RESUMO
Metabolism plays an essential role in cell fate decisions. However, the methods used for metabolic characterization and for finding potential metabolic regulators are still based on characterizing cellular metabolic steady-state which is dependent on the extracellular environment. In this work, we hypothesized that the response dynamics of intracellular metabolic pools to extracellular stimuli is controlled in a cell type-specific manner. We applied principles of process dynamics and control to human induced pluripotent stem cells (hiPSC) and human neural stem cells (hNSC) subjected to a sudden extracellular glutamine step. The fold-changes of steady-states and the transient profiles of metabolic pools revealed that dynamic responses were reproducible and cell type-specific. Importantly, many amino acids had conserved dynamics and readjusted their steady state concentration in response to the increased glutamine influx. Overall, we propose a novel methodology for systematic metabolic characterization and identification of potential metabolic regulators.
Assuntos
Células-Tronco Pluripotentes Induzidas , Redes e Vias Metabólicas/fisiologia , Células-Tronco Neurais , Reatores Biológicos , Células Cultivadas , Biologia Computacional , Espaço Extracelular/química , Espaço Extracelular/metabolismo , Glutamina/metabolismo , Glutamina/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismoRESUMO
Equine leptospirosis, although usually asymptomatic, has been associated with recurrent uveitis, abortion, and other systemic signs, constituting a major economic loss in the equine agribusiness sector. The occurrence of anti-Leptospira spp. agglutinins were investigated in 1200 serum samples of horses from 27 municipalities of the Recôncavo Baiano region, Bahia state (NE Brazil), besides the risk factors related to animals and their cattle farms. The microscopic agglutination test (MAT) was performed using 13 serogroups of Leptospira spp. as antigens. From 1200 analyzed equines, 97 (8%) were considered as positive, which obtained titer equal to or higher than 200. In 22 (78.6%) from the 28 properties, at least one animal was detected as seropositive. The most prevalent serogroup in animals, raised in stables, was the Australis (serovar Bratislava) 67%; in mounted police animals was the Sejroe (serovar Wolffi and Hardjobovis) 50%; and equestrian animals was the Australis (serovar Bratislava) 25% and Icterohaemorrhagiae (serovar Copenhageni) 25%. Equine leptospirosis is present in most cattle farms of the region studied. The risk factors of major importance regarded the semi-extensive livestock farming, the land topography of the cattle farm, the contact with animals in neighboring properties, and cohabitation with goats.
Assuntos
Doenças dos Cavalos/epidemiologia , Leptospirose/veterinária , Testes Sorológicos/veterinária , Testes de Aglutinação/veterinária , Animais , Antígenos de Bactérias/sangue , Brasil/epidemiologia , Bovinos , Fazendas , Cavalos , Fatores de Risco , Estudos Soroepidemiológicos , SorogrupoRESUMO
Regulation of tissue-type plasminogen activator (tPA) depends on fibrin binding and fibrin structure. tPA structure/function relationships were investigated in fibrin formed by high or low thrombin concentrations to produce a fine mesh and small pores, or thick fibers and coarse structure, respectively. Kinetics studies were performed to investigate plasminogen activation and fibrinolysis in the 2 types of fibrin, using wild-type tPA (F-G-K1-K2-P, F and K2 binding), K1K1-tPA (F-G-K1-K1-P, F binding), and delF-tPA (G-K1-K2-P, K2 binding). There was a trend of enzyme potency of tPA > K1K1-tPA > delF-tPA, highlighting the importance of the finger domain in regulating activity, but the differences were less apparent in fine fibrin. Fine fibrin was a better surface for plasminogen activation but more resistant to lysis. Scanning electron and confocal microscopy using orange fluorescent fibrin with green fluorescent protein-labeled tPA variants showed that tPA was strongly associated with agglomerates in coarse but not in fine fibrin. In later lytic stages, delF-tPA-green fluorescent protein diffused more rapidly through fibrin in contrast to full-length tPA, highlighting the importance of finger domain-agglomerate interactions. Thus, the regulation of fibrinolysis depends on the starting nature of fibrin fibers and complex dynamic interaction between tPA and fibrin structures that vary over time.
