Changes in protein expression in the sheep abomasum following trickle infection with Teladorsagia circumcincta.

Continual low-level exposure of sheep to the helminth Teladorsagia circumcincta elicits a temporary protective immunity, where factors in the immune abomasal mucosa prevent penetration of infective larvae, but which is essentially lost within 6 weeks of cessation of parasite challenge. Here, a proteomic approach was used to identify proteins that are differentially regulated in immune compared to naïve sheep, as potential key mediators of immunity. Six naïve sheep and 12 sheep trickle-infected with T. circumcincta were treated with anthelmintic, and the naïve (control) and 6 immune sheep were killed 7 days later. The remaining 6 sheep (immune waning) were killed 42 days after anthelmintic treatment. Abomasal tissue samples were subjected to 2D-gel electrophoresis and densitometric analysis. Selected spots (n=73) were identified by peptide mass fingerprinting and confirmatory Western blotting was carried out for 10 proteins. Spots selectively up-regulated in immune versus control, but not immune waning versus control sheep, included galectin-15 and thioredoxin, which were confirmed by Western blotting. In immune sheep, serum albumin was significantly down-regulated and albumin proteolytic cleavage fragments were increased compared to controls. Unexpectedly, albumin mRNA was relatively highly expressed in control mucosa, down-regulated in immune, and was immunolocalized to mucus-producing epithelial cells. Thus we have identified differential expression of a number of proteins following T. circumcincta trickle infection that may play a role in host protection and inhibition of parasite establishment.

Strain-specific copy number variation in the intelectin locus on the 129 mouse chromosome 1.

BACKGROUND: C57BL/6J mice possess a single intelectin (Itln) gene on chromosome 1. The function of intelectins is not well understood, but roles have been postulated in insulin sensitivity, bacterial recognition, intestinal lactoferrin uptake and response to parasites and allergens. In contrast to C57BL/6J mice, there is evidence for expansion of the Itln locus in other strains and at least one additional mouse Itln gene product has been described. The aim of this study was to sequence and characterise the Itln locus in the 129S7 strain, to determine the nature of the chromosomal expansion and to inform possible future gene deletion strategies. RESULTS: Six 129S7 BAC clones were sequenced and assembled to generate 600 kbp of chromosomal sequence, including the entire Itln locus of approximately 500 kbp. The locus contained six distinct Itln genes, two CD244 genes and several Itln- and CD244-related pseudogenes. It was approximately 433 kbp larger than the corresponding C57BL/6J locus. The expansion of the Itln locus appears to have occurred through multiple duplications of a segment consisting of a full-length Itln gene, a CD244 (pseudo)gene and an Itln pseudogene fragment. Strong evidence for tissue-specific distribution of Itln variants was found, indicating that Itln duplication contributes more than a simple gene dosage effect. CONCLUSIONS: We have characterised the Itln locus in 129S7 mice to reveal six Itln genes with distinct sequence and expression characteristics. Since C57BL/6J mice possess only a single Itln gene, this is likely to contribute to functional differences between C57BL/6J and other mouse strains.

Novel gene expression responses in the ovine abomasal mucosa to infection with the gastric nematode Teladorsagia circumcincta.

Infection of sheep with the gastric nematode Teladorsagia circumcincta results in distinct Th2-type changes in the mucosa, including mucous neck cell and mast cell hyperplasia, eosinophilia, recruitment of IgA/IgE producing cells and neutrophils, altered T-cell subsets and mucosal hypertrophy. To address the protective mechanisms generated in animals on previous exposure to this parasite, gene expression profiling was carried out using samples of abomasal mucosa collected pre- and post- challenge from animals of differing immune status, using an experimental model of T. circumcincta infection. Recently developed ovine cDNA arrays were used to compare the abomasal responses of sheep immunised by trickle infection with worm-naïve sheep, following a single oral challenge of 50 000 T. circumcincta L3. Key changes were validated using qRT-PCR techniques. Immune animals demonstrated highly significant increases in levels of transcripts normally associated with cytotoxicity such as granulysin and granzymes A, B and H, as well as mucous-cell derived transcripts, predominantly calcium-activated chloride channel 1 (CLCA1). Challenge infection also induced up-regulation of transcripts potentially involved in initiating or modulating the immune response, such as heat shock proteins, complement factors and the chemokine CCL2. In contrast, there was marked infection-associated down-regulation of gene expression of members of the gastric lysozyme family. The changes in gene expression levels described here may reflect roles in direct anti-parasitic effects, immuno-modulation or tissue repair.

The role of Intelectin-2 in resistance to Ascaris suum lung larval burdens in susceptible and resistant mouse strains.

