Exequibility of differential gene expression analysis by DDRT-PCR in murine bone marrow cells

Model of study: Experimental study. Introduction: Recently, stem cell research has generated great interest due to its applicability in regenerative medicine. Bone marrow is considered the most important source of adult stem cells and the establishment of new methods towards gene expression analysis regarding stem cells has become necessary. Thus Differential Display Reverse Transcription Polymerase Chain Reaction (DDRT-PCR) may be an accessible tool to investigate small differences in the gene expression of different stem cells in distinct situations. Aim: In the present study, we investigated the exequibility of DDRT-PCR to identify differences in global gene expression of mice bone marrow cells under two conditions. Methods: First, bone marrow cells were isolated fresh and a part was cultivated during one week without medium replacement. Afterwards, both bone marrow cells (fresh and cultivated) were submitted to gene expression analyses by DDRT-PCR. Results: Initially, it was possible to observe in one week-cultured bone marrow cells, changes in morphology (oval cells to fibroblastic-like cells) and protein profile, which was seen through differences in band distribution in SDS-Page gels. Finally through gene expression analysis, we detected three bands (1300, 1000 and 225 bp) exclusively expressed in the fresh bone marrow group and two bands (400 and 300 bp) expressed specifically in the cultivated bone marrow cell group. Conclusions: In summary, the DDRT-PCR method was proved efficient towards the identification of small differences in gene expression of bone marrow cells in two defined conditions. Thus, we expect that DDRT-PCR can be fast and efficiently designed to analyze differential gene expression in several stem cell types under distinct conditions.

Genotoxicity of the medicinal plant Maytenus robusta in mammalian cells in vivo.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cell Therapy with Bone Marrow Mononuclear Cells in Elastase-Induced Pulmonary Emphysema

Emphysema is characterized by destruction of alveolar walls with loss of gas exchange surface and consequent progressive dyspnea. This study aimed to evaluate the efficiency of cell therapy with bone marrow mononuclear cells (BMMC) in an animal model of elastase-induced pulmonary emphysema. Emphysema was induced in C57Bl/J6 female mice by intranasal instillation of elastase. After 21 days, the mice received bone marrow mononuclear cells from EGFP male mice with C57Bl/J6 background. The groups were assessed by comparison and statistically significant differences (p & 0. 05) were observed among the groups treated with BMMC and evaluated after 7, 14 and 21 days. Analysis of the mean linear intercept (Lm) values for the different groups allowed to observe that the group treated with BMMC and evaluated after 21 days showed the most significant result. The group that received no treatment showed a statistically significant difference when compared to other groups, except the group treated and evaluated after 21 days, evidencing the efficacy of cell therapy with BMMC in pulmonary emphysema. © 2012 Springer Science+Business Media New York.

C-terminal processing of the teneurin proteins: Independent actions of a teneurin C-terminal associated peptide in hippocampal cells

Many neuropsychiatric conditions have a common set of neurological substrates associated with the integration of sensorimotor processing. The teneurins are a recently described family of proteins that play a significant role in visual and auditory development. Encoded on the terminal exon of the teneurin genes is a family of bioactive peptides, termed teneurin C-terminal associated peptides (TCAP), which regulate mood-disorder associated behaviors. Thus, the teneurin-TCAP system could represent a novel neurological system underlying the origins of a number of complex neuropsychiatric conditions. However, it is not known if TCAP-1 exerts its effects as part of a direct teneurin function, whereby TCAP represents a functional region of the larger teneurin protein, or if it has an independent role, either as a splice variant or post-translational proteolytic cleavage product of teneurin. In this study, we show that TCAP-1 can be transcribed as a smaller mRNA transcript. After translation, further processing yields a smaller 15. kDa protein containing the TCAP-1 region. In the mouse hippocampus, immunoreactive (ir) TCAP-1 is exclusively localized to the pyramidal layers of the CA1, CA2 and CA3 regions. Although the localization of TCAP and teneurin in hippocampal regions is similar, they are distinct within the cell as most ir-teneurin is found at the plasma membrane, whereas ir-TCAP-1 is predominantly found in the cytosol. Moreover, in mouse embryonic hippocampal cell culture, FITC-labeled TCAP-1 binds to the plasma membrane and is taken up into the cytosol via dynamin-dependent caveolae-mediated endocytosis. Our data provides novel evidence that TCAP-1 is structurally and functionally distinct from the larger teneurins. © 2012.

