Magnesium incorporation in fibrinogen scaffolds promotes macrophage polarization towards M2 phenotype.

The host inflammatory response to biomaterials conditions their capacity to promote tissue repair, and macrophage polarization shift from M1 to M2 is determinant in this process. Previous work showed that extracts of a combination between fibrinogen and metallic magnesium materials acted synergistically to reduce macrophage inflammatory phenotype. The hypothesis underlying the current work was that the ability of magnesium-modified fibrinogen scaffolds to modulate macrophage phenotype depends on the concentration of magnesium. Thus, Fibrinogen (Fg) scaffolds incorporating precise concentrations of magnesium sulfate (Mg: 0, 10, 25, 50 mM) were developed and characterized. Mg incorporation in Fg scaffolds increased surface charge, while porosity decreased with increasing Mg concentrations, but only Fg scaffolds with 10 mM of Mg (FgMg10) had significantly improved mechanical properties. Human macrophages cultured on FgMg10 scaffolds, showed increased M2 and decreased M1 polarization, when compared to those cultured on scaffolds with 0, 25 and 50 mM of Mg. Macrophage polarization results were independent of the anion used (chloride or sulfate). Macrophage modulation by FgMg10 scaffolds involved reduced NF-κB p65 nuclear translocation, and impacted production of pro-inflammatory mediators (e.g. IFNγ, IL-12, TNF-⍺, IP-10). Importantly, FgMg10 scaffolds implanted in vivo increased the expression of M2 marker CD163, in macrophages from inflammatory exudates, compared to Sham and Fg-implanted animals, increasing the M2:M1 ratio. A cytokine/chemokine array showed that, while both Fg and FgMg10 scaffolds decreased inflammatory mediators, only FgMg10 decreased IL-1ß, IP-10, MIP-2, MDC and MIP-3⍺, compared to Sham-operated animals. This study demonstrated that incorporation of 10mM of Mg modulated inflammation, promoting M2 macrophage polarization in vitro and in vivo. STATEMENT OF SIGNIFICANCE: Developing biomaterials that can modulate inflammation and promote macrophage phenotype switch from M1 to M2 is crucial to promote a regenerative microenvironment. Our previous work showed that extracts of a combination between fibrinogen (Fg) and metallic magnesium (Mg) materials synergistically reduced macrophage pro-inflammatory phenotype. Herein, we tested the hypothesis that macrophage modulation was dependent on Mg concentration. A new family of Fg porous scaffolds incorporating different amounts of Mg (0, 10, 25 and 50 mM) was produced and characterized. We observed that only the combination of Fg scaffolds with 10 mM of Mg (FgMg10) significantly changed the scaffolds mechanical properties and directed macrophages towards a M2 phenotype, reducing the production of inflammatory mediators, both in vitro and in vivo.

IL-1ß-pre-conditioned mesenchymal stem/stromal cells' secretome modulates the inflammatory response and aggrecan deposition in intervertebral disc.

Mesenchymal stem/stromal cells (MSCs) have been increasingly used in clinical trials for low-back pain (LBP) and intervertebral disc (IVD) degeneration with promising results. Their action mechanisms are not fully understood, but they reduce IVD pro-inflammatory markers in a pro-inflammatory/degenerative IVD microenvironment. In this study the therapeutic potential of the MSC secretome, as an alternative cell-free approach for treating degenerated IVDs, was examined. Human bone marrow-derived MSC secretome (MSCsec) was collected after 48 h of preconditioning in IL-1ß (10 ng/mL) and low oxygen (6 % O2), mimicking the degenerative IVD. IL-1ß-pre-conditioning of MSCs increased secretion of pro-inflammatory markers hIL-6, hIL-8, hMCP-1, etc. The therapeutic effect of MSCsec was tested in a pro-inflammatory/degenerative IVD ex vivo model. MSCsec down-regulated IVD gene expression of pro-inflammatory cytokines (bIL-6, bIL-8) and matrix degrading enzyme bMMP1, while bMMP3 and bTIMP2 were up-regulated, at 48 h. After 14 d, MSCsec-treated IVDs revealed increased aggrecan deposition, although no differences in other ECM components were observed. Protein analysis of the MSCsec-treated IVD supernatant revealed a significant increase of CXCL1, MCP-1, MIP-3α, IL-6, IL-8 and GRO α/ß/γ (related to TNF, NOD-like receptor and neutrophil chemotaxis signalling), and a decrease of IFN-γ, IL-10, IL-4, IL-5 and TNF-α (associated with T-cell receptor signalling). MSCsec-treated IVD supernatants did not promote angiogenesis and neurogenesis in vitro. Overall, MSCsec can be a safe therapeutic approach, presenting a strong immunomodulatory role in degenerated IVD while potentiating aggrecan deposition, which can open new perspectives on the use of MSCsec as a cell-based/ cell-free therapeutic approach to LBP.

