Chiara Giudice

Role of the Molybdoflavoenzyme Aldehyde Oxidase Homolog 2 in the Biosynthesis of Retinoic Acid: Generation and Characterization of a Knockout Mouse†

ABSTRACT The mouse aldehyde oxidase AOH2 (aldehyde oxidase homolog 2) is a molybdoflavoenzyme. Harderian glands are the richest source of AOH2, although the protein is detectable also in sebaceous glands, epidermis, and other keratinized epithelia. The levels of AOH2 in the Harderian gland and skin are controlled by genetic background, being maximal in CD1 and C57BL/6 and minimal in DBA/2, CBA, and 129/Sv strains. Testosterone is a negative regulator of AOH2 in Harderian glands. Purified AOH2 oxidizes retinaldehyde into retinoic acid, while it is devoid of pyridoxal-oxidizing activity. Aoh2−/− mice, the first aldehyde oxidase knockout animals ever generated, are viable and fertile. The data obtained for this knockout model indicate a significant role of AOH2 in the local synthesis and biodisposition of endogenous retinoids in the Harderian gland and skin. The Harderian glands transcriptome of knockout mice demonstrates overall downregulation of direct retinoid-dependent genes as well as perturbations in pathways controlling lipid homeostasis and cellular secretion, particularly in sexually immature animals. The skin of knockout mice is characterized by thickening of the epidermis in basal conditions and after UV light exposure. This has correlates in the corresponding transcriptome, which shows enrichment and overall upregulation of genes involved in hypertrophic responses.

Feline injection-site sarcoma: recurrence, tumour grading and surgical margin status evaluated using the three-dimensional histological technique.

The three-dimensional histology technique is used in human medicine for the evaluation of complete lateral and deep surgical margins. In this study, the technique was applied to 48 excised feline injection-site sarcoma specimens. The predictive value of the histological margin status and tumour grading on local recurrence was investigated. In 32/48 cases, the margins were non-infiltrated, whilst in the remaining 16 cases, they were infiltrated. Overall, 6/32 (19%) tumours with non-infiltrated margins and 11/16 (69%) with infiltrated margins recurred. Tumours with infiltrated margins recurred about 10 times more frequently compared to tumours with non-infiltrated margins (P=0.0011). No statistically significant correlation was observed between grading and recurrence. The assessment of margin status using the 3D histology technique showed a good predictivity for post-surgical tumour recurrence. Extensive application of the 3D histology technique is recommended to standardise the evaluation of histological margins and to allow comparison between results from different laboratories.

Performances of different diagnostic tests for feline infectious peritonitis in challenging clinical cases

Objectives: Feline infectious peritonitis (FIP) can be difficult to diagnose. Histopathology is considered the gold standard test but immunohistochemistry (IHC) is mandatory to confirm/exclude the disease. This study aimed to assess the performances of tests carried out in vivo or at postmortem examination in challenging cases in which FIP was confirmed or excluded based on IHC or on adequate follow‐up. Methods: Twelve cases (four without FIP, eight with FIP) were retrospectively studied. Clinical findings, serum protein electrophoresis (SPE), analysis of the effusions (AE), antifeline coronavirus serology, serum concentration of α1‐acid glycoprotein (AGP) and histopathology were classified as consistent, doubtful or non‐consistent with FIP. Sensitivity, specificity and concordance (κ) with the final diagnosis were calculated. Results: Concordance was absent for serology (κ=−0·08) and AE (κ=−0·52), poor for histopathology (κ=0·09), fair for SPE (κ=0·25) and perfect for AGP (κ=1·00). Sensitivity was high for AGP (100%) and low for AE (50%), SPE (37·5%) and histopathology (37·5%). Specificity was high for AGP or histopathology (100%) and low for SPE (50%) and AE (0%). Clinical Significance: IHC must always be performed to confirm FIP. If this is not possible, when histopathology is controversial, elevated AGP concentrations may support the diagnosis of FIP.

Bilateral orbital and nasal aspergillosis in a cat.

