Giancarlo Avallone

Extra hepatic expression of the acute phase protein alpha 1-acid glycoprotein in normal bovine tissues.

Combined quantitative (real-time) polymerase chain reaction and immunohistochemistry were used to evaluate the expression of the minor acute phase protein alpha 1-acid glycoprotein (AGP, orosomucoid) in bovine extrahepatic tissues. AGP was produced mainly in the salivary glands and spleen, whereas minor expression was detected in all other tissues sampled, including lung, lymph nodes, uterus, ovary, kidney and tongue. The findings were consistent with immunohistochemical results. In view of the immunomodulatory and direct antibacterial activity of AGP, its expression in the salivary glands may signal an involvement in the regulation of the local immunity, even in non-pathological conditions.

Lipopolysaccharide-binding protein: Local expression in bovine extrahepatic tissues

Lipopolysaccharide-binding protein (LBP) is an acute phase protein involved in host response to Gram-negative and Gram-positive pathogens. It is synthesized by hepatocytes and released as 60-65kDa glycoprotein in plasma. Little is known about the distribution of LBP in non-pathological bovine tissues. The aim of the present study was to investigate the extra hepatic expression of LBP in different bovine tissues by qualitative and quantitative real time (RT) PCR. The presence of the protein was also confirmed by immunohistochemistry using an anti-human LBP antibody preliminarily validated by cross-reactivity in bovine tissues. While a wide panel of organs and tissues was investigated, the attention was focused on the digestive tract and mammary gland. Moderate amount of mRNA was detected in most of the tissues involved in this study. Extra hepatic LBP mRNA expression was particularly high in parotid and submandibular salivary glands. Remarkably, LBP mRNA was found in rumen, reticulum and omasum. High expression was also found in the mammary gland. Intensity of protein staining paralleled mRNA expression in most tissues, with the exception of lung, ovary and thyroid gland. The presence of LBP throughout epithelial mucosal tissues is indicative of an important role of LBP in mucosal immunity at sites of bacterial exposure. These results suggest that ruminant forestomachs may mount a local acute phase reaction.

Differential expression and secretion of alpha1-acid glycoprotein in bovine milk.

alpha1-Acid glycoprotein (AGP) is a lipocalin that is produced mainly by the liver and secreted into plasma in response to infections and injuries. In this study, we evaluated AGP isoforms that can be detected in bovine milk. We found that milk-AGP content is made up of at least two isoform groups, a low MW group (44 kDa) that is produced in the mammary gland (MG-AGP), and a higher MW group (55-70 kDa), that is produced by somatic cells (SC-AGP). Identical SC-AGP isoforms can be found both in milk and blood PMN cells. Analysis of the mammary tissue cDNA showed that the sequence of the MG-AGP isoform is identical to that of plasma AGP. Each group contains several proteins with different MWs and different isoelectric points, as shown by 2D-electrophoresis. The glycosylation patterns of these isoforms were analysed by means of specific lectin binding, to evaluate the degree of sialylation, fucosylation and branching. The MG-AGP glycan pattern was identical to plasma AGP produced by the liver. Several differences were detected, however, between plasma and SC-AGP isoforms, the most evident being the strong degree of fucosylation and the elevated number of di-antennary glycans in SC-AGP. Immunohistochemistry showed that AGP is found in all tissues that make up the mammary gland, but that it is most likely produced for the main part by the alveoli.

Splenic marginal zone lymphoma in 5 dogs (2001-2008).

BACKGROUNDnSplenic marginal zone lymphomas (MZL) in dogs arise from the marginal zone of B-cell follicles and can progress slowly.nnnOBJECTIVESnTo describe clinical features, treatment, and outcome of dogs with splenic MZL.nnnANIMALSnFive dogs with naturally occurring MZL.nnnMETHODSnClinical, laboratory, and follow-up data were retrospectively reviewed. Diagnosis was based on clinical, histopathological, and immunophenotypic features.nnnRESULTSnAll dogs had stage IV disease; among them, 2 were symptomatic (substage b) because of splenic rupture. Four dogs underwent splenectomy and adjuvant doxorubicin, and 1 dog underwent surgery only. Three out of the 4 dogs treated with surgery and chemotherapy died of causes unrelated to lymphoma, after 760, 939, and 1,825 days, whereas the remaining dog was alive and in complete remission after 445 days. The dog not receiving any adjuvant treatment had recurrence of the tumor after 180 days.nnnCONCLUSIONS AND CLINICAL IMPORTANCEnSplenic MZL appears indolent and can benefit from splenectomy, with or without systemic chemotherapy.

