Comparative analysis of swine leukocyte antigen gene diversity in European farmed pigs.

In Europe, swine represent economically important farm animals and furthermore have become a preferred preclinical large animal model for biomedical studies, transplantation and regenerative medicine research. The need for typing of the swine leukocyte antigen (SLA) is increasing with the expanded use of pigs as models for human diseases and organ-transplantation experiments and their use in infection studies and for design of veterinary vaccines. In this study, we characterised the SLA class I (SLA-1, SLA-2, SLA-3) and class II (DRB1, DQB1, DQA) genes of 549 farmed pigs representing nine commercial pig lines by low-resolution (Lr) SLA haplotyping. In total, 50 class I and 37 class II haplotypes were identified in the studied cohort. The most common SLA class I haplotypes Lr-04.0 (SLA-1*04XX-SLA-3*04XX(04:04)-SLA-2*04XX) and Lr-32.0 (SLA-1*07XX-SLA-3*04XX(04:04)-SLA-2*02XX) occurred at frequencies of 11.02 and 8.20% respectively. For SLA class II, the most prevalent haplotypes Lr-0.15b (DRB1*04XX(04:05/04:06)-DQB1*02XX(02:02)-DQA*02XX) and Lr-0.12 (DRB1*06XX-DQB1*07XX-DQA*01XX) occurred at frequencies of 14.37 and 12.46% respectively. Meanwhile, our laboratory has contributed to several vaccine correlation studies (e.g. Porcine Reproductive and Respiratory Syndrome Virus, Classical Swine Fever Virus, Foot-and-Mouth Disease Virus and Swine Influenza A Virus) elucidating the immunodominance in the T-cell response with antigen specificity dependent on certain SLA-I and SLA-II haplotypes. Moreover, these SLA-immune response correlations could facilitate tailored vaccine development, as SLA-I Lr-04.0 and Lr-32.0 as well as SLA-II Lr-0.15b and Lr-0.12 are highly abundant haplotypes in European farmed pigs.

Methane emission, metabolism, and performance of Holstein dairy cows with low, medium, and high lymphocyte proliferation during transition.

This study aimed to identify interactions between state of lactation (dry or early lactating) and immune responder group (low, medium, or high) for energy metabolism traits as well as metabolic and immunological traits in dairy cows. In early lactation, when the energy priority of cows shifts toward the mammary gland, the energy available to be partitioned toward the immune system may differ among individuals. The equilibrium between energy supply from feed, digestion, and body reserve mobilization and energy expenditure with milk, immune system, methane, and heat production is delicate in this stage. Seventeen Holstein cows entering their second to fifth lactation were kept under comparable feeding, housing, and management conditions and were studied from 14 ± 6 d before calving to 11 ± 3 d after calving. Feed intake, milk yield, body condition, blood metabolites, and cortisol as well as gaseous exchange in respiration chambers were measured. The latter was used to quantify methane emission and to calculate resting metabolic rate and heat production. Subsets of blood leukocytes and peripheral blood mononuclear cells (PBMC) were monitored. Activation and proliferation of the PBMC in response to the mitogen phytohemagglutinin ante- and postpartum were assessed using the oxygen consumption rate (24-h cell culture assay) and the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay (72-h cell culture assay). Cows were classified based on the in vitro proliferative response of the PBMC measured postpartum in low (n = 6), medium (n = 5), and high (n = 6) responders. We found no interaction of state of lactation with responder group for feed intake, milk yield, efficiency, metabolic traits, and immune cell activation ante- and postpartum. However, after calving, low-responder cows produced less methane per unit of body weight and per unit of energy-corrected milk compared with the other cows. This might be indicative of a low rumen fermentation intensity. Low responders might therefore suffer from a lower availability of digestible energy in early lactation and not be able to sustain the shift from immune cell activation to proliferation. If so, the selection of environmentally friendly low-methane emitters could promote phenotypes with a compromised immune response in the critical early lactation.

Assessing immune competence in pigs by immunization with tetanus toxoid.

