Mast cell tumours in dogs less than 12 months of age: a multi-institutional retrospective study.

OBJECTIVES: To describe the clinicopathological and genetic characteristics of mast cell tumours in dogs less than 12 months old. MATERIALS AND METHODS: Retrospective review of dogs aged less than 12 months when diagnosed with mast cell tumours at three referral hospitals in the UK. RESULTS: Sixteen pure-bred dogs were included, of which 11 were female. The median age at first presentation and diagnosis were 7.6 and 9 months, respectively. In 13 dogs the mast cell tumours were cutaneous and in three they were subcutaneous. Four cutaneous mast cell tumours were described as high-grade (Patnaik or Kiupel) and nine were Patnaik grade II; three had mitotic index of >5 in 10 high-power fields. Of the three subcutaneous tumours, two had an infiltrative growth pattern and one had mitotic index of 10 per 10 high-power fields. Of 10 tested dogs, seven had c-kit mutations in exon 11 and Ki-67 score was above the cut-off value in nine. Four of 12 cases showed evidence of metastasis in the regional lymph nodes. After varying treatment protocols, all patients were alive and disease free at a median of 1115 days after diagnosis. CLINICAL SIGNIFICANCE: The prognosis of mast cell tumours in dogs less than a year old appears better than the adult counterparts, even without extensive treatment.

Genome-wide analysis of canine oral malignant melanoma metastasis-associated gene expression.

Oral malignant melanoma (OMM) is the most common canine melanocytic neoplasm. Overlap between the somatic mutation profiles of canine OMM and human mucosal melanomas suggest a shared UV-independent molecular aetiology. In common with human mucosal melanomas, most canine OMM metastasise. There is no reliable means of predicting canine OMM metastasis, and systemic therapies for metastatic disease are largely palliative. Herein, we employed exon microarrays for comparative expression profiling of FFPE biopsies of 18 primary canine OMM that metastasised and 10 primary OMM that did not metastasise. Genes displaying metastasis-associated expression may be targets for anti-metastasis treatments, and biomarkers of OMM metastasis. Reduced expression of CXCL12 in the metastasising OMMs implies that the CXCR4/CXCL12 axis may be involved in OMM metastasis. Increased expression of APOBEC3A in the metastasising OMMs may indicate APOBEC3A-induced double-strand DNA breaks and pro-metastatic hypermutation. DNA double strand breakage triggers the DNA damage response network and two Fanconi anaemia DNA repair pathway members showed elevated expression in the metastasising OMMs. Cross-validation was employed to test a Linear Discriminant Analysis classifier based upon the RT-qPCR-measured expression levels of CXCL12, APOBEC3A and RPL29. Classification accuracies of 94% (metastasising OMMs) and 86% (non-metastasising OMMs) were estimated.

MicroRNAs in equine veterinary science.

MicroRNAs are small noncoding RNAs that play a pivotal role in diverse cellular processes through post-transcriptional regulation of gene expression. The dysregulation of specific microRNAs is associated with disease development and progression. In this review, we summarise how microRNAs modulate gene expression, and explain microRNA nomenclature. We discuss the potential applications of microRNAs in equine disease diagnosis and treatment, in the context of the sum of current knowledge about microRNA expression in normal and diseased equine tissues.

Obese asthmatics are characterized by altered adipose tissue macrophage activation.

BACKGROUND: Adipose tissue-derived inflammation is linked to obesity-related comorbidities. This study aimed to quantify and immuno-phenotype adipose tissue macrophages (ATMs) from obese asthmatics and obese non-asthmatics and to examine associations between adipose tissue, systemic and airway inflammation. METHODS: Visceral (VAT) adipose tissue and subcutaneous (SAT) adipose tissue were collected from obese adults undergoing bariatric surgery and processed to obtain the stromovascular fraction. Pro-inflammatory (M1) and anti-inflammatory (M2) macrophages were quantified by flow cytometry. Cytospins of induced sputum were stained for differential cell counts. Plasma C-reactive protein (CRP) and CD163 were measured by ELISA. RESULTS: VAT contained a higher number of ATMs compared to SAT. A higher percentage of M1 ATMs was observed in VAT of obese asthmatics compared to obese non-asthmatics. The M1:M2 ratio in VAT was negatively associated with FEV1 %. Sputum macrophage count was correlated positively with M1 ATMs and negatively with M2 ATMs in VAT. In obese asthmatics, CRP was positively associated with M1:M2 ratio in VAT. There were no associations with CD163. An elevated ratio of M1:M2 ATMs was observed in VAT of obese asthmatics with increased disease severity. CONCLUSIONS AND CLINICAL RELEVANCE: Visceral inflammation with increased pro-inflammatory macrophages (M1) occurs in obese asthma and may be a determinant of systemic inflammation and asthma severity.

