The effects of microchipping C57BL/6N mice on standard phenotyping tests.

The C57BL/6N inbred lines of mice are widely used in genetic research. They are particularly favoured in large scale studies such as the International Mouse Phenotyping Consortium (IMPC), where C57BL/6N mice are genetically altered to generate a collection of null alleles (currently more than 8500 null alleles have been generated). In this project, mice carrying null alleles are subjected to a pipeline of broad-based phenotyping tests to produce wide ranging phenotyping data on each model. We have previously described the development of a Home Cage Analysis system that automatically tracks the activity of group housed mice from a microchip inserted in the groin. This platform allows assessment of multiple biologically relevant phenotypes over long periods of time without experimenter interference, and therefore is particularly suited for high through-put studies. To investigate the impact of microchips on other tests carried out in the IMPC pipeline, we inserted microchips in 12 male and 12 female C57BL/6Ntac mice at seven weeks of age. Starting at nine weeks of age these mice underwent standard phenotyping tests, concurrently with 20 unchipped C57BL/6Ntac mice (10 females, 10 males). Tissues from a subset of the microchipped mice (six males and six females), chosen at random, were also sent for histopathological examination at the end of the phenotyping pipeline. No significant impact of insertion of microchip was observed in any of the phenotyping tests apart from bone mineral density measurement at DEXA due to the nature of the microchip. We therefore recommend that the microchip be inserted during the DEXA procedure, after the measurement is taken but before the mouse has recovered from the anaesthetic. This would avoid multiple anaesthetic exposures and prevent the potential variability in DEXA analysis output.

Podocyte GSK3 is an evolutionarily conserved critical regulator of kidney function.

Albuminuria affects millions of people, and is an independent risk factor for kidney failure, cardiovascular morbidity and death. The key cell that prevents albuminuria is the terminally differentiated glomerular podocyte. Here we report the evolutionary importance of the enzyme Glycogen Synthase Kinase 3 (GSK3) for maintaining podocyte function in mice and the equivalent nephrocyte cell in Drosophila. Developmental deletion of both GSK3 isoforms (α and ß) in murine podocytes causes late neonatal death associated with massive albuminuria and renal failure. Similarly, silencing GSK3 in nephrocytes is developmentally lethal for this cell. Mature genetic or pharmacological podocyte/nephrocyte GSK3 inhibition is also detrimental; producing albuminuric kidney disease in mice and nephrocyte depletion in Drosophila. Mechanistically, GSK3 loss causes differentiated podocytes to re-enter the cell cycle and undergo mitotic catastrophe, modulated via the Hippo pathway but independent of Wnt-ß-catenin. This work clearly identifies GSK3 as a critical regulator of podocyte and hence kidney function.

Modelling invasive group A streptococcal disease using bioluminescence.

BACKGROUND: The development of vaccines and evaluation of novel treatment strategies for invasive group A streptococcal (iGAS) disease requires suitable models of human infection that can be monitored longitudinally and are preferably non-invasive. Bio-photonic imaging provides an opportunity to reduce use of animals in infection modelling and refine the information that can be obtained, however the range of bioluminescent GAS strains available is limited. In this study we set out to develop bioluminescent iGAS strains for use in in vivo pneumonia and soft tissue disease models. RESULTS: Using clinical emm1, emm3, and emm89 GAS strains that were transformed with constructs carrying the luxABCDE operon, growth and bioluminescence of transformed strains were characterised in vitro and in vivo. Emm3 and emm89 strains expressed detectable bioluminescence when transformed with a replicating plasmid and light production correlated with viable bacterial counts in vitro, however plasmid instability precluded use in the absence of antimicrobial pressure. Emm89 GAS transformed with an integrating construct demonstrated stable bioluminescence that was maintained in the absence of antibiotics. Bioluminescence of the emm89 strain correlated with viable bacterial counts both in vitro and immediately following infection in vivo. Although bioluminescence conferred a detectable fitness burden to the emm89 strain during soft tissue infection in vivo, it did not prevent dissemination to distant tissues. CONCLUSION: Development of stably bioluminescent GAS for use in vitro and in vivo models of infection should facilitate development of novel therapeutics and vaccines while also increasing our understanding of infection progression and transmission routes.

