Lipopolysaccharide infusion up-regulates hepcidin mRNA expression in equine liver

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

Extramedullary hematopoiesis in a case of benign mixed mammary tumor in a female dog: cytological and histopathological assessment

Backgroud: Extramedullary hematopoiesis (EMH) is defined as the presence of hematopoietic stem cells such as erythroid and myeloid lineage plus megakaryocytes in extramedullary sites like liver, spleen and lymph nodes and is usually associated with either bone marrow or hematological disorders. Mammary EMH is a rare condition either in human and veterinary medicine and can be associated with benign mixed mammary tumors, similarly to that described in this case.Case presentation: Hematopoietic stem cells were found in a benign mixed mammary tumor of a 7-year-old female mongrel dog that presents a nodule in the left inguinal mammary gland. The patient did not have any hematological abnormalities. Cytological evaluation demonstrated two distinct cell populations, composed of either epithelial or mesenchymal cells, sometimes associated with a fibrillar acidophilic matrix, apart from megakaryocytes, osteoclasts, metarubricytes, prorubricytes, rubricytes, rubriblasts, promyelocytes, myeloblasts. Histological examination confirmed the presence of an active hematopoietic bone marrow within the bone tissue of a benign mammary mixed tumor.Conclusions: EMH is a rare condition described in veterinary medicine that can be associated with mammary mixed tumors. It's detection can be associated with several neoplastic and non-neoplastic mammary lesions, i.e. osteosarcomas, mixed tumors and bone metaplasia.

Molecular detection of Leishmania sp. in cats (Felis catus) from Andradina Municipality, São Paulo State, Brazil

The aim of this work was to molecularly detect Leishmania species in 52 cats from Andradina Municipality, São Paulo State, Brazil. The direct parasitological test was performed by using imprints of poplited lymph node, bone marrow and spleen to verify amastigote forms of Leishmania spp. The samples that were positive parasitological tests were subjected to molecular analysis (PCR) and sequencing. Infection was detected for 5.76% (3/52) of the examined cats and two had presence of amastigote forms of Leishmania spp. in lymph nodes. Polymerase chain reaction (PCR) of kinetoplast minicircle DNA, indicated positive amplification for samples of spleen and lymph nodes and the sequencing resulted in 97% similarity with Leishmania (L) chagasi. This study proved the occurrence of infection with Leishmania (L) chagasi in felines from Andradina municipality, São Paulo State. (C) 2010 Elsevier B.V. All rights reserved.

In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

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

Potencial de transdiferenciação neural das células-tronco mesenquimais da medula óssea de equino

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

Diferenciação in vitro de células-tronco mesenquimais da medula óssea de cães em precursores osteogênicos

O objetivo principal da nossa pesquisa foi avaliar o potencial de diferenciação osteogênica de células-tronco mesenquimais (MSC) obtidas da medula óssea do cão. As MSC foram separadas pelo método Ficoll e cultivadas sob duas condições distintas: DMEM baixa glicose ou DMEM/F12, ambos contendo L-glutamina, 20% de SFB e antibióticos. Marcadores de MSC foram testados, confirmando células CD44+ e CD34- através da citometria de fluxo. Para a diferenciação osteogênica, as células foram submetidas a quatro diferentes condições: Grupo 1, as mesmas condições utilizadas para a cultura de células primárias com os meios DMEM baixa glicose suplementado; Grupo 2, as mesmas condições do Grupo 1, mais os indutores de diferenciação dexametasona, ácido ascórbico e b-glicerolfosfato; Grupo 3, células cultivadas com meios DMEM/F12 suplementado; e Grupo 4, nas mesmas condições que no Grupo 3, mais indutores de diferenciação de dexametasona, ácido ascórbico e b-glicerolfosfato. A diferenciação celular foi confirmada através da coloração com alizarin red e da imunomarcação com o anticorpo SP7/Osterix. Nós observamos através da coloração com alizarin red que o depósito de cálcio foi mais evidente nas células cultivadas em DMEM/F12. Além disso, usando a imunomarcação com o anticorpo SP/7Osterix obtivemos positividade em 1:6 células para o Meio DMEM/F12 comparada com 1:12 para o meio DMEM-baixa glicose. Com base nos nossos resultados concluímos que o meio DMEM/F12 é mais eficiente para a indução da diferenciação de células-tronco mesenquimais caninas em promotores osteogênicos. Este efeito provavelmente ocorre em decorrência da maior quantidade de glicose neste meio, bem como da presença de diversos aminoácidos.

