The leukemia inhibitory factor receptor gene is not involved in the etiology of pituitary dwarfism in German shepherd dogs

Pituitary dwarfism in German shepherd dogs is characterized by combined pituitary hormone deficiency (CPHD) and intrapituitary cyst formation. Activation of the leukemia inhibitory factor (LIF)-LIF receptor (LIFR) signal transduction pathway results in a similar phenotype in (transgenic) mice. We therefore assessed the role of the LIFR in the etiology of pituitary dwarfism in German shepherd dogs. A polymorphic microsatellite marker (UULIFR) was used to analyze the segregation of the LIFR gene in 22 German shepherd dogs from 4 pedigrees, each including one dwarf. There was no allelic association between UULIFR and the dwarfism phenotype. Based on our findings LIFR was excluded as a candidate gene for CPHD.     

Ki-67 and minichromosome maintenance-7 (MCM7) expression in canine pituitary corticotroph adenomas

Pituitary-dependent hyperadrenocorticism (PDH) caused by pituitary corticotroph adenoma is a common endocrine disorder in dogs. The ratio between pituitary height and the area of the brain (P/B) has been used to evaluate the pituitary size. A P/B ratio > 0.31 indicates an enlarged pituitary, whereas a P/B ratio ≤ 0.31 indicates a nonenlarged pituitary. The aim of this study was to investigate the expression of proliferation markers Ki-67 and minichromosome maintenance-7 (MCM7) in canine corticotroph adenomas in enlarged and in nonenlarged pituitaries and to evaluate their relation with the size of canine pituitary corticotroph adenomas. Ki-67 and MCM7 expression in ACTH-positive tumor cells was determined by dual-labeling immunohistochemistry in resected corticotroph adenomas from 15 dogs with PDH. The mean ± SD Ki-67 labeling index (LI) was 0.55% ± 0.59% in corticotroph adenomas with nonenlarged pituitaries and 1.6% ± 0.6% in adenomas with enlarged pituitaries. The MCM7 LI in corticotroph adenomas with nonenlarged pituitaries and in adenomas with enlarged pituitaries was 2.9% ± 2.2% and 10.9% ± 3.7%, respectively. The Ki-67 LI and MCM7 LI were significantly greater in the adenomas with enlarged pituitaries than in the adenomas with nonenlarged pituitaries (P < 0.01 and P < 0.01, respectively). The MCM7 LI was significantly greater than the Ki-67 LI in adenomas (P < 0.01). The Ki-67 LI was positively correlated with the MCM7 LI (r = 0.820, P < 0.01), and the P/B ratio was positively correlated with the Ki-67 LI (r = 0.560, P = 0.03) and the MCM7 LI (r = 0.854, P < 0.01). In conclusion, canine corticotroph adenomas in enlarged pituitaries show greater proliferation potential than do adenomas in nonenlarged pituitaries. MCM7 expression was significantly greater than Ki-67 expression in canine pituitary corticotroph adenomas. Thus, MCM7 may be superior to Ki-67 as a proliferation marker in pituitary tumors.     

Expression of leukemia inhibitory factor and leukemia inhibitory factor receptor in the canine pituitary gland and corticotrope adenomas

