Immunohistochemistry with keratin,vimentin, desmin,and α‐smooth muscle actin monoclonal antibodies in canine mammary gland: Normal mammary tissue

Summary

Normal canine mammary gland tissue was studied immunohistochemically with monoclonal antibodies (MoAbs) directed against various human keratin types, vimentin, desmin, and α‐smooth muscle actin. Both ductal and alveolar luminal cells were immunoreactive with MoAbs recognizing respectively human keratins no. 7, 8, 18 and 19. In addition, some ductal luminal cells were labelled with a keratin 4 and a keratin 10 MoAb. Basal/myoepithelial cells were immunoreactive only with MoAbs directed against keratin 14, keratins 14 and 17, and a‐smooth muscle actin. The vimentin MoAb merely labelled solitary loose intraluminal cells representing macro‐phages or sloughed epithelial cells. These findings correspond largely to observations made in human breast tissue.     

Immunohistochemistry with keratin,vimentin, desmin,and α‐smooth muscle actin monoclonal antibodies in canine mammary gland: Malignant mammary tumours

Summary

Ten malignant canine mammary gland tumours and five metastases from three of these tumours were studied immunohistochemically with monoclonal antibodies (MoAbs) directed against different human keratin types (K), α‐smooth muscle actin, vimentin, and desmin.

In all tumours the neoplastic epithelium was rather homogeneously labelled with the keratin MoAbs RCK 102 (K 5 and 8) and CAM 5.2 (K 8). The adenocarcinomas (n=5), the solid carcinomas (n=2), and the carcinosarcoma (n=1) showed heterogeneous labelling with the MoAbs specific for luminal cell antigens in the normal canine mammary gland, i.e., K 18, K 7 and K 19 MoAbs. These cells were also immunoreactive with K 4 and K 10 MoAbs. The spindle cell carcinomas (n=2), however, did not react with these MoAbs.

All tumours except one adenocarcinoma were characterized by the absence of immunoreactive labelling with the α‐smooth muscle actin MoAb. In the solid carcinomas this was associated with the absence of labelling with one or both basal cell specific keratin MoAbs, i.e., 8.7 (K 14 and 17) and RCK 107 (K 14), respectively. In contrast, the other malignant tumours showed marked labelling of neoplastic epithelium with these MoAbs. Another remarkable finding was the labelling of a limited to moderate number of neoplastic epithelial cells with the vimentin MoAb. The presence of such labelling patterns in canine mammary gland tumours may be indicative of malignancy. Metastatic tumour tissues had a labelling pattern largely similar to that of the primary tumour, although also loss of reactivity for some keratin MoAbs was seen.     

Are complex carcinoma of the feline mammary gland and other invasive mammary carcinoma identical tumours? Comparison of clinicopathologic features,DNA ploidy and follow up

Feline mammary carcinomas are known for their unfavourable prognosis due to a strong tendency to local recurrence and metastasis. We studied 73 spontaneous primary mammary carcinomas and identified eight cases presenting a biphasic nature, with neoplastic epithelial and myoepithelial cells (complex carcinoma). These cases presented histopathologic features associated with a better prognosis; they were also associated with higher overall survival and disease-free survival rates compared to other common invasive mammary carcinomas of non-specified type. Complex carcinoma appears to be a low-grade malignancy.     

Pro-inflammatory and pro-apoptotic responses of TNF-α stimulated bovine mammary endothelial cells

Coliform mastitis may be severe in periparturient cows due to enhanced expression of pro-inflammatory cytokines that contribute to disease pathogenesis. Tumor necrosis factor (TNF)-α is implicated with the severity of coliform mastitis by provoking inflammatory responses in affected tissues. The endothelium is an integral organ in regulating inflammatory responses and loss of endothelial integrity may be fatal. Studies in humans suggest that endothelial cell apoptosis may be a consequence of TNF-α exposure and contributes to the development of sepsis, however, its impact on bovine mammary endothelial cells (BMEC) is unknown. We sought to determine the inflammatory and apoptotic responses of primary BMEC exposed to TNF-α in vitro. Stimulation of endothelial monolayers with TNF-α resulted in significant increase of toll-like receptor 4, interleukin-6 and -8, and intercellular adhesion molecule-1 and vascular cellular adhesion molecule-1 gene expression in a time-dependent manner. Caspase-8 and caspase-3 mRNA expression, as well as caspase enzyme activity, also increased significantly following TNF-α stimulation. Cell viability assessed by ATP activity and BMEC apoptosis determined by flow cytometry revealed no significant changes across time with TNF-α stimulation. Results suggest that TNF-α stimulation, at the dose used in this study, can elicit a pro-inflammatory response in BMEC, but not induce apoptosis. The impact of TNF-α on mammary vascular function and the subsequent impact on the pathophysiology of severe coliform mastitis warrant further investigation.     