Effects of neem oil (Azadirachta indica A. Juss) on midgut cells of predatory larvae Ceraeochrysa claveri (Navás, 1911) (Neuroptera: Chrysopidae)

The effects of ingested neem oil, a botanical insecticide obtained from the seeds of the neem tree, Azadirachta indica, on the midgut cells of predatory larvae Ceraeochrysa claveri were analyzed. C. claveri were fed on eggs of Diatraea saccharalis treated with neem oil at a concentration of 0.5%, 1% and 2% during throughout the larval period. Light and electron microscopy showed severe damages in columnar cells, which had many cytoplasmic protrusions, clustering and ruptured of the microvilli, swollen cells, ruptured cells, dilatation and vesiculation of rough endoplasmic reticulum, development of smooth endoplasmic reticulum, enlargement of extracellular spaces of the basal labyrinth, intercellular spaces and necrosis. The indirect ingestion of neem oil with prey can result in severe alterations showing direct cytotoxic effects of neem oil on midgut cells of C. claveri larvae. Therefore, the safety of neem oil to non-target species as larvae of C. claveri was refuted, thus the notion that plants derived are safer to non-target species must be questioned in future ecotoxicological studies. © 2012 Elsevier Ltd.

Primordial germ cells (spermatogonial stem cells) of bullfrogs express sex hormone-binding globulin and steroid receptors during seasonal spermatogenesis

In vertebrate species, testosterone seems to inhibit spermatogonial differentiation and proliferation. However, this androgen can also be converted, via aromatase, into estrogen which stimulates spermatogonial differentiation and mitotic activity. During seasonal spermatogenesis of adult bullfrogs Lithobates catesbeianus, primordial germ cells (PGCs) show enhanced testosterone cytoplasm immunoexpression in winter; however, in summer, weak or no testosterone immunolabelling was observed. The aim of this study was to confirm if PGCs express stem cell markers-alkaline phosphatase (AP) activity and GFRα1 (glial-cell-line-derived neurotrophic factor)-and verify whether testosterone is maintained in these cells by androgen receptors (ARs) and/or sex hormone-binding globulin (SHBG) in winter. Furthermore, regarding the possibility that testosterone is converted into estrogen by PGCs in summer, the immunoexpression of estrogen receptor (ER)β was investigated. Bullfrog testes were collected in winter and in summer and were embedded in glycol methacrylate for morphological analyses or in paraffin for the histochemical detection of AP activity. GFRα1, AR, SHBG and ERβ expression were detected by Western blot and immunohistochemical analyses. The expression of AP activity and GFRα1 in the PGCs suggest that these cells are spermatogonial stem cells. In winter, the cytoplasmic immunoexpression of ARs and SHBG in the PGCs indicates that testosterone is maintained by these proteins in these cells. The cytoplasmic immunoexpression of ERβ, in summer, also points to an ER-mediated action of estrogen in PGCs. The results indicate a participation of testosterone and estrogen in the control of the primordial spermatogonia during the seasonal spermatogenesis of L. catesbeianus. © 2012 S. Karger AG, Basel.

Photodynamic and Antibiotic Therapy Impair the Pathogenesis of Enterococcus faecium in a Whole Animal Insect Model

