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Tendons and ligaments injuries are frequent in sport horses and humans, representing a great therapeutic challenge. Tissue regeneration and function recovery are the paramount of tendon and ligament lesions management. Nowadays, several regenerative treatments are being developed, based on the use of stem cell and stem cell-based therapies. In the present study, the preparation of equine synovial membrane mesenchymal stem cells (eSM-MSCs) for clinical use - collection, transport, isolation, differentiation, characterization, and application is described. These cells are fibroblast-like and grow in clusters. They retain an osteogenic, chondrogenic and adipogenic differentiation potential. 16 clinical cases of tendonitis and desmitis, treated with allogenic eSM-MSCs and autologous serum are also presented, including their evaluation, treatment and follow-up. The use of autologous serum as a vehicle concern to a decreased immunogenic response after the administration of this combination and to its healing properties, due to the presence of growth factors and immunoglobulins. Most of the cases (14/16), healed in 30 days and presented a good outcome. Treatment of tendon and ligament lesions with the mixture of eSM-MSCs and autologous serum appears to be a promising clinical option for this category of lesions in equine patients.

Additive manufacturing or 3D printing applying polycaprolactone (PCL)-based medical devices represents an important branch of tissue engineering, where the sterilization method is a key process for further safe application in vitro and in vivo. In this study, the authors intend to access the most suitable gamma radiation conditions to sterilize PCL-based scaffolds in a preliminary biocompatibility assessment, envisioning future studies for airway obstruction conditions. Three radiation levels were considered, 25 kGy, 35 kGy and 45 kGy, and evaluated as regards their cyto- and biocompatibility. All three groups presented biocompatible properties, indicating an adequate sterility condition. As for the cytocompatibility analysis, devices sterilized with 35 kGy and 45 kGy showed better results, with the 45 kGy showing overall improved outcomes. This study allowed the selection of the most suitable sterilization condition for PCL-based scaffolds, aiming at immediate future assays, by applying 3D-customized printing techniques to specific airway obstruction lesions of the trachea.

This study evaluated the expression of vimentin and Ki-67 proliferative index (PI) by immunohistochemistry in 30 canine gastric carcinomas (GCs) and a possible association with clinical and pathological features and patient’s survival time. Vimentin immunoreactivity was assessed in neoplastic cells (in primary lesions, emboli, and metastases) and tumor-associated stroma (TAS) of canine GCs. Ki-67 PI was quantified in the neoplastic epithelial component. Vimentin immunolabeling in neoplastic cells was found in 30% of the primary lesions, in 82% of the neoplastic emboli, and in 50% of the metastases; in TAS, it was observed in all cases. A mean of 16% of the TAS was immunolabeled for vimentin. High vimentin immunolabeling in the TAS (>16%) was detected in 40% of cases. The average value of Ki-67 PI was 50%, and 80% of the lesions had Ki-67 PI above 20%. Vimentin immunolabeling in neoplastic cells was more frequent in less-differentiated carcinomas (diffuse [29%] and indeterminate types [75%]) than well-differentiated carcinomas (intestinal type [0%], P = .049). No significant differences were observed in vimentin immunolabeling in the TAS or Ki-67 PI according to histological diagnosis, depth of invasion, presence of neoplastic emboli or metastases. However, vimentin immunolabeling in the TAS was positively correlated with Ki-67 PI ( r = .394, P = .031). Furthermore, a moderate negative correlation was observed between Ki-67 PI and survival time ( r = −0.540). Our results suggest that vimentin and Ki-67 PI have potential for providing prognostic information in cases of canine GCs.

The development of biomaterials for application in advanced therapies requires thorough characterization of its biological behavior, which ultimately entails in vivo compatibility and performance assays. Electrospun fiber membranes of poly(l-lactic acid) (PLLA) and fish gelatin blends were produced and characterized, coupling the biomechanical features of PLLA with gelatin (GEL) biocompatibility. Fiber diameter was not affected by polymer blending, whereas the swelling degree increased with increasing GEL contents for values up to 566 ± 13%, behaving as a superhydrophilic material. Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) adhesion was favored in the PLLA-GEL membranes, and cell viability was not affected after 7 days in culture. Membranes were then evaluated for in vivo biocompatibility through subcutaneous implantation in a rat model, for up to 15 days. No significant differences between the biological behavior of PLLA, PLLA-GEL, and GEL electrospun membranes at 15 days postimplantation were verified, with attained inflammation scores supporting an acceptable tissue response, deeming them fit for further biological assays. This work demonstrates that fiber blends of PLLA and GEL present promising in vitro and in vivo characteristics to be explored for tissue engineering.