Commentary: the role of the toxicologic pathologist in academia.

Veterinary pathologists working as toxicologic pathologists in academic settings fill many vital roles, such as diagnosticians, educators, and/or researchers. These individuals have spent years investigating pathology problems that mainly or exclusively focus on the reactions of cells, organs, or systems to toxic materials. Thus, academic toxicologic pathologists are uniquely suited both to help trainees understand toxicity as a cause of pathology responses and also to provide expert consultation on toxicologic pathology. Most toxicologic pathologists in academia are employed at colleges of medicine or veterinary medicine, even though specific toxicologic pathology faculty appointments are uncommon in Europe and North America. Academic toxicologic pathologists typically receive lower financial compensation than do toxicologic pathologists in industry, but academic positions generally provide alternative rewards, such as higher workplace autonomy and scheduling flexibility, professional enrichment through student interactions, and enhanced opportunities for collaborative research and advanced diagnostic investigations. Regular participation by academic toxicologic pathologists in professional training opportunities (eg, as pathology and toxicology instructors in medical and veterinary medical courses, graduate programs, and residencies) offers an important means of engendering interest and inspiring veterinarians to select toxicologic pathology and toxicology as their own areas of future expertise.

Review of approaches to the recording of background lesions in toxicologic pathology studies in rats.

Pathological evaluation of lesions caused directly by xenobiotic treatment must always take into account the recognition of background (incidental) findings. Background lesions can be congenital or hereditary, histological variations, changes related to trauma or normal aging and physiologic or hormonal changes. This review focuses on the importance and correct approach to recording of background changes and includes discussion on sources of variability in background changes, the correct use of terminology, the concept of thresholds, historical control data, diagnostic drift, blind reading of slides, scoring and artifacts. The review is illustrated with background lesions in Sprague Dawley and Wistar rats.

Abdominal fibrosarcoma associated with a retained surgical swab in a dog.

An abdominal fibrosarcoma surrounding a retained surgical swab was identified in a 3-year-old neutered female rottweiler dog presented with chronic inappetence and lethargy. Laparotomy revealed a mass within the omentum, multiple hepatic masses and enlarged mesenteric lymph nodes. The dog was humanely destroyed and submitted for necropsy examination. Microscopically, the omental mass was consistent with a sarcoma surrounding centrally located fibres of foreign material and was infiltrated by epithelioid macrophages containing intracytoplasmic fibre fragments. Sarcoma tissue was also present in mesenteric lymph nodes, liver, spleen and lungs, and some affected lymph nodes contained intralesional epithelioid macrophages with fibre fragments. Immunohistochemical and electron microscopical examinations were consistent with a diagnosis of fibrosarcoma. By fibre analysis and electron microscopy, the intratumoural fibres were identified as cotton fibres with features identical to those obtained from a surgical swab. To our knowledge this is the first description of an abdominal fibrosarcoma associated with a retained surgical swab in a dog. Other examples of foreign body-associated sarcomas in the veterinary literature are vaccine- and implant-induced sarcomas.

Potentiation of the extracellular release of equine neutrophil elastase and alpha-1-proteinase inhibitor by a combination of two bacterial cell wall components: fMLP and LPS.

