2-Aminoethyl Dihydrogen Phosphate (2-AEH2P) Associated with Cell Metabolism-Modulating Drugs Presents a Synergistic and Pro-Apoptotic Effect in an In Vitro Model of the Ascitic Ehrlich Tumor.

The progression and maintenance of cancer characteristics are associated with cellular components linked to the tumor and non-cellular components with pro-tumoral properties. Pharmacological association with antagonists of the cellular components of the tumor, such as anti- and pro-apoptotic drugs, represents a novel adjuvant strategy. In this study, the antiproliferative, pro-apoptotic, and pharmacological effects of the combination of monophosphoester 2-AEH2P with Simvastatin, Coenzyme Q10, the chemotherapeutic drug paclitaxel, and colony-stimulating factor (GM-CSF) were evaluated. Tests were conducted to determine cytotoxic activity using the MTT method, cell cycle phases, and fragmented DNA by flow cytometry, mitochondrial membrane potential, expression of cell markers Bcl2, TNF-α/DR-4, Cytochrome c, caspase 3, and P53, and analysis of drug combination profiles using Synergy Finder 2.0 Software. The results showed a synergistic effect among the combinations, compared to individual treatments with the monophosphoester and other drugs. In addition, there was modulation of marker expression, indicating a pro-apoptotic and immunomodulatory effect of 2-AEH2P. Pharmacological analysis revealed that tumor cells treated with GM-CSF + 2-AEH2P exhibited a synergistic effect, while groups of tumor cells treated with paclitaxel, Coenzyme Q10, and Simvastatin showed additive effects. Furthermore, treatment with the paclitaxel + 2-AEH2P combination (12 h) resulted in a significant reduction in mitochondrial membrane potential. Pharmacological combinations for normal cells did not exhibit deleterious effects compared to mammary carcinomatosis tumor (EAT) cells.

The BR2 peptide associated with 2-aminoethyl dihydrogen phosphate is a formulation with antiproliferative potential for a triple-negative breast cancer model.

Triple-negative breast cancer is the most aggressive subtype of breast cancer, with worse clinical evolution and tumor-free survival, leading to the need to develop new effective therapies for its control. The present study evaluated the action of tumor-penetrating peptide BR2 associated with 2-aminoethyl dihydrogen phosphate (2-AEH2P) on triple-negative breast tumor cells. Cell viability was evaluated by the MTT colorimetric method, mitochondrial electrical potential, and proteins involved in cell proliferation and death control were evaluated by flow cytometry and structural and morphological analysis by confocal microscopy. The results obtained showed that the peptide BR2 and the association 2-AEH2P + BR2 promoted significant cytotoxicity in tumor lines, compared to 2-AEH2P alone. In addition, the association 2-AEH2P + BR2 promoted tumor cells arrest in the G0/G1 phases. Interestingly, both treatments modulated the expression of markers CD44, CD34, CD24, cyclin D1, and Bcl-2, increased p21, Bax, and released cytochrome c. The association proved to be more effective, providing modulation of proteins involved in cell death and senescence, more pronounced cytotoxicity for tumor cells compared to normal cells, and the reduction of markers related to aggressiveness profile, progression, and tumor metastasis.

Effects of chemical and physical methods on decellularization of murine skeletal muscles.

Volumetric muscle loss causes functional weakness and is often treated with muscle grafts or implant of biomaterials. Extracellular matrices, obtained through tissue decellularization, have been widely used as biological biomaterials in tissue engineering. Optimal decellularization method varies among tissues and have significant impact on the quality of the matrix. This study aimed at comparing the efficacy of four protocols, that varied according to the temperature of tissue storage and the sequence of chemical reagents, to decellularize murine skeletal muscles. Tibialis anterior muscles were harvested from rats and were frozen at -20°C or stored at room temperature, followed by decellularization in solutions containing EDTA + Tris, SDS and Triton X-100, applied in different sequences. Samples were analyzed for macroscopic aspects, cell removal, decrease of DNA content, preservation of proteins and three-dimensional structure of the matrices. Processing protocols that started with incubation in SDS solution optimized removal of cells and DNA content and preserved the matrix ultrastructure and composition, compared to those that were initiated with EDTA + Tris. Freezing the samples before decellularization favored cell removal, regardless of the sequence of chemical reagents. Thus, to freeze skeletal muscles and to start decellularization with 1% SDS solution showed the best results.

Comparison between placental and skeletal muscle ECM: in vivo implantation.

