Practical immunoregulation: neonatal immune response variation and prophylaxis of experimental food allergy in pigs.

The importance of environment in immune response is identified and the increase in prevalence of allergic, autoimmune and chronic inflammatory diseases reviewed. In particular, altered opportunity to acquire evolutionarily anticipated commensal microbiota is associated through the "hygiene hypothesis" with defective developmental and response signals to the innate and adaptive immune systems. Evidence of the detrimental effects of such environments is reviewed as is evidence for remediation using controlled exposure to bacteria or their active components such as LPS or peptidoglycan ligands for TLR and NOD-like receptors. Occurrence of major environmentally associated changes in porcine immune response phenotype are described. The prophylactic effects of heat-killed Escherichia coli given intramuscularly or of oral Lactococcus lactis on experimental ovomucoid-induced allergy in piglets are described in the context of altered immune response bias favouring reduced type-2 phenotypes. The high frequency of clinical tolerance to developing allergic signs even in the face of classical sensitization indicates possible function in this pig model of regulatory effectors such as Treg cells.

Immunoglobulin isotypes of lactating Holstein cows classified as high, average, and low type-1 or -2 immune responders.

Infectious diseases are detrimental to the health and economy of the livestock industry. Observations of cattle resistant to natural infections have implied the feasibility of breeding livestock for disease resistance. Studies of pigs selected for antibody (AMIR)- and cell (CMIR)-mediated immune responses have demonstrated increased immune responsiveness suggesting enhanced protection by both type 2 and type 1 responses, respectively. Additionally, natural or artificial infections of cattle suggest that the production of particular immunoglobulin (Ig) M, IgG1 and IgG2 isotypes are important for protecting against pathogens. In fact, IgG1/IgG2 ratios are often used to establish whether type 1 (CMIR) or type 2 (AMIR) responses predominate following immunization or infection. The objectives of this study were therefore; (1) to evaluate the Ig isotype bias responses to Candida albicans and hen-egg white lysozyme (HEWL) in cows classified as high responders (HR), average responders (AR) or low responders (LR) based on AMIR or CMIR; (2) to determine if ranking based on IFN-γ (a type 1 cytokine) and DTH responses were analogous in terms of ranking; and (3) to estimate IFN-γ, Ig isotypes, and DTH correlations. Antibody responses to HEWL and DTH to C. albicans were detected such that cows were phenotypically classified as HR, AR and LR for AMIR or CMIR with significant differences (p ≤ 0.05) among classified groups. C. albicans-induced IFN-γ allowed classification of cows, some of which had the same ranking as that of DTH response. The lowest IgG1/IgG2 ratio was to the C. albicans purified antigen (candin), but no differences were observed in anti-HEWL or anti-candin IgG1/IgG2 ratios between classified groups. Anti-HEWL IgG1 and IgG2 responses at day 21 post-immunization were negatively and significantly correlated with DTH to candin at 24h. There were no significant correlations between anti-HEWL or anti-candin IgG1 or IgG2 responses with IFN-γ. Based on Ig isotype bias, IFN-γ and DTH responses, it was concluded that immunization with C. albicans can be used to classify CMIR responder cows based on DTH read-out.

Porcine allergy and IgE.

Anaphylaxis was reported in 1963 in pigs experimentally sensitized with ovalbumin and was subsequently associated indirectly with IgE-related antibodies by functional assays to confirm heat-labile passive cutaneous anaphylaxis (PCA), reverse passive anaphylaxis (RPA) and Prausnitz-Küstner (PK) reactions to this and other allergens. The immunoglobulin mediating immediate hypersensitivity could be cross-adsorbed with anti-human IgE. Porcine IgE epsilon chain has been cloned and sequenced. Rabbit anti-pig IgE has been described by two groups, as has cross reactivity with pig IgE of various heterologous polyclonal and monoclonal anti-IgEs. Pigs develop transient post-weaning food allergy to soy allergens which can be prevented by pre-weaning feeding of soy proteins in sufficient quantity. Natural hypersensitivity also occurs to nematodes. Recently, experimental allergy has been induced in outbred pigs to peanut and to egg allergens which manifest as respiratory, cutaneous and enteric signs similar to those of human food allergy. These models are platforms for comparative allergy research as realistic alternatives to use of inbred mice or humans for investigation of pathogenesis, prophylaxis and therapy.