Canine cutaneous perivascular wall tumors at first presentation: clinical behavior and prognostic factors in 55 cases.

BACKGROUNDnCanine cutaneous perivascular wall tumors (c-PWT) are soft tissue sarcomas recently identified when hemangiopericytomas were reclassified. No previous clinical data are available for c-PWT.nnnHYPOTHESIS/OBJECTIVESnTo define the clinical behavior and prognostic role of clinical and pathological variables in a homogeneous population of c-PWT.nnnANIMALSnFifty-five c-PWT in 53 client-owned dogs at first presentation undergoing surgery.nnnMETHODSnRetrospective case series. The endpoint was the relapse of tumor (local and/or distant). The prognostic values of clinical (age, sex, weight, site and tumor size, adjuvant therapy) and pathological (status of surgical margins, histological grade, mitosis, percentage of tumor necrosis) variables were investigated by univariate and bivariate analyses (P < .05). The pattern of associations between variables was explored by multivariate correspondence analysis (MCA).nnnRESULTSnTwelve dogs had a relapse. Ten dogs had local recurrence, 1 had metastatic disease, and 1 had both. The estimated probability of local recurrence was 0.02, 0.08, 0.20, and 0.24 at 6 months, 1, 2, and 3 years, respectively. Size of the tumor was a significant prognostic factor while status of margins had only a clinically relevant hazard ratio. In MCA evaluation, young age, tumor size (< 5 cm), grade I, and location in the extremities were associated. Association was also observed for older age, tumor size (> 5 cm), grade II, and other location.nnnCONCLUSION AND CLINICAL IMPORTANCEnC-PWT tend to locally recur a long time after surgery. An early diagnosis of c-PWT associated with small tumor size (< 5 cm) and clean surgical margins ensures a good prognosis independently of histological grade.

Accuracy of cytology in distinguishing adrenocortical tumors from pheochromocytoma in companion animals.

BACKGROUNDnThe distinction between adrenocortical tumors and pheochromocytoma can be challenging using clinical findings, diagnostic imaging and laboratory tests. Cytology might be a simple, minimally invasive method to reach a correct diagnosis.nnnOBJECTIVESnThe purpose of this study was to assess the accuracy of cytology in differentiating cortical from medullary tumors of the adrenal glands in dogs and cats.nnnMETHODSnCytologic key features of adrenocortical tumors and pheochromocytoma were defined by one reference author. Cytologic specimens from primary adrenal tumors were submitted to 4 cytopathologists who were asked to classify the tumors based on the previously defined key features without knowledge of previous classification.nnnRESULTSnTwenty specimens from histologically confirmed adrenal tumors (Group 1) and 4 specimens from adrenal tumors causing adrenal-dependent Cushings syndrome (Group 2) were evaluated by the 4 cytopathologists. Accuracy in differentiating cortical from medullary origin ranged from 90% to 100%, with a Kappa coefficient of agreement between cytopathologists of 0.95.nnnCONCLUSIONSnThe origin of an adrenal tumor can be easily determined by cytology alone in many cases. However, cytology was not reliable in distinguishing benign from malignant neoplasia. Additional studies are needed to assess possible risks and complications associated with fine-needle biopsy of adrenal tumors in dogs and cats.

Canine Perivascular Wall Tumors: High Prognostic Impact of Site, Depth, and Completeness of Margins

Canine perivascular wall tumors (cPWTs) arise from vascular mural cells and are included among soft tissue sarcomas (STSs). Most prognostic studies are performed on canine STSs as a general group and regardless of their specific histotype. The aim of this study was to identify pathological parameters and profiles with prognostic impact for cutaneous/subcutaneous cPWTs. Anatomical location, type of growth, surgical margins, and size and depth of the tumor were collected in 56 cPWTs. The association between each pair of variables was evaluated by χ2 test. Multiple correspondence analysis (MCA) was performed to describe the multivariate association of variables and was followed by cluster analysis to identify specific pathological profiles. The prognostic impact of variables and profiles was assessed by Cox regression model. Size and depth were significantly associated with increased relapse probability. Cases with complete surgical margins did not recur. Other single variables were not significantly associated with relapse. Cluster analysis on MCA considering site, depth, margins, and type of growth identified 3 pathological profiles associated with PWT relapse and having a high prognostic impact. Major prognostic factors for cPWTs were tumor size, depth of growth, and pathological profiles.

