Extracellular vesicles in Alzheimer's disease / Vesículas extracelulares na doença de Alzheimer

Abstract Extracellular vesicles (EVs) are small vesicles released by cells that facilitate cell signaling. They are categorized based on their biogenesis and size. In the context of the central nervous system (CNS), EVs have been extensively studied for their role in both normal physiological functions and diseases like Alzheimer's disease (AD). AD is a neurodegenerative disorder characterized by cognitive decline and neuronal death. EVs have emerged as potential biomarkers for AD due to their involvement in disease progression. Specifically, EVs derived from neurons, astrocytes, and neuron precursor cells exhibit changes in quantity and composition in AD. Neuron-derived EVs have been found to contain key proteins associated with AD pathology, such as amyloid beta (Aß) and tau. Increased levels of Aß in neuron-derived EVs isolated from the plasma have been observed in individuals with AD and mild cognitive impairment, suggesting their potential as early biomarkers. However, the analysis of tau in neuron-derived EVs is still inconclusive. In addition to Aß and tau, neuron-derived EVs also carry other proteins linked to AD, including synaptic proteins. These findings indicate that EVs could serve as biomarkers for AD, particularly for early diagnosis and disease monitoring. However, further research is required to validate their use and explore potential therapeutic applications. To summarize, EVs are small vesicles involved in cell signaling within the CNS. They hold promise as biomarkers for AD, potentially enabling early diagnosis and monitoring of disease progression. Ongoing research aims to refine their use as biomarkers and uncover additional therapeutic applications.

Resumo As vesículas extracelulares (VEs) são pequenas estruturas liberadas pelas células que agem na sinalização celular. No sistema nervoso central (SNC), as VEs são estudadas em relação à doença de Alzheimer (DA), um distúrbio neurodegenerativo que cursa com declínio cognitivo e morte neuronal. As VEs podem ser biomarcadores potenciais para a DA devido ao seu papel na progressão da doença. As VEs derivadas de neurônios, astrócitos e células precursoras apresentam alterações na DA, contendo proteínas associadas à patologia da DA, como beta-amiloide (Aß) e tau. Níveis elevados de Aß foram observados nas VEs de neurônios de indivíduos com DA, sugerindo seu potencial como biomarcadores precoces. A análise de tau nas VEs de neurônios ainda é inconclusiva. Além disso, as VEs neurais carregam outras proteínas relacionadas à DA, incluindo proteínas sinápticas. As VEs podem ser promissoras como biomarcadores para o diagnóstico precoce e monitoramento da DA, porém mais pesquisas são necessárias para validar seu uso e explorar aplicações terapêuticas. Em resumo, as VEs são vesículas envolvidas na sinalização celular no SNC, com potencial como biomarcadores para a DA.

Campylobacter fetus releases S-layered and immunoreactive outer membrane vesicles

Abstract The study of outer membrane vesicles (OMVs) became relevant because of theirprobable important role in the transfer of virulence factors to host cells. Campylobacter fetusis mainly a mammal pathogen whose virulence characterization is still limited. The aim of thisstudy was to evaluate and to characterize the secretion of OMVs in this bacterium. By trans-mission electron microscopy, we confirmed the production of OMVs in all the strains assayed.Purified OMVs showed a spherical shape and variable size, although comparable to those ofother gram-negative bacteria. We also confirmed the presence of the S-layer on the surface ofthe OMVs of all the strains assayed with the exception of those derived from the NTCC referencestrain. In addition, we demonstrated their immunoreactivity by the dot-blot assay. Hence, C.fetus OMVs could contribute to the modulation of the host response and constitute a candidateto be evaluated as an adjuvant of current vaccines used in the veterinary field. This work rep-resents a platform to drive future studies towards the role of these subcellular structures in C.fetus-host interaction.

Resumen El estudio de las vesículas de membrana externa (VME) tomó un rol protagónico, yaque se las ha relacionado con la transferencia de factores de virulencia a la célula hospedadora.Campylobacter fetus es, principalmente, un patógeno de mamíferos cuya virulencia solo hasido caracterizada de forma limitada. El objetivo de este trabajo fue evaluar y caracterizar la secreción de VME en esta bacteria. Mediante microscopía electrónica de transmisión confir-mamos la producción espontánea de VME en todas las cepas estudiadas. Las VME purificadasmostraron una morfología esférica y un tama˜no variable, pero compatible con el reporte deotras bacterias gram negativas. Asimismo, hemos demostrado que estas vesículas conservanla capa S en todas las cepas, menos en la cepa de referencia NCTC y hemos confirmado suinmunorreactividad por dot-blot inmunoblot. Estas VME de C. fetus podrían contribuir a la mod-ulación de la respuesta del hospedador y constituir un buen candidato como adyuvante de lasactuales vacunas empleadas en el campo veterinario. Este trabajo representa una plataformapara impulsar estudios futuros en torno al rol de estas estructuras subcelulares en la interfaseC. fetus-hospedador.

Papel de los exosomas en la angiogénesis, revascularización y respuesta inmune / Role of exosomes in angiogenesis, revascularization and immune response

Introducción: Los exosomas son vesículas extracelulares de tamaño nanométrico, que se generan cuando los endosomas multivesiculares se fusionan con la membrana plasmática y el contenido de las vesículas intraluminales se libera en el espacio extracelular. Son producidos por casi todos los tipos de células, en condiciones fisiológicas y patológicas. Transportan proteínas, lípidos y ácido ribonucleico (ARN) no codificante, desde la célula madre hasta la célula receptora, estos son considerados un punto clave en la regeneración de tejidos, lo que se ha demostrado en una serie de estudios, con diferentes tejidos corporales, como piel, cartílago, pancreático y tejidos cardiovasculares. Objetivo: Explicar los aspectos generales y posibles usos de los exosomas en el campo médico. Métodos: Se realizó una búsqueda de información mediante consulta en las bases de datos SciELO PubMed, Science Direct y Lilacs, en los idiomas español e inglés, con diferentes combinaciones de palabras claves y términos MESH como: exosomes, neovascularization, wound healing, immunity, micro RNA, immunology, therapy, classification. Se efectuó un análisis y resumen de la información revisada. Conclusiones: En la actualidad, los exosomas se han convertido en objeto de investigación para diversos tratamientos, medicamentos y uso como marcadores moleculares. Se destacan en terapias contra el cáncer, la inmunomodulación, la estimulación o supresión de la angiogénesis, regeneración cutánea, cicatrización y curación de heridas; por lo que de forma general resultan prometedores en el ámbito de las ciencias médicas(AU)

Introduction: Exosomes are nano-sized extracellular vesicles, which are generated when multivesicular endosomes fuse with the plasma membrane and the content of intraluminal vesicles released into the extracellular space. Are produced by almost all types of cells, under physiological and pathological conditions and they transport proteins, lipids and non-coding RNA (ribonucleic acid), from the stem cell to the recipient cell, these are considered a key point in tissue regeneration, which has been shown in a series of studies, with different body tissues, such as skin, cartilage, pancreatic and cardiovascular tissues. Objective: To explain the general aspects and possible uses of exosomes in the medical field. Methods: A search for information was carried out by consulting the Scielo, PubMed, ScienceDirect and Lilacs databases, in Spanish and English, with different combinations of keywords and MESH terms such as: exosomes, neovascularization, wound healing, immunity, microRNA, immunology, therapy, classification. Then, an analysis and summary of the reviewed information was carried out. Conclusions: Currently, exosomes have become the object of research for various treatments, drugs, and their use as molecular markers. They stand out in cancer therapies, immunomodulation, stimulation or suppression of angiogenesis, skin regeneration, and wound healing, which is why they are generally promising in the field of medical sciences(AU)

Exosomes and cystic echinococcosis: systematic review / Exosomas y equinococosis quística: revisión sistemática

