Identification of potential plasma protein biomarkers for feline pancreatic carcinoma by liquid chromatography tandem mass spectrometry.

In both humans and cats, pancreatic carcinoma is an aggressive cancer with a grave prognosis. Proteomics techniques have successfully identified several blood-based biomarkers of human pancreatic neoplasia. Thus, this study aims to investigate whether similar biomarkers can be identified in the plasma of cats with FePAC by using liquid chromatography tandem mass spectrometry (LC-MS/MS). To facilitate evaluation of the low abundance plasma proteome, a human-based immunodepletion device (MARS-2) was first validated for use with feline plasma. Marked reduction and/or complete removal of albumin and immunoglobulins was confirmed by analysis of electrophoretograms and mass spectral data. Subsequently, plasma collected from 9 cats with pancreatic carcinoma (FePAC), 10 cats with symptomatic pancreatitis, and 10 healthy control cats was immunodepleted and subjected to LC-MS/MS. Thirty-seven plasma proteins were found to be differentially expressed (p < .05 in one-way ANOVA, FC >2 in fold change analysis). Among these proteins, ETS variant transcription factor 4 (p < .05) was overexpressed, while gelsolin (p < .01), tryptophan 2,3-dioxygenase (p < .05), serpin family F member 1 (p < .01), apolipoprotein A-IV (p < .01) and phosphatidylinositol-glycan-specific phospholipase D (p < .05) were down-regulated in cats with FePAC. Further studies on these potential biomarkers are needed to investigate their diagnostic value.

Comparison of Fine Arts- and Pathology-Based Observational Skills Training for Veterinary Students Learning Cytology.

Keen observational skills are essential for veterinarians; however, the development of these skills is not usually an explicit part of the veterinary curriculum. Fine arts-based (FAB) observation training has been shown to improve medical students' observational skills and might also improve veterinary students' observational skills. We compared FAB and pathology-based (PB) observation training in a veterinary cytology course. Students initially wrote a pre-test in which they described two cytology images and one art image, followed by participation in either FAB or PB observation training. Both groups completed a similar post-test immediately after training and a delayed post-test 4 weeks later following instruction in cytology. Differences between groups were noted only in the immediate post-test cytology descriptions. The PB group used significantly more specific vocabulary terms and significantly more accurate observations than the FAB group, suggesting an immediate benefit to the discipline-specific information gained in the PB observation training. In the delayed post-test, results for both groups were similar. The FAB group significantly increased their use of specific vocabulary terms and maintained but did not increase accurate observations following cytology instruction, while accurate observations decreased significantly for the PB group. The FAB group might have been able to generalize their observation skills to the discipline of cytology and to better retain these skills. Neither type of training resulted in both achievement and maintenance of the highest recorded scores for accurate observations. Both FAB and PB training led to improved observational skills, and explicit observation training may be useful for veterinary students.

Pharmacokinetics and pharmacodynamics of doxycycline in a Streptococcusequi subsp. zooepidemicus infection model in horses.

The objectives of this study were to investigate the pharmacokinetics (PK), pharmacodynamics (PD), and the efficacy of oral administration of doxycycline (DXC) in horses with Streptococcus zooepidemicus tissue infections. Tissue chambers (TC) were implanted subcutaneously in the cervical region of 7 horses and inoculated with a single S. zooepidemicus isolate with a minimum inhibitory concentration (MIC) of 0.25 µg/ml, determined by agar dilution. Doxycycline hyclate (10 mg/kg, orally, q 12 h, for 5 days) mixed with poloxamer gel was started following inoculation. The TC fluid was sampled prior to and following inoculation for cytology analysis, quantitative culture, and DXC determination. Plasma DXC concentrations were measured over 48 h following the last dose of DXC administered. The mean plasma peak concentration (Cmax ) of DXC was 0.32 µg/ml, and concentrations above the MIC were only reached in 3 TC samples. In plasma, mean T > MIC was 2.4 h, mean Cmax /MIC was 1.30, and mean AUClast /MIC was 11.63 h. These PK/PD indices did not reach the suggested targets for DXC treatments of infections, and the TC abscessed in all horses. This is the first study to evaluate the recommended dose of DXC in horse in an infection model.