Attenuation of allergy to ovomucoid in pigs by neonatal treatment with heat-killed Escherichia coli or E. coli producing porcine IFN-gamma.

Food allergy is epidemic and prompts investigation to reduce allergic predisposition. It was hypothesized that heat-killed Escherichia coli injected intramuscularly (im) with or without interferon gamma (IFN-gamma), reduces neonatal susceptibility to experimental egg allergy. Two litters of Yorkshire pigs were assigned to three intramuscular treatment groups (four/group): control (PBS), heat-killed E. coli with or without IFN-gamma-expressing plasmid. Pigs were sensitized to ovomucoid (Ovm) by intraperitoneal injection with cholera toxin. To assess induction of allergy, pigs were fed egg white in yoghurt and assigned scores for allergic signs. Significantly fewer pigs developed allergy and passive cutaneous anaphylaxis in E. coli and E. coli+IFN-gamma vs control groups. E. coli-treated pigs also had significantly lower frequency of mean clinical scores. E. coli and E. coli+IFN-gamma groups did not differ. Serum antibody associated with IgG (H & L), IgG(1), IgG(2) or IgE all correlated but did not differ by treatment groups. Thus, treatment of neonatal pigs with heat-killed E. coli by im injection reduced susceptibility to allergic sensitization with Ovm. Inclusion of the type-1 cytokine, IFN-gamma, had no additional effect. Results indicate a method for prophylaxis of allergy and suggest support for the "hygiene hypothesis".

Steering orally induced deviation of xenogeneic immunity with exogenous cytokines in rats fed porcine proteins.

Immune response to xenoantigens is a barrier to xenotransplantation. The objective of this study was to determine if rat cell-mediated immunity (CMI) and antibody (Ab) to discordant porcine xenoantigens could be suppressed by oral administration of porcine protein with adjunct systemic cytokine therapy. Based on principles of oral tolerance, it was hypothesized that: a. Feeding proteins from porcine blood mononuclear cells (PBMC) would induce a type 2 response, inhibiting CMI and type 1 Ab (associated with xenograft rejection) but increasing the amount of type 2 Ab. b. IL-4, a type 2 cytokine would exaggerate type 2 bias, enhancing immune deviation. c. IFN-gamma, a type 1 cytokine was expected to down-regulate overall Ab production, but increase CMI and type 1 Ab. DA rats fed porcine proteins with or without subcutaneous and intraperitoneal injections of IFN-gamma or IL-4 received PBMC by subcutaneous injection. Dermal delayed-type hypersensitivity to PBMC was the indicator of CMI. The IgG Ab to porcine proteins was quantified and type 2 (IgG(1)+IgG(2a)) to type 1 (IgG(2b)) Ab ratios indicated IR bias. Unfed, PBMC-challenged controls had a type 1 response bias but feeding PBMC induced a type 2 bias that suppressed CMI and type 1 Ab and increased type 2 Ab but not total IgG Ab. Contrary to the T(H)1/T(H)2 paradigm, IL-4 decreased oral-induced type 2 IR deviation and did not provide significant benefits relative to feeding alone. Treatment with IFN-gamma prevented a switch to type 2 IR but feeding-induced suppression of CMI was not significantly compromised. The IFN-gamma potently suppressed Ab, including, surprisingly, type 1 IgG isotypes. Contrary to the hypothesis tested, feeding Ag in combination with systemic delivery of recombinant IFN-gamma apparently created an immunoregulatory environment most favorable to xenograft survival.