Detection of biclonal gammopathy by capillary zone electrophoresis in a cat and a dog with plasma cell neoplasia

Gammopathies associated with plasma cell neoplasms in a 15-year-old female spayed domestic shorthaired cat and a 9-year-old female spayed Rottweiler dog were evaluated by serum protein electrophoresis. In the cat, the plasma cell neoplasm was found in the liver and spleen, and an evaluable sample of bone marrow was not obtained. Some of the plasma cells had the morphologic appearance of flame cells. The paraprotein was confirmed as IgG based on agar gel immunodiffusion precipitation and both immunocytochemical and immunohistochemical staining. The dog had multiple myeloma with production of IgG and IgA paraproteins. In both cases, serum proteins were evaluated by 2 methods of protein electrophoresis: cellulose acetate electrophoresis (CAE) and capillary zone electrophoresis (CZE). In the cat and the dog, CAE showed a single large oligoclonal-like peak, which occurred in the γ-region in the cat and the β-γ-region in the dog, whereas CZE showed a biclonal gammopathy with 2 very close narrow spikes in the γ- and β-γ-regions in the cat and dog, respectively. In selected cases, CZE may be more effective than routine CAE in distinguishing oligoclonal from monoclonal or biclonal paraproteinemia.

Use of Electron Microscopy to Classify Canine Perivascular Wall Tumors

The histologic classification of canine perivascular wall tumors (PWTs) is controversial. Many PWTs are still classified as hemangiopericytomas (HEPs), and the distinction from peripheral nerve sheath tumors (PNSTs) is still under debate. A recent histologic classification of canine soft tissue sarcomas included most histologic types of PWT but omitted those that were termed undifferentiated. Twelve cases of undifferentiated canine PWTs were evaluated by transmission electron microscopy. The ultrastructural findings supported a perivascular wall origin for all cases with 4 categories of differentiation: myopericytic (n = 4), myofibroblastic (n = 1), fibroblastic (n = 2), and undifferentiated (n = 5). A PNST was considered unlikely in each case based on immunohistochemical expression of desmin and/or the lack of typical ultrastructural features, such as basal lamina. Electron microscopy was pivotal for the subclassification of canine PWTs, and the results support the hypothesis that canine PWTs represent a continuum paralleling the phenotypic plasticity of vascular mural cells. The hypothesis that a subgroup of PWTs could arise from a pluripotent mesenchymal perivascular wall cell was also considered and may explain the diverse differentiation of canine PWTs.

The expression ratio of miR-17-5p and miR-155 correlates with grading in canine splenic lymphoma

In dogs as in humans, microRNAs (miRNAs) play a key role in normal and neoplastic hematopoiesis regulation. The general miRNA expression framework varies among different stages of development and differentiation of tumors, and miRNAs are widely investigated as new molecular tools for cancer diagnosis and classification. Canine lymphomas are currently classified according with the WHO classification, but a comprehensive grading study of clinical samples is still lacking, and molecular tools for quick grading are not yet available. In the present work, a retrospective study of the expression profile of a panel of miRNAs in canine primary splenic lymphomas was performed. The formalin fixed, paraffin embedded (FFPE) lymphoma samples were accurately classified according with the WHO classification, and were analyzed for miRNA expression using stem-loop TaqMan real time RT-PCR. For each miRNA investigated, relative and absolute quantification were performed after selecting the best housekeeping genes using the NormFinder and geNorm algorithms. The results of this study show a diversity in miRNA expression in low (L) grade lymphomas compared to intermediate-high (I-H) grade lymphomas. The molar ratio between miR-17-5p and miR-155 correlated with WHO grading. These results highlight the potential use of miR-17-5p/miR-155 molar ratio as a new molecular tool for grading of canine splenic lymphomas. The data here reported further support the utility of monitoring miRNA expression in canine hematopoietic malignancies diagnosis and prognosis.