Repurposing Benztropine, Natamycin, and Nitazoxanide Using Drug Combination and Characterization of Gastric Cancer Cell Lines.

Gastric cancer (GC) ranked as the fifth most incident cancer in 2020 and the third leading cause of cancer mortality. Surgical prevention and radio/chemotherapy are the main approaches used in GC treatment, and there is an urgent need to explore and discover innovative and effective drugs to better treat this disease. A new strategy arises with the use of repurposed drugs. Drug repurposing coupled with drug combination schemes has been gaining interest in the scientific community. The main objective of this project was to evaluate the therapeutic effects of alternative drugs in GC. For that, three GC cell lines (AGS, MKN28, and MKN45) were used and characterized. Cell viability assays were performed with the reference drug 5-fluororacil (5-FU) and three repurposed drugs: natamycin, nitazoxanide, and benztropine. Nitazoxanide displayed the best results, being active in all GC cells. Further, 5-FU and nitazoxanide in combination were tested in MKN28 GC cells, and the results obtained showed that nitazoxanide alone was the most promising drug for GC therapy. This work demonstrated that the repurposing of drugs as single agents has the ability to decrease GC cell viability in a concentration-dependent manner.

In Vitro and In Vivo Characterization of PLLA-316L Stainless Steel Electromechanical Devices for Bone Tissue Engineering-A Preliminary Study.

Bone injuries represent a major social and financial impairment, commonly requiring surgical intervention due to a limited healing capacity of the tissue, particularly regarding critical-sized defects and non-union fractures. Regenerative medicine with the application of bone implants has been developing in the past decades towards the manufacturing of appropriate devices. This work intended to evaluate medical 316L stainless steel (SS)-based devices covered by a polymer poly (L-lactic acid) (PLLA) coating for bone lesion mechanical and functional support. SS316L devices were subjected to a previously described silanization process, following a three-layer PLLA film coating. Devices were further characterized and evaluated towards their cytocompatibility and osteogenic potential using human dental pulp stem cells, and biocompatibility via subcutaneous implantation in a rat animal model. Results demonstrated PLLA-SS316L devices to present superior in vitro and in vivo outcomes and suggested the PLLA coating to provide osteo-inductive properties to the device. Overall, this work represents a preliminary study on PLLA-SS316L devices' potential towards bone tissue regenerative techniques, showing promising outcomes for bone lesion support.

Nipple Hibernoma in a Dog: A Case Report With Literature Review.

This report provides a clinical, histological, and immunohistochemical description of an unusual hibernoma (pale cell variant) in the subepidermal area of the nipple of a six-year-old bitch. Furthermore, an extensive literature review of hibernomas in animals was made. Physical examination revealed a nodular lesion in the subepidermal area of the third nipple of the left mammary chain. The histopathological findings included lobules of round to oval cells with abundant pale to eosinophilic cytoplasm, containing one or multiple optically empty vacuoles, consistent with nipple hibernoma. Immunohistochemically, the neoplastic cells were negative for cytokeratin AE1/AE3 and p53 but showed strong immunoreaction for vimentin and uncoupling protein-1, thus confirming the brown adipose tissue origin. Local recurrence was not detected after 18 months of follow-up. Hibernomas are rare and benign neoplastic lesions, originating from brown adipose tissue. Due to their histological and molecular resemblance with liposarcoma, a correct diagnosis of these neoplasms is required. In addition, the literature review suggests that hibernomas may present different features, according to species.

Mesenchymal Stem/ Stromal Cells metabolomic and bioactive factors profiles: A comparative analysis on the umbilical cord and dental pulp derived Stem/ Stromal Cells secretome.

Mesenchymal Stem/ Stromal Cells assume a supporting role to the intrinsic mechanisms of tissue regeneration, a feature mostly assigned to the contents of their secretome. A comparative study on the metabolomic and bioactive molecules/factors content of the secretome of Mesenchymal Stem/ Stromal Cells derived from two expanding sources: the umbilical cord stroma and the dental pulp is presented and discussed. The metabolic profile (Nuclear Magnetic Resonance Spectroscopy) evidenced some differences in the metabolite dynamics through the conditioning period, particularly on the glucose metabolism. Despite, overall similar profiles are suggested. More prominent differences are highlighted for the bioactive factors (Multiplexing Laser Bear Analysis), in which Follistatin, Growth Regulates Protein, Hepatocyte Growth Factor, Interleukin-8 and Monocyte Chemotactic Protein-1 dominate in Umbilical Cord Mesenchymal Stem/ Stromal Cells secretion, while in Dental Pulp Stem/ Stromal Cells the Vascular Endothelial Growth Factor-A and Follistatin are more evident. The distinct secretory cocktail did not result in significantly different effects on endothelial cell populations dynamics including proliferation, migration, tube formation capacity and in vivo angiogenesis, or in chemotaxis for both Mesenchymal Stem/ Stromal Cells populations.

