Detection of Pneumocystis and Morphological Description of Fungal Distribution and Severity of Infection in Thirty-Six Mammal Species.

Pneumocystis spp. are thought to adapt to the lungs of potentially all mammals. However, the full host range, fungal burden and severity of infection are unknown for many species. In this study, lung tissue samples originating from 845 animals of 31 different families of eight mammal orders were screened by in situ hybridization (ISH) using a universal 18S rRNA probe for Pneumocystis, followed by hematoxylin and eosin (H&E) staining for determining histopathological lesions. A total of 216 (26%) samples were positive for Pneumocystis spp., encompassing 36 of 98 investigated mammal species, with 17 of them being described for the first time for the presence of Pneumocystis spp. The prevalence of Pneumocystis spp. as assessed by ISH varied greatly among different mammal species while the organism load was overall low, suggesting a status of colonization or subclinical infection. Severe Pneumocystis pneumonia seemed to be very rare. For most of the Pneumocystis-positive samples, comparative microscopic examination of H&E- and ISH-stained serial sections revealed an association of the fungus with minor lesions, consistent with an interstitial pneumonia. Colonization or subclinical infection of Pneumocystis in the lung might be important in many mammal species because the animals may serve as a reservoir.

SiCoDEA: A Simple, Fast and Complete App for Analyzing the Effect of Individual Drugs and Their Combinations.

The administration of combinations of drugs is a method widely used in the treatment of different pathologies as it can lead to an increase in the therapeutic effect and a reduction in the dose compared to the administration of single drugs. For these reasons, it is of interest to study combinations of drugs and to determine whether a specific combination has a synergistic, antagonistic or additive effect. Various mathematical models have been developed, which use different methods to evaluate the synergy of a combination of drugs. We have developed an open access and easy to use app that allows different models to be explored and the most fitting to be chosen for the specific experimental data: SiCoDEA (Single and Combined Drug Effect Analysis). Despite the existence of other tools for drug combination analysis, SiCoDEA remains the most complete and flexible since it offers options such as outlier removal or the ability to choose between different models for analysis. SiCoDEA is an easy to use tool for analyzing drug combination data and to have a view of the various steps and offer different results based on the model chosen.

A case of generalised cutaneous apocrine cystomatosis in a Pekingese dog.

Clinical, histological and immunohistochemical examination of a 13-year-old male client-owned Pekingese dog revealed an uncommon presentation of apocrine cutaneous cystomatosis. This is a rare non-neoplastic condition of uncertain cause, characterised by multiple cystically dilated apocrine sweat glands. We aimed to describe the features of this unusual case of generalised cutaneous apocrine cystomatosis in the dog, which can be useful to distinguish it from multifocal benign cystic apocrine tumours.

Immunofluorescence Targeting PBP2a Protein: A New Potential Methicillin Resistance Screening Test.

The indiscriminate use of first-line drugs contributed to the spread of resistant bacteria, a major concern for both human and veterinary medicine. Methicillin resistance is acquired through the mecA gene, which encodes for the PBP2a protein and lends the resistance to ß-lactams. Verifying the correspondence between gene harboring and protein expression and accelerating methicillin resistance diagnosis is critical to improve the management of antimicrobial administration and to reduce the spread of drug resistances. We tested the applicability of immunofluorescence targeting PBP2a protein to identify a new potential methicillin resistance screening test, ancillary to conventional culture methods. We collected 26 clinical Staphylococcus pseudintermedius (SP) isolates: 25 from canine pyoderma and 1 from dermatitis in a dog owner. SP is one of the most important etiological agents in canine pyoderma and can harbor the mecA gene. We performed PCR for mecA gene detection, broth microdilution (BMD) for phenotypic methicillin resistance, and immunofluorescence targeting PBP2a protein. Compared to the PCR as the gold standard, immunofluorescence showed an apparent prevalence of 34.6% vs. a true prevalence of 53.8%, with 100% specificity, 64.3% sensitivity, and 80.8% diagnostic accuracy. PBP2a expression showed isolate-dependent variability: in some isolates, most of the bacterial cells showed an intense and clearly membranous pattern, while in others only a few of them could be detected. Performing the assay in duplicate improved the diagnostic accuracy. Since the mecA gene is shared among the members of the Staphylococcus genus, the test can be applied to identify methicillin resistance independently from the staphylococcal species, both in human and animal samples. Being a rapid and easy method and providing the unique possibility to study the expression of PBP2a by directly visualizing the morphology, it could represent a new interesting tool for both research and diagnostics. To accelerate methicillin resistance diagnosis, it would be worth further testing of its performance on cytological samples.