A preliminary investigation of the effect of sample collection technique on the cell and RNA content of fine-needle aspirates of five canine tumours.

OBJECTIVES: To evaluate the effect of syringe size, needle size, number of needle passes and operator experience on cell yield from tumour fine-needle aspirates, and the quantity and quality of extractable RNA. MATERIALS AND METHODS: Fine-needle aspirates were collected from canine lymphoma, cutaneous mast cell tumour, anal gland adenocarcinoma, fibrosarcoma and oral malignant melanoma using nine different techniques. RESULTS: There was a significant difference in cell yield between fine-needle aspirate techniques for melanoma, lymphoma and anal gland adenocarcinoma. The application of suction yielded the largest number of cells. Cell numbers in lymphoma and fibrosarcoma aspirates collected by different veterinary surgeons were not significantly different. Use of a smaller gauge needle and suction increased the quantity of RNA isolated from fibrosarcoma and anal gland adenocarcinoma aspirates, but did not influence RNA integrity. CLINICAL SIGNIFICANCE: Suction during fine-needle aspiration increases cell numbers obtained from five common canine tumours. Suction increases the quantity of RNA isolated from anal gland adenocarcinoma and fibrosarcoma aspirates without affecting RNA quality. Junior veterinary surgeons gain comparable cell numbers to senior staff.

Metastasis-associated microRNA expression in canine uveal melanoma.

BACKGROUND: Uveal melanoma (UM) is the most common primary intraocular tumour in dogs. There is no effective means of predicting whether a tumour will metastasize. microRNA (miRNA) metastasis signatures have been identified for several human cancers, including UM. AIMS: In this study we investigated whether metastasizing and non-metastasizing canine UMs can be distinguished by miRNA expression levels. MATERIALS AND METHODS: miRNA microarray profiling was used to compare miRNA expression in 8 metastasizing and 12 non-metastasizing formalin-fixed, paraffin-embedded (FFPE) primary UM biopsies. RESULTS: Fourteen miRNAs exhibited statistically significant differences in expression between the metastasizing and non-metastasizing tumours. Class prediction analysis pinpointed 9 miRNAs which categorized tumours as metastasizing or non-metastasizing with an accuracy of 89%. Of the discriminating miRNAs, 8 were up-regulated in metastasizing UM, and included 3 miRNAs implicated as potential "metastasis activators" in human cutaneous melanoma. The expression of 4 of the miRNAs was subsequently measured using the quantitative reverse transcription polymerase chain reaction (RT-qPCR), and their up-regulation in metastasizing tumours validated. CONCLUSION: miRNA expression profiles may potentially be used to identify UMs that will metastasize, and miRNAs that are up-regulated in metastasizing tumours may be targets for therapeutic intervention.

Targeting PP2A and proteasome activity ameliorates features of allergic airway disease in mice.