Abstract A 12‐year‐old, 4 kg, castrated male Persian cat was referred with a 2‐month history of sneezing and bilateral mucopurulent nasal discharge. Rhinoscopically acquired nasal biopsies at this time revealed bilateral lymphoplasmacytic rhinitis. A tapering dose of oral prednisone caused the complete remission of the clinical signs, but 2 months after discontinuation of the therapy, the rhinitis recurred and the OD became exophthalmic. Computed tomography showed a soft tissue mass in both sides of the nasal cavity, both frontal sinuses, the right orbit, and to a lesser extent the left orbit. A fine needle aspirate of the right orbit revealed pyogranulomatous inflammation and Aspergillus spp. hyphae. Repeat nasal biopsy demonstrated multi‐focal necrosis and a mixed inflammatory cell process which now included macrophages and scattered septate fungal hyphae. A few days later the cat became bilaterally blind and a contrast enhancing lesion involving the optic chiasm was found on magnetic resonance imaging. Despite a poor prognosis, therapy consisted of exenteration of the right orbit and trephination of both frontal sinuses before the planned initiation of medical antifungal therapy. Unfortunately, the cat died of cardiac arrest intraoperatively. Aspergillus fumigatus was cultured from both orbits at necropsy. Orbital aspergillosis has been rarely reported in cats and its relationship with lymphoplasmacytic rhinitis is unclear. In this patient lymphoplasmacytic rhinitis or previous antibiotic/corticosteroid therapy may have allowed secondary fungal invasion of the nasal mucosa and subsequently both orbits and the brain. Alternatively, Aspergillus infection may have preceded the lymphoplasmacytic rhinitis.

Immunohistochemical expression of the KIT protein (CD117) in normal and neoplastic canine testes.

The aim of the present study was to characterize the expression of the KIT protein (CD117) in normal and neoplastic canine testes. Archival samples of normal testis (n=5), interstitial cell tumours (ICTs; n=10), Sertoli cell tumours (SCTs; n=10) and seminomas (n=10) were selected. Seminomas were subclassified on the basis of expression of placental alkaline phosphatase (PLAP) as classical seminoma (SE; PLAP positive; n=5) or spermatocytic seminoma (SS; PLAP negative; n=5). In normal testes, KIT expression was observed in Leydig cells and in spermatogonia. All ICTs expressed KIT, but no SCT was positively labelled. Seven of 10 seminomas expressed KIT and these tumours were reclassified on this basis as SS (KIT negative) or SE (KIT positive). These findings are consistent with observations of SE in man where many of the neoplastic cells reach the stage of spermatogonia where PLAP expression is lost and that of KIT is maintained. It would therefore appear that immunolabelling for KIT expression is a more appropriate means of distinguishing between canine SE and SS.

Comparison of 2- and 3-category histologic grading systems for predicting the presence of metastasis at the time of initial evaluation in dogs with cutaneous mast cell tumors: 386 cases (2009–2014)

OBJECTIVE To compare the Kiupel (2 categories) and Patnaik (3 categories) histologic grading systems for predicting the presence of metastasis at the time of initial examination in dogs with cutaneous mast cell tumors (MCTs). DESIGN Retrospective case series. ANIMALS 386 client-owned dogs with cutaneous MCTs. PROCEDURES Medical records of dogs with newly diagnosed, histologically confirmed cutaneous MCTs that had undergone complete clinical staging were reviewed for clinical and histopathologic data. RESULTS All Patnaik grade 1 MCTs (n = 52) were classified as Kiupel low-grade MCTs, and all Patnaik grade 3 MCTs (43) were classified as Kiupel high-grade MCTs. Of the 291 Patnaik grade 2 MCTs, 243 (83.5%) were classified as Kiupel low-grade tumors, and 48 (16.5%) were classified as Kiupel high-grade MCTs. Dogs with Patnaik grade 3 MCTs were significantly more likely to have metastases at the time of initial examination than were dogs with grade 1 or 2 MCTs (OR, 5.46), and dogs with Kiupel high-grade MCTs were significantly more likely to have metastases than were dogs with Kiupel low-grade MCTs (OR, 2.54). However, 3 of 52 (5.8%) dogs with Patnaik grade 1 tumors, 48 of 291 (16.5%) dogs with Patnaik grade 2 tumors, and 44 of 295 (14.9%) dogs with Kiupel low-grade tumors had metastatic disease. CONCLUSIONS AND CLINICAL RELEVANCE Findings indicated that in dogs with cutaneous MCTs, prognostication should not rely on histologic grade alone, regardless of grading system used, but should take into account results of clinical staging.

Immunohistochemical Evaluation of the Expression of Anti-Müllerian Hormone in Mature, Immature and Neoplastic Canine Sertoli Cells

In mammals, the earliest specific protein expressed by Sertoli cells (SCs) is the anti-Müllerian hormone (AMH), which induces the regression of Müllerian ducts and is produced by SCs until the functional maturation of the testes. The aim of this study was to evaluate the expression of AMH by canine SCs during testicular maturation and neoplastic transformation. Testes from two fetuses, 18 newborn puppies, five puppies aged 43-180 days and six adult dogs, and 24 canine Sertoli cell tumours (SCTs) were studied immunohistochemically for expression of AMH. Fifteen of the 24 SCTs were classified as typical, eight as lipid-rich and one was considered malignant based on evidence of lymph node metastasis. SCs from fetuses and neonatal puppies and puppies up to 45 days old expressed AMH, while SCs from older puppies and adults were negative. All SCTs expressed AMH, suggesting that AMH expression is a useful marker of immature and neoplastic canine SCs.