Antibody to ovalbumin and delayed-type hypersensitivity to Candida albicans and mycobacteria in lactating Holstein cows using Quil A or Freund's complete adjuvant.

Antibody-mediated immune response (AMIR) to ovalbumin (OVA) or hen-egg white lysozyme (HEWL) and cell-mediated immune response (CMIR) such as delayed-type hypersensitivity (DTH) to mycobacteria have been proposed as quantitative traits for selective breeding to improve animal health. However, DTH to mycobacteria may confound diagnosis of tuberculosis in cattle. Candida albicans, a yeast also known to induce DTH, was tested as an alternative for DTH induction and testing since it is not a target of regulatory diagnostic tests. Other objectives were to determine if both AMIR and CMIR in cattle receiving Quil A as adjuvant were equivalent to corresponding responses induced by Freund's complete adjuvant (FCA). Forty lactating Holstein cows were randomly assigned to two treatment groups, which received ovalbumin (OVA) and C. albicans adjuvanted with FCA and Freund's incomplete adjuvant (FIA) on days 0 and 14, respectively, or Quil A on days 0 and 14. The FCA was used as adjuvant and as a source of Mycobacterium tuberculosis-induced DTH. Testing for DTH was performed on day 21 with killed C. albicans whole cell (CaWC), a purified extract from C. albicans (candin) and M. phlei purified protein (phlein). Both primary and secondary antibody responses to OVA were statistically significant and similar in both FCA and Quil A treatment groups. No significant differences were detected in immunoglobulin G (IgG) isotypic-mediated responses to OVA or candin between groups. C. albicans adjuvanted with Quil A induced DTH reaction similar to those induced by C. albicans and mycobacteria in FCA.

Porcine IgE in the context of experimental food allergy: purification and isotype-specific antibodies.

Measurement of allergen-specific immunoglobulin E (IgE) is a common practice in the investigation of allergy. It has not been possible to measure porcine IgE due to unavailability of anti-porcine IgE. This study was undertaken to purify and characterize porcine IgE from sera of allergic pigs, identify heterologous anti-IgE reactive with pig IgE and to use purified heavy (H) chain of porcine IgE to generate rabbit anti-IgE. A four-step process for the purification of porcine IgE is reported using ammonium sulphate precipitation, Protein G affinity chromatography, DEAE cellulose anion-exchange chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to obtain IgE H chain. The resultant IgE was evaluated for purity using SDS-PAGE and immunoreactivity was detected by Prausnitz-Küstner (PK) tests and passive cutaneous anaphylaxis with the allergen, crude peanut extract, used to induce experimental allergy. Cross-reactivity with anti-mouse and anti-human IgE antibodies were confirmed in western blot and enzyme-linked immunosorbent assays (ELISA). The H chain of IgE was excised from SDS-PAGE gels and used to develop rabbit anti-porcine IgE antisera. Antiserum obtained from rabbits immunized with porcine IgE, as well as heterologous murine and human-specific anti-IgE, induced reverse cutaneous anaphylaxis in pig skin and detected allergen-specific IgE in ELISA but did not react with IgG H chain in western blots. These results confirm allergy-associated bioactivity of porcine IgE and describe both homologous and heterologous anti-pig IgE suitable for use in allergen-specific and other assays. This will enhance utility of pig allergy models and provide an additional measure of type-2 immune response in pigs.

Expression of bioactive porcine interferon-gamma by recombinant Lactococcus lactis.

Lactococcus lactis is an immunomodulator and candidate live mucosal delivery vehicle for vaccine antigens and for biologically active molecules, including immunoregulatory cytokines such as interferon-gamma (IFN-gamma). To provide a tool for investigating downregulation of allergic predisposition of pigs to experimental food allergy, porcine IFN-gamma was cloned and expressed as a fusion protein with the usp45 secretion signal. Immunoblot analysis with polyclonal anti-pIFN-gamma-antibody demonstrated that the recombinant porcine IFN-gamma (rpIFN-gamma) protein was expressed in the L. lactis transformants as a secreted product. Activity of rpIFN-gamma was confirmed by ability to upregulate class II major histocompatibility complex (MHC) on cells of the porcine monocytic cell line 3D4/31. The L. lactis producing biologically active rpIFN-gamma will be used to investigate its possible ability to modulate the allergic immune response phenotype of pigs.

