Cytologic scoring of equine exercise-induced pulmonary hemorrhage: Performance of human experts and a deep learning-based algorithm.

Exercise-induced pulmonary hemorrhage (EIPH) is a relevant respiratory disease in sport horses, which can be diagnosed by examination of bronchoalveolar lavage fluid (BALF) cells using the total hemosiderin score (THS). The aim of this study was to evaluate the diagnostic accuracy and reproducibility of annotators and to validate a deep learning-based algorithm for the THS. Digitized cytological specimens stained for iron were prepared from 52 equine BALF samples. Ten annotators produced a THS for each slide according to published methods. The reference methods for comparing annotator's and algorithmic performance included a ground truth dataset, the mean annotators' THSs, and chemical iron measurements. Results of the study showed that annotators had marked interobserver variability of the THS, which was mostly due to a systematic error between annotators in grading the intracytoplasmatic hemosiderin content of individual macrophages. Regarding overall measurement error between the annotators, 87.7% of the variance could be reduced by using standardized grades based on the ground truth. The algorithm was highly consistent with the ground truth in assigning hemosiderin grades. Compared with the ground truth THS, annotators had an accuracy of diagnosing EIPH (THS of < or ≥ 75) of 75.7%, whereas, the algorithm had an accuracy of 92.3% with no relevant differences in correlation with chemical iron measurements. The results show that deep learning-based algorithms are useful for improving reproducibility and routine applicability of the THS. For THS by experts, a diagnostic uncertainty interval of 40 to 110 is proposed. THSs within this interval have insufficient reproducibility regarding the EIPH diagnosis.

Validation of digital microscopy: Review of validation methods and sources of bias.

Digital microscopy (DM) is increasingly replacing traditional light microscopy (LM) for performing routine diagnostic and research work in human and veterinary pathology. The DM workflow encompasses specimen preparation, whole-slide image acquisition, slide retrieval, and the workstation, each of which has the potential (depending on the technical parameters) to introduce limitations and artifacts into microscopic examination by pathologists. Performing validation studies according to guidelines established in human pathology ensures that the best-practice approaches for patient care are not deteriorated by implementing DM. Whereas current publications on validation studies suggest an overall high reliability of DM, each laboratory is encouraged to perform an individual validation study to ensure that the DM workflow performs as expected in the respective clinical or research environment. With the exception of validation guidelines developed by the College of American Pathologists in 2013 and its update in 2021, there is no current review of the application of methods fundamental to validation. We highlight that there is high methodological variation between published validation studies, each having advantages and limitations. The diagnostic concordance rate between DM and LM is the most relevant outcome measure, which is influenced (regardless of the viewing modality used) by different sources of bias including complexity of the cases examined, diagnostic experience of the study pathologists, and case recall. Here, we review 3 general study designs used for previous publications on DM validation as well as different approaches for avoiding bias.

Hypermethylation-Mediated Silencing of CIDEA, MAL and PCDH17 Tumour Suppressor Genes in Canine DLBCL: From Multi-Omics Analyses to Mechanistic Studies.

Gene expression is controlled by epigenetic deregulation, a hallmark of cancer. The DNA methylome of canine diffuse large B-cell lymphoma (cDLBCL), the most frequent malignancy of B-lymphocytes in dog, has recently been investigated, suggesting that aberrant hypermethylation of CpG loci is associated with gene silencing. Here, we used a multi-omics approach (DNA methylome, transcriptome and copy number variations) combined with functional in vitro assays, to identify putative tumour suppressor genes subjected to DNA methylation in cDLBCL. Using four cDLBCL primary cell cultures and CLBL-1 cells, we found that CiDEA, MAL and PCDH17, which were significantly suppressed in DLBCL samples, were hypermethylated and also responsive (at the DNA, mRNA and protein level) to pharmacological unmasking with hypomethylating drugs and histone deacetylase inhibitors. The regulatory mechanism underneath the methylation-dependent inhibition of those target genes expression was then investigated through luciferase and in vitro methylation assays. In the most responsive CpG-rich regions, an in silico analysis allowed the prediction of putative transcription factor binding sites influenced by DNA methylation. Interestingly, regulatory elements for AP2, MZF1, NF-kB, PAX5 and SP1 were commonly identified in all three genes. This study provides a foundation for characterisation and experimental validation of novel epigenetically-dysregulated pathways in cDLBCL.

