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Gestational diabetes mellitus (GDM) is associated with a high-risk for metabolic complications in offspring. However, exercise is recognized as a non-pharmacological strategy against metabolic disorders and is recommended in GDM treatment. This study aimed to investigate whether gestational exercise (GE) could modulate maternal high-fat high-sucrose (HFHS) diet-related hepatic metabolic and mitochondrial outcomes in female offspring of mothers with HFHS-induced GDM. Female Sprague-Dawley rats were fed with control or HFHS diet and kept sedentary or submitted to GE. Their female offspring were fed with control diet and kept sedentary. Hepatic lipid accumulation, lipid metabolism regulators, mitochondrial biogenesis and dynamics markers, and microRNAs associated to the regulation of these markers were evaluated. Female offspring of GDM mothers showed increased body weight at early age, whereas GE prevented this effect of maternal HFHS-feeding and reduced hepatic lipid accumulation. GE stimulated hepatic mRNA transcription and protein expression of mitochondrial biogenesis markers (peroxisome proliferator-activated receptor-gamma co-activator-1alpha and mitochondrial transcription factor A) and mRNA transcription of mitochondrial dynamics markers (mitofusin-1, mitofusin-2, and dynamin-related protein-1) that were altered by maternal GDM, while mitochondrial dynamics markers protein expression was not affected by maternal diet/GE except for optic atrophy-1. MicroRNAs associated with these processes (miR-122, miR-34a, miR-130b, miR-494), and the expression of auto/mitophagy- and apoptosis-related proteins were not substantially influenced by altered intrauterine environment. Our findings suggest that GE is an important regulator of the intrauterine environment positively affecting liver metabolism and promoting liver mitochondrial biogenesis in female offspring despite eventual effects of maternal HFHS-feeding and related GDM. Highlights: Maternal lifestyle can affect hepatic metabolic status of their female offspring; Female offspring hepatic metabolism is affected by maternal high-fat/sucrose diet; Gestational exercise mitigated maternal diet-related harmful outcomes on offspring liver; Gestational exercise is an important regulator of offspring mitochondrial biogenesis and dynamics; Exercise programs should be encouraged in clinical counseling during pregnancy. This work was supported by the EU's Horizon 2020 Research and Innovation program under the Marie Skłodowska-Curie Actions (No.722619, FOIE GRAS; No.734719, mtFOIE GRAS) and by the Por tuguese Foundation for Science and Technology (FCT) (FCT/UID/DTP/00617/2020-base; POCI-01-0145-FEDER-016690-PTDC/DTP-DES/7087/2014; POCI-01-0145-FEDER-016657-PTDC/DTP-DES/1082/2014), to JB (SFRH/BD/129645/2017). info:eu-repo/semantics/publishedVersion

Mammary tumors represent the second most common neoplasia in the canine species, where more than 50% of the cases are classified as malignant. The histological classification is used as a prognostic tool. Cadherins and catenins are responsible for cell adhesion and are intrinsic connected with the process of metastasis. E-cadherin expression in canine mammary tissues have been extensively studied. However, the studies with catenins are still scarce in the canine species. This study evaluated 74 canine mammary tissues by assessing the expression of E-cadherin and α, β and P-120 catenin molecules using the immunohistochemistry technique and their relationship with clinicopathological parameters. Three patterns of expression were identified in this study: membranous, cytoplasmic and both (membranous and cytoplasmic). In benign tumors, more than 80% of the cases had preserved expression and in malignant tumors 20% of the cases had reduced expression. A correlation between E-cadherin and P-120-catenin expression was found as well as a significant relationship between the histological type and the expression of α-catenin in malignant tumors.

Background: Gonadoblastoma (GB) is a rare mixed germ cell-sex cord-stromal tumour, first described in humans, commonly found in dysgenetic gonads of intersex patients that have a Y chromosome. However, this entity in not recognized in the WHO classification of tumours of genital system of domestic animals. Herein, we describe a case of ovarian gonadoblastoma with proliferation of dysgerminoma and sex cord-stromal tumour components, in a phenotypically and cytogenetically normal bitch. Case presentation: A 17-year-old cross-breed bitch had a firm, grey-white multinodular mass in the left ovary. The tumour was submitted to histopathological examination and Y chromosome detected through karyotype analysis and PCR studies. Microscopically, the ovary was almost replaced by an irregular neoplasm composed of three distinct, intermixed elements: dysgerminoma, mixed germ cell-sex cord-stromal tumour resembling human GB and a proliferative sex cord-stromal tumour component. The germ cells of gonadoblastoma and dysgerminoma components were immunoreactive for c-KIT. Sex cord-stromal cells of gonadoblastoma were immunoreactive for a-inhibin. The sex cord-stromal tumour was immunoreactive for AE1/AE3, occasionally for a-inhibin and negative for epithelial membrane antigen (EMA). The karyotype was 78, XX and PCR analysis confirmed the absence of the Y chromosome. Conclusion: Based on these findings, a diagnosis of gonadoblastoma with proliferation of dysgerminoma and sex cord-stromal tumour was made. This is the first case of ovarian gonadoblastoma in a female dog. Ana R. Flores (SFRH/BD/116373/2016) and João Lobo (SFRH/BD/132751/2017) acknowledge FCT, the Portuguese Foundation for Science and Technology, for financial support. IPATIMUP integrates the i3S Research Unit, which is partially supported by FEDER through the Operational Programme for Competitiveness Factors-COMPETE and National Funds through the Portuguese Foundation for Science and Technology (FCT), under the project number PEst-C/SAU/LA0003/2013. We kindly thank Professor Beatriz Porto and Mrs. Cláudia Oliveira from laboratory of Cytogenetics of the ICBAS-UP for the karyotype analysis and Msc. Patrícia Barradas who provided PCR technical support. We would like to thank Paulo Flores for help with scientific illustration.

Background: Treatment of schistosomiasis has relied on the anthelmintic drug praziquantel (PZQ) for more than a generation. Despite its celebrated performance for treatment and control of schistosomiasis and other platyhelminth infections, praziquantel has some shortcomings and the inability of this drug to counteract disease sequelae prompts the need for novel therapeutic strategies. Methods: Using a host-parasite model involving Biomphalaria glabrata and Schistosoma mansoni we established mechanical transformation of S. mansoni cercariae into newly transformed schistosomula (NTS) and characterized optimal culture conditions. Thereafter, we investigated the antischistosomal activity and ability of the antioxidants N-acetylcysteine (NAC) and resveratrol (RESV) to augment the performance of praziquantel and/or artesunate (AS) against larval stages of the parasite. Drug effects were evaluated by using an automated microscopical system to study live and fixed parasites and by transmission electron microscopy (TEM). Results: Transformation rates of cercariae to schistosomula reached ~ 70% when the manipulation process was optimized. Several culture media were tested, with M199 supplemented with HEPES found to be suitable for S. mansoni NTS. Among the antioxidants studied, RESV alone or combined with anthelminthic drugs achieved better results rather N-acetylcysteine (NAC). TEM observations demonstrated that the combination of AS + RESV induced severe, extensive alterations to the tegument and subtegument of NTS when compared to the constituent compounds alone. Two anthelmintic-antioxidant combinations, praziquantel-resveratrol [combination index (CI) = 0.74] and artesunate-resveratrol (CI = 0.34) displayed moderate and strong synergy, respectively. Conclusions: The use of viability markers including staining with propidium iodide increased the accuracy of drug screening assays against S. mansoni NTS. The synergies observed might be the consequence of increased action by RESV on targets of AS and PZQ and/or they may act through concomitantly on discrete targets to enhance overall antischistosomal action. Combinations of active agents, preferably with discrete modes of action including activity against developmental stages and/or the potential to ameliorate infection-associated pathology, might be pursued in order to identify novel therapeutic interventions. We express our deepest appreciation to Mrs. Maria Lurdes Delgado for expert maintenance of the schistosome life-cycle and her technical support NV acknowledges support from the Fundação para a Ciência e Tecnologia (FCT, Portugal) and FEDER (European Union) through Project Number IF/00092/2014/CP1255/CT0004. NV thanks FCT by IF position, Fundação Manuel António da Mota (FMAM, Portugal) and Pfizer Portugal for research group support. JMCC thanks FCT for UID/Multi/00211/2019 and Strategic Project UI211. PJB acknowledges support from award RO1CA164719 from the National Cancer Institute (NCI), National Institutes of Health (NIH), USA. The contents of this report are solely the responsibility of the authors and do not necessarily represent the official views of the FCT, FMAM, the NCI, or the NIH.

