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Background: The use of adrenocorticotropic hormone stimulation test as method to monitor efficacy of trilostane treatment of hypercortisolism (HC) in dogs has been questioned. Objectives: To evaluate and compare 12 methods with which to monitor efficacy of trilostane treatment in dogs with HC. Animals: Forty-five client-owned dogs with HC treated with trilostane q12h. Methods: Prospective cross-sectional observational study. The dogs were categorized as well-controlled, undercontrolled, and unwell through a clinical score obtained from an owner questionnaire. The ability to correctly identify trilostane-treatment control of dogs with HC with the following variables was evaluated: before trilostane serum cortisol (prepill), before-ACTH serum cortisol, post-ACTH serum cortisol, plasma endogenous ACTH concentrations, prepill/eACTH ratio, serum haptoglobin (Hp) concentration, serum alanine aminotransferase (ALT), gamma-glutamyl transferase (γGT) and alkaline phosphatase activity, urine specific gravity, and urinary cortisol:creatinine ratio. Results: Ninety-four re-evaluations of 44 dogs were included; 5 re-evaluations of 5 unwell dogs were excluded. Haptoglobin was significantly associated with the clinical score (P

Introduction: In this case study, a client-owned dog with a large pituitary tumor was experimentally treated by intratumoral injection of radioactive holmium-166 microspheres (166HoMS), named 166Ho microbrachytherapy. To our knowledge, this is the first intracranial intratumoral treatment through needle injection of radioactive microspheres.Materials and Methods: A 10-year-old Jack Russell Terrier was referred to the Clinic for Companion Animal Health (Faculty of Veterinary Medicine, Utrecht University, The Netherlands) with behavioral changes, restlessness, stiff gait, and compulsive circling. MRI and CT showed a pituitary tumor with basisphenoid bone invasion and marked mass effect. The tumor measured 8.8 cm3 with a pituitary height-to-brain area (P/B) ratio of 1.86 cm−1 [pituitary height (cm) ×10/brain area (cm2)]. To reduce tumor volume and neurological signs, 166HoMS were administered in the tumor center by transsphenoidal CT-guided needle injections.Results: Two manual CT-guided injections were performed containing 0.6 ml of 166HoMS suspension in total. A total of 1097 MBq was delivered, resulting in a calculated average tumor dose of 1866 Gy. At 138 days after treatment, the tumor volume measured 5.3 cm3 with a P/B ratio of 1.41 cm−1, revealing a total tumor volume reduction of 40%. Debulking surgery was performed five months after 166HoMS treatment due to recurrent neurological signs. The patient was euthanized two weeks later at request of the owners. Histopathological analysis indicated a pituitary adenoma at time of treatment, with more malignant characteristics during debulking surgery.Conclusion: The 40% tumor volume reduction without evident severe periprocedural side effects demonstrated the feasibility of intracranial intratumoral 166HoMS treatment in this single dog.

The COVID-19 pandemic raised concerns that companion animals might be infected with, and could become a reservoir of, SARS-CoV-2. As cats are popular pets and susceptible to Coronavirus, we investigated the seroprevalence of SARS-CoV-2 antibodies in shelter cats housed in Dutch animal shelters during the COVID-19 pandemic. In this large-scale cross-sectional study, serum samples of shelter cats were collected during the second wave of human COVID-19 infections in The Netherlands. Seroprevalence was determined by using an indirect protein-based ELISA validated for cats, and a Virus Neutralization Test (VNT) as confirmation. To screen for feline SARS-CoV-2 shedding, oropharyngeal and rectal swabs of cats positive for ELISA and/or VNT were analyzed using PCR tests. In 28 Dutch animal shelters, 240 shelter cats were convenience sampled. Two of these cats (0.8%, CI 95%: 0.1–3.0%) were seropositive, as evidenced by the presence of SARS-CoV-2 neutralizing antibodies. The seropositive animals tested PCR negative for SARS-CoV-2. Based on the results of this study, it is unlikely that shelter cats could be a reservoir of SARS-CoV-2 or pose a (significant) risk to public health.

Pituitary tumours are common in dogs and are being increasingly recognized in cats. Pituitary tumours are usually classified as adenomas and should only be classified as carcinomas when there is evidence of metastatic spread of the tumour, which is rare. Despite the benign nature of most pituitary tumours, they can still compress or invade neighbouring tissues. Pituitary tumours can be functional (hormonally active) or non-functional (hormonally silent). The aim of this review was to provide an overview of the different pituitary tumour types in dogs and cats that have been reported in the literature. In dogs, the most common pituitary tumour type is the corticotroph adenoma, which can cause pituitary-dependent hypercortisolism. In cats, the most common pituitary tumour is the somatotroph adenoma, which can cause hypersomatotropism, and the second-most common is the corticotroph adenoma. A lactotroph adenoma has been described in one dog, while gonadotroph, thyrotroph and null cell adenomas have not been described in dogs or cats. Hormonally silent adenomas are likely underdiagnosed because they do not result in an endocrine syndrome. Tools used to classify pituitary tumours in humans, particularly immunohistochemistry for lineage-specific transcription factors, are likely to be useful to classify canine and feline pituitary tumours of unknown origin. Future studies are required to better understand the full range of pituitary adenoma pathology in dogs and cats and to determine whether certain adenoma subtypes behave more aggressively than others. Currently, the mechanisms that underlie pituitary tumorigenesis in dogs and cats are still largely unknown. A better understanding of the molecular background of these tumours could help to identify improved pituitary-targeted therapeutics.