Immunization with Pentraxin3 prevents transition from subclinical to clinical lupus nephritis in lupus-prone mice: Insights from renal ultrastructural findings.

BACKGROUND: Pentraxin3 (PTX3) is an emerging player in lupus nephritis (LN). Anti-PTX3 antibodies showed to delay LN occurrence in vivo. AIM: To evaluate renal changes following immunization with PTX3 in a murine model of LN. MATERIALS AND METHODS: Twenty-two lupus-prone New Zealand Black/White (NZB/W)F1 mice were divided into two groups (n = 11) and subcutaneously injected with human recombinant (hr)PTX3 100 µg or phosphate buffer saline (PBS) 200 µl, three times 3 weeks apart, starting before development of proteinuria. Five mice from each group were scheduled for sacrifice at week 22 and 6 from each group at week 29. Renal lesions included electron-dense deposits (EDD), glomerular deposition of IgG, complement and PTX3 as markers of renal inflammation. They were evaluated by immunofluorescence (IF), confocal and immunoelectron microscopy (IEM). Validated semiquantitative scores were used when available to score renal lesions. Chi-squared test with Fisher exact test was used for comparison. RESULTS: Nineteen out of 22 mice were sacrificed as scheduled. Only hrPTX3-immunized mice developed anti-PTX3 antibodies. Compared to PBS-injected mice, they displayed a dramatic decrease in glomerular deposits of IgG, C1q and PTX3, as well as in the amount of EDD (p = 0.006) and podocyte effacement (p = 0.043). Importantly, PTX3 was pinpointed inside the EDD and co-localized with nuclear material. CONCLUSIONS: Immunization with PTX3 prevented progression from the preclinical to the clinical stage of LN, inciting anti-PTX3 antibodies and preventing renal PTX3 deposition. PTX3 is a novel component of EDD, submitting it as one initiating autoantigen in LN and as potential target for early treatment.

Diagnostic Validation of a Whole-Slide Imaging Scanner in Cytological Samples: Diagnostic Accuracy and Comparison With Light Microscopy.

Digital slides created by whole-slide imaging scanners can be evaluated by pathologists located in remote sites, but the process must be validated before this technology can be applied to routine cytological diagnosis. The aim of this study was to validate a whole-slide imaging scanner for cytological samples. Sixty cytological samples, whose diagnoses were confirmed by gold-standard examinations (histology or flow cytometry), were digitalized using a whole-slide imaging scanner. Digital slides and glass slides were examined by 3 observers with different levels of cytopathological expertise. No significant differences were noted between digital and glass slides in regard to the number of cases correctly diagnosed, or the sensitivity, specificity, or diagnostic accuracy, irrespective of the observers' expertise. The agreements between the digital slides and the gold-standard examinations were moderate to substantial, while the agreements between the glass slides and the gold-standard examinations were substantial for all 3 observers. The intraobserver agreements between digital and glass slides were substantial to almost perfect. The interobserver agreements when evaluating digital slides were moderate between observers 1 and 2 and between observers 1 and 3 while they were substantial between observers 2 and 3. In conclusion, our study demonstrated that the digital slides produced by the whole-slide imaging scanner are adequate to diagnose cytological samples and are similar among clinical pathologists with differing levels of expertise.

Relationship of diagnostic accuracy of renal cortical echogenicity with renal histopathology in dogs and cats, a quantitative study.

BACKGROUND: Renal cortical echogenicity is routinely evaluated during ultrasonographic investigation of the kidneys. Both in dog and cat previous ex-vivo studies have revealed a poor correlation between renal echogenicity and corresponding lesions. The aim of this study was to establish the in-vivo relationship between renal cortical echogenicity and renal histopathology. RESULTS: Thirty-eight dogs and fifteen cats euthanized for critical medical conditions were included in the study. Ultrasonographic images of both kidneys were acquired ante mortem at standardized ultrasonographic settings. The echogenicity was quantified by means of Mean Gray Value (MGV) of the renal cortex measured with ImageJ. A complete histopathological examination of both kidneys was performed. Five kidneys were excluded because histopathology revealed neoplastic lesions. Only samples affected by tubular atrophy showed statistically different values in dog, and histopathology explained 13% of the total variance. MGV was not correlated neither to the degeneration nor to the inflammation scores. However, significant differences were identified between mildly and severely degenerated samples. Overall, the classification efficiency of MGV to detect renal lesions was poor with a sensitivity of 39% and a specificity of 86%. In cats, samples affected by both tubular vacuolar degeneration and interstitial nephritis were statistically different and histopathology explained 44% of the total variance. A linear correlation was evident between degeneration and MGV, whereas no correlation with inflammation was found. Statistically significant differences were evident only between normal and severely degenerated samples with a sensitivity of 54.17% and a specificity of 83.3% and MGV resulted scarce to discriminate renal lesions in this species. CONCLUSIONS: Renal cortical echogenicity shows low relevance in detecting chronic renal disease in dog whereas it results worth to identify severe renal damage in cat.