Enterococcus faecium has emerged as one of the most important pathogens in healthcare-associated infections worldwide due to its intrinsic and acquired resistance to many antibiotics, including vancomycin. Antimicrobial photodynamic therapy (aPDT) is an alternative therapeutic platform that is currently under investigation for the control and treatment of infections. PDT is based on the use of photoactive dye molecules, widely known as photosensitizer (PS). PS, upon irradiation with visible light, produces reactive oxygen species that can destroy lipids and proteins causing cell death. We employed Galleria mellonella (the greater wax moth) caterpillar fatally infected with E. faecium to develop an invertebrate host model system that can be used to study the antimicrobial PDT (alone or combined with antibiotics). In the establishment of infection by E. faecium in G. mellonella, we found that the G. mellonella death rate was dependent on the number of bacterial cells injected into the insect hemocoel and all E. faecium strains tested were capable of infecting and killing G. mellonella. Antibiotic treatment with ampicillin, gentamicin or the combination of ampicillin and gentamicin prolonged caterpillar survival infected by E. faecium (P = 0.0003, P = 0.0001 and P = 0.0001, respectively). In the study of antimicrobial PDT, we verified that methylene blue (MB) injected into the insect followed by whole body illumination prolonged the caterpillar survival (P = 0.0192). Interestingly, combination therapy of larvae infected with vancomycin-resistant E. faecium, with antimicrobial PDT followed by vancomycin, significantly prolonged the survival of the caterpillars when compared to either antimicrobial PDT (P = 0.0095) or vancomycin treatment alone (P = 0.0025), suggesting that the aPDT made the vancomycin resistant E. faecium strain more susceptible to vancomycin action. In summary, G. mellonella provides an invertebrate model host to study the antimicrobial PDT and to explore combinatorial aPDT-based treatments.

Trafficking of phagocytic peritoneal cells in hypoinsulinemic-hyperglycemic mice with systemic candidiasis

Background: Candidemia is a severe fungal infection that primarily affects hospitalized and/or immunocompromised patients. Mononuclear phagocytes have been recognized as pivotal immune cells which act in the recognition of pathogens, phagocytosis, inflammation, polarization of adaptive immune response and tissue repair. Experimental studies have showed that the systemic candidiasis could be controlled by activated peritoneal macrophages. However, the mechanism to explain how these cells act in distant tissue during a systemic fungal infection is still to be elucidated. In the present study we investigate the in vivo trafficking of phagocytic peritoneal cells into infected organs in hypoinsulinemic-hyperglycemic (HH) mice with systemic candidiasis. Methods: The red fluorescent vital dye PKH-26 PCL was injected into the peritoneal cavity of Swiss mice 24 hours before the intravenous inoculation with Candida albicans. After 24 and 48 hours and 7 days of infection, samples of the spleen, liver, kidneys, brain and lungs were submitted to the microbiological evaluation as well as to phagocytic peritoneal cell trafficking analyses by fluorescence microscopy. Results: In the present study, PKH+ cells were observed in the peritoneum, kidney, spleen and liver samples from all groups. In infected mice, we also found PKH+ cells in the lung and brain. The HH condition did not affect this process. Conclusions: In the present study we have observed that peritoneal phagocytes migrate to tissues infected by C. albicans and the HH condition did not interfere in this process. © 2013 Fraga-Silva et al.; licensee BioMed Central Ltd.

Mechanism and effect of esculetin in an experimental animal model of inflammatory bowel disease

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Anticancer effects of geopropolis produced by stingless bees on canine osteosarcoma cells in vitro

Geopropolis is produced by indigenous stingless bees from the resinous material of plants, adding soil or clay. Its biological properties have not been investigated, such as propolis, and herein its cytotoxic action on canine osteosarcoma (OSA) cells was evaluated. OSA is a primary bone neoplasm diagnosed in dogs being an excellent model in vivo to study human OSA. spOS-2 primary cultures were isolated from the tumor of a dog with osteosarcoma and incubated with geopropolis, 70% ethanol (geopropolis solvent), and carboplatin after 6, 24, 48, and 72 hours. Cell viability was analyzed by the crystal violet method. Geopropolis was efficient against canine OSA cells in a dose- and time-dependent way, leading to a distinct morphology compared to control. Geopropolis cytotoxic action was exclusively due to its constituents since 70% ethanol (its solvent) had no effect on cell viability. Carboplatin had no effect on OSA cells. Geopropolis exerted a cytotoxic effect on canine osteosarcoma, and its introduction as a possible therapeutic agent in vivo could be investigated, providing a new contribution to OSA treatment. © 2013 Naiara Costa Cinegaglia et al.