Assuntos
Fibrina/metabolismo , Fibrina/ultraestrutura , Fibrinólise/fisiologia , Ativador de Plasminogênio Tecidual/metabolismo , Humanos , Cinética , Microscopia Confocal , Microscopia Eletrônica de Varredura , Plasminogênio/metabolismo , Ligação Proteica , Trombina/metabolismoRESUMO
OBJECTIVE: Arterial thrombi contain variable amounts of red blood cells (RBCs), which interact with fibrinogen through an eptifibatide-sensitive receptor and modify the structure of fibrin. In this study, we evaluated the modulator role of RBCs in the lytic susceptibility of fibrin. METHODS AND RESULTS: If fibrin is formed at increasing RBC counts, scanning electron microscopy evidenced a decrease in fiber diameter from 150 to 96 nm at 40% (v/v) RBCs, an effect susceptible to eptifibatide inhibition (restoring 140 nm diameter). RBCs prolonged the lysis time in a homogeneous-phase fibrinolytic assay with tissue plasminogen activator (tPA) by up to 22.7±1.6%, but not in the presence of eptifibatide. Confocal laser microscopy using green fluorescent protein-labeled tPA and orange fluorescent fibrin showed that 20% to 40% (v/v) RBCs significantly slowed down the dissolution of the clots. The fluorescent tPA variant did not accumulate on the surface of fibrin containing RBCs at any cell count above 10%. The presence of RBCs in the clot suppressed the tPA-induced plasminogen activation, resulting in 45% less plasmin generated after 30 minutes of activation at 40% (v/v) RBCs. CONCLUSIONS: RBCs confer lytic resistance to fibrin resulting from modified fibrin structure and impaired plasminogen activation through a mechanism that involves eptifibatide-sensitive fibrinogen-RBC interactions.
Assuntos
Eritrócitos/metabolismo , Fibrina/metabolismo , Fibrinólise , Trombose/sangue , Eptifibatida , Eritrócitos/efeitos dos fármacos , Fibrina/ultraestrutura , Fibrinolisina/metabolismo , Fibrinólise/efeitos dos fármacos , Fibrinolíticos/farmacologia , Humanos , Cinética , Microscopia de Força Atômica , Microscopia Confocal , Peptídeos/farmacologia , Plasminogênio/metabolismo , Receptores de Fibrinogênio/efeitos dos fármacos , Receptores de Fibrinogênio/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
Background: Activated cardiac fibroblasts (CF) play a central role in cardiac fibrosis, a condition associated with most cardiovascular diseases. Conversion of quiescent into activated CF sustains heart integrity upon injury. However, permanence of CF in active state inflicts deleterious heart function effects. Mechanisms underlying this cell state conversion are still not fully disclosed, contributing to a limited target space and lack of effective anti-fibrotic therapies. Materials and methods: To prioritize targets for drug development, we studied CF remodeling upon activation at transcriptomic and proteomic levels, using three different cell sources: primary adult CF (aHCF), primary fetal CF (fHCF), and induced pluripotent stem cells derived CF (hiPSC-CF). Results: All cell sources showed a convergent response upon activation, with clear morphological and molecular remodeling associated with cell-cell and cell-matrix interactions. Quantitative proteomic analysis identified known cardiac fibrosis markers, such as FN1, CCN2, and Serpine1, but also revealed targets not previously associated with this condition, including MRC2, IGFBP7, and NT5DC2. Conclusion: Exploring such targets to modulate CF phenotype represents a valuable opportunity for development of anti-fibrotic therapies. Also, we demonstrate that hiPSC-CF is a suitable cell source for preclinical research, displaying significantly lower basal activation level relative to primary cells, while being able to elicit a convergent response upon stimuli.