Detection of hereditary bisalbuminemia in bottlenose dolphins (Tursiops truncatus, Montagu 1821): comparison between capillary zone and agarose gel electrophoresis.

BACKGROUND: Hereditary bisalbuminemia is a relatively rare anomaly characterized by the occurrence of two albumin fractions on serum protein separation by electrophoresis. In human medicine, it is usually revealed by chance, is not been clearly associated with a specific disease and the causative genetic alteration is a point mutation of human serum albumin gene inherited in an autosomal codominant pattern. This type of alteration is well recognizable by capillary zone electrophoresis (CZE), whilst agarose gel electrophoresis (AGE) not always produces a clear separation of albumin fractions. The aims of this study is to report the presence of this abnormality in two separate groups of related bottlenose dolphins and to compare the results obtained with capillary zone and agarose gel electrophoresis. RESULTS: Serum samples from 40 bottlenose dolphins kept under human care were analyzed. In 9 samples a double albumin peak was evident in CZE electrophoresis while no double peak was noted in AGE profile. Since only an apparently wider albumin peaks were noted in some AGE electrophoretic profiles, the ratio between base and height (b/h) of the albumin peak was calculated and each point-value recorded in the whole set of data was used to calculate a receiver operating characteristic curve: when the b/h ratio of albumin peak was equal or higher than 0.25, the sensitivity and specificity of AGE to detect bisalbuminemic samples were 87 and 63 %, respectively. The bisalbuminemic dolphins belong to two distinct families: in the first family, all the siblings derived from the same normal sire were bisalbuminemic, whereas in the second family bisalbuminemia was present in a sire and in two out of three siblings. CONCLUSIONS: We report for the first time the presence of hereditary bisalbuminemia in two groups of related bottlenose dolphins identified by means of CZE and we confirm that AGE could fail in the identification of this alteration.

Digital Cytology in Veterinary Education: A Comprehensive Survey of Its Application and Perception among Undergraduate and Postgraduate Students.

The COVID-19 pandemic accelerated technological changes in veterinary education, particularly in clinical pathology and anatomic pathology courses transitioning from traditional methods to digital pathology (DP). This study evaluates the personal effectiveness and satisfaction, as well as the advantages and disadvantages, of DP, in particular digital cytology (DC), as a teaching method among European veterinary students, both at the undergraduate and postgraduate level, who attended digital pathology courses during and before the pandemic. A further aim is to discuss the differences between the two student groups. A Google Form survey consisting of 11 multiple-choice questions was emailed to pathology teachers and distributed to their students. Results indicated that undergraduate students showed greater digital pathology training, favouring DC as the most effective learning modality. In contrast, postgraduate students reported less digital slide training, and their preference for learning cytology was split between DC alone and DC integrated with traditional microscopy. All students experienced whole slide imaging for learning cytology slides prevalently, and they stated that DC enhanced their learning experience. While DC demonstrates personal effectiveness and satisfaction as a teaching method, it is important to not replace pathology training with light microscopy completely, as almost a third of the students indicated.

Evaluation of Galectin-3 in Dogs with Atrial Fibrillation.

Galectin-3 (Gal-3) is a lectin associated with fibrosis and inflammation, and increased circulating concentrations are considered a risk factor for atrial fibrillation (AF) in humans. This retrospective study aimed to evaluate the serum concentration of Gal-3 in dogs with cardiac disease, both with and without AF. Dogs with AF associated with acquired heart diseases were selected, while cardiac healthy dogs and dogs with heart diseases but without AF served as controls. We statistically compared the serum concentration of Gal-3, which was assessed using a commercial canine-specific ELISA kit, among healthy dogs and dogs with heart disease with and without AF. Additionally, associations between Gal-3 and clinical and echocardiographic variables were evaluated. A total of 73 dogs were included, of which 17/73 (23.3%) were cardiac healthy and 56/73 (76.7%) had heart disease, with 26/56 (46.4%) having AF. No significant difference in Gal-3 concentration was found between cardiac healthy dogs (3.90 ± 1.65 ng/mL) and dogs with heart disease, either with or without AF (3.37 ± 1.04 ng/mL, p = 0.436 and 4.68 ± 1.80 ng/mL, p = 0.332, respectively). Gal-3 showed a significant positive correlation with age (r = 0.47, p < 0.001) and a negative correlation with body weight (r = -0.45, p < 0.001). The results of this study suggest that Gal-3 does not have an important role in the development of AF in dogs, but it is associated with advanced age.