PURPOSE OF THE STUDY: Several tissues have been decellularized and their extracellular matrices used as allogeneic or xenogeneic scaffolds, either in orthotopic or heterotopic implantations, for tissue engineering purposes. Placentas have abundant matrix, extensive microvascular structure, immunomodulatory properties, growth factors and are discarded after birth, representing an interesting source of extracellular matrix. This study aimed at comparing decellularized canine placentas and murine skeletal muscles to regenerate skeletal muscles in a rat model. MATERIALS AND METHODS: Muscle pockets were created at the posterior limbs of male Wistar rats, where the muscle- and placenta-derived extracellular matrices were implanted. Macroscopic, histological, and immunohistochemical analyses were performed after 3, 15, and 45 days of surgeries. RESULTS: On the third day, intense inflammatory reaction, with macrophages (CD163+) and proliferative cells (PCNA+) being observed in control group and adjacent to the decellularized matrices. The percentage of proliferative cells was higher in placenta than in muscle matrices. Macrophages CD163+ high were higher in muscles than in placentas, whereas CD163+ low were higher in placentas than in muscle ECM, at days 3 and 15. Placental matrices were not completely degraded at day 15, as opposed to the muscular ones. After 45 days, both matrices were resorbed and morphologically normal myofibers, with reduction of cell infiltration, were observed. CONCLUSIONS: These results demonstrated that xenogeneic placental ECM, implanted heterotopically (representing a biologically critical and challenging microenvironment), induced local inflammatory reactions similar to the allogeneic muscle ECM, implanted orthotopically. Thus, placenta-derived extracellular matrix must be further explored in regenerative medicine.

Preclinical evaluation of Amblyomin-X, a Kunitz-type protease inhibitor with antitumor activity.

Amblyomin-X, a Kunitz-type protease inhibitor, is a recombinant protein that selectively induces apoptosis in tumor cells and promotes tumor reduction in vivo in melanoma animal models. Furthermore, Amblyomin-X was able to drastically reduce lung metastasis in a mice orthotopic kidney tumor model. Due to its antitumor activity, Amblyomin-X potential to become a new drug is currently under investigation, therefore the aim of the present study was to perform preclinical assays to evaluate Amblyomin-X toxicity in healthy mice. Exploratory toxicity assays have shown that treatment with 512 mg/kg of Amblyomin-X lead to animal mortality, therefore two groups of treatment were evaluated in the present work: in the acute toxicity assay, animals were injected once with doses ranging from 4 to 256 mg/kg of Amblyomin-X, while in the subacute toxicity assay, animals were injected with 0.25, 0.57 and 1 mg/kg of Amblyomin-X daily, during 28 days. Following this treatment regimens, Amblyomin-X did not cause any mortality; moreover, toxicity signs were discrete, reversible and observed only at the higher doses, thus establishing a safety profile for administration in mice, which can be further used to determine the dose translation of this novel drug candidate for treatment in other species.

Cell viability assessed in a reproducible model of human osteoblasts derived from human adipose-derived stem cells.

Human adipose tissue-derived stem cells (hASCs) have been subjected to extensive investigation because of their self-renewal properties and potential to restore damaged tissues. In the literature, there are several protocols for differentiating hASCs into osteoblasts, but there is no report on the control of cell viability during this process. In this study, we used osteoblasts derived from hASCs of patients undergoing abdominoplasty. The cells were observed at the beginning and end of bone matrix formation, and the expression of proteins involved in this process, including alkaline phosphatase and osteocalcin, was assessed. RANKL, Osterix, Runx2, Collagen3A1, Osteopontin and BSP expression levels were analyzed using real-time PCR, in addition to a quantitative assessment of protein levels of the markers CD45, CD105, STRO-1, and Nanog, using immunofluorescence. Rhodamine (Rho123), cytochrome-c, caspase-3, P-27, cyclin D1, and autophagy cell markers were analyzed by flow cytometry to demonstrate potential cellular activity and the absence of apoptotic and tumor cell processes before and after cell differentiation. The formation of bone matrix, along with calcium nodules, was observed after 16 days of osteoinduction. The gene expression levels of RANKL, Osterix, Runx2, Collagen3A1, Osteopontin, BSP and alkaline phosphatase activity were also elevated after 16 days of osteoinduction, whereas the level of osteocalcin was higher after 21 days of osteoinduction. Our data also showed that the cells had a high mitochondrial membrane potential and a low expression of apoptotic and tumor markers, both before and after differentiation. Cells were viable after the different phases of differentiation. This proposed methodology, using markers to evaluate cell viability, is therefore successful in assessing different phases of stem cell isolation and differentiation.

Cell internalization of 7-ketocholesterol-containing nanoemulsion through LDL receptor reduces melanoma growth in vitro and in vivo: a preliminary report.

