ADVANCED DIAGNOSTIC IMAGING AND PATHOLOGIC FINDINGS OF THYROID LESIONS IN TWO SHORT-BEAKED ECHIDNAS (TACHYGLOSSUS ACULEATUS).

Two zoo-maintained short-beaked echidnas (Tachyglossus aculeatus) had long histories of intermittent anorexia and lethargy. Case 1 presented with a recurrence of these signs after transfer to another facility and died shortly after arrival. A focal area of hyperattenuation within the paratracheal tissue of the cranial mediastinum was noted antemortem on CT. Postmortem, this corresponded with severe thyroid follicular hyperplasia with lymphoplasmacytic thyroiditis. Additional findings included a systemic fungal infection without an inflammatory response, suggesting underlying factors such as torpor or immunosuppression. In Case 2, an intrathoracic mass was identified during a preshipment examination. CT confirmed a contrast-enhanced mass compressing the cranial vena cava and right atrium, and the animal was euthanized. The mass was diagnosed histologically as thyroid adenocarcinoma. These cases report thyroiditis and thyroid adenocarcinoma in echidna and describe the use of IV contrast and CT as a diagnostic aid in this species.

POPULATION PHARMACOKINETICS OF CEFTAZIDIME AFTER A SINGLE SUBCUTANEOUS INJECTION AND NORMAL ORAL AND CLOACAL BACTERIAL FLORA SURVEY IN EASTERN HELLBENDERS (CRYPTOBRANCHUS ALLEGANIENSIS ALLEGANIENSIS).

Population pharmacokinetics utilizing sparse sampling were used to determine pharmacokinetics of ceftazidime in eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) due to their slow growth rate and the limited number of appropriately sized individuals in the zoo-housed population. Twenty-five eastern hellbenders received a single subcutaneous injection of ceftazidime at 20 mg/kg. Each animal had blood samples collected up to four times between 0 and 192 hr postinjection. Plasma samples were analyzed by high-pressure liquid chromatography. A nonlinear mixed-effects model was fitted to the data to determine typical values for population parameters, an ideal method due to the sampling limitation of each hellbender. Results indicate an elimination half-life of 36.63 hr and volume of distribution of 0.31 L/kg. Antibiotic concentrations were above a minimum inhibitory concentration (MIC) value of 8 µg/ml for 120 hr. Prior to antibiotic administration, six hellbenders had oral and six other individuals had cloacal swabs taken for aerobic culture. Fifty-five bacterial isolates were obtained (24 cloacal, 31 oral) with 10/12 (83%) individuals growing three or more different isolates and 11/12 (92%) growing Shewanella putrefaciens. Twelve isolates had susceptibility testing performed and all were susceptible to ceftazidime. These results indicate that ceftazidime is an appropriate choice of antibiotic in hellbenders and when given at a dosage of 20 mg/kg subcutaneously, maintains concentrations above the MIC of susceptible bacteria for up to 5 days.

T-cell tumour exclusion and immunotherapy resistance: a role for CAF targeting.

Recent studies have highlighted a major role for cancer-associated fibroblasts (CAFs) in promoting immunotherapy resistance by excluding T cells from tumours. Recently, we showed that CAFs can be effectively targeted by inhibiting the enzyme NOX4; this 'normalises' CAFs and overcomes immunotherapy resistance. Here we discuss our study and other strategies for CAF targeting.

Hyperglycemic hyperosmolar state in a chimpanzee (Pan troglodytes).

A 19-year-old female chimpanzee (Pan troglodytes) presented for cachexia, acute weakness, hyporexia, icterus, and polyuria. The animal was diagnosed with a hyperglycemic hyperosmolar state, which is a well-recognized syndrome in diabetic humans that is rarely diagnosed in animals. This case documents an important and likely under-reported syndrome in non-human primates.

Treatment of disseminated Strongyloides spp. infection in an infant Sumatran orangutan (Pongo abelii).

Strongyloides nematodes have been reported in all species of great apes with orangutans ≤5 years old most susceptible to severe clinical disease. This brief communication describes the first published case of antemortem diagnosis and treatment of disseminated strongyloidiasis in a clinically affected 5-month-old Sumatran orangutan (Pongo abelii).

Pro-migratory and TGF-ß-activating functions of αvß6 integrin in pancreatic cancer are differentially regulated via an Eps8-dependent GTPase switch.