BACKGROUND: Asthma is an allergic airway disease (AAD) caused by aberrant immune responses to allergens. Protein phosphatase-2A (PP2A) is an abundant serine/threonine phosphatase with anti-inflammatory activity. The ubiquitin proteasome system (UPS) controls many cellular processes, including the initiation of inflammatory responses by protein degradation. We assessed whether enhancing PP2A activity with fingolimod (FTY720) or 2-amino-4-(4-(heptyloxy) phenyl)-2-methylbutan-1-ol (AAL(S) ), or inhibiting proteasome activity with bortezomib (BORT), could suppress experimental AAD. METHODS: Acute AAD was induced in C57BL/6 mice by intraperitoneal sensitization with ovalbumin (OVA) in combination with intranasal (i.n) exposure to OVA. Chronic AAD was induced in mice with prolonged i.n exposure to crude house dust mite (HDM) extract. Mice were treated with vehicle, FTY720, AAL(S) , BORT or AAL(S) +BORT and hallmark features of AAD assessed. RESULTS: AAL(S) reduced the severity of acute AAD by suppressing tissue eosinophils and inflammation, mucus-secreting cell (MSC) numbers, type 2-associated cytokines (interleukin (IL)-33, thymic stromal lymphopoietin, IL-5 and IL-13), serum immunoglobulin (Ig)E and airway hyper-responsiveness (AHR). FTY720 only suppressed tissue inflammation and IgE. BORT reduced bronchoalveolar lavage fluid (BALF) and tissue eosinophils and inflammation, IL-5, IL-13 and AHR. Combined treatment with AAL(S) +BORT had complementary effects and suppressed BALF and tissue eosinophils and inflammation, MSC numbers, reduced the production of type 2 cytokines and AHR. AAL(S) , BORT and AAL(S) +BORT also reduced airway remodelling in chronic AAD. CONCLUSION: These findings highlight the potential of combination therapies that enhance PP2A and inhibit proteasome activity as novel therapeutic strategies for asthma.

A pathogenic role for tumor necrosis factor-related apoptosis-inducing ligand in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a life-threatening inflammatory respiratory disorder, often induced by cigarette smoke (CS) exposure. The development of effective therapies is impaired by a lack of understanding of the underlining mechanisms. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine with inflammatory and apoptotic properties. We interrogated a mouse model of CS-induced experimental COPD and human tissues to identify a novel role for TRAIL in COPD pathogenesis. CS exposure of wild-type mice increased TRAIL and its receptor messenger RNA (mRNA) expression and protein levels, as well as the number of TRAIL(+)CD11b(+) monocytes in the lung. TRAIL and its receptor mRNA were also increased in human COPD. CS-exposed TRAIL-deficient mice had decreased pulmonary inflammation, pro-inflammatory mediators, emphysema-like alveolar enlargement, and improved lung function. TRAIL-deficient mice also developed spontaneous small airway changes with increased epithelial cell thickness and collagen deposition, independent of CS exposure. Importantly, therapeutic neutralization of TRAIL, after the establishment of early-stage experimental COPD, reduced pulmonary inflammation, emphysema-like alveolar enlargement, and small airway changes. These data provide further evidence for TRAIL being a pivotal inflammatory factor in respiratory diseases, and the first preclinical evidence to suggest that therapeutic agents that target TRAIL may be effective in COPD therapy.

Mucosal production of uric acid by airway epithelial cells contributes to particulate matter-induced allergic sensitization.

Exposure to particulate matter (PM), a major component of air pollution, contributes to increased morbidity and mortality worldwide. PM induces innate immune responses and contributes to allergic sensitization, although the mechanisms governing this process remain unclear. Lung mucosal uric acid has also been linked to allergic sensitization. The links among PM exposure, uric acid, and allergic sensitization remain unexplored. We therefore investigated the mechanisms behind PM-induced allergic sensitization in the context of lung mucosal uric acid. PM10 and house dust mite exposure selectively induced lung mucosal uric acid production and secretion in vivo, which did not occur with other challenges (lipopolysaccharide, virus, bacteria, or inflammatory/fibrotic stimuli). PM10-induced uric acid mediates allergic sensitization and augments antigen-specific T-cell proliferation, which is inhibited by uricase. We then demonstrate that human airway epithelial cells secrete uric acid basally and after stimulation through a previously unidentified mucosal secretion system. Our work discovers a previously unknown mechanism of air pollution-induced, uric acid-mediated, allergic sensitization that may be important in the pathogenesis of asthma.

Prospective study to investigate the use of fine needle aspiration techniques in UK veterinary practice.