RESUMO
A selection of physicochemical and biological assays were investigated for their utility in detecting changes in preparations of Interferon alpha-2a and Interferon alpha-2b (IFN-alpha 2a, IFN-alpha 2b), which had been subjected to stressed conditions, in order to create models of biopharmaceutical products containing product-related impurities. The stress treatments, which included oxidation of methionine residues and storage at elevated temperatures for different periods of time, were designed to induce various degrees of degradation, aggregation or oxidation of the interferon. Biological activity of the stressed preparations was assessed in three different in vitro cell-based bioassay systems: a late-stage anti-proliferative assay and early-stage assays measuring reporter gene activation or endogenous gene expression by quantitative real time Reverse Transcription-Polymerase Chain Reaction (qRT-PCR). Relevant physicochemical methods such as SDS-PAGE, reverse phase (RP) chromatography, size-exclusion chromatography (SEC) and dynamic light scattering (DLS), proved their complementarity in detecting structural changes in the stressed preparations which were reflected by reductions in biological activity.
Assuntos
Bioensaio/métodos , Produtos Biológicos/química , Interferon-alfa/análise , Produtos Biológicos/metabolismo , Proliferação de Células , Cromatografia/métodos , Desenho de Fármacos , Eletroforese em Gel de Poliacrilamida , Humanos , Interferon alfa-2 , Interferon-alfa/metabolismo , Metionina/química , Oxigênio/química , Proteínas Recombinantes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Manejo de Espécimes , TemperaturaRESUMO
We have investigated the use of quantitative reverse transcription-polymerase chain reaction (qRT-PCR) as an alternative to a selection of late-stage functional bioassays for determination of the potency of preparations of vascular endothelial growth factor (VEGF). Responses were measured in cultures of human umbilical vein endothelial cells (HUVECs). Late-stage responses measured were cell survival and proliferation, and production of interleukin-8 (IL-8), interleukin-6 (IL-6), and tissue factor. The dose-response range was similar across the assays, increasing from 2 ng/mL VEGF and reaching a maximum between 30 ng/mL and 125 ng/mL VEGF. A number of VEGF-induced mRNA species demonstrated dose-response curves suitable for VEGF potency determination. IL-8 mRNA induction after 45 min incubation with VEGF, which showed maximal responses between 15.6 ng/mL and 62.5 ng/mL VEGF, was selected for further characterization. This gene-expression bioassay was robust across a range of cell seeding densities and could be used for samples processed immediately following incubation with VEGF and for cell lysates stored at -80 degrees C for 3 months. We also compared this gene-expression bioassay and the assays of late-stage responses in the potency measurement of the inhibitors of VEGF activity, anti-VEGF monoclonal antibody MAB293, and a VEGF soluble receptor VEGFsR1 preparation. We present a critical evaluation of the use of qRT-PCR in assaying the potency of VEGF and its inhibitors, and of the potential of this platform for measuring the potency of other biological therapeutics.
Assuntos
Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Anticorpos Monoclonais/farmacologia , Bioensaio , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Humanos , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Interleucina-8/genética , RNA Mensageiro/análise , Tromboplastina/biossíntese , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresRESUMO
Brain microenvironment plays an important role in neurodevelopment and pathology, where the extracellular matrix (ECM) and soluble factors modulate multiple cellular processes. Neural cell culture typically relies on heterologous matrices poorly resembling brain ECM. Here, we employed neurospheroids to address microenvironment remodeling during neural differentiation of human stem cells, without the confounding effects of exogenous matrices. Proteome and transcriptome dynamics revealed significant changes at cell membrane and ECM during 3D differentiation, diverging significantly from the 2D differentiation. Structural proteoglycans typical of brain ECM were enriched during 3D differentiation, in contrast to basement membrane constituents in 2D. Moreover, higher expression of synaptic and ion transport machinery was observed in 3D cultures, suggesting higher neuronal maturation in neurospheroids. This work demonstrates that 3D neural differentiation as neurospheroids promotes the expression of cellular and extracellular features found in neural tissue, highlighting its value to address molecular defects in cell-ECM interactions associated with neurological disorders.