Free circulating versus extracellular vesicle-associated microRNA expression in canine T-cell lymphoma.

Introduction: Canine lymphoma (cL) is one of the most frequent cancers in dogs. The T-cell lymphoma (TcL) is not the most common phenotype but presents an aggressive behavior. MicroRNAs (miRNAs), are small, single-stranded, non-coding RNA molecules which can circulate freely in blood or be associated with extracellular vesicles (EVs). The dysregulation of certain miRNAs has been identified in numerous types of human cancers and they have been largely investigated as possible tumors biomarkers in human medicine, while research in veterinary oncology is still scarce. The aim of this study was to compare the expression patterns of free circulating and EV-associated miRNAs in dogs with T-cell lymhoma (TcL) and healthy dogs. Methods: Eight dogs with TcL were selected as the lymphoma group (LG) and eight dogs were included as controls (Ctrl). Plasma samples were collected at the time of the diagnosis and EVs isolated with ultracentrifugation. miRNAs were extracted from both the circulating EVs and the plasma supernatant, obtaining EV-associated and free-miRNAs. Quantitative real-time PCR was performed to analyze the expression of 88 target miRNAs. Results: Ten and seven differentially expressed miRNAs between LG and Ctrl were detected in EV-associated and free-miRNAs, respectively. Among EV-associated and free-miRNAs, only has-miR-222-3p was overexpressed in both conditions. Discussion: All the differentially expressed miRNAs detected in this study, have been already described as dysregulated in other human or canine cancers. The EV-associated miRNAs, which appear to be more stable and better conserved than free-miRNAs, could be investigated in further larger studies to better assess their use as possible biomarkers for TcL.

Generation and characterization of cytochrome P450 3A74 CRISPR/Cas9 knockout bovine foetal hepatocyte cell line (BFH12).

In human, the cytochrome P450 3A (CYP3A) subfamily of drug-metabolizing enzymes (DMEs) is responsible for a significant number of phase I reactions, with the CYP3A4 isoform superintending the hepatic and intestinal metabolism of diverse endobiotic and xenobiotic compounds. The CYP3A4-dependent bioactivation of chemicals may result in hepatotoxicity and trigger carcinogenesis. In cattle, four CYP3A genes (CYP3A74, CYP3A76, CYP3A28 and CYP3A24) have been identified. Despite cattle being daily exposed to xenobiotics (e.g., mycotoxins, food additives, drugs and pesticides), the existing knowledge about the contribution of CYP3A in bovine hepatic metabolism is still incomplete. Nowadays, CRISPR/Cas9 mediated knockout (KO) is a valuable method to generate in vivo and in vitro models for studying the metabolism of xenobiotics. In the present study, we successfully performed CRISPR/Cas9-mediated KO of bovine CYP3A74, human CYP3A4-like, in a bovine foetal hepatocyte cell line (BFH12). After clonal expansion and selection, CYP3A74 ablation was confirmed at the DNA, mRNA, and protein level. The subsequent characterization of the CYP3A74 KO clone highlighted significant transcriptomic changes (RNA-sequencing) associated with the regulation of cell cycle and proliferation, immune and inflammatory response, as well as metabolic processes. Overall, this study successfully developed a new CYP3A74 KO in vitro model by using CRISPR/Cas9 technology, which represents a novel resource for xenobiotic metabolism studies in cattle. Furthermore, the transcriptomic analysis suggests a key role of CYP3A74 in bovine hepatocyte cell cycle regulation and metabolic homeostasis.

Serum Protein Concentration and Serum Protein Fractions in Bottlenose Dolphins (Tursiops truncatus) under Human Care Using Agarose Gel Electrophoresis.

