What's New in Old Horses? Postmortem Diagnoses in Mature and Aged Equids.

Postmortem findings in 241 equids admitted to a teaching hospital that were at least 15 years old at autopsy were reviewed (1) to determine disease prevalence, (2) to compare the cause of death (or euthanasia) in equids 15 to 19 years of age (n = 116) with that in equids ≥20 years of age (n = 125), and (3) to catalog coexisting lesions in equids with pituitary pars intermedia dysfunction (PPID). Breed and sex were evenly distributed between the age groups. Death or euthanasia was attributed to disease of the digestive system (41.5%), pituitary gland (12.9%), locomotor system (10.0%), nervous system (7.9%), cardiovascular system (4.6%), urinary system (4.6%), reproductive system (4.2%), respiratory system (4.2%), integumentary system (4.2%), lymphoid system (2.5%), liver (2.5%), or systemic neoplasia (1.2%). Nervous system disease was more common in the 15- to 19-year group; urinary tract disease was more common in the ≥20-year group. Neoplastic disease, regardless of systemic location, was the basis for death or euthanasia in 18.7% of all equids. Squamous cell carcinoma, lymphoma, and melanoma were the most common malignant neoplasms. PPID was the most common specific diagnosis, based on the postmortem presence of hyperplasia or adenoma, and was the reason for euthanasia in 47.7% of 65 equids with PPID. The most common nonpituitary causes for death or euthanasia in equids with PPID were colic, lameness, cancer, and spinal cord disease. Coexisting conditions in equids with PPID that were not considered the basis for euthanasia included neoplasms, infections, lameness, and recurrent airway obstruction.

Evaluation of field-testing protocols to diagnose insulin dysregulation in ponies using a Bayesian approach.

Field tests and their association with laminitis have not been evaluated in large cohorts. The objectives of this study were to evaluate the performance of basal insulin (BI), the oral sugar test (OST) and the insulin tolerance test (ITT) to diagnose ID and investigate their association with laminitis. Insulin dysregulation status was determined in 146 ponies using BI (insulin concentration >20 µIU/mL), an OST (insulin concentration >65 µIU/mL at 60 or 90 min after oral administration of 0.45 mL/kg corn syrup) and an ITT (< 50% reduction in glucose concentration 30 min after intravenous administration of 0.1 IU/kg insulin). Laminitis was identified using modified-Obel scores. A Bayesian approach was used to define the characteristics of the tests and receiver operating characteristic curves were used to assess their association with laminitis. All tests were well tolerated and laminitis was diagnosed in 9% of ponies. Insulin dysregulation was diagnosed in 15% of ponies using BI, 38% using the OST and 54% using the ITT with 11% of ponies positive for all three tests. The sensitivities and specificities of BI, the OST and the ITT to diagnose ID were 0.52 (95% confidence interval [CI], 0.35-0.79) and 0.97 (95% CI, 0.91 - 1.00), 0.84 (95% CI, 0.70 - 0.94) and 0.60 (95% CI, 0.49 - 0.71), and 0.85 (95% CI, 0.68-0.96) and 0.88 (95% CI, 0.75 - 0.97), respectively. Only BI and the OST were associated with laminitis (P = 0.003 and 0.015, respectively).

Investigation of breed differences in plasma adrenocorticotropic hormone concentrations among healthy horses and ponies.