Oxysterols are cholesterol oxygenated derivatives which possess several biological actions. Among oxysterols, 7-ketocholesterol (7KC) is known to induce cell death. Here, we hypothesized that 7KC cytotoxicity could be applied in cancer therapeutics. 7KC was incorporated into a lipid core nanoemulsion. As a cellular model the murine melanoma cell line B16F10 was used. The nanoparticle (7KCLDE) uptake into tumor cells was displaced by increasing amounts of low-density-lipoproteins (LDL) suggesting a LDL-receptor-mediated cell internalization. 7KCLDE was mainly cytostatic, which led to an accumulation of polyploid cells. Nevertheless, a single dose of 7KCLDE killed roughly 10% of melanoma cells. In addition, it was observed dissipation of the transmembrane potential, evidenced with flow cytometry; presence of autophagic vacuoles, visualized and quantified with flow cytometry and acridine orange; and presence of myelin figures, observed with ultrastructural microscopy. 7KCLDE impaired cytokenesis was accompanied by changes in cellular morphology into a fibroblastoid shape which is supported by cytoskeletal rearrangements, as shown by the increased actin polymerization. 7KCLDE was injected into B16 melanoma tumor-bearing mice. 7KCLDE accumulated in the liver and tumor. In melanoma tumor 7KCLDE promoted a >50% size reduction, enlarged the necrotic area, and reduced intratumoral vasculature. 7KCLDE increased the survival rates of animals, without hematologic or liver toxicity. Although more pre-clinical studies should be performed, our preliminary results suggested that 7KCLDE is a promising novel preparation for cancer chemotherapy.

Quantitative evaluation of collagen and elastic fibers after intense pulsed light treatment of mouse skin.

BACKGROUND AND OBJECTIVE: The aging of human skin includes intrinsic aging and photo-aging, which are characterized by a decrease in collagen and the deposition of abnormal elastic fibers. Intense pulsed light (IPL) sources are widely used in medicine to treat various cosmetic problems, including photo-damaged skin. Few studies have examined the microscopic changes produced by IPL. The objective of this study was to quantitatively evaluate the effects of IPL on collagen and elastic fibers in mice. MATERIALS AND METHODS: Forty female BALB/c mice were divided into four subgroups. Group 1 was the control group (n = 10), and groups 2, 3, and 4 were treatment groups (n = 10 in each group). Group 2 received one treatment, group 3 received two treatments, and group 4 received three treatments every 2 weeks. Skin tissue was obtained from irradiated areas 24 hours after the last treatment in each mouse. Collagen fibers were identified using the picrosirius red method. Elastic fibers were marked by Weigert-oxone stain. All samples were analyzed and quantified by a light microscope using analyzer system images. RESULTS: Group 4, which received three IPL treatments, showed significant quantitative increases in both collagen fibers (P < 0.05) and elastic fibers (P < 0.01). Collagen fibers demonstrated a better parallel distribution in relation to the epidermis. CONCLUSION: IPL treatment significantly increased the number of collagen and elastic fibers within the dermis and improved the parallel distribution of collagen fibers in relation to the epidermis. These results were evident after three IPL treatments. Lasers Surg. Med. © 2018 Wiley Periodicals, Inc.

Bovine central nervous system development / Desenvolvimento do sistema nervoso central de bovinos

Central nervous system (CNS) development researches are extremely important to the most common congenital disorders and organogenesis comprehension. However, few studies show the entire developmental process during the critical period. Present research can provide data to new researches related to normal development and abnormalities and changes that occur along the CNS organogenesis, especially nowadays with the need for preliminary studies in animal models, which could be used for experimental research on the influence of viruses, such as the influence of Zika virus on the development of the neural system and its correlation with microcephaly in human newborns. Then, present study describes CNS organogenesis in cattle according to microscopic and macroscopic aspects, identifying structures and correlating to gestational period. Fourteen embryos and nine bovine fetuses at different ages were collected and analyzed. All individuals were measured in order to detect the gestational period. Bovine embryo at 17 days age has its neural tube, cranial neuropore, caudal neuropore and somites developed. After 24 days of development, were observed in cranial part of neural tube five encephalic vesicles denominated: telencephalon, diencephalon, mesencephalon, metencephalon and myelencephalon. In addition, the caudal part of neural tube was identified with the primitive spinal cord. The primordial CNS differentiation occurred from 90 to 110 days. The five encephalic vesicles, primordial spinal cord and the cavities: third ventricule, mesencephalic aqueduct, fourth ventricle and central canal in spinal cord were observed. With 90 days, the main structures were identified: (1) cerebral hemispheres, corpus callosum and fornix, of the telencephalon; (2) interthalamic adhesion, thalamus, hypothalamus and epythalamus (glandula pinealis), of the diencephalon; (3) cerebral peduncles and quadruplets bodies, of the mesencephalon; (4) pons and cerebellum, of the metencephalon; (5) medulla oblongata or bulb, of the myelencephalon; and (6) spinal cord, of the primitive spinal cord. After 110 days of gestation, the five encephalic vesicles and its structures were completely developed. It was noted the presence of the spinal cord with the cervicothoracic and lumbossacral intumescences. In summary, the results describes the formation of the neural tube from the neural plate of the ectoderm, the encephalic vesicles derived from the neural tube and subsequent structural and cavities subdivisions, thus representing the complete embryology of the central nervous system.(AU)