The genus Helicobacter is composed of bacteria that colonize both the human and animal gastrointestinal tract. Helicobacter pylori infects half of the world’s population, causing various disorders, such as gastritis, duodenitis and gastric cancer. Additionally, non-Helicobacter pylori Helicobacter species (NHPH) are commonly found in the stomach of pigs, dogs and cats. Most of these species have zoonotic potential and prevalence rates of 0.2–6.0%, and have been described in human patients suffering from gastric disorders undergoing a gastric biopsy. The aim of this study was to determine the occurrence of Helicobacter spp. in the stomach of patients with gastric cancer (n = 17) and obese (n = 63) patients. Furthermore, the outcome of the Helicobacter eradication treatment and the current infection status was evaluated. Overall, based on the genus-specific PCR followed by sequencing, DNA from Helicobacter spp. was detected in 46.3% of the patients, including single infections with H. pylori in 43.8% of the patients and mixed infections with H. pylori and canine- or feline-associated H. felis in 2.5%. About 32.5% of the patients had been subjected to previous Helicobacter eradication therapy and the triple standard therapy was the most frequent scheme (42.3%). In 48.0% of the patients who received eradication treatment, bacteria were still detected, including one mixed infection. In 23.1% of the patients who reported that a subsequent test had been performed to confirm the elimination of the bacteria, Helicobacter were still detected. In conclusion, although in a smaller percentage, NHPH may also be present in the human stomach. Thus, specific NHPH screening should be included in the diagnostic routine. The continued presence of H. pylori in the stomach of patients recently subjected to eradication schemes raises questions about the efficacy of the current Helicobacter treatments.

The genus

Despite the progressive research and recent advances in drug therapy to treat solid tumours, the number of cases and deaths in patients with cancer is still a major health problem. Drug repurposing coupled to drug combination strategies has been gaining interest among the scientific community. Recently, our group proposed novel drug combinations for breast and colon cancer using repurposed drugs from different classes (antimalarial and central nervous system (CNS)) and chemotherapeutic agents such as 5-fluorouracil (5-FU), paclitaxel (PTX), and found promising results. Here, we proposed a novel drug combination using different CNS drugs and doxorubicin (DOX), an antineoplastic used in breast cancer therapy, and studied their anticancer potential in MCF-7 breast cancer cells. Cells were treated with each drug alone and combined with increasing concentrations of DOX and cell viability was evaluated by MTT and SRB assays. Studies were also complemented with morphological evaluation. Assessment of drug interaction was performed using the CompuSyn and SynergyFinder software. We also compiled our previously studied drug pairs and selected the most promising ones for evaluation of the expression of EMT biomarkers (E-cadherin, P-cadherin, vimentin, and β-catenin) by immunohistochemistry (IHC) to assess if these drug combinations affect the expression of these proteins and eventually revert EMT. These results demonstrate that combination of DOX plus fluoxetine, benztropine, and thioridazine at their IC50 can improve the anticancer effect of DOX but to a lesser degree than when combined with PTX (previous results), resulting in most of the drug interactions being antagonist or additive. This suggests that the choice of the antineoplastic drug influences the success of the drug combination. Collectively, these results also allow us to conclude that antimalarial drugs as repurposed drugs have enhanced effects in MCF-7 breast cancer cells, while combination with CNS drugs seems to be more effective in HT-29 colon cancer cells. The IHC results demonstrate that combination treatments increase E-cadherin expression while reducing P-cadherin, vimentin, and β-catenin, suggesting that these treatments could induce EMT reversal. Taken together, these results could provide promising approaches to the design of novel drug combinations to treat breast and colon cancer patients.

TFF1 expression is markedly reduced in human GCs, suggesting that TFF1 is a tumor suppressor for human gastric cancer. The present study evaluated the expression and distribution pattern of TFF1 in paraffin-embedded canine gastric tissue samples, including normal mucosa (n = 3), polyps (n = 8), carcinomas (n = 31) and their adjacent non-neoplastic mucosa (n = 30), neoplastic emboli (n = 14), and metastatic lesions (n = 9), by immunohistochemistry (IHC). All normal gastric tissues expressed TFF1 in the superficial foveolar epithelium and mucopeptic cells of the neck region. Most gastric polyps (GPs) displayed immunoreactivity for TFF1 in &gt, 75% of the epithelial component. In GCs, the expression of TFF1 was found reduced in 74.2% of the cases. The level of TFF1 expression had a decreased tendency from normal gastric mucosa to GPs and GCs (p &lt, 0.05). No significant differences in the expression of TFF1 were found in GCs, according to age, sex, histological type based on World Health Organization (WHO) and Lauren classification, tumor location, depth of tumor invasion, presence of neoplastic emboli or metastatic lesions. The median survival time of GC patients with preserved and reduced TFF1 immunoexpression were 30 and 12 days, respectively. Kaplan–Meier analysis revealed no significant survival differences between the two groups (p &gt, 0.05). These findings suggest that TFF1 protein may play a role in canine gastric carcinogenesis, and further studies are necessary to define its usefulness as a prognostic indicator in canine gastric carcinoma.

