Porcine Peripheral Blood Leukocytes Research Articles

Association of attenuated mutants of Salmonella enterica serovar Enteritidis with porcine peripheral blood leukocytes

In this study, we were interested in the association of attenuated mutants of Salmonella enterica serovar Enteritidis with subpopulations of porcine white blood cells (WBC). The mutants included those with inactivated aroA, phoP, rfaL, rfaG, rfaC and fliC genes and a mutant with five major pathogenicity islands removed (ΔSPI1-5 mutant). Using flow cytometry, we did not observe any difference in the interactions of the wild-type S. Enteritidis, aroA and phoP mutants with WBC. ΔSPI1-5 and fliC mutants had a minor defect in their association with granulocytes and monocytes, but not with T- or B-lymphocytes. All three rfa mutants associated with granulocytes, monocytes and B-lymphocytes more than the wild-type S. Enteritidis did. Electron microscopy confirmed that the association correlated with the intracellular presence of S. Enteritidis and that the Salmonella-containing vacuole in the WBC infected with the rfa mutants, unlike all other strains, did not develop into a spacious phagosome. Intact lipopolysaccharide, but not the type III secretion system encoded by SPI-1, SPI-2 or the flagellar operon, is important for the initial interaction of S. Enteritidis with porcine leukocytes. This information can be used for the design of live Salmonella vaccines preferentially targeting particular cell types including cancer or tumor cells.

Sensitivity of porcine peripheral blood leukocytes to gamma irradiation in vivo, in vitro and ex vivo

Purpose: The large white pig is a useful experimental model to compare in vivo, in vitro and ex vivo sensitivity of peripheral blood leukocytes to ionising radiation. Such studies are impossible to perform in humans and laboratory rodents due to ethical reasons and body size, respectively. We analysed dose- and time-dependent changes of lymphocyte and granulocyte absolute numbers in porcine peripheral blood after either whole-body irradiation (in vivo and ex vivo experiments) or exposure of porcine whole blood to γ-irradiation (in vitro experiments).Materials and methods: CytoCount™ absolute counting beads and light scatter analysis using a flow cytometer were used to determine major leukocyte subpopulation numbers in blood samples after red cell removal.Results: Similar to other species, lymphocyte numbers significantly decreased in pigs both in vivo and in vitro in a dose-dependent manner. Most importantly, our data clearly show that reduction of lymphocyte numbers after irradiation in vivo proceeds much faster than after irradiation in vitro and that granulocyte changes depend only on the time of analysis after irradiation.Conclusions: All three tested experimental arrangements demonstrated the radiosensitivity of lymphocytes and the radioresistance of peripheral blood granulocytes. These in vivo and in vitro approaches, as well as the newly introduced ex vivo observations, appear to be relevant to biodosimetry.

Gene expression profiling of porcine peripheral blood leukocytes after infection with Actinobacillus pleuropneumoniae

The gene expression profile of peripheral blood leukocytes (PBL) from extreme performing pigs after infection with Actinobacillus pleuropneumoniae was analysed using a custom complementary DNA (cDNA) microarray and quantitative reverse transcription-PCR (qRT-PCR). Four high performing animals with low disease-score (HP), three low performing animals with high disease-score (LP) and one medium performing animal with medium disease-score (MP) were selected for microarray profiling. PBL RNA from these eight pigs collected before and at 24 h after APP infection, was examined. The study identified 92 genes that were up-regulated and four genes that were down-regulated in PBL RNA from HP pigs compared to LP pigs. The majority of differentially expressed (DE) genes were identified by virtue of their elevated expression in the HP animals at 24 h post-infection. A large number of annotated DE genes are involved in innate immune response pathways. The gene expression profile of 10 DE candidate genes was further explored across the entire pig population in the same infection trial using qRT-PCR. Considerable animal-to-animal variation in PBL gene expression was observed, especially in the LP group. The qRT-PCR analysis suggested that only one true LP pig might be present in this study, which contributes significantly to the differential expression profile of the selected genes in HP animals following APP infection. This study has therefore identified a set of genes which could serve as molecular indicators for an effective immune response to APP in pigs and which could also serve as source for gene marker development in molecular genetics studies of heritable immune traits.