Assuntos
Diferenciação Celular , Microambiente Celular , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Biomarcadores , Técnicas de Cultura de Células , Imunofluorescência , Humanos , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
Collagen and calcium-binding EGF domain-1 (CCBE1) is a secreted protein critical for lymphatic/cardiac vascular development and regeneration. However, the low efficient production of the recombinant full-length CCBE1 (rCCBE1) has been a setback for functional studies and therapeutic applications using this protein. The main goal of this work was to implement a robust bioprocess for efficient production of glycosylated rCCBE1. Different bioprocess strategies were combined with proteomic tools for process/product characterization, evaluating the impact of process parameters on cell performance, rCCBE1 production and quality. We have shown that rCCBE1 volumetric yield was positively correlated with higher cell density at transfection (HDT), and under these conditions the secreted protein presented a mature glycosylated profile (complex N-glycans). Mild hypothermia was also applied to HDT condition that resulted in enhanced cell viability; however an enrichment of immature rCCBE1 variants was detected. Mass spectrometry-based tools allowed the identification of rCCBE1 peptides confirming protein identity in the affinity chromatography enriched product. rCCBE1 biological activity was validated by in vitro angiogenesis assay, where enhanced vessel formation was observed. Herein, we report a step forward in the production and characterization of human glycosylated rCCBE1, amenable for in vitro and in vivo studies to explore its regenerative therapeutic potential.
Assuntos
Indutores da Angiogênese/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Indutores da Angiogênese/farmacologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/farmacologia , Linhagem Celular , Glicosilação , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/farmacologiaRESUMO
The development of human cell models that can efficiently restore hepatic functionality and cope with the reproducibility and scalability required for preclinical development poses a significant effort in tissue engineering and biotechnology. Primary cultures of human hepatocytes (HHs), the preferred model for in vitro toxicity testing, dedifferentiate and have short-term viability in two-dimensional (2D) cultures. In this study, hepatocytes isolated from human liver tissue were co-cultured with human bone marrow mesenchymal stem cells (BM-MSCs) as spheroids in automated, computer-controlled, stirred-tank bioreactors with perfusion operation mode. A dual-step inoculation strategy was used, resulting in an inner core of parenchymal liver tissue with an outer layer of stromal cells. Hepatocyte polarization and morphology as well as the mesenchymal phenotype of BM-MSCs were maintained throughout the culture period and the crosstalk between the two cell types was depicted. The viability, compact morphology and phenotypic stability of hepatocytes were enhanced in co-cultures in comparison to monocultures. Gene expression of phase I and II enzymes was higher and CYP3A4 and CYP1A2 activity was inducible until week 2 of culture, being applicable for repeated-dose toxicity testing. Moreover, the excretory activity was maintained in co-cultures and the biosynthetic hepatocellular functions (albumin and urea secretion) were not affected by the presence of BM-MSCs. This strategy might be extended to other hepatic cell sources and the characterization performed brings knowledge on the interplay between the two cell types, which may be relevant for therapeutic applications. Copyright © 2015 John Wiley & Sons, Ltd.
