RESUMO

Disorders of gastrointestinal motility are the major physiologic problem in chagasic megacolon. The contraction mechanism is complex and controlled by different cell types such as enteric neurons, smooth muscle, telocytes, and an important pacemaker of the intestine, the interstitial cells of Cajal (ICCs). The role of ICCs in the progression of acute and chronic Chagas disease remains unclear. In the present work, we investigate the aspects of ICCs in a long-term model of Chagas disease that mimics the pathological aspects of human megacolon. Different subsets of ICCs isolated from Auerbach's myenteric plexuses and muscle layers of control and Trypanosoma cruzi infected animals were determined by analysis of CD117, CD44, and CD34 expression by flow cytometer. Compared with the respective controls, the results showed a reduced frequency of mature ICCs in the acute phase and three months after infection. These results demonstrate for the first time the phenotypic distribution of ICCs associated with functional dysfunction in a murine model of chagasic megacolon. This murine model proved valuable for studying the profile of ICCs as an integrative system in the gut and as a platform for understanding the mechanism of chagasic megacolon development.

Assuntos

RESUMO

Breast cancer is the most common cancer in women. Although chemotherapy is still broadly used in its treatment, adverse effects remain a challenge. In this scenario, aptamers emerge as a promising alternative for theranostic applications. Studies using breast cancer cell lines provide useful information in laboratory and preclinical investigations, most of which use cell lines established from metastatic sites. However, these cell lines correspond to cell populations of the late stage of tumor progression. On the other hand, studies using breast cancer cells established from primary sites make it possible to search for new theranostic approaches in the early stages of the disease. Therefore, this work aimed to select RNA aptamers internalized by MGSO-3 cells, a human breast cancer cell line, derived from a primary site previously established in our laboratory. Using the Cell-Internalization SELEX method, we have selected two candidate aptamers (ApBC1 and ApBC2). We evaluated their internalization efficiencies, specificities, cellular localization by Reverse Transcription-qPCR (RT-qPCR) and confocal microscopy assays. The results suggest that both aptamers were efficiently internalized by human breast cancer cells, MACL-1, MDA-MB-231, and especially by MGSO-3 cells. Furthermore, both aptamers could effectively distinguish human breast cancer cells derived from normal human mammary cell (MCF 10A) and prostate cancer cell (PC3) lines. Therefore, ApBC1 and ApBC2 could be promising candidate molecules for theranostic applications, even in the early stages of tumor progression.

Assuntos

Assuntos

RESUMO

Background/Aims: Chagasic megacolon is caused by Trypanosoma cruzi, which promotes in several cases, irreversible segmental colonic dilation. This alteration is the major anatomic-clinical disorder, characterized by the enteric nervous system and muscle wall structural damage. Herein, we investigate how T. cruzi -induced progressive colonic structural changes modulate the colonic contractile pattern activity. Methods: We developed a murine model of T. cruzi-infection that reproduced long-term modifications of the enlarged colon. We evaluated colonic and total intestinal transit time in animals. The patterns of motor response at several time intervals between the acute and chronic phases were evaluated using the organ bath assays. Enteric motor neurons were stimulated by electric field stimulation. The responses were analyzed in the presence of the nicotinic and muscarinic acetylcholine receptor antagonists. Western blot was performed to evaluate the expression of nicotinic and muscarinic receptors. The neurotransmitter expression was analyzed by real-time polymerase chain reaction. Results: In the chronic phase of infection, there was decreased intestinal motility associated with decreased amplitude and rhythmicity of intestinal contractility. Pharmacological tests suggested a defective response mediated by acetylcholine receptors. The contractile response induced by acetylcholine was decreased by atropine in the acute phase while the lack of its action in the chronic phase was associated with tissue damage, and decreased expression of choline acetyltransferase, nicotinic subunits of acetylcholine receptors, and neurotransmitters. Conclusions: T. cruzi -induced damage of smooth muscles was accompanied by motility disorders such as decreased intestinal peristalsis and cholinergic system response impairment. This study allows integration of the natural history of Chagasic megacolon motility disorders and opens new perspectives for the design of effective therapeutic.

