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In collaboration with the American College of Veterinary Pathologists

Respiratory disease is an important cause of morbidity and mortality in cetaceans, which are also threatened by environmental degradation caused by crude oil spills. Following oil spills, cetaceans at the water surface may inhale droplets of oil containing toxic polycyclic aromatic hydrocarbons (PAHs), which could potentially alter respiratory immunity via activation of the aryl hydrocarbon receptor (AHR) and its subsequent interaction with nuclear factor kappa B (NF-κB). β-defensins are antimicrobial peptides secreted by airway epithelial cells and their expression is known to be dependent on NF-κB. We hypothesized that PAHs may suppress the expression of β-defensins, and thereby contribute to the pathogenesis of pneumonia. This hypothesis was modeled by measuring the in vitro effects of benzo(a)pyrene (BAP), phenanthrene, and naphthalene on tracheal antimicrobial peptide (TAP) gene expression in bovine tracheal epithelial cells. Stimulation with lipopolysaccharide (LPS) induced 20 ± 17-fold (mean ± SD) increased TAP gene expression. Exposure of tracheal epithelial cells to 5 μM BAP for 4 or 8 h, followed by incubation with a combination of LPS and 5 μM BAP for another 16 h, significantly (P = 0.002) suppressed LPS-induced TAP gene expression by 40.6 ± 21.8% (mean ± SD) in tracheal epithelial cells from 9 calves tested. BAP-induced suppression of TAP gene expression coincided with induction of cytochrome P450 1A1 gene expression. In contrast, phenanthrene and naphthalene had no consistent effect, and exposure to PAHs did not significantly affect constitutive TAP gene expression (i.e. without LPS). These findings characterize the suppressive effects of BAP—a toxic pollutant found in crude oil—on this respiratory innate immune response.

Spermatogenesis is a highly regulated process leading to the development of functional spermatozoa through meiotic division and subsequent maturation. Recent studies have suggested that a novel class of Argonaute proteins, known as the PIWI clade, plays important roles in multiple stages of spermatogenesis. PIWI proteins bind specific small noncoding RNAs, called PIWI-interacting RNAs (piRNAs). These piRNAs guide the PIWI-piRNA complex to retrotransposon targets that become expressed during meiosis. Retrotransposons are subsequently silenced, either through PIWI "slicer" activity or through PIWI-directed methylation of the retrotransposon locus. Most mammalian studies have employed mouse models where sterility follows PIWI inactivation. The goal of this study was to characterize canine PIWIL1 to determine whether expression pattern and functional characteristics support a similar function in that species. Canine PIWIL1 cDNA is a 2.6-kb transcript that encodes an 861-amino acid protein showing high homology to other mammalian PIWIL1 proteins and containing features consistent with PIWI family members (PAZ, PIWI domains). Analysis of PIWIL1 protein and transcript levels revealed that PIWIL1 expression is limited to the testes and is associated with sexual maturity, with mature dogs showing higher levels of PIWIL1 expression. Immunohistochemistry demonstrated expression primarily in seminiferous tubules and confirmed higher levels of PIWIL1 in mature dogs. Functional characterization by RNA immunoprecipitation demonstrated that canine PIWIL1 binds short RNAs consistent in size with piRNAs (27-32 nucleotides). Together, these studies represent the first characterization of a PIWI protein in the dog and suggest that it is a functional piRNA-binding protein most highly expressed in the mature testes.

Spermatogenesis is a highly regulated process leading to the development of functional spermatozoa through meiotic division and subsequent maturation. Recent studies have suggested that a novel class of Argonaute proteins, known as the PIWI clade, plays important roles in multiple stages of spermatogenesis. PIWI proteins bind specific small noncoding RNAs, called PIWI-interacting RNAs (piRNAs). These piRNAs guide the PIWI-piRNA complex to retrotransposon targets that become expressed during meiosis. Retrotransposons are subsequently silenced, either through PIWI "slicer" activity or through PIWI-directed methylation of the retrotransposon locus. Most mammalian studies have employed mouse models where sterility follows PIWI inactivation. The goal of this study was to characterize canine PIWIL1 to determine whether expression pattern and functional characteristics support a similar function in that species. Canine PIWIL1 cDNA is a 2.6-kb transcript that encodes an 861-amino acid protein showing high homology to other mammalian PIWIL1 proteins and containing features consistent with PIWI family members (PAZ, PIWI domains). Analysis of PIWIL1 protein and transcript levels revealed that PIWIL1 expression is limited to the testes and is associated with sexual maturity, with mature dogs showing higher levels of PIWIL1 expression. Immunohistochemistry demonstrated expression primarily in seminiferous tubules and confirmed higher levels of PIWIL1 in mature dogs. Functional characterization by RNA immunoprecipitation demonstrated that canine PIWIL1 binds short RNAs consistent in size with piRNAs (27-32 nucleotides). Together, these studies represent the first characterization of a PIWI protein in the dog and suggest that it is a functional piRNA-binding protein most highly expressed in the mature testes.