Background Successful treatment of cats with hypersomatotropism by transsphenoidal hypophysectomy is described in small numbers of cats. Objectives To describe the endocrine profile, survival, and remission rates of hypersomatotropism and diabetes mellitus in a cohort of cats with hypersomatotropism that underwent hypophysectomy between 2008 and 2020. Animals Twenty-five client-owned cats with spontaneous hypersomatotropism. Methods Retrospective study. Diagnosis of hypersomatotropism was based on clinical signs, plasma insulin-like growth factor-1 (IGF-1) concentration, and imaging of the pituitary gland. Growth hormone (GH) and IGF-1 concentrations were measured repeatedly after surgery. Survival times were calculated based on follow-up information from owners and referring veterinarians. Results Median postoperative hospital stay was 7 days (range, 3-18 days). One cat died within 4 weeks of surgery. Median plasma GH concentration decreased significantly from 51.0 ng/mL (range, 5.0-101.0 ng/mL) before surgery to 3.8 ng/mL (range, 0.6-13.0 ng/mL) at 5 hours after surgery. Remission of hypersomatotropism, defined as normalization of plasma IGF-1 concentration, occurred in 23/24 cats (median, 34 ng/mL; range, 14-240 ng/mL) and 22/24 cats entered diabetic remission. Median survival time was 1347 days (95% confidence interval, 900-1794 days; range, 11-3180 days) and the overall 1-, 2-, and 3-year all-cause survival rates were 76%, 76%, and 52%, respectively. Conclusions and clinical importance This study shows the beneficial outcome of hypophysectomy in cats with hypersomatotropism, marked by low death rate and a high percentage of diabetic remission and definitive cure.

Objectives The aims of this study were to validate a commercially available chemiluminescent assay for measurement of feline plasma adrenocorticotropic hormone concentration (ACTH), to determine the normal reference interval (RI) of plasma ACTH in healthy cats, to assess plasma ACTH in cats with naturally occurring hypercortisolism (HC), primary hypoadrenocorticism (PH) and other diseases (OD), and to evaluate the effect of aprotinin on plasma ACTH degradation. Methods Forty healthy cats, 10 with HC, 11 with PH and 30 with OD, were included. The chemiluminescent enzyme immunometric assay was evaluated by measurement of intra-assay precision, interassay precision and linearity. The RI for plasma ACTH in healthy cats was established using robust methods. Plasma ACTH of samples collected with and without aprotinin, stored at 4°C and assayed over a 6-day period, was measured. Results The intra-assay coefficients of variance (CVs) ranged from 2.7% to 4.3% and interassay CVs from 3.3% to 10.7%. Dilution studies showed excellent accuracy (R2 >0.99). The RI for plasma ACTH in healthy cats was 32–370 pg/ml. Plasma ACTH was not significantly different between healthy cats and the OD group. Cats with pituitary-dependent hypercortisolism (PDH) and PH had significantly higher plasma ACTH than the other groups. Plasma ACTH did not show significant differences when samples collected with and without aprotinin were compared. Conclusions and relevance The Immulite chemiluminescent assay is a valid technique for measuring plasma ACTH in cats and the RI of plasma ACTH is quite wide. Owing to the low overlap between healthy or OD cats and cats with HC or PH, the measurement of plasma ACTH appears to be useful and should be included in the diagnostic work-up when HC or PH are suspected. Furthermore, the measurement of plasma ACTH may be an accurate test for differentiating PDH from adrenal-dependent hypercortisolism.

Contains fulltext : 235792.pdf (Publisher’s version ) (Closed access) BACKGROUND: Residential exposure to pesticides may occur via inhalation of airborne pesticides, direct skin contacts with pesticide-contaminated surfaces, and consumption of food containing pesticide residues. The aim was to study the association of dermal exposure to pesticides between the use and non-use periods, between farmer and non-farmer families and between dermal exposure and the excretion of metabolites from urine in residents living close to treated agricultural fields. METHODS: In total, 112 hand wipes and 206 spot urine samples were collected from 16 farmer and 38 non-farmer participants living within 50 m from an agricultural field in the Netherlands. The study took place from May 2016 to December 2017 during the use as well as the non-use periods of pesticides. Hand wipes were analysed for the parent compound and urines samples for the corresponding urinary metabolite of five applied pesticides: asulam, carbendazim (applied as thiophanate-methyl), chlorpropham, prochloraz and tebuconazole. Questionnaire data was used to study potential determinants of occurrence and levels of pesticides in hand wipes according to univariate and multivariate analysis. RESULTS: Carbendazim and tebuconazole concentrations in hand wipes were statistically significantly higher in the pesticide-use period compared to the non-use period. In addition, especially during the use periods, concentrations were statistically significantly higher in farmer families compared to non-farmer families. For asulam, chlorpropham and prochloraz, the frequency of non-detects was too high (57-85%) to be included in this analysis. The carbendazim contents in urine samples and hand wipes were correlated on the first and second day after taking the hand wipe, whereas chlorpropham was only observed to be related on the second day following the spray event. CONCLUSIONS: Concentrations in hand wipes were overall higher in pesticide use periods compared to non-use periods and higher in farmer families compared to non-farmer families. Only for carbendazim a strong correlation between concentrations in hand wipes and its main metabolite in urine was observed, indicating dermal exposure via contaminated indoor surfaces. We expect this to be related to the lower vapour pressure and longer environmental lifetime of carbendazim compared to the other pesticides studies.