B-mode and Doppler ultrasonography of abdominal structures of the peccary (Tayassu tajacu, Linnaeus 1758) / [Ultrassonografias Doppler e modo-B das estruturas abdominais de catetos (Tayassu tajacu Linnaeus, 1758)]

This study used B-mode and Doppler ultrasonography to characterize the abdominal structures of healthy peccaries raised in captivity. Fifteen peccaries were used for this study. The urinary vesicle appeared as an ovoid structure, located in the abdominal and pelvic transition, with a hyperechogenic, thin, smooth, and regular wall. The kidneys presented retroperitoneal topography and had similar sizes. The kidney/aorta ratio had an average value of 10.53±15cm (right) and 10.23±0.12 (left). The right adrenal gland had a length of 1.93±0.34cm and diameter of 0.56±0.16cm. The left adrenal gland had a length of 1.85±0.42cm and diameter of 0.52±0.11cm. The spleen had a diameter of 1.13±0.18cm. The hepatic vein demonstrated polyphasic flow in pulsed Doppler, with two retrograde peaks and an anterograde peak with a flow velocity of 25.7±0.83cm/s. The abdominal aorta had a diameter of 0.58±0.05cm and a flow velocity of 115.17±5.32cm/s. The morphological and hemodynamic study of the abdominal structures of the peccary, observed through B-mode and Doppler ultrasonography, aided in identifying the size, shape, position, echogenicity, and echotexture of the abdominal organs and in making inferences about the normal parameters for these structures in this species.(AU)

Este estudo teve como objetivo utilizar as ultrassonografias de modo-B e Doppler para caracterizar as estruturas abdominais de um cateto sadio criado em cativeiro. Quinze catetos foram utilizados para este estudo. A vesícula urinária apareceu como uma estrutura ovoide, localizada na transição entre as partes abdominal e pélvica, com uma parede hiperecogênica, fina, lisa e regular. Os rins apresentaram topografia retroperitoneal e tamanhos semelhantes. A relação rim/aorta teve um valor médio de 10,53 ± 15cm (direita) e 10,23 ± 0,12cm (esquerda). A glândula adrenal direita tinha um comprimento de 1,93 ± 0,34cm e um diâmetro de 0,56 ± 0,16cm. A glândula suprarrenal esquerda tinha um comprimento de 1,85 ± 0,42cm e um diâmetro de 0,52 ± 0,11cm. O baço tinha um diâmetro de 1,13 ± 0,18cm. A veia hepática demonstrou fluxo polifásico no Doppler pulsátil, com dois picos retrógrados e um pico anterógrado com velocidade de fluxo de 25,7±0,83cm/s. A aorta abdominal tinha um diâmetro de 0,58 ± 0,05cm e uma velocidade de fluxo de 115,17±5,32cm/s. Os estudos morfológico e hemodinâmico das estruturas abdominais do queixada, observadas por meio das ultrassonografias modo-B e Doppler, auxiliaram na identificação do tamanho, da forma, da posição, da ecogenicidade e da ecotextura dos órgãos abdominais e na realização de inferências sobre os parâmetros de normalidade para as estruturas nas espécies.(AU)

Echocardiographic reference ranges for sedated healthy peccaries ( Tayassu tajacu, Linnaeus, 1758 ) / Valores de referência para catetos saudáveis sedados

Peccaries are wild mammals belonging to the Tayassu genus that are found almost everywhere in the Americas and have demonstrated great potential as an experimental model for scientific investigations. Twelve healthy adult animals were sedated to perform echocardiographic examinations in B, M and Doppler mode. The variables that exhibited statistically significant correlation coefficients with weight were LVFWd, LVIDd, LVIDs, E wave, A'RV, MAM, and TAPSE. The HR exhibited a negative relationship with the IVRT. The LA variable showed a positive correlation with the AO. The MAM exhibited correlations with the LVIDd and LVIDs. The TAPSE showed positive correlations with the E'RV and A'RV. The present study provides the first reference values for echocardiographic measurements in B, M and Doppler modes from peccaries anesthetized with ketamine and midazolam. Echocardiography was easy to perform in collared peccaries, and the collected data revealed values that can aid in their clinical management and conservation.(AU)