Superovulation protocols have been described for different mouse strains, however the numbers of animals used are still high and still little information is known about hormone administration schedules and estrous cycle phases. In this study, we aimed to optimize a superovulation protocol by injecting 5 IU of pregnant mare serum gonadotropin followed by 5 IU of hCG 48 h later, using three different schedules related to the beginning of the dark cycle (3, 5 and 7 pm) in a light cycle of 7 am to 7 pm, with light on at 7 am. C57BL/6J mice at 3, 4 and 5 weeks of age were used and the estrous cycle phase for times of PMSG and hCG injections was also analyzed. Total oocyte number was counted in the morning after hCG injection. Hormones given at 3 weeks of age at 3 pm (59 ± 15 oocytes) and 7 pm (61 ± 10 oocytes) produced a significantly higher oocyte number compared with oocytes numbers collected from females at the same age at 5 pm (P = 0.0004 and

Background Maternal high-caloric nutrition and related gestational diabetes mellitus (GDM) are associated with a high-risk for developing metabolic complications later in life and in their offspring. In contrast, exercise is recognized as a non-pharmacological strategy against metabolic dysfunctions associated to lifestyle disorders. Therefore, we investigated whether gestational exercise delays the development of metabolic alterations in GDM mothers later in life, but also protects 6-week-old male offspring from adverse effects of maternal diet. Methods Female Sprague-Dawley rats were fed with either control (C) or high-fat high-sucrose (HFHS) diet to induce GDM and submitted to gestational exercise during the 3 weeks of pregnancy. Male offspring were sedentary and fed with C-diet. Results Sedentary HFHS-fed dams exhibited increased gestational body weight gain (p

Dogs are highly exposed to pathogens transmitted by ectoparasites. The Mediterranean climate of Southern Europe, together with the presence of stray and/or neglected pets in close proximity with humans, contribute for tick expansion and stand for increased risk to infections in humans due to the zoonotic potential of many of these agents. The aim of this study was to perform a molecular survey in dogs (suspected of tick-borne disease and/or infested with ticks), as well as in ticks collected from those animals, from 12 districts of Portugal to investigate the occurrence of Rickettsia spp. and other tick-borne pathogens (Babesia, Ehrlichia, Anaplasma and Hepatozoon). Additionally, a serological survey of spotted fever group Rickettsia in Portuguese dogs was performed using an in-house immunofluorescence assay (IFA). A total of 200 whole-blood samples and 221 Rhipicephalus sanguineus s. l. ticks were collected from dogs. A total of 14 (7 %) blood samples and 10 (4.5 %) ticks yielded presumptively positive 420-bp amplicons using the Rickettsia spp. partial ompB nested PCR. Screening of the ompB-positive samples using the gltA gene showed 8 positive ticks. All Rickettsia ompB and gltA sequences had the highest identity with R. massiliae. The Rickettsia-positive dogs were further tested for other tick-borne pathogens and were found to be infected with Babesia spp. (n = 5), but not with Ehrlichia, Anaplasma or Hepatozoon. Of the 149 dog serum specimens tested in the serological assay, 103 (69 %) were positive for IgG antibodies against spotted fever group Rickettsia. Antibodies were found in dogs from all the studied districts, in 55 (53 %) of the stray and in 48 (47 %) of the owned dogs. Our study detected and characterized for the first time R. massiliae in dogs from Portugal, broadening the geographical range of this canine pathogen and adding knowledge to the impact of this disease in dogs. P. Barradas (SFRH/BD/116449/2016) acknowledges the FCT - Portuguese Foundation for Science and Technology for financial support. IPATIMUP integrates the i3S Research Unit, which is partially supported by FEDER - European Regional Development Fund, Portugal funds through the Operational Programme for Competitiveness Factors-COMPETE and National Funds through the FCT, under the project number Pest-C/SAU/LA0003/2013. This paper was published under the framework of the European Social Fund, Human Resources Development Operational Programme 2007–2013 (POSDRU/159/1.5/S/136893).

Pseudomonas aeruginosa is one of the leading causes of nosocomial pneumonia and its associated mortality. Moreover, extensively drug-resistant high-risk clones are globally widespread, presenting a major challenge to the healthcare systems. Despite this, no vaccine is available against this high-concerning pathogen. Here we tested immunogenicity and protective efficacy of an experimental live vaccine against P. aeruginosa pneumonia, consisting of an auxotrophic strain which lacks the key enzyme involved in D-glutamate biosynthesis, a structural component of the bacterial cell wall. As the amounts of free D-glutamate in vivo are trace substances in most cases, blockage of the cell wall synthesis occurs, compromising the growth of this strain, but not its immunogenic properties. Indeed, when delivered intranasally, this vaccine stimulated production of systemic and mucosal antibodies, induced effector memory, central memory and IL-17A-producing CD4+ T cells, and recruited neutrophils and mononuclear phagocytes into the airway mucosa. A significant improvement in mice survival after lung infection caused by ExoU-producing PAO1 and PA14 strains was observed. Nearly one third of the mice infected with the XDR high-risk clone ST235 were also protected. These findings highlight the potential of this vaccine for the control of acute pneumonia caused by this bacterial pathogen., Author summary Pseudomonas aeruginosa is an opportunistic bacterium and one of the most common causes of healthcare-associated diseases, including acute pneumonia, causing high mortality within immunocompromised hosts. Most of these infections are strikingly difficult to treat using conventional antibiotic therapies, since this microorganism displays high intrinsic resistance to a wide range of antibiotics. Moreover, to date, no vaccine is available for prevention. Here we used a mutated bacterial strain, which is unable to replicate in vivo and to cause disease, as a live vaccine against acute pneumonia caused by this pathogen. When applied intranasally, this vaccine induced immunity both at local and distant body sites, activating immune cells which were recruited into the airway mucosa. This evoked immune response reduced the number of non-surviving mice after infection with two cytotoxic P. aeruginosa strains causing acute lung infection. Some protection was also observed against an internationally disseminated cytotoxic strain. These data indicate that this is a promising vaccine candidate against P. aeruginosa-pneumonia.

© 2020 Elsevier Ltd. All rights reserved., Radiotherapy (RT) is an essential treatment modality for several types of cancer. Despite its therapeutic potential, RT is frequently insufficient to overcome the immunosuppressive nature of the tumor microenvironment, failing to control tumor metastases. Innovative immunomodulatory strategies, like immunostimulatory biomaterials could be used to boost the immunogenic effects of RT. Herein, we addressed the synergistic potential of immunostimulatory chitosan/poly(γ-glutamic acid) nanoparticles (Ch/γ-PGA NPs) combined with RT to induce antitumor immunity in the 4T1 orthotopic breast tumor mouse model. Non-treated animals had progressive primary tumor growth and developed splenomegaly and lung metastases. While RT decreased primary tumor burden, Ch/γ-PGA NPs-treatment decreased systemic immunosuppression and lung metastases. The combination therapy (RT + Ch/γ-PGA NPs) synergistically impaired 4T1 tumor progression, which was associated with a significant primary tumor growth and splenomegaly reduction, a decrease in the percentage of splenic immunosuppressive myeloid cells and an increase in antitumoral CD4+IFN-γ+ population. Notably, animals from the combination therapy presented less and smaller lung metastatic foci and lower levels of the systemic pro-tumor cytokines IL-3, IL-4, IL-10, and of the CCL4 chemokine, in comparison to non-treated animals. Overall, these results evidenced that Ch/γ-PGA NPs potentiate and synergize with RT, headlining their promising role as adjuvant anticancer strategies., This work was financially supported by FEDER – Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 – Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, by Portuguese funds through FCT – Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e do Ensino Superior in the framework of the project “MAGICIAM-a MAcrophaGe Immunomodulatory-delivery system to prevent Cancer Invasion And Metastasis” (PTDC/BTM-SAL/31859/2017) and by Fund for Scientific Research Flanders (FWO-Vlaanderen). Some human resources were supported through the FCT PhD Programmes and by the Human Capital Operational Programme (POCH), specifically by the BiotechHealth Programme. F. Castro (PD/BD/114013/2015), M.L. Pinto (PD/BD/81103/2011), C.L. Pereira (SFRH/BD/85779/2012), K Serre (FCT Investigator Starting Grant IF/00004/2014); R.M. Gonçalves (FCT Investigator Starting Grant IF/00638/2014) and M.J. Oliveira (FCT2012-Investigator Program IF/01066/2012) also acknowledge FCT for their funding. F Castro also acknowledges the financial support provided by COST - European Cooperation in Science and Technology within the COST Action: CA15214, STSM reference number: 38043. The authors would also like to acknowledge Claúdia Machado and Nuno Mendes for the assistance with histological processing, Rui Fernandes for the support with TEM analysis, and the support of the i3S Scientific Platform Histology and Electron Microscopy Unit (HEMS), member of the PPBI (PPBI-POCI-01-0145-FEDER-022122).