Os estudos que descrevem o desenvolvimento do sistema nervoso central (SNC) são de suma importância para compreensão da organogênese e identificação dos prováveis eventos que resultam em malformações congênitas. Estes dados podem subsidiar novas pesquisas relacionadas ao desenvolvimento normal, e interpretação de malformações e alterações que ocorrem ao longo da organogênese do SNC, considerando neste momento a necessidade de estudos preliminares em modelos animais, os quais poderiam ser utilizados para pesquisas experimentais sobre a influência de agentes infecciosos como o Zika vírus, no desenvolvimento do sistema nervoso e suas relações com a microcefalia em humanos recém-nascidos. O presente estudo teve como objetivo descrever os aspectos morfológicos macro e microscópicos da organogênese do SNC de bovinos, buscando correlacionar os achados morfológicos com a idade gestacional. Todos os animais foram mensurados para detectar o período gestacional. Foram coletados e analisados 14 embriões e nove fetos de bovinos de diferentes idades gestacionais. No embrião bovino a partir do décimo sétimo dia de gestação, encontra-se a formação do tubo neural, o neuroporo cranial e neuroporo caudal, e formação dos somitos. Após 24 dias de desenvolvimento, são observadas na parte cranial do tubo neural cinco vesículas encefálicas denominadas: telencéfalo, diencéfalo, mesencéfalo, metencéfalo e mielencéfalo; e na parte caudal do tubo neural, encontra-se a medula espinhal primitiva. Entre 90 a 110 dias de gestação, observa-se a total diferenciação das cinco vesículas do SNC. Com 90 dias, são identificas as principais estruturas: (1) do telencéfalo, os hemisférios cerebrais, corpo caloso e fórnix; (2) do diencéfalo, a aderência intertalâmica, tálamo, hipotálamo e epitálamo (glândula pineal); (3) do mesencéfalo, os pedúnculos cerebrais e os corpos quadrigêmios; (4) do metencéfalo, a ponte e o cerebelo; (5) do mielencéfalo, a medula oblonga (ou bulbo); e (6) da medula espinhal primitiva, a medula espinhal. Após 110 dias, as cinco vesículas encefálicas e as suas subdivisões se encontram completamente desenvolvidas. Notou-se a presença da medula espinhal com as intumescências cervicotorácica e lombossacral. Em resumo, os resultados demonstram a formação do tubo neural a partir da placa neural do ectoderma, as vesículas encefálicas provenientes do tubo neural e posteriormente as subdivisões das estruturas e das cavidades, que representam a completa embriologia do sistema nervoso central.(AU)

Laser Photobiomodulation Over Teeth Subjected to Orthodontic Movement.

Background: Orthodontics of the 21st century requires aesthetic, painless, predictable, and quick treatments. This demand for faster results generated orthodontic movement acceleration protocols (OMAPs); among other OMAPs we present low-level laser (LLL) as a candidate. Objective: To evaluate levels of interleukin (IL)-1, IL-10, and type 1 collagen in the periodontal ligament of first molars of rats subjected to orthodontic traction with and without LLL irradiation, compared with untreated controls (CO), and to evaluate whether the dose of LLL used in this work is eligible as an OMAP. Materials and methods: A total of 35 male Wistar rats were distributed into three groups: group 1 NI (nonirradiated) n = 15, group 2 IR (laser irradiated using 5 J, 177 J/cm2, and 100 mW applied in contact to the vestibular mesial, vestibular distal, and palatal faces of gum tissue around molar region for 50 sec each point, for 3 consecutive days, immediately 24 and 48 h after orthodontic device placement.) n = 15, and group 3 CO n = 5; groups 1 and 2 were subjected to orthodontic force and each group was divided into three subgroups that were sacrificed after 3, 5, and 7 days, IL-1/10 and COL-1 levels were analyzed. Results: In the IR group, levels of IL-1/10 and COL-1 showed peak anticipation after LLL irradiation compared with those in the NI and CO groups. Conclusions: These results can also infer that this dose of LLL can be used as an OMAP.

Egg and fourth instar larvae gut of Aedes aegypti as a source of stem cells.

