Transcriptional responses consistent with perturbation in dermo-epidermal homeostasis in septic sole ulceration.

The aim of this study was to evaluate transcriptional changes in the sole epidermis and dermis of bovine claws with septic sole ulceration of the lateral claw. Assessment included changes in transcripts orchestrating epidermal homeostatic processes, including epidermal proliferation, differentiation, inflammation, and cell signaling. Sole epidermis and dermis samples were removed from region 4 of lesion-bearing lateral and lesion-free medial claws of pelvic limbs in multiparous, lactating Holstein cows. Control sole epidermis and dermis samples were obtained from region 4 of lateral claws of normal pelvic limbs. Transcript abundances were evaluated by real-time PCR, and relative expression analyzed by ANOVA. Relative to normal lateral claws, sole epidermis and dermis in ulcer-bearing claws exhibited downregulation of genes associated with growth factors, growth factor receptors, activator protein 1 (AP-1) and proto-oncogene (CMYC) transcription components, cell cycle elements, lateral cell-to-cell signaling elements, and structures of early and late keratinocyte differentiation. These changes were accompanied by upregulation of proinflammatory transcripts interleukin 1 α (IL1A), interleukin1 ß (IL1B), interleukin 1 receptor 1 (IL1R1), inducible nitric oxide synthase (NOS2), the inflammasome components NOD-like receptor protein 3 (NLRP3), pyrin and caspase recruitment domain (PYCARD), caspase-1 interleukin converting enzyme (CASPASE), the matrix metalloproteinases (MMP2 and MMP9), and the anti-inflammatory genes interleukin 1 receptor antagonist (IL1RN) and interleukin1 receptor 2 (IL1R2). Transcript abundance varied across epidermis and dermis from the ulcer center, margin, and epidermis and dermis adjacent to the lesion. Sole epidermis and dermis of lesion-free medial claws exhibited changes paralleling those in the adjacent lateral claws in an environment lacking inflammatory transcripts and downregulated IL1A, interleukin 18 (IL18), tumor necrosis factor α (TNFA), and NOS2. These data imply perturbations in signal pathways driving epidermal proliferation and differentiation are associated with, but not inevitably linked to epidermis and dermis inflammation. Further work is warranted to better define the role of crushing tissue injury, sepsis, metalloproteinase activity, and inflammation in sole ulceration.

A calcium-activated apyrase from Teladorsagia circumcincta: an excretory/secretory antigen capable of modulating host immune responses?

A cDNA representing the gene Teladorsagia circumcincta apyrase-1 (Tci-apy-1) was isolated, by PCR, from a T. circumcincta fourth-stage larval (L4) cDNA library. The closest orthologue of this gene is a Ca(2+)-dependent apyrase from Ostertagia ostertagi, with 92% amino acid identity across all 339 residues. Tci-apy-1 is transcribed in a stage-specific manner, the transcript being predominant in L4, detectable in the adult cDNA, but absent from eggs and infective third-stage larvae (L3). The protein, Tci-APY-1, was detected by immunoblotting in extracts of L4 nematodes and was present in excretory/secretory products from the same developmental stage. A recombinant version of Tci-APY-1 was expressed in bacteria as an active enzyme that hydrolysed nucleoside triphosphate substrates with a preference of ATP over other nucleoside triphosphates. Recombinant Tci-APY-1 hydrolysed ATP and ADP but not AMP. Apyrase activity was divalent cation-dependent, with no hydrolysis in the presence of Mg(2+), but activation in the presence of Ca(2+). Recombinant Tci-APY-1 was bound by IgG present in serum and both IgG and IgA present in abomasal mucus from trickle-infected, immune sheep but not in material derived from lambs exposed to a single infection. The potential immunomodulatory roles of this Tci-APY-1 are discussed in relation to purinergic signalling.

A calcium-activated nucleotidase secreted from Ostertagia ostertagi 4th-stage larvae is a member of the novel salivary apyrases present in blood-feeding arthropods.