Assuntos
Reatores Biológicos , Técnicas de Cultura de Células/métodos , Hepatócitos/citologia , Hepatócitos/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Técnicas de Cultura de Células/instrumentação , Células Cultivadas , Técnicas de Cocultura/instrumentação , Técnicas de Cocultura/métodos , HumanosRESUMO
To deliver the required cell numbers and doses to therapy, scaling-up production and purification processes (at least to the liter-scale) while maintaining cells' characteristics is compulsory. Therefore, the aim of this work was to prove scalability of an integrated streamlined bioprocess compatible with current good manufacturing practices (cGMP) comprised by cell expansion, harvesting and volume reduction unit operations using human mesenchymal stem cells (hMSC) isolated from bone marrow (BM-MSC) and adipose tissue (AT-MSC). BM-MSC and AT-MSC expansion and harvesting steps were scaled-up from spinner flasks to 2L scale stirred tank single-use bioreactor using synthetic microcarriers and xeno-free medium, ensuring high cellular volumetric productivities (50×106cellL-1day-1), expansion factors (14-16 fold) and cell recovery yields (80%). For the concentration step, flat sheet cassettes (FSC) and hollow fiber cartridges (HF) were compared showing a fairly linear scale-up, with a need to slightly decrease the permeate flux (30-50 LMH, respectively) to maximize cell recovery yield. Nonetheless, FSC allowed to recover 18% more cells after a volume reduction factor of 50. Overall, at the end of the entire bioprocess more than 65% of viable (>95%) hMSC could be recovered without compromising cell's critical quality attributes (CQA) of viability, identity and differentiation potential. Alongside the standard quality assays, a proteomics workflow based on mass spectrometry tools was established to characterize the impact of processing on hMSC's CQA; These analytical tools constitute a powerful tool to be used in process design and development.
Assuntos
Reatores Biológicos , Técnicas de Cultura de Células/métodos , Células-Tronco Mesenquimais/citologia , Proteoma , Regulação para Baixo , Humanos , Espectrometria de Massas , Células-Tronco Mesenquimais/metabolismo , Proteoma/análise , Proteoma/metabolismo , Proteômica , Regulação para CimaRESUMO
UNLABELLED: : Human embryonic stem cells (hESCs) have an enormous potential as a source for cell replacement therapies, tissue engineering, and in vitro toxicology applications. The lack of standardized and robust bioprocesses for hESC expansion has hindered the application of hESCs and their derivatives in clinical settings. We developed a robust and well-characterized bioprocess for hESC expansion under fully defined conditions and explored the potential of transcriptomic and metabolomic tools for a more comprehensive assessment of culture system impact on cell proliferation, metabolism, and phenotype. Two different hESC lines (feeder-dependent and feeder-free lines) were efficiently expanded on xeno-free microcarriers in stirred culture systems. Both hESC lines maintained the expression of stemness markers such as Oct-4, Nanog, SSEA-4, and TRA1-60 and the ability to spontaneously differentiate into the three germ layers. Whole-genome transcriptome profiling revealed a phenotypic convergence between both hESC lines along the expansion process in stirred-tank bioreactor cultures, providing strong evidence of the robustness of the cultivation process to homogenize cellular phenotype. Under low-oxygen tension, results showed metabolic rearrangement with upregulation of the glycolytic machinery favoring an anaerobic glycolysis Warburg-effect-like phenotype, with no evidence of hypoxic stress response, in contrast to two-dimensional culture. Overall, we report a standardized expansion bioprocess that can guarantee maximal product quality. Furthermore, the "omics" tools used provided relevant findings on the physiological and metabolic changes during hESC expansion in environmentally controlled stirred-tank bioreactors, which can contribute to improved scale-up production systems. SIGNIFICANCE: The clinical application of human pluripotent stem cells (hPSCs) has been hindered by the lack of robust protocols able to sustain production of high cell numbers, as required for regenerative medicine. In this study, a strategy was developed for the expansion of human embryonic stem cells in well-defined culture conditions using microcarrier technology and stirred-tank bioreactors. The use of transcriptomic and metabolic tools allowed detailed characterization of the cell-based product and showed a phenotypic convergence between both hESC lines along the expansion process. This study provided valuable insights into the metabolic hallmarks of hPSC expansion and new information to guide bioprocess design and media optimization for the production of cells with higher quantity and improved quality, which are requisite for translation to the clinic.