Characterization of new monoclonal antibodies against porcine lymphocytes: molecular characterization of clone 7G3, an antibody reactive with the constant region of the T-cell receptor δ-chains

A battery of mouse monoclonal antibodies (mAbs) reactive with porcine peripheral blood (PB) leukocytes was generated. Among the mAbs, 6F10 was found to react probably with cluster of differentiation (CD)8 alpha-chain, while 7G3 and 3E12 were found to recognize gammadelta T-cells, as revealed by two-color flow cytometric and immunoprecipitation studies. 7G3 was shown to react with the constant (C) region of the T-cell receptor (TCR) delta-chain by the following facts: (1) 7G3 immunoprecipitated full-length TCR delta-chain protein fused with glutathione S-transferase (GST) produced by Esherichia coli and (2) 7G3 reacted with TCR delta-chain expressing Cos-7 cells transfected with either full-length or N-terminal deleted mutant cDNA, but did not react with Cos-7 cells transfected with C-terminal deleted mutant TCR delta-chain cDNA. All three mAbs produced high-quality immunostaining results on frozen sections, revealing a distinct distribution of gammadelta T-cells and CD8(+) cells. This report precisely characterizes mAbs against porcine TCR for the first time, facilitating molecular biological investigations of the porcine immune system.

Shiga Toxin Binding to Isolated Porcine Tissues and Peripheral Blood Leukocytes

Shiga toxin (Stx) binding sites in porcine tissues and leukocytes were identified by the use of Stx overlay and anti-CD77/Gb3 immunoassays. Stx1 and Stx2 bound to similar tissue locations and leukocytes, although some differences were noted. Previously unreported Stx binding sites were identified in kidney tubules, intestinal lymphoid aggregates, sinusoidal liver cells, alveolar macrophages, and peripheral blood leukocytes.

Activation of human endothelial cells by mobilized porcine leukocytes in vitro: implications for mixed chimerism in xenotransplantation.

The induction of immunologic tolerance to pig antigens in primates may facilitate the development of successful clinical xenotransplantation protocols. The infusion of mobilized porcine peripheral blood leukocytes (PBPC, consisting of approximately 2% peripheral blood progenitor cells) into preconditioned baboons, intended to induce mixed hematopoietic cell chimerism, however, results in a severe thrombotic microangiopathy (TM) that includes vascular injury, microvascular thrombosis, and pronounced thrombocytopenia. Because the mechanisms responsible for TM are unclear, we have explored the effects of PBPC on human umbilical vein endothelial cell (HUVEC) activation. Confluent HUVEC monolayers were established in 96-well cell culture clusters. PBPC were mobilized from miniature swine with porcine interleukin 3 (pIL-3), porcine stem cell factor (pSCF), and human granulocyte-colony stimulating factor (hG-CSF) and were collected by leukapheresis. PBPC were added to HUVEC (0-1x10(7) PBPC/well) for 3- to 24-hr periods and, with cell-based ELISA techniques, surface levels of E-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) were measured. In some cases, peripheral blood leukocytes (PBL) were collected from pigs that did not receive pIL-3, pSCF, or hG-CSF and were added to HUVEC. PBPC were also sorted into subsets of CD2- cells, CD2+ cells, and cellular debris, each of which were added separately to HUVEC. Transwell permeable membrane inserts were placed over HUVEC to prevent direct cell-cell contact with PBPC in some instances. PBPC from different pigs (n=6) induced an increase in the expression of E-selectin, VCAM-1, and ICAM-1 to levels 5, 4, and 2 times greater than baseline, respectively. ICAM-1 expression reached maximum levels after the addition of 6x10(5) PBPC/well. Expression of E-selectin and VCAM-1 increased further with the addition of greater numbers of PBPC, reaching maximum levels after the addition of 1x10(7) PBPC/well. PBPC-induced up-regulation of E-selectin, VCAM-1, and ICAM-1 had a maximum effect after approximately 6 hr, 12 hr, and 6 to 9 hr, respectively (n=3). The effects of fresh and frozen PBPC on HUVEC were similar (n=2). Compared to PBPC, PBL induced higher levels of E-selectin, VCAM-1, and ICAM-1 on HUVEC (n=2). The addition of CD2- cells to HUVEC induced an increase in E-selectin and VCAM-1 to levels 4 times greater than baseline, whereas the addition of CD2+ cells or debris did not elicit a substantial effect (n=2). Transwell permeable membranes prevented PBPC-induced up-regulation of E-selectin, VCAM-1, and ICAM-1 on HUVEC (n=2), suggesting that the mechanism of activation requires direct cell-cell contact. Porcine PBPC activate HUVEC, as suggested by an increase in surface E-selectin, VCAM-1, and ICAM-1 levels, and have a maximum effect after 9 hr. Freezing of PBPC does not affect PBPC-induced activation of HUVEC. PBL induce greater activation of HUVEC than do PBPC. CD2- cells are primarily responsible for PBPC-induced activation of HUVEC and direct cell-cell contact is required. Removal of CD2- cells before the administration of PBPC or the use of agents that interrupt PBPC-endothelial cell interactions may prevent or treat TM in baboons.