The dissemination of vector arthropods harbouring zoonotic pathogens through the uncontrolled transboundary trade of exotic and pet animals poses an important threat to Public Health. In the present report, we describe the introduction of pathogenic Rickettsia africae and R. aeschlimanni in ticks removed from imported tortoises in Qatar. A total of 21 ticks were collected from pet spur‐thighed tortoises (Testudo graeca) from Doha, May 2018, and studied for species identification and characterization of Rickettsia spp. Morphological and molecular analysis of ticks allowed their identification as Hyalomma aegyptium. Molecular analysis of partial ompA and gltA genes showed that Rickettsia sequences found on these ticks clustered with sequences classified as R. aeschilimanii and R. africae. Since pre‐adult stages of H. aegyptium also feed on humans, this tick species may play a role in the transmission of R. aeschilimanii and R. africae. We alert for the introduction of non‐native pets as vehicles for tick importation, known vectors for animal and human pathogenic agents. Importation of exotic species into non‐autochthonous countries deserves strict control to enforce robust surveillance and mitigate potential exotic diseases epidemics. P. Barradas (SFRH/BD/116449/2016) acknowledges the Portuguese Foundation for Science and Technology (FCT) for financial support. IPATIMUP integrates the i3S Research Unit, which is partially supported by FEDER funds through the Operational Programme for Competitiveness Factors‐COMPETE and National Funds through the FCT, under the project number Pest‐C/SAU/LA0003/2013. This paper was published under the framework of the European Social Fund, Human Resources Development Operational Programme 2007–2013 (POSDRU/159/1.5/S/136893). info:eu-repo/semantics/publishedVersion

Malignant transformation is often associated with abnormal protein glycosylation expressed, amongst others, by the accumulation of simple mucin-type carbohydrates namely Tn and Sialyl-Tn (STn) antigens. These are usually limited in normal tissues and their increased expression has been associated with cancer progression and poor prognosis. This study aims to evaluate the role of Tn and STn antigens in the neoplastic transformation of the canine gastric mucosa and to correlate their putative immunoexpression alterations with some pathological features. Tn and STn antigens expression were immunohistochemically evaluated in canine normal gastric mucosa (n = 3), gastric polyps (n = 9) and gastric carcinomas (n = 25), neoplastic emboli (n = 12) and metastases (n = 8). In normal gastric mucosa, Tn antigen was detected in the gastric epithelial cells, while STn antigen was absent. Similarly, all gastric polyps expressed Tn antigen, but none displayed STn antigen immunostaining. In carcinomas, Tn antigen was expressed in 96% of the cases and STn antigen in 68% of the neoplasms. STn antigen was significantly higher in carcinomas compared with normal mucosa (P < .05). No correlation was found between each antigen and the different subtypes of tumours according to WHO classification, tumour differentiation, lymph vascular invasion or metastasis. All neoplastic emboli expressed both antigens, and the expression score was similar or higher than that displayed by the neoplastic cells of the primary tumour. The high prevalence of STn antigen in gastric carcinomas compared with normal mucosa highlights the cancer-associated nature of this antigen. Our results link STn antigen expression to neoplastic transformation and suggest that it may be a useful marker of gastric cancer progression in dogs.

Breast cancer is the most common cancer among women and is considered a developed country disease. Moreover, is a heterogenous disease, existing different types and stages of breast cancer development, therefore, better understanding of cancer biology, helps to improve the development of therapies. The conventional treatments accessible after diagnosis, have the main goal of controlling the disease, by improving survival. In more advance stages the aim is to prolong life and symptom palliation care. Surgery, radiation therapy and chemotherapy are the main options available, which must be adapted to each person individually. However, patients are developing resistance to the conventional therapies. This resistance is due to alterations in important regulatory pathways such as PI3K/AKt/mTOR, this pathway contributes to trastuzumab resistance, a reference drug to treat breast cancer. Therefore, is proposed the repurposing of drugs, instead of developing drugs de novo, for example, to seek new medical treatments within the drugs available, to be used in breast cancer treatment. Providing safe and tolerable treatments to patients, and new insights to efficacy and efficiency of breast cancer treatments. The economic and social burden of cancer is enormous so it must be taken measures to relieve this burden and to ensure continued access to therapies to all patients. In this review we focus on how conventional therapies against breast cancer are leading to resistance, by reviewing those mechanisms and discussing the efficacy of repurposed drugs to fight breast cancer.

Cancer is a disease that affects and kills millions of people worldwide. Breast cancer, especially, has a high incidence and mortality, and is challenging to treat. Due to its high impact on the health sector, oncological therapy is the subject of an intense and very expensive research. To improve this therapy and reduce its costs, strategies such as drug repurposing and drug combinations have been extensively studied. Drug repurposing means giving new usefulness to drugs which are approved for the therapy of various diseases, but, in this case, are not approved for cancer therapy. On the other hand, the purpose of combining drugs is that the response that is obtained is more advantageous than the response obtained by the single drugs. Using drugs with potential to be repurposed, combined with 5-fluorouracil, the aim of this project was to investigate whether this combination led to therapeutic benefits, comparing with the isolated drugs. We started with a screening of the most promising drugs, with verapamil and itraconazole being chosen. Several cellular viability studies, cell death and proliferation studies, mainly in MCF-7 cells (Michigan Cancer Foundation-7, human breast adenocarcinoma cells) were performed. Studies were also carried out to understand the effect of the drugs at the level of possible therapeutic resistance, evaluating the epithelial-mesenchymal transition. Combining all the results, the conclusion is that the combination of verapamil and itraconazole with 5-fluorouracil had benefits, mainly by decreasing cell viability and proliferation. Furthermore, the combination of itraconazole and 5-fluorouracil seemed to be the most effective, being an interesting focus in future studies. This work was financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operational Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and Portuguese funds through FCT-Fundação para a Ciência e a Tecnologia, in the framework of the projects “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274). NV also acknowledges support from FCT and FEDER (European Union), award number IF/00092/2014/CP1255/CT0004. Alexandra Correia was supported by FCT grant SFRH/BPD/91623/2012.

Cyclooxygenase-2 (COX-2) is overexpressed in several human and animal neoplasms, including the human endometrial carcinoma. It has been suggested as a prognostic marker and a potential therapeutic target. This study aimed to (i) clarify histological aspects of feline endometrial adenocarcinomas (FEA) of the papillary serous type and (ii) characterize COX-2 immunohistochemical expression in normal, hyperplastic and neoplastic endometrium in this species. Archived paraffin-embedded tissue samples of 33 FEA, eight cystic endometrial hyperplasias (CEHs) and 21 samples of normal, healthy endometrium in the follicular (FS; n = 10) and luteal (LS; n = 11) stages were evaluated. Histological evaluation of haematoxylin and eosin-stained sections of the FEA revealed a papillary proliferation of neoplastic cells of serous type, accompanied by clear and multinucleated cells. Other architectural arrangements mainly included solid and tubular growth. Randomly distributed areas of necrosis within the tumours were commonly observed. Invasion of the myometrium, of the serosa and of the vascular and/or lymphatic vessels was not constant features. The mean number of mitoses was higher in FEA compared to non-neoplastic endometrium. COX-2 scores were lower in FEA (p = 0.003) and CEH (p = 0.05) when compared to normal epithelium (NE). The loss of the membrane apical reinforcement in epithelial cells was observed in FEA samples, which was accompanied by the dislocation of COX-2 labelling into the cytoplasm and the perinuclear area; in contrast, in epithelial cells in the healthy and hyperplastic endometria, the immunoreaction showed the characteristic pattern of apical membrane reinforcement, suggestive of the membrane polarization. COX-2 epithelial scores were higher in the FS than in the LS. No differences were found in stromal COX-2 expression between normal, CEH and FEA groups, but it was higher in the LS than in the FS. In summary, loss of COX-2 compartmentalization in neoplastic epithelial cells might be one of the molecular events underlying endometrial carcinogenesis.