Comparison of parasitological, immunological and molecular methods for the diagnosis of leishmaniasis in dogs with different clinical signs

Aiming to improve the diagnosis of canine leishmaniasis (CanL) in an endemic area of the Northwest region of São Paulo State, Brazil, the efficacy of parasitological, immunological and molecular diagnostic methods were studied. Dogs with and without clinical sips of the disease and positive for Leishmania, by direct parasite identification on lymph node smears and/or specific antibody detection by ELISA, were selected for the study. According to the clinical signs, 89 dogs attending the Veterinary Hospital of UNESP in Aracatuba (SP, Brazil) were divided into three groups: symptomatic (36%), oligosymptomatic (22%) and asymptomatic (22%). Twenty-six dogs from an area non-endemic for CanL were used as negative controls (20%). Fine-needle aspiration biopsies (FNA) of popliteal lymph nodes were collected and Diff-Quick (R)-stained for optical microscopy. Direct immumofluorescence, immunocytochemistry and parasite DNA amplification by PCR were also performed. After euthanasia, fragments of popliteal lymph nodes, spleen, bone marrow and liver were collected and processed for HE and immunohistochemistry. Parasite detection by both HE and immunohistochemistry was specifically more effective in lymph nodes, when compared with the other organs. Immunolabeling provided higher sensitivity for parasite detection in the tissues. In the symptomatic group, assay sensitivity was 75.61% for direct parasite search on Diff-Quick (R)-stained FNAs, 92.68% for direct immunofluorescence, 92.68% for immunocytochemistry and 100% for PCR; the corresponding values in the other clinical groups were: 32, 60, 76 and 96% (oligosymptomatic), and 39.13, 73.91, 100 and 95.65% (asymptomatic). Results of the control animals from the CanL non-endemic area were all negative, indicating that the methods used were 100% specific. (C) 2006 Elsevier B.V. All rights reserved.

Clinical and parasitological evaluation of dogs naturally infected by Leishmania (Leishmania) chagasi submitted to treatment with meglumine antimoniate

Aiming to evaluate the efficacy of the treatment of canine visceral leishmaniasis, to verify the occurrence of a possible disease relapse, and to search for the presence of the parasites after the end of the treatment, seven dogs naturally infected by Leishmania (Leishmania) chagasi were used. The dogs were subjected to a treatment with 75 mg/kg meglumine antimoniate subcutaneously every 12 h for 21 days, and followed-up for a period of 6 months. During the whole experimental period the animals wore deltamethrin collars and were kept in a screened kennel to avoid reinfection. Lymph node and bone marrow aspiration biopsy was carried out to search for the parasite at seven moments: before the treatment, 30, 60, 90, 120 150 and 180 days after the start of the treatment. After the end of the experiment all dogs were humanely euthanized. Then, spleen and liver imprints and ill vitro cultures were carried out to search for amastigote forms of the parasite. During the treatment all animals presented remission of symptoms. However, two dogs were observed to present new symptoms in the course of the experiment. At the end of the experiment, the presence of amastigote forms of the parasite was evidenced in five of the seven dogs. This enabled us to conclude that the treatment promoted clinical cure but did not eliminate the parasites completely. (c) 2006 Elsevier B.V. All rights reserved.

Use of a titanium device in lateral sinus floor elevation: an experimental study in monkeys

Aim: To evaluate the effect of a space-maintaining device fixed to the lateral wall of the maxillary sinus after the elevation of the sinus mucosa on bone filling of the sinus cavity. Material and methods: Immediately after the elevation of the maxillary sinus Schneiderian membrane accomplished through lateral antrostomy in four monkeys, a titanium device was affixed to the lateral sinus wall protruding into the sinus cavity to maintain the mucosa elevated without the use of grafting material. The healing of the tissue around the implants was evaluated after 3 and 6 months. Ground sections were prepared and analyzed histologically. Results: The void under the elevated sinus membrane, originally filled with the blood clot, was reduced after 3 as well as after 6 months of healing of about 56% and 40.5%, respectively. In seven out of eight cases, the devices had perforated the sinus mucosa. The formation of mineralized bone and bone marrow amounted to about 42% and 69% after 3 and 6 months, respectively. The connective tissue represented about 53% and 23% of the newly formed tissue after 3 and 6 months, respectively. Conclusions: New bone formation was found below the devices. However, shrinkage of the newly formed tissue was observed both after 3 and 6 months of healing. Hence, the space-maintaining function of the devices used in the present study has to be questioned.