RESUMO

In recent years, stem cell therapy has shown promise in regenerative medicine. The lack of standardized protocols for cell isolation and differentiation generates conflicting results in this field. Mesenchymal stem cells derived from adipose tissue (ASC) and fibroblasts (FIB) share very similar cell membrane markers. In this context, the distinction of mesenchymal stem cells from fibroblasts has been crucial for safe clinical application of these cells. In the present study, we developed aptamers capable of specifically recognize ASC using the Cell-SELEX technique. We tested the affinity of ASC aptamers compared to dermal FIB. Quantitative PCR was advantageous for the in vitro validation of four candidate aptamers. The binding capabilities of Apta 2 and Apta 42 could not distinguish both cell types. At the same time, Apta 21 and Apta 99 showed a better binding capacity to ASC with dissociation constants (Kd) of 50.46 ± 2.28 nM and 72.71 ± 10.3 nM, respectively. However, Apta 21 showed a Kd of 86.78 ± 9.14 nM when incubated with FIB. Therefore, only Apta 99 showed specificity to detect ASC by total internal reflection microscopy (TIRF). This aptamer is a promising tool for the in vitro identification of ASC. These results will help understand the differences between these two cell types for more specific and precise cell therapies.

Assuntos

RESUMO

Human adipose-derived stromal/stem cells (ASC) have immunomodulatory properties and the potential to differentiate into several cell lines, important for application in regenerative medicine. However, the contamination with dermal fibroblasts (FIB) can impair the beneficial effects of ASC in cell therapy. It is then essential to develop new strategies that contribute to the distinction between these two cell types. In this study, we performed functional assays, high-throughput RNA sequencing (RNA-Seq) and quantitative PCR (qPCR) to find new markers that can distinguish ASC and FIB. We showed that ASC have adipogenic and osteogenic differentiation capacity and alkaline phosphatase activity, not observed in FIB. Gene expression variation analysis identified more than 2000 differentially expressed genes (DEG) between these two cell types. We validated 16 genes present in the list of DEG, including the alkaline phosphatase gene (ALPL). In conclusion, we showed that ASC and FIB have distinct biological properties as demonstrated by alkaline phosphatase activity and differentiation capacity, besides having different gene expression profiles. SIGNIFICANCE OF THE STUDY: Although many differences between stromal stem cells derived from human adipose tissue (ASC) and human dermal fibroblasts (FIB) are described, it is still difficult to find specific markers to differentiate them. This problem can interfere with the therapeutic use of ASC. This work aimed to find new markers to differentiate these two cell populations. Our findings suggest that these cells can be distinguished by biological and molecular characteristics, such as adipogenic and osteogenic differentiation, alkaline phosphatase activity and differential gene expression profiles. The DEG were related to the regulation of the cell cycle, development process, structural organization of the cell and synthesis of the extracellular matrix. This study helps to find new cellular markers to distinguish the two populations and to better understand the properties of these cells, which can improve cell therapy.

Assuntos

RESUMO

Two billion people are chronically infected with Toxoplasma gondii worldwide with unknown consequences. Important neurological diseases have been associated to the brain infection, making essential to understand the neurophysiological changes associated with the neuronal encystment. T. gondii may subvert neuronal functions modifying neurotransmitter concentration in chronically infected mice but the molecular mechanisms involved are still unclear. Parasites were observed inside neuronal cells in cultures from 24-192 hs. The rate of infection increased with time. Neurite density decreased affecting network functionality. Neuronal survival was affected and we detected the presence of cysts inside neuronal bodies and dilated portions of neurites in association with a relative increase of TH-positive neuritic area without noticeable changes in DA immunofluorescence pattern. These results advance our knowledge of the interaction between T. gondii and the neuronal network of the host.

