Efficacy and safety of neuroendoscopy surgery versus craniotomy for supratentorial intracerebral hemorrhage: an updated meta-analysis of randomized controlled trials.

Neuroendoscopy (NE) surgery emerged as a promising technique for the treatment of spontaneous intracerebral hemorrhage (ICH). A previous meta-analysis of randomized controlled trials (RCTs) analyzed the efficacy and safety of NE compared to craniotomy, but NE did not present a significant improvement in functional outcomes. However, a new study provided an opportunity to update the current knowledge. We searched PubMed, Embase, and Cochrane Central Register of Controlled Trials for RCTs reporting NE evacuation of spontaneous supratentorial ICH compared to craniotomy. The efficacy outcomes of interest were favorable functional outcome, functional disability, hematoma evacuation rate, and residual hematoma volume. The safety outcomes of interest were rebleeding, infection, and mortality. Seven RCTs were included containing 879 patients. The NE approach presented a significantly higher rate of favorable functional outcome compared with craniotomy (RR: 1.42; 95% CI 1.17, 1.73; p < 0.001). The evacuation rate was higher in patients who underwent the NE approach (MD: -8.36; 95% CI -12.66, -4.07; p < 0.001). NE did not show a benefit in improving the mortality rate (RR: 0.81, 95% CI 0.54, 1.22; p = 0.32). NE was associated with more favorable functional outcomes and lower rates of functional disabilities compared to craniotomy. Also, NE was superior regarding evacuation rate, while presenting a reduction in residual hematoma volume. NE might be associated with lower infection rates. Mortality was not improved by NE surgery. Larger, higher-quality randomized studies are needed to adequately evaluate the efficacy and safety of NE compared to craniotomy.

Chondrosarcoma secondary to hereditary multiple osteochondromas with spinal cord compression: A case report and systematic review.

Background: Hereditary multiple osteochondromas (HMOs) are a rare genetic disorder characterized by the formation of multiple benign osteochondromas that can undergo malignant transformation into chondrosarcoma. Case Description: A 24-year-old male with a history of HMO and osteochondroma surgery 4 years ago, presented with back pain and paresthesias. The magnetic resonance showed a right paravertebral infiltrating mass at the T12-L1 level causing spinal cord compression. Following en bloc resection of the tumor, the patient's symptoms/ signs resolved. The final pathological diagnosis was consistent with a chondrosarcoma. Conclusion: Chondrosarcomas secondary to HMO with spinal cord compression are rare. These patients often presenting with significant myelopathy/cord compression should undergo gross total resection where feasible to achieve the best outcomes.

Bioengineered Mesenchymal-Stromal-Cell-Derived Extracellular Vesicles as an Improved Drug Delivery System: Methods and Applications.

Extracellular vesicles (EVs) are cell-derived nano-sized lipid membranous structures that modulate cell-cell communication by transporting a variety of biologically active cellular components. The potential of EVs in delivering functional cargos to targeted cells, their capacity to cross biological barriers, as well as their high modification flexibility, make them promising drug delivery vehicles for cell-free therapies. Mesenchymal stromal cells (MSCs) are known for their great paracrine trophic activity, which is largely sustained by the secretion of EVs. MSC-derived EVs (MSC-EVs) retain important features of the parental cells and can be bioengineered to improve their therapeutic payload and target specificity, demonstrating increased therapeutic potential in numerous pre-clinical animal models, including in the treatment of cancer and several degenerative diseases. Here, we review the fundamentals of EV biology and the bioengineering strategies currently available to maximize the therapeutic value of EVs, focusing on their cargo and surface manipulation. Then, a comprehensive overview of the methods and applications of bioengineered MSC-EVs is presented, while discussing the technical hurdles yet to be addressed before their clinical translation as therapeutic agents.

Common mental disorder and related factors to the work of physiotherapists in the COVID-19 pandemic / Transtorno mental comum e fatores relacionados ao trabalho de fisioterapeutas na pandemia de COVID-19

