Morphologic, phenotypic, and genotypic similarities between primary tumors and corresponding 3D cell cultures grown in a repeatable system-preliminary results.

BACKGROUND: Three-dimensional (3D) cell cultures are the new frontier for reproducing the tumor micro-environment in vitro. The aims of the study were (1) to establish primary 3D cell cultures from canine spontaneous neoplasms and (2) to demonstrate the morphological, phenotypic and genotypic similarities between the primary canine neoplasms and the corresponding 3D cultures, through the expression of tumor differentiation markers. RESULTS: Seven primary tumors were collected, including 4 carcinomas and 3 soft tissue sarcomas. 3D cell cultures reproduced the morphological features of the primary tumors and showed an overlapping immunophenotype of the primary epithelial tumors. Immunohistochemistry demonstrated the growth of stromal cells and macrophages admixed with the neoplastic epithelial component, reproducing the tumor microenvironment. Mesenchymal 3D cultures reproduced the immunophenotype of the primary tumor completely in 2 out of 3 examined cases while a discordant expression was documented for a single marker in one case. No single nucleotide variants or small indel were detected in TP53 or MDM2 genes, both in primary tumors and in 3D cell cultures specimens. In one sample, MDM2 amplicons were preferentially increased in number compared to TP53 ones, indicating amplification of MDM2, detectable both in the primary tumor and in the corresponding cell culture specimen. CONCLUSION: Here we demonstrate a good cell morphology, phenotype and genetic profile overlap between primary tumors and the corresponding 3D cultures grown in a repeatable system.

Expression of Cannabinoid Receptors in the Trigeminal Ganglion of the Horse.

Cannabinoid receptors are expressed in human and animal trigeminal sensory neurons; however, the expression in the equine trigeminal ganglion is unknown. Ten trigeminal ganglia from five horses were collected post-mortem from an abattoir. The expression of cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and the cannabinoid-related receptors like transient receptor potential vanilloid type 1 (TRPV1), peroxisome proliferator-activated receptor gamma (PPARÉ£), and G protein-related receptor 55 (GPR55) in the trigeminal ganglia (TG) of the horse were studied, using immunofluorescence on cryosections and formalin-fixed paraffin-embedded (FFPE) sections. Neurons and glial cells were identified using fluorescent Nissl staining NeuroTrace® and an antibody directed against the glial marker glial fibrillary acidic protein (GFAP), respectively. Macrophages were identified by means of an antibody directed against the macrophages/microglia marker ionized calcium-binding adapter molecule 1 (IBA1). The protein expression of CB1R, CB2R, TRPV1, and PPARÉ£ was found in the majority of TG neurons in both cryosections and FFPE sections. The expression of GPR55 immunoreactivity was mainly detectable in FFPE sections, with expression in the majority of sensory neurons. Some receptors were also observed in glial cells (CB2R, TRPV1, PPARγ, and GPR55) and inflammatory cells (PPARγ and GPR55). These results support further investigation of such receptors in disorders of equine trigeminal neuronal excitability.

PDL1 immunohistochemistry in canine neoplasms: Validation of commercial antibodies, standardization of evaluation, and scoring systems.

Immuno-oncology research has brought to light the paradoxical role of immune cells in the induction and elimination of cancer. Programmed cell death protein 1 (PD1), expressed by tumor-infiltrating lymphocytes, and programmed cell death ligand 1 (PDL1), expressed by tumor cells, are immune checkpoint proteins that regulate the antitumor adaptive immune response. This study aimed to validate commercially available PDL1 antibodies in canine tissue and then, applying standardized methods and scoring systems used in human pathology, evaluate PDL1 immunopositivity in different types of canine tumors. To demonstrate cross-reactivity, a monoclonal antibody (22C3) and polyclonal antibody (cod. A1645) were tested by western blot. Cross-reactivity in canine tissue cell extracts was observed for both antibodies; however, the polyclonal antibody (cod. A1645) demonstrated higher signal specificity. Canine tumor histotypes were selected based on the human counterparts known to express PDL1. Immunohistochemistry was performed on 168 tumors with the polyclonal anti-PDL1 antibody. Only membranous labeling was considered positive. PDL1 labeling was detected both in neoplastic and infiltrating immune cells. The following tumors were immunopositive: melanomas (17 of 17; 100%), renal cell carcinomas (4 of 17; 24%), squamous cell carcinomas (3 of 17; 18%), lymphomas (2 of 14; 14%), urothelial carcinomas (2 of 18; 11%), pulmonary carcinomas (2 of 20; 10%), and mammary carcinomas (1 of 31; 3%). Gastric (0 of 10; 0%) and intestinal carcinomas (0 of 24; 0%) were negative. The findings of this study suggest that PDL1 is expressed in some canine tumors, with high prevalence in melanomas.