According to the World Health Organization, 2015 registered more than 1.206.172 cases of Dengue in the Americas. Recently, the Aedes aegypti has been not only related to Dengue, but also with cases of Zika virus and Chikungunya. Due to its epidemiological importance, this study characterized the morphology of the embryonated eggs of A. aegypti and provided a protocol to culture stem cells from eggs and digestive tract of fourth instar larvae in order to examine cell biology and expression of markers in these vectors. Cells were isolated and cultured in DMEM-High at 28°C, and their morphology, cell cycle and immunophenotyping were examined. Morphologically, embryos were at the end of the embryonic period and showed: head, thorax, and abdomen with eight abdominal segments. The embryonic tissues expressed markers related to cell proliferation (PCNA), pluripotency (Sox2 and OCT3/4), neural cells (Nestin), mesenchymal cells (Vimentin and Stro-1), and endosomal cells (GM130 and RAB5). In culture, cells from both tissues (eggs and larvae gut) were composed by a heterogeneous population. The cells had a globoid shape and small size. Cell cycle analysis on passage 1 (P1) showed 27.5%±2.0% of cell debris, 68% of cells on G0-G1 phase, 30.2% on S phase, 1.9%±0.5% on G2-M phase. In addition, cells on passage 2 showed: 10% of cell debris, 92.4% of cells on G0-G1 phase, 6.8% on S phase, 0.6% on G2-M phase. Embryonated eggs expressed markers involved with pluripotency (Sox2 and Oct 3/4), mesenchymal cells (vimentin and Stro-1), neural cells (Nestin), and cellular death by apoptosis (Caspase 3). Specific endosomal markers for insect cells (GM130 and RAB5) were also highly expressed. In cell culture of A. aegypti larvae gut the same labeling pattern was observed, with a small decrease in the expression of mesenchymal (vimentin and Stro-1) and neural (Nestin) markers. In summary, we were able to establish a protocol to culture embryonated eggs and larvae gut of A. aegypti, describing the characteristics of undifferentiated cells, as well as the cell cycle and expression of markers, which can be used for biotechnology studies for the biological control of this vector.

Animal-assisted therapy in early childhood schools in São Paulo, Brazil / Terapia assistida por animais em escolas de educação infantil de São Paulo

Since ancient times, humans and animals have interacted for different purposes. Animal Assisted Therapy (AAT) is used for the assistance and treatment in humans and educational projects where animals are used as co-therapists or co-educators. The use of animals facilitates the process of teaching and learning, and stimulates physical and therapeutic activities. So that knowledge on AAT could be expanded, current study analyzes the opinion of people directly involved in education on AAT implementation as an educational model in early childhood schools in São Paulo, Brazil. Questionnaires were handed out to 10 pedagogical coordinators, 32 teachers, 23 parents and 26 children aged 3-6 years. Results revealed that AAT is not well-known for most interviewees, including pedagogical coordinators, teachers and parents. However, interviewees believe in the benefits of child-pet interactions and are favorable to the implementation of AATs in schools. Projects should be interdisciplinary and must involve professionals from other areas, such as psychologists and veterinarians. Regarding the educational model, interviewees believe in the innovation capacity of AAT and in the possibilities of interdisciplinarity among teachers in the use of animals. Research also demonstrated that children like and support the use of animals in the school.(AU)

Desde a pré-história já existia a interação dos humanos com os animais com diferentes finalidades. A terapia assistida por animais (TAA) é utilizada para assistências e tratamentos em humanos e projetos pedagógicos, na qual os animais são utilizados como co-terapeutas ou co-educadores. O emprego de animais facilita o processo de ensino-aprendizagem e estimula atividades físicas e terapêuticas. Para contribuir com o conhecimento da TAA, o presente trabalho objetivou abordar a opinião de pessoas diretamente relacionadas à escola, a respeito da implantação da TAA, como modelo educacional nas escolas de educação infantil da cidade de São Paulo. Foram aplicados questionários em 10 coordenadores pedagógicos, 32 professores, 23 pais e 26 crianças de 3 a 6 anos de idade. Os resultados encontrados demonstraram que a TAA ainda não é bem conhecida por uma parcela dos entrevistados, incluindo coordenadores pedagógicos, professores e pais de alunos. No entanto, os entrevistados acreditam nos benefícios da interação criança-animal e defendem projetos voltados a implantação da TAA nas escolas, embora estes projetos devam ser interdisciplinares e envolver profissionais de outras áreas, tais como, pedagogos, psicólogos e médicos veterinários. Em relação ao modelo educacional, nossos entrevistados acreditam na capacidade inovadora da TAA, assim como, nas possibilidades de interdisciplinaridade entre os professores no uso dos animais. Também ficou demonstrado que as crianças gostam e apoiam o uso de animais na escola.(AU)

Simvastatin increases the antineoplastic actions of paclitaxel carried in lipid nanoemulsions in melanoma-bearing mice.

