Oestrogen and Progesterone Receptors and COX‐2 Expression in Endometrial Biopsy Samples During Maternal Recognition of Pregnancy in Llamas (Lama glama)

Endometrial expression of oestrogen receptor‐α (ERα), progesterone receptor (PR) and cyclooxigenase‐2 (COX‐2) was evaluated in non‐pregnant and pregnant llamas during the period when luteolysis/maternal recognition of pregnancy is expected to occur. Females (n = 28) were divided into two groups: non‐pregnant llamas were induced to ovulate with a Buserelin injection, and endometrial biopsies were obtained on day 8 (n = 5) or 12 (n = 5) post‐induction of ovulation. Animals of the pregnant group (n = 18) were mated with a fertile male. Pregnancy was confirmed by the visualization of the embryo collected by transcervical flushing in 5 of 9 animals on day 8 post‐mating and by progesterone profile on day 12 post‐mating in 4 of 9 animals, when endometrial biopsies were obtained. An immunohistochemical technique was used to evaluate receptors population and COX‐2 expression. Pregnant llamas showed a higher percentage of positive cells and stronger intensity for ERα than for non‐pregnant llamas in stroma on day 8 and in the luminal epithelium on day 12 post‐induction of ovulation, while a deep decrease in endometrial PR population was reported in pregnant llamas on that day in luminal and glandular epithelia and stroma. In the luminal epithelium, COX‐2 expression was lower in pregnant than in non‐pregnant animals. Briefly, the increase of ERα in pregnant llamas gives further support to the hypothesis that oestrogens are involved in the mechanism of maternal recognition of pregnancy. Endometrial PR decrease in pregnant llamas might be a necessary event to allow the expression of proteins involved in conceptus attachment, a mechanism widely accepted in other species. Moreover, embryo seems to attenuate maternal PGF_2α secretion during early pregnancy by decreasing the endometrial expression of COX‐2 in the luminal epithelium of pregnant llamas.     

Changes in c‐erbB‐2 Immunoexpression in Feline Endometrial Adenocarcinomas

Human epidermal growth factor receptor type 2 (c‐erbB‐2), an oncoprotein with potential prognostic marker and therapeutic use, is overexpressed in several human and animal tumours. But information regarding this molecule in feline tumours is scarce. This study aimed to assess the changes in the immunohistochemical expression of c‐erbB‐2 in feline endometrial adenocarcinomas (FEA) compared to normal endometrium. An immunohistochemistry assay using a specific antibody against c‐erbB‐2 was performed in FEA samples (n = 34) and in normal endometrium in the follicular (FS; n = 12) and luteal (LS; n = 11) stages. In FEA, the c‐erbB‐2 immunoexpression was assessed in neoplastic epithelial cells whilst in normal endometria it was individually evaluated in the surface and the superficial and deep glandular epithelia (SE, SGE and DGE, respectively). In FS and in LS, all the epithelia were positive for c‐erbB‐2; positivity was higher in the SE and the SGE than in DGE. Twenty of the 34 FEA samples (58.8%) were positive for c‐erbB‐2 immunolabelling. Nevertheless, its expression was higher in all the epithelia in the FS compared to FEA (p ≤ 0.0001) or the LS (p = 0.016). The results presented herein suggest that c‐erbB‐2 molecule is differently expressed in the feline endometrium through the oestrous cycle and though it may also be involved in feline endometrial carcinogenesis, a question remains unanswered on the importance of additional pathways of epithelial proliferation in the neoplastic changes in feline endometrium.     

Oestrogen,Progesterone and Oxytocin Receptors and COX‐2 Expression in Endometrial Biopsy Samples from the Induction of Ovulation to Luteolysis in Llamas (Lama glama)