Serum protein electrophoresis (SPE) is the most used and reliable method to determine the percentage of serum protein subfractions. The interpretation of the kinetics of total proteins and albumin and globulin fractions is receiving increased attention in wild animals, as well as in domestic animals, due to the possibility of identifying typical pathologic patterns. However, the interpretation of these data had to be performed in light of an appropriate method-and species- specific reference intervals (RIs). In marine mammals, as well as other non-domestic species, specific attention should also be given to the different environment (free ranging vs. human managed) and the associated different exposure to environmental stimuli. The aim of this report was to establish RIs for the serum protein fractions evaluated using agarose gel electrophoresis (AGE) in bottlenose dolphins under human care. Peripheral blood samples were collected from 40 bottlenose dolphins during standard veterinary procedures to evaluate their health status. Total protein concentration was determined using the biuret method while AGE was performed using an automated system. A pooled dolphin's serum sample was used to determine the intra-assay and inter-assay imprecision of AGE. The RIs were calculated using an Excel spreadsheet with the Reference Value Advisor set of macroinstructions. The intra and inter-assay imprecisions were 1.2% and 2.5%, respectively, for albumin; 2.9% and 5.7%, respectively, for α-globulins; 3.8% and 4.0%, respectively, for ß-globulins; and 3.4% and 4.8%, respectively, for γ-globulins. The total protein, albumin, α-globulin, ß-globulin, and γ-globulin concentrations were 65.5 ± 5.4 g/L, 45.5 ± 4.9 g/L, 8.0 ± 1.0 g/L, 5.0 ± 2.0 g/L, and 7.0 ± 2.0 g/L, respectively. We established the RIs for the total protein and serum protein fractions using AGE in bottlenose dolphins under human care.

Contrast-enhanced ultrasound features of adrenal lesions in dogs.

BACKGROUND: The contrast-enhanced ultrasound (CEUS) features of adrenal lesions are poorly reported in veterinary literature. METHODS: Qualitative and quantitative B-mode ultrasound and CEUS features of 186 benign (adenoma) and malignant (adenocarcinoma and pheochromocytoma) adrenal lesions were evaluated. RESULTS: Adenocarcinomas (n = 72) and pheochromocytomas (n = 32) had mixed echogenicity with B-mode, and a non-homogeneous aspect with a diffused or peripheral enhancement pattern, hypoperfused areas, intralesional microcirculation and non-homogeneous wash-out with CEUS. Adenomas (n = 82) had mixed echogenicity, isoechogenicity or hypoechogenicity with B-mode, and a homogeneous or non-homogeneous aspect with a diffused enhancement pattern, hypoperfused areas, intralesional microcirculation and homogeneous wash-out with CEUS. With CEUS, a non-homogeneous aspect and the presence of hypoperfused areas and intralesional microcirculation can be used to distinguish between malignant (adenocarcinoma and pheochromocytoma) and benign (adenoma) adrenal lesions. LIMITATIONS: Lesions were characterised only by means of cytology. CONCLUSIONS: CEUS examination is a valuable tool for distinction between benign and malignant adrenal lesions and can potentially differentiate pheochromocytomas from adenocarcinomas and adenomas. However, cytology and histology are necessary to obtain the final diagnosis.

Biological Variation and Reference Change Value of Routine Hematology Measurands in a Population of Managed Bottlenose Dolphins (Tursiops truncatus).

Hematological analyses are particularly useful in assessing a dolphin's health status. However, the creation of appropriate reference intervals for this species is difficult due to the low number of reference individuals. The implementation of individual reference intervals (iRIs) allows researchers to overcome this limitation and, moreover, also consider the within-individual variability. The aims of this study were (1) to evaluate the biological variations in some hematological measurands, including erythrocytes (RBC), hematocrit (Hct), mean cellular volume and hemoglobin content (MCV and MCHC, respectively), RBC distribution width (RDW), leukocytes (WBC), and platelets (PLT); and (2) to calculate the index of individuality (IoI) and reference change value (RCV), which enable the production of iRIs, in healthy managed bottlenose dolphins. Seven dolphins were included, and the results of six hematological exams were analyzed for each animal. Analytical imprecision (CVa), within-dolphin variation (CVi), and between-dolphins variations (CVg) were calculated, and the IoI and RCV were derived for each measurand. All the hematological measurands had intermediate IoI except WBC, for which Iol was low. The calculated RCV ranged from 10.33% (MCV) to 186.51% (WBC). The results reveal that the majority of hematological measurands have an intermediate level of individuality in dolphins, and thus the application of iRIs is appropriate. The calculated RCV can also be applied to other managed dolphins and could be useful in interpreting serial CBC exams.