PURPOSE: Lipid nanoemulsions (LDEs) that bind to low-density lipoprotein (LDL) receptors used as carriers of paclitaxel (PTX) can decrease toxicity and increase PTX antitumoral action. The administration of simvastatin (Simva), which lowers LDL-cholesterol, was tested as an adjuvant to commercial PTX and to PTX associated with LDE (LDE-PTX). MATERIALS AND METHODS: B16F10 melanoma-bearing mice were treated with saline solution or LDE (controls), Simva, PTX, PTX and Simva, LDE-PTX, and LDE-PTX and Simva: PTX dose 17.5 µmol/kg (three intraperitoneal injections, 3 alternate days): Simva 50 mg/kg/day by gavage, 9 consecutive days. RESULTS: Compared with saline controls, 95% tumor-growth inhibition was achieved by LDE-PTX and Simva, 61% by LDE-PTX, 44% by PTX and Simva, and 43% by PTX. Simva alone had no effect. Metastasis developed in only 37% of the LDE-PTX and Simva, 60% in LDE-PTX, and 90% in PTX and Simva groups. Survival rates were higher in LDE-PTX and Simva and in LDE-PTX groups. The LDE-PTX and Simva group presented tumors with reduced cellular density and increased collagen fibers I and III. Tumors from all groups showed reduction in immunohistochemical expression of ICAM, MCP-1, and MMP-9; LDE-PTX and Simva presented the lowest MMP-9 expression. Expression of p21 was increased in the Simva, LDE-PTX, and LDE-PTX and Simva groups. In the Simva and LDE-PTX and Simva groups, expression of cyclin D1, a proliferation and survival promoter of tumor cells, was decreased. Therapy with LDE-PTX and Simva showed negligible toxicity compared with PTX and Simva, which resulted in weight loss and myelosuppression. CONCLUSION: Simva increased the antitumor activity of PTX carried in LDE but not of PTX commercial presentation, possibly because statins increase the expression of LDL receptors that internalize LDE-PTX.

Comparative study of equine mesenchymal stem cells from healthy and injured synovial tissues: an in vitro assessment.

BACKGROUND: Bone marrow and adipose tissues are known sources of mesenchymal stem cells (MSCs) in horses; however, synovial tissues might be a promising alternative. The aim of this study was to evaluate phenotypic characteristics and differentiation potential of equine MSCs from synovial fluid (SF) and synovial membrane (SM) of healthy joints (SF-H and SM-H), joints with osteoarthritis (SF-OA and SM-OA) and joints with osteochondritis dissecans (SF-OCD and SM-OCD) to determine the most suitable synovial source for an allogeneic therapy cell bank. METHODS: Expression of the markers CD90, CD105, CD44, and CD34 in SF-H, SM-H, SF-OA, SM-OA, SF-OCD and SM-OCD was verified by flow cytometry, and expression of cytokeratin, vimentin, PGP 9.5, PCNA, lysozyme, nanog, and Oct4 was verified by immunocytochemistry. MSCs were cultured and evaluated for their chondrogenic, osteogenic and adipogenic differentiation potential. Final quantification of extracellular matrix and mineralized matrix was determined using AxioVision software. A tumorigenicity test was conducted in Balb-C(nu/nu) mice to verify the safety of the MSCs from these sources. RESULTS: Cultured cells from SF and SM exhibited fibroblastoid morphology and the ability to adhere to plastic. The time elapsed between primary culture and the third passage was approximately 73 days for SF-H, 89 days for SF-OCD, 60 days for SF-OA, 68 days for SM-H, 57 days for SM-OCD and 54 days for SM-OA. The doubling time for SF-OCD was higher than that for other cells at the first passage (P < 0.05). MSCs from synovial tissues showed positive expression of the markers CD90, CD44, lysozyme, PGP 9.5, PCNA and vimentin and were able to differentiate into chondrogenic (21 days) and osteogenic (21 days) lineages, and, although poorly, into adipogenic lineages (14 days). The areas staining positive for extracellular matrix in the SF-H and SM-H groups were larger than those in the SF-OA and SM-OA groups (P < 0.05). The positive mineralized matrix area in the SF-H group was larger than those in all the other groups (P < 0.05). The studied cells exhibited no tumorigenic effects. CONCLUSIONS: SF and SM are viable sources of equine MSCs. All sources studied provide suitable MSCs for an allogeneic therapy cell bank; nevertheless, MSCs from healthy joints may be preferable for cell banking purposes because they exhibit better chondrogenic differentiation capacity.