Catetos são mamíferos selvagens, pertencentes ao gênero Tayassu, encontrados em quase toda a América, os quais têm se destacado como modelos experimentais. Doze animais adultos saudáveis foram sedados para a realização de exames ecocardiográficos em modos B, M e Doppler. As variáveis que apresentaram coeficientes de correlação estatisticamente significativos em relação ao peso foram: LVFWd, LVIDd, LVIDs, onda E, A'RV, MAM, TAPSE. O HR mostrou correlação positiva com LA/AO, onda E, onda A, MAM, TAPSE e E/IVRT, e negativa com IVRT. Os valores de AVmax mostraram correlações positivas com onda E e onda A'. A variável LA mostrou correlação positiva com AO e correlação negativa com IVSd. A EF apresentou forte correlação com a relação de ondas FS, E/A, com A', E'RV. FS apresentou correlação positiva com a relação de onda E/A. O MAM mostrou correlação com LVIDd e LVIDs. TAPSE mostrou correlações positivas com E'RV e A'RV. O presente estudo forneceu os primeiros valores de referência para medições ecocardiográficas em modos B, M e Doppler de catetos anestesiados com ketamina e midazolam. O exame ecocardiográfico em catetos foi de fácil execução e os dados encontrados evidenciaram valores que podem auxiliar no seu manejo clínico e conservação.(AU)

Human Bone Marrow Mesenchymal Stem/Stromal Cells Preserve Their Immunomodulatory and Chemotactic Properties When Expanded in a Human Plasma Derived Xeno-Free Medium.

Due to their immunomodulatory and chemotactic properties, hMSC are being explored to treat immune-related diseases. For their use in human therapies, it is necessary to culture hMSC in xeno-free conditions. In this study, the impact that a xeno-free medium based on a human plasma derivate has on these properties was analysed. Bone marrow-derived hMSC preserved their immunosuppressive and immunostimulatory properties, as observed with in vitro assays with hMSC cocultured with mixed leukocyte reactions or with mitogen-stimulated leukocytes. Moreover, hMSC expanded in xeno-free medium were recruited by macrophages in both migration and invasion assays, which indicates that the cells maintained their chemotactic properties. These data suggest that xeno-free expanded hMSC preserved their immunomodulatory and chemotactic properties, indicating that the described xeno-free medium composition is a potential candidate to culture and expand hMSC for human cell therapies.

Decellularized human colorectal cancer matrices polarize macrophages towards an anti-inflammatory phenotype promoting cancer cell invasion via CCL18.

Macrophages are frequently identified in solid tumors, playing important roles in cancer progression. Their remarkable plasticity makes them very sensitive to environmental factors, including the extracellular matrix (ECM). In the present work, we investigated the impact of human colorectal tumor matrices on macrophage polarization and on macrophage-mediated cancer cell invasion. Accordingly, we developed an innovative 3D-organotypic model, based on the decellularization of normal and tumor tissues derived from colorectal cancer patients' surgical resections. Extensive characterization of these scaffolds revealed that DNA and other cell constituents were efficiently removed, while native tissue characteristics, namely major ECM components, architecture and mechanical properties, were preserved. Notably, normal and tumor decellularized matrices distinctly promoted macrophage polarization, with macrophages in tumor matrices differentiating towards an anti-inflammatory M2-like phenotype (higher IL-10, TGF-ß and CCL18 and lower CCR7 and TNF expression). Matrigel invasion assays revealed that tumor ECM-educated macrophages efficiently stimulated cancer cell invasion through a mechanism involving CCL18. Notably, the high expression of this chemokine at the invasive front of human colorectal tumors correlated with advanced tumor staging. Our approach evidences that normal and tumor decellularized matrices constitute excellent scaffolds when trying to recreate complex microenvironments to understand basic mechanisms of disease or therapeutic resistance.

Ovarian response is a better predictor of clinical pregnancy rate following embryo transfer than is thin endometrium or presence of an endometrioma.