Bronchoalveolar Lavage Cytology in Severe Equine Asthma: Cytocentrifugated versus Sediment Smear Preparations.

Equine asthma is a common respiratory disease that may affect horses of any age. The diagnosis of severe equine asthma (SEA) (historically referred as recurrent airway obstruction or RAO) is based mainly on the history of the animal and clinical signs, which are further supported by the cytological examination of the bronchoalveolar lavage (BAL). This can also be helpful in monitoring the inflammation of the lower airways in response to environmental management and medication. The cytocentrifugated preparation is usually considered the method of choice for BAL cytological interpretation. The aim of this study was to compare the results in terms of differential cell counts (DCC) in BAL cytology performed on sedimented smears and cytocentrifugated preparations. To carry this out, 48 BAL samples were collected from six horses with SEA that were subjected to a process of exacerbation of the disease by environmental stimuli, which was later followed by the appropriate treatment. Each collected BAL fluid was equally divided into duplicate portions: one processed by cytocentrifugation and one by sediment smear from simple centrifugation. Cytologic examination of all BAL by both methods showed poor concordance in DCC, although it was still able to allow diagnostic recognition of severe lung neutrophilic disorders. These results suggest that sediment smear preparation, although remaining a useful method in general equine practice associated with clinical assessments in the diagnosis of SEA under conditions where there is no possibility of using a cytocentrifuge, cannot be considered a comparable alternative.

GATA1-defective immune-megakaryocytes as possible drivers of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disorder with limited therapeutic options. Insufficient understanding of driver mutations and poor fidelity of currently available animal models has limited the development of effective therapies. Since GATA1 deficient megakaryocytes sustain myelofibrosis, we hypothesized that they may also induce fibrosis in lungs. We discovered that lungs from IPF patients and Gata1low mice contain numerous GATA1negative immune-poised megakaryocytes that, in mice, have defective RNA-seq profiling and increased TGF-ß1, CXCL1 and P-selectin content. With age, Gata1low mice develop fibrosis in lungs. Development of lung fibrosis in this model is prevented by P-selectin deletion and rescued by P-selectin, TGF-ß1 or CXCL1 inhibition. Mechanistically, P-selectin inhibition decreases TGF-ß1 and CXCL1 content and increases GATA1positive megakaryocytes while TGF-ß1 or CXCL1 inhibition decreased CXCL1 only. In conclusion, Gata1low mice are a novel genetic-driven model for IPF and provide a link between abnormal immune-megakaryocytes and lung fibrosis.

CXCL8/CXCR2 signaling mediates bone marrow fibrosis and is a therapeutic target in myelofibrosis.

Proinflammatory signaling is a hallmark feature of human cancer, including in myeloproliferative neoplasms (MPNs), most notably myelofibrosis (MF). Dysregulated inflammatory signaling contributes to fibrotic progression in MF; however, the individual cytokine mediators elicited by malignant MPN cells to promote collagen-producing fibrosis and disease evolution are yet to be fully elucidated. Previously, we identified a critical role for combined constitutive JAK/STAT and aberrant NF-κB proinflammatory signaling in MF development. Using single-cell transcriptional and cytokine-secretion studies of primary cells from patients with MF and the human MPLW515L (hMPLW515L) murine model of MF, we extend our previous work and delineate the role of CXCL8/CXCR2 signaling in MF pathogenesis and bone marrow fibrosis progression. Hematopoietic stem/progenitor cells from patients with MF are enriched for a CXCL8/CXCR2 gene signature and display enhanced proliferation and fitness in response to an exogenous CXCL8 ligand in vitro. Genetic deletion of Cxcr2 in the hMPLW515L-adoptive transfer model abrogates fibrosis and extends overall survival, and pharmacologic inhibition of the CXCR1/2 pathway improves hematologic parameters, attenuates bone marrow fibrosis, and synergizes with JAK inhibitor therapy. Our mechanistic insights provide a rationale for therapeutic targeting of the CXCL8/CXCR2 pathway among patients with MF.