Made available in DSpace on 2019-10-06T16:22:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01. Added 1 bitstream(s) on 2019-10-09T18:35:19Z : No. of bitstreams: 1 S0100-736X2019000100040.pdf: 2621138 bytes, checksum: aa859149f83aebb38f1b4c7f4dd8d6b8 (MD5) Canine prostate gland is a hormonal dependent organ and its imbalance of estrogen and androgen receptor expressions are directly associated with the development of different diseases. Due to the lack of information regarding the behavior of the aforementioned receptors in canine prostate cancer (PC), this study aimed to identify estrogen receptor alpha (ERα), androgen receptor (AR), Ki67 and phosphatase and tensin homolog (PTEN) protein expressions in canine PC by immunohistochemistry. We found nuclear expression of ERα and AR in the epithelial cells of normal canine samples and a loss of protein expression in PC samples. Normal samples showed Ki67 expression in a few basal cells and the PC samples showed the highest mean of positive cells (253.1). Canine prostate cancer showed a high proliferative index, which was associated with independence of hormonal actuation. PTEN showed positive nuclear and cytoplasmic expression in normal canine samples and a loss in PC. Loss of ERα, AR and PTEN indicated that canine PC exhibits the same immunohistochemical phenotype as in human patients with PC resistant to hormonal therapy. Therefore, canine PC should be considered as a model to study human PC resistant to hormonal therapy. Departamento de Clínica Veterinária Faculdade de Medicina Veterinária e Zootecnia (FMVZ) Universidade Estadual Paulista (Unesp), Rua Prof. Dr. Walter Mauricio Correra s/n, Rubião Junior Departamento de Anestesiologia Faculdade de Medicina de Botucatu, Av. Prof. Montenegro s/n Instituto de Ciências Biomédicas Abel Salazar (ICBAS) Universidade do Porto (UP), Rua Jorge de Viterbo Ferreira 228 Departamento de Clínica Veterinária Faculdade de Medicina Veterinária e Zootecnia (FMVZ) Universidade Estadual Paulista (Unesp), Rua Prof. Dr. Walter Mauricio Correra s/n, Rubião Junior Departamento de Anestesiologia Faculdade de Medicina de Botucatu, Av. Prof. Montenegro s/n

Background Ovine pulmonary adenocarcinoma (OPA) is a neoplastic disease caused by exogenous Jaagsiekte Sheep Retrovirus (exJSRV). The prevalence of JSRV-related OPA in Eastern European countries, including Romania is unknown. We aimed to investigate: the prevalence and morphological features of OPA (classical and atypical forms) in the Transylvania region (Romania), the immunophenotype of the pulmonary tumors and their relationships with exJSRV infection. A total of 2693 adult ewes slaughtered between 2017 and 2019 in two private slaughterhouses from Transylvania region (Romania) was evaluated. Lung tumors were subsequently assessed by cytology, histology, immunocytochemistry, immunohistochemistry, electron microscopy and DNA testing. Results Out of 2693 examined sheep, 34 had OPA (1.26% prevalence). The diaphragmatic lobes were the most affected. Grossly, the classical OPA was identified in 88.24% of investigated cases and the atypical OPA in 11.76% that included solitary myxomatous nodules. Histopathology results confirmed the presence of OPA in all suspected cases, which were classified into acinar and papillary types. Myxoid growths (MGs) were diagnosed in 6 classical OPA cases and in 2 cases of atypical form. Lung adenocarcinoma was positive for MCK and TTF-1, and MGs showed immunoreaction for Vimentin, Desmin and SMA; Ki67 expression of classical OPA was higher than atypical OPA and MGs. JSRV-MA was identified by IHC (94.11%) in both epithelial and mesenchymal cells of OPA. Immunocytochemistry and electron microscopy also confirmed the JSRV within the neoplastic cells. ExJSRV was identified by PCR in 97.05% of analyzed samples. Phylogenetic analysis revealed the presence of the exJSRV type 2 (MT809678.1) in Romanian sheep affected by lung cancer and showed a high similarity with the UK strain (AF105220.1). Conclusions In this study, we confirmed for the first time in Romania the presence of exJSRV in naturally occurring OPA in sheep. Additionally, we described the first report of atypical OPA in Romania, and to the best of our knowledge, in Eastern Europe. Finally, we showed that MGs have a myofibroblastic origin.