BACKGROUND Information on scintigraphy findings in dogs with thyroid neoplasia is scarce. The use of single-photon emission computed tomography (SPECT) could improve detection of metastatic disease. HYPOTHESIS/OBJECTIVES To describe planar and SPECT imaging findings in dogs with thyroid tumors, and to compare SPECT and thoracic radiography for metastasis detection. ANIMALS Sixty-eight dogs with thyroid neoplasia. METHODS Retrospective study, search of medical records for dogs with thyroid neoplasia (2008-2018). RESULTS Thyroid scintigraphy was available from 68 dogs, of which 6 presented after surgical resection. Radionuclide uptake was increased in 56% of dogs, decreased in 24%, and comparable to that of the salivary glands in 13%. The remainder had multiple masses with variable uptake. A homogeneous uptake pattern was present in 16% and a heterogeneous uptake pattern in 73%. In 11% (all dogs with multiple masses), various uptake patterns were present. Thyroid tumors were well delineated in 55%. There was a significant association between hormone status and uptake pattern (P = .009), with a heterogeneous uptake pattern in the majority of euthyroid dogs, and hormone status and tumor circumscription (P = .003), with well-circumscribed margins in the majority of hypothyroid and hyperthyroid dogs. Thoracic SPECT imaging was available in 39 dogs and identified metastatic lesions in 15 dogs. Thoracic radiographs were performed in 14 of these dogs, and detected metastases in 3 dogs. CONCLUSIONS AND CLINICAL IMPORTANCE SPECT imaging is a viable imaging technique to screen for thoracic metastasis and wider use of SPECT imaging is recommended in dogs with thyroid neoplasia.

Hypercortisolism is caused by a cortisol-secreting adrenocortical tumour (ACT) in approximately 15-20% of cases in dogs. Little is known about which molecular markers are associated with malignant behaviour of canine ACTs. The objective of this study was to identify molecular markers of prognosis, which could be useful to refine prognostic prediction and to identify potential treatment targets. Cortisol-secreting ACTs were included from 40 dogs, of which follow-up information was available. The ACTs were classified as low risk of recurrence tumours (LRT; n = 14) or moderate-high risk of recurrence tumours (MHRT; n = 26), based on the novel histopathological Utrecht score. Normal adrenals (NAs) were included from 11 healthy dogs as reference material. The mRNA expression of 14 candidate genes was analysed in the 40 ACTs and in 11 NAs with quantitative RT-PCR. The genes' expression levels were statistically compared between NAs, LRTs and MHRTs. Univariate and multivariate analyses were performed to determine the association of the genes' expression levels with survival. Seven genes were differentially expressed between NAs and ACTs, of which pituitary tumour-transforming gene-1 (PTTG1) and topoisomerase II alpha (TOP2A) were also differentially expressed between LRTs and MHRTs. In survival analyses, high expression levels of Steroidogenic factor-1 (SF-1), PTTG1 and TOP2A were significantly associated with poor survival. In conclusion, we have identified several genes that are part of the molecular signature of malignancy in canine ACTs. These findings can be used to refine prognostic prediction, but also offer insights for future studies on druggable targets. This article is protected by copyright. All rights reserved.

Background: In dogs, spontaneous Cushing’s syndrome is most often pituitary-dependent and caused by hypersecretion of adrenocorticotropic hormone (ACTH), resulting in increased adrenocortical glucocorticoid secretion similar to horses. In horses with Cushing’s syndrome (or pituitary pars intermedia dysfunction [PPID]) a thyrotropin-releasing hormone (TRH) stimulation test can be used for diagnosis, as TRH administration results in increased circulating ACTH and cortisol concentrations in affected horses. Objective: The aim of this study was to investigate the effect of TRH administration on the circulating ACTH and cortisol concentrations in dogs with pituitary-dependent hypercortisolism (PDH). Methods: Ten clinically normal control dogs and 10 dogs with PDH, all client owned, underwent a TRH stimulation test with measurement of plasma concentrations of ACTH and cortisol, before and after intravenous administration of 10 μg TRH/kg bodyweight. Results: Plasma ACTH concentration did not rise significantly after TRH stimulation, neither in PDH dogs nor in clinically normal dogs. In contrast, the plasma cortisol concentration did increase significantly after TRH stimulation in both groups (p = .003 in PDH and p

Previous studies have described that tumor organoids can capture the diversity of defined human carcinoma types. Here, we describe conditions for long-term culture of human mucosal organoids. Using this protocol, a panel of 31 head and neck squamous cell carcinoma (HNSCC)–derived organoid lines was established. This panel recapitulates genetic and molecular characteristics previously described for HNSCC. Organoids retain their tumorigenic potential upon xenotransplantation. We observe differential responses to a panel of drugs including cisplatin, carboplatin, cetuximab, and radiotherapy in vitro. Additionally, drug screens reveal selective sensitivity to targeted drugs that are not normally used in the treatment of patients with HNSCC. These observations may inspire a personalized approach to the management of HNSCC and expand the repertoire of HNSCC drugs.Significance:This work describes the culture of organoids derived from HNSCC and corresponding normal epithelium. These tumoroids recapitulate the disease genetically, histologically, and functionally. In vitro drug screening of tumoroids reveals responses to therapies both currently used in the treatment of HNSCC and those not (yet) used in clinical practice.See related commentary by Hill and D'Andrea, p. 828.This article is highlighted in the In This Issue feature, p. 813