OBJECTIVES: To document the fine needle aspiration methods used by UK veterinary practitioners for the assessment of cutaneous masses and relate this to the achievement of a representative sample. METHODS: An internet-based questionnaire was designed and publicised in the UK national veterinary press, at a national surgical meeting, and in letters to veterinary surgeons. RESULTS: One hundred and seventy respondents replied to the questionnaire: 58 · 2% sampled cutaneous masses on the basis of appearance or behaviour; 41 · 3% sampled every cutaneous mass. Practitioners with a greater oncological caseload or who graduated more recently were more likely to recommend fine needle aspiration for every cutaneous mass (P = 0 · 019 and P = 0 · 0002 respectively); 66 · 5% of respondents applied suction during fine needle aspiration; 89% of all respondents used a 2 or 5 mL syringe in combination with a 21 or 23 G needle. There was no statistically significant association between achievement of a representative sample and syringe (P = 0 · 64) or needle size (P = 0 · 63). CLINICAL SIGNIFICANCE: Fine needle aspiration is widely used in UK practice, but may be underutilised in practices with lower oncological caseloads. Survey participants reported a high rate of representative samples obtained using all the commonly used techniques. Further work is required to confirm these observations.

Tumor necrosis factor-related apoptosis-inducing ligand translates neonatal respiratory infection into chronic lung disease.

Respiratory infections in early life can lead to chronic respiratory disease. Chlamydia infections are common causes of respiratory disease, particularly pneumonia in neonates, and are linked to permanent reductions in pulmonary function and the induction of asthma. However, the immune responses that protect against early-life infection and the mechanisms that lead to chronic lung disease are incompletely understood. Here we identify novel roles for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in promoting Chlamydia respiratory infection-induced pathology in early life, and subsequent chronic lung disease. By infecting TRAIL-deficient neonatal mice and using neutralizing antibodies against this factor and its receptors in wild-type mice, we demonstrate that TRAIL is critical in promoting infection-induced histopathology, inflammation, and mucus hypersecretion, as well as subsequent alveolar enlargement and impaired lung function. This suggests that therapeutic agents that target TRAIL or its receptors may be effective treatments for early-life respiratory infections and associated chronic lung disease.

Social and ecological change over a decade in a village hunting system, central Gabon.

Despite widespread recognition of the major threat to tropical forest biological diversity and local food security posed by unsustainable bushmeat hunting, virtually no long-term studies tracking the socioecological dynamics of hunting systems have been conducted. We interviewed local hunters and collected detailed hunting data to investigate changes in offtake and hunter characteristics over 10 years (2001-2010) in Dibouka and Kouagna villages, central Gabon, in the context of hunter recollections of longer term trends since the 1950s. To control for changes in hunter behavior, such as trap location and characteristics, we report hunting offtake data per trap. Our results suggest the hunting area was already highly depleted by 2001; local hunters reported that 16 large-bodied prey species had become rare or locally extirpated over the last 60 years. Overall, we observed no significant declines in hunting offtake or changes in species composition from 2001 to 2010, and offtakes per trap increased slightly between 2004 and 2010. However, trapping distance from the villages increased, and there was a switch in hunting techniques; a larger proportion of the catch was hunted with guns in 2010. The number of hunters declined by 20% from 2004 to 2010, and male livelihood activities shifted away from hunting. Hunters with the lowest hunting incomes in 2004 were more likely than successful hunters to have moved away from the village by 2010 (often in response to alternative employment opportunities). Therefore, changes in trap success (potentially related to biological factors) were interacting with system-level changes in hunter number and composition (related to external socioeconomic factors) to produce a relatively static overall offtake. Our results highlight the importance of understanding the small-scale context of hunting to correctly interpret changes or apparent stasis in hunting effort and offtake over time.

Investigation of prognostic indicators for human uveal melanoma as biomarkers of canine uveal melanoma metastasis.

OBJECTIVE: To evaluate if 14 genes that discriminate metastasising and non-metastasising human uveal melanomas can differentiate metastasising and non-metastasising uveal melanomas in dogs. METHODS: Nineteen archival biopsies of eyes with a histopathological classification of primary benign (n = 9) and malignant (n = 10) uveal melanoma were selected. Thoracic and/or abdominal metastases confirmed metastatic spread of the primary tumour in seven dogs during the follow-up period. Gene expression was assayed by Reverse Transcription-quantitative Polymerase Chain Reaction. Genes displaying statistically significant differences in expression between the metastasising and non-metastasising tumours were identified. RESULTS: Four genes (HTR2B, FXR1, LTA4H and CDH1) demonstrated increased expression in the metastasising uveal melanomas. CLINICAL SIGNIFICANCE: This preliminary study illustrates the potential utility of gene expression markers for predicting canine uveal melanoma metastasis. The genes displaying elevated expression in the metastasising tumours are part of a 12-discriminating gene set used in a routine assay, performed on fine needle aspirate biopsies collected without enucleation, for predicting human uveal melanoma metastasis. Further work is required to validate the results.