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine of the IL-6 family that activates the hypothalamic-pituitary-adrenal axis and promotes corticotrope cell differentiation during development. The aim of this study was to investigate the expression of LIF and its receptor (LIFR) in the canine pituitary gland and in corticotrope adenomas, and to perform a mutation analysis of LIFR. Using immunohistochemistry, immunofluorescence, and quantitative expression analysis, LIF and LIFR expression were studied in pituitary glands of control dogs and in specimens of corticotrope adenoma tissue collected through hypophysectomy in dogs with pituitary-dependent hypercortisolism (PDH, Cushing's disease). Using sequence analysis, cDNA was screened for mutations in the LIFR. In the control pituitary tissues and corticotrope adenomas, there was a low magnitude of LIF expression. The LIFR, however, was highly expressed and co-localized with ACTH_1-24 expression. Cytoplasmatic immunoreactivity of LIFR was preserved in corticotrope adenomas and adjacent nontumorous cells of pars intermedia. No mutation was found on mutation analysis of the complete LIFR cDNA. Surprisingly, nuclear to perinuclear immunoreactivity for LIFR was present in nontumorous pituitary cells of the pars distalis in 10 of 12 tissue specimens from PDH dogs. These data show that LIFR is highly co-expressed with adrenocorticotropic hormone (ACTH) and α-melanocyte-stimulating hormone (α-MSH) in the canine pituitary gland and in corticotrope adenomas. Nuclear immunoreactivity for LIFR in nontumorous cells of the pars distalis may indicate the presence of a corticotrope adenoma.     

The use of 17-hydroxyprogesterone in the diagnosis of canine hyperadrenocorticism

A number of dogs are seen with clinical signs consistent with hyperadrenocorticism (HAC), supporting CBC and biochemical findings, but the disease cannot be confirmed with either the ACTH stimulation test or the low-dose dexamethasone suppression test (LDDST). Therefore, another screening test is required to aid diagnosis in these atypical cases of HAC. The aim of this study was to investigate whether measuring 17-hydroxyprogesterone (OHP) concentrations could be used in this role. Plasma cortisol and OHP concentrations were measured in dogs with clinical signs suggestive of HAC before and after administration of exogenous ACTH. In dogs with HAC, plasma OHP showed an exaggerated response to ACTH stimulation. This was seen in both typical cases of HAC with a positive cortisol response to ACTH administration and in atypical cases with negative screening test results. The test can be performed on plasma already taken for a conventional ACTH stimulation test. Post-ACTH OHP concentrations decreased after treatment with mitotane or adrenalectomy. These results suggest that OHP measurements can be used as an aid to diagnose and manage canine HAC.     

Pasireotide for the Medical Management of Feline Hypersomatotropism

Background

Feline hypersomatotropism (HST) is a cause of diabetes mellitus in cats. Pasireotide is a novel multireceptor ligand somatostatin analog that improves biochemical control of humans with HST.

Hypothesis/Objectives

Pasireotide improves biochemical control of HST and diabetes mellitus in cats.

Animals

Hypersomatotropism was diagnosed in diabetic cats with serum insulin‐like growth factor‐1 (IGF‐1) concentration >1,000 ng/mL by radioimmunoassay and pituitary enlargement.

Methods

Insulin‐like growth factor 1 was measured and glycemic control assessed using a 12‐hour blood glucose curve on days 1 and 5. On days 2, 3, and 4, cats received 0.03 mg/kg pasireotide SC q12h. IGF‐1, insulin dose, and estimated insulin sensitivity (product of the area under the blood glucose curve [BGC] and insulin dose) were compared pre‐ and post treatment. Paired t‐tests or Wilcoxon signed rank tests were employed for comparison where appropriate; a linear mixed model was created to compare BGC results.

Results

Insulin‐like growth factor 1 decreased in all 12 cats that completed the study (median [range] day 1: 2,000 ng/mL [1,051–2,000] and day 5: 1,105 ng/mL [380–1,727], P = .002, Wilcoxon signed rank test). Insulin dose was lower on day 5 than on day 1 (mean reduction 1.3 [0–2.7] units/kg/injection, P = .003, paired t‐test). The product of insulin dose and area under the BGC was lower on day 5 than day 1 (difference of means: 1,912; SD, 1523; u × mg/dL × hours, P = .001; paired t‐test). No clinically relevant adverse effects were encountered.

Conclusions

Short‐acting pasireotide rapidly decreased IGF‐1 in cats with HST and insulin‐dependent diabetes. The decrease in IGF‐1 was associated with increased insulin sensitivity.     