Abstract Introduction The physiotherapists were one of the health professional categories on the Coronavirus (COVID-19) pandemic front line, however little is known how the pandemic has affected the mental health of these professionals. Objective To analyze the relation-ship between the presence of common mental disorder (CMD) and elements related to the work of physiotherapists who have been assisting patients with COVID-19. Methods: This is a cross-sectional survey study composed by physiotherapists from four states of Brazil: Alagoas, Paraíba, Pernambuco and Rio Grande do Norte. Working conditions evaluated were type of contract, weekly worked hours, time working, job satisfaction, and psychosocial aspects related to work. The CMD assessment was performed using the Self-Reporting Questionnaire-20 (SRQ-20). Results A total of 288 physiotherapists answered the electronic form, out of which 76.2% had CMD. Fear of reinfection (OR 2.75, 95%CI: 1.35-5.63) and previous infection by COVID-19 (OR: 2.03, 95%CI: 1.09-3.78) were factors related to a higher chance of risk of CMD. However, those who reported being satisfied with the work (OR: 0.28, 95%CI 0.11-0.72) and being more than 30 years old (OR: 0.46, 95%CI 0.24-0.88) had a lower chance of CMD risk. Conclusion Physiotherapists showed a higher prevalence of CMD. Fear of reinfection and previous infection by COVID-19 were risk factors to CMD, while satisfaction with the work and being more than 30 years old were protective factors to CMD.

Resumo Introdução Os fisioterapeutas foram uma das categorias profissionais da linha de frente do combate à pandemia do coronavírus (COVID-19), todavia, pouco se sabe como esta pandemia afetou a saúde mental destes profissionais. Objetivo Analisar a relação entre a presença de transtorno mental comum (TMC) e elementos relacionados ao trabalho de fisioterapeutas que assistiram pacientes com COVID-19. Métodos Trata-se de um estudo transversal composto por fisioterapeutas de quatro estados do Brasil: Alagoas, Paraíba, Pernambuco e Rio Grande do Norte. As condições de trabalho avaliadas foram tipo de contrato, jornada semanal, tempo de trabalho, satisfação e aspectos psicossociais relacionados ao trabalho. A avaliação do TMC foi realizada por meio do Self-Reporting Questionnaire-20 (SRQ-20). Resultados Um total de 288 fisioterapeutas responderam ao formulário eletrônico, dos quais 76,2% tinham TMC. Medo de reinfecção (OR 2,75, IC 95%: 1,35-5,63) e infecção anterior por COVID-19 (OR: 2,03, IC 95%: 1,09-3,78) foram fatores relacionados a uma maior chance de risco de TMC. No entanto aqueles que relataram estarem satisfeitos com o trabalho (OR: 0,28, IC 95% 0,11-0,72) e os que tinham até 30 anos (OR: 0,46, IC 95% 0,24-0,88) tiveram menor chance de risco de TMC. Conclusão Os fisioterapeutas apresentaram maior prevalência de TMC. Medo de reinfecção e infecção prévia por COVID-19 foram considerados fatores de risco para TMC, enquanto a satisfação com o trabalho e ter mais que 30 anos foram fatores protetores ao TMC.

Mesenchymal Stromal Cells (MSCs): A Promising Tool for Cell-Based Angiogenic Therapy.

The Mesenchymal stromal cells (MSCs) are a diverse subset of adult multipotent precursors, known for their potential therapeutic properties in regenerative medicine mainly sustained by paracrine effects through secretion of a variety of biologically active molecules. MSC secretome includes a wide range of soluble protein factors, composed of growth factors and cytokines, and vesicular components, which transfer proteins and genetic material modulating the host microenvironment. In particular, MSC-derived secretome mediates the different steps of the angiogenic process, inducing endothelial cell functions in vitro and promoting angiogenesis in vivo. As a result, MSCs have been widely explored as a promising cell-based therapy in diseases caused by insufficient angiogenesis. Numerous studies of myocardial infarction, ischemic stroke, and critical limb ischemia in animals have shown that human MSCs can enhance angiogenesis and accelerate tissue regeneration. This extensive preclinical work encouraged the study of these remarkable cells for the treatment of these disorders in human clinical settings. The present review provides a comprehensive overview of the pro-angiogenic potential of MSCs and paracrine effectors of their secretome. In addition, bioengineering strategies, including ex vivo preconditioning and genetic modification approaches, to enhance MSC innate angiogenic properties, and thereby therapeutic potency, will be presented. Finally, an update on completed preclinical and clinical studies with MSCs for the treatment of ischemia-related diseases will be discussed.

Minicircle-based expression of vascular endothelial growth factor in mesenchymal stromal cells from diverse human tissues.