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 epithelial cells with the CD44+/CD24-/low phenotype possess tumor-initiating cells and epithelial-mesenchymal transition (EMT) capacity. Massive parallel sequencing can be an interesting approach to deepen the molecular characterization of these cells. We characterized CD44+/CD24-/cytokeratin(Ck)+/CD45- cells isolated through flow cytometry from 43 biopsy and 6 mastectomy samples harboring different benign and malignant breast lesions. The Ion Torrent Ampliseq Cancer Hotspot panel v2 (CHPv2) was used for the identification of somatic mutations in the DNA extracted from isolated CD44+/CD24-/Ck+/CD45- cells. E-Cadherin and vimentin immunohistochemistry was performed on sections from the corresponding formalin-fixed, paraffin-embedded (FFPE) blocks. The percentage of CD44+/CD24-/Ck+/CD45- cells increased significantly from non-malignant to malignant lesions and in association with a significant increase in the expression of vimentin. Non-malignant lesions harbored only a single-nucleotide polymorphism (SNP). Mutations in the tumor suppressor p53 (TP53), NOTCH homolog 1 (NOTCH1), phosphatase and tensin homolog (PTEN), and v-akt murine thymoma viral oncogene homolog 1 (AKT1) genes were found in isolated CD44+/CD24-/Ck+/CD45- cells from ductal carcinomas in situ (DCIS). Additional mutations in the colony-stimulating factor 1 receptor (CSF1R), ret proto-oncogene (RET), and TP53 genes were also identified in invasive ductal carcinomas (IDCs). The use of massive parallel sequencing technology for this type of application revealed to be extremely effective even when using small amounts of DNA extracted from a low number of cells. Additional studies are now required using larger cohorts to design an appropriate mutational profile for this phenotype.

Environmental risk assessment (ERA) based on effects caused by chronic and longer term exposure is highly relevant. Further, if mechanistic based approaches (e.g. omics) can be included, beyond apical endpoints (e.g. reproduction), the prediction of effects increases. For Cu NMs (and CuCl2) this has been studied in detail, covering multi-omics and apical effects using the soil standard species Enchytraeus crypticus. The intermediate level effects like cell/tissue and organ alterations represent a missing link. In the present study we aimed to: 1) perform long term exposure to Cu materials (full life cycle and multigeneration, 46 and 224 days) to collect samples; 2) perform histology and immunohistochemistry on collected samples at 12 time points and 17 treatments; 3) integrate all levels of biological organization onto an adverse outcome pathway (AOP) framework. CuO NMs and CuCl2 caused both similar and different stress response, either at molecular initiating events (MIE) or key events (KEs) of higher level of biological organization. Cell/Tissue and organ level, post-transcriptional and transcriptional mechanisms, through histone modifications and microRNA related protein, were similarly affected. While both Cu forms affected the Notch signalling pathway, CuCl2 also caused oxidative stress. Different mechanisms of DNA methylation (epigenetics) were activated by CuO NMs and CuCl2 at the MIE. published

A 12-year-old neutered female mixed-breed dog was referred for occasional vomiting that had increased progressively in frequency over the past 3 months. Palpation of the abdomen revealed a firm mass of unknown aetiology, located within the cranial to mid abdomen, while abdominal surgical exploration revealed a well-defined abdominal mass of pancreatic origin. The pancreatic mass caused lateral displacement of the duodenum as well as medial displacement of the pylorus, resulting in impairment of pyloric outflow. Further examination revealed a firm, poorly vascularized and coarsely lobulated structure. The histopathological findings were consistent with severe pancreatic lipomatosis and atrophy. Immunohistochemically, the remnant pancreatic cells were positive for cytokeratins AE1/AE3 and glucagon, and negative for insulin. Routine follow-up with the referring veterinarian showed no evidence of postoperative complications, but the dog continued to deteriorate further and died despite medical management. Pancreatic lipomatosis is a rare condition in small animal practice. The aetiology or predisposing factors have not been identified in animals.

The aim of this study was to evaluate of the efficacy of PRP employment associated with surgical sponges to improve the integration of the graft in the recipient bed. It was held at the Veterinary Hospital UNESP, Campus of Jaboticabal - SP, a study of 64 rabbits, divided into eight groups with eight animals. The groups were divided in control with saline solution 0,9%, control with PRP both without the sponge, surgical sponge with PRP, surgical sponge without PRP, and were used mesh and layer grafts in the respective groups. The data were submitted to statistical analysis (paired t-test, Kruskal-Wallis test, with subsequent use of the multiple comparison tests of Dunn, analysis of variance (F) test, Tukey test, P< 0.05). Edema and exudate with 3 and 3 and 7 days (P= 0,03 e P= 0,0049); coloring on the 14th day (P= 0,0001); cosmetic appearance on the 7th and 14th day (P= 0,0026 and P= 0,0001); mononuclear cells (P= 0,01) and polymorphonuclear (P= 0,01); fibroblast proliferation (P= 0,01); collagenous (P= 0,05); hemorrhage (P-007); necrosis and re-epithelialization (P= 0,2928 and P= 0,1). We concluded that the use of Platelet Rich Plasma Gel on skin grafts associated with a sponge as a compressive dressing promote the skin graft survival without a previous granulation tissue. RESUMO O objetivo deste estudo foi avaliar a eficácia do PRP associado com esponjas cirúrgicas na integração do enxerto ao leito receptor. Realizou-se, no Hospital Veterinário da Unesp, Jaboticabal, SP, um estudo com 64 coelhos, separados em oito grupos, com oito animais. Os grupos foram: Gprpc (PRP, sem esponja cirúrgica, enxerto camada), Gprpce (PRP, esponja cirúrgica, enxerto camada), Gcc (solução fisiológica 0,9%, sem esponja cirúrgica, enxerto camada), Gcce (solução fisiológica 0,9%, esponjas cirúrgicas, enxerto camada), Gprpm (PRP, sem esponja cirúrgica, enxerto malha), Gprpme (PRP, esponja cirúrgica, enxerto malha), Gcm (solução fisiológica 0,9%, sem esponja cirúrgica, enxerto malha) e Gcce (solução fisiológica 0,9%, esponjas cirúrgicas, malha). Os dados foram analisados pelo teste t emparelhado, Kruskal-Wallis, análise de variância, e teste de Tukey (P