Immune competence can be tested by challenging organisms with a set of infectious agents. However, disease control requirements impose restrictions on the infliction of infections upon domestic pigs. Alternatively, vaccinations induce detectable immune responses that reflect immune competence. Here, we tested this approach with tetanus toxoid (TT) in young domestic pigs. To optimize the vaccination protocol, we immunized the pigs with a commercial TT vaccine at the age of 21 or 35 days. Booster immunizations were performed either 14 or 21 days later. TT-specific antibodies in plasma as well as lymphoproliferative responses were determined both 7 and 14 days after booster immunization using ELISA and lymphocyte transformation tests, respectively. In addition, general IgG and IgM plasma concentrations and mitogen-induced proliferation were measured. The highest TT-specific antibody responses were detected when blood samples were collected 1 week after a booster immunization conducted 21 days after primary immunization. The pigs' age at primary immunization did not have a significant influence on TT-specific antibody responses. Similarly, the TT-specific proliferative responses were highest when blood samples were collected 1 week after booster immunization, while age and time of primary and booster immunization were irrelevant in our setup. While general IgG and IgM plasma levels were highly age dependent, there were no significant age effects for TT-specific immune responses. In addition, mitogen-induced proliferation was independent of immunization as well as blood sampling protocols. In summary, our model of TT vaccination provides an interesting approach for the assessment of immune competence in young pigs. The detected vaccination effects were not biased by age, even though our data were acquired from immune systems that were under development during our tests.

Phenotype analysis of male transgenic mice overexpressing mutant IGFBP-2 lacking the Cardin-Weintraub sequence motif: Reduced expression of synaptic markers and myelin basic protein in the brain and a lower degree of anxiety-like behaviour.

Brain growth and function are regulated by insulin-like growth factors I and II (IGF-I and IGF-II) but also by IGF-binding proteins (IGFBPs), including IGFBP-2. In addition to modulating IGF activities, IGFBP-2 interacts with a number of components of the extracellular matrix and cell membrane via a Cardin-Weintraub sequence or heparin binding domain (HBD1). The nature and the signalling elicited by these interactions are not fully understood. Here, we examined transgenic mice (H1d-hBP2) overexpressing a mutant human IGFBP-2 that lacks a specific heparin binding domain (HBD1) known as the Cardin-Weintraub sequence. H1d-hBP2 transgenic mice have the genetic background of FVB mice and are characterized by severe deficits in brain growth throughout their lifetime (p<0.05). In tissue lysates from brain hemispheres of 12-21day old male mice, protein levels of the GTPase dynamin-I were significantly reduced (p<0.01). Weight reductions were also found in distinct brain regions in two different age groups (12 and 80weeks). In the younger group, impaired weights were observed in the hippocampus (-34%; p<0.001), cerebellum (-25%; p<0.0001), olfactory bulb (-31%; p<0.05) and prefrontal cortex (-29%; p<0.05). At an age of 12weeks expression of myelin basic protein was reduced (p<0.01) in H1d-BP-2 mice in the cerebellum but not in the hippocampus. At 80weeks of age, weight reductions were similarly present in the cerebellum (-28%; p<0.001) and hippocampus (-31; p<0.05). When mice were challenged in the elevated plus maze, aged but not younger H1d-hBP2 mice displayed significantly less anxiety-like behaviour, which was also observed in a second transgenic mouse model overexpressing mouse IGFBP-2 lacking HBD1 (H1d-mBP2). These in vivo studies provide, for the first time, evidence for a specific role of IGFBP-2 in brain functions associated with anxiety and risk behaviour. These activities of IGFBP-2 could be mediated by the Cardin-Weintraub/HBD1 sequence and are altered in mice expressing IGFBP-2 lacking the HBD1.

Anticipation and frequency of feeding affect heart reactions in domestic pigs.