The underlying mechanism of predisposition to Ascaris infection is not yet understood but host genetics are thought to play a fundamental role. We investigated the association between the Intelectin-2 gene and resistance in F2 mice derived from mouse strains known to be susceptible and resistant to infection. Ascaris larvae were isolated from murine lungs and the number of copies of the Intelectin-2 gene was determined in F2 mice. Intelectin-2 gene copy number was not significantly linked to larval burden. In a pilot experiment, the response to infection in parental mice of both sexes was observed in order to address the suitability of female F2 mice. No overall significant sex effect was detected. However, a divergence in resistance/susceptibility status was observed between male and, female hybrid offspring. The responsiveness to Ascaris in mice is likely to be controlled by multiple genes and, despite a unique absence from the susceptible C57BL/6j strain, the Intelectin-2 gene does not play a significant role in resistance. The observed intra-strain variation in larval burden requires further investigation but we hypothesize that it stems from social/dominance hierarchies created by the presence of female mice and possibly subsequent hormonal perturbations that modify the intensity of the immune response.

Proteomic identification of interactions between histones and plasma proteins: implications for cytoprotection.

Extracellular histones released from cells during acute inflammation contribute to organ failure and death in a mouse model of sepsis, and histones are known to exert in vitro cytotoxicity in the absence of serum. Since addition of histones to serum and plasma is known to induce protein aggregation, we reasoned that plasma proteins may afford protection from cytotoxicity. We found that MODE-K mouse small intestinal epithelial cells were protected from histone-induced toxicity in the presence of 10% FCS. Therefore, the main aim of this study was to identify histone-interacting plasma proteins that might be involved in cytoprotection. The precipitate formed following addition of calf thymus histones to human EDTA plasma was characterised by shotgun proteomics, identifying a total of 36 protein subunits, including complement components, coagulation factors, protease inhibitors and apolipoproteins. The highly sulphated glycosaminoglycan heparin inhibited histone-induced plasma protein aggregation. Moreover, histones bound to heparin agarose were capable of pulling down plasma proteins from solution, indicating their effective cross-linking properties. It was particularly notable that inter-alpha-trypsin inhibitor was prominent among the histone-precipitated proteins, since it contains a chondroitin sulphate glycan chain, and suggests a potential role for this protein in histone sequestration during acute inflammation in vivo.

Chronic allergen challenge induces bronchial mast cell accumulation in BALB/c but not C57BL/6 mice and is independent of IL-9.

As genetically engineered mutant mice deficient in single genes are usually generated on a C57BL/6 background, to study mast cell trafficking in mutant mice, we initially investigated whether mast cells accumulated in bronchi in C57BL/6 mice challenged with OVA allergen acutely or chronically for 1 to 3 months. The total number of bronchial mast cells were quantitated using toluidine blue staining in airways of different sizes, i.e. , small (<90 microm), medium (90-155 microm), or large (>150 microm) airways. Non-OVA challenged and acute OVA challenged mice (C57BL/6 and BALB/c) had no detectable bronchial mast cells. Chronic OVA challenge in BALB/c mice for 1 or 3 months induced a significant increase in the number of bronchial mast cells in small-, medium-, and large-sized airways but minimal change in the number of bronchial mast cells in C57BL/6 mice. Both BALB/c and C57BL/6 mice developed significant lung eosinophilia following acute or chronic OVA challenge. Studies of IL-9-deficient mice on a BALB/c background demonstrated a significant increase in the number of bronchial mast cells in IL-9-deficient mice suggesting that IL-9 was not required for the bronchial accumulation of mast cells. Overall, these studies demonstrate that the chronic OVA challenge protocol we have utilized in BALB/c mice provides a model to study the mechanism of bronchial mast cell accumulation and that bronchial mast cell accumulation in chronic OVA challenged mice is independent of IL-9 in this model.

Expulsion of Trichuris muris is associated with increased expression of angiogenin 4 in the gut and increased acidity of mucins within the goblet cell.

BACKGROUND: Trichuris muris in the mouse is an invaluable model for infection of man with the gastrointestinal nematode Trichuris trichiura. Three T. muris isolates have been studied, the Edinburgh (E), the Japan (J) and the Sobreda (S) isolates. The S isolate survives to chronicity within the C57BL/6 host whereas E and J are expelled prior to reaching fecundity. How the S isolate survives so successfully in its host is unclear. RESULTS: Microarray analysis was used as a tool to identify genes whose expression could determine the differences in expulsion kinetics between the E and S T. muris isolates. Clear differences in gene expression profiles were evident as early as day 7 post-infection (p.i.). 43 probe sets associated with immune and defence responses were up-regulated in gut tissue from an E isolate-infected C57BL/6 mouse compared to tissue from an S isolate infection, including the message for the anti-microbial protein, angiogenin 4 (Ang4). This led to the identification of distinct differences in the goblet cell phenotype post-infection with the two isolates. CONCLUSION: Differences in gene expression levels identified between the S and E-infected mice early during infection have furthered our knowledge of how the S isolate persists for longer than the E isolate in the C57BL/6 mouse. Potential new targets for manipulation in order to aid expulsion have been identified. Further we provide evidence for a potential new marker involving the acidity of the mucins within the goblet cell which may predict outcome of infection within days of parasite exposure.

