Summary Early-life exposure to appropriate microbial flora drives expansion and development of an efficient immune system. Aberrant development results in increased likelihood of allergic disease or increased susceptibility to infection. Thus, factors affecting microbial colonization may also affect the direction of immune responses in later life. There is a need for a manipulable animal model of environmental influences on the development of microbiota and the immune system during early life. We assessed the effects of rearing under low- (farm, sow) and high-hygiene (isolator, milk formula) conditions on intestinal microbiota and immune development in neonatal piglets, because they can be removed from the mother in the first 24 h for rearing under controlled conditions and, due to placental structure, neither antibody nor antigen is transferred in utero. Microbiota in both groups was similar between 2 and 5 days. However, by 12–28 days, piglets reared on the mother had more diverse flora than siblings reared in isolators. Dendritic cells accumulated in the intestinal mucosa in both groups, but more rapidly in isolator piglets. Importantly, the minority of 2–5-day-old farm piglets whose microbiota resembled that of an older (12–28-day-old) pig also accumulated dendritic cells earlier than the other farm-reared piglets. Consistent with dendritic cell control of T cell function, the effects on T cells occurred at later time-points, and mucosal T cells from high-hygiene, isolator pigs made less interleukin (IL)-4 while systemic T cells made more IL-2. Neonatal piglets may be a valuable model for studies of the effects of interaction between microbiota and immune development on allergy.

The larynx is a mucosal organ positioned at the divergence of the respiratory and digestive tracts. It is exposed to a wide variety of environmental components, including foreign antigens, tobacco smoke, laryngopharyngeal reflux and pollutants. The mucosal immune system generates either active immune responses or tolerance, depending on the nature of the antigen and we hypothesize that the larynx is important organ for immunological decision-making in the airway. Because the pig is an ideal large animal model in which to explore laryngological research questions, such as those relating to laryngeal transplantation, we investigated the normal mucosal immunology of the porcine larynx. Pig larynges and tracheae were processed and prepared for bright-field microscopy and quantitative, multiple-colour immunofluorescence histology using pig-specific monoclonal antibodies. There was an abundance of immunologically active cells within the mucosa of the larynx and trachea of both the newborn and adult animal. Specifically, major histocompatibility complex class II (MHC class II+) cells, CD4(+) and CD8(+) cells were identified, although regional differences in numbers were apparent: specifically, the supraglottis contained fewer immunologically relevant cells than other sites sampled. There was a significant correlation between the numbers of MHC class II+ and CD4(+) cells indicating co-ordinate regulation and therefore functional local interactions. The presence of such an immunological structure suggests that the larynx may have important functions in respiratory immunology and that it may trigger strong alloresponses after laryngeal transplantation.

Laryngeal transplantation is an increasingly viable proposition for patients with irreversible diseases of the larynx. One human transplant has been performed successfully, but many questions remain before routine transplantation can begin. In order to measure the immunological changes in mismatched transplants, it is first necessary to know the immediate combined effects of ischaemia-reperfusion injury (IRI) plus the added insult of major surgery in a fully matched setting. We measured the changes in immunologically active mucosal cells following 3 h of cold ischaemia and 8 h of in situ reperfusion in a major histocompatibility complex (MHC)-matched minipig model (n = 4). Biopsies were prepared for quantitative, multiple-colour immunofluorescence histology. The number of immunologically active cells was significantly altered above (supraglottis) and below (subglottis) the vocal cords following transplantation and reperfusion (P < 0.05, P < 0.001, respectively). However, the direction of the change differed between the two subsites: cell numbers decreased post-transplant in the supraglottis and increased in the subglottis. Despite the statistical evidence for IRI, these changes were less than the large normal inter- and intrapig variation in cell counts. Therefore, the significance of IRI in exacerbating loss of function or rejection of a laryngeal allograft is open to question. Longer-term studies are required.

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.

Developments in immunohistology allow the routine simultaneous use on tissue sections of three monoclonal antibodies, tagged with different fluorochromes. Such staining can identify seven different cell populations and the limiting factor is rapid, reliable and reproducible analysis. Future reliance on computer-assisted analysis of digitised images depends on validation against manual counting, often viewed as the 'gold standard'. In this study images were digitised from sections of normal porcine skin, inflamed skin and tonsil, simultaneously stained with three monoclonal antibodies. Combinations of staining were quantified by four manual counts and by pixel-based area measurement. On individual images, the correlation between automated and manual measurements was poor. Despite this, the concordance between manual and automated measurements in the means and variances of tissues was good, and both techniques identified the same changes in inflamed versus normal tissues. In addition, pixel-based counting permitted statistical analysis of co-localisation of cell types in tissue sections. We conclude that automated counting is acceptable for the assessment of tissues, is faster and provides less opportunity for observer variation than manual counting. We also demonstrate that the technique is applicable where more than three fluorochromes are used such that manual counting becomes essentially impossible.