A neonatal swine model of allergy induced by the major food allergen chicken ovomucoid (Gal d 1).

BACKGROUND: Food allergy is a serious health problem for which a validated outbred large animal model would be useful in comparative investigations of immunopathogenesis and treatment and in testing hypotheses relevant to complex host-environmental interactions in predisposition to and expression of food allergy. OBJECTIVE: To establish a neonatal swine model of IgE-mediated allergy to the egg protein ovomucoid (Ovm) that may mimic human allergy. METHODS: In order to induce Ovm sensitivity, piglets at days 14, 21 and 35 of age were sensitized by intraperitoneal injection of 100 microg of crude Ovm and cholera toxin (50, 25 or 10 microg). Controls received 50 microg of cholera toxin in phosphate-buffered saline. The animals were challenged orally on day 46 with a mixture of egg white and yoghurt. Outcomes were reported as direct skin tests, clinical signs, IgG-related antibody and passive cutaneous anaphylaxis. RESULTS: Sensitized pigs developed immediate wheal and flare reactions, and after oral challenge, sensitized but not control animals displayed signs of allergic hypersensitivity. Serum IgG-related, Ovm-specific antibodies were detected only in the sensitized pigs and IgE-mediated antibody response to Ovm was confirmed by positive passive cutaneous anaphylaxis reactions induced by sera of sensitized but not by heat-treated sera from Ovm-sensitized pigs or sera of unsensitized control pigs. CONCLUSION: The present results confirm induction of Ovm-specific allergy in pigs and provide opportunity to investigate allergic predisposition and immunopathogenesis of IgE-induced Ovm allergy using outbred neonatal swine. This may better simulate allergic disease in humans and allow investigation of candidate prophylactic and therapeutic approaches.

Deviation of xenogeneic immune response and bystander suppression in rats fed porcine blood mononuclear cells.

Reducing or deviating xenogeneic immune response prior to xenotransplantation may enhance the efficacy of conventional immunosuppressive therapies in prolonging xenograft survival. The potential to suppress or steer immune responses by oral administration of xenoantigens was evaluated. Based on knowledge of oral tolerance, hypotheses tested were that feeding xenoantigens would inhibit cell-mediated immune response (CMIR) and production of antibodies associated with graft rejection and induce bystander suppression. DA and LEW rats, high and low responders to xenoantigens, respectively, were fed dead porcine blood mononuclear cells (PBMC) and subsequently received live PBMC and hen egg-white lysozyme (HEWL, a third-party antigen) by subcutaneous injection. Delayed-type hypersensitivity (DTH) to PBMC was an indicator of CMIR. Quantification of T(H)1 (IgG(2b)) and T(H)2 (IgG(1))-associated antibodies and their ratio measured magnitude and bias of the antibody-mediated response to PBMC and HEWL. Feeding PBMC reduced IgG(2b) antibody production by 90% (DA) and 71% (LEW) and increased IgG(1) antibodies by 116% in DA but not LEW rats (p

B-1 and B-2 B-cells in the pig cannot be differentiated by expression of CD5.