Cushing disease (CD) is a serious endocrine disorder that is most often caused by an ACTH-secreting pituitary adenoma. Patients can be treated medically when surgery is not an option or was unsuccessful. However, currently used pituitary-targeting drugs are effective in only 40% of patients. To efficiently identify new pituitary-targeting treatment options, we need an in vitro system that closely mimics in vivo conditions. We therefore aimed to establish organoid cultures of normal anterior pituitary and corticotroph adenomas. Organoids or tumoroids are miniature three-dimensional (3D) structures grown from stem cells, that closely resemble the organ or tumor they originate from. Because CD is a thousand times more prevalent in dogs than in humans, and hypophysectomy is the treatment of choice, we used canine tissues. Normal anterior pituitary glands were collected from three healthy dogs that were euthanized for reasons unrelated to the current study. Corticotroph adenomas were collected from six dogs that underwent transsphenoidal hypophysectomy at our University Clinic. The dogs were diagnosed with CD based on clinical signs, endocrine testing, and CT scan imaging. Normal anterior pituitary and corticotroph adenoma cells were cultured in a 3D matrix (basement membrane extract) with anterior pituitary organoid medium containing specific growth factors and ligands, which was refreshed twice a week. The organoids and tumoroids were characterized with histopathology and RT-qPCR. Structures resembling organoids or tumoroids grew from all nine samples (3 normal, 6 adenoma) that were put in culture. Both cystic and dense structures were observed. The organoids and tumoroids expanded rapidly, and could be passaged once every week. The organoids and tumoroids were successfully cultured up until passage number 10, and were then frozen down. Histopathology showed that the organoid or tumoroid cells morphologically resembled healthy anterior pituitary or corticotroph adenoma cells. All organoids cultures expressed mRNA of pituitary stem cell markers SOX2 and SOX9. This study shows that corticotroph adenomas can be cultured as tumoroids in vitro, something not previously published in any species. Based on the many opportunities in organoid culture (e.g., high-throughput drug screenings, gene editing, studying developmental processes), we expect that this in vitro model will pave the way to efficiently and reliably identify new treatment options for CD. Not only for humans, but also for our best friends: dogs.

Melanocortin 2 receptor antagonists in canine Cushing’s disease: in vitro studies Cushing’s disease (CD), caused by an ACTH-secreting pituitary adenoma, is one of the most common endocrinopathies in dogs. The current medical treatment options involve adrenocortical steroid synthesis inhibitors, but a selective targeted approach to block ACTH receptor at its receptor would be much more attractive. The objective of this study was to preclinically investigate the effect of MC2R antagonists on adrenocortical hormone production, cell viability, and mRNA expression of steroidogenic enzymes in canine primary adrenocortical cell cultures from adrenal glands of healthy dogs. Three different MC2R antagonists were used: CRN.1, CRN.2, and CRN.4. Canine primary adrenocortical cell cultures (n = 8) were incubated with 50 nM ACTH for 24h, to mimic CD. Thereafter, 10 nM (IC50) and 2 μM (maximal concentration) of CRN.1, CRN.2, and CRN.4 were added. The two concentrations were established based on preliminary studies. After 24 hours of incubation, adrenocortical hormone concentrations were measured in the culture medium using liquid chromatography-mass spectrometry. RNA was isolated from the cells using the RNeasy Microkit (Qiagen) for subsequent real-time quantitative PCR analysis. Cell viability was assessed after 24 hours of incubation using alamarBlue™ Cell Viability Reagent. All CRN compounds effectively inhibited cortisol concentrations, while leaving aldosterone concentrations unaffected. In incubations with a maximal concentration of the three compounds, cortisol concentration decreased to undetectable levels. The mRNA expression levels of steroidogenic enzymes StAR, CYP11A1, CYP17A1, HSD3B2, CYP21, and CYP11B were significantly inhibited in most conditions when compared to the ACTH-stimulated control. The mRNA expression of melanocortin 2 receptor accessory protein (MRAP) was suppressed as well. Cell viability was not affected by CNR.1 or CNR.4, but was slightly inhibited by CRN.2. In summary, canine adrenocortical cell culture is a useful model system for drug testing. Incubation with MC2R antagonists demonstrated the potential of CNR.1 and CNR.4 as new treatment options for CD. Future in vivo studies in dogs with spontaneous CD are indicated.