Preclinical studies on the use of a P-selectin-blocking monoclonal antibody to halt progression of myelofibrosis in the Gata1low mouse model.

The bone marrow (BM) and spleen from patients with myelofibrosis (MF), as well as those from the Gata1low mouse model of the disease contain increased number of abnormal megakaryocytes. These cells express high levels of the adhesion receptor P-selectin on their surface, which triggers a pathologic neutrophil emperipolesis, leading to increased bioavailability of transforming growth factor-ß (TGF-ß) in the microenvironment and disease progression. With age, Gata1low mice develop a phenotype similar to that of patients with MF, which is the most severe of the Philadelphia-negative myeloproliferative neoplasms. We previously demonstrated that Gata1low mice lacking the P-selectin gene do not develop MF. In the current study, we tested the hypothesis that pharmacologic inhibition of P-selectin may normalize the phenotype of Gata1low mice that have already developed MF. To test this hypothesis, we have investigated the phenotype expressed by aged Gata1low mice treated with the antimouse monoclonal antibody RB40.34, alone and also in combination with ruxolitinib. The results indicated that RB40.34 in combination with ruxolitinib normalizes the phenotype of Gata1low mice with limited toxicity by reducing fibrosis and the content of TGF-ß and CXCL1 (two drivers of fibrosis in this model) in the BM and spleen and by restoring hematopoiesis in the BM and the architecture of the spleen. In conclusion, we provide preclinical evidence that treatment with an antibody against P-selectin in combination with ruxolitinib may be more effective than ruxolitinib alone to treat MF in patients.

Diffuse Large B-Cell Lymphoma (DLBCL) in an Aged Raccoon (Procyon lotor).

A 15-years-old, captive, female raccoon (Procyon lotor) was necropsied after a one-week history of apathy and self-isolation. Gross changes consisted of the severe enlargement of the mesenteric lymph node; hepatosplenomegaly with multifocal to coalescing, white tan nodules in the spleen and liver,; and pale kidneys. Histologically, neoplastic CD79α-positive lymphocytes effaced the mesenteric lymph node and multifocally infiltrated the spleen, liver, and kidneys, and focally infiltrated the heart. Based on pathological and immunohistochemical findings, as well as the canine-adapted World Health Organization (WHO) diagnostic criteria, a diagnosis of diffuse large B-cell lymphoma (DLBCL) was made.

Cannabinoid receptors in the inflammatory cells of canine atopic dermatitis.

Background: Atopic dermatitis (AD) is one of the most common cutaneous inflammatory and pruritic diseases in dogs. Considering its multifactorial nature, AD can be a challenging disease to manage, and the therapeutic strategy must often be multimodal. In recent years, research has been moving toward the use of natural products which have beneficial effects on inflammation and itching, and no side effects. Cannabinoid receptors have been demonstrated to be expressed in healthy and diseased skin; therefore, one of the potential alternative therapeutic targets for investigating AD is the endocannabinoid system (ECS). Objective: To immunohistochemically investigate the expression of the cannabinoid receptor type 2 (CB2R), and the cannabinoid-related receptors G protein-coupled receptor 55 (GPR55), transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1) in mast cells (MCs), macrophages, dendritic cells (DCs), T cells, and neutrophils of the skin of dogs with AD. Animals: Samples of skin tissues were collected from eight dogs with AD (AD-dogs). Materials and methods: The immunofluorescent stained cryosections of the skins of 8 dogs with AD having antibodies against CB2R, GPR55, TRPV1, TRPA1 were semiquantitatively evaluated. The inflammatory cells were identified using antibodies against tryptase (mast cells), ionized calcium binding adaptor molecule 1 (IBA1) (macrophages/DCs), CD3 (T cells), and calprotectin (neutrophils). The proportions of MCs, macrophages/DCs, T cells, and neutrophils expressing CB2R, GPR55, TRPV1 and TRPA1 were evaluated. Results: The cells of the inflammatory infiltrate showed immunoreactivity (IR) for all or for some of the cannabinoid and cannabinoid-related receptors studied. In particular, MCs and macrophages/DCs showed CB2R-, GPR55-, TRPA1-, and TRPV1-IR; T cells showed CB2R-, GPR55- and TRPA1-IR, and neutrophils expressed GPR55-IR. Co-localization studies indicated that CB2R-IR was co-expressed with TRPV1-, TRPA1-, and GPR55-IR in different cellular elements of the dermis of the AD-dogs. Conclusions and clinical importance: Cannabinoid receptor 2, and cannabinoid-related receptors GPR55, TRPV1 and TRPA1 were widely expressed in the inflammatory infiltrate of the AD-dogs. Based on the present findings, the ECS could be considered to be a potential therapeutic target for dogs with AD, and may mitigate itch and inflammation.