OBJECTIVES: To examine whether endometrial thickness and the presence of endometrioma are independent predictors of clinical pregnancy rate or simply associated with poor ovarian response (POR). METHODS: This was a retrospective cohort study assessing the first cycle of all women undergoing in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) in a university hospital in Brazil between January 2011 and December 2012. Only the first cycle of each woman within the study period was considered. Women over 40 years of age and those who used clomiphene citrate during controlled ovarian stimulation (COS) or did not undergo embryo transfer were excluded from analysis. POR was defined as ≤ three oocytes retrieved and a thin endometrium was defined as endometrial thickness ≤ 7.0 mm on the day of human chorionic gonadotropin (hCG) administration. We performed a multiple regression analysis to identify which of the following parameters were independent predictors of clinical pregnancy: age, number of oocytes retrieved, endometrial thickness or the presence of endometrioma. RESULTS: Within the study period, 787 women began COS, but 270 were excluded from analysis. Among the 517 women analyzed, those who achieved pregnancy were younger and yielded more oocytes. The proportion of POR was higher in women with a thin endometrium (17/57 (29.8%) vs 80/460 (17.4%); P = 0.03) and in women with endometrioma (15/39 (38.5%) vs 82/478 (17.2%); P = 0.002). The results of regression analysis showed that only age and the number of oocytes retrieved were independent predictors of pregnancy. Additionally, we observed higher clinical pregnancy rates in women with a thin endometrium from whom ≥ seven oocytes were retrieved (11/25 (44.0%)) compared to women with normal endometrial thickness (99/241 (41.1%)). Considering only women from whom ≥ four oocytes were retrieved, we observed reasonable pregnancy rates in those with a thin endometrium (14/40 (35.0%)) and in those with endometrioma (9/24 (37.5%)). CONCLUSION: Both a thin endometrium and the presence of endometrioma are associated with POR but are not important independent predictors of clinical pregnancy. Good pregnancy rates can be observed when these conditions are present in women with a good ovarian response.

Desempenho, consumo e morfometria in vivo de cordeiros Santa Inês alimentados com rações contendo torta de girassol em substituição ao farelo de algodão / Performance, consumption and in vivo morphometry of Santa Inês lambs fed diets containing sunflower cake in place of cottonseed meal

Este estudo foi conduzido com o objetivo de determinar o melhor teor de substituição da proteína do farelo de algodão pela proteína da torta de girassol em dietas para cordeiros Santa Inês, por meio da avaliação do consumo, ganho de peso, conversão alimentar e medidas morfométricas. Foram confinados, por 60 dias, 30 cordeiros da raça Santa Inês, machos inteiros, com idade e peso médio no início do experimento de 80 dias e 21,45±2,16kg, respectivamente. O delineamento experimental foi completamente casualizado, sendo os animais divididos em 5 tratamentos, com 6 repetições por tratamento, de acordo com a quantidade de proteína do farelo de algodão substituída pela proteína da torta de girassol (0, 20, 40, 60 e 80% de substituição). As variáveis peso final, consumo de matéria seca e conversão alimentar não foram afetadas pelas dietas experimentais, embora o ganho médio diário tenha sido inferior para as dietas com maior teor de torta de girassol. Para os parâmetros de consumo, verificou-se diferença apenas no consumo de extrato etéreo em relação ao peso metabólico (g/kg PV0,75), em que cordeiros recebendo dieta com maior teor de torta de girassol ingeriram maiores quantidades de extrato etéreo. Em relação às medidas morfométricas, houve decréscimo linear da altura de dorso e largura de garupa com a inclusão da torta de girassol na dieta. A substituição da proteína do farelo de algodão pela proteína da torta de girassol afetou negativamente o ganho de peso de cordeiros Santa Inês em confinamento. Porém, teve pouca ou nenhuma influência nos parâmetros de consumo e nas medidas morfométricas in vivo.(AU)

This study was conducted with the aim to determine the optimal level of protein replacement of cottonseed meal protein by sunflower cake in diets for Santa Inês lambs, through the evaluation of consumption, weight gain, feed conversion and morphometric measurements. Thirty Santa Inês lambs, bulls, with age and weight at the beginning of the experiment of 80 days and 21.45±2.16kg, respectively, were confined for 60 days. A completely randomized experimental design was used, where the animals were divided into 5 treatments with 6 replicates per treatment. For the treatments, the protein from the cottonseed meal was replaced by the protein of the sunflower cake (0, 20, 40, 60 and 80% of substitutions). For morphometric determinations, the following measurements were taken: pre-slaughter body length, leg length, leg perimeter, height of the dorsum; hip height; chest girth, hip width and chest width. Final weight, dry matter intake and feed conversion were not affected by the experimental diets, although the average daily gain was lower for diets with higher content of sunflower cake. For consumption parameters, there was a difference only in the consumption of ether extract in relation to metabolic weight (g/kg PV 0, 75), where lambs receiving diets with higher levels of sunflower cake ingested larger amounts of ether extract. Regarding the morphometric measurements, there was a linear decrease of the height of the dorsum and hip width with the inclusion of sunflower cake in the diet. The replacement of cottonseed meal protein by sunflower cake protein negatively affected the weight gain of Santa Inês lambs. However, it affected little or nothing the parameters of consumption and in vivo morphometric measurements.(AU)