This retrospective study determined disease free survival (DFS) and progression free survival (PFS) in chemo-naïve dogs with multicentric or cranial mediastinal high-grade T-cell lymphoma, treated with a first-line CCNU-L(-chlorambucil)-CHOP protocol. Of thirteen dogs with multicentric lymphoma, 92.3% achieved a complete remission (CR), and the median DFS and PFS was 317 and 256 days, respectively. Three dogs had cranial mediastinal lymphoma, and achieved a CR with a median DFS and PFS of 978 and 1007 days, respectively. The oneand two-year DFS/PFS probability estimate for dogs with multicentric lymphoma was 0.50/0.46 and 0.42/0.38, respectively, for dogs with cranial mediastinal lymphoma 0.67/0.67. Neutropenia and thrombocytopenia were reported in 52.9% and 50% of the dogs, respectively, while 56.3% experienced grade 1-4 nephrotoxicity, hypothesized to be lomustine-induced. It was concluded that, compared to historical data, the currently described first-line CCNU-L(-chlorambucil)- CHOP protocol could benefit the survival of dogs with multicentric or cranial mediastinal highgrade T-cell lymphoma.

Health issues in purebred dogs are currently considered one of the biggest problems in companion animal health. The Labrador retriever (LR) is one of the most popular dog breeds. The aim of this study was to quantify LR breed health in comparison with mixed-breed dogs (MB), by using four different data sources: a veterinary practice management system (appr. 35,000 unique individuals LR + MB), data from two animal insurance companies (appr. 15,500 and 4500 individuals respectively), and a histopathological laboratory (appr. 4000 individuals). After extensive recoding of the data, health parameters utilised to quantify breed health were longevity, frequency of practice visits and insurance expense claims, and diagnostic codes. A Kaplan-Meier univariate and multivariable Cox proportional hazard model were used to evaluate longevity. A negative binomial model was used to analyse the frequency of visits, claims, and diagnostic codes in both sets of insurance data. Logistic regression was used to look into the categorical diagnostic codes in the laboratory data. The median lifespan of the LR was similar (12 years, practice data) or longer (10 versus 8 years, insurance data) than MB for individuals with a known birth and death date. When including censored individuals, survival time in the LR was comparable to MB individuals up to 10 years of age. Above 10 years of age, the LR lived a similar length as MB with a medium to large body size, but shorter than all MB. The LR visited the veterinary practice more often (risk ratio (RR) 1.2, 95% confidence interval 1.2-1.3), and also showed a higher frequency of insurance expense claims (RR 2.2 (2.1-2.3) and RR 1.2 (1.1-1.3) respectively for the two insurance data sets). The largest difference in organ systems between the LR and MB in insurance claims was related to ears (RR 5.3 (4.8-5.8) and RR 2.6 (2.3-3.1)), followed by airways (RR 2.6 (2.4-2.8)), tendons & muscles (RR 2.4 (2.2-2.6) and RR 1.4 (1.1-1.7)), and joints (RR 1.7 (1.3-2.1)), without a difference in median age at diagnosis. The data from the histopathological laboratory suggested a higher disease burden related to oncology for the LR compared to MB (OR 1.2, 95% CI 1.0-1.3). Oncological diagnoses were made at a younger age in the LR (8.8 versus 9.4 years). The disease burden was significantly higher for the LR than MB, but these results may suffer from substantial bias such as selection bias towards the database, and different behaviour of LR versus MB owners with regards to veterinary care. In the future, longer term population data can corroborate these results.

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of clinical efficacy, they are until now imperfect solutions. Stem cell therapy using ASC sheets could provide a solution to this problem. ASCs are considered as promising candidates for promoting tissue healing because of their trophic and immunomodulatory properties. Here, we provide methods to produce high-density ASC sheets, that are transplanted onto a colorectal anastomosis in a rat model to reduce the leakage. ASCs formed cell sheets in thermo-responsive culture dishes that could be easily detached. On the day of the transplantation, a partial colectomy with a 5-suture colorectal anastomosis was performed. Animals were immediately transplanted with 1 ASC sheet per rat. ASC sheets adhered spontaneously to the anastomosis without any glue, suture, or any biomaterial. Animal groups were sacrificed 3 and 7 days postoperatively. Compared to transplanted animals, the incidence of anastomotic abscesses and leakage was higher in control animals. In our model, the transplantation of ASC sheets after colorectal anastomosis was successful and associated with a lower leakage rate.

Hypercortisolism is one of the most commonly diagnosed endocrinopathies in dogs, and new targeted medical treatment options are desirable. Steroidogenic factor-1 (SF-1), an orphan nuclear hormone receptor, is a key regulator of adrenal steroidogenesis, development, and growth. In pituitary-dependent hypercortisolism (PDH), high plasma ACTH concentrations increase the transcriptional activity of SF-1. In adrenal-dependent hypercortisolism, SF-1 expression is significantly greater in dogs with recurrence after adrenalectomy than in those without recurrence. Inhibition of SF-1 could therefore be an interesting treatment option in canine spontaneous hypercortisolism. We determined the effects of 3 SF-1 inverse agonists, compounds IsoQ A, #31, and #32, on cortisol production, on the messenger RNA (mRNA) expression of steroidogenic enzymes and SFs, and on cell viability, in primary adrenocortical cell cultures of 8 normal adrenal glands and of 3 cortisol-secreting adrenocortical tumors (ATs). To mimic PDH, the normal adrenocortical cell cultures were stimulated with ACTH. The results show that only compound #31 inhibited cortisol production and SF-1 target gene expression in non-ACTH-stimulated and ACTH-stimulated normal adrenocortical cells but did not affect cell viability. In the AT cell cultures, the effects of #31 on cortisol production and target gene expression were variable, possibly caused by a difference in the SF-1 mRNA expressions of the primary tumors. In conclusion, inhibition of SF-1 activity shows much promise as a future treatment for canine hypercortisolism.