Tumor-infiltrating lymphocytes (TILs) in feline melanocytic tumors: A preliminary investigation.

The presence and the role of tumor-infiltrating lymphocytes (TILs) in different types of tumors, but particularly in melanoma, has become more and more investigated during the last decade, both in human and veterinary medicine. Melanocytic tumors are quite rare in cats, with diffuse iris melanoma being the most commonly diagnosed in this species. The aim of this study was to characterize the lymphocytic infiltration in feline melanocytic tumors and to analyze their association with the histological features of malignancy recognized in these tumors, as well as with the expression of the most commonly used immunohistochemical markers. Thirty-eight feline melanocytic tumors were retrospectively selected; histological and immunohistochemical characterization of the tumors (histologic criteria of malignancy; S100, Melan A, and PNL2 expression) and of TILs (presence/absence, density, distribution, and grade; CD3, CD20 expression) were performed and associations between them tested. Results showed that TILs grade increased with cellular pleomorphism (P < 0.05) and, within the group of cutaneous melanocytic tumors, also with the mitotic count (P < 0.05). On the other hand, TILs grade was inversely associated with the percentage of neoplastic cells positive for Melan A (P < 0.05) and PNL2 (P < 0.05). Both CD3+ and CD20+ lymphocytes increased significantly with TILs grade and in association with mitotic count, when stratified in low/high quantity. This preliminary study suggests that TILs in feline melanoma may be associated with histologic features of malignancy and loss of melanocytic-specific markers, such as Melan A and PNL2. Further studies, with a larger cohort and follow-up information, are recommended.

H2AFZ: A Novel Prognostic Marker in Canine Melanoma and a Predictive Marker for Resistance to CDK4/6 Inhibitor Treatment.

Uncontrolled proliferation is a key feature of tumor progression and malignancy. This suggests that cell-cycle related factors could be exploited as cancer biomarkers and that pathways specifically involved in the cell cycle, such as the Rb-E2F pathway, could be targeted as an effective anti-tumor therapy. We investigated 34 formalin-fixed paraffin-embedded (FFPE) tissue samples of canine cutaneous melanocytoma, cutaneous melanoma, and oral melanoma. Corresponding clinical follow-up data were used to determine the prognostic value of the mRNA expression levels of several cell cycle regulated E2F target genes (E2F1, DHFR, CDC6, ATAD2, MCM2, H2AFZ, GINS2, and survivin/BIRC5). Moreover, using four canine melanoma cell lines, we explored the possibility of blocking the Rb-E2F pathway by using a CDK4/6 inhibitor (Palbociclib) as a potential anti-cancer therapy. We investigated the expression levels of the same E2F target gene transcripts before and after treatment to determine the potential utility of these molecules as predictive markers. The E2F target gene H2AFZ was expressed in 91.43% of the primary tumors and H2AFZ expression was significantly higher in cases with unfavorable clinical outcome. Among the other tested genes, survivin/BIRC5 showed as well-promising results as a prognostic marker in canine melanoma. Three of the four tested melanoma cell lines were sensitive to the CDK4/6 inhibitor. The resistant cell line displayed higher expression levels of H2AFZ before treatment compared to the CDK4/6 inhibitor-sensitive cell lines. The present results suggest that CDK4/6 inhibitors could potentially be used as a new anti-cancer treatment for canine melanoma and that H2AFZ could serve as a prognostic and predictive marker for patient selection.