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.

Made available in DSpace on 2018-12-11T17:18:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-12-01. Added 1 bitstream(s) on 2019-10-09T18:31:33Z : No. of bitstreams: 1 S0100-736X2017001201519.pdf: 3105779 bytes, checksum: e7e2b1d54005cd3be7ef11220dbb4333 (MD5) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Histochemical staining consists of a set of specific chemical reactions of structures or tissue-endogenous substances. Immunohistochemistry allows verification of proteins in tissues related to biological and pathological factors. The standardization of methods to assess angiogenesis resulting from formation of new blood vessels in procedures with stimulants is important to facilitate the implementation of research as well as to assist interpretation of data. In rabbits some markers of angiogenesis antibodies in the skin are not standardized because of cross-reactions that may occur because the antibodies are made from such animals.The aim of this study was to analyze the immunohistochemical methods through dyes and immunohistochemical markers angiogenesis in rabbits (Oryctolagus cuniculus) having undergone reconstructive surgery with skin grafts associated with plasma angiogenesis stimulator rich in platelets, in order to evaluate which method would be better to visualize the vessels, as well as to evaluate which antibody would promote better immunostaining, and find the differences between the methods and to standardize the methodology to be applied in experiments using rabbits. Sixteen rabbits were used, split into two groups of eight animals: Gprp (plasma rich in platelets) and Gc (control, saline solution, 9%). The same technique of reconstructive surgery using graft mesh was performed on each rabbit. The groups differed only in the application of platelet-rich plasma before the surgical wound synthesis. Samples for evaluation of angiogenesis were collected 15 days after the surgical procedure. The dyes Hematoxylin & Eosin and Masson's Trichrome were used in the histochemical study to evaluate vascular proliferation. Markers CD31, CD34 and Caveolin-1 was used for the immunohistochemical study. The evaluation between the groups (Gprp and Gc) in regard to the categorical variable (vascular proliferation intensity) used the Kruskal-Wallis test with p values equal to or less than 0.05 being considered significant. The immunohistochemistry was subjected to analysis of variance for a completely randomized design, with two groups and five repetitions (medium) and 5% significance level. Multiple comparison of groups resulted in the Tukey test (p=0.05) used. The amount of vascular proliferation assessed by histochemical method HE and Masson's Trichrome was found to be a significant variable in Gprp when compared with group Gc. When evaluating the methods used, there was no significant difference. There was no difference in the three markers which were used for correlating microvessels; however, there was more intense staining of vessels when Caveolin-1 Antibody was used. This caused intense marking of the capillaries and small vessels, as well as of larger vessels. When using CD31 and CD34, the same was observed, but it was not as intense as with Caveolin-1; though some cases showed sincere and discreet marking. The results of this study demonstrated that the histochemical methods performed are effective for semi-quantitative assessment of angiogenesis. The immunohistochemical comparison of Caveolin-1, CD31, and CD34 as markers of angiogenesis in rabbits showed that both antibodies could immunostain the newly formed vessels; but the Caveolin-1 showed better immunostaining in small and medium-sized vessels, as well as a minor presence in the background. Although not specific markers for angiogenesis, they can be used as immunohistochemical markers of vascular endothelium in rabbits. Departamento de Clínica e Cirurgia Veterinária Faculdade de Ciências Agrárias e Veterinárias (FCAV) Universidade Estadual Paulista (Unesp), Via de Acesso Professor Paulo Donato Castellane s/n Departamento de Patologia Veterinária Instituto de Ciências Biomédicas Abel Salazar (ICBAS) Universidade do Porto (UP), Rua Jorge Viterbo Ferreira 228 Departamento de Clínica e Cirurgia Veterinária Faculdade de Ciências Agrárias e Veterinárias (FCAV) Universidade Estadual Paulista (Unesp), Via de Acesso Professor Paulo Donato Castellane s/n