SUMMARY: Exosomes are small and single-membrane secreted organelles, up to 200 nm in diameter that have the same topology as the cell, but are enriched in proteins, nucleic acids, lipids, and glycoconjugates. It can be found in any type of body fluids such as plasma, urine, saliva, sperm, bile, etc. On the other hand, cystic Echinococcosis (CE) has been studied from different points of view, among others, from genomics and proteomics, and the presence of CE exosomes in humans and other intermediate hosts has been reported in very few articles. The aim of this review was to report the evidence available regarding exosomes and CE. Systematic review. Data sources: Trip Database, SciELO, WoS, MEDLINE, EMBASE and SCOPUS. Eligibility criteria: Studies related to CEin any type of host and state of the parasite, without language restriction, published between 1966-2021. Variables: Year of publication, geographical origin, species isolated, location of exosomes. Forty-two studies were initially identified. After scrutinizing titles and abstracts, checking duplications and in-depth analysis of the studies selected, 12 articles including human, bovine, sheep, dog samples were also included. All were case reports or case series. The highest proportion of articles was published between 2019 and 2021 (58.3 %). Publications were predominantly from China (58.3 %). Evidence about exosomes and CE is scarce and reduced range of articles and cases.

RESUMEN: Los exosomas son orgánulos pequeños y secretados por una sola membrana, de hasta 200 nm de diámetro que tienen la misma topología que la célula, pero están enriquecidos en proteínas, ácidos nucleicos, lípidos y glicoconjugados. Se puede encontrar en cualquier tipo de fluidos corporales como plasma, orina, saliva, esperma, bilis, etc. Por otro lado, la equinococosis quística (CE) ha sido estudiada desde diferentes puntos de vista, entre otros, desde la genómica y proteómica, y la presencia de exosomas de CE en humanos y otros huéspedes intermediarios se ha informado en muy pocos artículos. El objetivo de esta revisión fue informar la evidencia disponible con respecto a los exosomas y la CE. Revisión sistemática. Fuentes de datos: Trip Database, SciELO, WoS, MEDLINE, EMBASE y SCOPUS. Criterios de elegibilidad: Estudios relacionados con la EC en cualquier tipo de hospedador y estado del parásito, sin restricción de idioma, publicados entre 1966-2021. Variables: año de publicación, origen geográfico, especie aislada, ubicación de exosomas. Se identificaron inicialmente 42 estudios. Después de examinar los títulos y resúmenes, verificar las duplicaciones y analizar en profundidad los estudios seleccionados, se incluyeron 12 artículos que incluían muestras de humanos, bovinos, ovinos y caninos. Todos fueron informes de casos o series de casos. La mayor proporción de artículos se publicó entre 2019 y 2021 (58,3 %). Las publicaciones fueron predominantemente de China (58,3 %). La evidencia sobre exosomas y CE es escasa y la gama de artículos y casos es reducida.

Retos y oportunidades en el estudio de vesículas extracelulares: contexto institucional a nivel mundial y situación actual en Colombia / Challenges and opportunities in the study of extracellular vesicles: Global institutional context and national state of the art

Resumen En la última década se ha incrementado el número de estudios y publicaciones sobre las vesículas extracelulares y los exosomas. En Colombia, ha habido interés y avances en su estudio, lo que se evidencia en el aumento de publicaciones y proyectos de investigación. Sin embargo, este es un campo de investigación aún en desarrollo, con desafíos analíticos y limitaciones técnicas, por lo cual, en el planteamiento de los proyectos de investigación y desarrollo, es necesario considerar cuál es el estado del campo científico a nivel mundial en cuanto a la nomenclatura y la clasificación de las vesículas extracelulares, las técnicas, recursos, requisitos y especificaciones de calidad y las instituciones que regulan el campo. La respuesta a esta pregunta permitirá desarrollar estudios que cumplan con los estándares internacionales, y las exigencias y recomendaciones institucionales. Sin embargo, la información científica disponible se encuentra dispersa y no todos los aspectos son tratados a cabalidad. En este actualización se condensa la información disponible y se presentan los términos oficiales para denominar las vesículas extracelulares y la nomenclatura aceptada actualmente, así como la evolución del campo, la homogenización de los parámetros experimentales, el establecimiento de autoridades científicas, instituciones y recursos, y las recomendaciones que se han generado a nivel mundial para el desarrollo de investigaciones en vesículas extracelulares, incluidos su aislamiento, caracterización y estudio funcional. Por último, se analiza el contexto nacional de una forma crítica, teniendo en cuenta las fortalezas institucionales, los errores usualmente cometidos, y las técnicas y tecnologías analíticas disponibles.

Abstract In the last decade, the number of studies and publications on extracellular vesicles (EV) and exosomes has boomed. Colombia has displayed interest and progress in their study as shown in the increase of research project publications and products. However, this research field is still developing and has its own analytical challenges and technical limitations. For planning research projects and developing EV studies it is necessary to consider what is the state of the scientific field worldwide concerning EV nomenclature and classification, available techniques, resources, requirements and quality specifications, and the institutions that regulate the field. Answering this question will elicit EV studies that comply with international standards and respond to institutional demands and recommendations. However, the scientific information available is scattered and not all the aspects are considered in full. In this update, the available information is condensed and the official terms and currently defined nomenclature is presented, as well as the evolution of the field, the homogenization of the experimental parameters, the establishment of scientific authorities, institutions, and resources, and the recommendations generated worldwide for their development and research including their isolation, characterization, and functional studies. Finally, I analyzed the national context in a critical way, considering institutional strengths, common mistakes, and available analytical techniques and technologies.

Bases inmunológicas de la enfermedad neumocócica y el candidato vacunal PVC7-TT / Immunological bases of pneumococcal disease and vaccine candidate PVC7-TT

Introducción: Las enfermedades infecciosas del tracto respiratorio se encuentran entre las primeras causas de entidades respiratorias en edades extremas de la vida. Objetivo: Describir las bases inmunológicas de la enfermedad y el nuevo candidato vacunal conjugado antineumocócico PCV7-TT desarrollado en Cuba. Métodos: Se realizó una búsqueda en las bases de datos Medline, Pubmed, SciELO, LILACS, Cochrane Library y Web of Science, de documentos publicados entre mayo del 2018 y marzo del 2020. Se seleccionaron los 64 artículos de mayor relevancia y novedad. Resultados: Streptococcus pneumoniae es el agente etiológico de la enfermedad neumocócica; se le atribuye alrededor de un millón de defunciones anuales, principalmente en países en vías de desarrollo. Es un coco Gram-positivo, anaerobio facultativo y encapsulado que se encuentra dividido en 48 serogrupos y 97 serotipos tipificados. Presenta varios factores de virulencia que garantizan su mecanismo de patogenicidad; uno de los más importantes es el polisacárido capsular que constituye la diana de las vacunas antineumocócicas conjugadas y no conjugadas existentes. En el presente artículo se consideró la proteína de superficie C del neumococo como un posible candidato en la investigación y desarrollo de vacunas preventivas. Asimismo, las vesículas extracelulares podría ser un posible candidato para adyuvante vacunal con fines preventivos y terapéuticos. Conclusiones: El neumococo es un problema de salud a nivel global y el uso de vacunas conjugadas antineumocócicas constituye la herramienta más eficaz para su prevención. El candidato vacunal PCV7-TT desarrollado en Cuba es seguro, bien tolerado, inmunogénico y no inferior a las vacunas actualmente registradas(AU)