Although there have been several studies on the use of immunohistochemical biomarkers of canine mammary tumors (CMTs), the results are difficult to compare. This article provides guidelines on the most useful immunohistochemical markers to standardize their use and understand how outcomes are measured, thus ensuring reproducibility of results. We have reviewed the biomarkers of canine mammary epithelial and myoepithelial cells and identified those biomarkers that are most useful and those biomarkers for invasion and lymph node micrometastatic disease. A 10% threshold for positive reaction for most of these markers is recommended. Guidelines on immunolabeling for HER2, estrogen receptors (ERs), and progesterone receptors (PRs) are provided along with the specific recommendations for interpretation of the results for each of these biomarkers in CMTs. Only 3+ HER2-positive tumors should be considered positive, as found in human breast cancer. The lack of any known response to adjuvant endocrine therapy of ER- and PR-positive CMTs prevents the use of the biological positive/negative threshold used in human breast cancer. Immunohistochemistry results of ER and PR in CMTs should be reported as the sum of the percentage of positive cells and the intensity of immunolabeling (Allred score). Incorporation of these recommendations in future studies, either prospective or retrospective, will provide a mechanism for the direct comparison of studies and will help to determine whether these biomarkers have prognostic significance. Finally, these biomarkers may ascertain the most appropriate treatment(s) for canine malignant mammary neoplasms.

Evaluation of the effectiveness of highly focalised thermotherapy (HFT) in a melanoma mouse model, using a ferrimagnetic cement (FC) and repeated low hyperthermia treatments.A melanoma mouse model was induced with B16F10 cells in C57BL6 mice. The FC, injected into the tumour, was used as the magnetic vehicle for HFT. FC location within the tumour was assessed by radiography and its capability to generate heat, when exposed to an external high frequency magnetic field (HFMF), monitored by thermal camera. The HFT treatment consisted of three HFMF exposures, with 48-h intervals, each one lasting 30 min, with a 5-6°C tumour temperature increase. At the end of the experiment, FC samples were characterised by scanning electron microscopy (SEM) and energy dispersion spectroscopy (EDS). The presence of iron contents was analysed in the tumour, lungs, liver and spleen. Histological evaluation and immunohistochemical staining for caspase-3 were performed. Tumour growth was monitored during the experiment.Surface analysis showed FC stabilisation within the tumour, and iron was absent. The thermal camera confirmed the localised temperature increase in the tumour. HFT treatments inhibited the tumour growth by ∼70% compared to controls. This was due to cell destruction by necrosis and apoptosis.The HFT, using the FC, proved to be a minimally invasive technique that statistically inhibited tumour growth. Results suggested that this methodology seems to be a promising technique for the treatment of solid tumours, allowing repeated low hyperthermia treatments, which can be easier and less traumatic than other hyperthermia techniques.

BACKGROUND/AIM Peripheral nerve sheath tumors (PNSTs) belong to a very heterogeneous group of neoplasms occurring both in dogs and humans. The aim of the present study was to evaluate the histological and immunohistochemical features of canine cutaneous PNSTs contributing to further refine their diagnosis. MATERIALS AND METHODS The histopathological phenotype and biological behavior of 40 canine cutaneous PNSTs were evaluated and vimentin, S-100, glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE), desmin, alpha-smooth muscle actin (α-SMA) and Ki-67 immunoreactivity were assessed. RESULTS Respectively, 17 and 23 lesions were classified as benign and malignant PNSTs. The malignant lesions were more often positive for S-100 and presented a proliferation index significantly higher when compared to the benign ones (p

Background: Ruthenium-based anti-cancer compounds are proposed as viable alternatives that might circumvent the disadvantages of platinum-based drugs, the only metallodrugs in clinical use for chemotherapy. Organometallic complexes in particular hold great potential as alternative therapeutic agents since their cytotoxicity involves different modes of action and present reduced toxicity profiles. Objective: During the last few years our research group has been reporting on a series of organometallic ruthenium(II)- cyclopentadienyl complexes with important cytotoxicity against several cancer cell lines, surpassing cisplatin in activity. We report herein preliminary in vivo studies with one representative compound of this family, with exceptional activity against several human cancer cell lines, including the glycolytic and highly metastatic MDAMB231 cell line used in this study. Method: The anti-tumor activity of our compound was studied in vivo on N:NIH(S)II-nu/nu nude female mice bearing triple negative breast cancer (TNBC) orthotopic tumors. Administration of 2.5 mg/kg/day during ten days caused cell death mostly by necrosis (in vitro and in vivo), inducing tumor growth suppression of about 50% in treated animals when compared to controls. Results: The most remarkable result supporting the effectiveness and potential of this drug was the absence of metastases in the main organs of treated animals, while metastases were present in the lungs of all control mice, as revealed by histopathological and immunohistochemical analysis. Conclusion: These in vivo studies suggest a dual effect for our drug not only by suppressing growth at the primary tumor tissue but also by inhibiting its metastatic behavior. Altogether, these results represent a benchmark and a solid starting point for future studies.

In humans, a germline mutation (c.309C>G) in the TWIST1 oncogene may predispose to breast cancer and its expression has been associated with tumour progression and metastasis. In this study, the feline TWIST1 gene was screened for sequence variations in 37 neoplastic and eight hyperplastic mammary gland lesions from cats. In addition, mRNA levels were examined in 15 mammary tumours and three cases of mammary hyperplasia by quantitative real-time reverse-transcriptase PCR. Feline mammary carcinomas had significantly lower levels of expression of TWIST1 mRNA than benign mammary tumours. No variations were identified in the TWIST1 coding region in feline mammary tumours and the mutation described in humans was not detected. However, two germline variants in the TWIST1 gene intron were identified in four and three carcinomas, respectively: GQ167299:g.535delG and GQ167299:g.460C>T. There was no association between these sequence alterations and TWIST1 mRNA levels.

Urokinase plasminogen activator (uPA) has been associated with aggressive behaviour and poor prognosis in human breast cancer, but there is no information on its expression in canine mammary tumours (CMT). uPA immunohistochemical expression was studied in 119 CMT (24 benign, 95 malignant) to investigate its relationship with clinical and histopathological parameters. Dogs with malignant mammary tumours (MMT) underwent a 2-year follow-up evaluation. MMT expressed significantly more uPA than benign tumours. In MMT, high uPA stromal expression was significantly associated with larger tumour size, high Ki-67 expression, invasive growth, high histological grade, regional lymph node metastases, development of distant metastases, and lower overall survival (OS) and disease-free survival (DFS). On the basis of these results, uPA could be considered a useful prognostic factor in dogs with MMT.

Studies focusing on the molecular basis of canine mammary tumors (CMT) have long been hampered by limited numbers of molecular tools specific to the canine species. The lack of molecular information for CMT has impeded the identification of clinically relevant tumor markers beyond histopathology and the introduction of new therapeutic concepts. Additionally, the potential use for the dog as a model for human breast cancer is debatable until questions are answered regarding cellular origin, mechanisms, and cellular pathways. During the past years, increasing numbers of canine molecular tools have been developed on the genomic, RNA, and protein levels, and an increasing number of studies have shed light on specific aspects of canine carcinogenesis, particularly of the mammary gland. This review summarizes current knowledge on the molecular carcinogenesis of CMT, including the role of specific oncogenes, tumor suppressors, regulators of apoptosis and DNA repair, proliferation indices, adhesion molecules, circulating tumor cells, and mediators of angiogenesis in CMT progression and clinical behavior. Whereas the data available are far from complete, knowledge of molecular pathways has a significant potential to complement and refine the current diagnostic and therapeutic approach to this tumor type. Furthermore, current data show that significant similarities and differences exist between canine and human mammary tumors at the molecular level. Clearly, this is only the beginning of an understanding of the molecular mechanisms of CMT and their application in clinical patient management.

Summary Caveolin-1 (Cav-1) is a major structural protein of caveolae, plasma membrane invaginations related to several cellular processes including regulation of signal transduction. In recent years there has been some controversy regarding the distribution of Cav-1 in normal and neoplastic mammary cell types, which may be attributed to different scoring systems adopted in different studies. The present study compares Cav-1 immunoexpression in normal ( n = 17) and neoplastic ( n = 79) canine mammary tissues assessed by two different scoring methods (previously reported by others with conflicting results) and associates Cav-1 expression with metastasis and overall survival (OS). Results obtained with both scoring methods were similar, revealing absence of immunoreactivity in normal luminal epithelium and in benign neoplasms and clearly associating Cav-1 expression with malignant transformation. The data suggest that Cav-1 expression is associated with highly malignant subtypes of mammary tumours (i.e. basal-like carcinoma), invasion and metastasis, thus supporting the hypothesis that it may play a major role in the epithelial–mesenchymal transition process. Furthermore, one of the scoring systems employed associated Cav-1 expression with unfavourable prognosis in canine mammary carcinomas, showing a strong correlation between Cav-1-positive carcinomas and shorter OS.