The integrin αvß6 is up-regulated in numerous carcinomas, where expression commonly correlates with poor prognosis. αvß6 promotes tumour invasion, partly through regulation of proteases and cell migration, and is also the principal mechanism by which epithelial cells activate TGF-ß1; this latter function complicates therapeutic targeting of αvß6, since TGF-ß1 has both tumour-promoting and -suppressive effects. It is unclear how these different αvß6 functions are linked; both require actin cytoskeletal reorganization, and it is suggested that tractive forces generated during cell migration activate TGF-ß1 by exerting mechanical tension on the ECM-bound latent complex. We examined the functional relationship between cell invasion and TGF-ß1 activation in pancreatic ductal adenocarcinoma (PDAC) cells, and confirmed that both processes are αvß6-dependent. Surprisingly, we found that cellular functions could be biased towards either motility or TGF-ß1 activation depending on the presence or absence of epidermal growth factor receptor pathway substrate 8 (Eps8), a regulator of actin remodelling, endocytosis, and GTPase activation. Similar to αvß6, we found that Eps8 was up-regulated in >70% of PDACs. In complex with Abi1/Sos1, Eps8 regulated αvß6-dependent cell migration through activation of Rac1. Down-regulation of Eps8, Sos1 or Rac1 suppressed cell movement, while simultaneously increasing αvß6-dependent TGF-ß1 activation. This latter effect was modulated through increased cell tension, regulated by Rho activation. Thus, the Eps8/Abi1/Sos1 tricomplex acts as a key molecular switch altering the balance between Rac1 and Rho activation; its presence or absence in PDAC cells modulates αvß6-dependent functions, resulting in a pro-migratory (Rac1-dependent) or a pro-TGF-ß1 activation (Rho-dependent) functional phenotype, respectively. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

RANDOMIZED CONTROLLED TRIAL COMPARING THE EFFECTS OF ALFAXALONE AND KETAMINE HYDROCHLORIDE IN THE HAITIAN GIANT GALLIWASP ( CELESTUS WARRENI).

The immobilization properties and cardiopulmonary effects following intramuscular administration of one of two chemical immobilization agents were compared in the Haitian giant galliwasp ( Celestus warreni) in a prospective, blinded, randomized controlled trial. Adult, clinically healthy galliwasps ( n = 30) were given a randomly assigned single intramuscular injection of either 15 mg/kg alfaxalone ( n = 15) or 40 mg/kg ketamine hydrochloride ( n = 15). Heart rate, respiratory rate, and depth classification stage were recorded every 5 min; cloacal temperature was recorded every 15 min to ensure maintenance within this species' preferred optimal temperature range (75-85°F, 24-29°C). Physical examination, radiographs, and phlebotomy were performed in all animals. Alfaxalone given intramuscularly resulted in reliable anesthetic induction, maintenance, and recovery (total duration of anesthesia 57.7 ± 23.6 min, recovery 7.9 ± 7.8 min). Ketamine hydrochloride resulted in variable levels of sedation or anesthesia and a longer recovery (total duration of anesthesia 14 ±17.5 min, recovery 47.9 ± 19.3 min). Heart and respiratory rates remained within clinically acceptable ranges in all lizards using both protocols; however, alfaxalone animals had lower heart rates and respiratory rates associated with increased anesthetic depth as compared to ketamine hydrochloride animals (heart rates: alfaxalone 59.6 ± 13.3 beats/min, ketamine hydrochloride 71.9 ± 7.9 beats/min; respiratory rates: alfaxalone 33.4 ± 16.8 breaths/min, ketamine hydrochloride 50.0 ± 16.2 breaths/min). Duration of anesthesia for alfaxalone-treated galliwasps was longer than previously reported in other studies. This study determined that a single injection of alfaxalone at 15 mg/kg administered intramuscularly can be used for consistent induction and maintenance of anesthesia and prompt recovery in the Haitian giant galliwasp, while ketamine hydrochloride even at 40 mg/kg was unreliable and is not recommended as a sole immobilization agent in the Haitian giant galliwasp.

COMPARISON OF PROPOFOL CONSTANT RATE INFUSION AND ISOFLURANE FOR MAINTENANCE OF ANESTHESIA IN SPEKE'S GAZELLE, GAZELLA SPEKEI.