Introduction: Infectious diseases of the respiratory tract are among the leading causes of respiratory conditions in patients at extreme ages. Objective: Describe the immunological bases of the disease and the new conjugate pneumococcal vaccine candidate PCV7-TT developed in Cuba. Methods: A search was conducted in the databases Medline, Pubmed, SciELO, LILACS, Cochrane Library and Web of Science for documents published from May 2018 to March 2020. The 64 most relevant and novel papers were selected. Results: Streptococcus pneumoniae is the causative agent of pneumococcal disease, a condition causing about one million deaths a year worldwide, mainly in developing countries. It is a Gram-positive facultative anaerobic encapsulated coccus divided into 48 serogroups and 97 typified serotypes. Several virulence factors ensure its pathogenicity mechanism. One of the most important of these is the capsular polysaccharide constituting the target of the existing conjugate and non-conjugate pneumococcal vaccines. The study considered pneumococcal surface protein C as a possible candidate for the research and development of preventive vaccines. On the other hand, extracellular vesicles could be a possible vaccine adjuvant candidate for preventive and therapeutic use. Conclusions: Pneumococcus is a global health problem, and the use of conjugate pneumococcal vaccines is the most effective tool for its prevention. The vaccine candidate PCV7-TT developed in Cuba is safe, well-tolerated, immunogenic and not inferior to the vaccines so far registered(AU)

Gingivoestomatitis herpética severa en paciente con cardiopatía congénita; reporte de caso clínico / Gengivoestomatite herpética grave em paciente com cardiopatia congênita; relato de caso clínico / Severe herpetic gingivostomatitis in a patient with congenital heart disease; clinical case report

La gingivoestomatitis herpética corres-ponde a la manifestación primaria de la infección por virus herpes simple tipo I que se presenta con mayor frecuencia en lactantes mayores y preescolares. Objeti-vo: describir abordaje y manejo de gingi-vo estomatitis herpética en una paciente con cardiopatía congénita y cuadro de desnutrición severa. El caso se refiere a paciente femenina de 16 meses de edad a quien le fue realizado anamnesis, exa-men clínicoe interconsultas con servicios de pediatría, patología, medicina bucal, infectología. El diagnóstico incluyó co-municación intraventricular, desnutrición severa y gingivoestomatitis herpética. Se realizó tratamiento paliativo para el dolor, terapia antiviral (aciclovir en suspensión 1cc cada 6 horas por 7 días) y tratamiento tópico (sucralfato en suspensión 1g/5ml mezclado en partes iguales con cetirizina en suspensión. 5 mg / 5 ml, 3 veces al día directamente sobre las lesiones) durante 14 días logrando reducción de la sintoma-tologia. Conclusiones: el correcto manejo multidisciplinario permitió lograr dismi-nución en tamaño y número de las lesio-nes en cavidad oral, orientación dietética y canalización apropiada.

A gengivoestomatite herpética correspon-de à manifestação primária da infecção pelo vírus herpes simplex tipo I, que ocorre com mais frequência em bebês e em idade pré-escolar. Objetivo: descrever a aborda-gem e o tratamento da estomatite herpética gengival em um paciente com cardiopatia congênita e desnutrição grave. O caso re-fere-se a uma paciente de 16 meses de ida-de, submetida a anamnese, exame clínico e interconsultas com serviços de pediatria, patologia, medicina bucal, infectologia. O diagnóstico incluiu comunicação intraven-tricular, desnutrição grave e gengivosto-matite herpética. Foram realizados trata-mento paliativo para dor, terapia antiviral (suspensão de aciclovir 1cc a cada 6 horas por 7 dias) e tratamento tópico (suspensão de sucralfato 1g / 5ml misturado em partes iguais com suspensão de cetirizina 5mg / 5ml, 3 vezes ao dia diretamente. lesões) por 14 dias, alcançando redução dos sintomas. Conclusões: o correto manejo multidisci-plinar permitiu diminuir o tamanho e o número de lesões na cavidade oral, orien-tação alimentar e canalização adequada.

Herpetic gingivostomatitis is the pri-mary manifestation of herpes simplex virus type I infection, common in older infants and preschoolers. Objective: to describe the approach and management of herpetic stomatitis in a patient with congenital heart disease and severe mal-nutrition. The case refers to a 16-mon-th-old female patient who underwent an anamnesis, clinical examination, and inter-consultations with pediatric, pa-thological, oral medicine services, and Diagnosis included intraventricular communication, severe malnutrition, and herpetic gingivostomatitis. Palliati-ve treatment for pain, antiviral therapy (acyclovir suspension 1cc every 6 hours for 7 days) and topical treatment (sucral-fate suspension 1g / 5ml mixed in equal parts with cetirizine suspension 5mg / 5ml, 3 times a day directly, were perfor-med. about injuries) for 14 days. Conclu-sions: multidisciplinary, management, allowed to obtain, clinical diagnosis and establish a treatment plan with positive outcome,decreasing oral cavity dietary guidance and appropriate channeling.

Exosomas: terapia celular regenerativa libre de células / Exosomes: "cell-free" regenerative cell therapy

La terapia celular basada en células mesenquimales/estromales se aplica ampliamente en la medicina moderna, aun cuando no todos los mecanismos de supervivencia y diferenciación están identificados. Sin embargo, hace pocos años se comenzaron a encontrar elementos extracelulares que generan nuevos paradigmas. En el presente trabajo se explican las principales características y funciones atribuidas a los exosomas, nanopartículas constituidas por microvesículas secretadas por las células con efecto en la matriz extracelular, y su repercusión como alternativa hacia una medicina regenerativa libre de células. Estas estructuras participan de forma notoria y crucial en la comunicación intercelular, lo que ha supuesto un cambio en el concepto de las funciones y el papel que desempeñan estas vesículas en los organismos vivos, en particular en la restauración de tejidos dañados y la respuesta inflamatoria e inmunológica. Se comentan algunos ejemplos de la repercusión biotecnológica de los exosomas en empresas y el mercado biofarmaceútico(AU)

Mesenchymal/stromal cell ;based therapy is widely applied in modern medicine, even though not all survival and differentiation mechanisms are identified. However, a few years ago, extracellular elements began to be found that generate new paradigms. The present work explains the main characteristics and functions attributed to exosomes, nanoparticles made up of microvesicles secreted by with an effect on the extracellular matrix, and their impact as an alternative towards cell-free regenerative medicine. These structures participate, notoriously and critically, in intercellular communication, which has led to a change in the concept of the functions and role that these vesicles play within living organisms, particularly in the restoration of damaged tissues and the inflammatory and immunological response. Some examples of the exosomes' biotechnological impact on companies and the biopharmaceutical market are discussed(AU)

Vesicles stage of camel brain development / Etapas del desarrollo de las vesículas cerebrales del camello

A successive embryonic developmental study was conducted on the brain of twenty eight embryos and fetuses of one humped camel (Camelus Dromedarius), whose crown vertebral rump lengths (CVRL) ranged from 9 to 80 mm, collected from the El-Basateen (Cairo) and Belbees (ElSharqya) Slaughterhouse. The current investigation revealed that camel brain was found to consist of fore, mid and hind brains. The fore brain is divided into telencephalon and diencephalon while the rhombencephalon divided into metencephalon and myelencephalon. Flexures appeared between the vesicles are cervical flexure between the rhomencephalon and the spinal cord, cephalic flexure in the mesencephalon and pontine flexure between the metencephalon, and the myelencephalon of the hind brain (rhombencephalon). The cavity of the rhombencephalon is the fourth ventricle, while that of the diencephalon is the third ventricle, and those of the telencephalon are the lateral ventricles but that of mid brain is the cerebral aqueduct. myelencephalon becomes medulla oblongata and metencephalon developed to pons and cerebellum while mesencephalon gives rise to the cerebral crura and anterior and a posterior colliculus. Diencephalon gives the thalamus, hypothalamus, mamillary body, infundibulum and pineal body while telencephalon becomes the cerebral hemispheres and corpus striatum.