Assessment of neutrophil function in canine cancer patients undergoing chemotherapy and correlation with neutrophil numbers.

Decreased neutrophil function following administration of chemotherapy has been reported in dogs with lymphoma. The first objective of our study was to determine if neutrophil oxidative burst and phagocytic activity are affected by chemotherapy 7 to 10 days following initiation of treatment in dogs with lymphoma and non-lymphoma malignancies. The second objective was to determine if there is a correlation between neutrophil numbers and neutrophil function before or after initiation of chemotherapy. Flow cytometric assessment of neutrophil oxidative burst and phagocytosis following stimulation with Escherichia coli was performed in 9 dogs diagnosed with lymphoma and 17 non-lymphoma tumor-bearing dogs pre- and post-chemotherapy, as well as 14 tumor-free control dogs. Spearman rank correlation was performed to determine if blood neutrophil numbers and neutrophil function were significantly correlated. Lymphoma patients showed significantly reduced percentage neutrophil oxidative burst post-chemotherapy compared to healthy controls as well as compared to pre-chemotherapy values (P = 0.0022 and P = 0.0020, respectively). Lymphoma patients also exhibited significantly reduced neutrophil phagocytosis activity post-chemotherapy compared to controls and pre-chemotherapy values (P = 0.0016 and P = 0.014, respectively). Dogs with non-lymphoma malignancies also showed a significant decrease in both percentage oxidative burst and phagocytosis post-chemotherapy compared to pre-chemotherapy values (P = 0.00040 and P = 0.029, respectively). Neutrophil numbers and function were not significantly correlated. The results of the study suggest that chemotherapeutic treatment decreases neutrophil oxidative burst and phagocytic activity 7 to 10 days post-treatment in dogs with various malignancies. Furthermore, neutrophil numbers cannot be used to predict neutrophil function.

Une diminution de la fonction des neutrophiles après l'administration d'une chimiothérapie a été rapportée chez des chiens atteints de lymphome. Le premier objectif de notre étude était de déterminer si la stimulation oxydative des neutrophiles et l'activité phagocytaire sont affectées par la chimiothérapie 7 à 10 jours après le début du traitement chez les chiens atteints de lymphomes et de tumeurs malignes non lymphomateuses. Le deuxième objectif était de déterminer s'il existe une corrélation entre les nombres de neutrophiles et la fonction des neutrophiles avant ou après le début de la chimiothérapie. L'évaluation par cytométrie en flux de la stimulation oxydative des neutrophiles et de la phagocytose après stimulation par Escherichia coli a été réalisée chez neuf chiens diagnostiqués avec un lymphome et 17 chiens avec des tumeurs non lymphomateuses avant et après la chimiothérapie, ainsi que 14 chiens témoins sans tumeur. Une corrélation des rangs de Spearman a été effectuée pour déterminer si les nombres de neutrophiles sanguins et la fonction des neutrophiles étaient significativement corrélés. Les patients atteints de lymphome ont montré un pourcentage significativement réduit de stimulation oxydative des neutrophiles après la chimiothérapie par rapport aux témoins sains ainsi que par rapport aux valeurs pré-chimiothérapie (P = 0,0022 et P = 0,0020, respectivement). Les patients atteints de lymphome ont également présenté une activité de phagocytose par les neutrophiles significativement réduite après la chimiothérapie par rapport aux témoins et aux valeurs pré-chimiothérapie (P = 0,0016 et P = 0,014, respectivement). Les chiens atteints de tumeurs malignes non lymphomateuses ont également montré une diminution significative du pourcentage de stimulation oxydative et de la phagocytose post-chimiothérapie par rapport aux valeurs pré-chimiothérapie (P = 0,00040 et P = 0,029, respectivement). Les nombres et la fonction des neutrophiles n'étaient pas significativement corrélés. Les résultats de l'étude suggèrent que le traitement chimiothérapeutique diminue la poussée oxydative des neutrophiles et l'activité phagocytaire 7 à 10 jours après le traitement chez les chiens atteints de diverses tumeurs malignes. De plus, les nombres de neutrophiles ne peuvent pas être utilisés pour prédire la fonction des neutrophiles.(Traduit par Docteur Serge Messier).

Tolerance of Ambiguity in Veterinary Students at the Beginning and End of a Second-year Clinical Pathology Course.