Modulation of platelet aggregation in baboons: implications for mixed chimerism in xenotransplantation. I. The roles of individual components of a transplantation conditioning regimen and of pig peripheral blood progenitor cells.

The induction of tolerance to pig antigens in primates may facilitate the development of successful clinical xenotransplantation protocols. The infusion of mobilized porcine peripheral blood leukocytes (PBPCs, comprised of approximately 2% peripheral blood progenitor cells) into splenectomized preconditioned baboons, intended to induce mixed hematopoietic cell chimerism, however, results in a severe thrombotic microangiopathy (TM) that includes pronounced thrombocytopenia. Because the mechanisms responsible for this phenomenon are unclear, we have explored the effects of individual components of the conditioning regimen, of therapeutic adjuncts, and of PBPCs on platelet aggregation. Groups of splenectomized baboons (n = at least 2 in each group) were treated with single components of the conditioning regimen--whole body irradiation (WBI), antithymocyte globulin (ATG), extracorporeal immunoadsorption (EI), mycophenolate mofetil (MMF), anti-CD40L monoclonal antibody (mAb), cobra venom factor (CVF), pig hematopoietic growth factors (interleukin-3 (pIL3) and stem cell factor (pSCF))--or with potential adjuncts, prostacyclin (PGI2), heparin, methylprednisolone, and eptifibatide (a GPIIb/IIIa antagonist). Blood samples were collected and platelet-rich plasma (PRP) was prepared. Using light transmission aggregometry, the extent of aggregation induced by platelet agonists (thrombin, adenosine diphosphate (ADP), collagen, ristocetin, and arachidonic acid) was determined in vitro. PRP was also prepared from untreated baboons, PBPCs were added, and platelet aggregation was measured in the absence of exogenous platelet agonists. WBI, ATG, MMF, anti-CD40L mAb, CVF, pIL3, pSCF, and PGI2 had no effect on purified baboon platelet aggregation profiles in vitro. Eptifibatide markedly inhibited platelet aggregation induced by all standard agonists. EI or heparin inhibited thrombin-induced platelet aggregation, and methylprednisolone inhibited ADP-induced aggregation to some extent. In vitro addition of PBPCs to PRP stimulated platelet aggregation in the absence of any agonists. Prior treatment of baboons with eptifibatide, however, inhibited this effect by 70% to 80%. Aggregation of baboon platelets and TM is directly induced by PBPCs, but not by individual components of the conditioning regimen. GPIIb/IIIa antagonists, such as eptifibatide, interfere directly with xenogeneic PBPC-platelet interactions and may further ameliorate TM in the pig-to-primate model.

Modulation of platelet aggregation in baboons: implications for mixed chimerism in xenotransplantation. II. The effects of cyclophosphamide on pig peripheral blood progenitor cell-induced aggregation.