Plasma adrenocorticotropic hormone (ACTH) concentration is commonly measured to diagnose pituitary pars intermedia dysfunction (PPID). Several intrinsic and extrinsic factors affect ACTH concentrations, including breed. The objective of this study was to prospectively compare plasma ACTH concentrations among different breeds of mature horses and ponies. Three breed groups comprised Thoroughbred horses (n = 127), Shetland ponies (n = 131) and ponies of non-Shetland breeds (n = 141). Enrolled animals did not show any signs of illness, lameness or clinical signs consistent with PPID. Blood samples were collected 6 months apart, around the autumn equinox and spring equinox, and plasma concentrations of ACTH were measured by chemiluminescent immunoassay. Pairwise breed comparisons within each season were performed on log transformed data using the Tukey test. Estimated mean differences in ACTH concentrations were expressed as fold difference with 95 % confidence intervals (CI). Reference intervals for each breed group per season were calculated using non-parametric methods. In autumn, higher ACTH concentrations were found among non-Shetland pony breeds compared with Thoroughbreds (1.55 fold higher; 95 % CI, 1.35-1.77; P < 0.001), and in Shetland ponies compared with Thoroughbreds (2.67 fold higher; 95 % CI, 2.33-3.08; P < 0.001) and non-Shetland pony breeds (1.73 fold higher; 95 % CI, 1.51-1.98; P < 0.001). In spring, no differences were identified among breed groups (all P > 0.05). Reference intervals were similar among breed groups in spring, but upper limits for ACTH concentrations were markedly different between Thoroughbred horses and pony breeds in autumn. These findings emphasise that breed should be accounted for when determining and interpreting reference intervals for ACTH concentrations among healthy horses and ponies in autumn.

Evaluation of postsale endoscopy as a predictor of future racing performance in an Australian thoroughbred yearling population.

INTRODUCTION: Upper airway endoscopy of thoroughbred (TB) yearlings is commonly used in an attempt to predict laryngeal function (LF) and its impact on future race performance. The aim of this study was to determine if different grading systems and laryngeal grades were correlated with future performance. MATERIALS AND METHODS: Postsale endoscopic recordings were obtained from an Australian TB yearling sale during a four-year period from 2008 to 2011. Horses were included if they had a diagnostic postsale video endoscopic recording and raced within Australia. Recordings were graded using the Havemeyer system and subsequently recategorised according to the Lane scale and two condensed scales. Performance data were sourced from Racing Australia and comparisons were made between groups. RESULTS: A total of 1244 horses met the inclusion criteria. There were no significant differences in sex or sales price between groups. There were no significant differences in the number of starts or wins between groups for any grading system. For the condensed Havemeyer scale, horses with intermediate LF were separated into two groups. Significant differences in earnings were found between 'normal' and 'abnormal' (P = 0.02) and 'intermediate-low' and 'abnormal' grades (P = 0.03). There were no significant differences between horses with 'intermediate-high' and 'abnormal' grades (P = 0.40). No significant differences were found between the two intermediate grades (P = 0.60) or between horses with normal LF and either 'intermediate-low' or 'intermediate-high' grades (P = 0.99). DISCUSSION: Resting LF in Australian yearling TBs assessed using a condensed Havemeyer grading scale had some predictive value for future racing performance. This information should be considered when performing yearling endoscopic examinations.

Uterine allotransplantation in ewes using an aortocava patch.

BACKGROUND: We investigated a novel allotransplantation model using an aortocava patch in ewes. METHODS AND RESULTS: We carried out 10 uterine orthotopic allotransplantations in ewes with end-to-side anastomosis of the aortocava donor patch on the left external iliac vessel recipient. The immunosuppressive protocol was a combination of cyclosporine (10 mg/kg/day) and mycophenolic acid (3 g/day). An estimation of the immunosuppressive therapy exposure was performed by measuring the area under the curve (AUC) of immunosuppressive plasma concentrations. The graft was assessed by vaginoscopy, magnetic resonance imaging (MRI) and second look laparotomy at 6, 8 and 10 weeks, respectively. The median (range) times for cold and warm ischemia were 95 min (75-130) and 91 min (55-165), respectively. All the vascular anastomoses were patent at the end of the surgery. The median AUC of cyclosporine and mycophenolic acid were 1.24 mg h/l (0.34-3.85) and 18.40 mg h/l (3.76-42.35), respectively. Of the 10 ewes receiving a transplant, 6 could be assessed. Cervical biopsies showed signs of necrosis in all six ewes. The MRI results correlated with the macroscopic observations of the 'second look' laparotomy. The aortocava vascular pedicles were thrombosed, adding to the peripheral neovascularization. Graft histology showed endometrial tissue in two out of six ewes. CONCLUSIONS: Mobility of the transplant within the pelvis, the length of the vascular pedicle and rejection can explain the high rate of transplant necrosis. The particular digestive anatomy and physiology of ruminants makes it difficult to administer an optimal immunosuppressive treatment. MRI appears to be a good non-invasive examination for graft estimation.