Tolerance of ambiguity (TOA) is essential for veterinarians because ambiguity and uncertainty are unavoidable aspects of veterinary practice. However, TOA has been little investigated in veterinarians or veterinary students. In this article, the 27-item Tolerance of Ambiguity of Veterinary Students (TAVS) scale, including eight additional clinical pathology-specific items, is used to evaluate TOA in veterinary students at the beginning and end of a clinical pathology course. Clinical pathology is often one of the first subjects in which students encounter ambiguity because real-life cases are used in teaching. The hypotheses are that TOA will increase across the course and that TOA will correlate with the final grade in the course. Analysis of the TAVS scale revealed very good inter-item reliability (α = 0.80) and a positive correlation between the original TAVS items and the new clinical pathology items (ρ = 0.63). Students demonstrated a significant increase in TOA across the course for TAVS items and a similar trend for clinical pathology items. Four items related to affinity for complexity and novice view showed significant increases in TOA. Two items related to discomfort from uncertainty showed significant decreases. There was no correlation between TOA and final grade in the course. Students rated their personal frustration with ambiguity in the course as low and did not think ambiguity in cases was problematic for teaching. The results suggest that the increased TOA at the end of the course might relate to students being taught-and learning how to cope with-ambiguity through the real-life cases used for teaching.

Using Fine Arts-Based Training to Develop Observational Skills in Veterinary Students Learning Cytology: A Pilot Study.

Arts-based training has been shown to improve medical students' observational skills. Veterinarians also need keen observational skills. Student veterinarians are expected to develop their observational skills; however, this training is usually not an explicit part of the veterinary curriculum. The impact of arts-based observation training has not been investigated in veterinary students learning cytology. In this pilot study, we compared student descriptions of art and cytology images before and immediately after receiving arts-based observation training. After 10 hours of cytology instruction, we again tested students' observational skills and asked for feedback via a survey. Pre-tests and post-tests were scored following a rubric based on expert descriptions of the images. Scores for art image descriptions were higher for both the immediate and delayed post-tests compared to the pre-test (p < .05). Scores for cytology image descriptions were higher for the immediate post-test than the pre-test, but this difference was not significant. Despite 10 hours of cytology instruction between post-tests, scores for cytology image descriptions were lower for the delayed post-test than the immediate post-test, but again, this difference was not significant. Student feedback on the arts-based observation training was positive. Overall, our results suggest that arts-based training may improve student observational skills, although context could be important, as the improvement in description was only significant for art images. Further investigation with a larger cohort of students and a control group that does not receive arts-based training would be valuable.

Prevalence and mutation analysis of the spike protein in feline enteric coronavirus and feline infectious peritonitis detected in household and shelter cats in western Canada.

Feline infectious peritonitis (FIP) is a fatal disease for which no simple antemortem diagnostic assay is available. A new polymerase chain reaction (PCR) test has recently been developed that targets the spike protein region of the FIP virus (FIPV) and can identify specific mutations (M1030L or S1032A), the presence of which indicates a shift from feline enteric coronavirus (FeCV) to FIPV. This test will only be useful in the geographical region of interest, however, if the FIP viruses contain these mutations. The primary objective of this study was to determine the presence of the M1030L or S1032A mutations in FeCV derived from stool samples from a selected group of healthy cats from households and shelters and determine how many of these cats excrete FeCV. The secondary objective was to evaluate how often these specific FIPV mutations were present in tissue samples derived from cats diagnosed with FIP at postmortem examination. Feline enteric coronavirus (FeCV) was detected in 46% of fecal samples (86/185), all were FeCV type 1, with no difference between household or shelter cats. Only 45% of the FIPV analyzed contained the previously reported M1030L or S1032A mutations. It should be noted that, as the pathological tissue samples were opportunistically obtained and not specifically obtained for PCR testing, caution is warranted in interpreting these data.