Comparative Performances of Vitek-2, Disk Diffusion, and Broth Microdilution for Antimicrobial Susceptibility Testing of Canine Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius is the primary cause of canine cutaneous infections and is sporadically isolated as a pathogen from humans. Rapidly emerging antibiotic-resistant strains are creating serious health concerns so that accurate and timely antimicrobial susceptibility testing (AST) is crucial for patient care. Here, the performances of the AST methods Vitek-2, disk diffusion (DD) and broth microdilution (BMD) were compared for the determination of susceptibility of 79 S. pseudintermedius isolates from canine cutaneous infections and one from human pyoderma to oxacillin (OXA), amoxicillin/clavulanate (AMC), cephalothin (CEF), gentamicin (GEN), enrofloxacin (ENR), doxycycline (DOX), clindamycin (CLI), inducible clindamycin resistance (ICR), mupirocin (MUP), and trimethoprim-sulfamethoxazole (SXT). Overall, the agreement of DD and Vitek-2 using the veterinary AST-GP80 card with reference BMD was ≥90%, suggesting reliable AST performances. While DD generated mainly minor errors and one major error for OXA, Vitek-2 produced one very major error for GEN, and it failed in identifying one ICR-positive isolate. Moreover, five bacteria were diagnosed as ICR-positive by Vitek-2, but they showed a noninduction resistance phenotype with manual methods. All S. pseudintermedius isolates were interpreted as susceptible or intermediately susceptible to DOX using CLSI breakpoints for human staphylococci that match the DOX concentration range included in AST-GP80. However, this could lead to inappropriate antimicrobial prescription for S. pseudintermedius infections in companion animals. Considering the clinical and epidemiological importance of S. pseudintermedius, we encourage updating action by the system manufacturer to address AST for this bacterium.

Characterization of Primary Cultures of Normal and Neoplastic Canine Melanocytes.

Although numerous animal models, especially mouse models, have been established for the study of melanoma, they often fail to accurately describe the mechanisms of human disease because of their anatomic, physiological, and immune differences. The dog, as a spontaneous model of melanoma, is nowadays considered one of the most valid alternatives due to the heterogeneity of clinical presentations and of histological and genetic similarities of canine melanoma with the human counterpart. The aim of the study was to optimize a protocol for the isolation and cultivation of healthy and neoplastic canine melanocytes derived from the same animal and obtained from cutaneous and mucosal (oral) sites. We obtained five primary tumor cell cultures (from 2 cutaneous melanoma, 2 mucosal melanoma and 1 lymph node metastasis) and primary normal melanocyte cell cultures (from normal skin and mucosa) from the same dogs. Immunocytochemical characterization with Melan A, PNL2 and S100 antibodies confirmed the melanocytic origin of the cells. This work contributes to expanding the case record of studies on canine melanoma cell cultures as suitable model to study human and canine melanoma. To the authors' knowledge, this is the first report of isolation of normal skin and mucosal canine melanocytes.

Immunoexpression of epithelial membrane antigen in canine meningioma: Novel results for perspective considerations.

Epithelial membrane antigen (EMA) is one of the most widely used diagnostic immunohistochemical markers for human meningioma. To date, no published study on EMA expression in formalin-fixed paraffin-embedded (FFPE) tissue samples of canine meningioma is available. Here, we describe the results of an immunohistochemical study on 25 FFPE canine meningiomas using a monoclonal anti-human EMA antibody. All meningiomas showed positive staining for EMA with cytoplasmic pattern, in nine cases associated with membranous staining. Area and intensity of staining were highly variable among cases. No clear relationships between tumour subtype/grade and area/intensity of staining were found. However, epithelial-like patterns showed a higher affinity for EMA compared to the mesenchymal one. The present study provides the basis to explore the potential diagnostic application of this marker in canine meningioma. To investigate EMA expression in other central nervous system tumours of dogs are necessary to assess the specificity of this marker.

FoxP3, CTLA-4, and IDO in Canine Melanocytic Tumors.