Distribution of Cannabinoid Receptors in Keratinocytes of Healthy Dogs and Dogs With Atopic Dermatitis.

It is commonly accepted that some form of skin barrier dysfunction is present in canine atopic dermatitis (AD), one of the most common cutaneous pruritic inflammatory diseases of dogs. The impaired skin barrier function facilitates the penetration of allergens and subsequently stronger sensitization responses. The role of the endocannabinoid system (ECS) in the physiology and pathology of the skin is becoming increasingly established. It has been demonstrated that cannabinoid receptors are expressed in healthy and diseased skin and, based on current knowledge, it could be stated that cannabinoids are important mediators in the skin. The present study has been designed to immunohistochemically investigate the expression of the cannabinoid receptors type 1 (CB1R) and 2 (CB2R) and the cannabinoid-related receptors G protein-coupled receptor 55 (GPR55), transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1), peroxisome proliferator-activated receptors alpha (PPARα), and serotoninergic receptor 1a (5-HT1aR) in keratinocytes of healthy dogs and of dogs with AD. Samples of skin tissues were collected from 7 healthy controls (CTRL-dogs) and from 8 dogs with AD (AD-dogs). The tissue samples were processed using an immunofluorescence assay with commercially available antibodies, and the immunolabelling of the receptors studied was quantitatively evaluated. The keratinocytes of the CTRL- and the AD-dogs showed immunoreactivity for all the receptors investigated with a significant upregulation of CB2R, TRPA1, and 5-HT1aR in the epidermis of the AD-dogs. The presence of cannabinoid and cannabinoid-related receptors in healthy keratinocytes suggested the possible role of the ECS in canine epidermal homeostasis while their overexpression in the inflamed tissues of the AD-dogs suggested the involvement of the ECS in the pathogenesis of this disease, having a possible role in the related skin inflammation and itching. Based on the present findings, the ECS could be considered a potential therapeutic target for dogs with AD.

Mutational Analysis of c-KIT and PDGFRA in Canine Gastrointestinal Stromal Tumors (GISTs).

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the canine gastrointestinal tract and are diagnosed by the immunohistochemical expression of the receptor tyrosine kinase (RTK) KIT. Activating mutations of the proto-oncogenes c-KIT and PDGFRA drive GIST oncogenesis and are used to predict the response to RTK-inhibitors in human oncology. Currently, the frequency and significance of these mutations in canine GIST have not been adequately explored. Therefore, we investigated the mutational status of c-KIT (exons 9, 11 and 13) and PDGFRA (exons 12 and 18) genes by PCR followed by fragment analysis for c-KIT deletions and PCR followed by screening with DHPLC and direct sequencing confirmation for single nucleotide variations in 17 formalin-fixed paraffin-embedded canine GISTs confirmed by KIT immunopositivity. c-KIT mutations were detected in 47% of cases, with a mutation detection rate significantly higher (p = 0.0004, Fisher's exact test) and always involving exon 11. A PDGFRA gene mutation (exon 18) was identified in one case. Even if follow-up data were not available for all cases, four cases with documented abdominal metastases displayed c-KIT mutations. These data confirm that c-KIT exon 11 mutations occur frequently in canine GISTs, and identify the presence of a PDGFRA mutation similar to human GISTs. This study also suggests a potential association of c-KIT mutation with more aggressive biological behavior.

The CXCR1/CXCR2 Inhibitor Reparixin Alters the Development of Myelofibrosis in the Gata1 low Mice.