La péritonite infectieuse féline (FIP) est une maladie fatale pour laquelle il n'existe pas de test diagnostique ante-mortem simple. Une nouvelle épreuve d'amplification en chaîne par la polymérase (PCR) a récemment été développée et qui vise la région de la protéine de spicule du virus FIP (FIPV) et peut identifier les mutations spécifiques (M1030L ou S1032A), la présence desquelles indique un glissement du coronavirus entérique félin (FeCV) vers le FIPV. Cette épreuve sera utile uniquement dans la région géographique d'intérêt, toutefois, si les virus FIP ont ces mutations. L'objectif premier de la présente étude était de déterminer la présence des mutations M1030L ou S1032A chez FeCV obtenu d'échantillons de fèces provenant d'un groupe sélectionné de chats en santé issus de maisonnée et refuges et de déterminer combien de ces chats excrètent FeCV. L'objectif secondaire était d'évaluer à quelle fréquence ces mutations spécifiques de FIPV étaient présentes dans des échantillons de tissu provenant de chats diagnostiqués avec FIP lors d'examen post-mortem. Le FeCV fut détecté dans 46 % des échantillons fécaux (86/185), tous de type FeCV 1, et aucune différence notée entre les chats de maisonnée ou de refuge. Seulement 45 % des FIPV analysés contenaient les mutations M1030L ou S1032A rapportées précédemment. Il faut noter que, étant donné que les échantillons de tissus pathologiques furent obtenus de manière opportuniste et non spécifiquement obtenus pour analyse par PCR, l'interprétation des résultats est à faire avec précaution.(Traduit par Docteur Serge Messier).

Tumor necrosis factor-alpha protein concentrations in bronchoalveolar lavage fluid from healthy horses and horses with severe equine asthma.

The objective of this study was to determine if TNF-α protein concentration differs in bronchoalveolar lavage fluid (BALF) obtained from healthy horses, horses with naturally occurring exacerbations of severe equine asthma and horses in remission from severe equine asthma. Tumor necrosis factor-alpha (TNF- α) protein concentrations were determined in BALF by commercial equine ELISA. Horses with naturally occurring exacerbation of severe equine asthma were found to have significantly lower BALF TNF-α protein concentrations than healthy horses (p = 0.0026). There was no significant difference in BALF TNF-α concentration between horses in exacerbation and remission from disease, but there was a decrease in median TNF-α concentration between healthy horses and horses with clinical exacerbation of severe equine asthma. These findings suggest, that similar to human asthma, the role of TNF-α in chronic lower airway inflammation of horses may differ between disease phenotypes and disease state. Furthermore, the method with which TNF-α is measured (mRNA expression vs. protein concentration) may affect results when studying the role of TNF-α in horses with severe equine asthma.

Does antimicrobial therapy improve outcomes in horses with severe equine asthma and a positive tracheal wash bacterial culture?

The objective of this study was to observe the outcomes of adding an antimicrobial treatment to a conventional treatment regime in horses with severe equine asthma in a clinical setting. Eleven client-owned horses with a history consistent with severe equine asthma, increased respiratory effort and nostril flaring, ≥ 20% neutrophils on bronchoalveolar lavage (BAL), and a positive tracheal wash (TW) bacterial culture were treated with environmental management, corticosteroids, and bronchodilators. Six horses were also treated with an antimicrobial (principal group), while the other 5 were administered saline as a placebo (control group). Treatment with antimicrobials significantly improved the post-treatment clinical score of the principal group compared with the pre-treatment score, whereas no significant difference occurred in the control group. The principal group also had significantly less neutrophil myeloperoxidase (MPO) activity post-treatment than pre-treatment, with a median difference of -0.39 units/[protein] in the principal group and a median difference of -0.21 units/[protein] in the controls. There was no difference in MPO activity pre- versus post-treatment in the control group. No differences were noted in the intra-group comparisons of pre- versus post-treatment BAL neutrophil counts, mucus scores, and concentrations of interleukin-8 (IL-8) or tumor necrosis factor-alpha (TNF-α) in bronchoalveolar lavage fluid (BALF) in either group. There were no differences found in the inter-group comparisons of the principal versus controls for each of the pre- and post-treatment time periods for BAL neutrophil count, mucus score, clinical scores, MPO activity, and IL-8 or TNF-α concentrations. The role of airway bacteria in horses with severe equine asthma requires further investigation as antimicrobial therapy improved post-treatment clinical scores and decreased MPO activity in the group of horses studied, but did not affect other measures of airway inflammation.