Epidermal Growth Factor Receptor (EGFR) has been extensively studied in human breast cancer; however, systematic studies of EGFR protein expression in canine mammary gland tumours are lacking. Therefore, we evaluated its immunohistochemical expression in a series of 136 canine mammary tumours and representative areas of adjacent normal and hyperplastic mammary tissue and investigated a possible correlation between EGFR overexpression and several clinicopathological parameters and survival. In normal and hyperplastic canine mammary glands, EGFR expression was consistently observed in myoepithelial cells, with luminal cells usually negative. In tumour tissues, EGFR overexpression was found in 9 benign (19.6%) and 38 malignant (42.2%) lesions, with EGFR positivity significantly related with malignancy. Besides animal age and tumour size, there were no significant associations between other clinicopathological parameters and EGFR overexpression. On survival analysis, tumours with EGFR overexpression showed a reduced disease-free and overall survival; however these associations failed to reach statistically significant levels.

Catechol-O-methyltransferase (COMT) is an important enzyme participating in inactivation of carcinogenic oestrogen metabolites. In humans there is a single nucleotide polymorphism in COMT gene (COMT va1158met) that has been associated with an increased risk for developing breast cancer. In dogs, there is a single nucleotide polymorphism in COMT gene (G482A), but its relation with mammary carcinogenesis has never been investigated. The aim of this study was to focus on the evaluation of such polymorphism as a risk factor for the development of mammary tumors in bitches and on the analysis of its relationship with some clinicopathologic features (dog's age and weight, number and histologic type of the lesions, lymph node metastasis) of canine mammary neoplasms. A case-control study was conducted analyzing 90 bitches with mammary tumors and 84 bitches without evidence of neoplastic disease. The COMT G482A polymorphism was analyzed by PCR-RFLP. We found a protective effect of the polymorphism in age of onset of mammary tumors, although we could not establish a significant association between COMT genotype and other clinicopathologic parameters nor with mammary tumor risk overall. Animals carrying the variant allele have a threefold likelihood of developing mammary tumors after 9 years of age in comparison with noncarriers. The Kaplan-Meier method revealed significant differences in the waiting time for onset of malignant disease for A allele carrier (12.46 years) and noncarrier (11.13 years) animals. This investigation constitutes the first case-control study designed to assess the relationship between polymorphic genes and mammary tumor risk in dogs. Our results point to the combined effect of COMT genotype with other genetic and/or environmental risk factors as important key factors for mammary tumor etiopathogenesis.

Galectin-3 is implicated in tumor progression and metastasis. High levels of galectin-3 have been reported in intravasated cells in primary and metastatic tumor sites of canine malignant mammary tumors (CMMT). Nevertheless, it is still unknown whether this increase is limited to the site of the lesion or if it is a systemic feature. To better understand the pattern of the expression of galectin-3 and to investigate the possibility of using serum galectin-3 levels as a relevant biomarker in this disease, galectin-3 concentrations were determined in a series of sera from CMMT-bearing female dogs. None of the dogs included in the study had detectable metastases at the time of presentation. Animals were retrospectively divided into two groups dependent on whether or not they developed metastatic lesions during a 25-month follow-up period. Samples were collected from all dogs before surgery, 1 month after resection of the primary tumor and every 3 months during the postoperative period. Galectin-3 levels were significantly higher 1 month after than at the time of surgery (p=0.0058). Higher galectin-3 was found in samples collected 7 (p=0.0007), 10 (p=0.0061) and 13 months (p=0.0052) after surgery from dogs of the metastatic group when compared to those remaining free of development of detectable metastases. In conclusion, increased serum galectin-3 levels seem to be present in both metastatic and non-metastatic cases during the postoperative period, however, while in non-metastatic cases the values tend to return to baseline levels after surgery, in metastatic cases, levels remain persistently elevated.

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.

Abnormal catenin expression has been related to mammary carcinogenesis in both human and canine species and they are considered tumor- and invasion-suppressor molecules; however, in feline mammary tissues they have been scarcely studied.The immunohistochemical expression of α-, β- and p120-catenin was studied in a series of normal feline mammary glands, hyperplastic/dysplastic lesions and benign and malignant mammary tumors. Their relationship with clinicopathological parameters and with E- and P-cadherin expression was assessed.Normal tissues, hyperplastic/dysplastic lesions and benign tumors expressed α-, β- and p120-catenin in the membrane of more than 75% of the luminal epithelial cells, while in malignant tumors, there was a reduction in their membranous expression and a p120-catenin cytoplasmic expression in 40%. Reduced α-catenin expression was related to tumor features with prognostic value, namely tumor size (p=0.0203) and necrosis (p=0.0205). The expression of α-, β- and p120-catenin were individually related to each other and collectively associated with E-cadherin expression.The results demonstrate a relationship between feline mammary carcinogenesis and decreased expression of catenins, suggesting that they may represent a valuable tool in the diagnosis of feline mammary neoplasms.

Background Endometrial adenocarcinomas are a rare type of tumour in cats. Though different morphologies have been reported, the most frequent histological type of feline endometrial adenocarcinoma (FEA) is the papillary serous. Characterization of molecular markers expression in FEA may contribute to clarify the pathogenesis of these tumours and to assess the differences between normal endometrium and FEA regarding the expression pattern of several proteins. Therefore, this study aimed to evaluate the immunohistochemical profile of a wide panel of antibodies (specific for ER-α, PR, Ki-67, CK7 and CK20) in twenty-four cases of FEA. Comparisons were made between FEA and feline normal cyclic endometrium in follicular (n = 13) and luteal (n = 10) stages. Except for Ki-67, all other molecular markers were assessed independently for the intensity of immunolabeling and for the percentage of cells expressing the protein. Results This study showed that in FEA a loss of expression occurs for ER-α (P ≤ 0.0001) and less markedly also for PR. The lost in sex steroid receptors concerns a decrease in both the proportion of labelled cells and the intensity of immunolabelling (P = 0.002 and P = 0.024, respectively). Proliferative activity, estimated via Ki-67 immunoreaction, significantly increased in FEA as compared to normal endometrium (P ≤ 0.0001). Feline endometrial adenocarcinomas maintained the CK7+/CK20+ status of normal endometrium. However, FEA showed decreased CK7 intensity of labelling compared to normal endometria (P ≤ 0.0001) and loss of CK20 expression, both in intensity (P ≤ 0.0001) and in percentage of positive cells (P = 0.01), compared to normal tissues. Conclusions Data gathered in this study suggest that proliferation in FEA accompanies ER-α down-regulation, possibly following activation of pathways mediated by local growth factors. Moreover, FEA retains combined expression of CK7 and CK20, as evidenced in normal endometrial epithelia, although a decrease in CK7 expression was observed.

This paper describes an immunohistochemical study of the expression of E-cadherin in four samples of normal, eight samples of hyperplastic and 19 samples of neoplastic feline mammary tissue. In the normal tissues, the luminal epithelial cells showed a strong pattern of staining for E-cadherin at the cell-cell boundaries, whereas the myoepithelium showed no immunoreactivity. In the hyperplastic tissues and the five benign neoplasms, there were disturbances in the expression of E-cadherin in the luminal epithelium, in the form of the coexistence of membranous and cytoplasmic staining, together with immunoreactivity in a small percentage of myoepithelial cells. In 11 of 14 carcinomas, there was a reduction or absence of E-cadherin expression and abnormalities in the pattern of immunostaining; these changes were more pronounced in cribriform and solid carcinomas.