Se llevó a cabo un estudio del desarrollo embrionario cerebral de veintiocho embriones y fetos de camello jorobado (Camelus dromedarius). Las muestras fueron recolectadas en los mataderos de El-Basateen (El Cairo) y Belbees (ElSharqya). La investigación reveló que el cerebro de camello posee un cerebro anterior, medio y posterior. El cerebro anterior se divide en telencéfalo y diencéfalo, mientras que el rombencéfalo se divide en metencéfalo y mielencéfalo. Las flexiones encontradas entre las vesículas son la flexión cervical entre el rombencéfalo y la médula espinal; la flexión cefálica en el mesencéfalo; y la flexión pontina entre el metencéfalo y el mielencéfalo del cerebro posterior (rombencéfalo). La cavidad del rombencéfalo conforma el cuarto ventrículo, la del diencéfalo forma el tercer ventrículo, y las del telencéfalo a los ventrículos laterales. En el cerebro medio, la cavidad corresponde al acueducto cerebral. El mielencéfalo se convierte en médula oblonga y el metencéfalo deriva en puente y cerebelo, mientras que el mesencéfalo da lugar a la crura cerebral y a los colículos anterior y posterior. El diencéfalo origina el tálamo, el hipotálamo, el cuerpo mamilar, el infundíbulo y la hipófisis, mientras que del telencéfalo se originan los hemisferios cerebrales y el cuerpo estriado.

Enfermedad boca-mano-pie atípica infantil con rasgos de eczema herpetico y de acrodermatitis / Infantile atypical hand-foot-mouth disease with features of eczema herpeticum and acrodermatitis

La enfermedad boca-mano-pie puede presentarse con formas atípicas, con lesiones más generalizadas y morfología diferente a la forma clásica. A veces, incluso simula otras enfermedades víricas. En las formas atípicas, existe la tendencia a afectar a las zonas de traumatismo o inflamación. Se presenta el caso de un niño de 2 años con antecedente de atopia, que consultó por presentar lesiones papulosas y vesiculosas umbilicadas que afectaban a la zona perioral, los miembros superiores e inferiores, con predominio en las zonas de presión y de dermatitis atópica previa. Aunque clínicamente se diagnosticó eczema herpético, las pruebas complementarias fueron negativas para herpes virus. Sin embargo, la reacción en cadena de la polimerasa del contenido de una vesícula, del exudado faríngeo y de heces fue positiva para enterovirus.

Hand-foot-mouth disease can present atypically, including forms with more numerous lesions and/or morphologically different from the classic presentation. It may even mimic other viral diseases. We present the case of a 2-year-old child previously diagnosed with atopic dermatitis, who presented with papules and umbilicated vesicles affecting the perioral area and limbs, predominantly in pressure areas, as well as in areas with previous atopic lesions. Although he was clinically diagnosed with herpetic eczema, tests results were negative for herpes virus. However, positive entorovirus polymerase chain reaction results were obtained from the content of a vesicle, a pharyngeal exudate and a stool sample.

Purificação e expressão de vesículas extracelulares humanas e a correlação com a toxoplasmose / Purification and expression of human extracellular vesicles and the correlation with toxoplasmosis

O objetivo deste estudo foi isolar e caracterizar as vesículas extracelulares (EVs) humanas e compará-las entre grupos de indivíduos com ou sem toxoplasmose, uma zoonose de distribuição mundial e de importância na saúde pública por seu papel na gestação e na co-infecção com o vírus da imunodeficiência humana (HIV). Oitenta e três amostras clínicas foram divididas em: grupo I, 20 plasmas de indivíduos saudáveis e gestantes (soronegativos para toxoplasmose); grupo II, 21 plasmas de pacientes com toxoplasmose sintomática; grupo III, 26 amostras de líquido cefalorraquidiano (LCR) de pacientes com toxoplasmose cerebral e co-infecção por HIV (TC/aids); e grupo IV, 16 amostras de LCR de pacientes sem toxoplasmose, mas com aids e outras infecções oportunistas (OI/aids). Após a recuperação das EVs por ultracentrifugação as amostras seguiram para análise de tamanho e concentração por Análise de Rastreamento de Nanopartícupas (NTA); morfologia, por Microscopia Eletrônica de Transmissão (TEM); perfil proteico, por SDS-PAGE/Imunoblot e expressão gênica de microRNAs (miRNAs), por PCR em tempo real (qPCR). A concentração das EVs-plasma do grupo II (2,4x1010 EVs/mL) foi estatisticamente maior do que as do grupo I (5,9x109 EVs/mL). Já nas EVs-LCR a concentração foi semelhante nos dois grupos, TC/aids (2,9x109 EVs/mL) e OI/aids (4,8x109 EVs/mL). Apenas a concentração das EVs foi capaz de distinguir os pacientes com...(AU)

This study aimed to isolate and characterize EVs and compare them between groups of individuals with or without toxoplasmosis, a worldwide distribution zoonosis, that plays an important role during pregnancy and co-infection with the human immunodeficiency virus (HIV). Eighty-three samples was divided as: group I, 20 plasma of health individuals and pregnant women (negatives for toxoplasmosis); group II, 21 plasma of toxoplasmosis patients; group III, 26 samples of cerebrospinal fluid (CSF) of patients with cerebral toxoplasmosis/aids (TC/AIDS) e group IV, 16 samples of CSF of patients without toxoplasmosis, but with AIDS and others opportunistic infections (OI/AIDS). After EV isolation by ultracentrifugation, size and concentration of were measured by Nano Tracking Analisys (NTA). In addition, morphology by Transmission Electronic Microscopy (TEM), protein profile by SDS-PAGE/Imunoblot and miRNAs expression, by Real time PCR (qPCR) were performed. Concentration of plasma EVs from group II patients (2.4x1010 EVs/mL) was statistically higher than those from group I (5.9x109 EVs/mL). Concentration od CSF EVs was similar in both groups, TC/AIDS (2.9x109 EVs/mL) and OI/AIDS (4,8x109 EVs/mL). Therefore, only the concentration was able to distinguish patients with toxoplasmosis than health individuals. Exosomes were also confirmed by TEM and CD-63 and CD-9 tetraspanina detection by immunoblot. EVs-plasma from group II had higher expression of miR-125b (4.9 times) and miR-146a (4.1 times) from control group...(AU)

Estandarización de un ELISA para la cuantificación de anticuerpos IgG contra vesículas de membrana externa de Salmonella Paratyphi A / Standardization of an ELISA for the quantification of IgG antibodies against outer membrane vesicle of Salmonella Paratyphi A

Salmonella Paratyphi A es un patógeno exclusivo de humanos, siendo la segunda causa más común de fiebre entérica en el sudeste asiático. Recientemente la incidencia en este continente ha aumentado, desplazando a Salmonella entérica serotipo Typhi como la primera causa de fiebre entérica. En la actualidad no existen vacunas licenciadas contra S. Paratyphi A. El Instituto Finlay de Vacunas se encuentra trabajando en la obtención de un candidato vacunal basado en vesículas de membrana externa (VME) contra S. Paratyphi A, por lo que se hizo necesario contar con una técnica para la evaluación de su inmunogenicidad. El objetivo de este trabajo fue la estandarización de un ELISA para la cuantificación de anticuerpos IgG contra VME de S. Paratyphi A. Para ello, se determinaron las mejores condiciones de este ensayo en cuanto a concentración óptima de recubrimiento y dilución de trabajo del conjugado. Además, se definió el intervalo y linealidad de la curva, la precisión intra e interensayo, la especificidad y el límite de detección. La curva de calibración se generó con un suero estándar interno y presentó un buen ajuste lineal con un R² =0.98. Los coeficientes de variación en los ensayos de precisión intra e interensayo estuvieron en los intervalos establecidos para cada uno (=10 por ciento, =20 por ciento respectivamente). Los resultados obtenidos avalan el empleo de este ELISA cuantitativo para la evaluación de la inmunogenicidad de formulaciones de VME de S. Paratyphi A en fases de investigación y desarrollo(AU)