Apyrases (ATP-diphosphohydrolase) comprise a ubiquitous class of glycosylated nucleotidases that hydrolyse extracellular ATP and ADP to orthophosphate and AMP. One class of newly-described, Ca2+-dependent, salivary apyrases known to counteract blood-clotting, has been identified in haematophagous arthropods. Herein, we have identified a gene (Oos-apy-1) encoding a protein that structurally conforms to the Ca2+-activated apyrase from the bed bug, Cimex lectularius, by immunologically screening an Ostertagia L4 cDNA expression library. The expressed protein (rOos-APY-1) was biochemically functional in the presence of Ca2+ only, with greatest activity on ATP, ADP, UTP and UDP. Host antibodies to the fusion protein appeared as early as 14 days post-infection (p.i.) and increased through 30 days p.i. Immunohistochemical and Western blot analyses demonstrated that the native Oos-APY-1 protein is present in the glandular bulb of the oesophagus and is confined to the L4. A putative signal sequence at the N-terminus and near 100% identity with a Teladorsagia circumcincta L4 secreted protein is consistent with the native protein being secreted at the cellular level. Predicated upon substrate specificity, the native protein may be used by the parasite to control the levels of host extracellular nucleotides released by locally-damaged tissues in an effort to modulate immune intervention and inflammation.

Age, segment, and horn disease affect expression of cytokines, growth factors, and receptors in the epidermis and dermis of the bovine claw.

The aim of this study was to examine changes in RNA expression for growth factors, cytokines, and receptors in epidermal-dermal tissues of the bovine claw relative to host age, claw segment, and disease state of the horn. Epidermal-dermal tissues were collected from the coronary, wall, sole, and bulb segments of 8- to 9-mo-old Holstein fetuses, normal adult cows, and adult cows with sole ulceration. Anatomic and pathologic characteristics were determined in tissues stained with eosin and hematoxylin, and RNA expression levels were evaluated using real-time, quantitative PCR. In normal tissues, certain RNA expression levels were clearly affected by host age: 290.0-, 610.0-, 53.4-, and 8.1-fold greater expression of granulocyte-macrophage colony stimulating factor was observed in fetal coronary, wall, sole, and bulb segment relative to adult tissues, respectively. A claw segment effect was also observed in that IL-1alpha expression was greater (1.59-fold) in the normal adult wall relative to the coronary segment, and IL-18 expression was greater (16.2-fold) in the normal adult sole compared with the coronary segment and 2.88 greater in the fetal sole relative to the bulb segment. Sole ulceration was associated with hemorrhage, thrombosis, inflammation, and striking increases in IL-1beta, IL-18, and inducible nitric oxide synthase, and with less dramatic, albeit measurable, changes in IL-1 type I receptor, IL-1 receptor antagonist, and tumor necrosis factor-alpha. Amidst striking increases in keratinocyte growth factor receptor (i.e., 21.0-fold, 10.4-fold, 0, and 21.6-fold in the coronary, wall, sole, and bulb segments, respectively), a concomitant decrease occurred in keratinocyte growth factor (i.e., 0.80-, 0.54-, 0.56-, and 0.72-fold, respectively). The results demonstrated changes in disease state and, to a lesser extent, claw segment and were accompanied by alterations in the RNA expression of several cytokines, growth factors, and receptors present in the normal claw.

Inhibition of bovine T lymphocyte responses by extracts of the stomach worm Ostertagia ostertagi.