RESUMO
Anchorage-dependent cell cultures are used for the production of viruses, viral vectors, and vaccines, as well as for various cell therapies and tissue engineering applications. Most of these applications currently rely on planar technologies for the generation of biological products. However, as new cell therapy product candidates move from clinical trials towards potential commercialization, planar platforms have proven to be inadequate to meet large-scale manufacturing demand. Therefore, a new scalable platform for culturing anchorage-dependent cells at high cell volumetric concentrations is urgently needed. One promising solution is to grow cells on microcarriers suspended in single-use bioreactors. Toward this goal, a novel bioreactor system utilizing an innovative Vertical-Wheel™ technology was evaluated for its potential to support scalable cell culture process development. Two anchorage-dependent human cell types were used: human lung carcinoma cells (A549 cell line) and human bone marrow-derived mesenchymal stem cells (hMSC). Key hydrodynamic parameters such as power input, mixing time, Kolmogorov length scale, and shear stress were estimated. The performance of Vertical-Wheel bioreactors (PBS-VW) was then evaluated for A549 cell growth and oncolytic adenovirus type 5 production as well as for hMSC expansion. Regarding the first cell model, higher cell growth and number of infectious viruses per cell were achieved when compared with stirred tank (ST) bioreactors. For the hMSC model, although higher percentages of proliferative cells could be reached in the PBS-VW compared with ST bioreactors, no significant differences in the cell volumetric concentration and expansion factor were observed. Noteworthy, the hMSC population generated in the PBS-VW showed a significantly lower percentage of apoptotic cells as well as reduced levels of HLA-DR positive cells. Overall, these results showed that process transfer from ST bioreactor to PBS-VW, and scale-up was successfully carried out for two different microcarrier-based cell cultures. Ultimately, the data herein generated demonstrate the potential of Vertical-Wheel bioreactors as a new scalable biomanufacturing platform for microcarrier-based cell cultures of complex biopharmaceuticals.
Assuntos
Adenoviridae/fisiologia , Reatores Biológicos , Técnicas de Cultura de Células/instrumentação , Células-Tronco Mesenquimais/citologia , Vírus Oncolíticos/fisiologia , Adenoviridae/isolamento & purificação , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Desenho de Equipamento , Humanos , Hidrodinâmica , Vírus Oncolíticos/isolamento & purificaçãoRESUMO
The integration of up- and downstream unit operations can result in the elimination of hold steps, thus decreasing the footprint, and ultimately can create robust closed system operations. This type of design is desirable for the bioprocess of human mesenchymal stem cells (hMSC), where high numbers of pure cells, at low volumes, need to be delivered for therapy applications. This study reports a proof of concept of the integration of a continuous perfusion culture in bioreactors with a tangential flow filtration (TFF) system for the concentration and washing of hMSC. Moreover, we have also explored a continuous alternative for concentrating hMSC. Results show that expanding cells in a continuous perfusion operation mode provided a higher expansion ratio, and led to a shift in cells' metabolism. TFF operated either in continuous or discontinuous allowed to concentrate cells, with high cell recovery (>80%) and viability (>95%); furthermore, continuous TFF permitted to operate longer with higher cell concentrations. Continuous diafiltration led to higher protein clearance (98%) with lower cell death, when comparing to discontinuous diafiltration. Overall, an integrated process allowed for a shorter process time, recovering 70% of viable hMSC (>95%), with no changes in terms of morphology, immunophenotype, proliferation capacity and multipotent differentiation potential.