Assuntos

RESUMO

O SAD português carateriza-se por um perfil tradicional. Reúne como principais caraterísticas funcionar de segunda a sexta-feira, com uma intensidade considerada baixa, com a prestação de cuidados voltadapara as AVD ́S, Atividades de vida diária dos idosos.Objetivos desteestudo: (i)identificar a oferta da estrutura de cuidados existentes,prestada pelo SAD, nas InstituiçõesParticulares de Solidariedade Social (IPSS)especificamentedo distrito de Braga (Portugal);e (ii) analisar seu perfil de funcionamento,com base nos comportamentos de gestãoe na existência ou não dos planos de cuidados no SAD.A metodologia utilizada foi a quantitativa, recorrendo-seà aplicação de inquéritos por questionário, por meiodo instrumento de avaliação,Protocolo de Serviço de Apoio Domiciliário, versão 2. A amostra foipor conveniência,composta pelos20 diretores técnicos das IPSS deBraga,Portugal.Como conclusões, oestudo aponta para um SAD tradicional, rural, com função assistencial, visando asatisfazer as AVD ́S, com declínio funcional, muito idosos,ou com idade inferior aos 65 anos,havendoa necessidade de completar a avaliação multidimensional realizada,e distribuir as tarefas executadas ainda centralizadas na figura do diretor técnico. São apontadas três prioridades:pensar numa boa gestão e rentabilização de recursos humanos; trabalhar os planos de cuidados com a área da saúde e a retaguarda familiar como principal enfoque;e uniformizar os critérios de exclusão e inclusão de acesso ao SAD,ostão importantes critérios de elegibilidade, dando lugar à implementação de um modelo de SAD que se baseienos cuidados pessoais individualizadoscom base na classificação do Rai Home Care.

The Portuguese SAD is characterized by a traditional profile. Its maincharacteristics are to operate from Monday to Friday, with a low intensity, with theprovision of care for ADLs, activities of daily living of the elderly. Objectives of this study:(i) to identify the provision of the existing care structure provided by SAD in the PrivateSocialSolidarity Institutions (IPSS) specifically in thedistrict of Braga (Portugal); and (ii) analyze itsoperating profile, based on managementbehaviors and the existence or otherwise ofcare plans in the DSS. The methodology usedwas quantitative, using surveys by means ofthe assessment instrument, Protocol of HomeSupport Service, version 2. The sample wasfor convenience, composed by the 20technical directors of Braga IPSS, Portugal. As conclusions, the study points to atraditional, rural DSS, with care function,aiming to satisfy the ADLs, with functionaldecline, very elderly, or under 65 years old,with the need to complete themultidimensional assessment performed, anddistribute the tasks performed still centered onthe technical director. Three priorities arepointed out: think about good managementand profitability of human resources; work withhealth care and family care plans as the mainfocus; and standardize the exclusion andinclusion criteria for access to SAD, the all-important eligibility criteria, leading to theimplementation of a model of SAD that isbased on individualized personal care basedon the Rai Home Care classification.

El SAD portugués se caracteriza por un perfil tradicional. Suscaracterísticas principales son operar de lunes a viernes, de baja intensidad, con laprestación de atención para las AVD, actividades de la vida cotidiana de los ancianos. Objetivos de este estudio: (i) identificar la provisión de la estructura de atención existenteproporcionada por SAD en las Instituciones Privadas de Solidaridad Social (IPSS)específicamente en el distrito de Braga (Portugal); y (ii) analizar su perfil operativo, enfunción de los comportamientos de gestión y la existencia o no de planes de atención en el DSS. La metodología utilizada fue cuantitativa, utilizando encuestas por medio delinstrumento de evaluación, Protocolo de Servicio de Apoyo a Domicilio, versión 2. La muestra fue por conveniencia, compuesta por los 20 directores técnicos de Braga IPSS,Portugal Como conclusiones, el estudio apunta a un DSS rural tradicional, con función asistencial, con el objetivo de satisfacer las AVD, con deterioro funcional, muy ancianoso menores de 65 años, con la necesidad de completar la evaluación multidimensionalrealizada, y distribuir las tareas aún en marcha centralizados en la figura del director técnico. Se identifican tres prioridades: pensar en una buena gestión y uso de los recursoshumanos; planes de trabajo de cuidar la salud y antecedentes familiares como el foco principal; y estandarizar los criterios de exclusión e inclusión para el acceso a SAD, loscriterios de elegibilidad más importantes, que conducen a la implementación de un modelo de SAD que se basa en el cuidado personal individualizado basado en laclasificación Rai Home Care.