Expression of hypoxia-inducible factor-1α and vascular density in mammary adenomas and adenocarcinomas in bitches

Background

The study aimed at examining hypoxia-inducible factor (HIF)1α expression in adenocarcinomas and adenomas in bitches in regard to tumour malignancy grade, proliferation, apoptosis and vascularisation. Therefore, paraffin sections of 15 adenomas and 64 adenocarcinomas sampled from 79 dogs aged 6 to 16 years were analysed.

Results

A significantly higher HIF-1α expression was noted in adenocarcinomas in comparison to adenomas (P < 0.0004). Moreover, HIF-1α expression in adenocarcinomas correlated positively with tumour malignancy grade (r = 0.59, P < 0.05), Ki-67 antigen expression (r = 0.43; P < 0.0005), TUNEL-positive cells (r = 0.62, P < 0001) and tumour vascularity measured by quantification of vessels characterized by the expression of von Willebrand Factor (r = 0.57, P < 0.05).

Conclusion

Results of this study indicate a similar biological role of HIF-1α in dogs and in humans, which may confirm suitability of the animal model in investigations on progression of tumours in humans.     

Relationship between NF-κB expression and malignancy of canine mammary gland tumor tissues

In this study, the nuclear expression of nuclear factor kappa B (NF-κB) in 48 tissues specimens from 25 canine spontaneous mammary gland tumor (MGT) patients was assessed by immunohistochemistry to compare their levels with clinical features, histological types, prognostic outcomes and proliferative activities, including the mitotic index (MI) and cylcinD1 expression. Twelve of eighteen (66.7%) malignant tumor tissues showed greater than 10% nuclear staining, while benign tumor and hyperplastic tissues showed less than 10% nuclear staining. Higher nuclear expression of NF-κB was positively correlated with larger tumor size, lymph node metastasis and higher MI; however, no correlation was observed with distant metastasis and cyclin D1 expression. Higher NF-κB nuclear expression correlated with shorter patient survival. These findings suggest that NF-κB is a useful prognostic factor for canine MGT patients.     

Expression of 14-3-3 σ protein in normal and neoplastic canine mammary gland

14-3-3 σ protein is a negative cell cycle regulator, with both reduced and elevated levels associated with cancer in humans. This study assessed the expression of this protein in canine mammary tissues using immunohistochemistry and Western blotting. 14-3-3 σ was detected in 97% of the mammary tissue samples examined and was found in both myoepithelial (MECs) and epithelial (ECs) cells. Expression levels were elevated and reduced in neoplastic ECs and MECs, respectively (P < 0.001). Intense expression of 14-3-3 σ was detected in neoplastic ECs infiltrating blood vessels and lymph nodes and suggests a possible role for this protein in the malignant transformation of mammary neoplasms. Moreover, double immunostaining for 14-3-3 σ and the MEC – specific marker p63, confirmed that 14-3-3 σ is a highly sensitive marker of MECs since all p63 – positive cells were also positive for 14-3-3 σ. However, this protein is not exclusive to MECs as ECs also labelled positively.     

Inducible expression of enhanced green fluorescent protein by interleukin-1α, interleukin-1β and Toll-like receptor 2 promoters in goat mammary epithelial cells in response to bacterial challenges

The development of a bacteria-inducible expression system has several advantages compared with persistent expression of anti-bacterial proteins in milk to prevent and treat mastitis. The present study determined whether mastitis responsive promoters could regulate enhanced green fluorescent protein (EGFP) expression in goat mammary epithelial cells (GMECs) in response to challenges with Escherichia coli, Staphylococcus aureus or Streptococcus agalactiae. The level of expression of interleukin (IL)-1α was significantly increased in GMECs challenged with E. coli, S. aureus or S. agalactiae compared with untreated GMECs. IL-1β was induced by E. coli and S. aureus, while Toll-like receptor 2 (TLR2) was induced by E. coli only.GMECs were transfected with IL-1α, IL-1β and TLR2 promoter-EGFP reporter gene lentiviral expression vectors and the levels of expression of EGFP were measured by flow cytometry and Western blot analysis after bacterial challenge. EGFP expression driven by the IL-1α and IL-1β promoters was higher in GMECs challenged with E. coli, S. aureus or S. agalactiae than in untreated GMECs. There were no differences in EGFP expression driven by the TLR2 promoter between GMECs challenged with S. aureus or S. agalactiae and untreated GMECs, but EGFP expression was significantly increased in GMECs challenged with E. coli. Overall, these results indicate that the promoters of some bacteria-inducible genes can regulate EGFP expression in GMECs in response to bacterial challenges. This bacteria-inducible expression strategy could be used for production of mastitis resistant animals by regulating the expression of anti-bacterial proteins in the mammary gland.     