Rapid molecular genetic assay for direct identification of Bordetella from patients specimens.

Biological diagnosis of whooping cough is increasingly necessary to confirm respiratory tract infection. Indeed, clinical symptoms are variable especially in adolescents and adults who contaminate newborns too young to be vaccinated. The PCR assay was proven highly sensitive for the diagnosis of pertussis. In this study, we reported the use of a new test (GenoQuick Bordetella [GQB], Hain Life Science, Germany) which permits the fast molecular genetic identification of Bordetella pertussis and parapertussis directly from patients specimens, i.e. swabs from nose or throat. The test was performed over a three months period on 40 specimens from patients (1 month to 65 years old), most of them were young children admitted in paediatric emergency with paroxysmal cough or prolonged cough.

Essential oil spray reduces clinical signs of insect bite hypersensitivity in horses.

OBJECTIVE: To assess the efficacy of an herbal spray combining various essential oils, with a claim of mast cell stabilisation, antipruritic, anti-inflammatory, and insect repellent effects on the clinical presentation of insect bite hypersensitivity (IBH) in horses. DESIGN: Double-blinded, placebo-controlled, randomised, cross-over clinical trial. METHODS: Twenty adult horses with clinical IBH were treated with a daily application of herbal spray or placebo for 28 days in a randomised, cross-over fashion, separated by a>28-day washout period. Horses were examined and scored prior to and after the completion of each treatment. Histopathology was performed on four horses. Owners kept daily diaries of observations. RESULTS: The herbal spray significantly reduced the severity of all assessed parameters (pruritus, excoriations, lichenification and alopecia; P < 0.05) compared with baseline values (pretreatment) and with placebo. Owners reported improvement of pruritus in 19/20 horses (95%) with complete resolution in 17 horses (85%) following treatment. Skin biopsies showed resolution of orthokeratosis in 4/4 horses, reduced thickness of the stratum spinosum in 2/4 horses and complete resolution of histopathological abnormalities in 1/4 horses after treatment, compared with either no change or deterioration of histopathologic lesions after placebo. No side effects were observed. CONCLUSIONS: The tested herbal spray may be an effective treatment for the management of equine IBH.

Diagnosis and treatment of confirmed and suspected primary hyperparathyroidism in equids: 17 cases (1999-2016).

BACKGROUND: Primary hyperparathyroidism is uncommon in equids. OBJECTIVES: To describe the diagnostic findings and efficacy of treatment in equids with primary hyperparathyroidism. STUDY DESIGN: Retrospective case series describing 16 horses and one mule. METHODS: Cases were identified by retrospective review of records at Cornell University and via an ACVIM listserv query. Inclusion criteria were an equid with hypercalcemia, normal renal function and high parathyroid hormone (PTH) or histopathological diagnosis of a parathyroid adenoma. Equids with normal PTH and PTH-related protein (PTHrP) in the face of hypercalcemia were included as suspect cases. RESULTS: The most common presenting complaints were weight loss (12/17) and hypercalcemia (10/17). PTH was above reference range in 12/17 cases. Suspected parathyroid tumours were localised in 12/14 equids imaged using ultrasonography alone (2/3), technetium 99m Tc sestamibi scintigraphy alone (1/1) or both modalities (9/10). Three horses did not have imaging performed. Surgical exploration successfully excised tumours in six of 10 cases. Five were located at the thoracic inlet, and surgery resulted in complete cure. One tumour was excised from the thyroid lobe, and the horse remained hypercalcemic. Four other cases explored surgically, four treated medically and three that were not treated also remained hypercalcemic. MAIN LIMITATIONS: The small study size prohibited statistical analysis. CONCLUSIONS: Parathyroid adenomas in equids can be successfully localised with ultrasonography and scintigraphy. Surgical excision appears more likely to be successful for single gland disease at the thoracic inlet.