Claudins (CLDNs) are tight junction proteins that have a role in regulating cell adhesion and polarity, paracellular permeability, proliferation and differentiation. Several immunohistochemical studies have shown reduced expression of CLDN-1 and CLDN-7 in human and canine mammary carcinomas, suggesting that these proteins may participate in mammary carcinogenesis, invasion and metastasis. The present study characterizes expression of CLDN-1 and CLDN-7 in feline mammary carcinomas (n = 52) and their metastases (n = 29). There was an inverse association between CLDN-7 expression and histological grade of tumour. Reduced expression of CLDN-7 was significantly associated with decreased tubule formation, high proliferative activity and metastasis. No significant associations were found between CLDN-1 expression and any of these features. Evaluation of expression of CLDN-7, but not CLDN-1, may therefore provide prognostic information, assisting in the diagnosis of a subgroup of aggressive feline mammary carcinomas that share some features with the recently described 'claudin-low' subgroup of human breast cancer.

The persistent organic pollutant p,p'-DDE, the major metabolite of the insecticide DDT, has displayed evidence of endocrine disruption through the inhibition of androgen binding to androgen receptors in different species. Although p,p'-DDE was continuously detected in wild fish with abnormal gonad development such as intersex, little is known about its mode of action during gonad development in fish. To elucidate the potential endocrine effects of this pollut- ant in zebrafish (Danio rerio), juveniles (30days post hatch) were exposed to p,p'-DDE during the critical window of sexual differentiation. Fish were exposed to sublethal concentrations ranging from 0.01 to 20 μgl -1 over 14days and were maintained in control water for an additional 4months. As core endpoints, the vitellogenin (vtg) concentration was measured at the end of exposure, and sex ratio and the gonadosomatic index were assessed 4months after the end of exposure. An increase in vtg production in whole body homogenate was observed in fish exposed to 0.2 and 2.0 μgl -1 p,p'-DDE. No significant differences were displayed in morphological parameters such as the gonadosomatic index of males and females or sex ratio. However, exposed females presented histopathological changes that include the reduction of the number of mature oocytes, which might impairtheir successfulreproduction. These results demonstrate the ability of p,p'-DDE to causeendocrinedisruption inzebrafish exposed during gonad differentiation of juvenile specimens. Furthermore, vtg induction by p,p'-DDE in juvenile zebrafish arises as a predictive marker for adverse effects of this DDT metabolite on the ovarian function of female zebrafish. Copyright © 2014 John Wiley & Sons, Ltd.

Claudins (CLDNs) are a family of tight junction (TJ) proteins that play an important role in maintaining cell polarity, in controlling paracellular ion flux and in regulating cell proliferation and differentiation. There is a growing body of evidence that associates changes in CLDN expression with the development of human breast cancer. In the present study CLDN-2 expression was examined immunohistochemically in samples of normal feline mammary tissue (n = 5) and mammary carcinomas (n = 52), including metastatic lesions (n = 29). Seventy-seven percent of carcinomas showed reduced CLDN-2 expression compared with that observed in normal mammary gland. Reduced expression of CLDN-2 was significantly associated with a high histological grade of carcinoma (P = 0.011), with 88.6% of grade II/III carcinomas showing decreased expression. Furthermore, CLDN-2 down-regulation was significantly associated with metastatic disease (P = 0.0027), with 93.1% of cases with signs of metastasis showing decreased expression of this protein. CLDN-2 may constitute a molecular marker for identification of a subgroup of feline mammary carcinomas characterized by high histological grade and the development of metastasis.

E-cadherin and beta-catenin are component of adherens junctions in epithelial cells. Loss of these proteins have been associated with progression of prostatic diseases. We performed immunohistochemistry for E-cadherin, beta-catenin and Ki-67 on canine prostatic lesions. We analyzed the expression of these antibodies in benign prostatic hyperplasia (BPH, n = 22), in pre neoplastic lesions Prostatic Intra-epithelial Neoplasia (PIN), n = 3 and Prostatic Inflammatory Atrophy (PIA), n = 7 and prostate carcinoma (PC, n = 10). In this study, a membranous expression of E-cadherin and beta-catenin and nuclear expression of Ki-67 antigen were demonstrated. The proliferative index was statistically different between carcinomas and BPH and carcinomas and pre-neoplastic lesions. Like in men, the reduction of E-cadherin and increase of Ki-67 expression in neoplastic lesions in dog prostate may be related to the carcinogenic process in this gland.