Assuntos
Reatores Biológicos , Células-Tronco Mesenquimais , Amônia/metabolismo , Adesão Celular , Contagem de Células , Técnicas de Cultura de Células , Proliferação de Células , Sobrevivência Celular , Fibroblastos/citologia , Prepúcio do Pênis/citologia , Glucose/metabolismo , Glutamina/metabolismo , Humanos , Ácido Láctico/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/fisiologia , Perfusão , Telomerase/metabolismoRESUMO
No período de janeiro de 2011 a dezembro de 2014 foram diagnosticados 9 surtos (A, B, C, D, E, F, G, H e I) de Oestrus ovis em pequenos ruminantes no estado da Bahia. No surto A obteve-se 0,5% (1/200); B 2,2% (2/90); C 0,8% (1/120); D 2% (2/100); E 1% (1/100); F 3% (1/33); G 0,6% (1/150); H 2,5% (5/200); I com 11,4% (8/70) em ovinos e 5% (2/40) em caprinos. Os sinais clínicos associados ao parasitismo pelas larvas nos surtos foram respiração ruidosa, espirro seguido de secreção nasal catarral, inquietação, movimentação excessiva da cabeça e andar em círculo. Macroscopicamente havia nos seios e conchas nasais hiperemia, edema da mucosa e presença de larvas. Todas as larvas coletadas dos cornetos e conchas nasais variavam desde o primeiro ao terceiro estágio de desenvolvimento. Algumas larvas L3 coletadas nas necropsias foram incubadas e o imago obtido das pupas mediram aproximadamente 10mm de cor acinzentada e abdômen escurecido. Realizada análise descritiva das condições climáticas, ano e positividade de casos de oestrose, demonstrou que a ocorrência tem tendência de crescimento com os anos (pË0,001) e que houve casos com menor média de temperatura mínima (pË0,001), possibilitando o desenvolvimento da mosca de O. ovis, demonstrando que houve a introdução da mosca enTtre o rebanho de ovinos e caprinos do estado da Bahia, e que as condições climáticas são ideais para perpetuação da espécie.(AU)
From January 2011 to December 2014 were diagnosed 9 outbreaks of Oestrus ovis infection in small ruminants (Outbreaks A-I) in the State of Bahia. The incidence of oestrosis in sheep in outbreak A was 0.5% (1/200), in B 2.2% (2/90), in C 0.8% (1/120), in D 2% (2/100), in E 1% (1/100), in F 3% (1/33), in G 0.6% (1/150), in H 2.5% (5/200), and in I 11.42% (8/70), and 5% (2/40) in goats. Clinical signs associated with parasitism were wheezing, sneezing followed by catarrhal nasal secretion, some restlessness, excessive head movement and walking in circles. The breasts and turbinates were hyperemic, with mucosal edema and presence of O. ovis larvae. All larvae collected from the turbinates ranged from the first to the third stage of development. Some L3 larvae collected at necropsy were incubated and the gray colored Imago with dark abdomen obtained from the pupae measured about 10mm. A descriptive analysis of the climatic conditions was carried out; in the year of investigation the incidence of O. ovis infection has grown (p<0.001), and the lowest mean minimum temperature (p<0.001) caused the development the O. ovis fly, so that there was an introduction of an increased number of these flies into the sheep and goat flocks in state of Bahia with the ideal climatic conditions for their perpetuation.(AU)
Assuntos
Animais , Larva/parasitologia , Miíase/veterinária , Obstrução Nasal/patologia , Obstrução Nasal/veterinária , Ovinos , Cavidade Nasal/patologia , RuminantesRESUMO
This paper describes recent progress made in the use of high pressure or supercritical fluids to process polymers into three-dimensional tissue engineering scaffolds. Three current examples are highlighted: foaming of acrylates for use in cartilage tissue engineering; plasticization and encapsulation of bioactive species into biodegradable polyesters for bone tissue engineering; and a novel laser sintering process used to fabricate three-dimensional biodegradable polyester structures from particles prepared via a supercritical route.
Assuntos
Materiais Biocompatíveis/química , Dióxido de Carbono/química , Cromatografia com Fluido Supercrítico/métodos , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Polímeros/química , Conformação Molecular , PressãoRESUMO
Um caso de linfangioleiomiomatose pulmonar diagnosticado por tomografia computadorizada de tórax com técnica de alta resolução e biópsia pulmonar aberta é apresentado...