Endometrial expression of oestrogen (ERα), progesterone (PR) and oxytocin receptor (OR) and cyclooxygenase‐2 (COX‐2) was evaluated from the induction of ovulation to luteolysis in llamas. Ovarian activity was daily assessed by ultrasonography in five females. Ovulation was induced immediately after the detection of an ovulatory follicle by a GnRH injection (Day 0). Endometrial samples were obtained by transcervical biopsies from the left and right horns on day 0 and days 4, 8, 10 and 12 post‐GnRH. Blood samples were collected daily for progesterone and estradiol‐17β determinations by RIA. An immunohistochemical technique was used to study receptors population and COX‐2 expression which were then evaluated by two independent observers. The expression of ERα and PR was highest on day 0 in the luminal epithelium and stroma in association with high plasma estradiol‐17β concentrations. Thereafter, a decrease in ERα population was registered on day 4 and a new increase of its expression was observed between days 8 and 12 in those cell types. Conversely, PR population was gradually down‐regulated until its lowest expression was reached on day 10 post‐GnRH in the luminal epithelium. Content of OR was similar throughout the study in all cell types. The expression of COX‐2 was highest from day 8 to 12 post‐GnRH in the luminal epithelium, in relation to the time of maximal PGF_2α release. Both steroid receptors populations and COX‐2 expression were similar between horns. Meanwhile, OR expression was higher in the right than in the left uterine horn. In summary, this study showed that the loss of endometrium sensitivity to progesterone by days 8–10 post‐induction of ovulation and the concomitant increase of COX‐2 expression could play a key role in the mechanism of luteolysis and somehow be related to the short corpus luteum lifespan of llamas.     

COX‐2 Expression in Feline Oral Squamous Cell Carcinoma (FOSCC)– An Immunohistochemical Study and Analysis of Survival

Introduction:  Cyclooxygenase‐2 (COX‐2) catalyses the rate‐limiting step in the conversion of arachidonic acid to eicosanoids and has been found to be overexpressed in many human and some animal cancers. Overexpression of COX‐2 in head and neck SCC in humans is associated with shorter survival times. Non‐resectable, FOSCC is a devastating disease with no effective therapy. Overexpression of COX‐2 in FOSCC may support the anecdotal use of NSAID therapy. Identification of a prognostic marker in cats may permit more effective, targeted therapy.
Methods:  Immunohistochemistry was performed on formalin‐fixed, paraffin‐embedded tissue from 59 FOSCC cases using an indirect staining technique and feline foetal kidney as positive control. Polyclonal antiserum specific to COX‐1 and COX‐2 were used after antigen retrieval with pH6 citrate buffer. Retrospective, observational data were collected by practitioner questionnaires. A Kaplan‐Meier survival plot was derived.
Results:  55/59 questionnaires were returned (93% response rate). Median age at presentation was 10.9 years (range 7–15.5). Median survival time was 44 days (95% CL: 31, 79). Preliminary results show that COX‐1 staining was positive in all tumour tissues and in 86.7%(13/15 cases) of normal tissues. COX‐2 staining was positive in 67.3%(37/55) of tumour tissues and absent in normal tissue. Results of proportional hazards regression for survival and multiple logistic regression for COX expression including age, sex, breed, prior NSAID administration, other medication and COX expression as potential explanatory variables will be presented.
Conclusions:  These results indicated that COX‐1 and COX‐2 expression is seen in FOSCC but may not be predictive for survival.     

Histopathologic and Immunohistochemical Exam in One Case of Canine Endometrial Adenocarcinoma

Canine endometrial carcinomas are rare, and mostly occur in geriatric bitches. In this work, the uterus of a 10‐year‐old female Boxer evidencing an endometrial carcinoma on the body of the uterus was used to describe the histopathological features of the tumour and to study its immunophenotype. In this work, a panel of immunomarkers (cytokeratins AE1/AE3 and 14, vimentin, CD10 and Ki‐67) was applied to the endometrial carcinoma to establish the staining patterns indicative of the tumour agressiveness and cellular differentiation. Additionally DNA ploidy was also performed. In this case, the tumour showed papillar pattern, with large pleomorphic, anaplastic cells and also some aberrant multinucleated and giant cells. In some areas of the tumour, it was also observed cytotrophoblastic‐like cells outlining the papillae. Cytokeratin AE1/AE3 expression was detected in the luminal neoplasic cells. Cytokeratin 14 positivity was sporadic and irregular, and was observed mainly in the luminal epithelium. Only stromal and aberrant cells showed a positive staining to vimentin. Positive membranous staining to CD10 was evidenced by clear epithelial, cytotrophoblastic‐like cells at the tumour surface but not by the stromal cells. The mitotic and Ki‐67 indices were low, suggestive of a weak aggressiveness of the tumour. The multinucleated and giant cells evidenced a positive immunostaining to CK AE1/AE3, and CD 10; its positivity to vimentin was sporadic. This study aims to contribute to the advancement of the knowledge in canine endometrial carcinoma immunophenotype.     