Natural killer cells induce neutrophil extracellular trap formation in venous thrombosis.

Essentials Neutrophil extracellular traps (NETs) are generated during deep vein thrombosis (DVT). The role of interferon γ (IFNγ) and natural killer (NK) cells in NET formation was studied. IFNγ promote venous thrombosis through NET formation. NK cell depletion reduces DVT. SUMMARY: Background Neutrophils contribute to venous thrombosis through the release of neutrophil extracellular traps (NETs), but the mechanism triggering their formation remains unclear. In vitro data show that interferon (IFN)-γ induces the formation of NETs. Objectives To determine whether IFN-γ and the transcription factor T-box expressed on T cells (Tbet) promote venous thrombosis through neutrophil activation. Methods Venous thrombosis was induced by flow restriction in the inferior vena cava in IFN-γ-/- , Tbet-/- or wild-type (WT) mice. After 48 h, thrombus size was measured by the use of high-frequency ultrasound. NET formation was determined by immunofluorescence. Results and Conclusions Thrombus formation was reduced in Tbet-/- and IFN-γ-/- mice, suggesting that Tbet/IFN-γ-expressing cells are required for venous thrombosis. The number of NETs formed during thrombosis was significantly lower in Tbet-/- and IFN-γ-/- mice. NET formation was also decreased in WT mice treated with an IFN-γ-blocking antibody. Injection of recombinant IFN-γ into IFN-γ-/- mice rescued the phenotype. Natural killer (NK) cells were specifically depleted prior to venous thrombosis induction. NK cell depletion results in decreased NET formation and smaller thrombi, suggesting that NK cells are required for thrombus development. In depleted mice, adoptive transfer of WT NK cells induced a similar thrombosis burden as in WT mice. In contrast, adoptive transfer of IFN-γ -/- NK cells resulted in thrombi similar in size to those in depleted mice. In vitro, we showed that WT neutrophils released fewer NETs when they were cocultured with IFN-γ-/- NK cells. This study demonstrates that NK cell-dependent IFN-γ production is crucial for thrombus development by promoting the formation of NETs by neutrophils.

Factors associated with survival, laminitis and insulin dysregulation in horses diagnosed with equine pituitary pars intermedia dysfunction.

BACKGROUND: Pituitary pars intermedia dysfunction (PPID) is a commonly described endocrine disorder in higher latitudes of the Northern hemisphere but the description of the disease at lower latitudes and in the Southern hemisphere is limited. OBJECTIVES: Document the clinical features of PPID at different Australian latitudes and climates, and investigate factors associated with survival, laminitis and insulin dysregulation (ID). STUDY DESIGN: Retrospective study of 274 equids from eight institutions across Australia. METHODS: A diagnosis of PPID was based on endogenous ACTH, overnight dexamethasone suppression test, thyrotropin-releasing hormone stimulation test or necropsy. Clinical and clinicopathologic characteristics of PPID and therapeutic responses were investigated. Laminitis was diagnosed by radiographic or histologic changes and ID was diagnosed based on endogenous insulin, an oral glucose test or a 2-step insulin-response test. RESULTS: Being a pony, having a higher body condition score and pergolide administration were associated with survival. The clinical presentation of PPID changed with latitude and climate, with anhidrosis and polyuria/polydipsia more commonly recognised at lower latitudes. Laminitis was diagnosed in 89.9% of cases and ID was present in 76.5% of cases in which they were investigated. MAIN LIMITATIONS: Despite the sample size, the lack of uniform testing at all locations (primary or referral cases) and the incompleteness of data sets limited the power of the statistical analyses. CONCLUSIONS: PPID can present with variable signs at different latitudes and climates, and ID should be investigated in equids diagnosed with PPID. Adequate body condition and administration of pergolide are fundamental in PPID management.

Continuous digital hypothermia prevents lamellar failure in the euglycaemic hyperinsulinaemic clamp model of equine laminitis.