A cortisol-secreting adrenocortical tumour (ACT) is the cause of naturally occurring canine hypercortisolism in approximately 15-20% of cases. The differentiation between an adrenocortical adenoma and carcinoma is usually based on histopathology. However, histopathological parameters have never been linked to the dogs' survival. Moreover, in human medicine the interobserver variability of some histopathological parameters that are used for ACTs is high. The objective of this study was to establish a reliable and easy-to-use histopathological scoring system for cortisol-secreting ACTs that can assess the prognosis of dogs after adrenalectomy. Cortisol-secreting ACTs of 50 dogs, collected between 2002 and 2015, were included in this study. Twenty histopathological features were assessed by one veterinary pathologist and one resident in veterinary pathology. In addition, the Ki67 proliferation index was assessed by two observers. Only parameters with intra- and interobserver agreement scores (intraclass correlation or Cohen's kappa coefficient) of ≥0.40 were included in survival analyses. Use of multivariate forward stepwise regression analysis with associated hazard ratios led us to a scoring system which we call the Utrecht score: the Ki67 proliferation index, +4 if more than 33% of the tumour cells have clear/vacuolated cytoplasm, and +3 if necrosis is present. Using cut-off values of 6 and 11, we could distinguish three groups that had significantly shorter survival times with increasing Utrecht scores. We conclude that the Utrecht score can be used to assess the prognosis of dogs with cortisol-secreting ACTs after adrenalectomy, which can help to select high-risk dogs that might benefit from adjuvant treatment or additional monitoring. This article is protected by copyright. All rights reserved.

Background In the last decade, a disorder characterized by episodes of involuntary movements and dystonia has been recognized in Border Terriers. Objectives To define clinical features of paroxysmal dyskinesia (PD) in a large number of Border Terriers and to study the genetics of the disease. Animals 110 affected and 128 unaffected client-owned Border Terriers. Methods A questionnaire regarding clinical characteristics of PD was designed at Utrecht University and the University of Helsinki. Thirty-five affected Border Terriers underwent physical examination and blood testing (hematology and clinical biochemistry). Diagnostic imaging of the brain was performed in 17 affected dogs and electroencephalograms (EEG) between episodes were obtained in 10 affected dogs. A genomewide association study (GWAS) was performed with DNA of 110 affected and 128 unaffected dogs. Results One hundred forty-seven questionnaires were included in the study. The most characteristic signs during episodes were dystonia, muscle fasciculations, and falling over. The majority of owners believed that their dogs remained conscious during the episodes. A beneficial effect of anti-epileptic therapy was observed in 29 of 43 dogs. Fifteen owners changed their dogs’ diet to a hypoallergenic, gluten-free diet, and all reported reasonable to good improvement of signs. Clinical examinations and diagnostic test results were unremarkable. The GWAS did not identify significantly associated chromosome regions. Conclusions and Clinical Importance The survey results and EEG studies provided further evidence that the observed syndrome is a PD rather than epilepsy. Failure to achieve conclusive results by GWAS indicates that inheritance of PD in Border Terriers probably is complex.

Background Transsphenoidal hypophysectomy is one of the treatment strategies in the comprehensive management of dogs with pituitary‐dependent hypercortisolism (PDH). Objectives To describe the influence of pituitary size at time of pituitary gland surgery on long‐term outcome. Animals Three‐hundred–and‐six dogs with PDH. Methods Survival and disease‐free fractions were analyzed and related to pituitary size; dogs with and without recurrence were compared. Results Four weeks after surgery, 91% of dogs were alive and remission was confirmed in 92% of these dogs. The median survival time was 781 days, median disease‐free interval was 951 days. Over time, 27% of dogs developed recurrence of hypercortisolism after a median period of 555 days. Dogs with recurrence had significantly higher pituitary height/brain area (P/B) ratio and pre‐operative basal urinary corticoid‐to‐creatinine ratio (UCCR) than dogs without recurrence. Survival time and disease‐free interval of dogs with enlarged pituitary glands was significantly shorter than that of dogs with a non‐enlarged pituitary gland. Pituitary size at the time of surgery significantly increased over the 20‐year period. Although larger tumors have a less favorable prognosis, outcome in larger tumors improved over time. Conclusions and Clinical Importance Transsphenoidal hypophysectomy is an effective treatment for PDH in dogs, with an acceptable long‐term outcome. Survival time and disease‐free fractions are correlated negatively with pituitary gland size, making the P/B ratio an important pre‐operative prognosticator. However, with increasing experience, and for large tumors, pituitary gland surgery remains an option to control the pituitary mass and hypercortisolism.