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.

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.

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.

The mucosal immune system expresses active responses against pathogens and also tolerance against harmless food and commensal bacterial antigens. The mechanisms that determine which of these outcomes occur after recognition of antigens by T-cells are not clear. One possibility is that it is determined by the initial interaction between a dendritic and a naïve T-cell in organised lymphoid tissue. However, such organised structures are, evolutionarily, quite recent and the original immune system must have made appropriate responses in more diffuse immunological architecture; a second possibility is that the critical interaction is between primed T-cells and their environment, in the lamina propria of the intestine. The mucosal immune system of neonates is poorly developed and inefficient at expressing appropriate immune responses. Development is influenced by a range of environmental factors including maternally derived antigen or antibody and commensal flora and pathogens. The intestine is a complex immunological structure in which the immune system and the macro- and microenvironment interact.

The mucosal immune system fulfils the primary function of defence against potential pathogens that may enter across vulnerable surface epithelia. However, a secondary function of the intestinal immune system is to discriminate between pathogen-associated and 'harmless' antigens, expressing active responses against the former and tolerance to the latter. Control of immune responses appears to be an active process, involving local generation of IgA and of regulatory and/or regulated T lymphocytes. Two important periods of maximum exposure to novel antigens occur in the young animal, immediately after birth and at weaning. In both cases the antigenic composition of the intestinal contents can shift suddenly, as a result of a novel diet and of colonisation by novel strains and species of bacteria. Changes in lifestyles of man, and husbandry of animals, have resulted in weaning becoming much more abrupt than previously in evolution, increasing the number of antigens that must be simultaneously evaluated by neonates. Thus, birth and weaning are likely to represent hazard and critical control points in the development of appropriate responses to pathogens and harmless dietary and commensal antigens. Neonates are born with relatively undeveloped mucosal immune systems. At birth this factor may prevent both expression of active immune responses and development of tolerance. However, colonisation by intestinal flora expands the mucosal immune system in antigen-specific and non-specific ways. At weaning antibody to fed proteins can be detected, indicating active immune responses to fed proteins. It is proposed that under normal conditions the ability of the mucosal immune system to mount active responses to foreign antigens develops simultaneously with the ability to control and regulate such responses. Problems arise when one or other arm of the immune system develops inappropriately, resulting in inappropriate effector responses to harmless food proteins (allergy) or inadequate responses to pathogens (disease susceptibility).

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.

European-wide directives are in place to establish a sustainable production of pigs without using production enhancers and chemotherapeutics. Thus, an economically-viable pig production is now only possible when the physiological mechanisms of defense against pathogens and tolerance against nutrients and commensal bacteria in the intestinal immune system are taken into account. During the postnatal period the piglet is facing first the time large amounts of new antigens and at weaning a second wave of nutritional antigens is entering the intestinal tract. The appropriate development of humoral and cellular functions of the intestinal immune system is essential for optimum growth and performance of the piglets. The integrity of the intestinal surfaces is a prerequisite of intestinal immunity and tolerance. Secretory IgA serves to exclude harmful antigens from uptake. The induction of intestinal immune reactions starts with antigen presentation by professional antigen presenting cells of Peyer's patches and mesenteric lymph nodes. In addition, the intestinal lamina propria serves as a mucosal compartment for regulation of immune responses. Here especially T regulatory cells (CD4(+) CD25(+)) have their function for maintaining intestinal homeostasis. The network of mucosal T and B cells develops after birth in a programmed sequence; it is almost completed at week 7 after birth. Weaning is associated with changes in the regulation of the lymphoid cells in the mucosa. In small and large intestine increases in pro- and anti-inflammatory cytokines were observed after weaning in lymphocytes. Epithelial cells were studied both in intestinal samples and in vitro. Here the cytokine patterns provide evidence that weaning is inducing a transient inflammation of the mucosa. Piglets weaned under conventional conditions have a thicker mucosa than pigs weaned from isolators. Cells of isolator-reared pigs show slightly higher levels of activation markers - probably reflecting the interaction of the foreign protein derived from bovine milk. The results presented in this overview demonstrate that further effort is necessary to elucidate the function of the porcine intestinal immune system in the postnatal period and at the time of weaning to provide criteria for porcine intestinal health.