The aims of this study were to determine if a propofol constant rate infusion (CRI) in Speke's gazelle, Gazella spekei, would serve as an effective alternative maintenance anesthetic, result in shorter recovery times, and improve anesthetic recovery quality when compared with isoflurane. Eight adult gazelle were enrolled in this complete crossover study with a minimum 3-wk washout period. All gazelle were induced with 10 mg/kg intravenous propofol and maintained with either propofol CRI (0.4 mg/kg/min) or isoflurane (1-3%) for 45 min. Animals were monitored for anesthetic depth and physiologic variables including heart and respiratory rates, oxygen saturation, end-tidal carbon dioxide, indirect blood pressure, and temperature every 5 min. Blood gas samples were analyzed within the first 10 min following anesthetic induction and within the last 10 min of anesthesia. Recovery times were recorded. Recovery quality was classified by a residual ataxia grading scale. Seven gazelle completed the study by undergoing both anesthetic treatments; one female (12 yr old) developed complications 2 days after isoflurane anesthesia, consisting of seizures, azotemia, leukocytosis, hypocalcemia, and hypomagnesemia but was treated successfully. Propofol anesthesia resulted in lower respiratory rates compared with isoflurane and a decrease in respiratory rate over time. Propofol CRI maintained blood pressure values closer to physiologically normal ranges compared with isoflurane for 45 min of anesthesia. Recovery times were comparable between propofol and isoflurane treatments. While individuals receiving propofol had higher residual ataxia scores compared with individuals receiving isoflurane, differences were not clinically important. This study demonstrated that propofol CRI (0.4 mg/kg/min) is an effective maintenance anesthetic agent in healthy adult Speke's gazelle for noninvasive procedures with endotracheal intubation and intermittent positive pressure ventilation.

Medical Management of Hypovitaminosis D With Cholecalciferol and Elastic Therapeutic Taping in Red-legged Seriema (Cariama cristata) Chicks.

Three hand-reared, 50-53 day-old, red-legged seriema (Cariama cristata) chicks were evaluated for acute lameness and reluctance to ambulate. Two of the 3 chicks presented with angular limb deformities of the proximal tarsometatarsi and external rotation of the legs. Radiographs demonstrated decreased opacity of the long bone of the legs, with poorly delineated cortices and deviation of the proximal tarsometarsi. Serum concentrations of 25-hydroxycholecalciferol revealed all 3 chicks were deficient in vitamin D(3) at presentation. The chicks were administered injectable vitamin D(3) (cholecalciferol), oral vitamin D(3), and an ultraviolet B (UV-B) light was placed in their enclosure. Elastic, therapeutic taping was used to correct angular limb deformities present in 2 of the 3 chicks. Taping was continued until the angular limb deformities were corrected and lameness resolved. Hypovitaminosis D is a common cause of metabolic bone disease in captive avian species. Cholecalciferol administration, UV-B light supplementation, and elastic, therapeutic taping were effective treatments for osteodystrophy and secondary angular limb deformities due to hypovitaminosis D. This multifaceted treatment may be useful in other long-legged juvenile birds with similar clinical signs.

MEDICAL MANAGEMENT OF LEIOMYOMATA AND SUSPECTED ENDOMETRIOSIS IN AN ALLEN'S SWAMP MONKEY (ALLENOPITHECUS NIGROVIRIDUS).

A 13-yr-old female nulliparous Allen's swamp monkey (Allenopitchecus nigroviridis) presented with intermittent excessive vaginal bleeding, cyclical lethargy, and a history of irregular menstrual cycles. Abdominal ultrasonography revealed a subjectively thickened, irregular endometrium, multiple leiomyomata (uterine fibroids), and bilateral anechoic foci on the ovaries. Treatment was initiated with leuprolide acetate i.m. monthly for 6 mo. Recheck ultrasound at 3 mo showed a decrease in leiomyoma diameter and no evidence of active follicles on the ovaries. Eleven months following completion of treatment, clinical signs recurred and the animal was treated with a deslorelin implant. Since implant placement, no vaginal bleeding has been noted.

MANAGEMENT OF TOXIC MASTITIS IN A BABIRUSA (BABYROUSA CELEBENSIS).

A 1 yr 8 mo-old, previously healthy, primiparous female babirusa (Babyrousa celebensis) presented acutely recumbent and minimally responsive approximately 36 hr after giving birth to a single piglet. Toxic mastitis was diagnosed based on physical examination and laboratory results. The mammary tissue was firm, discolored, and produced negligible amounts of milk. All of the teats were eventually affected, resulting in the inability to provide adequate nutrition to the piglet. Although toxic mastitis has a poor prognosis in domestic sows, this babirusa recovered completely with aggressive management, including antibiotics and supportive care.