Lowered immune responses during bovine ostertagiosis have been reported in both in vivo and in vitro assay systems. In the present study we have employed three different life cycle stages of the nematode Ostertagia ostertagi to determine if products of this economically important parasite inhibit in vitro proliferation of Con A-stimulated cells from uninfected animals. We have demonstrated an inhibitory effect upon the growth of Con A-stimulated lymphocytes after addition of fourth stage larval (L4) soluble extract (L4SE) to the cultures. In contrast, extracts from the third stage larvae (L3) had little or no inhibitory activity. The suppressive products were also shown to be secreted by the late L4. The suppressive activity is reversible if the L4 products are removed from culture. There is no immediate effect on proliferating cells and the L4SE must be in culture for 24-48 h before suppression is observable. The L4SE caused slight but not statistically significant decreases in the percentage of T cells and increases in B cell percentages in cultures when compared with cultures stimulated with Con A alone. No changes were seen in percentage of cells positive for markers for CD4, CD8, gammadelta T cells, or monocytes/macrophages as a consequence of the addition of L4SE. In contrast, there was a strong and significant reduction in the expression of the IL-2 receptors in cells cultured in the presence of the worm extract. There was no evidence of either necrosis or apoptosis resulting from the presence of L4 products in culture. The expression of messenger RNA for interleukin-2, -4, -13, tumor necrosis factor-alpha (TNF-alpha), and gamma-interferon (gamma-IFN) was decreased when L4SE was included in cultures of Con A-stimulated cells compared to cultures stimulated with Con A only. In contrast, messenger RNA expression of transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10) was increased in cells growing in the presence of L4 products. The potential role of these cytokines during ostertagiosis is discussed.

Cloning of a cDNA encoding bovine interleukin-18 and analysis of IL-18 expression in macrophages and its IFN-gamma-inducing activity.

Interleukin-18 (IL-18) is a recently described cytokine that enhances interferon-gamma (IFN-gamma) production, either independently or synergistically with IL-12. These properties identify IL-18 as an immunoregulatory cytokine that may be pivotal in host defense against intracellular pathogens. We have isolated and sequenced a cDNA encoding bovine IL-18. The open reading frame (ORF) is 582 bp in length, encoding a predicted 192 amino acid (aa) precursor protein. Multiple sequence alignment demonstrated that bovine IL-18 has 65% and 78% identity with the predicted amino acid sequences of murine and human IL-18, respectively. IL-18 mRNA was constitutively present in bovine peripheral blood monocyte-derived macrophages (MDM), with no upregulation on stimulation with lipopolysaccharide (LPS). IL-18 transcripts were weakly detected in B lymphocytes but inducible in the B cell line BL-3. Human recombinant IL-18 (rHuIL-18) induced IFN-gamma production by PHA-stimulated peripheral blood mononuclear cells (PBMC), which was potentiated by rHuIL-12. Further, rHuIL-12 and rHuIL-18 enhanced proliferation of untreated PBMC. Antigen-specific T cell lines demonstrated IL-18-dependent enhancement of IFN-gamma production, indicating that bovine T cells are one of the leukocyte subsets that respond to IL-18. Analysis of IL-18 expression and its ability to induce IFN-gamma production by bovine lymphocytes are important considerations for understanding mechanisms of protective immunity and designing vaccines for intracellular pathogens.

Development and in vitro characterization of recombinant vaccinia viruses expressing bovine leukemia virus gp51 in combination with bovine IL4 or IL12.

Type 1 and type 2 immune responses are modulated by IL12 or IL4, respectively, at the time of lymphocyte priming. Importantly, type 1 responses have been associated with resistance to retroviral infection in mice, humans, and ruminants. Specifically, vaccination of sheep with vaccinia virus expressing bovine leukemia virus (BLV) gp51 resulted in protective immunity with the characteristics of a type 1 response, whereas vaccination of cattle resulted in a non-protective type 2 response. In order to test the hypothesis that cattle inoculated with BLV gp51 and IL12 will respond with a type 1 response, a recombinant vaccinia virus expressing BLV gp51 together with bovine IL12 was developed and characterized in vitro. For induction of type 2 responses a recombinant vaccinia virus expressing gp51 with bovine IL4 was similarly constructed and characterized. In this study recombinant cassettes were developed containing either the BLVenv gene alone or in combination with bovine IL4 or the two genes, p35 and p40, encoding bovine IL12. Correct alignment with p7.5 or p11 vaccinia promoters and orientation was confirmed by complete sequencing. Recombinant vaccinia viruses were generated by homologous recombination, selected based on large plaque formation due to reconstitution of the vp37 gene, and structurally confirmed by Southern blotting. Transcription of recombinant BLVenv, bovine IL4, p35 and p40 was demonstrated by RT-PCR. Expression of BLVenv gp51 protein and bovine IL4 was shown by immunofluorescence and immunoblotting. Biologically active bovine IL4 expressed by vaccinia virus stimulated lymphoblast proliferation, B lymphocyte proliferation in the presence of CD40L, and inhibited IFN gamma secretion from PHA activated PBMC in a dose dependent fashion. Finally, bovine IL12 expression and biological function was confirmed by dose dependent induction of IFN gamma secretion by PHA activated PBMC and the moderate enhancement of lymphoblast proliferation. In conclusion, bovine IL12 and IL4 expressed by recombinant vaccinia virus in vitro clearly exhibited type 1-type 2 modulating properties.