Feline Endometrial Adenocarcinoma in Females <1 Year Old: A Description of Four Cases

Uterine neoplasms of epithelial origin are rare in cats and most often are described in older females. Yet, in less than 2 years, four ovariohysterectomy specimens were submitted from different practices to the Laboratory of Histology and Anatomical Pathology, at UTAD (Vila Real, Portugal), that emitted a diagnosis of feline endometrial adenocarcinoma. Untypically, all the females were aged <1 year old at the surgery. Access to the clinical files was requested to document the clinical features of the four cases, including any complementary data available, to construct the present case reports. The clinical situation developed with discrete signs, but vulvar discharge was present in three cases, ranging from bloody to brownish or colourless, and from purulent to mucous. The females were in dioestrus, although the oestrus remained unperceived in most cases. In this study, the four clinical situations are described and discussed on the basis of available literature, highlighting the aspects that may impair an early diagnosis and that may favour the progression of the disease and also that age should not be an excluding criteria when analysing the differential diagnosis list.     

COX‐2 and c‐kit expression in canine gliomas

Gliomas are among the most common primary neural tumours of dogs. Cyclooxygenase‐2 (COX‐2) and c‐kit overexpression are associated with increased aggressiveness of gliomas and decreased survival in human beings. COX‐2 is the inducible form of cyclooxygenase, which catalyzes prostaglandin formation and may increase tumour proliferation and angiogenesis. C‐kit is a tyrosine kinase receptor involved in normal cell physiology; c‐kit is upregulated in some canine tumours. In this retrospective study, 20 canine gliomas were identified: 11 (55%) oligodendrogliomas, including 1 anaplastic variant; 1 (5%) oligoastrocytoma; and 8 (40%) astrocytomas, of which 2 were glioblastoma multiforme. None of the gliomas expressed COX‐2. None of the gliomas were immunoreactive for c‐kit, although all three high‐grade tumours had intramural vascular expression. Consequently, COX‐2 inhibitors would likely be ineffective against canine gliomas. C‐kit inhibitors may have an anti‐angiogenic effect in high‐grade gliomas, but would likely be ineffective in low‐ and medium‐grade tumours.     

Effects of Hypo‐ and Hyperthyroidism on Proliferation,Angiogenesis, Apoptosis and Expression of COX‐2 in the Corpus Luteum of Female Rats

Although thyroid dysfunction occurs frequently in humans and some animal species, the mechanisms by which hypo‐ and hyperthyroidism affect the corpus luteum have not been thoroughly elucidated. This study evaluated the levels of proliferative activity, angiogenesis, apoptosis and expression of cyclooxygenase‐2 in the corpus luteum of female rats with thyroid dysfunction. These processes may be important in understanding the reproductive changes caused by thyroid dysfunction. A total of 18 adult female rats were divided into three groups (control, hypothyroid and hyperthyroid) with six animals per group. Three months after treatment to induce thyroid dysfunction, the rats were euthanized in the dioestrus phase. The ovaries were collected and immunohistochemically analysed for expression of the cell proliferation marker CDC‐47, vascular endothelial growth factor (VEGF), VEGF receptor Flk‐1 and cyclooxygenase‐2 (COX‐2). Apoptosis was evaluated using the TUNEL assay. Hypothyroidism reduced the intensity and area of COX‐2 expression in the corpus luteum (p < 0.05), while hyperthyroidism did not alter COX‐2 expression in the dioestrus phase. Hypothyroidism significantly reduced the expression of CDC‐47 in endothelial cells and pericytes in the corpus luteum, whereas hyperthyroidism did not induce a detectable change in CDC‐47 expression (p > 0.05). Hypothyroidism reduced the level of apoptosis in luteal cells (p < 0.05) and increased VEGF expression in the corpus luteum. In contrast, hyperthyroidism increased the level of apoptosis in the corpus luteum (p < 0.05). In conclusion, thyroid dysfunction differentially affects the levels of proliferative activity, angiogenesis and apoptosis and COX‐2 expression in the corpus luteum of female rats.     