In a number of species, such as mice, humans and cattle, B-cells can be differentiated into two populations based on the surface expression of CD5, a marker normally found on T-cells. These B-cell subsets have been found to differ with regard to location, development and phenotypic characteristics. The B-1 (CD5(+)) B-cells have also been shown to have a more restricted immunoglobulin isotype expression profile, limited combinatorial diversity in immunoglobulin heavy chains and lower somatic hyper-mutation. They are potent producers of IL-10. In the pig, CD5(+) and CD5(-) B-cell populations have previously been described in this laboratory. Here, we show that B-cells isolated and separated into CD5(+) and CD5(-) populations do not differ with regard to immunoglobulin isotype or IL-10 RNA expression, nor do the immunoglobulin heavy chain V(D)J re-arrangements differ in terms of gene usage, CDR3 length and composition or the frequency of hyper-mutations. In conclusion, expression of CD5 cannot be used to differentiate between pig blood B-1 and B-2 B-cells.

Rat genotype influences quantitative and qualitative aspects of xenogeneic immune response to pig blood mononuclear cells.

BACKGROUND: Xenotransplantation seeks to have cells with discordant genotypes co-exist. The hypothesis that host genotype modulates xenogeneic immune response (IR) was tested. METHODS: Two inbred rat strains [Dark Agouti (DA) and Lewis (LEW)] representing diverse IR phenotypes were immunized with porcine blood mononuclear cells (BMC). Delayed-type hypersensitivity (DTH), immunoglobulin (Ig), antibody (Ab) and isotype bias of Ab response were evaluated. RESULTS: DTH to pig BMC was greater in DA than in LEW rats. Natural Ab was qualitatively different between strains (IgM and IgA predominated in DA, IgM and IgG(2a) predominated in LEW). Twice as much IgG was elicited from DA than LEW rats and DA utilized all isotypes whereas LEW did not use IgG(2a) or IgG(2c). IR bias was diametrically opposed; type 1 in DA but type 2 in LEW. Strains even differed in Ig profiles and dermal responses to saline injections and mitogen. The DA rats were the higher responders to pig BMC. CONCLUSIONS: Recipient genotype had significant and broad effects on IR to porcine BMC and may influence xenograft rejection and xenotolerance induction. Moreover, this study suggests that caution should prevail when interpreting data derived from a single inbred strain, particularly given that humans, the target species, are genetically diverse.

Effects of antigen and recombinant porcine cytokines on pig dendritic cell cytokine expression in vitro.

To evaluate variables influencing in vitro immune response induction, pig monocyte-derived DCs (moDCs) were treated with putative type-1 and type-2 antigens (Ags, killed Mycobacterium tuberculosis (Mtb) and hen egg white lysozyme (HEWL)) and recombinant porcine cytokines (IL-6, IL-10, IL-12, IFN-gamma and TNF-alpha). Responses were measured as moDC cytokine mRNA expression. Treatment of moDCs with HEWL increased IL-13 but not IL-12, IFN-gamma or IL-10 mRNA, suggesting a DC2 phenotype. Addition of TNF-alpha, IFN-gamma or IL-12 to HEWL-treated moDCs increased IL-12p35 and reduced IL-13 mRNA; suggesting a DC1 phenotype. Mtb increased moDC IL-12p35, IFN-gamma and to a lesser extent IL-13 mRNA. This DC1 bias was enhanced by TNF-alpha, IFN-gamma or IL-12, which increased IL-12p35 and to a lesser extent IL-10 mRNA but reduced IL-13 mRNA. Addition of IL-10 to Mtb-pulsed moDCs reduced IL-12p35, IFN-gamma and IL-13, but increased IL-10 mRNA, suggesting diversion from DC1 to DC2. Thus porcine moDCs treated with Ag and/or cytokines alter moDC cytokine expression confirming their likely ability to initiate and steer acquired immune response.

Genetic selection for high and low immune response in pigs: effects on immunoglobulin isotype expression.