REASONS FOR PERFORMING STUDY: Lipopolysaccharide (LPS) and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-like peptides are Gram-negative bacterial cell wall components which, when released into the peripheral circulation in endotoxaemia, have the potential to activate leucocytes. In vitro, equine neutrophils require priming with LPS in order to generate reactive oxygen intermediates (ROI) in response to fMLP. OBJECTIVES: The aim of this study was to examine whether the release of other neutrophil products is similarly dependent on prior priming with LPS. In particular, neutrophil elastase (NE), a potent proteolytic enzyme, and its major inhibitor, alpha-1 proteinase inhibitor, were investigated. METHODS: Neutrophils were isolated from equine peripheral blood (n = 5) by discontinuous Percoll gradient preparative centrifugation and primed with LPS prior to stimulation with fMLP. ROI were measured by lucigenin dependent chemiluminescence (LDCL). Concentrations of NE and API were determined by ELISA on cell free supernatants taken at 0, 2, 10, 30, 60 and 90 mins post stimulus. Data was analysed by Kruskal-Wallis and Mann-Whitney Tests. RESULTS: Sequential exposure of Percoll purified equine blood neutrophils in vitro to LPS followed by fMLP resulted in the greatest release of NE from equine neutrophils and was required for ROI generation. However, LPS or fMLP stimulation alone resulted in an increase in NE release compared to unstimulated control cells. In contrast, significant API release was only induced by LPS stimulation or fMLP stimulation only after LPS priming, not fMLP on its own. CONCLUSIONS: These results suggest that different stimuli (fMLP or LPS) are capable of invoking similar responses from equine neutrophils with respect to NE release yet different ones with respect to API release. POTENTIAL RELEVANCE: In addition, demonstration of elastase release induced by LPS and/or fMLP suggests that monitoring serum elastase levels is a potential diagnostic tool for detecting the early onset of endotoxaemia in the horse.

Canine dysautonomia: two clinical cases.

Two clinical cases of canine dysautonomia are described. Two young female neutered dogs were presented with clinical signs including vomiting, diarrhoea, faecal tenesmus, dysphagia and urinary retention. Decreased tear production, dry mucous membranes, bilateral Horner's syndrome, decreased anal sphincter tone and gastrointestinal hypomotility were also observed. Presumptive diagnoses of dysautonomia were made based on the clinical presentation and investigations. Postmortem histopathological examination in one of the cases demonstrated marked depletion of neuronal cell bodies in the intestinal myenteric plexuses and parasympathetic ganglia, confirming the diagnosis in this case. Criteria for aiding the antemortem diagnosis of this rare condition based on clinical observations and diagnostic testing are proposed.

Characterisation of tryptase and a granzyme H-like chymase isolated from equine mastocytoma tissue.

Mast cell proteinases are important inflammatory mediators in man and other species, but until now there has been no investigation of the nature of equine mast cell proteinases. These studies describe the purification and characterisation of two proteolytic components from equine mastocytoma tissue, detected using chromogenic substrates for trypsin and chymotrypsin. Following chromatographic purification, the trypsin-like component was found to be equine mast cell tryptase by N-terminal amino acid sequencing, showing a close similarity with human tryptase-beta (85% identity over 20 residues). It also had similar subunit molecular size (34-36kDa by SDS-PAGE) and substantially similar cleavage specificity to human tryptase-beta with the substrates tested. A 32kDa chymotrypsin-like component was also purified from mastocytoma extract, and termed equine mast cell proteinase-1 (eqMCP-1). The N-terminal amino acid sequence of eqMCP-1 was very similar to human granzyme H (95% over 19 residues). Rabbit antisera directed against tryptase and eqMCP-1 both detected equine mast cells by immunohistochemistry, and will be of use in future clinical studies of the relevance of mast cell proteinases in equine allergic disease.

Sertoli cell tumour in an Amur tiger.

The histological and immunohistochemical characteristics of a malignant Sertoli cell tumour in a 17-year-old Amur tiger (Panthera tigris altaica) are described. Histological examination of the primary lesion in the right testis and metastatic lesions throughout the internal organs showed a variable cellular pattern with an admixture of tubular structures divided by fine stroma filled with fusiform to stellate cells, and sheets of polygonal cells with abundant vacuolated cytoplasm. Immunohistochemical techniques demonstrated strong positive staining for neuron-specific enolase and variable positive staining for vimentin in neoplastic cells, supporting a diagnosis of a tumour of Sertoli cell origin.

Canine hypertrophic osteoarthropathy associated with a malignant Sertoli cell tumour.