A major role for human (h)CXCL8 (interleukin-8) in the pathobiology of myelofibrosis (MF) has been suggested by observations indicating that MF megakaryocytes express increased levels of hCXCL8 and that plasma levels of this cytokine in MF patients are predictive of poor patient outcomes. Here, we demonstrate that, in addition to high levels of TGF-ß, the megakaryocytes from the bone marrow of the Gata1 low mouse model of myelofibrosis express high levels of murine (m)CXCL1, the murine equivalent of hCXCL8, and its receptors CXCR1 and CXCR2. Treatment with the CXCR1/R2 inhibitor, Reparixin in aged-matched Gata1 low mice demonstrated reductions in bone marrow and splenic fibrosis. Of note, the levels of fibrosis detected using two independent methods (Gomori and reticulin staining) were inversely correlated with plasma levels of Reparixin. Immunostaining of marrow sections indicated that the bone marrow from the Reparixin-treated group expressed lower levels of TGF-ß1 than those expressed by the bone marrow from vehicle-treated mice while the levels of mCXCL1, and expression of CXCR1 and CXCR2, were similar to that of vehicle-treated mice. Moreover, immunofluorescence analyses performed on bone marrow sections from Gata1 low mice indicated that treatment with Reparixin induced expression of GATA1 while reducing expression of collagen III in megakaryocytes. These data suggest that in Gata1low mice, Reparixin reduces fibrosis by reducing TGF-ß1 and collagen III expression while increasing GATA1 in megakaryocytes. Our results provide a preclinical rationale for further evaluation of this drug alone and in combination with current JAK inhibitor therapy for the treatment of patients with myelofibrosis.

In vivo imaging of mammary epithelial cell dynamics in response to lineage-biased Wnt/ß-catenin activation.

Real-time in vivo imaging provides an essential window into the spatiotemporal cellular events contributing to tissue development and pathology. By coupling longitudinal intravital imaging with genetic lineage tracing, here we capture the earliest cellular events arising in response to active Wnt/ß-catenin signaling and the ensuing impact on the organization and differentiation of the mammary epithelium. This enables us to interrogate how Wnt/ß-catenin regulates the dynamics of distinct subpopulations of mammary epithelial cells in vivo and in real time. We show that ß-catenin stabilization, when targeted to either the mammary luminal or basal epithelial lineage, leads to cellular rearrangements that precipitate the formation of hyperplastic lesions that undergo squamous transdifferentiation. These results enhance our understanding of the earliest stages of hyperplastic lesion formation in vivo and reveal that, in mammary neoplastic development, ß-catenin activation dictates a hair follicle/epidermal differentiation program independently of the targeted cell of origin.

Comparison of Trehalose/Hyaluronic Acid (HA) vs. 0.001% Hydrocortisone/HA Eyedrops on Signs and Inflammatory Markers in a Desiccating Model of Dry Eye Disease (DED).

BACKGROUND: Dry eye disease (DED) is a multifactorial disease where ocular surface inflammation and damage play key etiological roles. PURPOSE: To compare a combination of 3% trehalose (T) and 0.15% hyaluronic acid (HA) (Thealoz duo®, T/HA) with a tear substitute containing 0.001% hydrocortisone (I) and 0.2% HA (Idroflog®, I/HA), with respect to changes on signs and inflammatory markers in a mouse DED model. METHODS: Thirty 12-week-old C57BL/6 mice were exposed in a controlled-environment chamber as a desiccating stress model of DED for 35 days. At day 14 (T1), administration of 5 µL T or I in the right eye (RE) or NaCl 0.9% in the left eye (LE) started, twice a day. Animals were sacrificed after 7 (T2), 14 (T3), 21 (T4, endpoint) days from the beginning of treatment. Corneal fluorescein staining ratio (Image J), histological and histochemical assessment of ocular surface tissues (goblet cell GC density and characterization -PAS, Alcian blue pH 2.5, pH 1.0, and MUC4 expression-in the superior and inferior conjunctiva), and levels of inflammatory markers HLA-DR, IL-1ß and TNF-α in cornea and conjunctiva were measured. RESULTS: No animal fully recovered from DED signs at the endpoint. Difference between arms was observed at T3 and T4, with T treated eyes showing a higher corneal damage reduction, PAS-positive GC recovery, lower inflammatory marker expression as compared to the I treated ones. CONCLUSIONS: Data suggest that 21 days of treatment with T/HA improved signs, GC recovery and inflammatory markers in a DED mouse model, to a greater extent as compared to I/HA. Data suggest that 21 days of treatment with T/HA improved signs, GC recovery and inflammatory markers in a DED mouse model, to a greater extent as compared to I/HA.