Genetic parameters for growth, mortality and reproductive performances of Markhoz goats were estimated from data collected during 1993–2010 at Markhoz goat Performance Testing Station in Sanandaj, Iran. For kid performance traits 3763 records were available for birth weight (BW), 2931 for weaning weight (WW), average daily gain (ADG) and Kleiber ratio (KR) (approximated as ADW/WW0.75) and 3032 for pre-weaning mortality (PWM). For doe reproductive performance traits there were 2920 records available for litter size at birth (LSB), litter size at weaning (LSW), total litter weight at birth (TLWB) and litter mean weight per kid born (LMWKB), and 2182 for total litter weight at weaned (TLWW) and litter mean weight per kid weaned (LMWKW). Genetic parameters were estimated with univariate and bivariate models using restricted maximum likelihood (REML) procedures. Random effects were explored by fitting additive direct genetic effects, maternal additive genetic effects, maternal permanent environmental effects, the covariance between direct and maternal genetic effects, and common litter effects in different models for pre-weaning traits of kids. Also, in addition to an animal model, sire and threshold models, using a logit link function, were used for analyses of PWM. Models for LSB, LSW, TLWB, TLWW, LMWKB, and LMWKW included direct additive genetic effects, permanent environmental effects due to the animal as well as service sire effects. Estimated direct heritabilities were moderate for pre-weaning traits (0.22 for BW, 0.16 for WW, 0.21 for ADG, and 0.27 for KR and 0.29 for PWM), and low for reproduction traits (0.01 for LSB, 0.01 for LSW, 0.02 for TLWB, 0.03 for TLWW, 0.07 for LMWKB, and 0.06 for LMWKW). The estimates for the maternal additive genetic variance ratios were lower than direct heritability for BW (0.07) and KR (0.04). The estimate for the maternal permanent environmental variance ratios (c2) varied from 0.01 for KR to 0.07 for WW and ADG. The magnitude of common litter variance ratios (l2) was more substantial for BW (0.46) than the PWM (0.19) and KR (0.16). The estimate for the permanent environmental variance due to the animal (c2) ranged from 0.03 for LMWKB to 0.07 for TLWB and LMWKW, whereas service sire effects (s2) ranged from 0.02 to 0.04. The correlation between direct and maternal genetic effects were negative and high for BW (−0.51) and KR (−0.62). The genetic correlations between pre-weaning growth traits were positive and moderate to strong, as were genetic correlations between reproductive traits. Between BW and PWM the correlation was −0.35. Phenotypic and environmental correlations for all traits were generally lower than genetic correlations.

The survival of a laryngeal allograft will be dependent on the immunological composition of the donor larynx and, in particular, on the expression of major histocompatibility complex (MHC) class II antigens on professional and non-professional antigen-presenting cells. Laryngeal and tonsillar biopsies from normal individuals aged 18-78 years were processed and prepared for quantitative, multiple-colour immunofluorescence using mouse antihuman monoclonal antibodies to human leucocyte antigen (HLA)-DR, HLA-DQ and CD45. The laryngeal epithelium expressed HLA-DR locus products at variable levels, but expression of HLA-DQ was virtually absent. Tonsillar epithelial cells expressed HLA-DR at the basal layer only, while HLA-DQ was similarly not expressed. In contrast, both HLA-DR and -DQ locus products were present on lamina propria and intraepithelial leucocytes in both laryngeal and tonsillar mucosae, although at varying levels. The finding that laryngeal epithelial cells express MHC class II antigens has implications for the survival of laryngeal allografts and suggests that they may require significant immunomodulation. In addition, antigen presentation by epithelial cells has been hypothesized to contribute to the immunoregulatory function of mucosal tissues, and the finding that HLA-DQ locus products are only expressed at low levels by laryngeal epithelium raises questions about the repertoire of peptides to which the mucosal immune system can respond.

Leukocyte populations present in the discrete Peyer's patches (PP) of the pig were characterized from birth (Day 0) to day 35 after birth by immunohistochemistry and image analysis. Immediately after birth, cell membrane expression of CD2 and CD3, major histocompatibilty complex (MHC) class 11 (both SLA (swine leukocyte antigen) -DQ+ and SLA-DR+), CD21, 74-22-15 and surface immunoglobulin (sIg) were all demonstrable. Computer assisted morphometric techniques were used to confirm the significant expansion of these cell populations from birth onwards. The distribution of the cell types was not random but suggested a preferential retention of cells at specific sites. This implies a degree of organization of immunological cells within the discrete PP, enhancing the potential to mount immune responses in the most efficient manner.