Salmonella Paratyphi A, is an exclusive pathogen of humans, being the second most common cause of enteric fever in Southeast Asia. Recently the incidence of this disease in this continent has increased, displacing Salmonella enterica serotype Typhi as the first cause of enteric fever. Currently there are no vaccines licensed against S. Paratyphi A. The Finlay Institute of Vaccines is working on obtaining a vaccine candidate based on outer membrane vesicles (VME) against S. Paratyphi A, so it became necessary to develop a technique for the evaluation of its immunogenicity. The objective of this work was the standardization of an ELISA for the quantification of IgG antibodies against VME of S. Paratyphi A. The best conditions of this assay were determined in terms of optimum concentration of coating and working dilution of the conjugate. In addition, the interval and linearity of the curve, the intra- and inter-assay precision, the specificity and the limit of detection were defined. The calibration curve was generated with an internal standard serum and presented a good linear fit with an R² =0.98. The coefficients of variation in the intra- and interassay precision tests were in the intervals established for each one (=10 percent, =20 percent respectively). The results obtained support the use of this quantitative ELISA for the evaluation of the immunogenicity of VME formulations of S. Paratyphi A in research and development phases(AU)

Methanolic extract of Rhinella schneideri (Anura: Bufonidae) poison causes ultrastructural changes in nerve terminals of phrenic nerve-diaphragm preparations in mice / Extracto metanólico de Rhinella schneideri (Anura: Bufonidae) causa cambios ultraestructurales en las terminaciones nerviosas de preparaciones de nervio frénico de ratón

Abstract Rhinella schneideri (or Bufo paracnemis), popularly known in Brazil as cururu toad, is also found in other countries in South America. The cardiovascular effects of this poison are largely known and recently was shown that it is capable to affect the neuromuscular junction on avian and mice isolated preparation. In this work, we used transmission electron microscopy to investigate the ultrastructure of the motor nerve terminal and postsynaptic junctional folds of phrenic nerve-hemidiaphragm preparations incubated for either 5 or 60 min with the methanolic extract of R. schneideri (50 µg/mL). In addition, the status of the acetylcholine receptors (AChR) was examined by TRITC-α-bungarotoxin immunofluorescence location at the endplate membrane. The results show that 5 min of incubation with the gland secretion extract significantly decreased (32 %) the number of synaptic vesicles into the motor nerve terminal, but did not decrease the electron density on the top of the junctional folds where nicotinic receptors are concentrated; however, 60 min of incubation led to significant nerve terminal reloading in synaptic vesicles whereas the AChR immunoreactivity was not as marked as in control and after 5 min incubation. Muscle fibers were well-preserved but intramuscular motor axons were not. The findings corroborated pharmacological data since the decrease in the number of synaptic vesicles (5 min) followed by recovery (60 min) is in accordance with the transient increase of MEPPs frequency meaning increased neurotransmitter release. These data support the predominant presynaptic mode of action of the R. schneideri, but do not exclude the possibility of a secondary postsynaptic action depending on the time the preparation is exposed to poison. Rev. Biol. Trop. 66(3): 1290-1297. Epub 2018 September 01.

Resumen Rhinella schneideri (o Bufo paracnemis), conocido popularmente en Brasil como sapo cururu, también se encuentra en otros países de América del Sur. Los efectos cardiovasculares de este veneno son ampliamente conocidos y recientemente se demostró que es capaz de afectar la unión neuromuscular en la preparación aislada de aves y ratones. En este trabajo, utilizamos microscopía electrónica de transmisión para investigar la ultraestructura de la terminación nerviosa motora y pliegues de unión postsináptica de preparaciones de nervio frénico-hemidiafragma incubadas durante 5 o 60 min con el extracto metanólico de R. schneideri (50 μg/mL). Además, se examinó el estado de los receptores de acetilcolina (AChR) mediante la ubicación de inmunofluorescencia de TRITC-α-bungarotoxina en la membrana de la placa terminal. Los resultados muestran que 5 min de incubación con el extracto de secreción de glándula disminuyeron significativamente (32 %) el número de vesículas sinápticas en el terminal del nervio motor, pero no disminuyeron la densidad electrónica en la parte superior de los pliegues de unión donde se concentran los receptores nicotínicos. Sin embargo, 60 min de incubación condujeron a una recarga significativa de los terminales nerviosos en las vesículas sinápticas, mientras que la inmunorreactividad del AChR no fue tan marcada como en el control y después de 5 min de incubación. Las fibras musculares estaban bien conservadas, pero los axones motores intramusculares no. Los hallazgos corroboraron los datos farmacológicos ya que la disminución en el número de vesículas sinápticas (5 min) seguida de recuperación (60 min) está de acuerdo con el aumento transitorio de la frecuencia de MEPPs, lo que significa una mayor liberación de neurotransmisores. Estos datos apoyan el modo de acción presináptico predominante de R. schneideri, pero no excluyen la posibilidad de una acción postsináptica secundaria dependiendo del tiempo en que la preparación esté expuesta al veneno.

Bovine central nervous system development / Desenvolvimento do sistema nervoso central de bovinos

Central nervous system (CNS) development researches are extremely important to the most common congenital disorders and organogenesis comprehension. However, few studies show the entire developmental process during the critical period. Present research can provide data to new researches related to normal development and abnormalities and changes that occur along the CNS organogenesis, especially nowadays with the need for preliminary studies in animal models, which could be used for experimental research on the influence of viruses, such as the influence of Zika virus on the development of the neural system and its correlation with microcephaly in human newborns. Then, present study describes CNS organogenesis in cattle according to microscopic and macroscopic aspects, identifying structures and correlating to gestational period. Fourteen embryos and nine bovine fetuses at different ages were collected and analyzed. All individuals were measured in order to detect the gestational period. Bovine embryo at 17 days age has its neural tube, cranial neuropore, caudal neuropore and somites developed. After 24 days of development, were observed in cranial part of neural tube five encephalic vesicles denominated: telencephalon, diencephalon, mesencephalon, metencephalon and myelencephalon. In addition, the caudal part of neural tube was identified with the primitive spinal cord. The primordial CNS differentiation occurred from 90 to 110 days. The five encephalic vesicles, primordial spinal cord and the cavities: third ventricule, mesencephalic aqueduct, fourth ventricle and central canal in spinal cord were observed. With 90 days, the main structures were identified: (1) cerebral hemispheres, corpus callosum and fornix, of the telencephalon; (2) interthalamic adhesion, thalamus, hypothalamus and epythalamus (glandula pinealis), of the diencephalon; (3) cerebral peduncles and quadruplets bodies, of the mesencephalon; (4) pons and cerebellum, of the metencephalon; (5) medulla oblongata or bulb, of the myelencephalon; and (6) spinal cord, of the primitive spinal cord. After 110 days of gestation, the five encephalic vesicles and its structures were completely developed. It was noted the presence of the spinal cord with the cervicothoracic and lumbossacral intumescences. In summary, the results describes the formation of the neural tube from the neural plate of the ectoderm, the encephalic vesicles derived from the neural tube and subsequent structural and cavities subdivisions, thus representing the complete embryology of the central nervous system.(AU)