Desempenho de novilhos de corte em pastos de capim-tanzânia sob quatro alturas de desfolha / Performance of steers on pasture of Tanzania grass under four defoliation heights

Determinou-se a melhor altura de pastejo do capim-tanzânia (Panicum maximum Jacq. cv. Tanzânia) sob lotação contínua e com taxa de lotação variável na produção de bovinos de corte. A área experimental, de 12 hectares, foi dividida em piquetes de um hectare cada, com três repetições para cada altura - 20; 40; 60 e 80cm. Foram utilizados, em cada piquete, três bovinos machos, da raça Nelore, mais animais adicionais conforme necessidade de ajuste à altura pretendida. O delineamento experimental foi inteiramente ao acaso. A taxa de lotação apresentou comportamento linear negativo em razão das alturas de pastejo em todas as estações do ano, e o mesmo efeito foi observado com o ganho de peso vivo por hectare, enquanto o ganho médio diário apresentou comportamento contrário. Alturas de capim-tanzânia entre 40 e 60cm, sob lotação contínua, possibilitam bom ganho de peso médio diário em bovinos de corte e boa produção por hectare...

This study was carried out to evaluate the effects of four grazing heights under continuous stocking of Tanzânia grass (Panicum maximum Jacq. Cv. Tanzânia) on the stocking rate, performance and beef production per hectare. The experimental area was 12 hectares, divided into paddocks with one hectare each, with three replicates each (20, 40, 60 and 80 cm). There were three male Nellore bovines per paddock, adding animals as needed to adjust the desired heights in a completely randomized design. The stocking rate had a negative linear function of grazing heights in all seasons regarding yield per hectare, while the average daily gain showed opposite behavior. Apparently, Tanzânia grass heights between 40 and 60cm under continuous stocking enable good average daily weight gain in beef cattle, and high yield per hectare...

Macrophages stimulate gastric and colorectal cancer invasion through EGFR Y(1086), c-Src, Erk1/2 and Akt phosphorylation and smallGTPase activity.

The interactions between cancer cells and their microenvironment are crucial for malignant progression, as they modulate invasion-related activities. Tumor-associated macrophages are generally considered allies in the process of tumor progression in several types of cancer, although their role on gastric and colorectal carcinomas is still poorly understood. In this report, we studied the influence of primary human macrophages on gastric and colorectal cancer cells, considering invasion, motility/migration, proteolysis and activated intracellular signaling pathways. We demonstrated that macrophages stimulate cancer cell invasion, motility and migration, and that these effects depend on matrix metalloproteinase (MMP) activity and on the activation of epidermal growth factor receptor (EGFR) (at the residue Y(1086)), PLC-γ (phospholipase C-gamma) and Gab1 (GRB2-associated binding protein-1), as evidenced by siRNA (small interference RNA) experiments. Epidermal growth factor (EGF)-immunodepletion impaired macrophage-mediated cancer cell invasion and motility, suggesting that EGF is the pro-invasive and pro-motile factor produced by macrophages. Macrophages also induced gastric and colorectal cancer cell phosphorylation of Akt, c-Src and ERK1/2, and led to an increase of RhoA and Cdc42 activity. Interestingly, whereas macrophage-mediated cancer cell c-Src and ERK1/2 phosphorylation occurred downstream EGFR activation, Akt phosphorylation seems to be a parallel event, taking place in an EGFR-independent manner. The involvement of EGF, EGFR-downstream signaling partners and MMPs in macrophage-mediated invasion provides novel insights into the molecular crosstalk established between cancer cells and macrophages, opening new perspectives for the design of new and more efficient therapeutic strategies to counteract cancer cell invasion.

Adsorbed fibrinogen leads to improved bone regeneration and correlates with differences in the systemic immune response.

Designing new biomaterials that can modulate the inflammatory response instead of attempting just to reduce it constitutes a paradigm change in regenerative medicine. This work aimed to investigate the capacity of an immunomodulatory biomaterial to enhance bone regeneration. For that purpose we incorporated a molecule with well-established pro-inflammatory and pro-healing roles, fibrinogen, in chitosan scaffolds. Two different incorporation strategies were tested, leading to concentrations of 0.54±0.10mg fibrinogen g(-1) scaffold immediately upon adsorption (Fg-Sol), and 0.34±0.04mg fibrinogen g(-1) scaffold after washing (Fg-Ads). These materials were implanted in a critical size bone defect in rats. At two months post-implantation the extent of bone regeneration was examined by histology and the systemic immune response triggered was evaluated by determining the percentages of myeloid cells, T and B lymphocytes in the draining lymph nodes. The results obtained indicate that the fibrinogen incorporation strategy conditioned the osteogenic capacity of biomaterials. Fg-Ads scaffolds led to more bone formation, and the presence of Fg stimulated angiogenesis. Furthermore, animals implanted with Fg-Ads scaffolds showed significant increases in the percentages of B lymphocytes and myeloid cells in the draining lymph nodes, while levels of T lymphocytes were not significantly different. Finally, a significant increase in TGF-ß1 was detected in the plasma of animals implanted with Fg-Ads. Taken together the results presented suggest a potential correlation between the elicited immune response and biomaterial osteogenic performance.