A 15-year-old crossbred dog was presented with a severe cough of acute onset and an enlarged right testis. Symptomatic treatment for presumed 'kennel cough' failed to produce any improvement and at re-examination the dog had developed a swollen right forelimb. Radiographic examination suggested a diagnosis of hypertrophic pulmonary osteoarthropathy (Marie's disease) associated with pulmonary metastases from a testicular tumour. The dog was re-presented five days later with acute-onset severe vomiting and the owner elected for euthanasia. Necropsy was performed and histopathological assessment confirmed the presence of a Sertoli cell tumour in the right testis with multiple pulmonary and renal metastases. Hypertrophic pulmonary osteoarthropathy is a rare complication of metastatic canine Sertoli cell tumour. The authors know of no previously reported cases.

cDNA sequence of two sheep mast cell tryptases and the differential expression of tryptase and sheep mast cell proteinase-1 in lung, dermis and gastrointestinal tract.

BACKGROUND: Mast cell tryptases are a family of serine proteinases which are implicated in the proliferation of smooth muscle cells and fibroblasts, upregulation of interleukin-8 synthesis by endothelial cells, and recruitment of neutrophils and eosinophils. Trials in sheep showed that administration of a specific tryptase inhibitor reduced the late-phase response to inhaled allergen. OBJECTIVES: The aim of this study was to characterize the sequence and distribution of sheep tryptase(s), to validate the sheep model of allergic lung disease. METHODS: Reverse transcriptase PCR cloning was used to obtain cDNA sequences for two sheep tryptases. Lung and gut extracts were used as a source of tryptase for partial purification and characterization of the protein. The distribution of tryptase in skin, lung and gut was determined by immunohistochemistry, and compared with the distribution of sheep mast cell proteinase-1 (sMCP-1). RESULTS: Two highly similar cDNA sequences encoding sheep tryptase were found, indicating the presence of a 28 amino acid leader sequence, and a mature peptide of 245 amino acids. Partial purification of a putative sheep tryptase from lung and gut extracts was achieved using heparin-Sepharose affinity chromatography. Rabbit antihuman skin tryptase antiserum recognized the putative sheep tryptase on Western blot (approximate Mr 32-34 000) and paraformaldehyde-fixed tissue sections. Tryptase was detected in all lung, skin and gut mast cells by this antibody, and transcripts for tryptase were detected in all three tissues by RT PCR. Sheep mast cell proteinase-1, detected by a specific monoclonal antibody, was present in all intestinal and gastric mucosal mast cells, but was not found in mast cells of the muscularis, thus defining at least two mast cell phenotypes in the gut. Whereas all dermal and pulmonary mast cells were tryptase positive, only a low proportion in the lung, and almost none in the dermis, were positive for sMCP-1. CONCLUSION: In view of the structural and functional similarities of sheep and human tryptases, and their similarity in tissue distribution in normal sheep, the sheep lung appears to be a good model for in vivo studies relating to human tryptase.

Ultrasonographic diagnosis of pericardial effusion and atrial dilation in a spur-thighed tortoise (Testudo graeca).

Ultrasonography was used to diagnose pericardial effusion, atrial dilatation and liver masses in a spur-thighed tortoise which was more than 80 years old and suffering from posthibernation anorexia, lethargy, oedema and pneumonia. The tortoise was treated twice with frusemide and ceftazidime for the pneumonia, resulting each time in a temporary remission for about a month. After a further recurrence, the animal was euthanased and the lesions predicted by ultrasound were confirmed postmortem. It is suggested that ultrasound may be useful for the differentiation of cardiac problems from other causes of posthibernation lethargy in the tortoise.

Intestinal leiomyosarcoma in a cat.

Intramural ileocaecocolic leiomyosarcoma is described in an elderly neutered male domestic shorthaired cat with poor appetite and weight loss. Histological examination of the resected lesion revealed a poorly differentiated soft tissue sarcoma and a diagnosis of leiomyosarcoma was confirmed by positive immunohistochemical staining for vimentin and a-smooth muscle actin. Postoperative survival time was 102 days before local recurrence justified euthanasia.

Oral metastasis of renal cell carcinoma in a horse.

A 14-year-old hunter gelding presented with an ulcerated mass on the left premaxilla. Biopsy of the mass revealed a poorly differentiated carcinoma. Surgical excision was attempted, but local regrowth followed several months later, at which point radiotherapy was carried out. An initial improvement was followed by marked deterioration and the animal was humanely killed. Post-mortem examination revealed a massively enlarged right kidney and associated widespread metastases. A metastatic clear cell renal carcinoma was identified by histological examination.