Disponível em: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5264664/ The association of HFE (High Iron FE) major variants with breast cancer risk and behavior has been a matter of discussion for a long time. However, their impact on the expression of iron-related proteins in the breast cancer tissue has never been addressed. In the present study, hepcidin, ferroportin 1, transferrin receptor 1 (TfR1), and ferritin expressions, as well as tissue iron deposition were evaluated in a collection of samples from breast cancers patients and analyzed according to the patients’ HFE genotype. Within the group of patients with invasive carcinoma, those carrying the p.Cys282Tyr variant in heterozygosity presented a higher expression of hepcidin in lymphocytes and macrophages than wild-type or p.His63Asp carriers. An increased expression of TfR1 was also observed in all the cell types analyzed but only in p.Cys282Tyr/p.His63Asp compound heterozygous patients. A differential impact of the two HFE variants was further noticed with the observation of a significantly higher percentage of p.Cys282Tyr heterozygous patients presenting tissue iron deposition in comparison to p.His63Asp heterozygous. In the present cohort, no significant associations were found between HFE variants and classical clinicopathological markers of breast cancer behavior and prognosis. Although limited by a low sampling size, our results provide a new possible explanation for the previously reported impact of HFE major variants on breast cancer progression, i.e., not by influencing systemic iron homeostasis but rather by differentially modulating the local cellular expression of iron-related proteins and tissue iron deposition. OM is a recipient of the PhD grant SFRH/BD/2011/78184 from Fundação para a Ciência e Tecnologia (FCT). The authors also acknowledge financial support from ICBAS/AI&NSUMIB and by national funds through FCT and Ministério da Educação e Ciência (MEC) and when applicable co-funded by FEDER funds within the partnership agreement PT2020 related with the research unit number 4293. info:eu-repo/semantics/publishedVersion

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 Cancer stem cells are tumor cells that present self-renewal, clonal tumor initiation capacity and clonal long-term repopulation potential. We have previously demonstrated that the co-expression of the breast cancer stem cell (BCSC) markers hyaluronan receptor (CD44) and aldehyde dehydrogenase-1 (ALDH1) in ductal carcinomas in situ could be determinant for disease progression. Combining these established BCSC markers with Ki-67 to evaluate quiescence we sought to identify, evaluate the distribution and estimate the mean percentages of CD44(+)ALDH1(+)Ki-67(-) breast cells. MATERIALS AND METHODS Triple-immunohistochemistry for CD44, ALDH1 and Ki-67 was applied in a series of 16 normal, 54 non-malignant and 155 malignant breast tissues. Clinical relevance was inferred by associations with markers of breast cancer behavior, progression and survival. RESULTS The mean percentages of cells with this phenotype increased significantly from non-malignant lesions to high-grade ductal carcinomas in situ, decreasing in invasive ductal carcinomas, as also evidenced by an inverse correlation with histological grade and tumor size. The mean percentage of CD44(+)ALDH1(+)Ki-67(-) cells was also significantly higher in women who developed distant metastasis and died due to breast cancer, and a significant association with human epidermal growth factor type 2 (HER2) negativity was observed. CONCLUSION Our novel findings indicate that CD44(+)ALDH1(+)Ki-67(-) tumor cells may favor distant metastasis and can predict overall survival in patients with ductal carcinomas of the breast. More importantly, quiescence may have a crucial role for tumor progression, treatment resistance and metastatic ability of BCSCs.

The functional and structural performance of a 5cm synthetic small diameter vascular graft (SDVG) produced by the copolymerization of polyvinyl alcohol hydrogel with low molecular weight dextran (PVA/Dx graft) associated to mesenchymal stem cells (MSCs)-based therapies and anticoagulant treatment with heparin, clopidogrel and warfarin was tested using the ovine model during the healing period of 24 weeks. The results were compared to the ones obtained with standard expanded polyetetrafluoroethylene grafts (ePTFE graft). Blood flow, vessel and graft diameter measurements, graft appearance and patency rate (PR), thrombus, stenosis and collateral vessel formation were evaluated by B-mode ultrasound, audio and color flow Doppler. Graft and regenerated vessels morphologic evaluation was performed by scanning electronic microscopy (SEM), histopathological and immunohistochemical analysis. All PVA/Dx grafts could maintain a similar or higher PR and systolic/diastolic laminar blood flow velocities were similar to ePTFE grafts. CD14 (macrophages) and α-actin (smooth muscle) staining presented similar results in PVA/Dx/MSCs and ePTFE graft groups. Fibrosis layer was lower and endothelial cells were only detected at graft-artery transitions where it was added the MSCs. In conclusion, PVA/Dx graft can be an excellent scaffold candidate for vascular reconstruction, including clinic mechanically challenging applications, such as SDVGs, especially when associated to MSCs-based therapies to promote higher endothelialization and lower fibrosis of the vascular prosthesis, but also higher PR values.

Breast cancer epithelial cells with the CD44

The existing standard enchytraeid reproduction test (ERT) concerns the assessment of effects on survival and reproduction. In the present study we optimized and propose an embryotoxicity test using Enchytraeus crypticus. Cadmium (Cd) was used as a test substance. Endpoints evaluated were embryo development, number of embryonic structures, Calcium (Ca) channels quantification and hatching success with macroscopic monitoring, histological and immunohistochemistry analysis. Results showed that Cd is embryotoxic for this species, causing a decrease in the hatching success (EC50=3.1mg/kg), a delay or disruption in formation of embryonic structures depending on concentrations (5mg Cd/kg or ⩾16mg Cd/kg). Results from immunohistochemistry suggest a competitive binding between Cd and Ca for Ca channels, resulting in changes in Ca homeostasis. The use of the E. crypticus embryotoxicity test with the combination of histological and immunohistological tools provided a good option towards mechanistic information enhancing the importance of these tests to evaluate the hazard of chemicals and possible use in risk assessment.