BACKGROUND: Mesenchymal stromal cells (MSC) have been exploited for the treatment of ischemic diseases given their angiogenic potential. Despite bone marrow (BM) being the most studied tissue source, cells with similar intrinsic properties can be isolated from adipose tissue (AT) and umbilical cord matrix (UCM). The present study aims to compare the angiogenic potential of MSC obtained from BM, AT and UCM that were genetically modified with vascular endothelial growth factor (VEGF)-encoding minicircle (MC) vectors. The overexpression of VEGF combined with the intrinsic properties of MSC could represent a promising strategy towards angiogenic therapies. METHODS: We established a microporation-based protocol to transfect human MSC using VEGF-encoding MC (MC-VEGF). VEGF production levels were measured by an enzyme-linked immunosorbent assay and a quantitative polymerase chain reaction. The in vitro angiogenic potential of transfected cells was quantified using cell tube formation and migration functional studies. RESULTS: MSC isolated from BM, AT or UCM showed similar levels of VEGF secretion after transfection with MC-VEGF. Those values were significantly higher when compared to non-transfected cells, indicating an effective enhancement of VEGF production. Transfected cells displayed higher in vitro angiogenic potential than non-transfected controls, as demonstrated by functional in vitro assays. No significant differences were observed among cells from different sources. CONCLUSIONS: Minicircles can be successfully used to transiently overexpress VEGF in human MSC, regardless of the cell tissue source, representing an important advantage in a clinical context (i.e., angiogenic therapy) because a standard protocol might be applied to MSC of different tissue sources, which can be differentially selected according to the application (e.g., autologous versus allogeneic settings).

Marsupialização de cisto dentígero após traumatismo / Marsupialization of dentigerous cyst after traumatism

Objetivo: as causas mais comuns das alterações na dentição permanente são os traumatismos alvéolo-dentários (TAD) na dentição decídua, ultrapassando, inclusive, as incidências de cárie ou doença periodontal. Estes podem levar a hipoplasia de esmalte, alterações na morfologia dentária e desenvolvimento de cistos, como o cisto dentígero, considerando ainda que o tratamento desta ocorrência diverge ao tratar da dentição decídua e não da permanente. O objetivo deste trabalho é ilustrar essas características e como elas podem ser manejadas através de um relato de caso. Relato de caso: no caso em questão, ocorreu o desenvolvimento de um cisto dentígero de grandes dimensões após traumatismo alvéolo-dentário em dentição decídua na região anterior da maxila com retenção de três dentes permanentes. Foram discutidas as formas terapêuticas para tal situação clínica, bem como a possibilidade de uma abordagem multidisciplinar da cirurgia-ortodontia. Considerações finais: os TADs na dentição decídua devem ser bem diagnosticados e tratados, visando a prevenção de sequelas na dentição permanente.(AU)

Objective: the most common causes of changes in permanent dentition are alveolar-dental trauma (TAD) in deciduous dentition, with occurrence rates even higher than decays or periodontal disease, which can lead to enamel hypoplasia, changes in dental morphology and the development of cysts such as the dentigerous cyst, considering treatment differs when dealing with deciduous dentition instead of permanent dentition. The objective of this work is to illustrate these characteristics and propose a possible treatment, through a case report. Case report: in the case at hand occurred a development of a large dentigerous cyst occurred after TAD in primary dentition, in the anterior region of the maxilla with retention of three permanent teeth, discussing the therapeutic forms for such clinical situation, as well as the possibility of a multidisciplinary approach to surgery-orthodontics. Final considerations: TAD in deciduous teeth should be well diagnosed and treated aiming to prevent sequelae in permanent dentition.(AU)

Conditioned Medium From Azurin-Expressing Human Mesenchymal Stromal Cells Demonstrates Antitumor Activity Against Breast and Lung Cancer Cell Lines.

Recently, cell-based therapies have been explored as a strategy to enhance the specificity of anticancer therapeutic agents. In this perspective, human mesenchymal stromal cells (MSC) hold a promising future as cell delivery systems for anticancer proteins due to their unique biological features. In this study, we engineered human MSC to secrete a human codon-optimized version of azurin (hazu), a bacterial protein that has demonstrated anticancer activity toward different cancer models both in vitro and in vivo. To this end, microporation was used to deliver plasmid DNA encoding azurin into MSC derived from bone marrow (BM) and umbilical cord matrix (UCM), leading to expression and secretion of hazu to the conditioned medium (CM). Engineered hazu-MSC were shown to preserve tumor tropism toward breast (MCF-7) and lung (A549) cancer cell lines, comparable to non-modified MSC. Azurin was detected in the CM of transfected MSC and, upon treatment with hazu-MSC-CM, we observed a decrease in cancer cell proliferation, migration, and invasion, and an increase in cell death for both cancer cell lines. Moreover, expression of azurin caused no changes in MSC expression profile of cytokines relevant in the context of cancer progression, thus suggesting that the antitumoral effects induced by hazu-MSC secretome might be due to the presence of azurin independently. In conclusion, data shown herein indicate that MSC-produced azurin in a CM configuration elicits an anticancer effect.