Despite promising immunotherapy strategies in human melanoma, there are few studies on the immune environment of canine melanocytic tumors. In humans, the activation of immunosuppressive cell subpopulations, such as regulatory T cells (Tregs) that express forkhead box protein P3 (FoxP3), the engagement of immunosuppressive surface receptors like cytotoxic T lymphocyte antigen (CTLA-4), and the secretion of molecules inhibiting lymphocyte activation, such as indoleamine-pyrrole 2,3-dioxygenase (IDO), are recognized as immunoescape mechanisms that allow tumor growth and progression. The aim of our study was to investigate the expression of these immunosuppression markers in canine melanocytic tumors and to postulate their possible role in melanoma biology and progression. Fifty-five formalin-fixed, paraffin-embedded canine melanocytic tumors (25 oral melanomas; 20 cutaneous melanomas; 10 cutaneous melanocytomas) were selected to investigate the expression of FoxP3, CTLA-4, and IDO by immunohistochemistry and RT-qPCR (real-time quantitative polymerase chain reaction). All of the tested markers showed high gene and protein expression in oral melanomas and were differently expressed in cutaneous melanomas when compared to their benign counterpart. IDO expression was associated with an increased hazard of death both in univariable and multivariable analyses (P < .05). FoxP3 protein expression >6.9 cells/HPF (high-power field) was an independent predictor of death (P < .05). CTLA-4 gene and protein expressions were associated with a worse prognosis, but only in the univariable analysis (P < .05). FoxP3, CTLA-4, and IDO likely play a role in canine melanoma immunoescape. Their expression, if supported by future studies, could represent a prognostic tool in canine melanoma and pave the way to future immunotherapeutic approaches in dogs.

E-Cadherin Expression in Canine Melanocytic Tumors: Histological, Immunohistochemical, and Survival Analysis.

E-cadherin, a glycoprotein involved in cell-cell adhesion, has a pivotal role in epithelial-mesenchymal transition, a process through which neoplastic epithelial cells develop an invasive phenotype. In human cutaneous melanomas, decreased E-cadherin expression is associated with shorter survival and increased Breslow thickness, whereas in the dog its role is poorly understood. Tumor thickness and modified Clark level were recently proposed as useful features to assess canine melanocytic tumors, but no studies investigated their association with E-cadherin expression. We performed immunohistochemistry on 77 formalin-fixed, paraffin-embedded primary canine melanocytic tumors. A 3-tier and a 2-tier classification system for assessing E-cadherin expression were tested, with the latter being more informative for the assessment of canine melanocytic tumors. E-cadherin expression was lower in cutaneous melanomas than melanocytomas, as well as in amelanotic tumors compared to pigmented tumors. In amelanotic melanomas, absent E-cadherin expression was associated with an unfavorable outcome, suggesting a potential use of this marker in defining the prognosis of amelanotic melanomas. E-cadherin expression was lower in tumors with greater tumor thickness and modified Clark level ≥IV, suggesting its possible utility in identifying the most invasive tumors. The expression of E-cadherin in oral melanomas was heterogeneous, but was associated with pigmentation and clinical outcome; thus, E-cadherin evaluation could be advantageous to detect the most aggressive neoplasms. However, cutaneous melanomas without E-cadherin expression frequently had a favorable clinical outcome. Hence, its importance as prognostic factor should be carefully considered depending on the tumor origin.

Tumour-infiltrating lymphocytes in canine melanocytic tumours: An investigation on the prognostic role of CD3+ and CD20+ lymphocytic populations.

The study of the immune response in several types of tumours has been rapidly increasing in recent years with the dual aim of understanding the interactions between neoplastic and immune cells and their importance in cancer pathogenesis and progression, as well as identifying targets for cancer immunotherapy. Despite being considered one of the most immunogenic tumour types, melanoma can progress in the presence of abundant lymphocytic infiltration, therefore suggesting that the immune response is not able to efficiently control tumour growth. The purpose of this study was to investigate whether the density, distribution and grade of tumour-infiltrating lymphocytes (TILs) in 97 canine melanocytic tumours is associated with histologic indicators of malignancy and can be considered a prognostic factor in the dog. As a further step in the characterization of the immune response in melanocytic tumours, an immunohistochemical investigation was performed to evaluate the two main populations of TILs, T-lymphocytes (CD3+ ) and B-lymphocytes (CD20+ ). The results of our study show that TILs are present in a large proportion of canine melanocytic tumours, especially in oral melanomas, and that the infiltrate is usually mild. The quantity of CD20+ TILs was significantly associated with some histologic prognostic factors, such as the mitotic count, the cellular pleomorphism and the percentage of pigmented cells. Remarkably, a high infiltration of CD20+ TILs was associated with tumour-related death, presence of metastasis/recurrence, shorter overall and disease-free survival, increased hazard of death and of developing recurrence/metastasis, hence representing a potential new negative prognostic factor in canine melanocytic tumours.