Claudins (CLDNs) are crucial structural and functional components of tight junctions playing an important role in maintaining cell polarity, controlling paracellular diffusion and regulating cell growth and differentiation of epithelial cells. CLDNs are differentially expressed in neoplastic lesions compared to the corresponding healthy tissues; therefore, they are thought to play a role in tumorigenesis and tumor progression. Reduced expression of CLDN-1 has been observed in several types of human cancers, including thyroid tumors. There are no reports of CLDN-1 immunoexpression in normal and neoplastic canine thyroid tissues.CLDN-1 immunoexpression was investigated in normal canine thyroid gland (n=2), benign (n=1) and malignant (n=10) tumors, as well as neoplastic emboli (n=6).CLDN-1 was constitutively expressed in normal canine follicular epithelium. Ninety percent of the malignant canine thyroid lesions showed absence or reduced CLDN-1 expression compared to that of normal thyroid gland. Additionally, a cytoplasmic subcellular location of CLDN-1 was recorded in the malignant epithelial cells and neoplastic emboli.These findings link altered expression of CLDN-1 to neoplastic transformation and suggest that CLDN-1 expression is associated with malignant canine thyroid tumors and their vascular invasion.

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.

The authors describe their experience with an automated immunohistochemical system applied to canine tissue samples. Twenty human cellular markers specific monoclonal and polyclonal antibodies and two different antigen retrieval methods were used in normal and neoplastic breast tissue, as well as skin samples obtained from female dogs of pure and mixed breeds. The antibodies tested were the most frequently used in human and veterinary medicine studies, employed with diagnostic purposes in breast pathology, as well as in cancer research. Most of them may be used to study other normal and abnormal tissues and included cytokeratins, progesterone receptor, c-erbB2, p53, MIB-1, PCNA, EMA, vimentin, desmin, alpha-actin, S-100, pan-cadherin, and E-cadherin. The results demonstrated that using an automated staining system it is possible to use different human markers in veterinary pathology. The advantages of automated immunohistochemistry are improved quality, reproducibility, speed, and standardisation. Os autores descrevem sua experiência com um sistema automático de imunoistoquímica aplicada à amostras de tecido canino. Foram utilizados 20 anticorpos humanos monoclonais e policlonais e dois diferentes métodos de recuperação antigênica em tecido mamário normal e neoplásico, bem como em amostras de pele obtidas de cadelas. Os anticorpos testados estão entre os mais usados em estudos de medicina humana e veterinária, com finalidade de diagnóstico em patologia mamária, bem como na pesquisa do câncer. Muitos deles podem ser usados para estudar outros tecidos normais e com alterações e incluem citoqueratinas, receptor de progesterona, c-erbB2, p53, MIB-1, PCNA, EMA, vimentina, desmina, alfa-actina, S-100, pan-caderina e E-caderina. Os resultados demonstraram que usando um sistema automático de imunoistoquímica é possível usar diferentes marcadores humanos em patologia veterinária. As vantagens da imunoistoquímica automatizada são melhora da qualidade, reprodutibilidade, rapidez e padronização.

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 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 Cancer Stem Cell (CSC) model proposes that cancer is driven by a cellular component which possesses stem cell (SC) properties, cancer stem cells (CSCs), a distinct cell-type which is tumorigenic and capable of invasion and metastasis. Enzymatic activity of aldehyde dehydrogenase-1 (ALDH1), a de-toxifying enzyme that oxidizes intracellular aldehydes, has been used as a marker of normal and malignant breast stem cells (BSCs). CD44-transmembrane protein has already been shown to possess the ability to identify breast epithelial cells with stem properties.In order to compare two of the currently most reliable BSCs markers, ALDH1 and CD44, and to correlate their expression within different breast lesions, 190 samples from breast cancer specimens were analyzed by tissue microarrays.ALDH1 expression was observed in 85.43% and CD44 in 90.3% of all samples. No overexpression was observed for ALDH1 between invasive tumors, ductal carcinomas in situ and non-malignant lesions of breast, although ALDH1 had a significant negative correlation with estrogen-receptor (ER) and progesterone-receptor (PR) status (p=0.002 and p=0.001, respectively) and a positive correlation with CD44 (p0.001). Moreover, combined overexpression of ALDH1 and CD44 was observed in ductal in situ tumors (p0.001).The combined overexpression of these markers in ductal carcinomas in situ is in agreement with the CSC model in breast cancer.