Immunoglobulin (Ig) function varies by isotype and antibody activity is best mediated by isotypes most able to control the inciting infection. In pigs, a high ratio of IgG1:IgG2 is associated with resistance to disease caused by the extra-cellular bacterium Actinobacillus pleuropneumoniae. This ratio is controlled by type 1/type 2 cytokines in vitro, reflecting cell- (CMI) or antibody-mediated immune (AMI) responses, respectively. Animals were used which had been previously selectively bred for high (HIR) or low (LIR) combined AMI and CMI and had been immunized with hen eggwhite lysozyme (HEWL) in Quil A (days 0 and 14) while Bacillus Calmette Guérin was given on day 9. To test the hypothesis that lines do not differ in IgG isotype expression as antibody to HEWL, the ratio of anti-HEWL associated with IgG1 and IgG2 was determined at days 0, 9, 14 and 21. The ratio of IgG1:IgG2-associated antibody was always <1.0 indicating a type 1 response and differed significantly over time in HIR and LIR animals. After primary and secondary immunizations, the HIR animals' IgG1:IgG2-associated antibody ratio increased and approached 1 while for LIR animals the ratio decreased. Thus anti-HEWL antibody in HIR, but not LIR, approached balance in type 2:type 1 expression. Individual variation in immune response was frequently significant within each immune response group. Thus, proportional production of anti-HEWL antibody associated with IgG isotypes varies by individual and differs over time as a function of genotype in pigs selectively bred for HIR or LIR.

Toll-like receptor, MHC II, B7 and cytokine expression by porcine monocytes and monocyte-derived dendritic cells in response to microbial pathogen-associated molecular patterns.

To evaluate effects of treatment with pathogen-associated molecular patterns (PAMPs) on toll-like receptor (TLR), MHC II, B7 and cytokine expression, pig monocytes and monocyte-derived DCs (moDCs) were treated with LPS, CpG, lipoteichoic acid (LTA), poly IC or peptidoglycan (Pep). Monocytes and moDCs treated with LPS, CpG, LTA, poly IC or Pep altered expression of at least one TLR (4, 5 and 9) and up-regulated MHC II and/or B7. The mRNA for IL-4 was not detected after any treatment. Treatment with LPS or LTA tended to up-regulate mRNA for TLR 4, Th-1 (IFN-gamma and IL-12p35) and Th-2 cytokines (IL-10 and IL-13). Poly IC or CpG tended to up-regulate TLR 9 and Th-1 cytokines. Porcine monocytes and moDCs like those of humans and mice responded to microbial PAMPs by altering TLR expression, up-regulating MHC II and B7 and altering cytokine expression toward Th-1 and/or Th-2, which may steer immune response. Hence, porcine moDCs and monocytes are likely able to discriminate between microorganisms using TLRs which determine cytokine expression and immune response bias.

Evaluation of bovine cutaneous delayed-type hypersensitivity (DTH) to various test antigens and a mitogen using several adjuvants.

The Bacillus Calmette Guerin (BCG)-induced/purified protein derivative (PPD)-elicited tuberculin skin test is a reliable measure of cell-mediated immune response (CMIR), specifically delayed-type hypersensitivity (DTH); however, its use in livestock may confound diagnosis of Mycobacterium tuberculosis. Therefore, various alternative antigen/adjuvant combinations were evaluated as inducers of DTH that were compared to the BCG/PPD test system with the purpose of finding a skin DTH protocol that does not cross-react with the tuberculin test and allows identification of high and low CMIR responder phenotypes. Specifically, 30 non-lactating cows (five/treatment) were sensitized on day 0 with mycobacteria [BCG, M. tuberculosis or Mycobacterium phlei cell wall extract (MCWE)], and ovalbumin (OVA) emulsified in Freund's complete adjuvant (FCA), non-ulcerative Freund's adjuvant (NUFA), complete NUFA or MCWE. On day 21, cows were injected intradermally with various test antigens including PPD tuberculin, phlein, and OVA. Phosphate buffered saline was included as the negative control and the T-cell mitogen phytohemagglutinin (PHA) was also administered. Double skin-fold thickness was evaluated before and at 6, 24, and 48 h post-injection. Skin biopsies were taken at 24 and 48 h to assess oedema, necrosis, and inflammatory cell infiltration. BCG/PPD and M. phlei/phlein treatments when given with a Freund's adjuvant induced equivalent DTH with peak reactions at 24-48 h after antigen injection. Cows receiving NUFA had fewer injection site granulomas than FCA or CNUFA treatments. The change in skin thickness response to PHA peaked at 6 h. Only cows receiving mycobacteria in NUFA had skin response to OVA, which peaked 6-24 h post-injection. Only sites tested with PPD or phlein had significantly higher lymphocyte infiltration than control, whereas neutrophils were significantly higher at PHA test sites and eosinophils predominated at the PHA test sites. Macrophages were significantly more numerous at the PPD and/or phlein test sites in treatment groups that received killed mycobacteria in a Freund's adjuvant and/or with BCG, and at the PHA test sites in all treatment groups. It was concluded that the M. phlei/phlein system did induce DTH and was similar to the DTH induced by the BCG/PPD system when MCWE was administered with a Freund's adjuvant. Therefore, this protocol is suitable for detecting high/low CMIR responders in research herds. However, cross-reaction to PPD was evident following induction of DTH using M. phlei. Hence, this protocol does not alleviate the problem of artificial induction of DTH cross-reactivity and would not be suitable for commercial herds where tuberculin testing is required.