Adipose tissue-derived stem cells (ASCs) are known to be tissue-healing promoters due to their cellular plasticity and secretion of paracrine factors. Cultured ASC sheets provide a novel method of ASCs application and can retain ASCs at the targeted tissue. The purpose of this systematic review is to evaluate preclinical studies using adipose tissue-derived cell (ASC) sheet transplantation therapy for promoting tissue healing. Firstly, we searched databases to identify studies of ASC sheet therapy in different experimental animal models, and then determined the quality score of studies using SYRCLE's risk bias tool. A total of 18 included studies examined the role of ASC sheets on tissue healing and function in models for myocardial infarction, dilated cardiomyopathy, full-thickness skin wounds, hind limb ischemia, esophageal strictures and oral ulcers. ASC sheet application after myocardial infarction improved survival rate, cardiac function and capillary density and reduced the extent of fibrosis. Application of ASC sheets to a full thickness skin wound decreased the wound size and stimulated wound maturation. In the hind limb ischemia model, ASC sheet application improved limb perfusion, and capillary density and decreased the amount of ischemic tissue and inflammation. ASC sheet application to mucosal wounds of the gastrointestinal system accelerated wound healing and decreased the degree of stricture and fibrosis. Taken together, transplanted ASC sheets had a positive effect on tissue healing and reconstruction in these pre-clinical studies. The reported favorable effects of ASC sheet therapy in various tissue healing applications may be implemented in future translational studies. It is suggested that future preclinical animal model studies of ASC sheet therapy should concern standardization of culture techniques and investigate the mechanisms of action. In addition, clearly indicated experimental set-ups according to the SYRCLE's guidelines should improve study quality and validity.

Morphologically, canine soft-tissue sarcomas (STSs) resemble human STSs. In humans, proper classification of STSs is considered essential to improve insight in the biology of these tumors, and to optimize diagnosis and therapy. To date, there is a paucity of data published on the significance of detailed classification of STSs in the dog. We revised a cohort ( n = 110) of proliferative lesions obtained from a study in Golden Retrievers that were considered “soft tissue sarcoma, not otherwise specified or of uncertain subtype” in order to optimize the diagnoses of these lesions. The criteria according to the veterinary WHO classification, recent veterinary literature, and the WHO classification for humans were applied. Revision was initially based on morphologic characteristics of hematoxylin and eosin–stained histologic sections of the neoplasms. If considered necessary ( n = 76), additional immunohistochemistry was applied to aid characterization. The diagnosis of STS was confirmed in 75 neoplasms (68%). Of this group, diagnosis of a specific subtype of the STSs was possible in 58 neoplasms. Seven neoplasms had morphologic characteristics that were suggestive for sarcoma subtypes only described in the WHO classification for humans. Seventeen neoplasms remained “unclassified STSs.” Thirty-one lesions (28%) were diagnosed “neoplasm, not being STS.” Four lesions (4%) were considered nonneoplastic. Because incorrect classification of a tumor could lead to inappropriate therapeutic intervention and prognostication, the results of our study clearly illustrate the importance of revision and further diagnosis of “unclassified STSs” in dogs.

International audience; BackgroundSerum cystatin C (sCysC) is used as biomarker for glomerular filtration rate (GFR). The effects of diabetes mellitus (DM) on renal function in dogs are unclear. Some renal variables have been evaluated in dogs with hyperadrenocorticism (HAC), but not sCysC. ObjectivesThe purpose of this study was the validation of a particle-enhanced nephelometric immunoassay (PENIA) for measuring canine sCysC, and to assess renal function in dogs with DM or HAC. MethodsA PENIA was analytically validated for canine sCysC by determining imprecision and linearity. In a longitudinal 6-month study, renal function of 14 DM dogs was assessed, using serum creatinine, GFR, urinary protein-to-creatinine (UPC) ratio, urinary markers, systolic blood pressure (SBP), and sCysC, and compared to 17 healthy dogs at baseline. Furthermore, sCysC was measured at initial presentation and during a 12-month follow-up in 22 HAC dogs. ResultsThe sCysC intra- and inter-assay variation coefficients were < 8% and highly linear (r = .997). About 33% and 67% of DM dogs had persistent proteinuria and systemic hypertension, respectively, but there were no significant differences in GFR, UPC, and urinary markers over time, and compared with healthy dogs at initial presentation. Serum CysC decreased significantly (P < .05) over time within the DM group. It did not change significantly over time within the HAC group. ConclusionsA PENIA measured sCysC linearly and precisely. There were no clinically relevant renal alterations over time in dogs with DM, although persistent proteinuria was observed. In dogs with HAC, sCysC measurement was not useful, although significant GFR changes occurred over time.

Pseudopregnancy is a frequently diagnosed reproductive disorder in (dairy) goats. This cross-sectional study evaluates the incidence, possible risk factors and therapies for pseudopregnancy on Dutch dairy goat farms. Two questionnaires, one for farmers and one for veterinarians, were designed and included questions about general farm demographics, breeding management, hormonal oestrous induction, treatment, measures for reduction and stress moments in dairy goats in the period June 1, 2016-May 31, 2017. In total, 43 farmers (21.5 per cent response rate) and 27 veterinarians (22.5 per cent response rate) completed the questionnaire. The annual incidence of pseudopregnancy varied between 1 and 54 per cent per farm, with a mean annual incidence of 17 per cent (95 per cent CI 0.14 to 0.21). In this study, we found a significant association between incidence of pseudopregnancy and a higher percentage of goats with an extended lactation (p

Spontaneous cortisol-secreting adrenocortical tumours in pet dogs are an attractive animal model for their human counterparts. Adrenal morphology and function are similar in dogs and humans, and adrenocortical tumours have comparable clinical and pathological characteristics. Their relatively high incidence in pet dogs represents a potential source of adrenocortical tumour tissue to facilitate research. The molecular characteristics of canine cortisol-secreting adrenocortical tumours suggest that they will be useful for the study of angiogenesis, the cAMP/protein kinase A pathway, and the role of Steroidogenic Factor-1 in adrenal tumourigenesis. Pet dogs with spontaneous cortisol-secreting adrenocortical tumours may also be useful in clinical testing of new drugs and in investigating the molecular background of adrenocortical tumours.