Resident Self-Tissue of Proinflammatory Cytokines Rather Than Their Systemic Levels Correlates with Development of Myelofibrosis in Gata1low Mice.

Serum levels of inflammatory cytokines are currently investigated as prognosis markers in myelofibrosis, the most severe Philadelphia-negative myeloproliferative neoplasm. We tested this hypothesis in the Gata1low model of myelofibrosis. Gata1low mice, and age-matched wild-type littermates, were analyzed before and after disease onset. We assessed cytokine serum levels by Luminex-bead-assay and ELISA, frequency and cytokine content of stromal cells by flow cytometry, and immunohistochemistry and bone marrow (BM) localization of GFP-tagged hematopoietic stem cells (HSC) by confocal microscopy. Differences in serum levels of 32 inflammatory-cytokines between prefibrotic and fibrotic Gata1low mice and their wild-type littermates were modest. However, BM from fibrotic Gata1low mice contained higher levels of lipocalin-2, CXCL1, and TGF-ß1 than wild-type BM. Although frequencies of endothelial cells, mesenchymal cells, osteoblasts, and megakaryocytes were higher than normal in Gata1low BM, the cells which expressed these cytokines the most were malignant megakaryocytes. This increased bioavailability of proinflammatory cytokines was associated with altered HSC localization: Gata1low HSC were localized in the femur diaphysis in areas surrounded by microvessels, neo-bones, and megakaryocytes, while wild-type HSC were localized in the femur epiphysis around adipocytes. In conclusion, bioavailability of inflammatory cytokines in BM, rather than blood levels, possibly by reshaping the HSC niche, correlates with myelofibrosis in Gata1low mice.

Airway Remodeling in Feline Lungs.

Airway remodeling encompass structural changes that occur as the result of chronic injury and lead to persistently altered airway structure and function. Although this process is known in several human respiratory conditions such as asthma and chronic obstructive pulmonary disease (COPD), airway remodeling is poorly characterized in the feline counterpart. In this study, we describe the spontaneous pulmonary changes in 3 cats paralleling the airway remodeling reported in humans. We observed airway smooth muscle cells (ASMCs) hyperplasia (peribronchial and interstitial), airway subepithelial and interstitial fibrosis, and vascular remodeling by increased number of vessels in the bronchial submucosa. The hyperplastic ASMCs co-expressed α-SMA, vimentin and desmin suggesting that vimentin, which is not normally expressed by ASMCs, may play a role in airway thickening, and remodeling. ASMCs had strong cytoplasmic expression of TGFß-1, which is known to contribute to tissue remodeling in asthma and in various bronchial and interstitial lung diseases, suggesting its involvement in the pathogenesis of ASMCs hyperplasia. Our findings provide histologic evidence of airway remodeling in cats. Further studies on larger caseloads are needed to support our conclusions on the value of this feline condition as an animal model for nonspecific airway remodeling in humans.

The Role of Pathology in the Diagnosis of Swine Respiratory Disease.

The definition "porcine respiratory disease complex" (PRDC) is used to indicate the current approach for presenting respiratory pathology in modern pig farming. PRDC includes pneumonias with variable pictures, mixed with both aerogenous and hematogenous forms with variable etiology, often multimicrobial, and influenced by environmental and management factors. The notion that many etiological agents of swine respiratory pathology are ubiquitous in the airways is commonly understood; however, their isolation or identification is not always associable with the current pathology. In this complex context, lung lesions registered at slaughterhouse or during necropsy, and supplemented by histological investigations, must be considered as powerful tools for assigning a prominent role to etiologic agents. In recent years, the goal of colocalizing causative agents with the lesions they produce has been frequently applied, and valid examples in routine diagnostics are those that indicate pulmonary involvement during porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) infections.

A Double Histochemical/Immunohistochemical Staining for the Identification of Canine Mast Cells in Light Microscopy.

