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Int. j. morphol ; 40(1): 157-167, feb. 2022. ilus, tab
Article in English | LILACS-Express | LILACS | ID: biblio-1385584


SUMMARY: Carbon tetrachloride (CCl4) is a manufactured chemical and does not occur naturally in the environment. CCl4 is a clear liquid that evaporates very easily. It has a sweet odor. CCl4 is toxic to the mammalian liver and is hepatocarcinogenic in both rats and mice. Rosemary (Rosmarinus Officinalis) is commonly used as a spice and flavoring agent in food processing. It is known for its antioxidant properties. The present study aims to investigate the antioxidant activity of rosmarinic acid (RA) on CCl4-induced liver toxicity in adult male albino rats. Forty adult male albino rats were divided into 4 groups with 10 rats in each group. Group I (control group). Group II animals received RA at a dose of 50 mg/kg/day by oral gavage for 4 weeks. Group III animals received CCl4 intraperitoneally at a dose of 3ml/kg twice weekly for 4 weeks. Group IV animals received CCl4 Plus RA. At the end of the experiment, liver specimens are processed for histological, immunohistochemical, EM and biochemical studies. Administration of RA deceased the elevated serum liver enzymes (AST, ALT, and ALP), elevated MDA level and immunoexpression of the proapoptotic protein (Bax) induced by CCl4. It increased reduced glutathione (GSH), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and immunoexpression of the antiapoptotic protein (Bcl2). It also improved the histological and ultrastructural changes induced by CCl4. It appears that Rosmarinic acid has protective effects against CCl4-induced hepatotoxicity as indicated by biochemical, histological, immunohistochemical and ultrastructural results.

RESUMEN: El tetracloruro de carbono (CCl4) es un producto químico fabricado y no se encuentra de forma natural en el medio ambiente. CCl4 es un líquido transparente que se evapora fácilmente; tiene un olor dulce. CCl4 es tóxico para el hígado de los mamíferos y es hepatocarcinogénico tanto en ratas como en ratones. El romero (Rosmarinus officinalis) se usa comúnmente como condimento y agente aromatizante en el procesamiento de alimentos. Es conocido por sus propiedades antioxidantes. El presente estudio tuvo como objetivo investigar la actividad antioxidante del ácido rosmarínico (RA) sobre la toxicidad hepática inducida por CCl4 en ratas albinas macho adultas. Se dividieron cuarenta ratas albinas macho adultas en 4 grupos con 10 ratas en cada grupo. Grupo I (grupo control). Los animales del grupo II recibieron AR a una dosis de 50 mg / kg / día por sonda oral durante 4 semanas. Los animales del grupo III recibieron CCl4 por vía intraperitoneal a una dosis de 3 ml / kg dos veces por semana durante 4 semanas. Los animales del grupo IV recibieron CCl4 Plus RA. Al final del experimento, las muestras de hígado se procesaron para estudios histológicos, inmunohistoquímicos, EM y bioquímicos. La administración de AR eliminó las enzimas hepáticas séricas elevadas (AST, ALT y ALP), el nivel elevado de MDA y la inmunoexpresión de la proteína proapoptótica (Bax) inducida por CCl4. Aumentó el glutatión reducido (GSH), glutatión peroxidasa (GSH-Px), la superóxido dismutasa (SOD) y la inmunoexpresión de la proteína antiapoptótica (Bcl2). También mejoró los cambios histológicos y ultraestructurales inducidos por CCl4. El ácido rosmarínico puede tener efectos protectores contra la hepatotoxicidad inducida por CCl4, tal como lo indican los resultados bioquímicos, histológicos, inmunohistoquímicos y ultraestructurales.