BACKGROUND: Continuous digital hypothermia can prevent the development and progression of laminitis associated with sepsis but its effects on laminitis due to hyperinsulinaemia are unknown. OBJECTIVES: To determine the effects of continuous digital hypothermia on laminitis development in the euglycaemic hyperinsulinaemic clamp model. STUDY DESIGN: Randomised, controlled (within subject), blinded, experiment. METHODS: Eight clinically normal Standardbred horses underwent laminitis induction using the euglycaemic hyperinsulinaemic clamp model (EHC). At initiation of the EHC, one forelimb was continuously cooled (ICE), with the other maintained at ambient temperature (AMB). Dorsal lamellar sections (proximal, middle, distal) were harvested 48 h after initiation of the EHC and were analysed using histological scoring (0-3) and histomorphometry. Cellular proliferation was quantified by counting epidermal cell nuclei staining positive with an immunohistochemical proliferation marker (TPX2). RESULTS: Severe elongation and disruption of SEL with dermo-epidermal separation (score of 3) was observed in all AMB feet at one or more section locations, but was not observed in any ICE sections. Overall 92% of the AMB sections received the most severe histological score (grade 3) and 8% were grade 2, whereas ICE sections were classified as either grade 1 (50%) or grade 2 (50%). Relative to AMB feet, ICE sections were 98% less likely to exhibit grades 2 or 3 (OR: 0.02, 95% CI 0.001, 0.365; P<0.01). Histomorphometry measurements of total and nonkeratinised primary epidermal lamellar length were significantly increased (P<0.01) in AMB limbs compared with ICE. TPX2 positive cell counts were significantly increased (P<0.01) in AMB limbs compared with ICE. MAIN LIMITATIONS: Continuous digital hypothermia was initiated before recognition of laminitis and therefore the clinical applicability requires further investigation. CONCLUSIONS: Continuous digital hypothermia reduced the severity of laminitis in the EHC model and prevented histological lesions compatible with lamellar structural failure.

First case of sternum replacement with a bioceramic prosthesis after radio-induced sarcoma.

Objectives: To date, no "gold standard" technique has been developed for sternum replacement in cases of radioinduced sarcoma, which is a rare and aggressive disease. Current techniques rely on metallic prostheses, meshes, or bone grafts-procedures that that are associated with several complications. We therefore tried a new solution that might simplify and optimize this surgery. Methods: We used a porous alumina ceramic prosthesis (Ceramil: i.ceram, Limoges, France) that has several interesting characteristics, such as osseointegration, biocompatibility, radiolucency, and high mechanical strength. Results: We report the first case of sternal replacement surgery involving the implantation of a ceramic prosthesis after radio-induced sternal sarcoma. In 2005, a 54-year-old woman was diagnosed with local breast cancer for which she underwent all appropriate treatment. Ten years later, she developed radio-induced sarcoma of the sternum. A complete sternal replacement was performed on 24 April 2015, with no postoperative complications. Imaging by 18F-flurodeoxyglucose positron-emission tomography-computed tomography performed 26 months after the surgery showed no local recurrence. The patient seems to have fully recovered and has resumed normal activity. Conclusions: This new technique is promising. For the first time, we highlight the feasibility, safety, and efficacy of sternal replacement using a porous alumina ceramic prosthesis.

The diagnosis of equine insulin dysregulation.

Insulin dysregulation is the hallmark of equine metabolic syndrome and has received attention because of its direct association with laminitis. In the absence of an adequate treatment for laminitis, a focus on prophylaxis is needed, making early detection of individuals at risk of developing laminitis one of the main challenges in equine endocrinology. Recent studies have shown that insulin dysregulation goes beyond tissue insulin resistance and it is now demonstrated that the equine enteroinsular axis plays a major role in insulin secretion and equine hyperinsulinaemia. In this review, we discuss the different tests currently available to diagnose insulin dysregulation in horses: the ones investigating tissue insulin resistance and those investigating the enteroinsular axis, detailing their goals, practicalities and limitations. This review supports the contention that the diagnosis of equine insulin dysregulation should now be based on the investigation of both tissue insulin resistance and the equine enteroinsular axis. Regardless of the tests used many factors of variation, such as breed, diet, fasting state or season, have been identified and could potentially confound the results of a specific test. Therefore, careful interpretation of the results of a given test in each individual situation is required to optimise the detection of horses at risk of laminitis.