The effect of adsorbed fibronectin and osteopontin on macrophage adhesion and morphology on hydrophilic and hydrophobic model surfaces.

Macrophages play a crucial role in the host response to biomaterials. Here we investigated the effect of adsorbed fibronectin (FN) and osteopontin (OPN), two important proteins for tissue repair, on macrophage adhesion and morphology. Since cell-biomaterial interactions are modulated via proteins adsorbed onto biomaterial surfaces, FN and OPN were adsorbed on model self-assembled monolayers (SAMs) of alkanethiols on gold with different functional terminal groups (CH(3), OH and tetra(ethylene-glycol)). The initial interaction of inflammatory cells with a biomaterial is crucial for the ensuing phases of an inflammatory reaction. For this reason short-term cultures of primary human macrophages were performed. To account for the competitive adsorption of other proteins serum was added to the culture medium and the effect compared with serum-free medium cultures. In the presence of serum hydrophilic surfaces increased macrophage adhesion. In particular, FN induced a higher cell density, while OPN tended to decrease it. In serum-free medium cell adhesion was greater on hydrophobic surfaces, except for OPN-coated SAMs. Importantly, FN no longer enhanced macrophage adhesion, while OPN maintained its inhibitory effect. Cell polarization studies indicated that macrophage morphology variations induced by surface chemistry are overcome by pre-adsorbed OPN. Taken together our results show that in the presence of serum macrophage adhesion is promoted by FN hydrophilic surfaces, but impaired on OPN-coated surfaces. The effects of inhibited macrophage adhesion on macrophage fusion, and its relevance to the initial stages of the inflammatory response to biomaterials are discussed.

Cytokine gene expression in the tissues of dogs infected by Leishmania infantum.

Canine leishmaniosis (CanL) caused by the protozoan parasite Leishmania infantum is a chronic systemic disease that is endemic in certain parts of the world. The domestic dog is the most important reservoir of L. infantum and is the main source of infection for other animals and for the human population. The aim of this study was to evaluate and compare the level of expression of genes encoding particular cytokines (interleukin [IL]-12, interferon [IFN]-γ, IL-2 and IL-4) in different tissues and organs of 53 adult dogs with or without clinical signs of leishmaniosis and after treatment for the disease. Asymptomatic dogs showed high expression of genes encoding IL-4 in blood leucocytes and of genes encoding IL-12 and IL-2 in lymph nodes. Blood leucocytes from symptomatic dogs had a mixed Th1 and Th2 cytokine gene expression profile, but lymph nodes from these animals had dominant IL-2 and IFN-γ gene expression, while bone marrow appeared to be unresponsive. The predominance of IL-4 gene expression in the blood of asymptomatic dogs may favour parasite replication, while the balance between Th1 and Th2 cytokine gene expression in the blood of symptomatic dogs may be important in reducing parasite replication and delaying the dissemination of Leishmania to other organs. The drugs used to treat CanL do not completely eliminate the parasite, so the high expression of the gene encoding IL-4 in blood leucocytes and the high expression of IL-12 and IL-4 mRNA in lymph nodes may reflect the persistence of residual Leishmania amastigotes. L. infantum appears able to regulate the host immune response in order to ensure its survival, but also to prevent the host from succumbing to infection. This guarantees its transmission and the completion of its life cycle.