L'objectif de la présente étude était d'observer dans un contexte clinique les résultats de l'ajout d'un traitement antimicrobien au traitement conventionnel de chevaux souffrant d'asthme sévère. Onze chevaux appartenant à des propriétaires et ayant une histoire correspondant avec de l'asthme sévère, un effort inspiratoire augmenté et un élargissement des narines, ≥ 20 % de neutrophiles dans le lavage bronchoalvéolaire (LBA), et une culture bactérienne positive à partir du lavage trachéal (LT) ont été traités par gestion de leur environnement, des corticostéroïdes, et des broncho-dilatateurs. Six chevaux ont également été traités avec un antimicrobien (groupe principal) alors que les cinq autres chevaux ont reçu de la saline à titre de placebo (groupe témoin). Le traitement avec les antimicrobiens améliora de manière significative le score clinique post-traitement du groupe principal comparativement au score pré-traitement, alors qu'aucune différence significative ne fut notée dans le groupe témoin. Dans le groupe principal on nota également qu'il y avait significativement moins d'activité myéloperoxydase (MPO) des neutrophiles post-traitement comparativement à pré-traitement, avec une différence médiane de −0,39 unités/[protéine] dans le groupe principal et une différence médiane de −0,21 unités/[protéine] dans le groupe témoin. Il n'y avait pas de différence de l'activité MPO pré- versus post-traitement dans le groupe témoin. Aucune différence ne fut notée dans les comparaisons intra-groupe pré- versus posttraitement du dénombrement de neutrophiles dans les LAB, du score de mucus, et des concentrations d'interleukine-8 (IL-8) ou du facteuralpha nécrosant des tumeurs (TNF-α) dans les liquides de lavage broncho-alvéolaire (LLBA) d'un groupe ou l'autre. Aucune différence ne fut trouvée dans les comparaisons inter-groupes du principal versus les témoins pour chacune des périodes de temps pré- et post-traitement pour le dénombrement des neutrophiles des LAB, le score de mucus, les scores cliniques, l'activité MPO, et les concentrations d'IL-8 ou de TNF-α. Le rôle des bactéries dans les voies respiratoires des chevaux souffrant d'asthme sévère nécessite des études supplémentaires étant donné que les thérapies antimicrobiennes ont améliorés les scores cliniques post-traitement et ont diminué l'activité MPO dans le groupe de chevaux étudiés, mais n'affecta pas d'autres mesures de l'inflammation des voies respiratoires.(Traduit par Docteur Serge Messier).

Differential circulating concentrations of adipokines, glucagon and adropin in a clinical population of lean, overweight and diabetic cats.

BACKGROUND: Dyslipidemia, dysregulated adipokine secretion and alteration in glucagon and adropin concentrations are important obesity-related factors in the pathophysiology of human Type 2 diabetes; however, their roles in the pathophysiology of feline diabetes mellitus are relatively unknown. Here, we determined the concentrations of circulating leptin, adiponectin, pro-inflammatory cytokines, glucagon, adropin, triglycerides, and cholesterol, in non-diabetic lean and overweight cats and newly diagnosed diabetic cats. Client-owned cats were recruited and assigned into 3 study groups: lean, overweight and diabetic. Fasting blood samples were analyzed in lean, overweight and diabetic cats at baseline and 4 weeks after consumption of high protein/low carbohydrate standardized diet. RESULTS: Serum concentrations of triglycerides were greater in diabetics at baseline and were increased in both diabetic and overweight cats at 4 weeks. Plasma leptin concentrations were greater in diabetic and overweight at baseline and 4 weeks, whereas adiponectin was lower in diabetics compared to lean and overweight cats at baseline and 4 weeks. Diabetics had greater baseline plasma glucagon concentrations compared to lean, lower adropin than overweight at 4 weeks, and lower IL-12 concentrations at 4 weeks than baseline. CONCLUSIONS: Our results suggest that feline obesity and diabetes mellitus are characterized by hypertriglyceridemia and hyperleptinemia; however, diabetic cats have significantly lower adiponectin and adropin compared to overweight cats. Thus, despite having similar body condition, overweight and diabetic cats have differential circulating concentrations of adiponectin and adropin.

A comparative proteomic study of plasma in feline pancreatitis and pancreatic carcinoma using 2-dimensional gel electrophoresis to identify diagnostic biomarkers: A pilot study.