Antitumor Effect of Berberine Analogs in a Canine Mammary Tumor Cell Line and in Zebrafish Reporters via Wnt/ß-Catenin and Hippo Pathways.

The heterogeneous nature of human breast cancer (HBC) can still lead to therapy inefficacy and high lethality, and new therapeutics as well as new spontaneous animal models are needed to benefit translational HBC research. Dogs are primarily investigated since they spontaneously develop tumors that share many features with human cancers. In recent years, different natural phytochemicals including berberine, a plant alkaloid, have been reported to have antiproliferative activity in vitro in human cancers and rodent animal models. In this study, we report the antiproliferative activity and mechanism of action of berberine, its active metabolite berberrubine, and eight analogs, on a canine mammary carcinoma cell line and in transgenic zebrafish models. We demonstrate both in vitro and in vivo the significant effects of specific analogs on cell viability via the induction of apoptosis, also identifying their role in inhibiting the Wnt/ß-catenin pathway and activating the Hippo signals with a downstream reduction in CTGF expression. In particular, the berberine analogs NAX035 and NAX057 show the highest therapeutic efficacy, deserving further analyses to elucidate their mechanism of action more in detail, and in vivo studies on spontaneous neoplastic diseases are needed, aiming at improving veterinary treatments of cancer as well as translational cancer research.

Impaired reproduction, energy reserves and dysbiosis: The overlooked consequences of heatwaves in a bivalve mollusc.

Extreme events like Marine Heatwaves (MHWs) are becoming more intense, severe, and frequent, threatening benthic communities, specifically bivalves. However, the consequences of non-lethal MHWs on animals are still poorly understood. Here, we exposed the Manila clam Ruditapes philippinarum to non-lethal MHW for 30 days and provided an integrative view of its effects. Our result indicated that albeit non-lethal, MHW reduced clam's energy reserves (by reducing their hepato-somatic index), triggered antioxidant defenses (particularly in males), impaired reproduction (via the production of smaller oocytes in females), triggered dysbiosis in the digestive gland microbiota and altered animals' behaviour (by impacting their burying capacity) and filtration rate. Such effects were seen also at RNA-seq (i.e. many down-regulated genes belonged to reproduction) and metabolome level. Interestingly, negative effects were more pronounced in males than in females. Our results show that MHWs influence animal physiology at multiple levels, likely impacting its fitness and its ecosystem services.

A Machine Learning-Based Approach for Classification of Focal Splenic Lesions Based on Their CT Features.

The aim of the study was to describe the CT features of focal splenic lesions (FSLs) in dogs in order to predict lesion histotype. Dogs that underwent a CT scan and had a FSL diagnosis by cytology or histopathology were retrospectively included in the study. For the statistical analysis the cases were divided into four groups, based on the results of cytopatholoy or hystopathology, namely: nodular hyperplasia (NH), other benign lesions (OBLs), sarcoma (SA), round cell tumour (RCT). Several qualitative and quantitative CT features were described for each case. The relationship occurring between each individual CT feature and the histopathological groups was explred by means of c chi-square test for the count data and by means of Kruskal-Wallis or ANOVA for the continuous data. Furthermore, the main features of each group were described using factorial discriminant analysis, and a decision tree for lesion classification was then developed. Sarcomas were characterised by large dimensions, a cystic appearance and an overall low post contrast-enhancement. NH and OBLs were characterised by small dimensions, a solid appearance and a high post-contrast enhancement. OBLs showed higher post-contrast values than NH. Lastly, RCTs did not exhibit any distinctive CT features. The proposed decision tree had a high accuracy for the classification of SA (0.89) and a moderate accuracy for the classification of OBLs and NH (0.79), whereas it was unable to classify RCTs. The results of the factorial analysis and the proposed decision tree could help the clinician in classifying FSLs based on their CT features. A definitive FSL diagnosis can only be obtained by microscopic examination of the spleen.

Contrast-enhanced ultrasound features of focal pancreatic lesions in dogs.