Composição química e perfil de ácidos graxos do leite e muçarela de búfalas alimentadas com diferentes fontes de lipídeos / Chemical composition and fatty acids profile in milk and mozzarella cheese of water buffalo fed different lipid sources

Avaliaram-se a composição química e o perfil de ácidos na gordura do leite de búfalas alimentadas com fontes de lipídeos. Foram utilizadas nove búfalas em lactação, multíparas e com produção média diária de leite de 6,5kg. O delineamento experimental foi quadrado latino 3x3, triplo, composto por três tratamentos, três períodos de 21 dias e nove repetições. Os tratamentos foram dietas sem lipídeo adicional, com grão de soja e com óleo de soja. A proporção de silagem de milho variou entre 70 e 75 por cento. A inclusão de óleo de soja elevou os teores de gordura no leite e no queijo tipo muçarela. As concentrações de ácidos graxos saturados (AGS) variaram de 62,8 a 69,8 por cento. Não houve diferenças nos teores de AGS entre os tratamentos sem lipídeo adicional e com grão de soja. O tratamento com óleo de soja resultou em queda de 10 por cento no teor de AGS. As fontes lipídicas reduziram as concentrações de AGS e aumentaram as concentrações de ácidos graxos insaturados. Ácidos graxos encontrados na muçarela, em ordem decrescente, foram: palmítico, oleico, láurico e esteárico. O óleo de soja apresentou maior capacidade de elevar as concentrações do ácido linoleico conjugado (CLA) e do ácido vaccênico no leite e no queijo muçarela.

The chemical composition of milk and fatty acids profile in milk fat of water buffaloes fed different lipid sources were evaluated. Nine lactating multiparous water buffaloes, averaging 6.5kg of milk daily, were used. The statistical design was 3x3 triple Latin Square, three periods of 21 days and nine repetitions. The treatments were diet without additional fat, diet with soybean grain, and diet with soybean oil. Corn silage content in the diets ranged from 70 to 75 percent. The inclusion of soybean oil increased fat content in milk and mozzarella. Saturated fatty acid (SFA) concentrations in milk ranged from 62.8 to 69.8 percent. SFA content was similar in milk for the diets without additional fat and with soybean grain. However, in the diet with soybean oil, SFA decreased about 10 percent. Lipid sources reduced SFA and increased unsaturated fatty acids (UFA). The main fatty acids found in mozzarella fat, in decreasing array, were: palmitic, oleic, lauric, and estearic. Diet with soybean oil presented higher capacity of increasing conjugated linoleic acid (CLA) and vaccenic acid concentrations in milk and mozzarella.

Ácido linoléico conjugado e perfil de ácidos graxos no músculo e na capa de gordura de novilhos bubalinos alimentados com diferentes fontes de lipídios / Linoleic conjugated acid and fatty acids profile in the muscle and fat layer of water buffalo steers fed different fat sources

Avaliaram-se o teor de ácido linoléico conjugado (CLA) e o perfil de ácidos graxos no músculo e na capa de gordura de novilhos bubalinos alimentados com diferentes fontes de lipídios. Utilizaram-se 12 animais castrados, da raça Murrah, com peso vivo de 306±8kg, submetidos aos tratamentos sem lipídeo adicional, grão de soja e óleo de soja, confinados durante 84 dias. Após o abate a carcaça foi resfriada a 5ºC, durante 24 horas. Foi feita secção entre a nona e a 11ª costelas da meia carcaça direita, de onde se separou músculo e capa de gordura, analisadas as concentrações de CLA e de ácidos graxos, por cromatografia gasosa. O fornecimento de óleo de soja resultou em maior concentração de CLA no músculo e na capa de gordura, e a adição de óleo de soja menores concentrações de ácidos graxos saturados, principalmente os ácidos mirístico e palmítico. Os animais que receberam a dieta com grão de soja integral também apresentaram menor teor de CLA e redução nas concentrações de mirístico e palmítico. Esses efeitos foram observados somente na capa de gordura e em menor intensidade.

The effect of different fat sources on fatty acid concentrations in the muscle and in the fat layer of water buffalo steers was studied. Twelve water buffalo steers weighting 306±8kg, fed without additional fat, soybean grain or soybean oil and confined during 84 days were used. The animals were slaughtered after 16-hours-fasting and the carcass was cooled at 5ºC, for 24 hours. A section was extracted between 9th and 11th ribs from the right half carcass and this section was separated in bone, muscle and fat layer. In the last two fractions, the fatty acids, including conjugated linoleic acid (CLA), were quantified by gaseous chromatography. The soybean oil provided higher CLA concentrations in the steers muscle and fat layer. Moreover, the soybean oil inclusion promoted lower saturated fatty acids contents, mainly miristic and palmitic acids. There was also increase in CLA concentrations and decrease in miristic and palmitic acids contents in the fat layer of the animals fed soybean grain diet. However, these effects were more moderate than those observed for the diet with soybean oil.

Attachment, spreading and short-term proliferation of human osteoblastic cells cultured on chitosan films with different degrees of acetylation.