Expression of Ki-67, PCNA,and p27kip1 in canine pituitary corticotroph adenomas

Pituitary-dependent hypercortisolism (PDH), which is caused by adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas, is a common endocrinopathy in dogs. Dogs with non-enlarged pituitaries harboring a microadenoma have a better prognosis than those with enlarged pituitaries. The aim of this study was to investigate the expression of the proliferation markers Ki-67 and proliferating cell nuclear antigen (PCNA) and the cell-cycle inhibitor p27kip1 in corticotroph adenomas in enlarged and non-enlarged pituitaries. The expression of Ki-67, PCNA, and p27kip1 was analyzed by immunohistochemical staining of 17 pituitary adenoma samples harvested during pituitary surgery in dogs with PDH. The labeling index was calculated by counting the number of immunopositive cells per 1,000 cells. The mean (± standard deviation) labeling index for Ki-67 was 8.4% ± 14.2% for the group with enlarged pituitaries, and 8.8% ± 5.5% for the group with non-enlarged pituitaries; that for PCNA was 35.5% ± 12.2% and 37.0% ± 15.5%; and that for p27kip1 was 29.3% ± 22.6% and 42.5% ± 27.9%, respectively. No significant differences in Ki-67, PCNA, and p27kip1 labeling indices were found between enlarged and non-enlarged pituitaries. However, a trend toward significance was observed when comparing the expression of p27kip1 in enlarged pituitaries versus normal pituitary tissue. It is concluded that Ki-67 and PCNA are not useful as proliferative markers for studying the pathobiology of pituitary corticotroph adenomas in dogs.     

High-resolution fluorodeoxyglucose positron emission tomography and magnetic resonance imaging findings of a pituitary microtumor in a dog

A 16-year-old, castrated, male English cocker spaniel dog was presented due to generalized alopecia. Routine clinical pathology, endocrine and abdominal ultrasonography results were consistent with a diagnosis of pituitary-dependent hyperadrenocorticism. The adenohypophyseal lesion was clearly visualized on both 3 T and 7 T magnetic resonance imaging (MRI) of the pituitary gland. Although biochemical and MRI findings were consistent with a functional pituitary microtumor, a pituitary lesion was not detected using ¹⁸F-fluorodeoxyglucose positron emission tomography (FDG-PET). This report firstly describes the application of high-resolution FDG-PET to a spontaneous pituitary microtumor in a dog.     

Negative energy balance increases periprandial ghrelin and growth hormone concentrations in lactating dairy cows

The reported effects of feeding on growth hormone (GH) secretion in ruminants have been inconsistent, and are likely influenced by energy status of animals. High-producing dairy cows in early lactation and late lactation were used to assess the effects of energy balance on temporal variation of plasma metabolites and hormones. Cows were fed a single diet once daily, and feed was withdrawn for 90 min prior to feeding. Beginning at the time of feed withdrawal, plasma samples were collected via jugular catheters hourly for 24h. Concentrations of non-esterified fatty acids and GH were measured for all samples, while insulin, glucose, and acylated (active) ghrelin were quantified for four sample times around feeding. As expected, calculated energy balance was significantly lower in early lactation than late lactation cows (-43.5 MJ retained/day versus 7.2 MJ retained/day). Following the primary meal of the day, a GH surge was observed in early lactation but not in late lactation cows. This difference was not explained by temporal patterns in non-esterified fatty acid, insulin, or glucose concentrations. However, a preprandial ghrelin surge was observed in early lactation only, suggesting that ghrelin was responsible for the prandial GH surge in this group. Results of a stepwise regression statistical analysis showed that both preprandial ghrelin concentration and energy balance were significant predictors of prandial GH increase over baseline. Adaptations to negative energy balance in lactating dairy cattle likely include enhanced ghrelin secretion and greater GH response to ghrelin.     