It is widely established that angiogenesis is required during tumor progression. Emerging data, suggests that estrogens can mediate endothelial proliferation and differentiation. We investigated the role of estrogens in the formation and stabilization of capillary-like structures, and identified 17 beta -estradiol-driven pathways involved in vessel assembly. We show that estrogens induce MCF7 breast cancer cells to secrete TGF beta 1. In addition, TGF beta cross talks with EGFR signaling pathway with concomitant up-regulation of EGFR ligand, TGF alpha, promoting cord-like formations in HUVEC cultures. The action of 17 beta -estradiol was not restricted to endothelium, since 17 beta -estradiol also stimulated recovery and migration of a smooth muscle cell line (FLTR) to injured areas again by the cross talk between these two signaling pathways. Finally, given the relevant role of 17 beta -estradiol in vessel stabilization, co-cultures of HUVEC and FLTR cells were established in the presence of 17 beta -estradiol or TGF beta 1. By blocking TGF beta or EGFR signaling, we demonstrate that 17 beta -estradiol promoted vessel stabilization through the interplay of TGF beta 1 and EGFR signaling transduction pathways. Our data suggest that estrogen mediates endothelial cell stabilization and vessel assembly. These vessel protective effects involve TGF beta 1 and EGFR signaling transduction pathways.

Mammalian target of rapamycin (mTOR) is a serine-threonine kinase involved in cell growth, proliferation and survival. Activation of mTOR has been reported in various tumour types, including human breast cancer; however, the expression of mTOR in canine mammary tumours has not been examined. In the present study, expression of the activated form of mTOR (phospho-mTOR [p-mTOR]) was examined immunohistochemically in five normal canine mammary glands, 45 canine mammary carcinomas and their corresponding metastatic lesions (n = 15). Phospho-mTOR was not expressed in normal canine mammary tissue, but cytoplasmic labelling was observed in 78% of canine mammary carcinomas. Two carcinomas had both cytoplasmic and nuclear labelling. No significant relationship was found between p-mTOR cytoplasmic expression and histological type or grading of carcinomas, degree of tubular formation, anisokaryosis, mitotic activity or lymph node metastasis. In all except one case, the expression pattern of p-mTOR in lymph node metastases was similar or decreased when compared with the primary lesion. The findings suggest that p-mTOR is involved in mammary carcinogenesis in dogs. However, p-mTOR cytoplasmic expression does not appear to be a prognostic indicator in canine mammary carcinomas, which may be related to its subcellular location in the neoplastic cells. Canine mammary tumours may provide a model for the development of innovative medical strategies involving mTOR inhibitors in human breast cancer.

Galectin-1 and galectin-3 are carbohydrate-binding proteins that have been implicated in the pathobiology of several types of cancer. The aim of the present study was to investigate the expression pattern of both these galectins in canine non-neoplastic mammary tissues and mammary tumors (CMT).Protein and mRNA expression of galectin-1 and -3 were assessed in 12 benign and 41 malignant CMT.Galectin-1 was overexpressed in the majority of malignant CMT cases in tumor cells and stroma. Its expression in malignant tumor cells was associated with smaller-sized tumours. Distant metastases presented a strong intensity of galectin-1 and reduced galectin-3 expression, while the opposite was observed in circulating tumor cells. Interestingly intravascular tumor cells presented galectin-3 up-regulation at the mRNA level. Double-labelling further made it clear that galectin-3 and galectin-1 expression did not overlap in normal-adjacent mammary and CMT cells.Taken together, our data suggest that malignant CMT cell sub-populations have alternating expression of galectin-1 or -3. This might confer survival advantage to tumour cells in different phases of tumour progression.

Background Gastric polyps (GP) are characterised by luminal overgrowths projecting above the plane of the mucosal surface that can be classified as non-neoplastic and neoplastic lesions. In humans, recent studies have drawn attention to the malignant potential of some of these lesions. However, gastric polyps are uncommon lesions in dogs. Findings In this study, the presence of Helicobacter spp., the cellular proliferative activity, potential phenotypic alterations, COX-2 and p53 expression in canine spontaneous gastric polyps were investigated. The expression of these molecules was also studied in normal canine gastric mucosa in order to gain further knowledge of the significance of their loss or overexpression in gastric lesions. Conclusions The normal expression of almost all the factors evaluated, along with the reduced proliferative activity is strongly suggestive that, in dogs, spontaneous gastric polyps are not only a rare finding but also of benign nature. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_166

The expression of hormone receptors and their relationship to cell proliferation in six samples of normal canine mammary tissue, and 11 benign and 10 malignant mammary neoplasms from female dogs were assessed by immunohistochemistry in formalin-fixed paraffin-embedded samples, by using monoclonal antibodies against progesterone and oestrogen receptors, and nuclear antigen Ki-67 (MIB-1). Malignant tumours negative for progesterone receptors proliferated at higher rates than progesterone receptor-positive tumours, suggesting that the progression towards malignancy in spontaneous mammary tumours is accompanied by a decrease in hormonal steroid dependency. Only one malignant tumour was positive for oestrogen receptors.

DNA measurement by image cytometry, and a detailed immunohistochemical study using monoclonal antibodies directed against different human cytokeratin types, muscle-specific actin, vimentin and S100 protein were carried out on normal canine mammary tissue (n =4), benign canine mammary mixed tumours (n =20) and malignant canine mammary mixed tumours (n =13). The results showed that ductal and alveolar luminal cells in normal and neoplastic tissue were immunoreactive with CAM5.2 and AE1/AE3 antibodies recognizing human keratins.Basal/myoepithelial cells were clearly differentiated from ductal and alveolar epithelial cells, since the latter only expressed cytokeratins, whereas the former also expressed vimentin and muscle-specific actin. This immunohistochemical study showed that there is loss of expression of muscle-specific actin and cytokeratins in areas of myoepithelial proliferation, and enhanced expression of vimentin and S100 protein in proliferative areas with osseous and/or chondroid metaplasia. The ploidy studies revealed that 20% (4/20) of benign and 54% (7/13) of malignant mixed tumours of canine mammary gland were aneuploid and that the epithelial and myoepithelial components of the mixed tumours had identical DNA content. Our results reinforce the role of myoepithelial cells in mesenchymal metaplasia in mixed mammary tumours and suggest the possibility of a common origin of both components from a totipotential stem cell with capacity for divergent differentiation.

Background Although several histopathological and clinical features of canine mammary gland tumours have been widely studied from a prognostic standpoint, considerable variations in tumour individual biologic behaviour difficult the definition of accurate prognostic factors. It has been suggested that the malignant behaviour of tumours is the end result of several alterations in cellular physiology that culminate in tumour growth and spread. Accordingly, the aim of this study was to determine, using a multivariable model, the independent prognostic value of several immunohistochemically detected tumour-associated molecules, such as MMP-9 and uPA in stromal cells and Ki-67, TIMP-2 and VEGF in cancer cells. Results Eighty-five female dogs affected by spontaneous malignant mammary neoplasias were followed up for a 2-year post-operative period. In univariate analysis, tumour characteristics such as size, mode of growth, regional lymph node metastases, tumour cell MIB-1 LI and MMP-9 and uPA expressions in tumour-adjacent fibroblasts, were associated with both survival and disease-free intervals. Histological type and grade were related with overall survival while VEGF and TIMP-2 were not significantly associated with none of the outcome parameters. In multivariable analysis, only a MIB-1 labelling index higher than 40% and a stromal expression of MMP-9 higher than 50% retained significant relationships with poor overall and disease-free survival. Conclusions The results of this study indicate that MMP-9 and Ki-67 are independent prognostic markers of canine malignant mammary tumours. Furthermore, the high stromal expressions of uPA and MMP-9 in aggressive tumours suggest that these molecules are potential therapeutic targets in the post-operative treatment of canine mammary cancer.

Human ERBB2 is a proto-oncogene that codes for the erbB-2 epithelial growth factor receptor. In human breast cancer (HBC), erbB-2 protein overexpression has been repeatedly correlated with poor prognosis. In more recent works, underexpression of this gene has been described in HBC. Moreover, it is also recognised that oncogenes that are commonly amplified or deleted encompass point mutations, and some of these are associated with HBC. In cat mammary lesions (CMLs), the overexpression of ERBB2 (27%–59.6%) has also been described, mostly at the protein level and although cat mammary neoplasias are considered to be a natural model of HBC, molecular information is still scarce. In the present work, a cat ERBB2 fragment, comprising exons 10 to 15 (ERBB2_10–15) was achieved for the first time. Allelic variants and genomic haplotype analyses were also performed, and differences between normal and CML populations were observed. Three amino acid changes, corresponding to 3 non-synonymous genomic sequence variants that were only detected in CMLs, were proposed to damage the 3D structure of the protein. We analysed the cat ERBB2 gene at the DNA (copy number determination), mRNA (expression levels assessment) and protein levels (in extra- and intra protein domains) in CML samples and correlated the last two evaluations with clinicopathological features. We found a positive correlation between the expression levels of the ERBB2 RNA and erbB-2 protein, corresponding to the intracellular region. Additionally, we detected a positive correlation between higher mRNA expression and better clinical outcome. Our results suggest that the ERBB2 gene is post-transcriptionally regulated and that proteins with truncations and single point mutations are present in cat mammary neoplastic lesions. We would like to emphasise that the recurrent occurrence of low erbB-2 expression levels in cat mammary tumours, suggests the cat mammary neoplasias as a valuable model for erbB-2 negative HBC. POCI/CVT/62940/2004 and by the PhD grants (SFRH/BD/23406/2005 and SFRH/BD/31754/2006, of the Science and Technology Foundation (FCT) from Portugal.