MANAGEMENT OF ACUTE RENAL FAILURE WITH DELAYED HYPERCALCEMIA SECONDARY TO SARCOCYSTIS NEURONA-INDUCED MYOSITIS AND RHABDOMYOLYSIS IN A CALIFORNIA SEA LION (ZALOPHUS CALIFORNIANUS).

A 3-yr-old captive-born California sea lion (Zalophus californianus) developed Sarcocystis neurona-induced myositis and rhabdomyolysis that led to acute renal failure. The sea lion was successfully managed with fluid therapy, antiprotozoals, antibiotics, anti-inflammatories, antiemetics, gastroprotectants, and diuretics, but developed severe delayed hypercalcemia, a syndrome identified in humans after traumatic or exertion-induced rhabdomyolysis. Treatment with calcitonin was added to the management, and the individual recovered fully. The case emphasizes that animals with rhabdomyolysis-induced renal failure risk developing delayed hypercalcemia, which may be life threatening, and calcium levels should be closely monitored past the resolution of renal failure.

PRESUMPTIVE DYSGERMINOMA IN AN ORANGE-SPOT FRESHWATER STINGRAY (POTAMOTRYGON MOTORO).

A captive-born, 13-yr-old female orange-spot freshwater stingray, (Potamotrygon motoro), presented with an acute caudodorsal swelling. Ultrasonography revealed an intracoelomic mass of mixed echogenicity containing fluid pockets. The ray was euthanatized and gross postmortem examination confirmed the presence of a fluid-filled coelomic mass in the region of the reproductive tract. The mass was identified histologically as a malignant round cell tumor of the ovary. Although immunohistochemistry for protein gene product 9.5 (PGP9.5), octamer-3/4 (OCT-3/4), and inhibin was attempted, antibodies that had been validated in mammalian species did not cross-react with stingray control tissues and did not label neoplastic cells. The final diagnosis was a presumptive dysgerminoma.

Differentiation signals induce APOBEC3A expression via GRHL3 in squamous epithelia and squamous cell carcinoma.

Two APOBEC (apolipoprotein-B mRNA editing enzyme catalytic polypeptide-like) DNA cytosine deaminase enzymes (APOBEC3A and APOBEC3B) generate somatic mutations in cancer, driving tumour development and drug resistance. Here we used single cell RNA sequencing to study APOBEC3A and APOBEC3B expression in healthy and malignant mucosal epithelia, validating key observations with immunohistochemistry, spatial transcriptomics and functional experiments. Whereas APOBEC3B is expressed in keratinocytes entering mitosis, we show that APOBEC3A expression is confined largely to terminally differentiating cells and requires Grainyhead-like transcription factor 3 (GRHL3). Thus, in normal tissue, neither deaminase appears to be expressed at high levels during DNA replication, the cell cycle stage associated with APOBEC-mediated mutagenesis. In contrast, we show that in squamous cell carcinoma tissues, there is expansion of GRHL3 expression and activity to a subset of cells undergoing DNA replication and concomitant extension of APOBEC3A expression to proliferating cells. These findings indicate a mechanism for acquisition of APOBEC3A mutagenic activity in tumours.

Single-cell analysis reveals prognostic fibroblast subpopulations linked to molecular and immunological subtypes of lung cancer.

Fibroblasts are poorly characterised cells that variably impact tumour progression. Here, we use single cell RNA-sequencing, multiplexed immunohistochemistry and digital cytometry (CIBERSORTx) to identify and characterise three major fibroblast subpopulations in human non-small cell lung cancer: adventitial, alveolar and myofibroblasts. Alveolar and adventitial fibroblasts (enriched in control tissue samples) localise to discrete spatial niches in histologically normal lung tissue and indicate improved overall survival rates when present in lung adenocarcinomas (LUAD). Trajectory inference identifies three phases of control tissue fibroblast activation, leading to myofibroblast enrichment in tumour samples: initial upregulation of inflammatory cytokines, followed by stress-response signalling and ultimately increased expression of fibrillar collagens. Myofibroblasts correlate with poor overall survival rates in LUAD, associated with loss of epithelial differentiation, TP53 mutations, proximal molecular subtypes and myeloid cell recruitment. In squamous carcinomas myofibroblasts were not prognostic despite being transcriptomically equivalent. These findings have important implications for developing fibroblast-targeting strategies for cancer therapy.

Surgical removal of a thymoma in a burrowing owl (Athene cunicularia).