Governamentalidade e práticas psicológicas: modos de gestão nos artigos brasileiros de psicologia do trabalho (1949 a 1965) / Governamentality and psychological practices: the styles of gestion on Brazilian Work psychology (1949-1965) / Gubernamentalidad y prácticas psicológicas: estilos de gestión brasilenõs en textos de psicología del trabajo (1949-1965)

Esta pesquisa objetivou, tendo como base os desenvolvimentos de Foucault e Rose acerca da governamentalidade liberal, avaliar como certas práticas específicas da psicologia contemporânea se engajam nos modos de governo atuais. Valendo-nos dos resultados produzidos por Rose, acerca dos períodos de estabelecimento da psicologia do trabalho em contexto britânico e norte-americano, buscamos entender como se deu esse processo no Brasil. Para tal, investigamos produções acadêmicas publicadas nacionalmente que tratassem sobre a questão do trabalho: os Arquivos Brasileiros de Psicotécnica e materiais publicados no Instituto de Psicologia da Universidade do Brasil. Não foi possível adequar os documentos investigados à periodização de Rose, levando-nos a desenvolver nosso próprio esquema classificatório. Ademais, os resultados parciais da pesquisa ainda em início apontaram para uma predominância nos anos 1949-1965 de artigos de estilo disciplinar e centrado na produção

This research's objective was, based on Foucault's and Rose's discussion on liberal governamentality, to inquire on how certain specific contemporary psychological practices engage in nowadays modes of government. Having Rose's results about the periods of establishment of work psychology in North American and British contexts in mind, we sought to understand how this process occurred in Brazil. For that, we investigated national academic productions that treated about the issue of work: the Arquivos Brasileiros de Psicotécnic and material published on the Institute of Psychology of University of Brasil. It was not possible to adequate the investigated documents to Rose's periodization, what led us to develop our own classification. Furthermore, the partial results of this research that is still in the beginning pointed out for a predominance of disciplinar and centred in the production style in the years 1949-1965

Basada en los desarrollos de Foucault y Rose sobre la gubernamentalidad liberal, esta investigación tuvo como objetivo evaluar cómo ciertas prácticas específicas de la psicología contemporánea se involucran en los modos de gobierno actuales. A partir de los resultados producidos por Rose sobre los períodos de establecimiento de la psicología del trabajo en el contexto británico y estadounidense, buscamos comprender cómo se llevó a cabo este proceso en Brasil. Con este fin, investigamos las producciones académicas publicadas a nivel nacional que trataron el tema del trabajo: los Archivos Brasileños de Psicotécnica y los materiales publicados en el Instituto de Psicología de la Universidad de Brasil. No fue posible adaptar los documentos investigados a la periodización de Rose, lo que nos llevó a desarrollar nuestro propio esquema de clasificación. Además, los resultados parciales de las primeras investigaciones apuntaron a un predominio en los artículos de estilo disciplinario y centrado en la producción de 1949-1965

Engineering of Human Mesenchymal Stem/Stromal Cells with Vascular Endothelial Growth Factor-Encoding Minicircles for Angiogenic Ex Vivo Gene Therapy.

Peripheral artery disease (PAD) is a debilitating and prevalent condition characterized by blockage of the arteries, leading to limb amputation in more severe cases. Mesenchymal stem/stromal cells (MSC) are known to have intrinsic regenerative properties that can be potentiated by the introduction of pro-angiogenic genes such as the vascular endothelial growth factor (VEGF). Herein, the use of human bone marrow MSC transiently transfected with minicircles encoding for VEGF is proposed as an ex vivo gene therapy strategy to enhance angiogenesis in PAD patients. The VEGF gene was cloned in minicircle and conventional plasmid vectors and used to transfect bone marrow-derived MSC ex vivo. VEGF expression was evaluated both by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The number of VEGF transcripts following MSC transfection with minicircles increased 130-fold relative to the expression in non-transfected MSC, whereas for the plasmid (pVAX1)-based transfection, the increase was 50-fold. Compared to the VEGF basal levels secreted by MSC (11.1 ± 3.4 pg/1,000 cells/day), significantly higher values were detected by enzyme-linked immunosorbent assay after both minicircle and pVAX1 transfection (644.8 ± 82.5 and 508.3 ± 164.0 pg/1,000 cells/day, respectively). The VEGF overexpression improved the angiogenic potential of MSC in vitro, as confirmed by endothelial cell tube formation and cell migration assays, without affecting the expansion potential ex vivo, as well as multilineage differentiation capacity or immunophenotype of MSC. Although preclinical in vivo studies are required, these results suggest that minicircle-mediated VEGF gene delivery, combined with the unique properties of human MSC, could represent a promising ex vivo gene therapy approach for an improved angiogenesis in the context of PAD.