The induction of tolerance to pig antigens in primates may facilitate the development of successful clinical xenotransplantation protocols. The infusion of mobilized porcine peripheral blood leukocytes (PBPCs, comprised of approximately 2% peripheral blood progenitor cells) into splenectomized preconditioned (whole body irradiation (WBI)-based) baboons, intended to induce mixed hematopoietic cell chimerism, however, results in a severe thrombotic microangiopathy (TM) that includes pronounced thrombocytopenia. Previous studies have indicated that the infused PBPCs initiate platelet aggregation, but that the various individual components of the conditioning regimen are not associated with the development of aggregation. We have now investigated the effects of cyclophosphamide (CPP) as an alternative to WBI on platelet aggregation. Splenectomized baboons (n=3) were treated with CPP. Blood samples were collected and platelet-rich plasma (PRP) was prepared. Using light transmission aggregometry, the extent of aggregation induced by platelet agonists (thrombin, adenosine diphosphate (ADP), collagen, ristocetin, and arachidonic acid) was determined in vitro. PRP was also prepared from untreated baboons and from baboons receiving CPP, PBPCs were added, and platelet aggregation was measured in the absence of exogenous platelet agonists. CPP markedly inhibited platelet aggregation induced by all standard agonists. In vitro addition of PBPCs to PRP stimulated platelet aggregation in the absence of any agonists. Prior treatment of baboons with CPP, however, inhibited this effect by 55% to 65%. TM was not evident in baboons receiving a conditioning regimen that included CPP instead of WBI. Aggregation of baboon platelets and TM is directly induced by PBPCs. CPP has direct anti-aggregatory properties and may provide an alternative strategy to WBI in this pig-to-primate model intended to induce mixed hematopoietic cell chimerism.

Primary structure and functional characterization of a soluble, alternatively spliced form of B7-1.

Recent studies have suggested that soluble forms of B7-1 and B7-2 may exist, but transcripts that code for these molecules have not been previously described. In this study, we report the cloning and characterization of an alternatively spliced soluble form of porcine B7-1 (sB7-1) that lacks exons coding for both the transmembrane and cytoplasmic domains. Northern blot analysis of RNA from alveolar macrophages revealed an approximate 3:1 ratio of the transmembrane form of B7-1 mRNA relative to sB7-1 mRNA. Porcine B7-1 was present on the surface of both B and T cells following stimulation with PMA/ionomycin. A histidine-tagged form of porcine sB7-1 (sB7-1-His) interacted with both CD28 and CTLA-4, and effectively blocked IL-2 production from human responder cells stimulated with PHA and either porcine or human stimulator cells. In addition, sB7-1-His inhibited human T cell proliferation in response to porcine or human peripheral blood leukocytes. This study is the first report of an alternatively spliced form of B7 that codes for a soluble protein. Furthermore, these data demonstrate that porcine B7-1 interacts with the human receptors CD28 and CTLA-4, suggesting a potential role for this molecule in pig to human xenotransplantation. Possible physiological functions for the soluble form of B7-1 are discussed.

Preliminary characterization of protein binding factor for porcine reproductive and respiratory syndrome virus on the surface of permissive and non-permissive cells.

Summary. In its natural host, porcine reproductive and respiratory syndrome virus (PRRSV) has been reported to have a restricted tropism for cells of the monocyte/macrophage lineage. To date, cloned monkey kidney cell lines, such as MARC-145 and CL2621 cells which have been established from MA-104 cells, are the only non-porcine cells known to support PRRSV replication. In the present study, a binding assay was set up to follow by flow cytometry the attachment of PRRSV on the surface of porcine and non-porcine cells. PRRSV was found to be able to bind permissive cells like porcine alveolar macrophages and MARC-145. Further binding assays with porcine peripheral blood leukocytes showed that only monocytes can attach the virus. By their lack of binding factor, lymphocytes appeared to be refractory to PRRSV infection. Pre-incubation of MARC-145 cells with chymotrypsin and pronase E, but not neuraminidase, blocked their binding activity for PRRSV. The binding activity of the protease-treated cells was regenerated 8 hours after treatment, but cells remained unable to bind PRRSV if maintained in the presence of cycloheximide, thus confirming the proteinic nature of the specific binding factor(s). Experiments conduced with cells that have been previously characterized as non-permissive to PRRSV infection showed that many of them were able to bind the virus. Data obtained suggest that interaction of PRRSV with a specific binding factor on the surface of some cells is not sufficient to lead to a productive infection, and that a second putative receptor or other phenomena are probably required to pursue later events.