Luteolysis and associated interrelationships among circulating PGF2α, progesterone, LH, and estradiol in mares

The changing concentrations and temporal relationships among a PGF2α metabolite (PGFM), progesterone (P₄), LH, and estradiol-17β (E₂) before, during, and after luteolysis were studied in 10 mares. Blood samples were collected every hour for ≥4 d beginning on day 12 after ovulation. The luteolytic period extended from a decrease in P₄ at a common transitional hour (Hour 0) at the end of preluteolysis and beginning of luteolysis to a defined ending when P₄ reached 1 ng/mL. The length of luteolysis was 22.9 ± 0.9 h, contrasting with 2 d in published P₄ profiles from sampling every 6 to 24 h. In mares with complete data for Hours −40 to −2 (n = 6), PGFM concentrations remained below assay sensitivity (n = 2) or two or three small pulses (peak, 29 ± 4 pg/mL) occurred. During luteolysis, the pulses became more prominent (peak, 193 ± 36 pg/mL). Rhythmicity of PGFM pulses was not detected by a pulsatility program during preluteolysis but was detected in seven of nine mares during luteolysis and postluteolysis combined. The nadir-to-nadir interval for LH pulses and the peak-to-peak interval between adjacent pulses were longer (P < 0.05) during preluteolysis than during luteolysis (nadir to nadir, 5.2 ± 0.3 h vs 3.6 ± 0.4 h; peak to peak, 9.4 ± 1.0 h vs 4.7 ± 0.5 h). Unlike reported findings in cattle, concentrations of P₄ decreased linearly within the hours of each PGFM pulse during luteolysis, and a positive effect of an LH pulse on P₄ and E₂ concentration was not detected. The reported balancing of P₄ concentrations between a negative effect of PGF2α and a positive effect of LH in heifers was not detected in mares.     

Diagnostic orientation values for ACTH and other parameters for clinically healthy donkeys and mules (insulin,triglycerides, glucose,fructosamines, and ɣ-GT)

Pituitary pars intermedia dysfunction is the most prevalent endocrine disease in horses. Although donkeys and mules may also be affected, only a few data have been published. Reference values for diagnostic parameters, such as adrenocorticotropic hormone (ACTH), are especially scarce or even lacking. Therefore, in the present study, available data from the literature have been verified and completed to facilitate a reliable diagnosis. Clinical inspections and haematological and biochemical examinations were carried out four times in a three-month interval (February to November) in 44 donkeys and 31 mules. Data from clinically healthy animals were used as an orientation. Plasma ACTH concentrations showed seasonal changes in both animal groups. However, it was generally higher in donkeys than mules. Although blood glucose (EDTA plasma) showed no difference between groups, serum insulin concentrations were consistently higher in donkeys. Serum fructosamine levels were slightly higher in mules, whereas, in some cases, serum triglyceride levels were considerably higher in donkeys. Serum gamma-glutamyltransferase showed a striking peak in mules in August, whereas the remaining gamma-glutamyltransferase values were lower compared to donkeys. By comparing donkeys and mules, the present work reveals differences in various blood parameters which should be considered for diagnoses and future studies.     

Choosing among correlative,mechanistic, and hybrid models of species’ niche and distribution

Different models are available to estimate species’ niche and distribution. Mechanistic and correlative models have different underlying conceptual bases, thus generating different estimates of a species’ niche and geographic extent. Hybrid models, which combining correlative and mechanistic approaches, are considered a promising strategy; however, no synthesis in the literature assessed their applicability for terrestrial vertebrates to allow best-choice model considering their strengths and trade-offs. Here, we provide a systematic review of studies that compared or integrated correlative and mechanistic models to estimate species’ niche for terrestrial vertebrates under climate change. Our goal was to understand their conceptual, methodological, and performance differences, and the applicability of each approach. The studies we reviewed directly compared mechanistic and correlative predictions in terms of accuracy or estimated suitable area, however, without any quantitative analysis to support comparisons. Contrastingly, many studies suggest that instead of comparing approaches, mechanistic and correlative methods should be integrated (hybrid models). However, we stress that the best approach is highly context-dependent. Indeed, the quality and effectiveness of the prediction depends on the study's objective, methodological design, and which type of species’ niche and geographic distribution estimated are more appropriate to answer the study's issue.     

Prolactin – to be reconsidered in canine mammary tumourigenesis?

Mammary tumours represent the most common neoplastic disease of the female dog, and the incidence in female dogs is much higher than in women. Whereas the influence of sexual steroids on breast cancer (BC) development in dogs has been studied, very little is known about the role of prolactin (PRL). New studies show that until recently, the importance of PRL in human BC development and progression has been highly underestimated. PRL plays a role in promoting benign as well as malignant neoplastic cell growth in BC in vitro and in vivo. Sporadic publications proposed a tumour promotor role in the dog. The goal of this review is to summarize our knowledge about PRL and human BC as well as canine mammary tumourigenesis, and propose future research in this area.