Molecular cloning of cDNA encoding porcine interleukin-15.

Interleukin-15 (IL-15) is a recently identified growth and differentiation factor with an important potential role in the initial immune responses to infection. To enable the study of the role of this cytokine in the protective immune-mechanisms generated against parasitic diseases of swine, cDNA was generated from a macrophage enriched adherent cell population from peripheral blood mononuclear cells (PBMC). This cDNA was used for the enzymatic amplification of the porcine IL-15 sequence using human IL-15-derived primers. The open-reading frame of the porcine IL-15 cDNA is 486 base pairs (bp) in length and encodes a 162-amino-acid (aa) protein. Comparisons of the predicted swine protein sequence with those predicted from human, bovine and mouse IL-15 sequences indicate similarities of 82.1, 84.6, and 71.6%, respectively.

Immunological responses in the mouse host to a cloned antigen of Taenia crassiceps.

Adult female Swiss-Webster mice were immunized either intraperitoneally (IP) or subcutaneously (SQ) with cyst fluid or a genetically engineered fusion protein, Taenia crassiceps antigen 2-maltose binding protein (TCA2-MBP) from Taenia crassiceps metacestodes, or with live, non-budding cysts SQ, and then challenged IP with T. crassiceps metacestodes and necropsied 9 weeks later. Numbers of peripheral blood eosinophils were increased after IP immunization, but were not increased after SQ immunization or with SQ cysts given before the challenge infection. Eosinophil numbers gradually decreased over the course of the experiment, and were not found in increased numbers in the blood or peritoneal cavity at necropsy. Antigen-specific antibody responses were seen at day 14 or 28 in IP and SQ immunized groups: IgG1 and IgG3 isotypes continued to increase over the course of the experiment. A significant protective response was induced by immunization with the cyst fluid (15 +/- 4, X +/- SE recovered larvae) or the TCA2-MBP (22 +/- 12) given IP, but not SQ (122 +/- 36; 207 +/- 53, respectively) as measured by the numbers of larvae recovered at necropsy. Live cysts given SQ resulted in reduced numbers of cysts in the peritoneal cavity (188 +/- 66), but was not as effective as cyst fluid or TCA2-MBP given IP. Locally (IP) induced immune responses may be involved in the development of the protective response to a challenge infection with T. crassiceps metacestodes.

A Taenia crassiceps cDNA sequence encoding a putative immunodiagnostic antigen for bovine cysticercosis.