A novel function for CUGBP2 in controlling the pro-inflammatory stimulus in H9c2 cells: Subcellular trafficking of messenger molecules

Accumulating evidence demonstrates that chronic inflammation plays an important role in heart hypertrophy and cardiac diseases. However, the fine-tuning of cellular and molecular mechanisms that connect inflammatory process and cardiac diseases is still under investigation. Many reports have demonstrated that the overexpression of the cyclooxygenase-2 (COX-2), a key enzyme in the conversion of arachidonic acid to prostaglandins and other prostanoids, is correlated with inflammatory processes. Increased level of prostaglandin E2 was also found in animal model of left ventricle of hypertrophy. Based on previous observations that demonstrated a regulatory loop between COX-2 and the RNA-binding protein CUGBP2, we studied cellular and molecular mechanisms of a pro-inflammatory stimulus in a cardiac cell to verify if the above two molecules could be correlated with the inflammatory process in the heart. A cellular model of investigation was established and H9c2 was used.We also demonstrated a regulatory connection between COX-2 and CUGBP2 in the cardiac cells. Based on a set of different assays including gene silencing and fluorescence microscopy, we describe a novel function for the RNA-binding protein CUGBP2 in controlling the pro-inflammatory stimulus: subcellular trafficking of messenger molecules to specific cytoplasmic stress granules to maintain homeostasis. © 2013 International Federation for Cell Biology.

Equine tendonitis therapy using mesenchymal stem cells and platelet concentrates: A randomized controlled trial

Introduction. Tendon injury is a major cause of lameness and decreased performance in athletic equines. Various therapies for tendonitis have been described; however, none of these therapies results in complete tissue regeneration, and the injury recurrence rate is high even after long recovery periods involving rest and physiotherapy. Methods. A lesion was induced with collagenase gel in the superficial digital flexor tendon in the center portion of the metacarpal region of eight equines of mixed breed. After two weeks, the lesions of the animals in the treated and control groups were treated through the intralesional administration of mesenchymal stem cells derived from adipose tissue (adMSCs) suspended in platelet concentrate (PC) and with phosphate buffered saline (PBS), respectively. Serial ultrasound analyses were performed every two weeks. After 16 weeks of therapy, a biopsy was performed for histopathological, immunohistochemical and gene expression (type I collagen (COL1A1), type III collagen (COL3A1), tenascin-C (TNC), tenomodulin (TNMD), and scleraxis (SCX)) analyses. Results: Differences in the ultrasound and histopathological analyses were observed between the groups. Improved results were reported in the group treated with adMSCs suspended in PC. There was no difference in the gene expression levels observed after the different treatments. The main results observed from the histopathological evaluation of the treated group were as follows: a prevention of the progression of the lesion, a greater organization of collagen fibers, and a decreased inflammatory infiltrate. A lack of progression of the lesion area and its percentage was observed in the ultrasound image, and increased blood flow was measured by Power Doppler. Conclusions: The use of adMSCs combined with PC for the therapy of experimentally induced tendonitis prevented the progression of the tendon lesion, as observed in the ultrasound examination, and resulted in a greater organization and decreased inflammation, as observed in the histopathological evaluation. These data demonstrate the therapeutic potential of this therapy for the treatment of equine tendonitis. © 2013 Carvalho et al.; licensee BioMed Central Ltd.

Avaliação da atividade antimutagênica de alguns produtos naturais de origem animal por meio de ensaios com células HepG2

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Isolation and characterization of equine peripheral blood-derived multipotent mesenchymal stromal cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Culture of equine bone marrow mononuclear fraction and adipose tissue-derived stromal vascular fraction cells in different media

The objective of this study was to evaluate the culture of equine bone marrow mononuclear fraction and adipose tissue - derived stromal vascular fraction cells in two different cell culture media. Five adult horses were submitted to bone marrow aspiration from the sternum, and then from the adipose tissue of the gluteal region near the base of the tail. Mononuclear fraction and stromal vascular fraction were isolated from the samples and cultivated in DMEM medium supplemented with 10% fetal bovine serum or in AIM-V medium. The cultures were observed once a week with an inverted microscope, to perform a qualitative analysis of the morphology of the cells as well as the general appearance of the cell culture. Colony-forming units (CFU) were counted on days 5, 15 and 25 of cell culture. During the first week of culture, differences were observed between the samples from the same source maintained in different culture media. The number of colonies was significantly higher in samples of bone marrow in relation to samples of adipose tissue.