Immunohistochemistry (IHC) is a widely used technique in diagnostic pathology, but the simultaneous analysis of more than one antibody at a time with different chromogens is rather complex, time-consuming, and quite expensive. In order to facilitate the identification of mast cells (MCs) during immunohistochemical analysis of membrane and/or nuclear markers, we propose a new staining method that includes the association of IHC and toluidine blue as a counterstain. To achieve this goal, we tested c-kit, Ki67, and cannabinoid receptor 2 on several cases of cutaneous canine mast cell tumors (MCTs), cutaneous mastocytosis, and atopic dermatitis. The results obtained show how this double staining technique, although limited to non-cytoplasmic markers and of little use in poorly differentiated MCTs in which MC metachromasia is hard to see, can be used during the evaluation of nuclear and/or membranous immunohistochemical markers in all canine cutaneous disorders, especially if characterized by the presence of a low number of MCs. It can help to evaluate those MCTs in which neoplastic MCs must be clearly distinguished from inflammatory cells that can infiltrate the tumor itself, in facilitating the calculation of the Ki67 index. Moreover, it can be used to study the expression of new markers in both animal and human tissues containing MCs and in MC disorders.

High Intrinsic Expression of P-glycoprotein and Breast Cancer Resistance Protein in Canine Mammary Carcinomas Regardless of Immunophenotype and Outcome.

P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are major actors in multidrug resistance (MDR) phenomenon in both human and canine mammary carcinomas (CMCs). The aim of this study was to investigate an association between the intrinsic expression of P-gp and BCRP compared to the immunophenotypes and outcome in CMCs. Fifty CMCs were evaluated at immunohistochemistry (IHC) for P-gp, BCRP, Estrogen receptor alpha (ER), Progesterone receptors (PR), Human Epidermal Growth Factor Receptor type 2 (HER2), basal cytokeratins 5/6 (CK5/6), Epidermal Growth Factor Receptor 1 (EGFR), and Ki67 proliferation index. P-gp and BCRP positive cases were, respectively, 52% and 74.5%, with a significantly higher expression of BCRP than P-gp. Five immunophenotypes were defined in 37 out of 50 CMCs: 9 (24.3%) Luminal A, 5 (13.5%) Luminal B, 9 (24.3%) HER2 overexpressing, 9 (24.3%) Triple-negative basal-like, and 5 (13.5%) Triple-negative non-basal-like. In all CMCs at least one marker was expressed. Follow-up data were available for 25 animals. The average cancer-specific survival was 739 ± 444 days. A number of CMCs bear a high expression of P-gp and BCRP but no significant association was found between their expression and the immunophenotypes, Ki67 index, the histological grade, and tumor-related death.

Novel targets to cure primary myelofibrosis from studies on Gata1low mice.

In 2002, we discovered that mice carrying the hypomorphic Gata1low mutation that reduces expression of the transcription factor GATA1 in megakaryocytes (Gata1low mice) develop myelofibrosis, a phenotype that recapitulates the features of primary myelofibrosis (PMF), the most severe of the Philadelphia-negative myeloproliferative neoplasms (MPNs). At that time, this discovery had a great impact on the field because mutations driving the development of PMF had yet to be discovered. Later studies identified that PMF, as the others MPNs, is associated with mutations activating the thrombopoietin/JAK2 axis raising great hope that JAK inhibitors may be effective to treat the disease. Unfortunately, ruxolitinib, the JAK1/2 inhibitor approved by FDA and EMEA for PMF, ameliorates symptoms but does not improve the natural course of the disease, and the cure of PMF is still an unmet clinical need. Although GATA1 is not mutated in PMF, reduced GATA1 content in megakaryocytes as a consequence of ribosomal deficiency is a hallmark of myelofibrosis (both in humans and mouse models) and, in fact, a driving event in the disease. Conversely, mice carrying the hypomorphic Gata1low mutation express an activated TPO/JAK2 pathway and partially respond to JAK inhibitors in a fashion similar to PMF patients (reduction of spleen size but limited improvement of the natural history of the disease). These observations cross-validated Gata1low mice as a bona fide animal model for PMF and prompted the use of this model to identify abnormalities that might be targeted to cure the disease. We will summarize here data generated in Gata1low mice indicating that the TGF-ß/P-selectin axis is abnormal in PMF and represents a novel target for its treatment.