[Diagnosis of lung cancer. Role of PET/CT fusion scan in lung cancer]. / Diagnostic des CBP. L'apport de la fusion TEP-TDM dans le diagnostic des cancers bronchiques.

Whole-body positron emission tomography (PET) with radiolabeled [18F]-fluoro-2-deoxy-D-glucose (18FDG) plays an important role in the diagnosis, staging, and management of lung cancer. The preferential accumulation of FDG in malignant cells assists in the differentiation of benign and malignant tissue. However, PET alone does not allow an accurate anatomic localisation of FDG uptake. The combination of PET with CT images improves the spatial resolution, sensitivity and specificity of the test. PET/CT, as with PET alone, is indicated for the diagnosis of pulmonary nodules as well as the locoregional and extrathoracic staging of non-small-cell lung cancer (NSCLC). In many published studies, PET/CT has been shown to be superior to CT alone, to PET alone, or to both imaging techniques used separately to evaluate pulmonary nodules or for staging NSCLC. Now, PET/CT imaging is being investigated in the staging and management of small-cell lung cancer, in radiation treatment planning, in response prediction following treatment and in the detection of lung cancer recurrence in NSCLC. PET/CT is likely to have an important role in the management of lung cancer. New machines are in development and new radiopharmaceutical agents must be assessed.

[Pleural metastases of sclerosing epithelioid fibrosarcoma]. / Métastases pleurales d'un fibrosarcome épithélioïde sclérosant.

Sclerosing epithelioid fibrosarcoma is a rare tumor recently described. The histological presentation can be confused with certain soft tissue benign tumors and certain sarcomas. Metastatic spread is usually late in the natural course of the disease. We report a case of recurrent sclerosing epithelioid fibrosarcoma with pleural metastases which developed ten years after surgical resection of the primary tumor. The tumor was formed by small uniform round epithelioid cells with a clear cytoplasm. The tumor cells were strongly positive for vimentin. This clinical case is discussed in light of other cases reported in the literature.

Cerebrospinal Nematodiasis in 20 Camelids.

BACKGROUND: Information about the clinical and clinicopathologic aspects of cerebrospinal nematodiasis (CN) in camelids is limited. HYPOTHESIS: Clinical and therapeutic variables will be identified as factors predictive of survival. ANIMALS: Client-owned camelids suspected of having CN admitted to Purdue University between 1995 and 2015. METHODS: A retrospective study was performed. A diagnosis of CN was based on cerebrospinal fluid (CSF) eosinophilic pleocytosis or postmortem findings. RESULTS: Eleven alpacas and 9 llamas met the inclusion criteria. Seventy-five percent of the camelids were male (27% castrated and 73% intact). Common clinical abnormalities included proprioceptive deficits (100% of animals), recumbency (55%), tachypnea (55%), and ataxia (40%). Among the 85% of treated animals, 100% received PO fenbendazole, and 88% received a nonsteroidal anti-inflammatory drug. The survival rate to discharge was 45%. Plasma fibrinogen concentration, creatine kinase activity, and serum creatinine concentration were significantly higher in nonsurvivors. Blood eosinophil count, platelet count, and total CO2 were significantly lower in nonsurvivors. Factors associated with survival were species, sex, absence of treatment with corticosteroids, and clinical improvement. There was no association between recumbency at admission and survival. A plasma fibrinogen concentration above >266 mg/dL was an excellent diagnostic test to predict survival in the presence of neurological signs or CSF eosinophilia. CONCLUSIONS: Although prognosis for CN in camelids is guarded, presence of recumbency at admission is not predictive of nonsurvival. Male camelids and llamas appear more likely to die from CN. Corticosteroid treatment is contraindicated in animals diagnosed with CN.