Evaluation of the Proliferative Effects Induced by Low-Level Laser Therapy in Bone Marrow Stem Cell Culture.

OBJECTIVE: The objective of this study was to evaluate the effect of laser irradiation on dog bone marrow stem cells. BACKGROUND DATA: Low doses of low-level red laser positively affect the viability of mesenchymal stem cells, and also increase proliferation. METHODS: Low-level laser (wavelength, 660 nm; power output, 50 mW), was applied to dog bone marrow stem cell cultures (DBMSC). The energy densities delivered varied from 1 to 12J/cm(2). The effect of the laser irradiation was evaluated on cell proliferation measured with the MTT colorimetric test, cell cycle phase, and on lipidic peroxidation (free radical production). RESULTS: The results indicate that laser irradiation to DBMSC did not change the morphology of the cells, but significantly increased their viability and the number of cells at the G2/M phase with 6, 10, and 12 J/cm(2). On the other hand, malonaldehyde production was significantly enhanced with 8 J/cm(2). CONCLUSIONS: The parameters used to irradiate DBMSC increased significantly proliferation without producing high levels of reactive oxygen species (ROS).

Characterization of adipose-derived stem cells of anatomical region from mice.

BACKGROUND: Stem cells constitute a group of great capacity for self-renewal, long-term viability, and multi-lineage potential. Several studies have provided evidence that adipose tissue represents an alternative source of stem cells, with the main benefit of adipose-derived stem cells being that they can be easily harvested from patients by a simple minimally invasive method and can be easily cultured. The aim of this study was to establish a culture protocol for obtaining and characterizing adipose-derived stem cells (ADSCs) from C57BL/6 J mice. RESULTS: The results showed that the yield, viability, and cell morphology obtained differ according to the age of isolated anatomic regions of the adipose tissue from ovarian and epididymis. The results of determination of cyclin D1 showed uniformity in the expression between different populations of ADSCs. A significant increase in the expression of caspase-3 active, was also observed in large cell populations from mice after 120 days. ADSCs were positive for mesenchymal markers CD90 and CD105, Nanog, SSEA-1, CD106, and VEGFR-1, and negative for hematopoietic markers CD34 and CD45. A large number of cells in S + G2/M phases was also observed for both sexes, demonstrating high proliferative capacity of ADSCs. CONCLUSIONS: We observed that the adipose tissue of C57BL/6 J mice, isolated from the studied anatomic regions, is a promising source for obtaining pluripotent mesenchymal stem cells with high viability and proliferative response.

Association of daunorubicin to a lipid nanoemulsion that binds to low-density lipoprotein receptors enhances the antitumour action and decreases the toxicity of the drug in melanoma-bearing mice.

OBJECTIVES: To test the toxicity and antitumoral activity of the compound N-oleyl-daunorubicin (oDNR) with a cholesterol-rich nanoemulsion (LDE) formulation. METHODS: LDE-oDNR was prepared by high-pressure homogenisation of lipid mixtures. B16F10 melanoma cells and NIH/3T3 fibroblasts were used for cytotoxicity tests. The maximum tolerated dose (MTD) of both commercial and LDE-oDNR was determined in mice, and melanoma-bearing mice were used for the antitumoral activity tests. KEY FINDINGS: CC50 for LDE-oDNR and DNR in melanoma cells were 200 µm and 15 µm, respectively, but LDE-oDNR was less toxic against fibroblasts than DNR. MTD for LDE-oDNR was 65-fold higher than commercial DNR. In tumour-bearing mice, LDE-oDNR (7.5 µmol/kg) reduced tumour growth by 59 ± 2%, whereas the reduction by DNR was only 23 ± 2%. LDE-oDNR increased survival rates (P < 0.05), which was not achieved by DNR treatment. The number of mice with metastasis was only 30% in LDE-oDNR-treated mice, compared with 82% under DNR treatment. By flow cytometry, there were 9% viable cells in tumours of animals treated with LDE-oDNR compared with 27% in DNR-treated animals. Less haematological toxicity was observed in LDE-oDNR-treated mice. CONCLUSIONS: Compared with DNR, LDE-oDNR improved tumour growth inhibition and survival rates with pronouncedly less toxicity, and thus may become a new tool for cancer treatment.

Angiogenesis enhanced by Phyllocaulis boraceiensis mucus in human cells.