Bovine intelectins: cDNA sequencing and expression in the bovine intestine.

Intelectins (Itlns) are lectins with potential roles in innate immunity, capable of binding bacteria via galactofuranose residues. Itlns also function as intestinal receptors for the antimicrobial glycoprotein lactoferrin (Lf). Since Lf binds strongly to enterohemorrhagic Escherichia coli O157:H7 (EHEC), we aimed to determine the expression of Lf receptor in terminal rectum, the site of predilection of EHEC in cattle. We sequenced two bovine intelectins (Itln1 and Itln2) and showed that both were expressed in abomasum and rectum, but expression appeared minimal in the jejunum. There was significantly higher expression of Itln2 in terminal rather than proximal rectum. Lactoferrin was expressed in all samples examined. Thus, we have demonstrated two novel bovine Itlns and shown that they are expressed along with Lf in the gastrointestinal tract, where they may interact with microbial pathogens.

Extended cleavage specificity of mMCP-1, the major mucosal mast cell protease in mouse-high specificity indicates high substrate selectivity.

Mucosal mast cells are in the mouse predominantly found in the epithelium of the gastrointestinal tract. They express the beta-chymases mMCP-1 and mMCP-2. During nematode infections these intraepithelial mast cells increase in numbers and high amounts of mMCP-1 appear in the jejunal lumen and in the circulation. A targeted deletion of this enzyme leads to decreased ability to expel the intraepithelial nematode Trichinella spiralis. A suggested role for mMCP-1 is alteration of epithelial permeability by direct or indirect degradation of epithelial and endothelial targets, however, no such substrates have yet been identified. To enable a screening for natural substrates we performed a detailed analysis of the extended cleavage specificity of mMCP-1, using substrate phage display technology. In positions P1 and P1' distinct preferences for Phe and Ser, respectively, were observed. In position P2 a high selectivity for large hydrophobic amino acids Phe, Trp and Leu was detected, and in position P2' aliphatic amino acids Leu, Val and Ala was preferred. In positions P3 and P4, N-terminal of the cleaved bond, mMCP-1 showed specificity for aliphatic amino acids. The high selectivity in the P2, P1, P1' and P2' positions indicate that mMCP-1 has a relatively narrow set of in vivo substrates. The consensus sequence was used to screen the mouse protein database for potential substrates. A number of mouse extracellular or membrane proteins were identified and cell adhesion and connective tissue components were a dominating subfamily. This information, including the exact position of potential cleavage sites, can now be used in a more focused screening to identify which of these target molecules is/are responsible for the increased intestinal permeability observed in parasite infected mice.

The proteome of gastric lymph in normal and nematode infected sheep.

Lymph node cannulation allows the collection of lymph draining from a defined anatomical region. Proteomic analysis of that lymph offers a potentially valuable insight into the immunoinflammatory response of that particular region. In this study, ovine gastric lymph has been used to monitor the proteomic changes occurring in the tissue fluid of the abomasum, in response to infection with the parasitic nematode, Teladorsagia circumcincta. Lymph, collected temporally over an experimental infection period, was analysed by means of 2-DE and subsequent gel analysis using densitometry software. In addition, the composition of the lymphatic proteome was further explored by means of MALDI-TOF and MS/MS analyses. The concentration of gelsolin, alpha-1 beta glycoprotein and haemopexin were altered significantly (p<0.05) with infection.

Up-regulation of intelectin in sheep after infection with Teladorsagia circumcincta.

A novel intelectin molecule designated sheep intelectin 2 (sITLN2) was detected in sheep abomasal mucosa. The full sequence shared 76-83% homology with other mammalian intelectins. Intelectins are mucus-associated proteins that have been shown to be up-regulated in gastrointestinal nematode infections in rodents and in human asthma. Expression of sheep abomasal ITLN2 mRNA was significantly up-regulated on day 10 post-challenge of worm-free sheep with Teladorsagia circumcincta and at day 2 in previously infected, immune sheep. Increased expression of ITLN protein following challenge was confirmed by Western blot and was immunolocalised to the mucous neck cells of the abomasal mucosa. Infection with T. circumcincta was also associated with increased levels of abomasal transcripts encoding sheep mast cell protease-1, ovine galectin-14 and IL4, which collectively suggested a Th2 type response. Intelectin may play an important role in the mucosal response to gastrointestinal nematode infections in ruminants.