A cDNA expression library was constructed in lambda gt11 using poly A mRNA from the metacestode stage of Taenia crassiceps. The library was screened with rabbit antiserum to a previously defined protein fraction from Taenia hydatigena immunodiagnostic for bovine cysticercosis and with sera from cattle with experimentally induced cysticercosis. One clone (lambda TcA2) containing a 279-bp cDNA insert, reacted strongly with both antisera. A second clone (lambda TcA5.5) revealed the full-length cDNA sequence to be 361 bp. Data from Southern blots and enzymatically amplified genomic DNA segments were consistent with multiple copies or a gene family within the genome. The lambda TcA2 cDNA insert was subcloned into the plasmid pPR987 which generated a 47-kDa maltose-binding fusion protein (TcA2-MBP). Affinity-purified TcA2-MBP antigen reacted positively by ELISA with sera from cattle with experimentally induced T. saginata infections but not with sera from cattle with Fasciola hepatica or common gastrointestinal parasite infections. Rabbit polyclonal, monospecific antisera to TcA2-MBP recognized a 10-kDa protein in the cyst fluid, body wall and excretory/secretory products of the metacestode stage of T. crassiceps and immonolocalized this protein to organelles within the matrix of the cyst wall.

A recombinant immunodiagnostic antigen for bovine cysticercosis.

The 70% ammonium sulfate-soluble fraction of the cyst fluid of Taenia hydatigena (designated ThFAS) was previously shown to have potential as an immunodiagnostic reagent for bovine cysticercosis. Western blot analysis indicated that the specific reactivity with antibodies in sera of T. saginata-infected cattle was associated with a 10 kDa component. Rabbit antiserum to ThFAS identified a homologous antigenic protein from the cestode Taenia crassiceps. Consequently, a cDNA expression library was constructed in lambda gt11 using poly A mRNA purified from T. crassiceps metacestodes and screened with rabbit antiserum to ThFAS. One strongly reactive clone (designated lambda TCA-2) produced a 123 kDa beta-galactosidase fusion protein which reacted in Western blot with sera from calves experimentally-infected with T. saginata and did not react with sera from uninfected calves or from cattle infected with Fasciola hepatica or with common gastrointestinal cattle parasites.

Characterization of a noncyst-forming isolate of Trichinella from a wild boar in Yugoslavia.

An isolate of Trichinella obtained from a wild boar in Yugoslavia did not form cysts in the musculature of its natural host. Subsequent inoculation into experimental hosts demonstrated that some larvae became encysted only after extended time periods, whereas others remained unencapsulated. Histological staining of larvae in the musculature demonstrated no deposition of collagen typically seen for Trichinella spiralis spiralis, Trichinella spiralis nativa, or Trichinella spiralis nelsoni. The Yugoslavian isolate, given the name of Zagreb isolate after the University where it was first studied, had low infectivity for pigs and mice. Isozyme analysis demonstrated greater homology with T. s. nelsoni than with other subspecies of Trichinella. Restriction fragment length polymorphisms and dot blot analyses further demonstrated the distinctive nature of this isolate. These results suggest that lack of cyst formation might be characteristic of isolates other than those designated Trichinella pseudospiralis and that this character might be important in the classification of Trichinella.

Identification of interferon-modulated proliferation-related cDNA sequences.

To identify genes mediating the antiproliferative action of interferon (IFN), two cDNA libraries were constructed with mRNA from IFN-treated and untreated human fibrosarcoma (HT1080) cells previously shown to be highly sensitive to the antiproliferative effects of IFN. Differential screening of these two libraries identified cloned sequences whose expression was either induced or repressed with IFN treatment. Rescreening of these sequences with cDNA probes constructed from proliferating or quiescent cells led to the identification of one IFN-induced and three IFN-repressed sequences whose expressions also appeared to be modulated by cell proliferation. Blot-hybridization analysis revealed that RNA levels corresponding to the three repressed genes decreased when HT1080 cells were treated with IFN or when proliferation of normal CUA foreskin fibroblast cells became naturally arrested by contact inhibition. Levels of RNA corresponding to the induced gene increased in HT1080 cells within 24 hr after IFN-treatment but declined below basal levels by 48 hr. Expression of these genes was unaffected or only slightly affected by IFN treatment in variant cells resistant to the antiproliferative effects of IFN. Collectively, these results suggest that the identified cDNAs correspond to genes that are involved in the antiproliferative action of IFN. Moreover, these results also suggest that IFN's antiproliferative action may be exerted through genes that contribute to arresting cell proliferation during contact inhibition.