Identification of peptides associated with chicken major histocompatibility complex class II molecules of B21 and B19 haplotypes.

Chicken major histocompatibility complex (MHC) molecules present peptides to T cells to initiate immune response. Some variants of the chicken MHC, such as B19 and B21 haplotypes, are strongly associated with susceptibility and resistance to Marek's disease, respectively. The objective of the present study was to characterize the repertoire and origin of self-peptides presented by chicken MHC class II (B-L) molecules of B19 and B21 haplotypes. Following immunoaffinity purification of B21 and B19 B-L molecules from transformed B cell lines, their associated peptides were eluted, high performance liquid chromatography-fractionated, and sequenced by tandem mass spectrometry. Four peptides were identified associated with B21 B-L molecules. These ranged from 16 to 21 residues in length and had originated from membrane-bound, cytosolic, and mitochondrial proteins. Two of these peptides were present in form of an overlapping set, which is a common characteristic of MHC II-associated peptides. The single B19-associated peptide was 17 residues long and had originated from a cytosolic source. Presentation of endogenous peptides, such as those derived from cytosolic and mitochondrial proteins, by B-L molecules is indicative of cross-sampling between MHC class I and II antigen presentation pathways. These findings facilitate future studies aimed at elucidating mechanisms of chicken MHC association with disease resistance.

Dietary 18:3omega3 influences immune function and the tissue fatty acid response to antigens and adjuvant.

Alpha-linolenic acid (18:3omega3) has many important physiological functions including being beta-oxidized, serving a precursor to the synthesis of other lipids and it has immunomodulation properties. The objective of the present study was to test the effects of immunization and dietary 18:3omega3 on immune function and the fatty acid profile of immunized pig tissues. Piglets suckled from sows consuming either a control or high 18:3omega3 diet until 14 days old when they were weaned onto a similar diet as the sow and were moved to a segregated nursery for the remainder of the study. At 35 days of age, pigs on both diets (2 x 2 factorial design) received either an injection containing hen eggwhite lysozyme (HEWL), killed Mycobacterium tuberculosis and Freund's complete adjuvant (immunized) or phosphate buffered saline (PBS) (non-immunized) into the neck followed by a booster injection 2 weeks later and induction of delayed-type hypersensitivity (DTH) one week later. Immunization increased (compared to non-immunized) while the high 18:3omega3 diet decreased haptoglobin by 30% compared to pigs consuming the control diet. Immunized pigs had a seven-fold increase in antibodies to HEWL and pigs consuming the high 18:3omega3 diet also had transiently higher levels of serum antibodies. There was a diet by immunization interaction on the DTH reaction such that immunized pigs consuming the high 18:3omega3 had the largest DTH reaction. The neck muscle proximal to the site of injection of immunized pigs had 10-30% lower levels of triglyceride and phospholipid linoleic (18:2omega6) and 18:3omega3 compared to non-immunized pigs. Thus, a high 18:3omega3 intake in pigs modulates immune function and tissue fatty acids in response to immunization.

