p62/Sequestosome-1: Mapping Sites of Protein-Handling Stress in Canine Cutaneous Mast Cell Tumors.

Canine cutaneous mast cell tumors (MCT) are common, frequently malignant neoplasms that are currently graded histologically for provision of prognostic information. Continuing evidence of subsets of MCT within certain grades (with differing survival times) indicate the need for biomarkers that will facilitate better patient stratification and also provide further information on the biological processes involved in progression. We decided to investigate the expression of p62/sequestosome-1 (p62/SQSTM1), a stress-inducible "hub protein" found in all cell types that shuttles rapidly between the nucleus and cytoplasm and is known to play important roles in protein handling and tumorigenesis. The identity of canine p62/SQSTM1 was confirmed in silico and by validation of a commercial antibody using both Western blotting and functional (pharmaceutical-based) analyses in cell culture. Using immunohistochemistry, 3 patterns of p62 expression were identified based on the predominant intracellular localization, that is, nuclear, mixed (nuclear and cytoplasmic), and cytoplasmic. There was a highly significant association with the 2-tier (Kiupel) grade (P < .0001), with all p62-nuclear immunoreactivity being associated with low grade and most p62-cytoplasmic immunoreactivity (93%) with high grade. Most but not all mixed nuclear-cytoplasmic labeling occurred in low-grade MCT; in other (human) tumor types, this pattern has been interpreted as borderline malignant. These data indicate that there is a shift in protein-handling stress from the nucleus to the cytoplasm in association with increasing malignancy in MCT. Studies to identify the processes and drug-able targets involved in this progression are ongoing.

Canine paediatric oncology: retrospective assessment of 9522 tumours in dogs up to 12 months (1993-2008).

Little information is available on the occurrence of neoplasms in dogs up to the age of 12 months. This is a retrospective review of histopathological diagnoses of neoplasia in dogs up to the age of 12 months based on biopsy specimens submitted to a commercial veterinary diagnostic laboratory in the United Kingdom between 1993 and 2008. In 20 280 histological submissions, 9522 neoplasms were identified. Canine cutaneous histiocytoma (n = 8465; 89%) was the most common histological type. Neoplasms other than histiocytoma (n = 1057; 11%) were grouped as benign epithelial (n = 375; 4%), haematopoietic (n = 229; 2%), benign mesenchymal (n = 145; 2%), miscellaneous (n = 118; 1%), non-hematopoietic malignant mesenchymal (n = 118; 1%) or malignant epithelial tumours (n = 72; <1%). Excluding canine cutaneous histiocytoma, 52% of tumours (n = 547) were benign, and 66% were from the skin or soft tissues. These data provide valuable epidemiological information on neoplasms occurring in juvenile dogs in the United Kingdom.

Feline paediatric oncology: retrospective assessment of 233 tumours from cats up to one year (1993 to 2008).

OBJECTIVES: To determine which types of tumour occur in cats up to the age of 12 months based on biopsies submitted to Idexx Laboratories, Wetherby, UK. METHODS: Retrospective review of histopathological diagnoses of tumours in cats up to the age of 12 months from biopsies received between September 1993 and March 2008. RESULTS: A total of 4196 submissions from cats 12 months old or younger were identified; 233 biopsies (6%) were neoplastic and fulfilled the search criteria. Tumours were categorised as haematopoietic (n=73, 31%), malignant epithelial (n=44; 19%), malignant mesenchymal (n=38; 16%), benign epithelial (n=37; 16%), benign mesenchymal (n=30, 13%) and miscellaneous (n=11; 5%). The most frequent tumours were lymphoma (n=51; 22%), soft-tissue sarcoma (n=34; 15%), mast cell tumour (n=22; 9%) and squamous cell carcinoma (n=16; 7%). The most common tumour site was the skin and soft tissues (41% of tumours). In all, 164 neoplasms (70%) were malignant or had malignant potential. CLINICAL SIGNIFICANCE: These data provide unique epidemiological information on a poorly characterised subgroup of feline cancer patients in the UK.

Ulcerative pododermatitis in a cat associated with Anatrichosoma sp.

A 9-year-old castrated male Chartreaux cat was presented for an ulcerative pododermatitis of all 4 paws. A clinical exam was inconclusive and supportive therapy did not improve the condition. Histologic examination revealed an ulcerative and eosinophilic dermatitis associated with epidermal and dermal nematodes and ova consistent with the aphasmid Anatrichosoma sp. Treatment with ivermectin completely resolved the skin lesions. Anatrichosomiasis should be included in the differential diagnosis of ulcerative pododermatitis in cats, at least in the southwestern United States.

Fusobacteriosis in captive wild-caught pronghorns (Antilocapra americana).

An outbreak of Fusobacterium necrophorum-induced septicemia occurred in a group of 40 captive wild-caught pronghorns (Antilocapra americana). Primary pododermatitis or necrotic stomatitis progressed to produce fatal septicemia with metastatic lesions in the forestomachs, lung, liver, and cecum in 38 of the animals. Two remaining animals were euthanatized because of chronic pododermatitis. Housing the animals in a pasture previously used by bovids and heavy rains with persistence of ground water pools in the pasture were contributing factors in the pathogenesis of this outbreak.