Phyllocaulis boraceiensis mucus is known to be a compound capable of inducing cell proliferation and enhancing the wound healing process. The process of angiogenesis is a chain of mechanisms responsible for the formation of new vessels, which are are involved in cell proliferation, and factors that will act in the healing process. Our aim was to demonstrate that the angiogenesis process is enhanced in cultures of endothelial cells and fibroblasts treated with P. boraceiensis mucus. Experiments were carried out with 10(5) cells·mL(-1) of endothelial cells and fibroblasts treated with P. boraceiensis mucus in concentrations that have significant effects in proliferation assays, i.e. 0.012 µg·µL(-1) and 0.18 µg·µL(-1) , both of which cause extreme responses. Aliquots of 100 µL of cell suspensions were incubated for 1 h at 4 °C with 1 µL of antibodies specific for the cell markers vascular endothelial growth factor receptor 1 and cluster of differentiation 34, and negative isotype controls. Reading and expression analysis of cell markers was performed on a FACSCalibur flow cytometer. Expression levels of vascular endothelial growth factor receptor 1 and cluster of differentiation 34 expression were significantly increased in endothelial cells cultivated with 0.012 µg·µL(-1) P. boraceiensis mucus, suggesting that this compound is capable of enhancing angiogenesis.

Azidothymidine is effective against human multiple myeloma: a new use for an old drug?

Azidothymidine (AZT) is an antiretroviral drug that affects cell proliferation, apoptosis, and the NF-κB pathway. As multiple myeloma (MM) presents with constitutive activation of NF-κB, we analyzed the effect of AZT on human MM cell lines. We evaluated the cytotoxic effect of AZT in human MM cell lines sensitive (8226/S) or resistant to doxorubicin (8226/DX5) and human T cell lymphoblast-like cells, uterine sarcoma cells, and HUVEC using MTT assay. Cytotoxicity was also evaluated in vivo in nude mice xenografted with 8226/S tumor. The effect of AZT on the expression of genes involved in cell proliferation, apoptosis, angiogenesis, and the NF-κB pathway was analyzed in the xenografts using real-time polymerase chain reaction. AZT was effective against both 8226/S and 8226/DX5 cells in a dose and time-dependent manner (p = 0.02) in vitro and promoted cell cycle arrest in S phase in these cells. The tumor volume was lower in mice treated with AZT compared to untreated mice (p = 0.0003). AZT down-regulated the pro-proliferative genes encoding AKT1, MYC, STAT1, MAPK8, MAPK9, CCL-3, Bcl-3, and cyclin D2; pro-angiogenenic genes encoding VEGF and IL8; and genes involved in cell adhesion (ICAM1 and FN1) and the NF-κB pathway. AZT up-regulated the expression of tumor suppressor gene FOXP1 and the pro-apoptotic genes encoding BID, Bcl-10, and caspase-8. Thus, we demonstrated the cytotoxic effect of AZT in human MM cell lines for the first time. Our data may provide the rationale for future clinical trials of AZT for treating MM.

Drug-targeting in combined cancer chemotherapy: tumor growth inhibition in mice by association of paclitaxel and etoposide with a cholesterol-rich nanoemulsion.

BACKGROUND: Lipid nanoemulsions (LDE) may be used as carriers of paclitaxel (PTX) and etoposide (ETP) to decrease toxicity and increase the therapeutic action of those drugs. The current study investigates the combined chemotherapy with PTX and ETP associated with LDE. METHODS: Four groups of 10-20 B16F10 melanoma-bearing mice were treated with LDE-PTX and LDE-ETP in combination (LDE-PTX + ETP), commercial PTX and ETP in combination (PTX + ETP), single LDE-PTX, and single LDE-ETP. PTX and ETX doses were 9 µmol/kg administered in three intraperitoneal injections on three alternate days. In two control groups mice were treated with saline solution or LDE alone. Tumor growth, metastasis presence, cell-cycle distribution, blood cell counts and histological data were analyzed. Toxicity of all treatments was evaluated in mice without tumors. RESULTS: Tumor growth inhibition was similarly strong in all treatment groups. However, there was a greater reduction in the number of animals bearing metastases in the LDE-PTX + ETP group (30 %) in comparison to the PTX + ETP group (82 %, p < 0.05). Reduction of cellular density, blood vessels and increase of collagen fibers in tumor tissues were observed in the LDE-PTX + ETP group but not in the PTX + ETP group, and in both groups reduced melanoma-related anemia and thrombocytosis were observed. Flow cytometric analysis suggested that LDE-PTX + ETP exhibited greater selectivity to neoplastic cells than PTX-ETP, showing arrest (65 %) in the G(2)/M phase of the cell cycle (p < 0.001). Toxicity manifested by weight loss and myelosuppression was markedly milder in the LDE-PTX + ETP than in the PTX + ETP group. CONCLUSION: LDE-PTX + ETP combined drug-targeting therapy showed markedly superior anti-cancer properties and reduced toxicity compared to PTX + ETP.