Inhibition of complement and CD14 attenuates the Escherichia coli-induced inflammatory response in porcine whole blood.

The innate immune response is a double-edged sword in systemic inflammation and sepsis. Uncontrolled or inappropriate activation can damage and be lethal to the host. Several studies have investigated inhibition of downstream mediators, including tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta). Emerging evidence indicates that upstream inhibition is a better therapeutic approach for attenuating damaging immune activation. Therefore, we investigated inhibition of two central innate immune pathways, those of complement and CD14/Toll-like receptor 4 (TLR4)/myeloid differentiation protein 2 (MD-2), in a porcine in vitro model of Escherichia coli-induced inflammation. Porcine whole blood anticoagulated with lepuridin, which did not interfere with the complement system, was incubated with E. coli lipopolysaccharide (LPS) or whole bacteria. Inhibitors of complement and CD14 and thus the LPS CD14/TLR4/MD-2 receptor complex were tested to investigate the effect on the inflammatory response. A broad range of inflammatory readouts were used to monitor the effect. Anti-CD14 was found to saturate the CD14 molecule on granulocytes and completely inhibited LPS-induced proinflammatory cytokines in a dose-dependent manner. Anti-CD14 significantly reduced the levels of the E. coli-induced proinflammatory cytokines TNF-alpha and IL-1beta, but not IL-8, in a dose-dependent manner. No effect on bacterial clearance was seen. Vaccinia complement control protein and smallpox inhibitor of complement enzymes, two Orthopoxvirus-encoded complement inhibitors, completely inhibited complement activation. Furthermore, these agents almost completely inhibited the expression of wCD11R3, which is associated with CD18 as a beta2 integrin, on porcine granulocytes and decreased IL-8 levels significantly in a dose-dependent manner. As expected, complement inhibition reduced bacterial clearance. We conclude that inhibition of complement and CD14 attenuates E. coli-induced inflammation and might be used as a therapeutic regimen in gram-negative sepsis along with appropriate treatment with antibiotics.

Effect of oral antigen and antibody exposure at birth on subsequent immune status. A study in neonatal pigs.

BACKGROUND: The purpose of this work was to investigate the effects of early low-level exposure to either antigen or antibody alone on subsequent immune responses in entirely immunologically naïve animals. This is impossible in species with a permeable placenta such as rodents or humans, where both antigen and antibody can be transferred in utero. It is, however, possible in pigs, due to the impermeable placenta of the sow. Thus, neonatal piglets were used for this study. METHODS: Newborn piglets were exposed to ovalbumin (OVA) at dosages similar to those used in rodents to sensitise, as well as to serum containing anti-OVA antibodies. RESULTS: Both single low doses of OVA (10 and 1,000 mg per animal) induced classical oral tolerance following a systemic challenge: both doses reduced specific systemic IgG responses and tertiary in vitro recall proliferative responses by splenocytes and especially by mesenteric lymph node (MLN) cells. Additionally, dietary challenge had phenotypic effects on helper T cells in MLN, which could be reversed by OVA at birth. In contrast, giving antibody as serum collected from hyperimmune or orally tolerant pigs had no functional effects. CONCLUSIONS: Overall, our data support the hypothesis that contrary to previous work in rodents, very early exposure of neonatal pigs to a single small dose of antigen can reduce subsequent immune responses. This may have implications for human health. However, although these data point to a reducing/regulatory effect of low doses of antigen in very young animals, they cannot be extrapolated directly to allergy.

Effects of Bacillus cereus var. toyoi on immune parameters of pregnant sows.