Expression of porcine endogenous retrovirus in peripheral blood leukocytes from ten different breeds.

The expression of porcine endogenous retroviruses (PERV) was investigated in primary porcine peripheral blood leukocytes (PBL) of ten different pig breeds. The data suggest that PERV exists in all porcine PBL. A new retroviral element, a foamy-like pol-related sequence, was also detected in PBL. Three types of PERV were detected in almost every animal. The breeding of PERV-free pigs is likely to be difficult. Further studies are required to assess the infectious disease risks associated with xenotransplantation.

Influence of cold ischemia time, pretransplant anti-porcine antibodies, and donor/recipient size matching on hyperacute graft rejection after discordant porcine to cynomolgus kidney transplantation.

Organs transplanted between phylogenetically disparate species, such as from the pig into the primate, are subject to hyperacute rejection (HAR). This form of xenograft rejection is mediated by preformed natural antibodies and is believed to occur invariably in discordant xenografts thus leading to rapid destruction and complete thrombosis of the graft. Recent data, however, have shown that in the porcine to cynomolgus monkey setting, HAR is not inevitably seen after porcine kidney transplantation. The influence of preoperative antiporcine antibody levels in the recipient, cold ischemia time, and donor organ weight on the onset of HAR was investigated by using unmodified large white pigs (aged 3-12 weeks) as organ donors and adult cynomolgus monkeys (aged 1.5-3.5 years) as recipients. Porcine kidney xenotransplantation was performed in either a non-life-supporting model (n=7) or in a life-supporting model (n=8). In both models, no correlation was found between cold ischemia time and HAR. When preoperative anti-porcine antibody levels were investigated, a significant increase in incidence of HAR was observed in animals with elevated anti-porcine IgM (P<0.05) but not IgG levels (P=NS). Interestingly, although 5 of 12 grafts with an organ weight of less than 50 g underwent HAR, none of three grafts with a donor organ weight of more than 70 g showed signs of HAR. In addition, all three larger grafts showed intraoperative and postoperative urine production, although only in 1 (48 g) of the 12 grafts weighing less than 50 g primary graft function was observed. In one animal, a second porcine kidney (23 g) was successfully transplanted (without HAR) immediately after HAR and subsequent removal of a first porcine kidney (20 g). These results indicate that in the porcine to cynomolgus monkey setting anti-porcine IgM rather than IgG anti-porcine antibody levels seem to be of predominant importance for the induction of HAR. By increasing the donor organ size and weight the frequency of the onset of HAR can be at least reduced. This is most likely due to immunoabsorption of the recipients preformed antibodies in the porcine kidney without lethal damage for the graft.

Interaction of pseudorabies virus with porcine peripheral blood lymphocytes.

To examine effects of pseudorabies virus (PrV) on immune cells, we investigated the ability of PrV to infect and replicate in porcine peripheral blood leukocytes (PBLs). Flow cytometric analysis revealed a leukopenia after challenge, with loss of 40% of circulating monocytes and 50% of circulating lymphocytes. Virus was isolated from PBLs of challenged pigs by cocultivation with porcine kidney cells, indicating that PBLs were infected in vivo. Presence of virus in PBLs coincided with the appearance of neurological signs 1 to 2 days prior to death. Lymphocytes stimulated with mitogens and infected in vitro sustained a low-level infection (10(5) median tissue culture infective dose per 2 x 10(6) cells). In vivo challenge perturbed the CD4/CD8 ratio of circulating lymphocytes. Survival was associated with low CD4/CD8 ratios and high levels of CD8+ cells. Mortality was associated with low levels of CD8+ cells and CD4/CD8 ratios greater than one. A maturational deficiency of CD8+ cells was found in young pigs. Our results support a mechanism of PrV immunosuppression through direct infection of circulating lymphocytes, with CD8+ T lymphocytes being important for survival.