Periodontal Tissue Engineering After Tooth Replantation

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

Effect of cyclosporin A on alveolar bone homeostasis in a rat periodontitis model

Objectives: the administration of cyclosporin A has been associated with significant bone loss and increased bone remodeling. The present investigation was designed to evaluate the effects of cyclosporin A on alveolar bone of rats subjected to experimental periodontitis, using serum, stereometric and radiographic analysis.Methods: Twenty-four rats were divided into one of the following groups with six animals each: group I, control rats; group II, in which the animals received a cotton ligature around the lower first molars; group III, in which the rats received a cotton ligature around the lower first molars and were treated with 10 mg/(kg body weight day) of cyclosporin A; group IV, in which the rats were treated with 10 mg/(kg body weight day) of cyclosporin A. At the end of experimental period, at 30 days, animals were killed and the serum calcium and alkaline phosphatase levels were measured in all groups. The distance from the alveolar bone crest to the cemento-enamel junction was measured radiographically for each mesial surface of the lower first molars of each rat. After histological processing, the stereological parameters: volume densities of multinucleated osteoclasts (V-o), alveolar bone (V-b), marrow (V-m), and relation of eroded surface/bone surface (Es/Bs) were assessed at the mesial region of the alveolar bone.Results: Significant decreases in serum calcium were observed in those groups that received cyclosporin A therapy. No significant changes in serum alkaline phosphatase were observed. The therapy with cyclosporin A combined with the ligature placement decreased the V-b and increased the V-o, V-m and Es/Bs at the mesial surface of lower first molars. on the other hand, the radiographic data showed that cyclosporin A therapy diminished the alveolar bone loss at the mesial surface of the lower first molars.Conclusions: Therefore, within the limits of this study, we suggest that cyclosporin A at immunosuppressive levels can bring about an imbalance in the alveolar bone homeostasis in rats. However, in the presence of inflammatory stimulation, the inhibition of the immune system by cyclosporin A may decrease the initial periodontal breakdown.

iNOS-Derived Nitric Oxide Stimulates Osteoclast Activity and Alveolar Bone Loss in Ligature-Induced Periodontitis in Rats

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

Jaw and Long Bone Marrows Have a Different Osteoclastogenic Potential

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Green Brazilian propolis action on macrophages and lymphoid organs of chronically stressed mice

Stress is a generic term that summarizes how psychosocial and environmental factors influence physical and mental well-being. The interaction between stress and immunity has been widely investigated, involving the neuroendocrine system and several organs. Assays using natural products in stress models deserve further investigation. Propolis immunomodulatory action has been mentioned and it has been the subject of scientific investigation in our laboratory. The aim of this study was to evaluate if and how propolis activated macrophages in BALB/c mice submitted to immobilization stress, as well as the histopathological analysis of the thymus, bone marrow, spleen and adrenal glands. Stressed mice showed a higher hydrogen peroxide (H2O2) generation by peritoneal macrophages, and propolis treatment potentiated H2O2 generation and inhibited nitric oxide (NO) production by these cells. Histopathological analysis showed no alterations in the thymus, bone marrow and adrenal glands, but increased germinal centers in the spleen. Propolis treatment counteracted the alterations found in the spleen of stressed mice. New research is being carried out in order to elucidate propolis immunomodulatory action during stress.

Ectopic mineralization of articular cartilage in the bullfrog Rana catesbeiana and its possible involvement in bone closure

Mineralization of the articular cartilage is a pathological condition associated with age and certain joint diseases in humans and other mammals. In this work, we describe a physiological process of articular cartilage mineralization in bullfrogs. Articular cartilage of the proximal and distal ends of the femur and of the proximal end of the tibia-fibula was studied in animals of different ages. Mineralization of the articular cartilage was detected in animals at 1 month post-transformation. This mineralization, which appeared before the hypertrophic cartilage showed any calcium deposition, began at a restricted site in the lateral expansion of the cartilage and then progressed to other areas of the epiphyseal cartilage. Mineralized structures were identified by von Kossa's staining and by in vivo incorporation of calcein green. Element analysis showed that calcium crystals consisted of poorly crystalline hydroxyapatite. Mineralized matrix was initially spherical structures that generally coalesced after a certain size to occupy larger areas of the cartilage. Alkaline phosphatase activity was detected at the plasma membrane of nearby chondrocytes and in extracellular matrix. Apoptosis was detected by the TUNEL (TDT-mediated dUTP-biotin nick end-labeling) reaction in some articular chondrocytes from mineralized areas. The area occupied by calcium crystals increased significantly in older animals, especially in areas under compression. Ultrastructural analyses showed clusters of needle-like crystals in the extracellular matrix around the chondrocytes and large blocks of mineralized matrix. In 4-year-old animals, some lamellar bone (containing bone marrow) occurred in the same area as articular cartilage mineralization. These results show that the articular cartilage of R. catesbeiana undergoes precocious and progressive mineralization that is apparently stimulated by compressive forces. We suggest that this mineralization is involved in the closure of bone extremities, since mineralization appears to precede the formation of a rudimentary secondary center of ossification in older animals.