O uso do correio eletrônico na comunicação entre usuários e uma equipe de saúde da família: relato de experiência / The use of electronic mail between users and a primary health care team: a case report / El uso del correo electronico en la comunicación entre usuarios y un equipo de atención primaria: relato de experiencia

Este relato descreve o uso do correio eletrônico por uma equipe de saúde da família para comunicação com os pacientes e agendamento. Tal uso surgiu como consequência de um processo de facilitação do acesso presencial e não presencial. No período de 25 meses houve crescimento no número de mensagens e de remetentes. Das 1437 mensagens recebidas, 61,5% foram para agendar consultas, 27,8% solicitavam informações em saúde e 7,6% eram sobre serviços oferecidos. As mulheres enviaram 87% das mensagens (63,3% delas tinham 20-40 anos). Em 60% das vezes, o contato referia-se à demanda do remetente e mais de 30% à de familiares. A equipe respondeu a 83% das mensagens em até 8 horas úteis. Na percepção da equipe, o uso do correio eletrônico melhorou a comunicação e o acesso e foi bem aceito pelos usuários, não prejudicando grupos mais vulneráveis. Investigações mais aprofundadas serão necessárias para avaliar a contribuição dessa ferramenta na Atenção Primária à Saúde.

This report describes the use of electronic mail by a primary health care team (PHT) in order to communicate with patients and aid in scheduling. Such use has emerged as a result of a process of facilitating access, both electronically and face-to-face. During a 25-month study period, users sent a total of 1437 messages with the purpose of scheduling appointments (61.5%), asking health-related questions (27.8%), and inquiring about services offered (7.6%). Women submitted 87% of the messages (63.3% of them were 20­40 years of age), with 60% of these messages concerning themselves and over 30% of them concerning their families. The team responded to 83% of messages within 8 business hours. According to the team, the use of electronic mail has improved communication and access, and has been well received by users without harming the most vulnerable groups. Further investigation is needed to assess the full contribution of this tool to primary health care.

Este texto describe la utilización del correo electronico por un equipo de salud de la familia para comunicación con los pacientes y marcación de citas. El correo surgió como consecuencia de un proceso para facilitar el acceso presencial y no presencial. En el periodo de 25 meses hubo crecimiento en el numero de mensajes y de remitentes. De las 1.437 mensajes recibidas, el 61,5% fueron para la marcación de citas, el 27,8% solicitaban informaciones de salud y el 7,6% eran sobre servicios ofrecidos. Las mujeres enviaron el 87% de los correos (el 63,3% de ellas tenían entre 20 y 40 años). En el 60% de las veces, el contacto se refería a la demanda del remitente y más del 30% a las de sus familiares. El equipo contestó al 83% de los mensajes dentro de 8 horas útiles. Según el equipo, el uso del correo electrónico mejoró la comunicación y el acceso y fue bien acepto por los usuarios, sin perjudicar a los grupos más vulnerables. Investigaciones más profundas serán necesarias para evaluar la contribución de esta herramienta en la Atención Primaria a la Salud

Imagens de uma nova velhice: considerações a partir do filme E se vivêssemos todos juntos? / Images of a new aging: considerations from the film All together?

O objetivo do artigo é refletir sobre novas imagens da velhice construídas e compartilhadas no filme E se vivêssemos todos juntos?, de Stéphane Robelin, em meio às perspectivas socioetárias atuais. Para isso, colocamos em diálogo estudos e análises da imagem, especialmente da cinematografia, com reflexões políticas e psicossociais acerca dos diferentes modos de envelhecer no mundo hoje, difundidas pelos estudos antropológicos em interface com o campo da Gerontologia.