COX‐2, mPGES‐1 and EP2 receptor immunohistochemical expression in canine and feline malignant mammary tumours

Prostaglandin (PG) signalling is involved in human and animal cancer development. PG E₂ (PGE₂) tumour‐promoting activity has been confirmed and its production is controlled by Cyclooxygenase‐2 (COX‐2) and microsomal PGE synthase‐1 (mPGES‐1). Evidence suggests that mPGES‐1 and COX‐2 contribute to carcinogenesis through the EP2 receptor. The aim of our study was to detect by immunohistochemistry COX‐2, mPGES‐1 and EP2 receptor expression in canine (n = 46) and feline (n = 50) mammary tumours and in mammary non‐neoplastic tissues. COX‐2 positivity was observed in 83% canine and 81% feline mammary carcinomas, mPGES‐1 in 75% canine and 66% feline mammary carcinomas and the EP2 receptor expression was observed in 89% canine and 54% feline carcinomas. The frequency of COX‐2, EP2 receptor and mPGES‐1 expression was significantly higher in carcinomas than in non‐neoplastic tissues and adenomas. COX‐2, mPGES‐1 and EP2 receptor expression was strongly associated. These findings support a role of the COX‐2/PGE2 pathway in the pathogenesis of these tumours.     

Nonlinear mixed‐effects pharmacokinetic modeling of the novel COX‐2 selective inhibitor vitacoxib in dogs

The objective of this study was to develop a nonlinear mixed‐effects model of vitacoxib disposition kinetics in dogs after intravenous (I.V.), oral (P.O.), and subcutaneous (S.C.) dosing. Data were pooled from four consecutive pharmacokinetic studies in which vitacoxib was administered in various dosing regimens to 14 healthy beagle dogs. Plasma concentration versus time data were fitted simultaneously using the stochastic approximation expectation maximization (SAEM) algorithm for nonlinear mixed‐effects as implemented in Monolix version 2018R2. Correlations between random effects and significance of covariates on population parameter estimates were evaluated using multiple samples from the posterior distribution of the random effects. A two‐compartment mamillary model with first‐order elimination and first‐order absorption after P.O. and S.C. administration, best described the available pharmacokinetic data. Final parameter estimates indicate that vitacoxib has a low‐to‐moderate systemic clearance (0.35 L hr^?1 kg^?1) associated with a low global extraction ratio, but a large volume of distribution (6.43 L/kg). The absolute bioavailability after P.O. and S.C. administration was estimated at 10.5% (fasted) and 54.6%, respectively. Food intake was found to increase vitacoxib oral bioavailability by a fivefold, while bodyweight (BW) had a significant impact on systemic clearance, thereby confirming the need for BW adjustment with vitacoxib dosing in dogs.     

Expression of Matrix Metalloproteinases (MMP‐2, MMP‐9) and their Inhibitors (TIMP‐1, TIMP‐2) in Canine Testis,Epididymis and Semen

This study aims to investigate the role of matrix metalloproteinases (MMPs) in determining semen quality and to evaluate the expression and cellular localization of MMP‐2, MMP‐9, tissue inhibitor of metalloproteinase‐1 (TIMP‐1) and TIMP‐2 in the testes, epididymis and ejaculated spermatozoa. Gelatinase activities between normal (n = 21) and abnormal (n = 25) semen samples showed a significant, sixfold increase in proMMP‐2 and MMP‐2 activity in high than low sperm concentration samples (p < 0.001). ProMMP‐9 and MMP‐9 levels were significantly elevated in samples with low sperm counts compared to those with high sperm density (p < 0.001). High levels of proMMP‐2 and MMP‐2 were associated with high sperm motility (≥70%, p < 0.001). Sperm‐rich fraction showed significantly (eight‐fold) higher proMMP‐9 enzymatic activity compared with prostatic fraction. The mRNA expressions of MMP‐2, MMP‐9, TIMP‐1 and TIMP‐2 were confirmed in testicular and epididymal tissues. Immunohistochemical staining illustrated the MMP‐2‐specific strong immunoreactivity in the head of mature spermatids during spermatogenesis, whereas MMP‐9, TIMP‐1 and TIMP‐2 were absent in these cells. Matrix metalloproteinase‐9 immunoreactivity was observed in the spermatocyte and round spermatid, whereas TIMP‐1 was only exhibited in the residual bodies. Immunolabeling of epididymal and ejaculated sperm demonstrated MMP‐2 localization along acrosomal region of sperm, while MMP‐9, TIMP‐1 and TIMP‐2 localization was merely limited to the flagella. In conclusion, spermatozoa initially acquire MMP‐2 during their formation at testicular level, and the presence of this protein persists through the epididymal transit and up to ejaculate. The enzymatic activity of MMP‐2 and MMP‐9 may serve as an alternative biomarker in determining semen quality.     