Background Mesenchymal stromal cells (MSC) are increasingly investigated for their clinical utility in dogs. Fetal bovine serum (FBS) is a common culture supplement used for canine MSC expansion. However, FBS content is variable, its clinical use carries risk of an immune response, and its cost is increasing due to global demand. Platelet lysate (PL) has proven to be a suitable alternative to FBS for expansion of human MSC. Hypothesis and Objectives We hypothesized that canine adipose tissue (AT) and bone marrow (BM) MSC could be isolated and expanded equally in PL and FBS at conventionally-used concentrations with differentiation of these MSC unaffected by choice of supplement. Our objectives were to evaluate the use of canine PL in comparison with FBS at four stages: 1) isolation, 2) proliferation, 3) spontaneous differentiation, and 4) directed differentiation. Results 1) Medium with 10% PL was unable to isolate MSC. 2) MSC, initially isolated in FBS-supplemented media, followed a dose-dependent response with no significant difference between PL and FBS cultures at up to 20% (AT) or 30% (BM) enrichment. Beyond these respective peaks, proliferation fell in PL cultures only, while a continued dose-dependent proliferation response was noted in FBS cultures. 3) Further investigation indicated PL expansion culture was inducing spontaneous adipogenesis in concentrations as low as 10% and as early as 4 days in culture. 4) MSC isolated in FBS, but expanded in either FBS or PL, maintained ability to undergo directed adipogenesis and osteogenesis, but not chondrogenesis. Conclusions/Significance Canine PL did not support establishment of MSC colonies from AT and BM, nor expansion of MSC, which appear to undergo spontaneous adipogenesis in response to PL exposure. In vivo studies are warranted to determine if concurrent use of MSC with any platelet-derived products such as platelet-rich plasma are associated with synergistic, neutral or antagonistic effects.

Additional File 12. Additional methods for cytokine analysis and mass spectrometry.

Additional file 5. Bronchoalveolar lavage fluid protein concentrations in calves prior to and 24 and 96 h post-aerosolization of bacterial lysate or saline.

Additional file 7. PANTHER classification of 19 upregulated proteins and 26 downregulated proteins in bronchoalveolar lavage fluid following aerosolization of bacterial lysate in 4 calves.

Additional file 8. Reactome pathway analysis for differently expressed proteins in bronchoalveolar lavage fluid in 4 calves.

Additional file 9. Gross lung lesions at 24 h after aerosol administration of Staphylococcus aureus and Escherichia coli lysate.

Additional file 3. Bronchoalveolar lavage fluid cytology before and 24 h after aerosolization of bacterial lysate.

Additional file 3. Bronchoalveolar lavage fluid cytology before and 24 h after aerosolization of bacterial lysate.

Additional file 10. Histologic lesions in respiratory tissues from 4 calves treated with aerosolized Staphylococcus aureus and Escherichia coli lysate.

Additional file 11. Clinical scoring system.

Additional file 2. Bronchoalveolar lavage fluid cells pre- and post- administration of differing doses of bacterial lysate or saline in Holstein bull calves.

Additional File 12. Additional methods for cytokine analysis and mass spectrometry.

Additional file 2. Bronchoalveolar lavage fluid cells pre- and post- administration of differing doses of bacterial lysate or saline in Holstein bull calves.

Additional file 6. Mass spectrometry data for proteins in bronchoalveolar lavage fluid in 4 calves (A4, A3, A5, A6) before and 24 h after aerosolization with heat-killed lysate of Escherichia coli and Staphylococcus aureus.

Additional file 10. Histologic lesions in respiratory tissues from 4 calves treated with aerosolized Staphylococcus aureus and Escherichia coli lysate.

Additional file 11. Clinical scoring system.

Additional file 8. Reactome pathway analysis for differently expressed proteins in bronchoalveolar lavage fluid in 4 calves.

Additional file 7. PANTHER classification of 19 upregulated proteins and 26 downregulated proteins in bronchoalveolar lavage fluid following aerosolization of bacterial lysate in 4 calves.

Additional file 1. Clinical parameters over time following aerosolization in 6 Holstein calves following aerosolization of bacterial lysate.

Additional file 4. Surface area of macrophages before and after aerosolization of bacterial lysate.

Additional file 4. Surface area of macrophages before and after aerosolization of bacterial lysate.

Additional file 6. Mass spectrometry data for proteins in bronchoalveolar lavage fluid in 4 calves (A4, A3, A5, A6) before and 24 h after aerosolization with heat-killed lysate of Escherichia coli and Staphylococcus aureus.

Additional file 5. Bronchoalveolar lavage fluid protein concentrations in calves prior to and 24 and 96 h post-aerosolization of bacterial lysate or saline.