Maria C. Peleteiro

Mitochondrial genetic diversity, selection and recombination in a canine transmissible cancer

Canine transmissible venereal tumour (CTVT) is a clonally transmissible cancer that originated approximately 11,000 years ago and affects dogs worldwide. Despite the clonal origin of the CTVT nuclear genome, CTVT mitochondrial genomes (mtDNAs) have been acquired by periodic capture from transient hosts. We sequenced 449 complete mtDNAs from a global population of CTVTs, and show that mtDNA horizontal transfer has occurred at least five times, delineating five tumour clades whose distributions track two millennia of dog global migration. Negative selection has operated to prevent accumulation of deleterious mutations in captured mtDNA, and recombination has caused occasional mtDNA re-assortment. These findings implicate functional mtDNA as a driver of CTVT global metastatic spread, further highlighting the important role of mtDNA in cancer evolution. DOI: http://dx.doi.org/10.7554/eLife.14552.001

Fatal cases of Theileria annulata infection in calves in Portugal associated with neoplastic-like lymphoid cell proliferation

This study was carried out to investigate fifteen cases of acute lethal infection of calves (≤ 4 months of age) by the protozoan parasite Theileria (T.) annulata in the south of Portugal. Calves developed multifocal to coalescent nodular skin lesions, similar to multicentric malignant lymphoma. Infestation with ticks (genus Hyalomma) was intense. Theileria was seen in blood and lymph node smears, and T. annulata infection was confirmed by isolation of schizont-transformed cells and sequencing of hypervariable region 4 of the 18S rRNA gene. At necropsy, hemorrhagic nodules or nodules with a hemorrhagic halo were seen, particularly in the skin, subcutaneous tissue, skeletal and cardiac muscles, pharynx, trachea and intestinal serosa. Histologically, nodules were formed by large, round, lymphoblastoid neoplastic-like cells. Immunohistochemistry (IHC) identified these cells as mostly CD3 positive T lymphocytes and MAC387 positive macrophages. A marker for B lymphocytes (CD79αcy) labeled very few cells. T. annulata infected cells in these nodules were also identified by IHC through the use of two monoclonal antibodies (1C7 and 1C12) which are diagnostic for the parasite. It was concluded that the pathological changes observed in the different organs and tissues were caused by proliferation of schizont-infected macrophages, which subsequently stimulate a severe uncontrolled proliferation of uninfected T lymphocytes.

Allergic conjunctivitis and conjunctival provocation tests in atopic dogs

INTRODUCTION Canine atopic dermatitis (cAD) is a very common disease, but little is known about eye involvement. The conjunctival provocation test (CPT) is used in human to study the ocular response to allergenic stimuli and to evaluate anti-allergic therapy. To our knowledge it has not been used in dogs. OBJECTIVES To evaluate the prevalence of ocular signs in a population of atopic dogs and relate these with clinical cAD scores; and the usefulness of CPT for dust mites in atopic dogs with itchy eyes. PROCEDURES Sixty cAD patients were evaluated for (i) ocular signs of allergic conjunctivitis including conjunctival hyperemia, chemosis, epiphora, ocular discharge, pruritus and corneal involvement, graded 0 to 3 according to severity, and (2) cAD Extent and Severity Index (CADESI-03). Additionally, CPTs for Dermatophagoides farinae (n = 12) and Dermatophagoides pteronyssinus (n = 12) were performed in sensitized atopic dogs and 24 control dogs. RESULTS Periocular and ocular signs of allergy were present in 60% (36/60) of cases. Conjunctival hyperemia (90%) was the most common sign. Severity of ocular signs was significantly correlated with eye pruritus (r(s)  = 0.690, P = <0.001) and skin lesions score for head region (r(s) = 0.261, P = 0.04). A highly significant difference (P < 0.001, Fisher test) was found in CPTs between the test and the control groups. CONCLUSION  Allergic conjunctivitis signs associated with cAD seem under valuated so these patients would benefit from an ophthalmologic evaluation. Furthermore, we found CPT to be a reliable, easy to perform and safe test for the etiologic diagnosis of allergic conjunctivitis in the dog.