Assuntos

RESUMO

Este estudo exploratório aborda o papel do Serviço de Apoio Domiciliário no quotidiano dos idosos e suas famílias, em função do contexto urbano e rural, e o percurso de vida dos utentes de dois SAD do norte de Portugal. Recorreu-se a uma metodologia qualitativa, com a aplicação de 11 entrevistas semiestruturadas. O SAD mantém uma função assistencialista/tradicional, tendo um papel de substituição em meio urbano e complementar em meio rural. Este estudo aponta para a necessidade de apostar numa lógica de prevenção, educação e reabilitação, em torno da criação de um plano de cuidados personalizado, baseado num sistema de classificação dos utentes.

This exploratory study deals with the role of the Domiciliary Support Service in the daily life of the elderly and their families, depending on the urban and rural context and the life course of the users of two SADs in the north of Portugal. A qualitative methodology was used, with the application of 11 semi-structured interviews. The SAD maintains a welfare / traditional function, having primarily a substitution role in an urban and complementary environment in rural areas. This study points to the need of betting on a logic of prevention, education and rehabilitation, around the creation of a personalized care plan, based on a classification system of the users.

Este estudio exploratorio aborda el papel de la atención domiciliaria de la vida cotidiana de las personas mayores y sus familias, de acuerdo con el contexto urbano y rural y el modo de vida de los usuarios del SAD dos norte de Portugal. Se recurrió a una metodología cualitativa, con la aplicación de 11 entrevistas semiestructuradas. El SAD mantiene una función asistencialista / tradicional, teniendo un papel de sustitución en medio urbano y complementario en medio rural. Este estudio apunta a la necesidad de apostar en una lógica de prevención, educación y rehabilitación, en torno a la creación de un plan de cuidados personalizado, basado en un sistema de clasificación de los usuarios.

Assuntos

RESUMO

Mesenchymal stem/stromal cells (MSCs) are promising tools in cell therapy. They secrete extracellular vesicles (EVs) that carry different classes of molecules that can promote skin repair, but the mechanisms are poorly understood. Skin wound healing is a complex process that requires the activity of several signaling pathways and cell types, including keratinocytes and fibroblasts. In this study, we explored whether adipose tissue MSC-derived EVs could accelerate migration and proliferation of keratinocytes and fibroblasts, activate the AKT pathway, and promote wound healing in vivo. Furthermore, we evaluated if EV effects are miR-205 dependent. We found that MSC EVs had an average diameter of 135 nm. Keratinocytes and fibroblasts exposed to EVs exhibited higher levels of proliferation, migration, and AKT activation. Topical administration of EVs accelerated skin wound closure. Knockdown of miR-205 decreased AKT phosphorylation in fibroblasts and keratinocytes, whereas migration was decreased only in keratinocytes. Moreover, knockdown of miR-205 failed to inhibit AKT phosphorylation in fibroblasts and keratinocytes exposed to EVs. About the mechanism of EV effects, we found that incubation with EVs prevented inhibition of AKT activation by miR-205 knockdown, suggesting that EVs activate AKT independently of miR-205. In conclusion, we demonstrated that EVs are a promising tool for wound healing.