Measuring heart reactions has become a widely used method for the assessment of emotions. Heart rate and its variability, which can quite easily be noninvasively recorded, reflect the inputs of the sympathetic and parasympathetic branches of the autonomous nervous system. We tested the hypothesis that frequent anticipation of a positive event results in an increased state of welfare in pigs, expressed as positive arousal in anticipation of announced feeding as well as lowered heart rate and augmented heart rate variability during resting periods. We used a controlled paradigm with 3 groups of young domestic pigs (Sus scrofa domestica). We compared frequent acoustic announcement of feed delivery (group 1: 3 feedings between 0730 h and 1030 h plus 3 feedings between 1200 h and 1530 h) with the same number of feedings as in group 1 but without a temporal relation to the sound (group 2) and with a fixed-schedule feeding (group 3: 2 feedings at 0600 h and 1500 h). Specific cardiac and behavioral reactions indicated short-term (1 min) anticipation in the conditioned group. In this group, heart rate increased (P < 0.001) mainly through vagal withdraw and behavior became more active (P < 0.001). Only the conditioned group displayed changing heart rate characteristics during the sound. Pigs in the frequent unpredictable feed group reacted to feed delivery with increased heart rates (P < 0.001), whereas the heart-rate characteristics of pigs with the fixed schedule were unchanged during the sound and while the other 2 treatment groups were feeding. Clear evidence for long-term anticipation (over the course of hours) was not present in the data. Comparisons between the 3 treatment groups suggested that in housing conditions where pigs cannot obtain feed by their actions but must wait for feed delivery, feeding at 2 fixed times would be preferred. Animals in this treatment group presented lower resting heart rates at the end of the experiment than animals in the other 2 groups (P < 0.01). Therefore, merely announcing a positive stimulus without giving control to its access is apparently not suitable for increasing welfare.

A decrease of intracellular ATP is compensated by increased respiration and acidification at sub-lethal parathion concentrations in murine embryonic neuronal cells: measurements in metabolic cell-culture chips.

We present a label-free in vitro method for testing the toxic potentials of chemical substances using primary neuronal cells. The cells were prepared from 16-day-old NMRI mouse embryos and cultured on silicon chips (www.bionas.de) under the influence of different parathion concentrations with sensors for respiration (Clark-type oxygen electrodes), acidification (pH-ISFETs) and cell adhesion (interdigitated electrode structures, IDES). After 12 days in vitro, the sensor readouts were simultaneously recorded for 350 min in the presence of parathion applying a serial 1:3 dilution. The parathion-dependent data was fitted by logistic functions. IC(50) values of approximately 105 µM, 65 µM, and 54 µM were found for respiration, acidification, and adhesion, respectively. An IC(50) value of approximately 36 µM was determined from the intracellular ATP-levels of cells, which were detected by an ATP-luminescence assay using micro-well plates. While the intracellular ATP level and cell adhesion showed no deviation from a simple logistic decay, increases of approximately 29% in the respiration and 15% in the acidification rates above the control values were found at low parathion concentrations, indicating hormesis. These increases could be fitted by a modified logistic function. We believe that the label-free, continuous, multi-parametric monitoring of cell-metabolic processes may have applications in systems-biology and biomedical research, as well as in environmental monitoring. The parallel characterization of IC(50) values and hormetic effects may provide new insights into the metabolic mechanisms of toxic challenges to the cell.

Differential expression of costimulatory molecules B7-1 and B7-2 on microglial cells induced by Th1 and Th2 cells in organotypic brain tissue.