The objective of this article is to reflect upon new images of old age built and shared in the movie All together?, of Stéphane Robelin, among the current social aging prospects. To do this, we put in dialogue image studies and analyzes, especially cinema images, with political and psicossocial reflections on the different modes of aging in the world today, broadcasted by anthropological studies in interface with the gerontological field.

Plasmid DNA production with Escherichia coli GALG20, a pgi-gene knockout strain: fermentation strategies and impact on downstream processing.

The market development of plasmid biopharmaceuticals for gene therapy and DNA vaccination applications is critically dependent on the availability of cost-effective manufacturing processes capable of delivering large amounts of high-quality plasmid DNA (pDNA) for clinical trials and commercialization. The producer host strain used in these processes must be designed to meet the upstream and downstream processing challenges characteristic of large scale pDNA production. The goal of the present study was to investigate the effect of different glucose feeding strategies (batch and fed-batch) on the pDNA productivity of GALG20, a pgi Escherichia coli strain potentially useful in industrial fermentations, which uses the pentose phosphate pathway (PPP) as the main route for glucose metabolism. The parental strain, MG1655ΔendAΔrecA, and the common laboratory strain, DH5α, were used for comparison purposes and pVAX1GFP, a ColE1-type plasmid, was tested as a model. GALG20 produced 3-fold more pDNA (∼141 mg/L) than MG1655ΔendAΔrecA (∼48 mg/L) and DH5α (∼40 mg/L) in glucose-based fed-batch fermentations. The amount of pDNA in lysates obtained from these cells was also larger for GALG20 (41%) when compared with MG1655ΔendAΔrecA (31%) and DH5α (26%). However, the final quality of pDNA preparations obtained with a process that explores precipitation, hydrophobic interaction chromatography and size exclusion was not significantly affected by strain genotype. Finally, high cell density fed-batch cultures were performed with GALG20, this time using another ColE1-type plasmid, NTC7482-41H-HA, in pre-industrial facilities using glucose and glycerol. These experiments demonstrated the ability of GALG20 to produce high pDNA yields of the order of 2100-2200 mg/L.

Enhancement of DNA vaccine efficacy by intracellular targeting strategies.

Immune response against an encoded antigenic protein can be elicited by including targeting sequences to DNA vaccines that promote protein sorting to processing pathways, related with antigen presentation by major histocompatibility complexes (MHC). Candidate DNA vaccines coding for neuraminidase 3 of the avian influenza virus were designed to encode different sequences that direct the protein to specific cellular compartments such as endoplasmic reticulum (i.e., adenovirus E1A), lysosomes (i.e., LAMP), and the combination of protein targeting to the endoplasmic reticulum and lysosome (i.e., E1A-LAMP). The DNA vaccine prototypes were engineered by biomolecular techniques and subsequently produced in E. coli cells. The biological activity of the vaccines was tested firstly in vitro, in Chinese hamster ovary cells, through flow cytometry and real-time polymerase chain reaction analysis. Then, an essential in vivo study was performed in chickens, in order to evaluate the efficacy of DNA prototype vaccines, by measuring the antibody production by enzyme-linked immunosorbent assay.

Plasmid DNA size does affect nonviral gene delivery efficiency in stem cells.

Genetic modification of stem cells, prior to transplantation, can enhance their survival and can improve their function in cell therapy settings. Mesenchymal stem cells (MSC) are considered one of the most promising tools for cell-based gene therapy, due to their multipotency, ease of isolation, as well as their high ex vivo expansion potential. Neural stem cells (NSC) may also present an ideal route for gene therapy and have been considered for use in cell replacement therapies in various neurodegenerative diseases. Gene therapy-based applications require the transfer of genetic material, either by viral or nonviral gene delivery methods, although the latter has been associated with low efficiencies, especially within hard to transfect cells as stem cells. Herein, we present results on the influence of plasmid size in gene delivery to human MSC and mouse NSC. We used minimized plasmids encoding a fluorescent protein but lacking the antibiotic resistance gene. This work shows that (1) for smaller plasmids the intracellular plasmid copy number can be up to 2.6-fold higher, and (2) the number of cells presenting fluorescence can be twice the number obtained for larger plasmids. Furthermore, by using plasmid constructs containing different polyA signals, we also demonstrated that differences between the plasmids depend largely on the transgene mRNA level. Based on our data we demonstrate that plasmid size severely affects the efficiency of nuclear uptake and we propose that it can also affect the rate of heterochromatin associated gene silencing in stem cells.

Comparative analysis of antigen-targeting sequences used in DNA vaccines.