Computed tomography characteristics of fibrosarcoma: a histological subtype of feline injection-site sarcoma

Feline injection-site sarcoma (FISS) may be a consequence of subcutaneous injection. In the present study, the medical records and the computed tomography (CT) features of 22 cats with a FISS, histopathological subtype fibrosarcoma, were used. The majority of the fibrosarcomas (45%) were located in the interscapular region. All fibrosarcomas, except one with mild enhancement, showed strong contrast uptake, characterised as ring (42%), heterogeneous (36%), homogeneous (9%), heterogeneous/ring (6.5%) or mixed heterogeneous/homogeneous enhancement (6.5%). The longest axis of the mass was in a cranio-caudal (68%) or dorso-ventral (32%) direction. The median volume calculated on CT was 7.57 cm3. Common features were a marked local invasiveness of the musculature and heterogeneity of the tissue in the periphery of the neoplasia. When the fibrosarcoma was interscapular, performing an additional post-contrast scan with the forelimbs positioned caudally along the body, in addition to the standard protocol with the forelimbs extended cranially, allowed better evaluation of the actual relationship between the tumour and the surrounding tissues. The mean number of muscles involved with the tumour was 2.09 with extended and 1.95 with flexed forelimbs. When a lower number of structures was considered infiltrated through the double positioning, a less invasive surgical approach to underlying muscles and scapula was performed.

Amniotic membrane transplantation for the treatment of feline corneal sequestrum: pilot study.

OBJECTIVES To describe and evaluate the use of equine amniotic membrane trans-plantation after lamellar keratectomy for the treatment of corneal sequestrum in cats. METHODS Six cats (seven eyes) of various breed and ages with corneal sequestra were treated surgically with lamellar keratectomy and amniotic membrane transplantation. All the sequestra and a small piece of the amniotic membranes used for each surgery were submitted for histopathologic examination. RESULTS Five of the seven eyes showed minimal level of scarring in the cornea and good transparency. No recurrences of the sequestra have been noted during the follow-up period (3-9 months). One eye had necrosis of the amniotic membrane 2 weeks after the surgery. The sequestrum of this eye showed a high level of bacterial contamination on histopathology. Three months later the same cat developed a descemetocele in the area where the necrotic amniotic membrane was rejected. A second eye developed a perforation under the amniotic membrane two weeks after the surgery. The sequestrum of this eye was deep and without vascularization. CONCLUSION Amniotic membrane transplantation after lamellar keratectomy was a valid procedure for surgical treatment of corneal sequestrum in cats. The procedure resulted in excellent cosmesis and functional vision in five of seven eyes; although case selection is important, particularly to exclude the very deep and non-vascularized sequestra.

Invasive mould infections of the naso-orbital region of cats: a case involving Aspergillus fumigatus and an aetiological review.

Case and context This report describes a cat diagnosed with sinonasal-orbital Aspergillus fumigatus infection using advanced imaging, histopathology and culture. Aetiology, clinical aspects and treatment of this rare and devastating infection are discussed with reference to a literature review of invasive mould (ie, filamentous fungal) infections involving tissues of the naso-orbital region of cats. Presentations Invasive fungal infections can present with different localisations (nasal passages, sinuses, orbits, subcutaneous space, palate, etc) depending on the species involved and its means of introduction into the tissues. Localised subcutaneous lesions (swellings, ulcerations, masses, nodules, etc), without concomitant signs of nasal/orbital disease, generally result from traumatic injuries and subsequent inoculation of fungal spores into the subcutaneous space. In contrast, naso-ocular involvement and concurrent signs of nasal disease (nasal discharge, sneezing, masses protruding from the nostrils) generally result from inhalation of spores, with subsequent spread of infection into the nasal planum or penetration of overlying bone and invasion of the subcutaneous space. Aspergillus species typically show such an invasion mechanism and frequently affect orbital tissues. Dematiaceous fungi (ie, filamentous fungi with brown/black hyphae) are reported to cause solitary, less invasive, slowly developing lesions, probably as a result of traumatic injuries with inoculation of fungal propagules. Accordingly, the subcutaneous space is more frequently primarily involved. Treatment and outcome Whatever the mould species, reported treatment options include surgery and a series of antifungal drugs. The outcome is frequently poor, especially for Aspergillus infections, although various measures can be taken to maximise the chances of success, as discussed in this report.