Influence of temperature and humidity manipulation on chicken embryonic development

BackgroundTemperature and relative humidity (RH) are very important factors affecting embryo development, hatchability, and posthatch performance. This study aimed at characterizing embryonic metabolic and behavioural response to a harsh incubation environment generated by manipulations (elevations and drops) in these two key factors. This study was aimed at establishing patterns of metabolic and behavioural response, as well as mortality and the development of malformations, all of which can potentially be used in monitoring incubating operations and diagnosing problems with faulty equipment.ResultsOf all the parameters monitored throughout embryonic development the ones shown to be most affected were: albumen-weight to egg-weight ratio (AR); yolk-weight to egg-weight ratio (YR); embryo-weight to egg-weight ratio (ER); heart rate (HR); voluntary movements per minute (VMM); mortality rates; malformation prevalence and type.The most significant changes in the evolution of AR and YR throughout incubation involved delay and reduction in the amplitude of the expected drop in albumen and yolk levels, reflecting lower nutrient consumption by the embryo. ER tended to grow more slowly and remain lower than the established normal, especially in embryos challenged with temperature treatments. HR and VMM were considered to be strong indicators of embryonic stress, as all treatments applied resulted in elevated heart rate and decreased embryo movement.Mortality rates for both temperature-related treatments were higher during the first four days of incubation. Changes in relative humidity have produced less radical effects on mortality. Malformation rates were higher for embryos subjected to high incubation temperatures and were most prominently related to the abdominal wall, head, skull and limbs.Overall, manipulations in environmental (incubator) temperature during incubation produced more drastic changes in embryo development than humidity-related manipulations, especially where mortality and malformation rates were concerned.ConclusionsThis paper describes changes in embryonic metabolism and behaviour, as well as in mortality and malformation rates, in response to manipulations in environmental temperature and relative humidity. Together with further studies, these patterns may prove helpful in the diagnosis of equipment malfunctions relating to temperature or relative humidity.

Immunohistochemical Evaluation of Vascular Urinary Bladder Tumors from Cows with Enzootic Hematuria

Fifty-six endothelial-derived urinary bladder tumor samples collected from 26 animals with bovine enzootic hematuria were selected for immunohistochemical studies. Expression of factor VHI-related antigen (FVIIIra), CD31, muscle-specific actin, uroplakin III (UPIII), and the cell cycle-related proteins cyclin Dl and p53 was evaluated in hemangiomas, “hemangioendotheliomas” (a vascular tumor that histologically is intermediate in appearance between a hemangioma and a conventional hemangiosarcoma), and hemangiosarcomas. Although CD31 expression was seen in all endothelial tumors tested, FVIIIra was not expressed in poorly differentiated endothelial tumor cells from solid areas or in 7 muscle-invasive hemangiosarcomas. Cyclin Dl overexpression was seen in 53% of hemangiomas, 82% of hemangioendotheliomas, and 95% of hemangiosarcomas. P53 immunoreac-tivity was only seen in muscle-invasive hemangiosarcomas. The UPIII staining pattern, normally very intense on the apical aspect and cytoplasm of superficial urothelial cells, was altered in the urothelium in an estimated 25% of hemangiomas, most hemangioendotheliomas, and most hemangiosarcomas. In conclusion, CD31 is a better marker than FVIIIra in the characterization of bovine endothelial tumors. The cell cycle regulatory pathways involving cyclin Dl and p53 seem to be impaired in endothelial urinary bladder tumors, p53 immunoreactivity positively correlating with enhanced invasion.

Notch signaling dynamics in the adult healthy prostate and in prostatic tumor development.

The Notch signaling pathway has been implicated in prostate development, maintenance and tumorigenesis by its key role in cell‐fate determination, differentiation and proliferation. Therefore, we proposed to analyze Notch family members transcription and expression, including ligands (Dll1, 3, 4 and Jagged1 and 2), receptors (Notch1–4) and effectors (Hes1, 2, 5 and Hey1, 2, L), in both normal and tumor bearing mouse prostates to better understand the dynamics of Notch signaling in prostate tumorigenesis.

Detection and quantification of bovine papillomavirus type 2 in urinary bladders and lymph nodes in cases of Bovine Enzootic Hematuria from the endemic region of Azores.