Plasmid vectors can be optimized by including specific signals that promote antigen targeting to the major antigen presentation and processing pathways, increasing the immunogenicity and potency of DNA vaccines. A pVAX1-based backbone was used to encode the Green Fluorescence Protein (GFP) reporter gene fused either to ISG (Invariant Surface Glycoprotein) or to TSA (trans-sialidase) Trypanosoma brucei genes. The plasmids were further engineered to carry antigen-targeting sequences, which promote protein transport to the extracellular space (secretion signal), lysosomes (LAMP-1) and to the endoplasmic reticulum (adenovirus e1a). Transfection efficiency was not affected by differences in the size between each construct as no differences in the plasmid copy number per cell were found. This finding also suggests that the addition of both ISG gene and targeting sequences did not add sensitive regions prone to nuclease attack to the plasmid. Cells transfected with pVAX1GFP had a significant higher number of transcripts. This could be a result of lower mRNA stability and/or a lower transcription rate associated with the bigger transcripts. On the other hand, no differences were found between transcript levels of each ISG-GFP plasmids. Therefore, the addition of these targeting sequences does not affect the maturation/stability of the transcripts. Microscopy analysis showed differences in protein localization and fluorescent levels of cells transfected with pVAX1GFP and ISG constructs. Moreover, cells transfected with the lamp and secretory sequences presented a distinct distribution pattern when compared with ISG protein. Protein expression was quantified by flow cytometry. Higher cell fluorescence was observed in cells expressing the cytoplasmic fusion protein (ISG-GFP or TSA-GFP) compared with cells where the protein was transported to the lysosomal pathway. Protein transport to the endoplasmic reticulum does not lead to a decrease in the mean fluorescence values. The secretion signal was only effective when used in conjunction with TSA gene. Therefore, the characteristics of each protein (e.g., presence of transmembrane domains) might influence the efficacy of its cellular transport. This analysis constitutes a useful tool for the optimization of the design of DNA vaccines.

Influência da monofenilbutazona associada ou não ao omeprazol sobre o sistema digestório e renal de pôneis hígidos / Influence of mofebutazone associated or not to omeprazole on the digestive and renal tracts of healthy ponies

Os objetivos deste trabalho foram averiguar se a monofenilbutazona causa efeitos colaterais no trato digestório e lesões renais em pôneis hígidos e verificar a capacidade do omeprazol em inibir a gênese de úlceras gástricas. O experimento foi executado em duas etapas. Na primeira foram utilizados seis pôneis, sendo três deles tratados diariamente por via intravenosa (IV) com as doses de 3, 4,5 ou 6mg kg-1 de monofenilbutazona durante 12 dias. Os demais, além de antiinflamatório, também receberam 3mg kg-1 de omeprazol. Já na segunda etapa foram incluídos quatro pôneis hígidos, sendo dois tratados com doses diárias de 4,5mg kg-1 de monofenilbutazona durante 12 dias e os demais com 5mL de NaCl a 0,9 por cento, por via IV. Todos os pôneis foram submetidos à gastroscopia antes e após cada etapa experimental. Adicionalmente, na primeira etapa, foram realizadas urinálise e determinação dos valores de variáveis hematológicas (hematócrito e proteína plasmática total) e bioquímicas (creatinina, albumina, Ca+2 e P+3). Na primeira etapa, apenas os dois pôneis tratados com 6mg kg-1 de monofenilbutazona apresentaram úlceras na região aglandular, ao longo da margo plicatus. Na segunda etapa, dois animais também apresentaram úlceras gástricas, sendo que um deles havia recebido apenas NaCl a 0,9 por cento. A ocorrência das úlceras não foi influenciada (P>0,05) pela administração e pela dose da monofenilbutazona, nem pela presença do omeprazol. O efeito da monofenilbutazona sobre as variáveis hematológicas e bioquímicas foi inexpressivo (P+3) ou ausente (hematócrito, proteína plasmática total, creatinina, albumina, Ca+2) (P>0,05). Os resultados obtidos permitem concluir que: a ocorrência de úlceras na região aglandular de pôneis hígidos não sofre a influência da aplicação e da dose de monofenilbutazona, quando administrada durante 12 dias; úlceras em grau 4 na região aglandular de pôneis não necessariamente estão acompanhadas de sintomatologia clínica...