The pharmacokinetics of mavacoxib,a long‐acting COX‐2 inhibitor,in young adult laboratory dogs

Cox, S.R., Lesman, S.P., Boucher, J.F., Krautmann, M.J., Hummel, B.D., Savides, M., Marsh, S., Fielder, A., Stegemann, M.R. The pharmacokinetics of mavacoxib, a long‐acting COX‐2 inhibitor, in young adult laboratory dogs. J. vet. Pharmacol. Therap. 33 , 461–470. The pharmacokinetics of mavacoxib were evaluated in an absolute bioavailability study, a dose‐proportionality study and a multi‐dose study in young healthy adult laboratory Beagle dogs and in a multi‐dose safety study in Beagle‐sized laboratory Mongrel dogs. When administered as the commercial tablet formulation at 4 mg/kg body weight (bw) to fasted dogs, the absolute bioavailability (F) of mavacoxib was 46.1%; F increased to 87.4% when mavacoxib was administered with food. Following intravenous administration, the total body plasma clearance of mavacoxib was 2.7 mL·h/kg, and the apparent volume of distribution at steady‐state was 1.6 L/kg. The plasma protein binding of mavacoxib was approximately 98% in various in vitro and ex vivo studies. The dose‐normalized area under the plasma concentration–time curve was similar in Beagle and Beagle‐sized Mongrel dogs when mavacoxib was administered with food. Mavacoxib exhibited dose‐proportional pharmacokinetics for single oral doses of 2–12 mg/kg in Beagle dogs and for multiple oral doses of 5–25 mg/kg in Beagle‐sized Mongrel dogs. Only minor accumulation occurred when mavacoxib was administered at doses of 2–25 mg/kg bw orally to laboratory dogs with a 2‐week interval between the 1st two doses but with a monthly interval thereafter. Across all three Beagle studies (n = 63) the median terminal elimination half‐life (t_½) was 16.6 days, with individual values ranging 7.9–38.8 days. The prolonged t_½ for mavacoxib supports the approved regimen in which doses are separated by 2–4 weeks.     

Endometrial Expression of IFNAR‐1 and Oxytocin Receptor (OTR) is not Improved by Prostaglandin Analogues when Compared to Progestagens in Ewes

The objective of this study was to investigate differences on the endometrial immunoexpression of type I IFN receptor subunit 1 (IFNAR1) and oxytocin receptor (OTR) during the time of maternal recognition of pregnancy in sheep, when oestrus is synchronized with either prostaglandin analogues (group PG) or conventional progestagens (group P). Plasma progesterone was measured from day 0 to 21 post‐coitus (pc) (day 0 = day of oestrus). Immunohistochemistry was performed in samples of uterine horns from pregnant sheep on days 9pc, 13pc, 15pc, 17pc and 21pc to locate IFNAR1 and OTR expression in different endometrial compartments. Mean levels of plasma progesterone were different between treatments, obtaining higher levels in the PG group than in the P group (p < 0.05). Comparing days of pregnancy, IFNAR1 protein expression was different in the luminal epithelium (LE) (p < 0.05), while OTR was different in the LE and in the superficial glandular epithelium (SG) (p < 0.05). Temporal variation on the expression of both proteins from day 9pc to 21pc has been evidenced. IFNAR1 and OTR expression did not show significant differences between treatments. However, the response observed in the endometrium was highly inconsistent when prostaglandin analogues were used. Therefore, the protocol based on prostaglandin analogues still needs to be optimized before being considered as a better alternative to progestagens for oestrous synchronization in sheep.     

Immunohistochemical study of matrix metalloproteinase‐3 (MMP‐3) at the articular cartilage in osteochondrotic pigs

In order to understand the mechanism of osteochondrosis in the pig, articular cartilage was taken from the distal femoral condyles of Duroc pigs exhibiting leg weakness and then examined immunohistochemically for the localization of matrix metalloproteinases‐3 (MMP‐3), one of the enzymes involved in the resolution of cartilage matrix. The articular cartilage had the typical characteristics of osteochondrotic lesions, such as abnormal calcification, clefts of cartilage, disappearance of proteoglycan, and necrotic chondrocytes. The immunoreaction of MMP‐3 was observed in chondrocytes at the boundary between normal cartilage and proteoglycan‐deficient area. Moreover, chondrocytes expressing MMP‐3 showed normal morphology, but the surrounding cartilage matrix did not stain with toluidine blue, which indicated a lack of glycosaminoglycans. These results suggest that MMP‐3 is highly involved in the appearance and expansion of osteochondrotic lesions.