Changing immune parameters during pregnancy have previously been reported in humans and cattle, and have been suggested to contribute to increased susceptibility to infections. However, data regarding immune parameters during pregnancy in sows are rare. In this study, we investigated the peripartal immune status of sows using phenotypical (FACS analysis) as well as functional (proliferation assays, cytokine analysis) parameters of peripheral blood mononuclear cells (PBMCs) in pregnant sows. In previous studies, we reported a modulation of the immune system after feed supplementation of the probiotic Bacillus cereus var. toyoi in piglets [Schierack, P., Wieler, L.H., Taras, D., Herwig, V., Tachu, B., Hlinak, A., Schmidt, M.F., Scharek, L., 2007. Bacillus cereus var. toyoi enhanced systemic immune response in piglets. Vet. Immunol. Immunopathol. 118, 1-11]. Here, we extended these previous studies to include investigations of possible probiotic effects on the peripartal immune status of sows and their reproductivity. We show that immune parameters of sows change during pregnancy, the proliferative response of PBMCs to several bacterial antigens in control animals decreased from days 90 to 30 ante partum. Relative numbers (%) of CD3+CD8+, CD4+, cytotoxic T, CD14+ and CD21+ cells were reduced compared to non-pregnant sows. In contrast, the proliferative response of PBMCs of probiotic-treated sows increased during pregnancy. Bacterial antigens primarily stimulated the proliferation of naïve CD21+ cells and the relative CD21+ cell numbers were elevated in the probiotic group in the absence of effects on other immune cell populations. The clinical and microbial status of both control and probiotic sows was similar, excluding pre-existing health problems or infections as responsible for the immunological changes, and feed supplementation also had no significant effects on reproductivity. The results suggest that the probiotic B. cereus var. toyoi can alter the proliferative response of lymphocytes and affects the immune cell population ratios of pregnant sows. How and to what extent this may affect health and reproductivity should be the focus of further studies.

Immune development in jejunal mucosa after colonization with selected commensal gut bacteria: a study in germ-free pigs.

The immunological structure of the porcine jejunal lamina propria in germ-free piglets was compared with that of their counterparts associated with two strains of commensal Escherichia coli, A0 34/86 serotype O83:K24:H31 and the O86 E. coli strain, up to 20 days post-colonization. In the antigen-presenting compartment, both dendritic cells (DC) and cells expressing CD163, probably macrophages were investigated. In addition we also assessed the number of CD2+/CD3+ (T) cells. In contrast to some previous reports, we show a total lack of both DC and T cells for germ-free animals in the diffuse lymphoid tissue of villi and crypts of the jejunum. Association with either strain of commensal E. coli had a profound effect on the immune structure and resulted in extensive recruitment of DC to the lamina propria and of T cells to epithelium and lamina propria. The data suggest that the earliest immigrant cells were monocytes, which soon acquired the phenotype of mucosal DC. T cells migrated in at a slightly slower rate. Nevertheless, the response could be extremely rapid: within 3 days of colonization with O83, the magnitude of this response was comparable to that observed 20 days post-colonization.

Progress in the discovery and definition of monoclonal antibodies for use in feline research.

The practice of veterinary medicine and research into both animal diseases and animal models of human disease are restricted by the scarcity of monoclonal antibodies (mAb) that react with animal proteins. One way to enlarge the repertoire of mAb to animal leukocyte differentiation antigens (LDA) is to test mAb specific to other species for cross-reactivity to the species of interest. We have tested a panel of 380 commercially available anti-human mAb for cross-reactivity to feline LDA. Twenty-six of these mAb cross-react with cat LDA and 19 others are of questionable cross-reactivity. Definition of mAb specificity in the cat is being investigated by multi-color flow cytometry (FCM) to compare test mAb specificity with that of mAb to known feline LDA. The addition of these cross-reactive mAb to the anti-feline mAb currently available will enhance studies in comparative medicine.

Interactions between Streptococcus suis serotype 2 and cells of the myeloid lineage in the palatine tonsil of the pig.

Streptococcus suis is a major pathogen in pigs and causes significant morbidity and mortality in herds world-wide. A major problem with S. suis is the presence of asymptomatic carrier animals which can spread the organism within and between herds. The palatine tonsil is one of the main sites where the organism can be recovered, both in infected and carrier animals. The use of multiple-colour immunohistology allowed identification of the cell types associated with bacteria in the tonsils of infected gnotobiotic piglets. Bacteria were never associated with T-cells or B-cells but were always associated with cells of the myeloid lineage. Expression of CD16 and CD163 on these leukocytes suggested an association with mature macrophages in tonsil, which may lead to clearance or control of the micro-organism.

Ontogeny of systemic cellular immunity in the neonatal pig: correlation with the development of post-weaning multisystemic wasting syndrome.