This research aimed to investigate whether mofebutazone causes gastrointestinal and renal side effects in healthy ponies as well as to verify the capacity of omeprazole to inhibit the genesis of gastric ulcers. The experiment was carried out in two phases. In the first, six ponies were used, with three of them being treated daily with different doses (3, 4.5 and 6mg kg-1) of intravenous (IV) mofebutazone for 12 days. The other ponies were given 3mg kg-1 of omeprazole in addition to the anti-inflammatory drug. In the second phase, four ponies were included, with two of them being treated daily with 4.5mg kg-1 of mofebutazone for 12 days and the two remainders with 5mL of IV NaCl at 0.9 percent. All ponies underwent gastroscopy before and after each experimental phase. Additionally, in the first phase, urine, hematological (hematocrit, and total plasma protein) and biochemical (creatinine, albumin, Ca+2 and P+3) analysis were determined. In the first phase, only the two ponies treated with 6mg kg-1 of mofebutazone presented ulcers in the aglandular region along the margo plicatus. In the second phase, two animals also presented gastric ulcers, with one having received only NaCl at 0.9 percent solution. Ulcers occurrence was neither influenced (P>0.05) by the administration and dose of mofebutazone, nor by the presence of omeprazole. Mofebutazone effect on the hematological and biochemical variables was unremarkable (P+3) or absent (hematocrit, total plasma protein, creatinine, albumin, Ca+2) (P>0.05). Based on these results the following conclusions could be drawn: the occurrence of gastric ulcers in the aglandular region of healthy ponies was not influenced by application and dose of mofebutazone when administered for 12 days; grade four ulcers in the aglandular region of ponies may not be accompanied by clinical signs; healthy ponies tolerate application of up to 6mg kg-1 of IV mofebutazone for 12 days without the occurrence of renal...

Development of a candidate DNA vaccine against Maedi-Visna virus.

DNA vaccine candidates against Maedi-Visna virus (MVV) infection in ovines were developed as an alternative to conventional vaccines. Candidates were constructed by cloning genes encoding the MVV gag polyprotein and gag proteins p16 and p25 fused to a beta-galactosidase reporter in a plasmid backbone. Transfection of different ovine cells showed a higher protein expression with plasmid lacZp16, which was hence further optimised by (i) removing a putative inhibitory sequence via reduction of the AU-content in the p16 gene or by (ii) introducing a secretory signal (Sc) to promote antigen secretion and increase its presentation to APCs. Unexpectedly, plasmids constructed on the basis of the first strategy by mutagenesis of lacZp16 (lacZp16mut(24)), led to a reduction in the expression of the antigen/reporter fusion in cultured ovine cells. This indicates that the high AU content in MVV does not inhibit protein expression. However, mice primed with lacZp16mut(24) and boosted with MVV protein displayed higher humoral response when compared with control lacZp16. The addition of the Sc signal (Sc-p16) led to lower amounts of intracellular antigen/reporter fusion in transfected ovine cells, thus confirming secretion. These findings correlate with in vivo experiments, which showed that mice primed with Sc-p16 and boosted with MVV exhibited stronger antibody responses when compared with control mice primed with lacZp16 and boosted with MVV. Stronger humoral responses were recorded by immunising mice with (i) Sc-p16 and lacZp16mut(24) plasmids together or with (ii) one plasmid containing both the mutations and the Sc signal.

Pênfigo foliáceo em um eqüino / Pemphigus foliaceus in a horse

Pênfigo foliáceo é uma rara afecção cutânea auto-imune que acomete várias espécies. Relata-se o caso de uma égua de quatro anos de idade, sem raça definida, apresentando áreas de alopecia, exsudação e crostas localizadas na face, região peitoral e membros pélvicos, além de prurido. Os exames laboratoriais para pesquisa de ácaros, bactérias e estruturas fúngicas foram negativos. O exame histopatológico revelou uma dermatite pustular intra-epidérmica com acantólise subcorneal, assim como dermatite perivascular superficial, com infiltração de eosinófilos, sendo o quadro compatível com pênfigo foliáceo.

Pemphigus foliaceus is an uncommon autoimmune skin disorder affecting various species. A case of a 4-year-old mare of undefined breed, displaying alopecic areas, exsudation and crusty lesions on the face, breast region, and hindlimbs as well as pruritus, is reported. Laboratory exams for mite, bacteria and fungal structures were negative. Histopathological exam revealed an intra dermal pustule dermatitis with acantholysis, as well as superficial perisvascular dermatitis with infiltration of eosinophyles, conditions compatible with pemphigus foliaceus.