Constitutive production of IL-13 promotes early-life Chlamydia respiratory infection and allergic airway disease.

Deleterious responses to pathogens during infancy may contribute to infection and associated asthma. Chlamydia respiratory infections in early life are common causes of pneumonia and lead to reduced lung function and asthma. We investigated the role of interleukin-13 (IL-13) in promoting early-life Chlamydia respiratory infection, infection-induced airway hyperresponsiveness (AHR), and severe allergic airway disease (AAD). Infected infant Il13(-/-) mice had reduced infection, inflammation, and mucus-secreting cell hyperplasia. Surprisingly, infection of wild-type (WT) mice did not increase IL-13 production but reduced IL-13Rα2 decoy receptor levels compared with sham-inoculated controls. Infection of WT but not Il13(-/-) mice induced persistent AHR. Infection and associated pathology were restored in infected Il13(-/-) mice by reconstitution with IL-13. Stat6(-/-) mice were also largely protected. Neutralization of IL-13 during infection prevented subsequent infection-induced severe AAD. Thus, early-life Chlamydia respiratory infection reduces IL-13Rα2 production, which may enhance the effects of constitutive IL-13 and promote more severe infection, persistent AHR, and AAD.

Programming of the lung by early-life infection.

Many important human diseases, such as asthma, have their developmental origins in early life. Respiratory infections in particular may alter the course of asthma and may either protect against or promote the development of this disease. It is likely that the nature of the effects depends on the type and age of infection and is determined by the impact of infection on the immune and respiratory systems. Immunity in early life is plastic and can be moulded by antigen encounter, which may enhance or reinforce the asthmatic phenotype of early life, or induce protective responses. Chlamydial respiratory infections have specific effects and may increase asthma severity in early life by promoting systemic interleukin 13 responses and causing permanent changes in lung structure. Respiratory viral infections, such as those of respiratory syncytial virus and rhinovirus, promote pro-asthmatic responses in early life that contribute to the induction of asthma. By contrast, probiotics or infection or exposure to certain bacteria, such as Streptococcus pneumoniae, may have protective effects in asthma by increasing the numbers and activity of regulatory T cells. Here, we review the impact of infections on the developmental origins of asthma. Understanding these effects may lead to new therapeutic approaches for asthma that either target deleterious infections or utilize beneficial ones.

Streptococcus pneumoniae infection suppresses allergic airways disease by inducing regulatory T-cells.

An inverse association exists between some bacterial infections and the prevalence of asthma. We investigated whether Streptococcus pneumoniae infection protects against asthma using mouse models of ovalbumin (OVA)-induced allergic airway disease (AAD). Mice were intratracheally infected or treated with killed S. pneumoniae before, during or after OVA sensitisation and subsequent challenge. The effects of S. pneumoniae on AAD were assessed. Infection or treatment with killed S. pneumoniae suppressed hallmark features of AAD, including antigen-specific T-helper cell (Th) type 2 cytokine and antibody responses, peripheral and pulmonary eosinophil accumulation, goblet cell hyperplasia, and airway hyperresponsiveness. The effect of infection on the development of specific features of AAD depended on the timing of infection relative to allergic sensitisation and challenge. Infection induced significant increases in regulatory T-cell (Treg) numbers in lymph nodes, which correlated with the degree of suppression of AAD. Tregs reduced T-cell proliferation and Th2 cytokine release. The suppressive effects of infection were reversed by anti-CD25 treatment. Respiratory infection or treatment with S. pneumoniae attenuates allergic immune responses and suppresses AAD. These effects may be mediated by S. pneumoniae-induced Tregs. This identifies the potential for the development of therapeutic agents for asthma from S. pneumoniae.

Using lymph node fine needle aspirates for gene expression profiling of canine lymphoma.