Elevated parathyroid hormone-related protein and hypercalcemia in two dogs with schistosomiasis.

Two adult dogs were evaluated for hypercalcemia. Diagnostic evaluation identified elevated parathyroid hormone-related protein (PTHrP) and presumptive humoral hypercalcemia of malignancy. At necropsy, schistosomiasis was diagnosed. North American schistosomiasis is caused by Heterobilharzia americana. Clinical findings may include dermatitis, coughing, diarrhea, and anorexia. Clinicopathological findings may include hypercalcemia, hyperglobulinemia, hypoalbuminemia, anemia, and eosinophilia. Diagnosis by fecal examination is difficult. Praziquantel or fenbendazole treatment may be curative or palliative. These are the first reported cases of hypercalcemia with elevated PTHrP in animals without diagnosed malignancy. Elevation of PTHrP has not been previously reported in hypercalcemic humans or in animals with granulomatous inflammation.

Small intestine large granular lymphoma in a horse.

A 12-year-old Appaloosa gelding was referred to the Texas Veterinary Medical Center with a history of chronic diarrhea and weight loss. At necropsy, numerous oval, craterlike ulcers were observed throughout the small intestine. Histologically, these lesions were composed of a neoplastic proliferation of round cells with intracytoplasmic phosphotungstic acid-hematoxylin-positive granules. The tumor cells stained positively for the CD3 antigen and negatively for a B-cell marker. A diagnosis of large granular lymphoma was based on the morphologic and immunohistochemical characteristics of the neoplasm. The postmortem presentation of this case depicted unusual multifocal, ulcerative lymphomatous lesions throughout the small intestine without involvement of the regional lymph nodes. The histologic and ultrastructural morphology of the neoplastic lymphocytes was similar to that in previously reported cases of abdominal equine large granular lymphomas, but in this case the neoplasm was restricted to the small intestine.

Synergistic interactions of antibodies in rate of virus neutralization.

Antibodies and antibody combinations are often evaluated only by their potency in inactivating a known quantity of virus in dose-effect assays. However, a crucial additional parameter is the rate at which neutralization takes place, or kinetics. Synergism of certain antibody combinations in dose-effect assays has been previously demonstrated. In the present report, using a battery of murine monoclonal antibodies to herpes simplex virus (HSV), we investigated whether antiviral antibodies can also synergize in neutralization kinetics. To determine whether synergism in dose-effect assays can predict synergism in neutralization rate, the ability of neutralizing antibodies to synergize in neutralization rate (kinetics) was compared to their ability to synergize in dose-effect assays (potency) in cell-free assays. Although certain antibody combinations synergized in both neutralization rate and potency, combinations that did not clearly synergize in potency could still significantly synergize in neutralization rate. Weak neutralizing antibodies could also greatly increase the neutralization rate of more potent antibodies. These results suggest that evaluating antibody combinations in dose-effect assays but not in neutralization kinetics provides a partial picture of neutralizing antibody dynamic interactions and may prevent the identification of certain favorable antibody combinations. These findings also support the importance of establishing defined antibody cocktails for prophylactic and therapeutic purposes. A simple strategy to evaluate antibody interactions in neutralization kinetics is proposed in which a quantitative prediction of additivity is made on the basis of the neutralization rate constants of the individual antibodies in the combination.

Macrophage tropism of rabbit hemorrhagic disease virus is associated with vascular pathology.

To delineate the interactions between rabbit hemorrhagic disease virus (RHDV) and host cells, organ and cellular targets of infection were identified in vivo. Viral specific antigens were detected by immunohistochemistry in liver, lung, spleen and lymph nodes cells. Also, intravascular infected cells were detected in most organs including kidneys, myocardium, thymus and central nervous system. To further characterize infected target cells, viral proteins and cell-specific surface antigens were identified simultaneously in double labeling experiments. Numerous lymphoid organ macrophages, from the splenic red pulp, circulating monocytes, alveolar macrophages and Kupffer cells were double labeled, demonstrating that cells of the mononuclear phagocyte lineage are major hosts for RHDV. Double labeling for other specific cell markers were negative. The distribution of viral antigens in these tissues coincided with those areas where cells presented morphology of apoptosis. Association of intravascular monocyte infection and apoptosis, could represent a possible mechanism to develop disseminated intravascular coagulation.

Characterization of a type-common human recombinant monoclonal antibody to herpes simplex virus with high therapeutic potential.