The human ERBB2 proto-oncogene is widely considered a key gene involved in human breast cancer onset and progression. Among spontaneous tumors, mammary tumors are the most frequent cause of cancer death in cats and second most frequent in humans. In fact, naturally occurring tumors in domestic animals, more particularly cat mammary tumors, have been proposed as a good model for human breast cancer, but critical genetic and molecular information is still scarce. The aims of this study include the analysis of the cat ERBB2 gene partial sequences (between exon 17 and 20) in order to characterize a normal and a mammary lesion heterogeneous populations. Cat genomic DNA was extracted from normal frozen samples (n = 16) and from frozen and formalin-fixed paraffin-embedded mammary lesion samples (n = 41). We amplified and sequenced two cat ERBB2 DNA fragments comprising exons 17 to 20. It was possible to identify five sequence variants and six haplotypes in the total population. Two sequence variants and two haplotypes show to be specific for cat mammary tumor samples. Bioinformatics analysis predicts that four of the sequence variants can produce alternative transcripts or activate cryptic splicing sites. Also, a possible association was identified between clinicopathological traits and the variant haplotypes. As far as we know, this is the first attempt to examine ERBB2 genetic variations in cat mammary genome and its possible association with the onset and progression of cat mammary tumors. The demonstration of a possible association between primary tumor size (one of the two most important prognostic factors) and the number of masses with the cat ERBB2 variant haplotypes reveal the importance of the analysis of this gene in veterinary medicine.

Galectin-3 is involved both in facilitating detachment of cells from primary tumour sites and favouring cancer cell adhesion and survival to anoikis in the blood stream. The mechanisms behind these apparently contradictory roles of the lectin have not yet been resolved. In order to investigate possible interplays between galectin-3 and its ligands underlying their role in the metastatic process, we examined mucin-1 (MUC1) and epidermal growth factor receptor (EGFR), well-known galectin-3 ligands, as well as galectin-3-binding site expression in a series of spontaneous canine malignant mammary tumours (CMMT) and a metastatic CMMT cell line. Despite the fact that CMMT cells expressed MUC1 and EGFR homogeneously over their plasma membrane, intravascular tumour cells, positive for galectin-3, expressed MUC1 and EGFR in a more focal membrane localization. Moreover, MUC1 overexpression in primary CMMT was present in parallel with down-regulation of galectin-3. Furthermore, in the CMT-U27 cell line, galectin-3 knock-down led to increased MUC1 expression, while MUC1 knock-down led to down-regulation of the lectin. Finally, removal of sialic acid from both CMMT and CMT-U27 xenograft samples exposed galectin-3-ligands throughout the tumour tissue, whereas these ligands were only present in galectin-3-positive invading cells in untreated samples. Interestingly indeed, we show that in vessel-invading cells, there is interaction between galectin-3 and the T antigen in vivo. We therefore hypothesized that loss of galectin-3 and sialylation-related masking of its ligands, in conjunction with their overexpression in specific tumour cell subpopulations, are crucial in regulating adhesive/de-adhesive events in the progression and invasive capacity of metastatic cells.

The enormous biologic complexity of human cancer has stimulated the development of more appropriate experimental models that could resemble in a natural and spontaneous manner the physiopathologic aspects of cancer biology. Companion animals have many desired characteristics that fill the gap between in vitro and in vivo studies, and these characteristics have proven to be important in understanding many complex molecular aspects of human cancer. Spontaneous tumors in dogs share a wide variety of epidemiologic, biologic, and clinical features with human cancer, which makes this animal model both attractive and underused in oncology research. In this review, we summarize the importance of naturally occurring canine tumors as valuable tools for studying numerous aspects of human cancer as well as the potential use of this animal model for the development of new cancer treatments. We address specifically the use of canine mammary tumors as an increasingly powerful model to study human breast cancer.

Overexpression of mucins is known to decrease cell-to-cell adhesion and thus to facilitate the invasion of cancer cells through the extracellular matrix. Mucin 6 (MUC6) is overexpressed and aberrantly O-glycosylated in human breast cancer, serving as a carrier for one of the most specific cancer-associated antigens, Tn antigen. Despite its relevance in breast cancer, MUC6 expression has not yet been characterized in canine mammary tumours (CMTs). The aims of this study were to assess the expression of MUC6 and Tn antigen in 55 benign and 77 malignant CMTs of different histological types and to investigate possible correlations with pathological features. MUC6 and Tn antigen were found to be significantly overexpressed in malignant compared with benign CMTs. MUC6 was significantly overexpressed in simple and complex carcinomas compared with simple and complex adenomas, respectively. When considering only the epithelial population, significant MUC6 overexpression was observed in carcinosarcomas when compared with benign mixed tumours. In addition, MUC6 was significantly overexpressed in simple compared with complex carcinomas. Finally, double-labelling immunofluorescence performed on seven malignant CMTs showed MUC6 and Tn co-expression. Therefore, MUC6 and Tn antigen overexpression is associated with malignant phenotypes of CMTs.

Canine renal cell carcinomas (RCCs) are uncommon aggressive tumors that occur mainly in middle-aged male dogs. Their histologic classification bears no relationship with prognosis, and little information is available concerning their immunohistochemical properties. In this retrospective study, formalin-fixed, paraffin-embedded tissues from 13 canine RCCs were retrieved from the archives, classified histologically, and evaluated immunohistochemically. The dogs were 7 males and 6 females (1 spayed) of 10 different breeds, averaging 8 years in age. The tumors were classified as papillary, tubulopapillary, papillary–cystic, solid, or sarcomatoid. All 13 tumors were immunohistochemically positive for uromodulin, 12 for c-KIT, 11 for vimentin, 9 for wide-spectrum-screening cytokeratins, 7 for cytokeratins AE1/AE3 and carcinoembryonic antigen, 4 for cytokeratins CAM 5.2, and 3 for CD10. All 3 solid RCCs expressed vimentin, c-KIT, and carcinoembryonic antigen and were negative for cytokeratins. All 7 papillary and tubulopapillary tumors expressed vimentin; 6 (86%), cytokeratins; and 6 (86%), c-KIT. Both papillary–cystic RCCs were positive for cytokeratins and c-KIT and negative for vimentin. These results indicate that the different histologic types of RCC have characteristic immunohistochemical profiles and that c-KIT may be involved in the pathogenesis of canine RCC.

Major discrepancies exist between histological predictions and actual metastatic potential of canine mammary tumors. Detection of circulating tumor cells (CTC) has a proven prognostic value for human breast cancer but similar markers for canine CTC are lacking. In the present study a panel of 16 human CTC markers was tested for their ability to specifically and sensitively detect canine carcinoma cells in peripheral blood. PCR assays for CK19, ERBB2, EGFR, CLDN7 and ELF3 were able to sensitively detect one carcinoma cell in up to 10(7) peripheral blood leukocytes. These CTC markers are thus candidate markers for identifying canine mammary CTC in the peripheral blood and may serve as prognostic factors for metastatic behavior in the future.