Chitosan (Ch) is being actively investigated as a non-protein template for the growth of an increasing number of anchorage-dependent cells, including chondrocytes and bone cells. In the present work, Ch films with degrees of N-acetylation (DAs) in the range of 4 to 49% were evaluated with respect to the attachment, spreading and short-term proliferation of osteoblasts, using human osteoblastic MG-63 cells. The films were characterized in terms of surface morphology and surface charge by atomic force microscopy and streaming potential measurements, respectively. Cell attachment was assessed after 3 and 24 h of cell culture. After 24 h of incubation, cell attachment was found to be dependent on the DA, lower DAs favouring cell adhesion. With time, cell spreading and cytoskeleton organization were only attained for DAs

Leptin effect on RANKL and OPG expression in MC3T3-E1 osteoblasts.

Recent studies have suggested that leptin hormone may play a pivotal role on bone remodeling through a direct effect by modulating positively the OPG/RANKL balance. Here, we investigate the effect of leptin hormone on RANKL and OPG expression in MC3T3-E1 osteoblasts using RT-PCR and ELISA measurements. We have at first identified the expression of Ob-Rb and Ob-Ra leptin receptor isoforms in MC3T3-E1 and observed that these cells respond to mrleptin treatments. We then investigated the effect of mrleptin on RANKL and OPG expression. We show that mrleptin dose-dependently regulated the expression of RANKL mRNA with complete inhibition observed at concentrations higher than 12 ng/ml. This effect was confirmed with sRANKL protein measurements. However, the exposure of MC3T3-E1 to mrleptin had no effect on OPG mRNA. Taken together, these results suggest that leptin modulates positively OPG/RANKL balance by inhibiting the expression of RANKL gene.

Mineralization of regenerated cellulose hydrogels induced by human bone marrow stromal cells.

The proliferation of cultured human bone marrow stromal cells (HBMSC) on regenerated cellulose hydrogels was assessed. Regenerated cellulose hydrogels showed good rates of HBMSC proliferation, the cells exhibiting a flattened morphology, and after 22 days in culture, the cells had homogeneously colonized the surface of the materials. Moreover, since the early days in culture, between the surface of the materials and attached cells a continuous granulated hydroxyapatite layer was formed. It has been previously demonstrated in vitro, but without cells, that these materials did not mineralize. Hence, it seems that HBMSC promoted the mineralization of the surface.

Rat bone marrow stromal cell osteogenic differentiation and fibronectin adsorption on chitosan membranes: the effect of the degree of acetylation.

Cell adhesion, migration, and proliferation of a few anchorage-dependent cells cultured on chitosan (Ch) matrices are influenced by the degree of N-acetylation (DA) of Ch. In the present work, we examined the influence of the DA on the attachment, spreading, proliferation, and osteogenic differentiation of rat bone marrow stromal cells (rBMSCs). Ch membranes were characterized in terms of surface morphology, roughness, and wettability, and in terms of adsorption of an adhesive protein, fibronectin (Fn). Chs with DAs in the range of 4 to 49% were used. Among the Ch samples, the DA of 4% led to the highest Fn surface concentration, both from single protein solution and from diluted serum. Furthermore, the levels of Fn adsorbed from serum found for this DA were threefold higher than for the tissue culture polystyrene control, indicating that in the presence of competitive proteins Ch is more specific toward Fn adsorption than tissue culture polystyrene. rBMSCs cultured on Ch carrying a DA of 4% were able to spread, proliferate, and differentiate, reaching a higher level of osteogenic differentiation than on the control, despite the lower cell attachment observed for all Ch samples. Because the Ch sample with a DA of 4% showed the highest Fn adsorption from serum, we suggest that cell adhesion, spreading, and osteogenic differentiation of rBMSCs on Ch may be mediated by the adsorbed layer of Fn.

Fabrication of alternating polycation and albumin multilayer coating onto stainless steel by electrostatic layer-by-layer adsorption.

Multilayer films consisting of polyethylenimine (PEI) and albumin were successfully prepared on biomedical 316L stainless steel surface via electrostatic self-assembly of the PEI and albumin. The process of electrostatic self-assembly of PEI/albumin was monitored by125I radiolabeling, electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM). The EIS data revealed that the multilayer coating was stable in Tris-HCl (pH 7.35) buffer solution for 21 days. 125I radiolabeling experiments indicated that less than 10% albumin was eluted by PBS in 45 days. Static platelet adhesion experiments indicated that the PEI/albumin deposited on stainless steel could resist platelet adhesion effectively. Such an easy processing and shape-independent method may have good potential for surface modification of cardiovascular devices.