Autoreactive T-cells are involved in demyelination, neurodegeneration, and the recruitment of peripheral macrophages and nonspecific activated T-cells in autoimmune diseases such as multiple sclerosis (MS). The ligation of costimulatory B7 molecules on microglia with CD28/CTLA-4 on T-cells is thought to be crucial to the onset and course of MS and its rodent model experimental autoimmune encephalomyelitis (EAE). It is currently unclear as to how far the nature of infiltrating T-cells has an impact on the expression of the B7 molecules on microglia, the resident antigen-presenting cells (APCs) of the brain. We studied the expression of B7-1 and B7-2 on microglia after encounter with preactivated Th1 and Th2 cells from transgenic mice whose T-cells express a receptor (TCR) either specific to myelin basic protein (MBP) or ovalbumin (OVA) using murine organotypic entorhinal-hippocampal slice cultures (OEHSC). Our main finding was that Th1 cells downregulate the constitutive expression of B7-2 and induce B7-1 expression while Th2 cells do not induce this B7-1 upregulation. The main difference between MBP- and OVA-specific cells was seen in experiments were Th1 cells had direct contact to APCs but not to brain tissue. In contrast to MBP-specific Th1 cells, OVA-specific Th1 cells required the addition of antigen to upregulate B7-1 and downregulate B7-2. When the cells were allowed to have contact to brain tissue, no difference was seen in the pattern of B7 regulation between OVA- and MBP-specific T-cells. Our data suggest that T-cells are able to modulate B7 expression on microglial cells in the brain independent of antigen presentation through TCR/MHC-II ligation but presumably by soluble mediators.

Th2 cells support intrinsic anti-inflammatory properties of the brain.

In experimental autoimmune encephalomyelitis (EAE), Th1 cells are responsible for disease induction while Th2 cells can be protective. To address the mechanisms of this differential behavior, we utilized organotypic murine entorhinal-hippocampal slice cultures to analyze interactions between myelin basic protein-specific Th1 and Th2 cells with microglial cells. While both Th1 and Th2 cells induced CD40 expression, only Th1 cells induced intercellular adhesion molecule-1 (ICAM-1) expression on microglia. Moreover, Th2 cells prevented or even reversed Th1-induced ICAM-1 upregulation. Evidently, Th2 cells could diminish Th1-induced inflammatory reactions and actively support the resting state of microglia, which could be one mechanism of Th2-mediated remission of neuroinflammation during EAE.

Turnover of rat brain perivascular cells.

Brain perivascular spaces harbor a population of cells which exhibit high phagocytic capacity. Therefore, these cells can be labeled by intraventricular injection of tracers. Such perivascular cells at the interface between blood and brain are believed to belong to the monocyte/macrophage lineage and to be involved in antigen presentation. Currently, it is unclear whether these cells undergo a continuous turnover by entering and leaving the bloodstream. Using bone-marrow-chimeric animals, migration of donor macrophages into brain perivascular spaces has been reported. On the other hand, following intracerebral injection of india ink into nontransplanted animals, ink-labeled perivascular cells were still found 2 years after injection, suggesting a high stability of this cell pool. Thus, the turnover of perivascular cells observed in chimeras might be a result of bone marrow transplantation rather than a physiological occurrence. To address this issue, we monitored de novo invasion of macrophages into perivascular spaces of apparently healthy adult rats by applying techniques other than bone marrow transplantation, (i) consecutive injections of different tracers and (ii) ex vivo isolation of macrophages from the blood, cell labeling, and reinjection into the same animal to avoid MHC mismatch. Both approaches revealed vivid de novo invasion of macrophages into perivascular spaces, but not into brain parenchyma, rendering untenable the concept of perivascular cells forming a stable population of macrophages in the brain. Thus, brain perivascular spaces are under permanent immune surveillance of blood borne macrophages in normal adult rats.

Reactive astrocytes upregulate Fas (CD95) and Fas ligand (CD95L) expression but do not undergo programmed cell death during the course of anterograde degeneration.

Tissue homeostasis is determined by a balance between proliferation and apoptosis. Various lesions in the brain are accompanied by proliferation and subsequent death of glial cells, but the mechanisms that limit this expansion of glial populations remains unknown. One possible candidate is the death ligand, FasL, and its receptor Fas, because the expression of both proteins was reported on glial cells. To elucidate the expression and putative function of Fas and FasL on proliferative glial cells, we performed stereotactic lesion of the entorhinal cortex of adult rats. Such lesions induce proliferation of astrocytes and microglial cells in the hippocampal fields of anterograde degeneration. Subsequently, the total number of both cell types returns to pre-lesion counts. We found that Fas and FasL is strongly upregulated on astrocytes in the zone of anterograde degeneration with a peak 5 days postlesion (dpl) and a return to control levels at 10 dpl. However, evidence for astrocytic cell death was neither detected by TUNEL staining, immunocytochemistry for c-Jun, and apoptosis-specific protein (ASP), nor by staining for morphologic hallmarks of apoptotic or necrotic cell death at the light and electron microscopic level. Thus, increased expression of Fas and FasL is not accompanied by cell death of reactive astrocytes during anterograde degeneration.