Computerized morphometry of mean nuclear diameter and nuclear roundness in canine mammary gland tumors on cytologic smears

BACKGROUND: The use of computer-based image analysis systems in veterinary oncology has increased. Computerized morphometry is a part of image analysis that describes geometric figures of cellular structures in any dimension. Most investigators have performed morphometric analysis on histologic specimens. Computer-assisted nuclear cytomorphometry can provide important preoperative information on neoplastic lesions in animals. OBJECTIVES: The aim of this study was to define whether the morphometric parameters of mean nuclear diameter and nuclear roundness could be used to differentiate benign from malignant canine mammary gland tumors on cytologic specimens. METHODS: Mean nuclear diameter and nuclear roundness were determined by computer-assisted morphometry of epithelial cells in Hemacolor-stained cytologic smears from normal canine mammary gland (n = 7) and from canine mammary adenomas (n = 8), tubulopapillary carcinomas (n = 9), and solid carcinomas (n = 6). Data were analyzed by the Mann-Whitney U test. RESULTS: Significant differences (P <.001) were found in mean nuclear diameter and nuclear roundness among all tumor types and in comparison with normal canine mammary gland epithelial cells (except for nuclear roundness between tubulopapillary carcinomas and solid carcinomas). CONCLUSIONS: The morphometric parameters of mean nuclear diameter and nuclear roundness can be used in the preoperative differentiation of benign from malignant canine mammary gland tumors.     

Adenohypophyseal Function in Dogs with Primary Hypothyroidism and Nonthyroidal Illness

Background: A recent study of dogs with induced primary hypothyroidism (PH) demonstrated that thyroid hormone deficiency leads to loss of thyrotropin (TSH) hypersecretion, hypersomatotropism, hypoprolactinemia, and pituitary enlargement with large vacuolated "thyroid deficiency" cells that double-stained for growth hormone (GH) and TSH, indicative of transdifferentiation of somatotropes to thyrosomatropes.
Hypothesis: Similar functional changes in adenohypophyseal function occur in dogs with spontaneous PH as do in dogs with induced PH, but not in dogs with nonthyroidal illness (NTI).
Animals: Fourteen dogs with spontaneous PH and 13 dogs with NTI.
Methods: Adenohypophyseal function was investigated by combined intravenous administration of 4 hypophysiotropic releasing hormones (4RH test), followed by measurement of plasma concentrations of ACTH, GH, luteinizing hormone (LH), prolactin (PRL), and TSH. In the PH dogs this test was repeated after 4 and 12 weeks of thyroxine treatment.
Results: In 6 PH dogs, the basal TSH concentration was within the reference range. In the PH dogs, the TSH concentrations did not increase with the 4RH test. However, TSH concentrations increased significantly in the NTI dogs. Basal and stimulated GH and PRL concentrations indicated reversible hypersomatotropism and hyperprolactinemia in the PH dogs, but not in the NTI dogs. Basal and stimulated LH and ACTH concentrations did not differ between groups.
Conclusions and Clinical Importance: Dogs with spontaneous PH hypersecrete GH but have little or no TSH hypersecretion. Development of hyperprolactinemia (and possible galactorrhea) in dogs with PH seems to occur only in sexually intact bitches. In this group of dogs with NTI, basal and stimulated plasma adenohypophyseal hormone concentrations were not altered.     

Characterization of spheres derived from canine mammary gland adenocarcinoma cell lines

There is increasing evidence for the presence of cancer stem cells in several solid tumors, and these cancer stem cells have a potential role in tumor initiation, aggression, and recurrence. The stem cell-like properties of spheres derived from canine mammary tumors remain largely elusive. We attempted to induce sphere formation using four cell lines of canine mammary adenocarcinoma, and characterized the spheres derived from a CHMp line in vitro and in vivo. The CHMp-derived spheres showed predominantly CD44⁺CD24⁻ population, higher expression of stem cell-related genes, such as CD133, Notch3 and MDR, and higher resistance to doxorubicin compared with the CHMp-derived adherent cells. Xenograft transplantations in nude mice demonstrated that only 1 × 10⁴ sphere cells were sufficient for tumor formation. Use of the sphere assay on these sphere-derived tumors showed that sphere-forming cells were present in the tumors, and were maintained in serial transplantation. We propose that spheres derived from canine mammary adenocarcinoma cell lines possess a potential characteristic of cancer stem cells. Spheres derived from canine mammary tumors could be a powerful tool with which to investigate novel therapeutic drugs and to elucidate the molecular and cellular mechanisms that underlie tumorigenesis.     