Tumor Thickness and Modified Clark Level in Canine Cutaneous Melanocytic Tumors.

Breslow thickness and Clark level are prognostic factors for human cutaneous melanomas. Breslow thickness is measured with an ocular micrometer from the top of the granular layer of the epidermis to the deepest invasive cell across the broad base of the tumor, while Clark level is based on the anatomical level of invasion through the layers of the dermis. Because of the anatomical differences between humans and dogs, we evaluated the tumor thickness and a modified Clark level in 77 canine primary cutaneous melanocytic tumors. Tumor thickness (using both a traditional and a more convenient system) and modified Clark level were measured and associated with histological diagnosis and clinical outcome. Tumor thickness was a prognostic factor, being greater in animals with shorter overall survival and disease-free time. Cutoffs of 0.95 cm and 0.75 cm defined a higher hazard for an unfavorable outcome and to develop recurrence/metastasis, respectively. Because of an excellent agreement between the 2 methods, it was concluded that tumor thickness could be measured with a ruler when an ocular micrometer is not available. Modified Clark level was not found to be relevant for prognosis. However, we suggest that both tumor thickness and a modified Clark level can be valid additional parameters when histological diagnosis is uncertain. Further studies, including a wider sample population, would be worthwhile to confirm the prognostic significance of these 2 parameters.

FoxP3 and IDO in Canine Melanocytic Tumors.

Human melanoma is one of the deadliest forms of cancer, with poor prognosis and high resistance to chemotherapy and radiotherapy. The discovery of immunosuppressive mechanisms in the human melanoma microenvironment led to the use of new prognostic markers and to the development of immunotherapies targeting immune checkpoint molecules. Immunoescape mechanisms in canine melanoma have not yet been investigated, and no such immunotherapy has been tested. The aim of this study was to provide preliminary data on the expression of transcription factor forkhead box protein P3 (FoxP3) and indoleamine 2,3-dioxygenase (IDO) in primary canine melanocytic tumors and to investigate their prognostic role. Formalin-fixed, paraffin-embedded samples from 74 canine melanocytic tumors (26 oral melanomas, 23 cutaneous melanomas, and 25 cutaneous melanocytomas) were retrospectively evaluated by immunohistochemistry to explore the expression of FoxP3 and IDO. An increased risk of death due to melanoma was associated with a higher number of FoxP3+ cells per high-power field (FoxP3+/HPF), a higher percentage of CD3+ cells that were also FoxP3+ infiltrating and surrounding the tumor (%FoxP3), and a higher number of IDO+ cells/HPF (IDO+/HPF). A prognostic value for FoxP3 and IDO is suggested by our study, with optimal cutoffs of 14.7 FoxP3+ cells/HPF, 6.1 IDO+ cells/HPF, and 12.5% FoxP3+ cells. Both markers were also associated with tumor type. Multivariable analysis identified IDO+/HPF ( P < .001) as an independent prognostic marker. Even though stratification by diagnosis caused a loss of significance, results from the present study suggest a prognostic role for IDO and FoxP3, possibly related to the establishment of an immunosuppressive microenvironment.

Comparison of 2 differently sized endoscopic biopsy forceps in the evaluation of intestinal disease in cats.