Phagocytosis of neuronal or glial debris by microglial cells: upregulation of MHC class II expression and multinuclear giant cell formation in vitro.

Most CNS pathologies are accompanied by the occurrence of activated, phagocytic microglial cells. We intended to investigate whether (1) isolated microglial cells removed from the CNS cytokine network sustain their capacity to acquire an activated phenotype when challenged with cellular or noncellular debris; and (2) different substrates lead to different patterns of microglial activation. It was observed that although removed from their usual surroundings microglial cells preserve their ability to transform to an amoeboid morphology, form multinucleated giant cells, and enhance their expression of MHC class II when exposed to membranes of neuronal or glial origin. Furthermore, cellular substrates derived from primary hippocampal neuronal cultures, neuroblastic cells (B50), or glial cells were all able to induce similar morphological changes and enhanced expression of MHC class II. In contrast, phagocytosis of Latex beads induced an amoeboid morphology but no increase in the expression of immunologically relevant molecules. Interferon-beta (IFN-beta), a substance clinically used in the treatment of the relapsing-remitting form of multiple sclerosis, was shown to inhibit the phagocytosis-induced upregulation of MHC-class II. In summary, phagocytic microglial cells are independent from the CNS cytokine network in their transition from a resting to an activated phenotype; and different cellular substrates, regardless whether they are of neuronal, glial, or even malignant origin, result in similar morphological and functional changes.

Axonal damage induced by invading T cells in organotypic central nervous system tissue in vitro: involvement of microglial cells.

Neuroinflammation in the course of multiple sclerosis and experimental autoimmune encephalomyelitis results in demyelination and, recently demonstrated, axonal loss. Invading neuroantigen specific T cells are the crucial cellular elements in these processes. Here we demonstrate that invasion of activated T cells induces a massive microglial attack on myelinated axons in entorhinal-hippocampal slice cultures. Flow cytometry analysis of activation markers revealed that the activation state of invading MBP-specific T cells was significantly lower in comparison to PMA-activated T cells. Moreover, MBP-specific T cells showed a significantly lower secretion of IFN-gamma. Conversely, MBP-specific T cells displayed a significantly higher potential to trigger activation of microglial cells, i.e. upregulation of MHC class II and ICAM-1 expression, and, most importantly, microglial phagocytosis of pre-traced axons. Our data suggest that this was mediated via specific cellular interactions of T cells and microglial cells since IFN-gamma alone was not sufficient to induce axonal damage while such damage was apparent in response to TNF-alpha which is released by activated microglial cells. TNF-alpha secretion by both T cell populations was negligible. Thus, MBP-specific T cells which invade nervous tissue in the course of neuroinflammation are more effective in axon-damaging recruiting microglial cells than activated T cells of other specificities.

Inhibitors of Src-family tyrosine kinases favour Th2 differentiation.

Two potent pyrazolo-pyrimidine inhibitors of Src-family kinases were found to enhance interleukin 4 (IL-4) and reduce interferon gamma(IFN-gamma) production in cultures of splenocytes from ovalbumin-specific TCR-transgenic BALB/c mice. The effect was increased by addition of a monoclonal antibody binding to domains 3/4 of CD4, while other antibodies binding to domain 1 had the opposite effect. The inhibitors suppressed CD40 ligand (CD40L) expression on activated CD4 T cells, which may explain this Th2 differentiating effect. More generally, the effect fits within the overall framework of signal attenuation in T cells driving this form of differentiation. The inhibitors did not revert previously induced Th1 differentiation in serial cultures of the TCR-transgenic cells. Drugs with this activity are of obvious interest as probes and potential therapeutic agents in autoimmune disease.