Methods for diagnosis of canine leishmaniasis and immune response to infection

Canine leishmaniasis (CanL) caused by Leishmania infantum (syn. L. chagasi, in Latin America), which is transmitted by the bite of phlebotomine sand flies, is endemic and affects millions of dogs in Europe, Asia, North Africa and South America. It is an emergent disease in North America. Early detection and treatment of infected animals may be critical in controlling the spread of the disease and is an essential part of human zoonotic visceral leishmaniasis control. The laboratory diagnosis of CanL still poses a challenge, despite progress made in the development of several direct and indirect methods. An effective diagnosis test, apart of being able to confirm a clinical suspicion in a single patient as well as to detect infection in asymptomatic dogs, should have high sensitivity, specificity and reproducibility; it must be simple, easy to perform, non-expensive, feasible in regional laboratories or adaptable for field conditions. Ideally, it should detect all Leishmania-infected dogs, preferentially using non-invasive collection of biological samples. In this paper we review the advantages and shortcomings of the available procedures for CanL diagnosis in the different phases, e.g. pre-patent and patent period of the infection and methods to determine the related immune response.     

Analytical performances of d-ROMs test and BAP test in canine plasma. Definition of the normal range in healthy Labrador dogs

An high level of ROS (Reactive Oxygen Species), due to an increased production of oxidant species and/or a decreased efficacy of antioxidant system, can lead to oxidative stress, an emerging health risk factor involved in the aging and in many diseases, including inflammatory, infectious and degenerative disorders, either in humans or in animals. In the last years some assays panels have been developed to globally evaluate the oxidative balance by means of the concomitant assessment of ROS production and antioxidant system capability. In this report, the validation trials of d-ROMs (Reactive Oxygen Metabolites— derived compounds) and BAP (Biological Antioxidant Potential) tests in canine specie are described and also the specific referral ranges are calculated in a Labrador population. The results of linearity, precision and accuracy trials show that both tests exhibit good to excellent analytical performances. The possibility of measuring oxidative stress in vivo with simple, cheap and accurate tests, d-ROMs test and BAP test, provides for the veterinarians a very suitable tool to monitor oxidative stress and to correctly choice of eventual antioxidant supplementations in diseases proven related to oxidative stress in animals and particularly in dogs. Further studies will be useful to confirm this possibility.     

Use of Oil Red O stain in the cytologic diagnosis of canine liposarcoma

BACKGROUND: Oil Red O, a stain commonly used to demonstrate lipid in frozen tissue, also may be used to stain air-dried cytologic specimens. OBJECTIVE: The purpose of this study was to prospectively evaluate the value of Oil Red O in identifying lipid to aid in the differentiation of liposarcomas from other types of sarcoma. METHODS: Twelve tumor specimens from dogs were evaluated. The tumors were included in the study if initial cytologic evaluation indicated a sarcoma, and if histologic confirmation was available. Oil Red O was applied to all cytologic specimens. RESULTS: Tumor specimens were diagnosed histologically as liposarcoma (3 well-differentiated, 1 pleomorphic), hemangiopericytoma (n = 3), fibrosarcoma (n = 3), malignant fibrous histiocytoma (n = 1), and undifferentiated sarcoma (n = 1). Cytologic specimens from all liposarcomas showed strong positive staining of cytoplasmic vacuoles for lipid. Specimens from other sarcomas stained negative for Oil Red O, with the exception of weak, irregular positive staining in 1 hemangiopericytoma. CONCLUSION: Our results suggest that Oil Red O staining may be an easy, inexpensive, and useful diagnostic tool for the differentiation of liposarcoma from other mesenchymal neoplasms.     