Conventional classification schemes for canine lymphomas do not discriminate between phenotypically indistinct tumours that may exhibit differences in behaviour. Transcriptional profiling has the potential to afford objective clinically relevant stratification of canine lymphomas, and its sensitivity means that prognostic assays could be performed on tumour needle aspirates collected without anaesthesia. In this pilot study, we compared the expression profiles derived from surgical biopsies and fine needle aspirates of five lymphomas. The aspirates yielded expression profiles of equivalent complexity and strong similarity (median correlation Coefficient = 0.911) to those generated from corresponding surgical biopsies. Differences in gene expression observed between the two tissue sources suggest that the aspirates represent a purer source of lymphocytes. Despite the absence of a standardized sample collection protocol, the aspirates yielded expression profiles of consistently high quality suggesting that they represent a robust source of tumour tissue for a potential transcriptional profile-based prognostic assay for canine lymphomas.

Quorum sensing differentially regulates Pseudomonas aeruginosa type VI secretion locus I and homologous loci II and III, which are required for pathogenesis.

Pseudomonas aeruginosa harbours three type VI secretion (T6S) loci. Although HSI-I has been partially studied, limited knowledge is available on the homologous loci HSI-II and HSI-III. We show that quorum sensing (QS) differentially regulates the expression of genes at all three loci. HSI-I-associated gene expression is suppressed by both the homoserine lactone transcription factor LasR and the 4-hydroxy-2-alkylquinoline (HAQ) transcriptional regulator MvfR. Conversely, both HSI-II and HSI-III loci are positively controlled by LasR and MvfR. PqsE, a key component of the MvfR regulon, is required for the expression of part of HSI-III but not HSI-II, and previously identified inhibitors of HAQ biosynthesis significantly downregulate HSI-II and -III gene expression. Animal and plant infection studies reveal that both HSI-II and -III play important roles in pathogenesis. Furthermore, analysis of a double DeltaHSI-II : : III mutant suggests that these loci functionally compensate for one another in virulence. This study illustrates the contribution of the QS systems to T6S gene regulation and reveals the importance of HSI-II and -III in mediating P. aeruginosa pathogenesis. Moreover, this work provides new insights into the design and development of selective compounds that may restrict human P. aeruginosa and possibly other clinical infections.

Preliminary analysis of genomic abnormalities in canine meningiomas.

Cytogenetic detection of unbalanced genomic aberrations in tumours is a strategy for the identification of tumour suppressor genes and oncogenes. When considered in concert with clinical data, the approach also represents a means of identifying markers of prognosis. In a preliminary investigation of the molecular basis of canine meningioma tumorigenesis, we profiled three tumours by comparative genomic hybridization. Distinct patterns of sub-chromosomal deletions were identified suggesting alternative mechanisms of tumour initiation. The deleted chromosomal segments encompass two regions (10q23.1 and 17q22-q23) that are syntenic to the chromosomes (22 and 1p) most often deleted in human meningiomas. A number of genes associated with DNA repair, cell cycle progression and apoptosis are located on both the deleted canine chromosomal segments and the syntenic regions deleted in human meningiomas. This study represents the first report of chromosomal copy number abnormalities in non-cultured canine brain tumour tissue.

Extensive coloured identification of dog chromosomes to support karyotype studies: the colour code.

The identification of individual dog chromosomes is problematic because the 38 pairs of autosomes are small and acrocentric. Here we describe the design and application of a FISH tool that enables definitive identification of each dog autosome in a normal karyotype, without relying on subjective interpretation of DAPI banding patterns. From a high-resolution physical map of the canine genome, we have chosen a panel of 80 canine chromosome-specific BAC clones. DNA from each clone is labeled with one of five different fluorochrome-conjugated nucleotides. By selecting one to three spatially separated BACs per chromosome, and labelling them with a distinctive combination of colours, each autosome can be identified objectively and orientated accurately, irrespective of the quality of DAPI chromosome banding. This tool, or part of it, can be used for any purpose where accurate identification of canine autosomes in a normal karyotype is essential. In this study, we demonstrate use of the 'colour code' for chromosome identification following CGH analysis of unbalanced genomic aberrations in a canine brain tumour. Our method is an improvement of an earlier procedure, featuring chromosome-specific BACs and sequential FISH hybridisations, as it enables simultaneous identification of all chromosomes in a single hybridisation.