Equine ocular mast cell tumor: histopathological and immunohistochemical description.

This report describes an ocular mast cell tumor in a 13-year-old female sport horse. Clinical examination revealed a solitary firm mass located in the ocular mucosa, protruding from behind the left lower eyelid. The lesion was surgically removed and submitted to histopathology. Microscopically, the mass was composed of sheets of well-differentiated neoplastic round cells circumscribed by delicate connective tissue. Positive Giemsa and Toluidine Blue staining confirmed the presence of cytoplasmic granules. Neoplastic cells showed strong membranous and mild diffuse cytoplasmic immunoreactivity for c-KIT and a low KI-67 proliferative index. Based on these findings, a diagnosis of ocular mast cell tumor was made. Six months after surgical removal, no evidence of ocular lesion recurrence was detected.

Histochemical and immunohistochemical evaluation of angiogenesis in rabbits (Oryctolagus cuniculus) submitted to skin grafts associated with platelet-rich plasma / Histoquímica e imunohistoquíca na avaliação da angiogênese em coelhos submetidos ao emprego de enxertos cutâneos associado com plasma rico em plaquetas

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.(AU)

Colorações histoquímicas consistem de um conjunto de reações químicas específicas das estruturas ou substâncias endógenas do tecido. Logo a Imunohistoquímica permite observar proteínas presentes nos tecidos relacionadas com fatores determinantes do comportamento biológico e patológico. A padronização dos métodos que avaliam a angiogênese decorrente de procedimentos que utilizam substâncias estimulantes à formação de novos vasos são importantes, a fim de facilitar a execução das pesquisas, bem como auxiliar na interpretação dos dados, visto que em coelhos alguns anticorpos marcadores de angiogênese na pele ainda não são padronizadas em virtude das reações cruzadas que podem ocorrer devido aos anticorpos serem confeccionados a partir de tais animais. Objetivou-se analisar os métodos histoquímicos por meio das colorações e imunohistoquímicas com marcadores de angiogênese em coelhos (Oryctolagus cuniculus) submetidos ao emprego de enxertos cutâneos associado com estimulador de angiogênese plasma rico em plaquetas, a fim de avaliar qual método seria melhor para visualização dos vasos, bem como avaliar qual anticorpo promoveria melhor imunomarcação, buscando-se assim encontrar a diferenças entre os métodos e padronizar a metodologia a ser aplicada em experimentos que utilizem coelhos. Utilizou-se 16 coelhos, separados em dois grupos com oito animais, compreendendo os grupos Gprp (plasma rico em plaquetas) e Gc (controle, solução fisiológica 0,9%). Em todos os animais foi realizada a mesma técnica de cirurgia reconstrutiva de enxertia do tipo malha, os grupos diferiram apenas a aplicação do plasma rico em plaquetas antes da síntese da ferida cirúrgica. As amostras para avaliação da angiogênese foram coletadas após 15 dias do procedimento cirúrgico. Utilizou-se no estudo histoquímico as colorações Hematoxilina & Eosina e Tricrômico de Masson para avaliação da proliferação vascular, e os anticorpos CD31 e CD34 e Caveolina - 1 para avaliação imunohistoquímica. A comparação entre os grupos (Gprp e Gc) em relação à variável categórica (intensidade de proliferação vascular) foi utilizado o teste de Kruskal-Wallis, com valores de p iguais ou inferiores a 0,05 foram considerados significativos. Os dados imuno-histoquímico foram submetidos à análise de variância para um delineamento inteiramente ao acaso, com 2 grupos e 5 repetições (médias) e nível de significância de 5%. Nas comparações múltiplas das médias dos grupos, utilizou-se o teste de Tukey (α = 0,05). A intensidade de proliferação vascular avaliada pelo método histoquímico HE e Tricômico de Masson encontrou-se que tal variável foi significativa no Gprp, quando comparado com o Gc. Avaliando os métodos utilizados não houve diferença significativa. A contagem microvascular (MVC) realizada com os diferentes marcadores (Caveolina-1, CD31 e CD34) foi significativa no Gprp. Correlacionando a contagem microvascular dos três marcadores utilizados não houve diferença significativa, no entanto observou-se marcação mais intensa dos vasos utilizando o anticorpo Caveolina-1, sendo intensa a marcação dos capilares, vasos de pequeno calibre, bem como em vasos maiores. Nas avaliações de CD31 e CD34 observou que houve imunomarcação dos vasos, porém não foi intensa como a Caveolina-1, alguns casos apresentaram fundo, bem como marcação discreta. Os resultados encontrados neste estudo evidenciaram os métodos histoquímicos são eficazes para avaliação semiquantitativa da angiogênese. A comparação imunohistoquímicas da Caveolina-1, CD31 e CD34 como marcadores de angiogênese em coelhos evidenciaram que ambos os anticorpos são capazes de imunomarcar os vasos neoformados, porém a Caveolina-1 apresentou melhor imunomarcação de vasos de pequeno e médio calibre, bem como menor presença de fundo, embora não seja um marcador específico para angiogênese pode ser utilizada como marcador imunohistoquímico de endotélio vascular em coelhos.(AU)

Molecular Heterogeneity of Canine Cutaneous Peripheral Nerve Sheath Tumors: A Drawback in the Diagnosis Refinement.