The aetiology of porcine post-weaning multisystemic wasting syndrome (PMWS) is poorly understood. Porcine circovirus type 2 (PCV-2) is an essential component of the experimental disease model for PMWS: however, evidence from experimental and field studies indicates that additional factors play a critical role in the aetiopathogenesis of PMWS. Current candidates include (1) immune stimulation (for example, via co-infection or vaccination), and (2) a novel infectious agent. A prospective, longitudinal case-control study was designed to investigate molecular triggers in leucocytes of neonatal piglets that may predispose to the development of PMWS. Blood samples were collected weekly from pigs (n=125) within five farms, from 1 week to 8 weeks of age: that is, before the appearance of clinical signs. Four colour flow cytometry was used to investigate changes in subsets of peripheral blood mononuclear cells, using monoclonal antibodies against the following cell associated markers; sIgG, CD3, MHCII dR, CD14, CD4a, CD8a, CD45RC, CD25, SWC3a, SWC8, CD163 and CD45. Sampling and laboratory analysis was supported by monitoring of clinical signs from 1 week to 20 weeks of age, or until disease supervened. At the conclusion of the study, 68 pigs (54%) were classified in Group 1 (no signs of clinical disease), 34 pigs (27%) in Group 2 (signs of clinical disease but not characteristic of PMWS), 17 pigs (14%) in Group 3 (suspect PMWS case) and 5 pigs (4%) in Group 4 (PMWS case). A single case of Porcine Dermatitis and Nephropathy syndrome (PDNS) was also diagnosed. Significant changes with age were demonstrated in clinically normal, neonatal pigs (Group 1), including an increase in B-cells and T-cells, and an increase in the proportion of total T-cells expressing MHCII. Within the T-cell subset, the proportion of CD8(+high) CD4(-) T-cells increased, in addition to the proportion of CD4(+) T-cells co-expressing CD8. Of the factors recorded, farm was found to have a highly significant effect on immune system development in the neonate. Comparison of Groups 1 and 4 cases identified significant differences between pigs which remained normal and those which subsequently developed PMWS. Pigs which went on to develop PMWS had a greater proportion of T-cells expressing MHCII in early life, higher mean intensity of expression of MHCII on T-cells, higher mean intensity of expression of MHCII on B cells and higher expression of CD25 on CD45RC(-) T-cells. These findings suggest that lymphocyte activation may be a key early event in the aetiology of PMWS.

Development of the palatine tonsil in conventional and germ-free piglets.

Palatine tonsils, like the Peyer's patches, are considered to be major inductive sites for the mucosa-associated lymphoid tissue (MALT), providing sampling and effector functions for the upper respiratory tract. Consistent with this, they have the architecture required of a classic inductive site (B-cell follicles, immunoglobulin class switching and the presence of naïve and memory T-cells). Here we show that much of this architecture develops after birth in the neonatal piglet, the numbers of T-cells, B-cells and accessory cells increasing with age. Conventional piglets also had higher levels of activated and memory T-cell subsets than germ-free piglets, consistent with development occurring as a result of microbial stimulus. The results suggest that the microbial environment influences the development of the tonsil immunological architecture. Given the role of the tonsil in induction of mucosal responses, this raises questions as to the effectiveness of the tonsil in dealing with colonising organisms in the neonate.

An ex vivo model for reperfusion of laryngotracheal grafts.

OBJECTIVE: To describe the development of an ex vivo model to facilitate the study of ischemia-reperfusion injury in laryngotracheal grafts taken from Minnesota minipigs. STUDY DESIGN: This is a descriptive study. METHODS: Laryngotracheal grafts from Minnesota minipigs were harvested and placed in cold storage for 3 hours. Autologous blood was used to reperfuse the graft for up to 8 hours using the described reperfusion model. RESULTS: By altering retrieval technique and pressures within the graft, we demonstrated healthy grafts after 8 hours of reperfusion. CONCLUSIONS: Ex vivo reperfusion offers a range of experimental advantages over in vivo reperfusion including close control of interventions, ease of outcome measurement, and reduction in animal use. This model is now ready to be used for further studies of interventions to reduce ischemia-reperfusion injury in these grafts.

Antigen presenting cells in mucosal sites of veterinary species.