Differentiation of stem progenitor CD9/SOX2-positive cells is promoted with increased prolactin-producing and endothelial cells in the pituitary

Sex-determining region Y-box 2 (SOX2)-positive cells are stem/progenitor cells in the adenohypophysis, comprising the anterior and intermediate lobes (AL and IL, respectively). The cells are located in the marginal cell layer (MCL) facing Rathke’s cleft (primary niche) and the parenchyma of the AL (secondary niche). We previously demonstrated in vitro that the tetraspanin superfamily CD9 and SOX2 double-positive (CD9/SOX2-positive) cells in the IL-side MCL migrate to the AL side and differentiate into hormone-producing and endothelial cells in the AL parenchyma. Here, we performed in vivo studies to evaluate the role of IL-side CD9/SOX2-positive cells in pregnancy, lactation, and treatment with diethylstilbestrol (DES; an estrogen analog) when an increased population of prolactin (PRL) cells was observed in the AL of the rat pituitary. The proportions of CD9/SOX2-, CD9/Ki67-, and PRL/TUNEL-positive cells decreased in the primary and secondary niches during pregnancy and DES treatment. In contrast, the number of CD9/PRL-positive cells increased in the AL-side MCL and AL parenchyma during pregnancy and during DES treatment. The proportion of PRL/Ki67-positive cells increased in the AL-side MCL and AL parenchyma in response to DES treatment. Next, we isolated CD9-positive cells from the IL-side MCL using an anti-CD9 antibody. During cell culture, the cells formed free-floating three-dimensional clusters (pituispheres). Furthermore, CD9-positive cells in the pituisphere differentiated into PRL cells, and their differentiation potential was promoted by DES. These findings suggest that CD9/SOX2-positive cells in the IL-side MCL may act as adult stem cells in the AL parenchyma that supply PRL cells under the influence of estrogen.     

Acromegaly due to a somatroph adenoma in a dog

A 10-year-old uncastrated male Dalmatian dog was referred for gait abnormalities consisting of chronic progressive stiffness and rigidity. Other symptoms were polyphagia associated with weight gain, polyuria and polydipsia, excessive panting, and an inspiratory stridor. The owner had noticed progressive thickening of the skin and enlargement of the tongue over the last 3 years. Physical examination revealed thickening of the skin, redundant skin folds, and enlargement of the tongue. The only remarkable abnormalities found on routine laboratory examination were mild anaemia and an increased serum fructosamine concentration. Circulating concentrations of total thyroxine, free thyroxine, and cTSH, and the results of an ACTH stimulation test were all within reference ranges. The basal serum growth hormone (GH) concentration was markedly elevated (23microg/l) and did not decrease during a glucose tolerance test or after somatostatin administration. The serum insulin-like growth factor-1 concentration was also markedly elevated (1254microg/l). Basal serum insulin concentration was high (95mU/l) and insulin concentrations increased considerably after glucose loading, consistent with insulin resistance. Abdominal ultrasonography showed no abnormalities. Survey radiographs of the vertebral column showed severe spondylosis deformans extending from the cervical to the lumbosacral spine. CT scanning of the skull showed an enlarged pituitary gland with normal enhancement pattern. On post-mortem examination, the entire vertebral column appeared as a single and inflexible structure due to the presence of multiple fused osteophytes. The pituitary gland contained an acidophilic adenoma that immunostained positively for GH (and negatively for ACTH and alpha-MSH). In conclusion, this Dalmatian dog with acromegaly and insulin resistance represents the first case of GH hypersecretion proven to be due to a somatotroph adenoma.