Os estudos que descrevem o desenvolvimento do sistema nervoso central (SNC) são de suma importância para compreensão da organogênese e identificação dos prováveis eventos que resultam em malformações congênitas. Estes dados podem subsidiar novas pesquisas relacionadas ao desenvolvimento normal, e interpretação de malformações e alterações que ocorrem ao longo da organogênese do SNC, considerando neste momento a necessidade de estudos preliminares em modelos animais, os quais poderiam ser utilizados para pesquisas experimentais sobre a influência de agentes infecciosos como o Zika vírus, no desenvolvimento do sistema nervoso e suas relações com a microcefalia em humanos recém-nascidos. O presente estudo teve como objetivo descrever os aspectos morfológicos macro e microscópicos da organogênese do SNC de bovinos, buscando correlacionar os achados morfológicos com a idade gestacional. Todos os animais foram mensurados para detectar o período gestacional. Foram coletados e analisados 14 embriões e nove fetos de bovinos de diferentes idades gestacionais. No embrião bovino a partir do décimo sétimo dia de gestação, encontra-se a formação do tubo neural, o neuroporo cranial e neuroporo caudal, e formação dos somitos. Após 24 dias de desenvolvimento, são observadas na parte cranial do tubo neural cinco vesículas encefálicas denominadas: telencéfalo, diencéfalo, mesencéfalo, metencéfalo e mielencéfalo; e na parte caudal do tubo neural, encontra-se a medula espinhal primitiva. Entre 90 a 110 dias de gestação, observa-se a total diferenciação das cinco vesículas do SNC. Com 90 dias, são identificas as principais estruturas: (1) do telencéfalo, os hemisférios cerebrais, corpo caloso e fórnix; (2) do diencéfalo, a aderência intertalâmica, tálamo, hipotálamo e epitálamo (glândula pineal); (3) do mesencéfalo, os pedúnculos cerebrais e os corpos quadrigêmios; (4) do metencéfalo, a ponte e o cerebelo; (5) do mielencéfalo, a medula oblonga (ou bulbo); e (6) da medula espinhal primitiva, a medula espinhal. Após 110 dias, as cinco vesículas encefálicas e as suas subdivisões se encontram completamente desenvolvidas. Notou-se a presença da medula espinhal com as intumescências cervicotorácica e lombossacral. Em resumo, os resultados demonstram a formação do tubo neural a partir da placa neural do ectoderma, as vesículas encefálicas provenientes do tubo neural e posteriormente as subdivisões das estruturas e das cavidades, que representam a completa embriologia do sistema nervoso central.(AU)

Purificação e caracterização de vesículas extracelulares de taquizoítos de toxoplasma gondii / Purification and characterization of extracellular vesicles of taquizoítos of toxoplasma gondii

Toxoplasma gondii, protozoário causador da toxoplasmose, é transmitido dos animais para os seres humanos pela ingestão de carne contaminada ou por oocistos liberados nas fezes de felinos no ambiente. Nos seres humanos, a infecção é normalmente assintomática, mas em casos em que a infecção primária ocorre durante a gravidez ou a reativação da infecção latente ocorre em pacientes imunosuprimidos pode ser grave. Nas últimas décadas tem se estudado pequenas estruturas secretadas pelas células procarióticas e eucarióticas denominadas de vesículas extracelulares (EVs). As EVs podem transportar biomarcadores de doenças, macromoléculas biorreativas contribuindo para a patogênese e sendo uma forma de diagnóstico não invasivo. Estas pequenas estruturas podem ser isoladas por ultracentrifugação, cromatografia e observadas por microscopia eletrônica. Elas participam na comunicação entre as células, na transferência de proteínas, lipídios e ácidos nucléicos. Diante do exposto, o presente estudo teve como objetivo estabelecer um protocolo para isolar e caracterizar vesículas extracelulares produzidas e excretadas por taquizoítos da cepa RH de T. gondii. Taquizoítos provenientes de culturas de células VERO foram isolados dos sobrenadantes das culturas e centrifugados em cinco séries de lavagens. A seguir, foram incubados em meio de cultura por 24 horas para secreção das EVs. Em seguida, investigou-se o tamanho e concentração das EVs isoladas por análise de varredura de partículas (NTA) no equipamento NanoSight. Paralelamente, a morfologia e a liberação das EVs também foram investigadas por microscopia eletrônica de transmissão e de varredura. Então, as EVs foram purificadas por cromatografia em gelexclusão em alíquotas de 1 mL (24-32 frações) e imuno-selecionadas por ELISA utilizando um "pool" de soros reagente para toxoplasmose. A seguir foi investigado a presença de miRNA nas EVs; e finalmente, foi avaliado o perfil proteico das EVs de T. gondii para verificar por testes sorológicos se estas partículas poderiam ser reconhecidas pelo sistema imune hospedeiro. As análises realizadas por NTA permitiram determinar que cerca de 1 x 106 taquizoítos secretaram de 4 a 8 x 108 EVs/mL num período de 24 horas de incubação em meio de cultura celular. Adicionalmente, estas vesículas apresentaram morfologia e tamanho de 165-175 nm de diâmetro, tamanho correspondente às microvesículas. Estes resultados também foram confirmados pela avaliação das imagens fornecidas pelas microscopias eletrônicas de transmissão e varredura. Tambem foi possível determinar a presenção de small RNAs e micro RNAs através de uma corrida eletroforética micro fluídica. As análises por SDS-PAGE mostram que as proteínas que compõem as EVs apresentaram um perfil eletroforético com espectro de 15 a 70 kDa. Soros de camundongos cronicamente infectados (com 2 diferentes cepas de T. gondii) e soros humanos reconheceram distintos padrões eletroforéticos no immunoblotting. As vesículas liberadas por T. gondii podem ser um mecanismo importante pelo qual os parasitas apresentam seus antígenos ao hospedeiro e, portanto, podem ter um papel importante na patogênese da toxoplasmose

Toxoplasma gondii, a protozoan that causes toxoplasmosis, is transmitted from animals to humans through ingestion of infected meat or oocysts released by felines into the environment. In humans, infection is usually asymptomatic, but in cases where primary infection occurs during pregnancy or the reactivation of latent infection occurs in immunosuppressed patients it can be serious. In the last decades, small structures secreted by prokaryotic and eukaryotic cells called extracellular vesicles (EVs) were studied. These small structures can be isolated by ultracentrifugation, chromatography and examined by electron microscopy. EVs participate in the communication between cells, transfer of proteins, lipids and nucleic acids. In addition, them can carry disease biomarkers, bioreactive macromolecules contributing to the pathogenesis of diseases. In view of the above, the present study aimed to establish a protocol to isolate and characterize extracellular vesicles produced and excreted by tachyzoites of T. gondii RH strain. To achieve this goal, tachyzoites from VERO cell cultures were isolated from the culture supernatants and centrifuged in five sets of washes. Next, they were incubated in culture medium for 24 hours for secretion of the EVs. The size and concentration of the isolated EVs by particle scan analysis (NTA) were investigated on the NanoSight (NTA) equipment. In parallel, the morphology and the release of the EVs were also investigated by transmission and scanning electron microscopy. Then, EVs were purified by gel-exclusion chromatography in 1 mL aliquots (24-32 fractions) and immuno-selected by ELISA using a pool of reagent sera for toxoplasmosis. Next, the presence of miRNA in the EVs was investigated; and finally, the protein profile of T. gondii EVs was evaluated and verify by serological tests if these particles could be recognized by the host immune system. The results showed that the protocol for recovery of T. gondii EVs was established from 1 to 1010 tachyzoites obtained from cultured cells. Analyzes performed by NTA allowed us to determine that about 1 x 106 tachyzoites secreted 4 to 8 x 108 EVs / mL within 24 hours of incubation in culture medium. Additionally, these vesicles presented morphology and size of 165-175 nm of diameter, corresponding microvesicles size. These results were also confirmed by the evaluation of images provided by transmission and scanning electron microscopy. The miRNA purifications from the EVs and subsequent microfluidic electrophoretic run confirmed the presence of smallRNA and miRNA in the vesicles. SDS-PAGE analyzes show that the proteins carried by the EVs showed an electrophoretic profile with a spectrum of 15 to 70 kDa. Sera from chronically infected mice (with 2 different strains of T. gondii) and humam serum recognized distinct electrophoretic patterns in immunoblotting

Desenvolvimento de vesículas poliméricas de poli(etileno glicol)-b-poli(ε-caprolactona) (PEG-PCL) para veiculação de L-asparaginase / Development of polyethylene glycol-polycaprolactone polymer vesicles for L-Asparaginase