The Effect of Fasting Duration on Baseline Blood Glucose Concentration, Blood Insulin Concentration, Glucose/Insulin Ratio, Oral Sugar Test, and Insulin Response Test Results in Horses.

OBJECTIVES: Published descriptions of the oral sugar test (OST) and insulin response test (IRT) have been inconsistent when specifying the protocol for fasting horses before testing. The purpose of our study was to examine the effect of fasting duration on blood glucose concentration, blood insulin concentration, glucose/insulin ratio, OST, and IRT results in horses. ANIMALS: Ten healthy adult horses. PROCEDURES: Both OST and IRT were performed on horses without fasting and after fasting for 3, 6, and 12 hours. Thus, 8 tests were performed per horse in a randomized order. Blood collected at the initial time point of the OST was analysed for both blood glucose and serum insulin concentrations so that baseline concentrations and the glucose/insulin ratio could be determined. Unless fasted, horses had free-choice access to grass hay. RESULTS: There was no effect of fasting and fasting duration on blood glucose concentration, serum insulin concentration, glucose/insulin ratio, or the OST. Response to insulin in the IRT was decreased in fasted horses. The effect increased with fasting duration, with the least response to insulin administration after a 12-hour fast. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicate that insulin sensitivity is not a fixed trait in horses. Fasting a horse is not recommended for a glucose/insulin ratio or IRT, and fasting a horse for 3 hours is recommended for the OST.

Simple sequence repeat polymorphism within the p53 gene.

This report describes a new polymorphism, in intron 3 of the p53 gene, which consists of a single repeat of 16 nucleotides, absent in the published wild-type p53 gene sequence. In the Caucasian population tested (n = 82), 28% of individuals were heterozygotes for this polymorphism. Using PCR-based analysis, we were able to demonstrate p53 allelic losses in three of six breast tumors from heterozygote patients tested.

Growth arrest-specific 6 regulates thrombin-induced expression of vascular cell adhesion molecule-1 through forkhead box O1 in endothelial cells.

BACKGROUND: Growth arrest-specific 6 (Gas6)-deficient mice are protected against venous thromboembolism (VTE), suggesting a role for Gas6 in this disorder. We previously demonstrated that Gas6 induces forkhead box O1 (FoxO-1) phosphorylation through the phosphoinositide 3-kinase-Akt pathway. FoxO-1 regulates the expression of vascular cell adhesion molecule-1 (VCAM-1), a molecule that has been implicated in VTE. OBJECTIVES: To assess the role of FoxO-1 in Gas6-dependent VCAM-1 expression. METHODS: Thrombin was used to stimulate endothelial cells (ECs). Wild-type (WT) and Gas6(-/-) ECs were transfected with small interfering RNA targeting Axl or FoxO-1, a luciferase-coupled plasmid containing the FoxO-1 consensus sequence, and a phosphorylation-resistant FoxO-1 mutant, or treated with an Akt inhibitor. VCAM-1 mRNA expression was measured by real time-qPCR. VCAM-1 protein expression and FoxO-1 and Akt phosphorylation were assessed by western blot analysis. FoxO-1 localization was assessed by immunofluorescence. Adhesion of bone marrow mononuclear cells (BM-MCs) on ECs was assessed by fluorescence. RESULTS AND CONCLUSIONS: Thrombin induces both VCAM-1 expression and FoxO-1 phosphorylation and nuclear exclusion in WT ECs only. Silencing of FoxO-1 enhances VCAM-1 expression in both WT and Gas6(-/-) ECs. Inhibition of Akt or FoxO-1 phosphorylation prevents VCAM-1 expression in WT ECs. These data show that Gas6 induces FoxO-1 phosphorylation, leading to derepression of VCAM-1 expression. BM-MC-EC adhesion is increased by thrombin in WT ECs. BM-MC-EC adhesion is further increased when FoxO-1 is silenced, but decreased when FoxO-1 phosphorylation is inhibited. These results demonstrate that the Gas6-FoxO-1 signaling axis plays an important role in VCAM-1 expression in the context of VTE by promoting BM-MC-EC adhesion.