Modulating the Th1/Th2 balance in inflammatory arthritis.

The balance between Th1 and Th2 cells regulates the choice between inflammatory and antibody-mediated immune responses. To an increasing extent this balance is thought to involve the participation of antigen-presenting cells, rather than the entirely autonomous activity of T cells and their cytokines. Here we survey current opinion concerning the working of this balance, and its condition in rheumatoid arthritis and the other inflammatory arthritides. The contrast between Lyme arthritis and reactive arthritis is particularly illuminating, since one is triggered by extracellular and the other by intracellular infection. We describe current approaches to the modulation of this balance. Guided by the principles that genetic polymorphism is likely to identify relevant genes, that any cytokine gene picked up by a virus must matter and that natural immunosuppressive activity at mucosal surfaces should be worth exploiting, we identify as particularly worthy of attention: (i) IL-10, (ii) inhibitors of IL-12 production, (iii) inhibitors of CD40 ligand expression and (iv) oral and nasal tolerance. Other protective T cell subsets are touched on, and the impact of oligonucleotide arrays mentioned.

Determination of viral neuraminidase specificity for membrane-bound sialic acids by cell electrophoresis.

The ability of the influenza virus neuraminidase (NA) to cleave specific sialic acids was measured by cell electrophoresis. Most of the surface charge of human erythrocytes can be attributed to sialic acids. Therefore cleavage of sialic acids reduces the surface charge density which is measurable as a reduced cell electrophoretic mobility (EPM). For experiments specifically sialylated, erythrocytes were used. Their EPM was significantly decreased after incubation with virus strains possessing the corresponding NA specificity, even when the viral haemagglutinin (HA) was unable to bind to the erythrocyte's surface. Thus, the limited applicability of elution experiments, which requires virus binding, is overcome. An additional advantage of this procedure is that it is non-radioactive. In our model system the erythrocyte's surface resembles the natural situation of viral interaction with membrane-bound receptors.

Type II collagen serology: a guide to clinical responsiveness to oral tolerance?

A double-blind placebo-controlled randomized trial of oral collagen type II (CII) treatment in rheumatoid arthritis was completed in Berlin. Anti-CII antibody titres were measured before and after the treatment. They showed that: (1) the titre prior to treatment did not identify a responder subgroup, (2) the treatment reduced CII antibody titres, but only in those patients making a clinical response and (3) administration of 10 mg CII per day reduced the titre in these subsets more effectively than 1 mg per day. Although the data are limited, they suggest that a titre drop may be useful for identifying those patients who respond to this form of treatment and that the drop may be a valid parameter for detecting the impact of the treatment on the immune system.

Two evolutionary strategies of influenza viruses to escape host non-specific inhibitors: alteration of hemagglutinin or neuraminidase specificity.

The porcine serum inhibitor alpha 2-macroglobulin prevents influenza virus from entering host cells by competing for the SA alpha 2, 6Gal-binding site of the hemagglutinin (HA). We studied a series of inhibitor-sensitive and inhibitor-resistant human and porcine influenza virus isolates of the H3N2 subtype, all of which contained HAs, which initially bound only to SA alpha 2, 6Gal oligosaccharides. When their neuraminidase was inhibited, the naturally resistant viruses, as a result of no longer being able to elute from the inhibitor, became sensitive. Evidently it is the neuraminidase which enabled these viruses to grow in hosts which possess the inhibitor. Escape-mutants selected under laboratory conditions in the presence of porcine serum became inhibitor-resistant by two alternative mechanisms: they changed either their HA-specificity or their neuraminidase-specificity. The study thus disclosed two evolutionary strategies for acquiring resistance to a host neuraminidase-sensitive inhibitor: (i) acquisition of an HA able to bind to oligosaccharides not present on the inhibitor; or (ii) acquisition of a neuraminidase able to cleave the oligosaccharide bound by the HA.