A L-Asparaginase (ASNase) é um importante agente quimioterapêutico utilizado para o tratamento da leucemia linfoblástica aguda (ALL) há mais de 40 anos. No entanto, devido à origem biológica da ASNase, enzima produzida por Escherichia coli, problemas como a imunogenicidade e baixa meia vida-plasmática devem ser considerados. Com o objetivo de minimizar essas desvantagens, várias ASNases homólogas bem como formulações de ASNase de E. coli foram investigadas. Nenhuma das formulações desenvolvidas, entretanto, foi capaz de resolver definitivamente esses problemas associados à sua origem. Nesse sentido, considerando os recentes avanços na ciência de polímeros com a possibilidade do obtenção de vesículas poliméricas usando copolímeros, este trabalho concentrou-se no desenvolvimento de polimerossomos de poli(etileno glicol)-b-poli(ε-caprolactona) (PEG-PCL) para encapsular a ASNase. Diversas condições experimentais foram investigadas e, ao final, os polimerossomos foram produzidos pela técnica de hidratação do filme polimérico utilizando a centrifugação como técnica de pós-filme para remoção de copolímero precipitado, produzindo assim vesículas polímericas de 120 a 200nm com PDI de aproximadamente 0,250. A eficiência de encapsulação da ASNase, utilizando as metodologias de centrifugação ou cromatografia de exclusão molecular, revelou taxas de encapsulação de 20-25% e 1 a 7%, repectivamente. Esses resultados apontam a importância de se determinar a eficiência de encapsulação por cromatografia de exclusão molecular ou método direto no caso de nanoestruturas auto-agregadas formadas por copolímeros, devido a valores superestimados com o emprego da centrifugação. Ainda que estudos complementares se façam necessários para liberação da enzima encapsulada ou penetração da L-asparagina nas vesículas, nossos resultados demonstram o potencial de polimerossomos para veiculação de ASNase, bem como de outras proteínas terapêuticas

L-Asparaginase (ASNase) is an important chemotherapeutic agent used for the treatment of acute lymphoblastic leukemia (ALL) for more than 40 years. However, due to the biological origin of ASNase (produced by Escherichia coli) some drawbacks such as immunogenicity and low plasma half life are present. In order to minimize the disadvantages, several ASNases proteoforms and formulations of E. coli ASNase were investigated. However, none of this formulations completely solved the main drawbacks of ASNase. In this sense, considering the recents advances in polymers science with the possibility to develop polymeric vesicles using copolymers, this work aimed at the development of poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-PCL) vesicles to encapsulate ASNase. Different experimental conditions were investigated and, the final polymersomes formulation was prepared by film hydratation using centrifugation as a post-film technique to remove the bulky coplymer. Polymeric vesicles of 120 to 200nm with PDI of approximately, 0.250 were obtained. The encapsulation efficiency of ASNase was determined indirectly by centrifugation and directly by size exclusion chromatography, resulting in encapsulation rates of 20-25% and 1 to 7%, respectively. These results indicate the importance of determining the efficiency of encapsulation by size exclusion chromatography or direct method in the case of self-aggregated nanostructures formed by copolymers, due to values overestimated with the use of centrifugation. Our results point to the potential of polymersomes for ASNase delivery, as well as other therapeutic proteins. Nonetheless, complimentary studies are still necessary for ASNase release or L-asparagine penetration into the vesicles

Vesículas extracelulares derivadas de macrófagos alteram o potencial de invasão, proliferação e migração de linhagens celulares do carcinoma de células escamosas de língua oral / Macrophage-derived extracellular vesicles alter the potential for invasion, proliferation, and migration of oral tongue squamous cell carcinoma cell lines

O câncer é composto pelas células malignas em proliferação associadas às diferentes células circunjacentes, formando o microambiente tumoral (TME), onde há uma constante troca de informações. Uma das formas de comunicação entre os diferentes tipos celulares do TME se dá por meio da liberação de vesículas extracelulares (EVs), um campo de estudo ainda pouco explorado. O presente estudo se propôs a avaliar os efeitos das EVs liberadas por macrófagos do TME, células altamente plásticas em seu fenótipo (M1 ­ perfil antitumoral; M2 ­ perfil pró-tumoral), em diferentes linhagens do carcinoma de células escamosas de língua oral (CCELO) no tocante à capacidade invasiva, proliferativa e migratória. Foi observado que as amostras de EVs extraídas dos macrófagos eram relativamente puras em EVs, porém subtipo inespecíficas. No ensaio de invasão em miomas, quando colocadas as células inflamatórias em cocultura com as células HSC-3, as células M1 inibiram a invasão e M2 aumentaram a capacidade invasiva das células malignas. Por outro lado, o tratamento com M1 EVs aumentou a capacidade invasiva das células HSC-3 e o tratamento com EVs de M2 inibiu a invasão dessas células, sendo observado um perfil semelhante nas células SCC-25 e SAS quando submetidas aos mesmos tratamentos. Na análise do marcador Ki-67 nos miomas, tanto as células HSC-3 quanto SCC-25 e SAS apresentaram o mesmo padrão de proliferação independentemente do tratamento utilizado, quando comparados com os respectivos controles negativos. Quando analisada a proliferação das células malignas no IncuCyte®, tratadas com EVs dos diferentes tipos de macrófagos em diferentes concentrações, foi identificado um aumento na capacidade proliferativa de células HSC-3 e SAS tratadas com M1 EVs em um padrão dose dependente. Um aumento da capacidade proliferativa seguindo um padrão dose dependente também ocorreu quando as células SAS foram tratadas com M2 EVs. Nos demais ensaios de proliferação no IncuCyte® também foram identificados efeitos na capacidade proliferativa, no entanto um padrão dose dependente não foi observado. No ensaio de migração no IncuCyte®, foram identificadas diferenças significativas na capacidade migratória de células SCC-25 e SAS tratadas com diferentes tipos de EVs nas diferentes concentrações, quando comparadas ao controle negativo. Os achados deste estudo sugerem que as EVs derivadas de macrófagos são fatores importantes na tumorigênese do CCELO, bem como abre discussões sobre os diferentes efeitos das células inflamatórias no TME a depender do tipo de comunicação celular executada (AU).

Cancer is an entity composed of proliferating malignant cells associated with the different types surrounding cells, forming the tumor microenvironment (TME), where there is a constant exchange of information. One of the ways of communicating between different types of TME cells is through the release of extracellular vesicles (EVs), a field of study that remains poorly understood. The aim of the present study was to evaluate the effects of EVs released from TME macrophages, which are cells highly plastic in their phenotype (M1 showing an anti-tumor profile and M2 exhibiting a pro-tumor profile) in different cell lines of tongue squamous cells carcinoma (TSCC) regarding to invasive, proliferative and migratory capacity. It was observed that EVs samples obtained from macrophages were relatively pure in EVs, although they were non-specific subtypes. In the myoma invasion assay, it was observed that when inflammatory cells were co-cultured with HSC-3 cells, M1 cells inhibited invasion and M2 increased the invasive ability of the malignant cells. On the other hand, treatment with M1 EVs increased the invasive capacity of HSC-3 cells, and treatment with M2 EVs inhibited the invasion of these malignant cells, and a similar profile was observed in SCC-25 and SAS cells when they were submitted to the same treatments. In the analysis of the Ki-67 marker in myomas, HSC-3, SCC-25 and SAS cells showed the same proliferation pattern regardless the type of the treatment used when compared to the respective negative controls. When it was analyzed the proliferation of malignant cells in IncuCyte® treated with EVs derived from different types of macrophages at different concentrations, an increase in the proliferative ability of HSC-3 and SAS cells treated with M1 EVs was observed in a dosedependent pattern. An increase in proliferative ability in dose-dependent profile was also observed when SAS cells were treated with M2 EVs. In the other proliferation assays performed in IncuCyte®, effects on proliferative capacity were also highlighted, however a dose-dependent pattern was not observed. In the IncuCyte® migration assay, significant differences were observed in the migration capacity of SCC-25 and SAS cells treated with different types of EVs at different concentrations when compared to the negative control. The findings of this study suggest that macrophages-derived EVs are pivotal factors in TSCC tumorigenesis, as well as permits discussions on the different effects of inflammatory cells on TME depending on the type of cell communication performed (AU).