Bovine Enzootic Hematuria (BEH) is a disease with a severe impact on production indexes and characterized by the development of bovine urinary bladder tumors, particularly in the Azores archipelago. The purpose of this study was to investigate and quantify BPV2 tissue distribution in bovine urinary bladder tumors, normal bladders, and iliac lymph nodes of cattle from the Azores. A real-time PCR system targeting the L1 gene was developed and allowed for the specific detection of the virus. BPV2 DNA was detected in a large proportion of the samples tested, both from neoplastic and healthy tissues, indicating that this virus is very prevalent in the bovine population of the Azores. Moreover, all types of tissues tested were positive, confirming a wide viral distribution within the infected animal. Bovine cutaneous papillomas sampled from Portuguese mainland dairy cattle were used as controls. Viral load ranged between 2.2×10(4) copies/cell in the skin papillomas, and 0.0002 copies/cell in the urinary bladders tumors from the Azores. This is the first report presenting quantitative data on BPV2 infection in bovine urinary bladder lesions from the Azores. This approach will provide a useful tool to evaluate the role of BPV2 not only in the pathogenesis BEH but also in cell transformation mechanisms.

p63 and E-cadherin Expression in Canine Oral Squamous Cell Carcinoma:

The expression of p63 and E-cadherin was studied in 22 oral squamous cell carcinomas in the dog according to immunohistochemical techniques. The association between these markers and clinicopathologic parameters was assessed. All tumor cells studied showed enhanced p63 expression. Regarding E-cadherin expression, 17 of 22 cases (77.3%) showed decreased immunoreactivity, and in 13 of 22 cases (59.1%), its expression was cytoplasmic. Neither p63 nor E-cadherin expression patterns were associated with tumor size, bone invasion, or lymph node metastasis. p63 score was related to proliferating cell nuclear antigen proliferative index (P = .020). A statistically significant correlation between the expression patterns of these 2 markers was noted (P = .026). Furthermore, they were related with tumor grade. An atypical p63 labeling and a cytoplasmic E-cadherin staining were statistically related with a higher tumor grade (P = .022 and P = .017, respectively). These findings suggest that changes in p63 and E-cadherin expression are frequent events in oral squamous cell carcinoma in dogs.

PCNA and grade in 13 canine oral squamous cell carcinomas: association with prognosis.

This study evaluated the prognosis factors of age, tumour size, anatomic location, histological grade and proliferating cell nuclear antigen (PCNA) expression in 13 dogs with oral squamous cell carcinoma (OSCC) with bone invasion and without signs of lymph node or distant metastasis. All animals were treated with radical excision performed with at least 1 cm margin, based on computed tomography images. In the 2-year follow-up, median disease-free survival was 138 days for dogs with grade 3 tumours and was not reached for those with grade 2 tumours. Grade 3 tumours and PCNA labelling index ≥65% were related with a shorter disease-free survival time and consequently poor prognosis (p = 0.003 and p = 0.034, respectively). Mean PCNA labelling index was significantly higher in recurrent cases (p = 0.011). Histological grade and PCNA expression may be important prognosis factors in canine OSCC.

Ki-67 as a Prognostic Factor in Feline Mammary Carcinoma: What Is the Optimal Cutoff Value?

Ki-67 is a nuclear protein and a proliferation marker frequently used in establishing the prognosis for breast cancer patients. To investigate the prognostic value of the Ki-67 proliferation index in female cats with mammary carcinoma, a prospective study was conducted with 96 animals. The Ki-67 index of primary tumors (n = 96) was initially determined, and whenever possible, the Ki-67 index of regional lymph node metastasis (n = 38) and distant metastasis (n = 16) was also estimated. The optimal cutoff value for the Ki-67 index was determined by univariate and multivariate analysis. Ki-67 indices ≥14% were detected in 72.9% (70 of 96) of the tumors. Tumors with a Ki-67 index ≥14% were significantly associated with large size (P = .022), poor differentiation (P = .009), presence of necrotic areas (P = .008), estrogen receptor-negative status (P < .0001), fHER2-negative status (P = .003), and shorter overall survival (P = .012). Moreover, Ki-67 expression in the primary tumor was strongly and positively correlated with both regional metastasis (P < .0001; r = 0.83) and distant metastasis (P < .0001; r = 0.83), and was significantly higher in distant metastases when compared with the primary tumor (P = .0009). A similar correlation was also observed between regional and distant metastasis (P < .0001; r = 0.75). On the basis of the above results, the authors propose the adoption of the 14% value as the optimal cutoff for Ki-67 to identify tumors with high risk of disease progression.