Th-1/Th-2 type cytokine profiles of pig T-cells cultured with antigen-treated monocyte-derived dendritic cells.

To examine the effects of cytokine environment at the time of antigenic exposure on T-cell cytokine profiles following T-cell-antigen presenting cell (APC) interaction, pig monocyte-derived dendritic cells (mDCs) were treated with hen egg white lysozyme (HEWL) or killed Mycobacterium tuberculosis (Mtb) alone or with a recombinant pig cytokine (TNF-alpha, interleukin (IL)-12, IL-10, interferon (IFN)-gamma or IL-6) and then incubated with autologous T-cell-enriched lymphocytes. Messenger RNA was isolated from the T-cells and used to evaluate the effects of treatment on IL-12p35, IFN-gamma, IL-4, IL-10 and IL-13 expression using RT-PCR. T-cells exposed to HEWL-treated mDCs expressed high IL-13 and moderate IL-10 and IFN-gamma, suggesting T-helper 2 (Th-2) bias. Addition of any cytokine during HEWL treatment of mDCs reduced subsequent expression of IL-10 and IL-13 by T-cells. Added IL-12 increased IFN-gamma mRNA. T-cells exposed to Mtb-treated mDCs expressed increased IFN-gamma and decreased IL-10 suggesting Th-1 bias. Addition of cytokines to mDCs treated with Mtb altered T-cell cytokine mRNA expression such that TNF-alpha, IFN-gamma or IL-12 increased IFN-gamma; IL-12 and IFN-gamma suppressed IL-10, while IL-10 and IL-12 enhanced IL-13. Messenger RNA for IL-4 and IL-12p35 was not detected in the T-cells. Results suggest Th-1/Th-2 type response bias in pigs T-cells as a function of antigen type and that cytokine environment at the time of antigen-mDC interaction alters cytokine profiles of T-cells responding to antigen-pulsed mDCs. Hence, cytokines may allow designed steering of porcine immune response.

Immunoglobulin G subisotype responses of pneumonic and healthy, exposed foals and adult horses to Rhodococcus equi virulence-associated proteins.

Rhodococcus equi causes severe pyogranulomatous pneumonia in foals and in immunocompromised humans. Replication of virulent isolates within macrophages correlates with the presence of a large plasmid which encodes a family of seven virulence-associated proteins (VapA and VapC to VapH), whose functions are unknown. Although cell-mediated immunity is thought to be crucial in eliminating R. equi infection, antibody partially protects foals. The antibody response to both VapA and VapC was similar in six adult horses and six naturally exposed but healthy foals, as well as in eight foals with R. equi pneumonia. The immunoglobulin G (IgG) subisotype response of pneumonic foals to Vap proteins was significantly IgGb biased and also had a trend toward higher IgGT association compared to the isotype association of antibody in adult horses and healthy exposed foals. This suggests that in horses, IgGb and IgGT are Th2 isotypes and IgGa is a Th1 isotype. Furthermore, it suggests that foals which develop R. equi pneumonia have a Th2-biased, ineffective immune response whereas foals which become immune develop a Th1-biased immune response. Pneumonic foals had significantly more antibody to VapD and VapE than did healthy exposed foals. This may indicate a difference in the expression of these two Vap proteins during persistent infection. Alternatively, in pneumonic foals the deviation of the immune response toward VapD and VapE may reflect a bias unfavorable to R. equi resistance. These data indicate possible age-related differences in the equine immune response affecting Th1-Th2 bias as well as antibody specificity bias, which together favor the susceptibility of foals to R. equi pneumonia.