We report the characterization of a type-common human recombinant monoclonal antibody previously isolated by antigen selection from a phage-displayed combinatorial antibody library established from a herpes simplex virus (HSV)-seropositive individual. Competition with well-characterized murine monoclonal antibodies and immunodetection of gD truncations revealed that this antibody recognizes the group Ib antigenic site of glycoprotein D, a highly conserved and protective type-common determinant. To our knowledge, this is the first human group Ib monoclonal antibody ever described. The antibody also displayed first-order neutralization kinetics and a high neutralization rate constant, was capable of completely inhibiting syncytium formation by a fusogenic strain of HSV type 1, and efficiently neutralized low-passage clinical isolates of both HSV serotypes. Taken together with our earlier observations of the in vivo antiviral activities of this human recombinant antibody in animal models of HSV infection, the present results support the high therapeutic potential of this antibody.

Modulation of immune cell populations and activation markers in the pathogenesis of African swine fever virus infection.

African swine fever (ASF) virus induces immune cell alterations that may be detected by changes in peripheral blood cells phenotypic antigens and activation markers which were examined by flow cytometry, analyzing both cell proportion and/or expression intensity of superficial antigens. These studies were conducted in pigs with experimental acute of chronic ASF infection to determine whether changes among important surface activation markers and phenotypic antigens, and their correlative lymph node status, reflected similar or disparate aspects of immune pathology. In acute infection produced by virulent viruses, macrophage and B lymphocyte populations decreased in peripheral blood after a short activation period at the beginning of the infection. A significative decrease of interleukin 2 receptor (IL 2R) expression was also observed in those pigs. These variations correlated with lymph node cell depletion due to an intense lymphoid cell death by apoptosis, affecting mainly the B lymphocyte subpopulation as determined by immunohistochemistry. Nevertheless, pigs infected with an attenuated isolate undergoing chronic persistent infection, presented a distinct pattern of modification, according with a different clinicopathological evolution. Changes consisted in systemic immune activation coincident with the highest viremia titer, with an augmentation in CD8+ T lymphocyte, macrophage, and B cell populations, and MHC (major histocompatibility complex) antigens. Percentage elevation of circulating immune subpopulations was accompanied by cell accumulation with lymphoid hyperplasia but a conserved distribution of B lymphocytes in lymphoid organs of chronically infected pigs.

Apoptosis: a mechanism of cell killing and lymphoid organ impairment during acute African swine fever virus infection.

Induction of programmed cell death has been described during infection with many different viruses. We have investigated the influence of African swine fever virus (ASFV) on apoptosis of different cell populations during in vitro and in vivo infection. We observed apoptosis in ASFV-infected monocyte/macrophage and peripheral blood mononuclear cell cultures. Apoptosis was demonstrated in these cells by DNA fragmentation, DNA staining and DNA-associated histone fraction detection assays. Flow cytometry analysis of infected cultures also showed morphological and functional alterations, including changes in the cell cycle and percentage of cell fractions stained with propidium iodide. After in vivo infection with three different virulent strains of ASFV, apoptosis of infected cells from the mononuclear phagocytic system and closely related elements from different tissues was observed. Additionally, infected pigs showed an intense degree of apoptosis of lymphocytes, which are not infected by the virus. In lymph nodes and other lymphoid organs, broad bands of apoptotic cells presented typical nuclear changes under light microscopy. The occurrence of DNA fragmentation was confirmed in these tissues using terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling. These findings, together with the pathological observations in infected pigs of a depletion in cell populations in lymphoid organs, suggest that virus interference with programmed cell death plays a central role in pathogenesis of this disease, being responsible for lymphoid organ impairment in acute ASFV infection.

Neutralizing antibodies to different proteins of African swine fever virus inhibit both virus attachment and internalization.

African swine fever virus induces in convalescent pigs antibodies that neutralized the virus before and after binding to susceptible cells, inhibiting both virus attachment and internalization. A further analysis of the neutralization mechanisms mediated by the different viral proteins showed that antibodies to proteins p72 and p54 are involved in the inhibition of a first step of the replication cycle related to virus attachment, while antibodies to protein p30 are implicated in the inhibition of virus internalization.

Application of a monoclonal antibody recognizing protein p30 to detect African swine fever virus-infected cells in peripheral blood.

Monoclonal antibody (MAb) 174F11.8 recognizes an epitope of the African swine fever (ASF) virus-induced protein, p30, a protein expressed on the plasma membrane of infected cells. This MAb has been used to analyze infected cell populations in peripheral blood of experimentally inoculated pigs with a virulent or attenuated ASF virus. Flow cytometric analysis of peripheral blood at different days postinfection using this MAb, showed expression of p30 mainly in the monocyte/macrophage cell lineage. Additionally, a small percentage of granulocytes also expressed p30 during infection. This methodology allowed the quantification of fluctuations in the pool of infected monocyte/macrophage cells in the inoculated pigs, maximum percentages ranging between 6 and 31%. Significant differences in the percentages of cell populations expressing p30 were not found between virulent or attenuated virus infection. However, a 2- to 4-day delay in the maximum percentage of cells expressing p30 was observed during infection with the attenuated virus when compared to virulent virus infection. Percentages of infected cells detected by the expression of p30 and viral titres obtained in peripheral blood showed positive correlation. Consequently, MAb 174F11.8 constitutes a marker to follow evolution of ASF virus infection, allowing quantification of percentages of infected cells in peripheral blood.