While pancreatitis is now recognized as a common ailment in cats, the diagnosis remains challenging due to discordant results and suboptimal sensitivity of ultrasound and specific feline pancreatic lipase (Spec fPL) assay. Pancreatitis also shares similar clinical features with pancreatic carcinoma, a rare but aggressive disease with a grave prognosis. The objective of this pilot study was to compare the plasma proteomes of normal healthy cats (n = 6), cats with pancreatitis (n = 6), and cats with pancreatic carcinoma (n = 6) in order to identify potential new biomarkers of feline pancreatic disease. After plasma protein separation by 2-dimensional gel electrophoresis, protein spots were detected by Coomassie Brilliant Blue G-250 staining and identified by mass spectrometry. Alpha-1-acid glycoprotein (AGP), apolipoprotein-A1 (Apo-A1), and apolipoprotein-A1 precursor (Pre Apo-A1) appeared to be differentially expressed, which suggests the presence of a systemic acute-phase response and alteration of lipid metabolism in cats with pancreatic disease. Future studies involving greater case numbers are needed in order to assess the utility of these proteins as potential biomarkers. More sensitive proteomic techniques may also be helpful in detecting significant but low-abundance proteins.

Bien que la pancréatite soit maintenant reconnue comme un problème peu fréquent chez les chats, le diagnostic demeure un défi étant donné les résultats discordants et la sensibilité sous-optimale de l'échographie et de l'épreuve spécifique de la lipase pancréatique féline (Spec fPL). La pancréatite partage également des similarités cliniques avec le carcinome pancréatique, une maladie rare mais agressive ayant un pronostic grave. L'objectif de cette étude pilote était de comparer les protéomes plasmatiques de chats normaux en santé (n = 6), de chats avec une pancréatite (n = 6), et de chats avec un carcinome pancréatique (n = 6) afin d'identifier de nouveaux biomarqueurs potentiels de maladie pancréatique féline. Après séparation des protéines plasmatiques par électrophorèse en gel en deux dimensions, les taches de protéines furent détectées par coloration avec du bleu brillant de Coomassie G-250 et identifiées par spectrométrie de masse. La glycoprotéine acide alpha-1 (AGP), l'apolipoprotéine A1 (Apo-A1), et le précurseur de l'apolipoprotéine A1 (Pre Apo-A1) apparaissent comme étant exprimées de manière différentielle, ce qui suggère la présence d'une réponse de phase-aiguë systémique et une altération du métabolisme des lipides chez les chats avec une maladie pancréatique. Des études additionnelles regroupant un plus grand nombre de cas sont nécessaires afin d'évaluer l'utilité de ces protéines comme biomarqueurs potentiels. Des techniques plus sensibles de protéomique pourraient également être utiles pour détecter des protéines significatives mais de faible abondance.(Traduit par Docteur Serge Messier).

Utility of nuclear morphometry in the cytologic evaluation of canine cutaneous soft tissue sarcomas.

Cytopathologists lack reliable criteria to distinguish neoplastic from reactive spindle cells; however, with computer-based nuclear morphometry, it is now possible to more objectively and precisely quantify differences between selected populations of cells. Forty-four cutaneous soft tissue sarcomas and 5 cases of reactive spindle cell proliferations in the dog were morphometrically analyzed with regard to median and standard deviation (SD) of nuclear area, diameter (max, min, mean), radius (max, min), perimeter, and roundness. Overall, nuclei from reactive spindle cells were larger, with greater variation in nuclear size and shape. Significant differences (P < 0.05) were found for several nuclear parameters, including the median and SD of maximum diameter and radius, as well as the SD of roundness. No significant differences were found in nuclear parameters between soft tissue sarcomas divided by histologic grade, mitotic index, or tumor necrosis score. Analysis of the sources of variation indicated near-perfect intraobserver and substantial interobserver agreement. The largest source of variation was due to selection of different measurement fields, reflecting the inherent biological variation in nuclear size within the tumor cell population. The results indicate that nuclear morphometry on cytologic preparations is a reproducible method that may be able to differentiate cutaneous soft tissue sarcomas from reactive mesenchymal lesions in the dog. Further studies, including a larger number of cases, are warranted to assess repeatability of results.