Trichinella spiralis induces de novo expression of group IVC phospholipase A2 in the intestinal epithelium.

Phospholipase A2 (PLA2) enzymes play a central role in the initiation, propagation and resolution of inflammation. Here, we describe de novo expression of group IVC PLA2 (PLA2g4c) within the intestinal epithelium of Trichinella spiralis parasitised mice. This mouse mast cell protease-1 sensitive, calcium-independent PLA2 is not detectable in the jejunal epithelium of uninfected mice but becomes highly expressed within the epithelial compartment within days of nematode establishment. We propose that epithelial PLA2g4c accounts for the increased lysophospholipase activity observed during intestinal nematodiasis and that it plays a major role in the inflammatory response to nematodes.

The involvement of mast cells and mast cell proteinases in the intestinal response to equine cyathostomin infection.

Cyathostomins (Cyathostominae) are regarded as the most pathogenic equine nematode worldwide. These nematodes are difficult to control in equine populations due to emerging anthelmintic resistance and evasion of encysted larval cyathostomins to regular modern anthelmintics. Mast cells and their proteinases have been shown to play a role in the mammalian immune response to nematode infections. Involvement of mast cells and mast cell proteinases in the equine immune response to cyathostomin infection is proposed. A technique was established to perform immunohistochemical staining using polyclonal rabbit anti-equine mast cell proteinase-1 (eqMCP-1) and anti-equine tryptase on formalin-fixed large intestinal sections, from horses classified as cyathostomin positive and negative at the time of death based upon larval enumeration. Quantitative analysis of antibody labelled mast cells was used to detect mast cell proteinases in equine large intestinal sections positive and negative for cyathostomin larvae. This demonstrated an increase in equine tryptase labelled mucosal and submucosal mast cells in cyathostomin positive horses. This study has established an immunohistochemical technique to demonstrate mast cell proteinases in formalin-fixed large intestinal sections. This technique may be used to determine possible involvement of mast cells and their proteinases in the equine immune response to cyathostomin larvae. Further studies are required to define a specific role.

The expression of intelectin in sheep goblet cells and upregulation by interleukin-4.

Upregulation of intelectin (ITLN) transcript and protein has previously been shown in intestinal nematode infections of resistant mice strains with immunolocalisation of protein to goblet cells and paneth cells. In man, intelectin expression has been shown in respiratory tract epithelium, with upregulation occurring in bronchoalveolar lavage fluid of asthmatic individuals. This study describes the expression of intelectin in the respiratory tract of sheep and the immunolocalisation to goblet cells using a novel affinity-purified chicken anti-intelectin peptide antibody. Furthermore we show that when sheep tracheal explants were cultured for 48 h+/- recombinant sheep IL-4, sheep ITLN transcripts were upregulated compared with controls. Putative roles for intelectin have included an antibacterial role and an alteration of the character of mucus. Our data suggest ITLNs may play an important role in the mucosal response in allergy and parasitic infections.

Primary antibody-Fab fragment complexes: a flexible alternative to traditional direct and indirect immunolabeling techniques.

Immunolabeling with immune complexes of primary and secondary antibodies offers an attractive method for detecting and quantifying specific antigen. Primary antibodies maintain their affinity for specific antigen after labeling with Fab fragments in vitro. Incubation of these immune complexes with excess normal serum from the same species as the primary antibody prevents free Fab fragments from recognizing immunoglobulin. Effectively a hybrid between traditional direct and indirect immunolabeling techniques, this simple technique allows primary antibodies to be non-covalently labeled with a variety of reporter molecules as and when required. Using complexes containing Fab fragments that recognize both the Fc and F(ab')2 regions of IgG, we show that this approach prevents nonspecific labeling of endogenous immunoglobulin, can be used to simultaneously detect multiple antigens with primary antibodies derived from the same species, and allows the same polyclonal antibody to be used for both antigen capture and detection in ELISA.

Characterisation of an extracellular serine protease gene (nasp gene) from Dermatophilus congolensis.