Analyses of the primary in vitro responsiveness of non-immune porcine peripheral blood mononuclear cells with reference to immunization by African swine fever virus antigen and treatment with leucine methyl ester.

Peripheral blood mononuclear cells (PBMC) from non-immune pigs were immunized in vitro using African swine fever (ASF) virus antigen with concomitant mitogenic stimulations known to have varying effects on B and T lymphocyte activity. None of these conditions, including those previously reported as being successful for the in vitro immunization of non-immune porcine PBMC with ASF virus antigen, supported the induction of specific antibody. Due to the reports on in vitro immunization of human PBMC, attempts were made to control suppressor cell activity in the porcine PBMC from non-immune pigs through L-leucine methyl ester (Leu-OMe) treatment. Upon immunization of the Leu-OMe treated PBMC with ASF virus antigen, concomitant with mitogen or cytokine stimulations, no specific antibody production was detected. Nevertheless, aspecific porcine immunoglobulin secretion was observed. Further analysis of the PBMC responsiveness demonstrated that 2.5 mM Leu-OMe (the dose recommended for use with human PBMC) suppressed mitogen-induced porcine lymphocyte proliferation, but in the absence of any detectable cytotoxicity. In fact, both anti-ASF virus antigen specific immunization and stimulation with "T-lymphocyte" mitogens were suppressed, whereas pokeweed mitogen stimulation of B lymphocyte aspecific immunoglobulin secretion was unaffected. Consequently, it was not possible to immunize in vitro non-immune porcine PBMC with ASF virus antigen as had been previously reported, nor was it possible to transfer the technology successfully used with non-immune human PBMC to the in vitro stimulation/immunization of non-immune porcine PBMC. The furtherance of this work will require greater insight into the immunobiological parameters and dynamics of the stimulation of non-immune porcine peripheral blood leukocytes, which are not as simple as previously reported, nor the same as identified with human PBMC.

Molecular cloning of a gene encoding porcine interferon-beta.

A gene encoding the porcine interferon-beta (poIFN-beta) was cloned from a genomic library. The sequence of a potential intronless coding region as well as 1,265 bp of the 5'- and 277 bp of the 3'-flanking regions is presented. The gene is predicted to encode a mature protein of 165 amino acids and a signal peptide of 21 amino acids. This probable poIFN-beta shows high homology (63%) with human (hu) IFN-beta at the amino acid level, but less with porcine (po) IFN-alpha 1 (32%). It contains three cysteines and three potential N-glycosylation sites. A region of the 5' flank (-116 to -159) of the gene is homologous to the IFN gene regulatory element (IRE) of the huIFN-beta gene which mediates virus inducibility. Southern blot analysis indicates that the poIFN-beta gene is present as a single copy in the porcine genome. Its expression in porcine peripheral blood leukocytes stimulated in vitro by pseudorabies virus (PRV) was demonstrated at the RNA level both by Northern blot analysis and by in situ hybridization. The latter approach in addition detected only about one IFN-beta mRNA-containing cell per 2,000 PRV-stimulated porcine leukocytes, a frequency in the same range as that for leukocytes containing IFN-alpha mRNA.

Effects of opiates on transforming growth factor beta (TGF-β) expression in porcine peripheral blood leukocytes

Effects of opiates on transforming growth factor beta (TGF-β) expression in porcine peripheral blood leukocytes

Flow Cytometric Analysis of Porcine Peripheral Blood Leukocytes Infected With Pseudorabies Virus