A 12-year-old male burrowing owl (Athene cunicularia) was presented for evaluation of a mass in the right cervical region. A thymoma was diagnosed after surgical resection and histopathologic evaluation. Extensive adherence of the thymoma to the esophagus and suspected invasion into the right jugular vein contributed to a poor postsurgical outcome. Diagnosis and treatment of thymomas in avian species is similar to that in mammals. Surgical removal of noninvasive thymomas is usually curative. Thymomas are rarely reported in avian species and this is the first report in a strigiform bird.

Targeting cancer-associated fibroblasts: Challenges, opportunities and future directions.

Cancer-associated fibroblasts (CAFs) are a common cell in the tumour microenvironment with diverse tumour-promoting functions. Their presence in tumours is commonly associated with poor prognosis making them attractive therapeutic targets, particularly in the context of immunotherapy where CAFs have been shown to promote resistance to checkpoint blockade. Previous attempts to inhibit CAFs clinically have not been successful, however, in part due to a lack of understanding of CAF heterogeneity and function, with some fibroblast populations potentially being tumour suppressive. Recent single-cell transcriptomic studies have advanced our understanding of fibroblast phenotypes in normal tissues and cancers, allowing for a more precise characterisation of CAF subsets and providing opportunities to develop new therapies. Here we review recent advances in the field, focusing on the evolving area of therapeutic CAF targeting.

ATM Regulates Differentiation of Myofibroblastic Cancer-Associated Fibroblasts and Can Be Targeted to Overcome Immunotherapy Resistance.

Myofibroblastic cancer-associated fibroblast (myoCAF)-rich tumors generally contain few T cells and respond poorly to immune-checkpoint blockade. Although myoCAFs are associated with poor outcome in most solid tumors, the molecular mechanisms regulating myoCAF accumulation remain unclear, limiting the potential for therapeutic intervention. Here, we identify ataxia-telangiectasia mutated (ATM) as a central regulator of the myoCAF phenotype. Differentiating myofibroblasts in vitro and myoCAFs cultured ex vivo display activated ATM signaling, and targeting ATM genetically or pharmacologically could suppress and reverse differentiation. ATM activation was regulated by the reactive oxygen species-producing enzyme NOX4, both through DNA damage and increased oxidative stress. Targeting fibroblast ATM in vivo suppressed myoCAF-rich tumor growth, promoted intratumoral CD8 T-cell infiltration, and potentiated the response to anti-PD-1 blockade and antitumor vaccination. This work identifies a novel pathway regulating myoCAF differentiation and provides a rationale for using ATM inhibitors to overcome CAF-mediated immunotherapy resistance. SIGNIFICANCE: ATM signaling supports the differentiation of myoCAFs to suppress T-cell infiltration and antitumor immunity, supporting the potential clinical use of ATM inhibitors in combination with checkpoint inhibition in myoCAF-rich, immune-cold tumors.

Targeting cancer associated fibroblasts to enhance immunotherapy: emerging strategies and future perspectives.

Cancer associated fibroblasts are a prominent component of the tumour microenvironment in most solid cancers. This heterogeneous population of cells are known to play an important role in tumour progression and recent studies have demonstrated that CAFs may confer resistance to checkpoint immunotherapy, suggesting that targeting these cells could improve response rates. However, effective clinical strategies for CAF targeting have yet to be identified. In this editorial, we highlight current limitations in our understanding of CAF heterogeneity, and discuss the potential and possible approaches for CAF-directed therapy.

Cancer-Associated Fibroblasts in Oral Cancer: A Current Perspective on Function and Potential for Therapeutic Targeting.

The role of the tumour microenvironement (TME) in cancer progression and resistance to therapies is now widely recognized. The most prominent non-immune cell type in the microenvironment of oral cancer (OSCC) is cancer-associated fibroblasts (CAF). Although CAF are a poorly characterised and heterogenous cell population, those with an "activated" myofibroblastic phenotype have been shown to support OSCC progression, promoting growth, invasion and numerous other "hallmarks of malignancy." CAF also confer broad resistance to different types of therapy, including chemo/radiotherapy and EGFR inhibitors; consistent with this, CAF-rich OSCC are associated with poor prognosis. In recent years, much CAF research has focused on their immunological role in the tumour microenvironment, showing that CAF shield tumours from immune attack through multiple mechanisms, and particularly on their role in promoting resistance to anti-PD-1/PD-L1 checkpoint inhibitors, an exciting development for the treatment of recurrent/metastatic oral cancer, but which fails in most patients. This review summarises our current understanding of CAF subtypes and function in OSCC and discusses the potential for targeting these cells therapeutically.