BACKGROUND: In clinical practice, histopathological diagnosis of chronic intestinal disease is challenging because of difficulty in obtaining adequate duodenal samples. At present, no studies have investigated the influence of biopsy forceps size on sample quality in cats. OBJECTIVES: Duodenal biopsy using larger biopsy forceps (2.4 mm) will provide higher quality samples. ANIMALS: Fifty client-owned cats underwent endoscopy of the upper gastrointestinal tract for evaluation of chronic gastrointestinal signs, with inflammatory bowel disease (IBD) or intestinal lymphoma as differential diagnoses. METHODS: For each cat, duodenal biopsy specimens were obtained using both small (1.8 mm) and large (2.4 mm) forceps and evaluated for adequacy, orientation, the presence of artifacts, villi morphology, the presence of inflammation, and neoplastic infiltration. RESULTS: The percentage of adequate and evaluable biopsy specimens obtained using the larger forceps was significantly higher than that collected using the smaller forceps. Agreement between the forceps was variable for histological features and substantial in the case of lymphoma. However, in case of disagreement, the proper diagnosis usually was achieved only with the larger biopsy forceps. CONCLUSIONS AND CLINICAL IMPORTANCE: Use of a larger biopsy forceps allows collection of a higher percentage of adequate and evaluable biopsy specimens compared to the commonly used smaller forceps and indirectly decreases the percentage of artifacts and increases the percentage of samples with evaluable villi. The use of a larger forceps could be helpful to obtain high-quality samples and improve diagnostic accuracy.

Transcriptome Analysis of Canine Cutaneous Melanoma and Melanocytoma Reveals a Modulation of Genes Regulating Extracellular Matrix Metabolism and Cell Cycle.

Interactions between tumor cells and tumor microenvironment are considered critical in carcinogenesis, tumor invasion and metastasis. To examine transcriptome changes and to explore the relationship with tumor microenvironment in canine cutaneous melanocytoma and melanoma, we extracted RNA from formalin-fixed, paraffin-embedded (FFPE) specimens and analyzed them by means of RNA-seq for transcriptional analysis. Melanocytoma and melanoma samples were compared to detect differential gene expressions and significant enriched pathways were explored to reveal functional relations between differentially expressed genes. The study demonstrated a differential expression of 60 genes in melanomas compared to melanocytomas. The differentially expressed genes cluster in the extracellular matrix-receptor interaction, protein digestion and absorption, focal adhesion and PI3K-Akt (phosphoinositide 3-kinase/protein kinase B) signaling pathways. Genes encoding for several collagen proteins were more commonly differentially expressed. Results of the RNA-seq were validated by qRT-PCR and protein expression of some target molecules was investigated by means of immunohistochemistry. We hypothesize that the developing melanoma actively promotes collagen metabolism and extracellular matrix remodeling as well as enhancing cell proliferation and survival contributing to disease progression and metastasis. In this study, we also detected unidentified genes in human melanoma expression studies and uncover new candidate drug targets for further testing in canine melanoma.

CD4+ T Cell and NK Cell Interplay Key to Regression of MHC Class Ilow Tumors upon TLR7/8 Agonist Therapy.

One of the next challenges in cancer immunotherapy is the resistance of tumors to T-cell-based treatments through loss of MHC class I. Here, we show that under these circumstances, the Toll-like receptor (TLR)-7/8 ligand imiquimod, but not the TLR3 ligand poly I:C or TLR9 ligand CpG, mediated an effective antitumor response. The rejection of these immune-escaped cancers was mediated by NK cells and CD4+ T cells, whereas activated CD8+ T cells were dispensable. Application of the innate immune stimulator at a distant site activated NK cells and thereby elicited tumor-specific T-cell responses in tumor-bearing mice. Mechanistically, imiquimod activated NK cells to kill tumor cells, resulting in release of tumor antigens and induction of tumor-specific CD4+ T cells. These T helper cells provoked a strong induction of CXCL9 and CXCL10 in the tumor environment. Simultaneously, imiquimod induced the expression of the cognate chemokine receptor CXCR3 on peripheral lymphocytes. This ignited intratumoral CD4+ T-cell infiltration and accumulation, which was critical for tumor rejection; CXCR3 blocking antibodies mitigated the clinical response. In the effector phase, NK cell recruitment to tumors and their activation depended on CD4+ T cells. Together, we have uncovered a potent immune axis of tumor-specific CD4+ T cells and NK cells that eliminates escaped MHC-Ilow tumors. Cancer Immunol Res; 5(8); 642-53. ©2017 AACR.