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<0.05). CONCLUSION: The differential diagnosis of PNST on routine stained samples is difficult and the immunohistochemical examination may contribute to the final diagnosis. However, these lesions present a complex histogenesis and show very variable individual features; thus, an unequivocally immunohistochemical panel that could have supported the PNST diagnostic was not achieved. Nevertheless, we concluded that Ki-67 can be a useful marker helping to discriminate the biological behavior of canine PNST.

Inflammatory response to dextrin-based hydrogel associated with human mesenchymal stem cells, urinary bladder matrix and Bonelike® granules in rat subcutaneous implants.

Increasing relevance has been attributed to hydrogels due to their ability to provide an extracellular matrix (ECM)-like environment for cellular adhesion and proliferation, acting as mechanical scaffolds for tissue remodeling or as delivery matrices. In vivo biocompatibility of a hybrid dextrin hydrogel produced from oxidized dextrin and adipic acid dihydrazide and its combinations with human mesenchymal stem cells (hMSCs), ECM from a porcine bladder (urinary bladder matrix) and ceramic granules (Bonelike®), was evaluated following ISO 10993 after subcutaneous implantation in a rat model. Histological analysis after 3 and 15 d showed typical acute and chronic inflammatory responses, respectively, with a more severe reaction exhibited whenever the ceramic granules were present. However, the dextrin hydrogel was able to stabilize granules in the implant site. Dextrin hydrogel was scored as slight irritant after 3 d, similar to its combination with UBM, and as non-irritant after 15 d. The presence of viable hMSCs in the subcutaneous tissue could be confirmed by the presence of anti-human nuclei antibody (HuNu+) cells. The production of growth factors and inflammatory and immunomodulatory cytokines by these cells was also quantified in peripheral blood confirming the successful encapsulation of hMSCs into the hydrogel matrix for cell survival promotion. The presence of hMSCs seemed to modulate the inflammatory response by accelerating its progression when compared to the acellular experimental groups. Dextrin hydrogel has proven to be a biocompatible multifunctional matrix for minimally invasive biomedical procedures, including orthopedic surgeries when associated with bone substitutes and also as a possible encapsulation matrix for cell-based therapies.

Local iron homeostasis in the breast ductal carcinoma microenvironment.

BACKGROUND: While the deregulation of iron homeostasis in breast epithelial cells is acknowledged, iron-related alterations in stromal inflammatory cells from the tumor microenvironment have not been explored. METHODS: Immunohistochemistry for hepcidin, ferroportin 1 (FPN1), transferrin receptor 1 (TFR1) and ferritin (FT) was performed in primary breast tissues and axillary lymph nodes in order to dissect the iron-profiles of epithelial cells, lymphocytes and macrophages. Furthermore, breast carcinoma core biopsies frozen in optimum cutting temperature (OCT) compound were subjected to imaging flow cytometry to confirm FPN1 expression in the cell types previously evaluated and determine its cellular localization. RESULTS: We confirm previous results by showing that breast cancer epithelial cells present an 'iron-utilization phenotype' with an increased expression of hepcidin and TFR1, and decreased expression of FT. On the other hand, lymphocytes and macrophages infiltrating primary tumors and from metastized lymph nodes display an 'iron-donor' phenotype, with increased expression of FPN1 and FT, concomitant with an activation profile reflected by a higher expression of TFR1 and hepcidin. A higher percentage of breast carcinomas, compared to control mastectomy samples, present iron accumulation in stromal inflammatory cells, suggesting that these cells may constitute an effective tissue iron reservoir. Additionally, not only the deregulated expression of iron-related proteins in epithelial cells, but also on lymphocytes and macrophages, are associated with clinicopathological markers of breast cancer poor prognosis, such as negative hormone receptor status and tumor size. CONCLUSIONS: The present results reinforce the importance of analyzing the tumor microenvironment in breast cancer, extending the contribution of immune cells to local iron homeostasis in the tumor microenvironment context.

An immunohistochemical study on the expression of sex steroid receptors, Ki-67 and cytokeratins 7 and 20 in feline endometrial adenocarcinomas.

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.