RATIONALE: Despite extensive studies, the genetic and epigenetic mechanisms that mediate initiation and progression of lung cancers have not been fully elucidated. Previously, we have demonstrated that via complementary mechanisms, including DNA methylation, polycomb repressive complexes, and noncoding RNAs, cigarette smoke induces stem-like phenotypes that coincide with progression to malignancy in normal respiratory epithelia as well as enhanced growth and metastatic potential of lung cancer cells. OBJECTIVES: To further investigate epigenetic mechanisms contributing to stemness/pluripotency in lung cancers and potentially identify novel therapeutic targets in these malignancies, induced pluripotent stem cells were generated from normal human small airway epithelial cells. METHODS: Lung induced pluripotent stem cells were generated by lentiviral transduction of small airway epithelial cells of OSKM (Yamanaka) factors (octamer-binding transcription factor 4 [Oct4], sex-determining region Y box 2 [SOX2], Kruppel-like factor 4 [KLF4], and MYC proto-oncogene, bHLH transcription factor [MYC]). Western blot, real-time polymerase chain reaction, and chromatin immunoprecipitation sequencing analysis were performed. RESULTS: The lung induced pluripotent stem cells exhibited hallmarks of pluripotency, including morphology, surface antigen and stem cell gene expression, in vitro proliferation, and teratoma formation. In addition, lung induced pluripotent stem cells exhibited no chromosomal aberrations, complete silencing of reprogramming transgenes, genomic hypermethylation, upregulation of genes encoding components of polycomb repressive complex 2, hypermethylation of stem cell polycomb targets, and modulation of more than 15,000 other genes relative to parental small airway epithelial cells. Additional sex combs like-3 (ASXL3), encoding a polycomb repressive complex 2-associated protein not previously described in reprogrammed cells, was markedly upregulated in lung induced pluripotent stem cell as well as human small cell lung cancer lines and specimens. Overexpression of the additional sex combs like-3 gene correlated with increased genomic copy number in small cell lung cancer lines. Knock-down of the additional sex combs like-3 gene inhibited proliferation, clonogenicity, and teratoma formation by lung induced pluripotent stem cells and significantly diminished in vitro clonogenicity and growth of small cell lung cancer cells in vivo. CONCLUSIONS: Collectively, these studies highlight the potential utility of this lung induced pluripotent stem cell model for elucidating epigenetic mechanisms contributing to pulmonary carcinogenesis and suggest that additional sex combs like-3 is a novel target for small cell lung cancer therapy.

Cortisol-secreting adrenocortical tumours (AT) in dogs are characterised by uncontrolled growth and excessive cortisol secretion. Dysregulated hormone receptor expression might be involved in tumour growth and hypersecretion of cortisol. The relative mRNA expression of growth hormone receptor, progesterone receptor, somatostatin receptors (SSTR1-3) and dopamine receptors (DRD1-2 and DRD5) was evaluated in 36 canine ATs and 15 adrenal glands obtained from healthy dogs. Compared with normal adrenal tissue, DRD2 mRNA expression was relatively lower in carcinomas, while SSTR1 mRNA expression was lower in both adenomas and carcinomas. Both of these features might contribute to loss of inhibition of tumour growth and upregulation of cortisol secretion. In canine ATs that had recurred within 30 months of surgical adrenalectomy, a marked increase in expression of DRD1 mRNA was observed. Targeting of specific hormone receptors, expressed by ATs, might be exploited for therapy.

Nanobody-targeted photodynamic therapy (NB-PDT) has been developed as a potent and tumor-selective treatment, using nanobodies (NBs) to deliver a photosensitizer (PS) specifically to cancer cells. Upon local light application, reactive oxygen species are formed and consequent cell death occurs. NB-PDT has preclinically shown evident success and we next aim to treat cats with oral squamous cell carcinoma (OSCC), which has very limited therapeutic options and is regarded as a natural model of human head and neck SCC. Immunohistochemistry of feline OSCC tissue confirmed that the epidermal growth factor receptor (EGFR) is a relevant target with expression in cancer cells and not in the surrounding stroma. Three feline OSCC cell lines were employed together with a well-characterized human cancer cell line (HeLa), all with similar EGFR expression, and a low EGFR-expressing human cell line (MCF7), mirroring the EGFR expression level in the surrounding mucosal stroma. NBA was identified as a NB binding human and feline EGFR with comparable high affinity. This NB was developed into NiBh, a NB-PS conjugate with high PS payload able to effectively kill feline OSCC and HeLa cell lines, after illumination. Importantly, the specificity of NB-PDT was confirmed in co-cultures where only the feline OSCC cells were killed while surrounding MCF7 cells were unaffected. Altogether, NiBh can be used for NB-PDT to treat feline OSCC and further advance NB-PDT towards the human clinic.