Galectin-3 is a glycan-binding protein that mediates cell-cell and/or cell-extracellular matrix (ECM) interactions. Although galectin-3 is implicated in the progression of various types of cancers, the mechanisms by which galectin-3 enhances metastasis remain unclear. In order to elucidate the role of galectin-3 in the complex multistage process of cancer metastasis, we examined galectin-3 and galectin-3-binding site expression in a series of 82 spontaneous canine mammary tumors (CMT) and two CMT cell lines. Benign CMT tumors exhibited strong nuclear/cytoplasmic galectin-3 immunostaining, whereas malignant CMT tumors and metastases exhibited dramatically decreased galectin-3 expression with the majority of the immunostaining confined to the cytoplasm. Interestingly, intravascular tumor cells overexpressed galectin-3 regardless of their location. CMT-U27 xenografts displayed the same pattern of galectin-3 expression found in spontaneous malignant CMT. In parallel with the downregulation of galectin-3, malignant CMT displayed an overall loss of galectin-3-binding sites in the ECM and focal expression of galectin-3-binding sites mainly detected in intravascular tumor cells and endothelium. Furthermore, loss of galectin-3-binding sites was correlated with the downregulation of GLT25D1, a β (1-O) galactosyltransferase that modifies collagen, and upregulation of stromal galectin-1. Finally, GLT25D1 mRNA expression was strikingly downregulated in malignant CMT-U27 compared with the benign cell line, and its expression was further decreased in a galectin-3 knockdown CMT-U27 cell line. We therefore hypothesized that the loss of galectin-3-binding sites in the ECM in conjunction with the overexpression of galectin-3 in specific tumor cell subpopulations are crucial events for the development of mammary tumor metastases.

Canine mammary tumours (CMTs) are a very heterogeneous group of neoplasms with variable prognosis. Their aggressiveness is mainly due to their ability to invade locally and to metastasize. The degradation of extracellular matrix components is an important determinant of the invasive phenotype. The aims of this study were to analyse by immunohistochemistry and double immunofluorescence the expression of metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in eight normal canine mammary glands and 118 CMTs (24 benign, 94 malignant) and to investigate relationships with metastatic disease and survival. MMP-2 and TIMP-2 expression was higher in malignant tumours than in normal canine mammary tissue and benign tumours. The main difference between benign and malignant CMTs was the pattern of expression of both molecules: benign tumours presented TIMP-2 and MMP-2 immunoreactivity in the myoepithelial cells lining the basement membrane of tubuloalveolar structures, while malignant tumours showed mainly diffuse expression in neoplastic cells. In malignant tumours, increased TIMP-2 expression was significantly associated with the development of distant metastases, lower overall survival and lower disease-free survival. MMP-2 expression was not significantly associated to any of these parameters. These results suggest that the immunohistochemical expression of TIMP-2 is a useful prognostic factor in CMTs.

Canine Merkel cell tumours are rare neuroendocrine neoplasms that show a relatively benign biological behaviour when compared with their human counterparts. To date, little information is available on their immunohistochemical properties. This report describes the histopathological and immunohistochemical features of two such tumours. The tumours' immunoreactivity profile was studied with respect to different cellular molecules including chromogranin A (CGA), neurone-specific enolase (NSE), S100 protein, c-KIT, the cytokeratins (CKs) detected by pancytokeratin (AE1/AE3) antibodies (i.e. high molecular weight CKs 1, 2, 3, 4, 5, 6, 10, 14, 15 and 16, and low molecular weight CKs 7, 8 and 19) and three markers proposed to correlate with increased malignancy in human tumours: E-cadherin, beta-catenin and p63 protein. In both lesions, tumour cells were positive for cytokeratins, CGA, NSE, S100 and c-KIT. No immunostaining was observed for p63 protein, and there was no loss or change in E-cadherin or beta-catenin immunoexpression. These results suggest that the generally benign behaviour of canine Merkel cell tumours, when compared with their human counterparts, may be partly explained by the conservation of important intercellular adhesion molecules such as E-cadherin and beta-catenin. Additionally, expression of S100 but not of the p63 protein suggests that these canine tumours present a trend towards neural, rather than basal, epithelial differentiation and do not readily compare with human Merkel cell tumours.

Tissue regeneration over a large defect with a subsequent satisfactory functional recovery still stands as a major problem in areas such as nerve regeneration or bone healing. The routine technique for the reconstruction of a nerve gap is the use of autologous nerve grafting, but still with severe complications. Over the last decades several attempts have been made to overcome this problem by using biomaterials as scaffolds for guided tissue regeneration. Despite the wide range of biomaterials available, functional recovery after a serious nerve injury is still far from acceptable. Prior to the use of a new biomaterial on healing tissues, an evaluation of the host's inflammatory response is mandatory. In this study, three chitosan membranes were tested in vitro and in vivo for later use as nerve guides for the reconstruction of peripheral nerves submitted to axonotmesis or neurotmesis lesions. Chitosan membranes, with different compositions, were tested in vitro, with a nerve growth factor cellular producing system, N1E-115 cell line, cultured over each of the three membranes and differentiated for 48h in the presence of 1.5% of DMSO. The intracellular calcium concentrations of the non-differentiated and of the 48h-differentiated cells cultured on the three types of the chitosan membranes were measured to determine the cell culture viability. In vivo, the chitosan membranes were implanted subcutaneously in a rat model, and histological evaluations were performed from material retrieved on weeks 1, 2, 4 and 8 after implantation. The three types of chitosan membranes were a viable substrate for the N1E-115 cell multiplication, survival and differentiation. Furthermore, the in vivo studies suggested that these chitosan membranes are promising candidates as a supporting material for tissue engineering applications on the peripheral nerve, possibly owing to their porous structure, their chemical modifications and high affinity to cellular systems.

BackgroundThe process of metastasis involves a series of steps and interactions between the tumor embolus and the microenvironment. Key alterations in adhesion molecules are known to dictate progression from the invasive to malignant phenotype followed by colonization at a distant site. The invasive phenotype results from the loss of expression of the E-cadherin adhesion molecule, whereas the malignant phenotype is associated with an increased expression of the carbohydrate ligand-binding epitopes, (e.g. Sialyl Lewis (x/a)) that bind endothelial E-selectin of the lymphatics and vasculature.MethodologyOur study analyzed the expression of two adhesion molecules, E-cadherin and Sialyl Lewis x (sLe(x)), in both a canine mammary carcinoma and human inflammatory breast cancer (IBC) model, using double labelled immunofluorescence staining.ResultsOur results demonstrate that canine mammary carcinoma and human IBC exhibit an inversely correlated cellular expression of E-cadherin and sLe(x) within the same tumor embolus.ConclusionsOur results in these two comparative models (canine and human) suggest the existence of a biologically coordinated mechanism of E-cadherin and sLe(x) expression (i.e. molecular plasticity) essential for tumor establishment and metastatic progression.

Catechol-O-methyltransferase (COMT) is an important enzyme involved in inactivation of catechol estrogens, which are metabolites with carcinogenic properties. Some investigations in human breast cancer associate a genetic polymorphism in the COMT gene (COMT val158met) with an increased risk and poor clinical progression of the disease. In dogs, there are 2 recognized single nucleotide polymorphisms in the COMT gene (COMTG216A and COMTG482A); however, their influence on the outcome of mammary neoplasms has never been investigated. The purpose of this study is to investigate the influence of COMT in the clinical progression of canine mammary tumors, namely in recurrence, metastasis and survival by testing 2 SNPs (G216A and G482A), and 2 genotypes of the COMT gene. A case series was conducted analyzing genomic DNA samples by polymerase chain reaction-restriction fragment length polymorphism from 80 bitches with mammary tumors. Animals were submitted to an active follow-up study for a period of 24 months after surgery. We observed that bitches carrying both genetic variations simultaneously are more likely to develop recurrence of mammary lesions. Our results demonstrate a possible role for COMT genotypes in the outcome of mammary neoplasms in the dog. Identifying a genetic factor predictive of recurrence may be useful in selecting the most effective surgical approach for canine mammary neoplasms.

Secretory carcinoma is an uncommon variant of breast cancer, characterized by the presence of intracellular and extracellular eosinophilic secretion. Here, we report the cytologic, histologic, and immunohistochemical findings of a secretory carcinoma diagnosed in the left inguinal mammary gland of a 3-year-old female German Shepherd Dog. The fine-needle aspiration cytology showed numerous large branching sheets of neoplastic cells and isolated cells with cytoplasmic vacuoles. Histologically, the tumor was composed of cells with clear cytoplasm and prominent vacuoles that pushed the nuclei to the periphery, resembling signet ring cells. These cells were arranged in solid or tubular structures with lumenal spaces filled with eosinophilic secretion. Immunohistochemical reactions to cytokeratin (CAM 5.2) and α-lactalbumin were strongly positive in all neoplastic cells, and staining for vimentin and S100 protein was negative. The cytomorphologic and immunohistochemical features of this tumor are similar to those seen in tumors in women, hence enabling the diagnosis of a rare case of primary secretory carcinoma of the canine mammary gland.