A partial amino acid sequence of a serine protease from Dermatophilus congolensis allowed the design of oligonucleotide primers that were complemented with additional ones from previously published partial sequences of the gene encoding the enzyme. The polymerase chain reaction (PCR), using combinations of specific and degenerate oligonucleotide primers, allowed the amplification of a 1738-bp internal fragment of the gene, which was finally characterised by inverse PCR as the first full-length sequenced serine protease gene (nasp) from Dermatophilus congolensis. The deduced amino acid sequence of this enzyme, probably involved in the pathogenesis of dermatophilosis, links it to the subtilisin family of proteases.

The goblet cell is the cellular source of the anti-microbial angiogenin 4 in the large intestine post Trichuris muris infection.

BACKGROUND: Mouse angiogenin 4 (Ang4) has previously been described as a Paneth cell-derived antimicrobial peptide important in epithelial host defence in the small intestine. However, a source for Ang4 in the large intestine, which is devoid of Paneth cells, has not been defined. METHODOLOGY/PRINCIPAL FINDINGS: Analysis was performed on Ang4 expression in colonic tissue by qPCR and immunohistochemistry following infection with the large intestine dwelling helminth parasite Trichuris muris. This demonstrated an increase in expression of the peptide following infection of resistant BALB/c mice. Further, histological analysis of colonic tissue revealed the cellular source of this Ang4 to be goblet cells. To elucidate the mechanism of Ang4 expression immunohistochemistry and qPCR for Ang4 was performed on colonic tissue from T. muris infected mouse mutants. Experiments comparing C3H/HeN and C3H/HeJ mice, which have a natural inactivating mutation of TLR4, revealed that Ang4 expression is TLR4 independent. Subsequent experiments with IL-13 and IL-4 receptor alpha deficient mice demonstrated that goblet cell expression of Ang4 is controlled either directly or indirectly by IL-13. CONCLUSIONS: The cellular source of mouse Ang4 in the colon following T. muris infection is the goblet cell and expression is under the control of IL-13.

Sheep intelectin-2 co-purifies with the mucin Muc5ac from gastric mucus.

Secretion of gastric mucins plays an essential role in host protection, and modifications in mucus properties are characteristic of the protective immune responses to pathogens. This study describes the purification and characterisation of sheep gastric mucins, and identification of those proteins that co-purify with mucins, with the potential to modify mucus properties. Gastric mucus was collected and pooled from four abattoir sheep and separated by CsCl density gradient centrifugation. Proteomic analysis of the mucin-containing fraction indicated the presence of gastric mucin (Muc5ac) and several co-purifying proteins, including intelectin-2 (Itln2). Further experimentation indicated that a combination of denaturation and reduction was required to fully release Itln2 from gastric mucin. A putative correlation was found between mucin-bound intelectin concentration and rheological properties in further sheep gastric mucus samples. In conclusion, this study provides the first characterisation of sheep gastric mucins and their purification partners, revealing potentially important mucin-intelectin interactions.

Genome-wide transcriptomic analysis of intestinal tissue to assess the impact of nutrition and a secondary nematode challenge in lactating rats.

BACKGROUND: Gastrointestinal nematode infection is a major challenge to the health and welfare of mammals. Although mammals eventually acquire immunity to nematodes, this breaks down around parturition, which renders periparturient mammals susceptible to re-infection and an infection source for their offspring. Nutrient supplementation reduces the extent of periparturient parasitism, but the underlying mechanisms remain unclear. Here, we use a genome wide approach to assess the effects of protein supplementation on gene expression in the small intestine of periparturient rats following nematode re-infection. METHODOLOGY/PRINCIPAL FINDINGS: The use of a rat whole genome expression microarray (Affymetrix Gene 1.0ST) showed significant differential regulation of 91 genes in the small intestine of lactating rats, re-infected with Nippostrongylus brasiliensis compared to controls; affected functions included immune cell trafficking, cell-mediated responses and antigen presentation. Genes with a previously described role in immune response to nematodes, such as mast cell proteases, and intelectin, and others newly associated with nematode expulsion, such as anterior gradient homolog 2 were identified. Protein supplementation resulted in significant differential regulation of 64 genes; affected functions included protein synthesis, cellular function and maintenance. It increased cell metabolism, evident from the high number of non-coding RNA and the increased synthesis of ribosomal proteins. It regulated immune responses, through T-cell activation and proliferation. The up-regulation of transcription factor forkhead box P1 in unsupplemented, parasitised hosts may be indicative of a delayed immune response in these animals. CONCLUSIONS/SIGNIFICANCE: This study provides the first evidence for nutritional regulation of genes related to immunity to nematodes at the site of parasitism, during expulsion. Additionally it reveals genes induced following secondary parasite challenge in lactating mammals, not previously associated with parasite expulsion. This work is a first step towards defining disease predisposition, identifying markers for nutritional imbalance and developing sustainable measures for parasite control in domestic mammals.