BACKGROUND: Contrast-enhanced ultrasound (CEUS) features of pancreatic lesions are poorly reported in veterinary literature. METHODS: Qualitative and quantitative features of pancreatic benign (nodular hyperplasia [NH], cyst and abscess) and malignant (adenocarcinoma and insulinoma) lesions during B-mode and CEUS examinations are described in 75 dogs. RESULTS: Adenocarcinomas (n = 23) had mixed echogenicity at B-mode, and they were hypoenhancing or non-enhancing at CEUS, with a non-homogeneous and cystic enhancement pattern. Insulinomas (n = 23) appeared as hypoechoic lesions at B-mode, and as hyperenhancing, homogeneous and solid lesions at CEUS. NH (n = 17) had an constant appearance, being hypoechoic at ultrasound (US) and isoenhancing at CEUS. Cysts (n = 7) were all anechoic, with acoustic enhancement clearly detectable at US, but were non-enhancing at CEUS. Lastly, abscesses (n = 5) had mixed echogenicity, and they showed both hyperenhancement and non-enhancement at CEUS. Hypoenhancement and non-homogeneous appearance had a moderate diagnostic accuracy in the detection of adenocarcinomas. In particular, hyperenhancement was evident only in malignant lesions (adenocarcinomas and insulinomas). CONCLUSION: CEUS, in combination with B-mode US features, is a valuable tool for distinction of benign and malignant abnormalities of the pancreas and can potentially differentiate insulinomas from adenocarcinomas.

Deepening the Whole Transcriptomics of Bovine Liver Cells Exposed to AFB1: A Spotlight on Toll-like Receptor 2.

Aflatoxin B1 (AFB1) is a food contaminant metabolized mostly in the liver and leading to hepatic damage. Livestock species are differently susceptible to AFB1, but the underlying mechanisms of toxicity have not yet been fully investigated, especially in ruminants. Thus, the aim of the present study was to better characterize the molecular mechanism by which AFB1 exerts hepatotoxicity in cattle. The bovine fetal hepatocyte cell line (BFH12) was exposed for 48 h to three different AFB1 concentrations (0.9 µM, 1.8 µM and 3.6 µM). Whole-transcriptomic changes were measured by RNA-seq analysis, showing significant differences in the expression of genes mainly involved in inflammatory response, oxidative stress, drug metabolism, apoptosis and cancer. As a confirmatory step, post-translational investigations on genes of interest were implemented. Cell death associated with necrosis rather than apoptosis events was noted. As far as the toxicity mechanism is concerned, a molecular pathway linking inflammatory response and oxidative stress was postulated. Toll-Like Receptor 2 (TLR2) activation, consequent to AFB1 exposure, triggers an intracellular signaling cascade involving a kinase (p38ß MAPK), which in turn allows the nuclear translocation of the activator protein-1 (AP-1) and NF-κB, finally leading to the release of pro-inflammatory cytokines. Furthermore, a p38ß MAPK negative role in cytoprotective genes regulation was postulated. Overall, our investigations improved the actual knowledge on the molecular effects of this worldwide relevant natural toxin in cattle.

Contrast-enhanced ultrasound features of focal pancreatic lesions in cats.

A large overlap in the ultrasound (US) features of focal pancreatic lesions (FPLs) in cats is reported. Furthermore, only a small number of studies describing the contrast-enhanced ultrasound (CEUS) features of FPLs in cats have been conducted today. The aim of this study is to describe the B-mode US and CEUS features of FPLs in cats. Ninety-eight cats cytologically diagnosed with FPL were included. The lesions were classified as adenocarcinoma (n = 40), lymphoma (n = 11), nodular hyperplasia (n = 17), other benign lesion (OBL) (n = 20), cyst (n = 4) or other malignant lesion (OML) (n = 6). Several qualitative and quantitative B-mode and CEUS features were described in each case. OMLs and cysts were not included in the statistical analysis. A decision tree to classify the lesions based on their B-mode and CEUS features was developed. The overall accuracy of the cross-validation of the decision tree was 0.74 (95% CI: 0.63-0.83). The developed decision tree had a very high sensitivity and specificity for nodular hyperplasia (1 and 0.94, respectively) as well as good sensitivity and specificity for both adenocarcinomas (0.85 and 0.77, respectively) and OBLs also (0.70 and 0.93, respectively). The algorithm was unable to detect any specific feature for classifying lymphomas, and almost all the lymphomas were classified as adenocarcinomas. The combination between CEUS and B-mode US is very accurate in the classification of some FPLs, especially nodular hyperplasia and adenocarcinomas. Cytopathology and or histopathology is still a fundamental step FPL diagnostic workflow.