RESUMO

Infections are a major complication in end-stage renal disease (ESRD) patients undergoing peritoneal dialysis (PD). Because the oral cavity may act as a source of systemic pathogens, some authors advocated specific measures when these patients are submitted to oral interventions, such as the administration of prophylactic antibiotics. Oral protozoa colonization may vary significantly with geographic distribution and to our knowledge no studies were performed in Portugal. The aim of the present study was to evaluate protozoa colonization in the saliva of ESRD patients undergoing PD and of their family members, living in the north of Portugal. Saliva was collected from 39 PD patients with a mean time on PD therapy of 12.7 - 15.9 months, and from 18 healthy volunteers (ESRD family members) for microscopic evaluation of protozoa by Lugols’ direct smear and specific staining techniques (Giemsa, Trichrome and Kinyoun). After the analysis of 456 smears obtained from 57 participants, only one PD patient (2.6%) presented an amoeba trophozoite in saliva. In conclusion, very low oral protozoa colonization was found, both on PD patients and family controls, suggesting that the oral protozoa colonization of Portuguese population is low and not significantly modified by the presence of end-stage chronic kidney disease. Further studies are required to address this issue.

Las infecciones son la principal complicación en pacientes renales del último estadio (ESRD) y que necesitan de diálisis del peritoneo (PD). Como la cavidad oral puede funcionar como una fuente de patógenos sistémicos, algunos autores indican medidas específicas cuando esos pacientes son sometidos a intervenciones orales, como la administración de antibióticos profilácticos. La colonización oral puede variar significativamente con la distribución geográfica. Según nuestros conocimientos, no han sido realizados estudios similares en Portugal. El principal objetivo fue evaluar la colonización de protozoos en saliva de pacientes ESRD del Norte de Portugal que hacían PD y, también, de sus familiares. Muestras de saliva fueron recogidas de 39 pacientes PD, con tiempo medio de terapia de PD de 12,7-15,9 meses y, también de 18 voluntarios saludables (familiares de ESRD). Las mismas utilizadas para evaluación microscópica de protozoos en laminas con lugol y tinciones especificas (Giemsa, Trichrome and Kinyoun). Después del análisis de 456 laminas, obtenidas de los 57 participantes, solamente en un paciente PD (2.6%) se observó un trofozoíto del ameba. En conclusión, se encontró una baja prevalencia de colonización oral de protozoos en el grupo estudiado. Así, la colonización oral de la población Portuguesa por protozoos es baja y no se cambia con la evolución de la enfermedad. Para mejor analizar esta situación, futuros estudios son necesarios.

RESUMO

Giardia duodenalis (syn. lamblia; syn. intestinalis) susceptibility testing is not routinely performed because the classical culture methods are very time-consuming and laborious. We developed a novel flow cytometry (FC) assay to evaluate the susceptibility of G. duodenalis trophozoites to metronidazole (MTZ). Different concentrations of MTZ were added to cultures of trophozoites (10 5 /mL) and the cultures were incubated for different periods. The 50% inhibitory concentration was calculated and propidium iodide (PI) was used to quantify the number of dead cells. After treatment, PI-positive trophozoites increased with increasing drug concentration and exposure time. An excellent correlation was found between FC and the classical method. A novel, accurate and reliable method is now available to evaluate G. duodenalis viability. .

Assuntos

RESUMO

Giardia duodenalis (syn. lamblia; syn. intestinalis) susceptibility testing is not routinely performed because the classical culture methods are very time-consuming and laborious. We developed a novel flow cytometry (FC) assay to evaluate the susceptibility of G. duodenalis trophozoites to metronidazole (MTZ). Different concentrations of MTZ were added to cultures of trophozoites (10 5 /mL) and the cultures were incubated for different periods. The 50% inhibitory concentration was calculated and propidium iodide (PI) was used to quantify the number of dead cells. After treatment, PI-positive trophozoites increased with increasing drug concentration and exposure time. An excellent correlation was found between FC and the classical method. A novel, accurate and reliable method is now available to evaluate G. duodenalis viability.