Kinetics of equine neutrophil elastase release and superoxide anion generation following secretagogue activation: a potential mechanism for antiproteinase inactivation.

Man and horses both suffer from neutrophil mediated pulmonary diseases however there are striking species differences in the underlying pathology. In particular while pulmonary emphysema is a common pathological sequel to human respiratory disease it is not a major feature of the common equine neutrophil mediated condition, chronic obstructive pulmonary disease (COPD). The proposed reason for this difference is that equine neutrophils contain less elastase than equivalent human cells and therefore there is a reduced risk of excess and/or uninhibited elastase activity, which is considered the major cause of pulmonary emphysema in man, in the horse lung. In previous studies equine neutrophil elastase (ENE) has been assayed by measuring elastinolytic activity whereas human neutrophil elastase content has been determined using immunological techniques. Neutrophils contain several intracellular protease inhibitors therefore measurement of elastase activity may underestimate the total NE content. The aim of the current study was to develop immunological techniques to allow investigation of the cellular content, distribution and release of ENE from purified equine neutrophils. Equine neutrophil elastase 2A (ENE 2A), the most abundant elastase in equine neutrophils, and equine alpha-1-proteinase inhibitor (API), the main inhibitor of elastase were found to be present at 0.813 pg +/- 0.179 and 0.021 pg +/- 0.003 (mean +/- SEM, n = 11 individual horses) per neutrophil, respectively. This represents twice as much elastase as previously found in the equine neutrophil and a comparable amount to that reported in human neutrophils. Immunolocalisation demonstrated that ENE 2A has a granular distribution within the cytosol of neutrophils, whereas API exhibits a uniform non-granular cytoplasmic appearance. In addition the kinetics of simultaneous generation and release of superoxide anions (SOA) and release of ENE 2A from equine neutrophils, stimulated in vitro by zymosan-activated serum (ZAS) in the presence and absence of the cation chelator ethylene glycol-N,N,N',N'-tetraacetic acid (EGTA), showed a close relationship between total SOA generation and total ENE 2A release during the initial 90 min post-ZAS stimulation and the dependence of both events on extracellular cations. In conclusion these studies have shown that horse and human neutrophil elastase content and mediator release functions are more closely matched than was previously thought. This suggests that the species differences in pathology resulting from neutrophil-mediated respiratory disease are determined by other factors such as differences in the abundance and function of intra- and extra-cellular protease inhibitors.

Histochemical and ultrastructural modification of mucosal mast cell granules in parasitized mice lacking the beta-chymase, mouse mast cell protease-1.

The soluble beta-chymases mouse mast cell protease-1 (mMCP-1) and rat mast cell protease-II are predominantly expressed by intestinal mucosal mast cells (IMMCs) and may promote mucosal epithelial permeability when released during intestinal allergic hypersensitivity responses. To study the function of these chymases, we generated mice with a homozygous null mutation of the mMCP-1 gene and investigated their response to infection with the intestinal nematode Nippostrongylus brasiliensis. Whereas mMCP-2, -4, and -5 were transcribed normally, there was no transcription of the mMCP-1 gene in null (-/-) mice, nor was mature mMCP-1 protein detected in (-/-) jejunal mucosa. In contrast, levels of mMCP-1 in wild-type (+/+) jejunal mucosa increased 200- to 350-fold from 0.66 microg mMCP-1/g wet weight in uninfected mice to 129 and 229 microg/g wet weight on days 8 and 10 of infection, respectively. The kinetics of IMMC recruitment differed in -/- mice compared with +/+ controls on days 8 (P < 0.05) and 10 (P < 0.03) of infection. The IMMCs in infected -/- mice stained poorly, if at all, for esterase with naphthol AS-D chloroacetate compared with the intense staining observed in +/+ controls. Ultrastructurally, the prominent crystal intragranular structures that are found in intraepithelial +/+ IMMCs were absent from -/- IMMCs. These data show that disruption of the mMCP-1 gene leads to profound histochemical and ultrastructural changes in IMMC granules.