Circulating Cell-Free DNA in Dogs with Mammary Tumors: Short and Long Fragments and Integrity Index

Circulating cell-free DNA (cfDNA) has been considered an interesting diagnostic/prognostic plasma biomarker in tumor-bearing subjects. In cancer patients, cfDNA can hypothetically derive from tumor necrosis/apoptosis, lysed circulating cells, and some yet unrevealed mechanisms of active release. This study aimed to preliminarily analyze cfDNA in dogs with canine mammary tumors (CMTs). Forty-four neoplastic, 17 non-neoplastic disease-bearing, and 15 healthy dogs were recruited. Necrosis and apoptosis were also assessed as potential source of cfDNA on 78 CMTs diagnosed from the 44 dogs. The cfDNA fragments and integrity index significantly differentiated neoplastic versus non-neoplastic dogs (P<0.05), and allowed the distinction between benign and malignant lesions (P<0.05). Even if without statistical significance, the amount of cfDNA was also affected by tumor necrosis and correlated with tumor size and apoptotic markers expression. A significant (P<0.01) increase of Bcl-2 in malignant tumors was observed, and in metastatic CMTs the evasion of apoptosis was also suggested. This study, therefore, provides evidence that cfDNA could be a diagnostic marker in dogs carrying mammary nodules suggesting that its potential application in early diagnostic procedures should be further investigated

HISTOPATHOLOGICAL AND IMMUNOHISTOCHEMICAL STUDY OF EXOCRINE AND ENDOCRINE PANCREATIC LESIONS IN AVIAN INFLUENZA A EXPERIMENTALLY-INFECTED TURKEYS

In order to investigate the lesions caused by two (H7N1, H7N3) low-pathogenicity avian influenza (LPAI) viruses isolated in turkeys during epidemics in Italy, 28 turkeys experimentally-infected by oro-nasal route and 2 negative controls were submitted to histopathological analysis. Hematobiochemical tests were also performed. Animals euthanized 4 days post-infection (DPI) revealed a moderate multifocal acute necrotizing pancreatitis (3/8). In animals euthanized 7-10 DPI major findings were a moderate-to-severe multifocal-to-diffuse chronic sclerosing pancreatitis and ductular hyperplasia (9/12). In turkeys euthanized 17-39 DPI (8/8) a multifocal moderate-to-severe lymphocytic fibrosing pancreatitis was observed. No lesions were detected in controls. Double-immunolabeled immunohistochemistry for glucagon and avian influenza virus revealed an occasional viral positivity of exocrine pancreas in turkeys euthanized 4-7 DPI whilst the glucagon presence was detected throughout the post-infection course. Immunohistochemistry for insulin revealed positivity in all sections taken from day 4 to 39 post-infection. Insulin-producing cells were more widely distributed than glucagon+ cells. Three out of six turkeys euthanized at 39 DPI were mildly hyperglycemic at 14 DPI and lowered blood glucose levels from 18 to 39 DPI; 4/6 turkeys at 7 DPI showed a marked increase of lipase level that lowered at 14 DPI. One of these 4 animals was also hyperglycemic and showed unusual and marked glucagon/insulin positivity within pancreatic interstitium. qRT-PCR confirmed the viral presence in pancreas in 2/8 animals euthanized at 4 DPI, in 5/5 turkeys euthanized at 7 DPI and in 1/6 turkeys euthanized at 9 or 10 DPI. In conclusion, we describe the post infection progression of specific pancreatic morphologic features and of endocrine cell expression, in turkeys experimentally infected with Italian LPAI viruses

Histopathological and immunohistochemical study of exocrine and endocrine pancreatic lesions in avian influenza A experimentally-infected turkeys showing evidence of pancreatic regeneration

In order to investigate the pancreatic lesions caused by the infection with either H7N1 or H7N3 low-pathogenicity avian influenza viruses, 28 experimentally infected turkeys were submitted for histopathology, immunohistochemistry, haematobiochemistry and real-time reverse transcriptase polymerase chain reaction after different days post-infection (DPI). The localization of viral antigen and the measurement of insulin and glucagon expression in the pancreas were assessed to verify the progression from pancreatitis to metabolic disorders, such as diabetes. At the early infection phase (4\u20137 DPI), a severe acute necrotizing pancreatitis was recognized. During the intermediate phase (8\u201317 DPI), a mixed acute/chronic change associated with regenerative ductular proliferation was observed. A loss of pancreatic islets was detected in most severe cases and viral antigen was found in the pancreas of 11/28 turkeys (4\u201310 DPI) with the most severe histological damage. In turkeys euthanized at 39 DPI (late phase), a chronic fibrosing pancreatitis was observed with the reestablishment of both the exocrine and the endocrine pancreas. Insulin and glucagon expression manifested a progressive decrease with subsequent ductular positivity. Haematobiochemistry revealed increased lipasemia in the first week post-infection and hyperglycaemia in the second, with a progressive normalization within 21 DPI. This study allowed the identification of progressive virus-associated exocrine and endocrine pancreatic damage, suggesting that influenza virus might be responsible for metabolic derangements. Moreover, it highlighted a remarkable post-damage hyperplastic and reparative process from a presumptive common exocrine/endocrine precursor. This potential regeneration deserves further investigation for its relevance in a therapeutic perspective to replace lost and non-functional cells in diabetes mellitus

Signalment and morphological features of the canine mammary tumors included in the study.

<p>Signalment and morphological features of the canine mammary tumors included in the study.</p

Circulating cell-free (cf) DNA concentrations in healthy dogs and dogs with neoplastic and non-neoplastic diseases (diseased).

<p>(A) Scatterplot of cfDNA short and long fragments. (B) Box and Whiskers of cfDNA integrity index (long/short fragments) in healthy dogs and dogs with neoplastic and non-neoplastic diseases (diseased). (C) Box and Whiskers of cfDNA integrity index in neoplastic dogs with benign or malignant tumors and in healthy dogs. Error bars represent standard deviation. *P<0.05; ** P<0.01.</p

Immunohistochemistry results (mean ± standard deviation) of Bcl-2, Bax, and Bad expression in luminal epithelial cells of canine mammary tumors.

<p>Immunohistochemistry results (mean ± standard deviation) of Bcl-2, Bax, and Bad expression in luminal epithelial cells of canine mammary tumors.</p