by single-cell gel electrophoresis (comet assay) after exposure to the chemical warfare nerve agent (CWNA), soman, at doses ranging from 0.1 LD50 to 0.4 LD50, once per day for either 5 or 10 days. Post-exposure recovery periods ranged from 0 to 17 days. Leukocytes were imaged from each animal, and the images analyzed by computer. Data obtained for exposure to soman demonstrated significant increases in DNA fragmentation in circulating leukocytes in CWNA-treated guinea pigs compared with saline-injected control animals at all doses and time points examined. Notably, significantly increased DNA fragmentation was observed in leukocytes 17 days after cessation of soman exposure. Our findings demonstrate that leukocyte DNA fragmentation assays may provide a sensitive biomarker for low-dose CWNA exposure.

Upregulation of interleukin-10 gene expression in the leukocytes of pigs infected with porcine reproductive and respiratory syndrome virus.

Recent studies suggest that porcine reproductive and respiratory syndrome virus (PRRSV) may have immunomodulatory effects on the host immune system by upregulating interleukin (IL)-10 gene expression. To determine the effect of PRRSV on porcine cytokine gene expression in vivo, we infected pigs with either the European or North American strain of PRRSV and monitored cytokine gene expression in peripheral blood mononuclear cells (PBMC) and bronchoalveolar lavage cells (BALC) using a multiplex PCR assay. Our results showed that both European and North American strains of PRRSV significantly upregulated IL-10 gene expression in PBMC of infected pigs from 5 days post-infection (p.i.). In addition, upregulation of IL-10 and interferon (IFN)-gamma gene expression was observed in BALC starting from 9 days p.i. The upregulation of cytokine gene expression in BALC was observed concurrent with an increased percentage of lymphocytes in the BALC population, suggesting a role for peripheral leukocytes in cytokine production in lungs. Our results showed that PRRSV infection resulted in an upregulation of IL-10 gene expression in vivo and that both European and North American strains induced comparable levels of IL-10 gene expression in the infected pigs, despite differences in the clinical signs. Our data support the notion that induction of IL-10 production may be one of the strategies used by PRRSV to modulate the host's immune responses, and this may contribute to the unique clinical picture observed following PRRSV infection.

Release of pig leukocytes during pig kidney perfusion and characterization of pig lymphocyte carbohydrate xenoantigens.

The Galalpha1-3Gal (alphaGal) antigen is considered the main xenoantigen in the pig to human species combination but other porcine antigens have to be considered such as the swine lymphocyte antigen (SLA), the blood group A/O and the Hanganutziu-Deicher (H-D) antigens. The H-D antigens are N-glycolyl-neuraminic acid (NeuGc) terminated gangliosides that are widely distributed in mammalian species but absent in humans. Upon exposure to a vascularized pig organ, the human recipient can be immunized by direct interaction with the pig tissue or/and by transfer of tissue/cells from the organ into the recipient. In the present work, we describe the release of cells from porcine kidneys upon perfusion and the expression of glycolipid based alphaGal, blood group A/O and H-D antigens in pig lymphocytes. Pig kidneys were flushed with 20 ml of NaCl or Lidocain containing 5000 U heparin, and thereafter perfused with 3000-ml perfusion solution and the cells released were counted and examined microscopically. Neutral glycolipid and ganglioside fractions were extracted from purified pig lymphocytes. The extracted components were characterized by thin layer chromatography, degradation and mass spectrometry. The expression of alphaGal and H-D epitopes on cells released from pig kidneys and purified pig lymphocytes were studied by immune electron microscopy. A total amount of about 300 x 106 leukocytes, mainly lymphocytes were released in the perfusate from the kidneys, of which about 100 x 106 cells were eluated in the 600 to 2400 ml perfusate fraction. Immunelectron microscopical analysis with Griffonia simplicifolia isolectin B4 showed staining of pig leukocytes and other cells, morphologically similar to endothelial cells, released in the perfusate. The purified porcine lymphocytes contained 930 microg neutral glycolipid (4.2 microg/mg cell protein) of which 95% was glycolipids with one to four sugar residues. Immunostaining of the neutral glycolipid fractions revealed alphaGal terminated compounds migrating in the five and 10 to 12 sugar regions and blood group A compounds in the six and eight sugar regions. Two major gangliosides NeuGc-GM3 and NeuGc-GD3 were found in the pig lymphocytes. In a patient extracorporeally xenoperfused with a pig kidney, an increased staining of both alphaGal terminated structures as well as the H-D reactive gangliosides were found in the post-perfusion serum samples. In summary, leukocytes, mainly lymphocytes are released from pig kidneys during perfusion which may contribute to immunization of human xenograft recipients.

Molecular cloning and structural analysis of the porcine homologue to CD97 antigen

CD97 is a member of a novel subfamily of leukocyte proteins that are characterized by the presence of tandemly repeated extracellular epidermal growth factor (EGF)-like domains and a seven-span transmembrane region, known as EGF–TM7. We here report the cloning of cDNA encoding the pig homologue of CD97. A pig CD97 specific probe was generated by PCR amplification of pig leukocyte cDNA, using primers based on consensus regions among the known sequences of mouse and human CD97. Screening of a pig aorta smooth muscle cDNA library identified one clone containing an open reading frame (ORF) that encoded an 18 amino acid putative signal peptide, a 141 amino acid sequence consisting of three EGF domains, a mucin-like spacer region of 276 amino acid, containing a G-protein coupling motif of 52 amino acids, followed by a 250 amino acid region containing seven membrane spanning domains and a 47 amino acid cytoplasmic tail. The amino acid sequence of the clone was 75, 67 and 59% homologous to cattle, human and mouse CD97 antigen, respectively. Therefore, it was termed pig CD97. Pig CD97 antigen shares many structural features with human, cattle and mouse CD97. RT-PCR analysis of cDNA from different pig cells and tissues showed that CD97 was highly expressed in leukocytes and lymph node cells. This is the first report describing the identification of a member of the EGF–TM7 family in the pig.