Caracterização do secretoma de cepas de Xylella fastidiosa / Characterization of the secretome Xylella fastidiosa strains

As doenças causadas pelo fitopatógeno Xylella fastidiosa, uma bactéria Gram-negativa, devem-se aos seus múltiplos fatores de virulência, tais como formação de biofilme, secreção de enzimas de degradação da parede celular do xilema (CWDE), expressão de proteínas de adesão e produção de vesículas de membrana externa (OMVs). Esses fatores de virulência são controlados por uma via de sinalização mediada por DSF (fatores de sinalização difusíveis de natureza lipídica) e relacionada com percepção de quórum. Nesse trabalho, tivemos como objetivo ampliar a caracterização do secretoma de cepas selvagens e mutantes de X. fastidiosa para evidenciar proteínas e metabólitos potencialmente associados à adaptação ao hospedeiro, virulência e patogenicidade. Desenvolvemos, paralelamente, três estudos empregando como abordagens metodológicas a proteômica, a metabolômica e a transcritômica. No primeiro estudo, comparamos o secretoma (exoproteoma) da cepa Temecula1 selvagem (WT) e do mutante no gene da sintase de DSF (ΔrpfF), o qual exibe fenótipo de hipervirulência em videiras. A este estudo associamos a comparação dos transcritomas dessas cepas. Os resultados mostraram que, mesmo no cultivo in vitro, X. fastidiosa expressa e secreta fatores de virulência previamente conhecidos (lipases-esterases e proteases), além de toxinas (microcinas) que, supostamente, teriam papel de controlar bactérias competidoras pelo mesmo nicho. No segundo estudo caracterizamos a composição de OMVs secretadas no cultivo in vitro por X. fastidiosa Fb7 e 9a5c (cepas isoladas de laranjeiras) e Temecula1 (cepa isolada de videira). Demonstramos que Fb7 produz até 57% mais OMVs que 9a5c e Temecula1 e identificamos um total de 202 proteínas distintas nas OMVs produzidas pelas 3 cepas, ampliando consideravelmente o número de proteínas secretadas por meio de OMVs descrito, até então, para X. fastidiosa. Entre as proteínas enriquecidas, citamos adesinas afimbriais, porinas, lipoproteínas, hidrolases (lipases/esterases, proteases e peptidases) e uma pectina-liase putativa. Destacamos a detecção da enzima L-ascorbato oxidase nas OMVs e sugerimos que esta enzima poderia atuar na depleção do ascorbato produzido pelo hospedeiro vegetal. Além disso, demonstramos, pela primeira vez, que OMVs de X. fastidiosa transportam ácidos graxos da família DSF, sugerindo um papel adicional para OMVs nesse fitopatógeno. Finalmente, no terceiro estudo verificamos alterações relevantes no perfil de metabólitos secretados por X. fastidiosa em resposta a sua interação com metabólitos secretados por Burkholderia phytofirmans, proposta como uma cepa para o biocontrole da doença de Pierce de videiras. Confirmamos que o sobrenadante de B. phytofirmans possui um composto de natureza apolar que induz a formação de biofilme em X. fastidiosa, contudo ainda não foi possível decifrar a natureza química deste composto

The diseases caused by the phytopathogen Xylella fastidiosa, a Gram-negative bacterium, are due to multiple virulence factors, such as biofilm formation, secretion of xylem cell wall degradation enzymes (CWDE), expression of adhesion proteins and production of outer membrane vesicles (OMVs). These virulence factors are controlled by a DSF (diffusible signaling factors of a lipidic nature) mediating signaling pathway and related to quorum sensing perception. In this work, we aimed to extend the characterization of the secretoma of wild type and mutants strains of X. fastidiosa to uncover proteins and metabolites potentially associated to host adaptation, virulence and pathogenicity. We developed three studies in parallel using proteomics, metabolomics and transcriptomics as methodological approaches. In the first study, we compared the secretome (exoproteome) of the wild type strain Temecula1 (WT) and of DSF synthase mutant (ΔrpfF) which exhibits hypervirulence phenotype in grapevines. We also compared the transcriptomes of these strains. Our results showed that, even in in vitro culture, X. fastidiosa expresses and secretes previously known virulence factors (lipasesesterases and proteases), as well as toxins (microcins) that might play a role in controlling competing bacteria in the same niche. In the second study, we characterized the composition of OMVs secreted by in vitro cultures of X. fastidiosa Fb7 and 9a5c (strains isolated from orange trees) and Temecula1 (strain isolated from grapevine). We have shown that Fb7 produces up to 57% more OMVs than the 9a5c and Temecula1. Moreover we identified a total of 202 distinct proteins in the OMVs produced by these three strains, increasing considerably the number of OMVs secreted proteins so far described for X. fastidiosa. Among the proteins enriched in OMVs, we point out afimbrial adhesins, porins, lipoproteins, hydrolases (lipases/esterases, proteases and peptidases) and a putative pectin-lyase. We highlight the detection of the enzyme L-ascorbate oxidase in the OMVs and we suggest that this enzyme could act in the depletion of ascorbate produced by the plant host. In addition, we have demonstrated, for the first time, that X. fastidiosa OMVs transport fatty acids from the DSF family, suggesting an additional role for OMVs in this phytopathogen. Finally, in the third study we verified relevant changes in the profile of metabolites secreted by X. fastidiosa in response to the interaction with metabolites secreted by Burkholderia phytofirmans that has been sugested as a biocontrol strain for Pierce's disease in grapevines. We confirm that the B. phytofirmans supernatant has a non-polar compound that induces biofilm formation in X. fastidiosa, but it has not yet been possible to elucidate the chemical nature of this compound

Enfermedad de Hailey-Hailey: A case report / Comunicación de un caso / Hailey-Hailey disease

La enfermedad de Hailey-Hailey (EHH) también conocida como pénfigo crónico familiar benigno (PCFB), es una genodermatosis muy infrecuente sin predilección de sexos; se caracteriza por ampollas y vesículas fláccidas, pruriginosas y dolorosas, localizadas en áreas intertriginosas. El diagnóstico suele tardarse debido a la diversidad de su presentación clínica. Presentamos el caso atípico de un paciente de 86 años de edad, que presentaba hace seis meses vesículas dolorosas erosionadas en cuello, axilas, ingles y mucosa oral; tratado anteriormente con corticoides, sin cambios, motivo por el que se hace biopsia de las lesiones, siendo la histopatología compatible con la enfermedad de Hailey-Hailey.

Hailey-Hailey disease or familial benign chronic pemphigus, is a very rare genodermatosis, autosomal dominant inheritance, has no sex and race predilection. It is caused by mutations in the ATP2C1 gene, which causes dysfunction calcium channel, key in regulating epidermal differentiation and processing desmosomal proteins involved in cell adhesion, as a result there is a defect in adherence of keratinocytes and acantholysis occurs and generating alterations in the epidermis. It´s clinical manifestations are characterized by flaccid, pruritic and painful ampules and vesicles located in intertriginous areas and rarely in mucosas; chronic and recurrent evolution. We report the unusual case of a male patient aged 86, presenting six months ago eroded and painful vesicles on the neck, armpits, groin and oral mucosa; previously treated with corticosteroids, unimproved, whereby lesions are biopsied, being histopathology compatible with Hailey-Hailey disease.