The susceptibility of fractionated porcine peripheral blood leukocytes (PBL) to pseudorabies virus (PRV) was studied by flow cytometry and defined by viral antigen expression. Viral antigens on the surface of infected cells and cell viability were evaluated by forward angle light scatter (FALS), 90-degree light scatter (90LS), green fluorescence (FITC-anti-PRV), and red fluorescence (propidium iodide). Approximately 10% of infected mononuclear cells from healthy pigs expressed cell-surface PRV antigen. Cell-surface fluorescence and cell type were confirmed by sorting live positive cells for microscopy. In sorted positive samples, the lymphocyte versus monocyte ratio was approximately 50%:50%, defined by morphology. Positive lymphocytes represent 5.75% of total mononuclear cells. When cells were stimulated with phytohemagglutinin (PHA) and lipopolysaccharide (LPS) before infection, mitogen-stimulated T-lymphoblasts showed increased susceptibility to PRV (40.7% positive) and died of infection. Monocytes, particularly adherent monocytes, were highly susceptible (40% to 71.4% positive). Granulocytes appeared to be refractory. The relative susceptibility of various PBL populations was compared by normalizing lymphocyte susceptibility to 1 as follows: resting total lymphocytes (1); B-lymphocytes (0.67); T-lymphoblasts (7.08); total monocytes (4.27); adherent cells (4.03 to 10.88); adherent monocytes (6.95 to 12.42); granulocytes (0.24). These findings suggest a possible mechanism by which PRV could have an immunosuppressive effect as well as a pathway for dissemination of PRV.

Inhibition of transmissible gastroenteritis coronavirus (TGEV) multiplication in vitro by non-immune lymphocytes

SummaryIn vitro studies were undertaken to examine the effects of non-immune porcine peripheral blood leukocytes (PBL) on a Coronavirus infection due to transmissible gastroenteritis virus (TGEV). The assay consisted of TGEV-infected epithelial cells expressing viral antigens on the cell surface and producing low amounts of interferon (IFN). Non-immune PBL were found to limit virus replication at an effector-to-target ratio of 100/1 even when effector cells were depleted of phagocytic cells. Neutralizing anti-IFN antibodies did not abrogate the effect. PBL from newborn animals were as effective as adult cells, whereas fibroepithelial cells, human and mouse lymphoid cells did not exert antiviral effects. Under similar conditions, PBL from adult animals could lyse TGEV-infected cells even in the presence of anti-IFN antibodies. However, newborn PBL were not cytotoxic. Moreover, depletion of NK cells by monoclonal antibodies plus complement did not alter the inhibitory effect. These latter observations suggest that virus multiplication-inhibition effects and cytotoxic (or NK) activities are unrelated.

Biotechnological production and functional characterization of leukocytic cytokine(s) with cytotoxic effect on resting but not activated thymocytes

In the present report we describe the analysis of suppressive and cytotoxic factor(s) derivedfrom Con A-induced porcine leukocyte culture supernatant solution(s) (PCAS). The supernatant solution(s) had been produced on a biotechnological scale using porcine peripheral blood leukocytes isolated from 200 to 1000 l of porcine blood. The supernatant solution(s) were tested for their effect during murine T cell activation. We found that crude supernatant solution(s) contain suppressive and cytotoxic factor(s) (SF) which have a similar selectivity for non-activated thymocytes as suppressive factor(s) previously detected in the supernatant solution(s) of allo-antigen- stimulated murine spleen cell cultures. SF was obtained from crude serum-free culture supernatants by fractionated ammonium-sulphate precipitation between 35 and 45% saturation. SF has the following characteristics: (i) its release into the culture supernatant is dependent on mitogenic stimulation, (ii) it acts during early stages of T lymphocytic activation, (iii) the activity towards naive thymocytes is most likely caused by an irreversible cytolytic mechanism, and (iv) phenotypically and functionally immature thymocytes are preferentially affected through exposure to SF.

6,7,4′-trihydroxyisoflavan: A potent and selective inhibitor of 5-lipoxygenase in human and porcine peripheral blood leukocytes

The effect of 6,74′-trihydroxyisoflavan on human platelet 12-lipoxygenase and human and porcine PMNL 5-lipoxygenase activities has been studied. 6,7,4′-Trihydroxyisoflavan was found to inhibit 5-lipoxygenase more strongly than 12-lipoxygenase; its concentration for 50% inhibition (IC 50) was 1.6 μm for human and porcine 5-lipoxygenase adn 22 μM for human platelet 12-lipoxygenase. Inhibition of microsomal cyclooxygenase from ram seminal vesicles is exhibited at much higher concentrations of 6,7,4′-trihydroxyisoflavan (IC 50 = 200 μM).