Assuntos

RESUMO

Es un hecho conocido que las infecciones oportunistas por protozoos y hongos han aumentado en los últimos años, debido especialmente al aumento de las infecciones por VIH. Cryptosporidium spp., Giardia lamblia y Encephalitozoon intestinalis son protozoos y hongo, respectivamente, mundialmente reconocidos como agentes oportunistas emergentes, responsables de brotes epidémicos provocados por la ingestión de agua potable contaminada, incluso después de una correcta desinfección. La ingestión de estos protozoos puede provocar diferentes grados de enfermedad, entre aguda o leve (población sana) hasta situaciones más graves y agresivas, hasta a veces mortales (pacientes inmunocomprometidos y/o inmunodeprimidos). A pesar de ser responsables de muchos brotes epidémicos, su diagnóstico de laboratorio permanece arduo y trabajoso, incluso utilizando las nuevas técnicas desarrolladas en los últimos años. En esta revisión se resumen las consideraciones generales de estos oportunistas emergentes, así como los métodos de diagnóstico más usuales, incluso los más recientes y específicos.

Epidemiological data, regarding parasitic and fungi opportunist infections, have changed in the last years, especially due to HIV infection. Cryptosporidium spp., Giardia lamblia and Encephalitozoon intestinalis are protozoan and fungi, respectively, worldwide known as opportunistic emergent agents, being responsible by epidemic outbreaks after ingestion of contaminated water, even following a correct disinfection treatment. Its ingestion can cause different effects on individuals’ health, from light or acute among the healthy population, to serious, aggressive or even deadly among the immunodepressed or immunocompromised patients. Contaminated water ingestion can result in outbreaks but protozoa laboratory diagnosis still remains very laborious, even after the development of more sensitive and specific techniques in the last years. In this paper, a revision of these emergent opportunists, their main characteristics and diagnostic tools are described, including the most recent and specific techniques.

Assuntos

RESUMO

Este trabalho teve como objetivo a construção de tábuas de mortalidade de inválidos dos segurados de clientela urbana do Regime Geral da Previdência Social. Assumindo que o número de mortes em cada idade segue uma distribuição de Poisson, as taxas de mortalidade por idade simples foram graduadas por meio de métodos estatísticos bayesianos, pelo modelo paramétrico de Gompertz-Makehan, utilizando inferência estatística bayesiana. Foram construídas tábuas de mortalidade para homens e mulheres e intervalos de credibilidade para os parâmetros e componentes do modelo, bem como para as taxas de mortalidade e funções da tábua. Uma aplicação foi feita calculando-se uma anuidade e o passivo atuarial.

Este trabajo tuvo como objetivo la construcción de tablas de mortalidad de inválidos procedentes de clientela urbana asegurada con el régimen general de la Seguridad Social. Asumiendo que el número de muertes en cada edad sigue una distribución de Poisson, las tasas de mortalidad por edad simple se graduaron mediante métodos estadísticos bayesianos, por el modelo paramétrico de Gompertz-Makehan, utilizando la inferencia estadística bayesiana. Se construyeron tablas de mortalidad para hombres y mujeres e intervalos de credibilidad para los parámetros y componentes del modelo, así como para las tasas de mortalidad y funciones de la tabla. Una aplicación se realizó calculando una anualidad y el pasivo actuarial.

The objective of this article is to construct life tables of invalids among urban beneficiaries of the General Regime of the National Brazilian Social Security System. Assuming that the number of deaths in each table follows a Poisson distribution, the death rates by simple age were graduated through Bayesian statistical methods, by using the Gompertz-Makehan parametric model and using Bayesian statistical inferences. Life tables were constructed for death rates of men and women, and credibility intervals were set up for the parameters and components of the model, as well as for the death rates and functions of the board. An application was made by calculating an annuity and the actuarial liabilities.

Assuntos