The ability of antigen presenting cells, in particular dendritic cells, to integrate a variety of environmental signals, together with their ability to respond appropriately by initiating either tolerance or defensive immune responses make them cells of particular relevance and importance in the mucosal environment. They have been demonstrated in a variety of mucosal tissues in veterinary species and have been characterized to varying degrees, showing that fundamental immunological principles apply throughout all species, but also highlighting some species differences. A major advantage of carrying out immunological research in veterinary species is their size: it is possible to cannulate lymphatic ducts and obtain information about cell migration between different tissues. It is also possible to obtain pure populations of relatively rare cell types such as the plasmacytoid dendritic cells or mucosal dendritic cells ex vivo for the study of immune responses to diseases in their natural host and for other thorough functional studies. Two major myeloid antigen presenting cell (APC) (dendritic cells, DC) cell populations have been described in gut draining lymph and other mucosal sites in ruminants and pigs, characterised by the presence or absence of surface molecules, their enzyme profiles, their ability to phagocytose and their different potential as APC. There is evidence that one of these subsets has migrated from the diffuse mucosal tissue, where it is found as a phagocytic as well as stimulatory APC population, which in turn may be derived from blood macrophages. In addition, the presence and role in viral infection of the IFN-alpha producing plasmacytoid DC in mucosal tissue is discussed, based on studies in pigs.

The postnatal development of the mucosal immune system and mucosal tolerance in domestic animals.

The mucosal immune system is exposed to a range of antigens associated with pathogens, to which it must mount active immune responses. However, it is also exposed to a large number of harmless antigens associated with food and with commensal microbial flora, to which expression of active, inflammatory immune responses to these antigens is undesirable. The mucosal immune system must contain machinery capable of evaluating the antigens to which it is exposed and mounting appropriate effector or regulatory responses. Since the immune system is likely to have evolved initially in mucosal tissues, the requirement to prevent damaging allergic responses must be at least as old as the adaptive immune system, and studies of the mechanisms should include a range of non-mammalian species. Despite the importance for rational design of vaccines and for control of allergic reactions, the mechanisms involved are still largely unclear. It is not clear that the classical experimental protocol of "oral tolerance" is, in fact, measuring a biologically important phenomenon, nor is it clear whether tolerance is regulated in the evolutionarily recent organised lymphoid tissue (the lymph nodes) or the more ancient, diffuse architecture in the intestine. The capacity of the immune system to discriminate between "dangerous" and "harmless" antigens appears to develop with age and exposure to microbial flora. Thus, the ability of an individual or a group of animals to correctly regulate mucosal immune responses will depend on age, genetics and on their microbial environment and history. Attempts to manipulate the mucosal immune system towards active immune responses by oral vaccines, or towards oral tolerance, are likely to be confounded by environmentally-induced variability between individuals and between groups of animals.

Smoking influences the immunological architecture of the human larynx.

Little is known about the effects of demographic and lifestyle factors on laryngeal mucosal immunology. Pinch biopsies of laryngeal mucosa were studied from 63 patients without laryngeal disease. Areas of positive staining for HLA-DR, HLA-DQ, HLA-DP, CD45, CD45RA, CD45RO, CD4, CD8, and CD79 were calculated. Patients were stratified according to gender and smoking status. Analysis of covariance showed current cigarette smokers had increased numbers of CD4+ T cells and there was an association between older age and greater CD4+ T cell numbers in both epithelium and lamina propria. Older age and female gender were associated with decreased lamina propria CD4+ CD45RO+ T cells and an increase in CD4+ CD45RO- T cells. T cell populations in the larynx may therefore be influenced by smoking, age and gender. We hypothesize that smoking induces changes in normal immunological function of the larynx, which may contribute to the etiology of inflammatory disease and cancer.

Effects of infection with transmissible gastroenteritis virus on concomitant immune responses to dietary and injected antigens.

Normal piglets weaned onto soy- or egg-based diets generated antibody responses to fed protein. Concurrent infection with transmissible gastroenteritis virus (TGEV) did not affect the responses to dietary antigens at weaning, nor did it affect the subsequent development of tolerance. However, TGEV infection did enhance the primary immunoglobulin M (IgM) and IgG1, but not IgG2, antibody responses to injected soy in comparison to those of uninfected animals. Paradoxically, TGEV-infected animals showed an enhanced primary IgG1 antibody response to injected soy at 4 weeks of age, but they subsequently showed a reduced secondary response after an intraperitoneal challenge at 9 weeks of age in comparison to uninfected animals. The results suggest that an enteric virus, either used as a vaccine vector or present as a subclinical infection, may not have significant effects on the development of dietary allergies but may have effects both on the primary response and on the subsequent recall response to systemic antigens to which the animal is exposed concurrently with virus antigens.