Animals , Male , Mice , Carbon Tetrachloride/toxicity , Cinnamates/administration & dosage , Depsides/administration & dosage , Chemical and Drug Induced Liver Injury/drug therapy , Antioxidants/administration & dosage , Superoxide Dismutase/analysis , Immunohistochemistry , Cinnamates/pharmacology , Oxidative Stress/drug effects , Microscopy, Electron, Transmission , Depsides/pharmacology , Glutathione Peroxidase/analysis , Malondialdehyde/analysis , Antioxidants/pharmacology
An. bras. dermatol ; 97(1): 58-62, Jan.-Feb. 2022. graf
Article in English | LILACS | ID: biblio-1360094


Abstract McCune - Albright syndrome is a genetic disease with cutaneous mosaicism caused by post-zygotic activating mutations in GNAS locus, it has a triad of fibrous bone dysplasia, café-au-lait macules and precocious puberty. We examined a 22-year-old female patient with café au lait spot in right side of the abdomen, with a chessboard - like distribution, extending to right thigh with geographical contours, she has also an ovarian cyst, scoliosis and truncal obesity. Biopsies were taken from the hyperpigmented area and processed for light microscopy and for transmission electron microscopy. Light microscopy showed increased melanin pigment with HE staining. Immunohistochemistry with melanocytic markers (HMB-45 and Melan-A) revealed a normal number of melanocytes. Transmission electron microscopy demonstrated normal epidermal structures, such as desmosomes, cytokeratin filaments and hemidesmosomes. With high magnifications an irregular melanossomal contour was seen, with some indentations in their outline.

Humans , Female , Adult , Young Adult , Puberty, Precocious , Fibrous Dysplasia of Bone , Fibrous Dysplasia, Polyostotic/diagnosis , Cafe-au-Lait Spots , Microscopy, Electron, Transmission
Braz. J. Pharm. Sci. (Online) ; 58: e19106, 2022. tab, graf
Article in English | LILACS | ID: biblio-1374575


Abstract In this work, polystyrene-b-poly (acrylic acid) (PS-b-PAA) nanovesicles were coated by modified chitosans aiming at studying its physicochemical parameters. The chitosan (CS) was chemically modified to add hydrophilic and/or hydrophobic groups, obtaining three modified chitosans. The PS-b-PAA nanovesicles were obtained by organic (1,4-dioxane) cosolvent method in water, resulting in nanovesicles with less than 150 nm of diameter (polydispersibility index - PDI at 90° = 0.106), measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM), and negative zeta potential (-37.5 ± 3.2 mV), allowing the coating of its surface with oppositely charged polysaccharides, such as the CS and the modified chitosans. The coating process was made by mixing the colloidal suspensions with the CS and the modified chitosans at specific ENT#091;CS-xENT#093;/ENT#091;PS-b-PAAENT#093; ratios (0.001 to 1.0 wt %) and measuring the change in size and surface charge by DLS and zeta potential. Upon reaching maximum adsorption, the zeta potential parameter was positively stabilized (+26.7 ± 4.1 mV) with a hydrodynamic diameter slightly longer (< 200 nm of diameter). The encapsulation efficiency (EE) of minoxidil, quantified by capillary electrophoresis, was 50.7%, confirming their potential as drug delivery carriers and the coating process showed the possibility of controlling the surface charge nature of these nanovesicles

Chitosan/metabolism , Minoxidil/analogs & derivatives , Microscopy, Electron, Transmission/methods , Efficiency/classification , Dynamic Light Scattering/instrumentation , Methods
Braz. J. Pharm. Sci. (Online) ; 58: e191132, 2022. tab, graf
Article in English | LILACS | ID: biblio-1394049


Abstract To explore the effects and mechanisms of benzoylaconitine and paeoniflorin on collagen-induced arthritis (CIA) rats. Weight, paw swelling, arthritis index and joint pathologic changes were examined in each group after CIA induction. PGE2, IL-1ß, IL-6, IL-10, TNF-α, VEGF, MMP-3, IgG and anti-CII Ab were assessed by ELISA; STAT1 and STAT3 expressions were analyzed immunohistochemically, and the ultrastructure of synovial cells was observed by transmission electron microscopy. Therapeutic effects were determined in CIA rats via injecting benzoylaconitine and paeoniflorin, which could alleviate the degree of swelling and arthritis index (AI) and pathological lesions of the sacroiliac gland; decrease the levels of PGE2, IL-1ß, TNF-α, VEGF and IgG in serum; reduce STAT1 and STAT3 expression in the membrane tissue; and inhibit the secretion and proliferation of synovial cells. These results showed that benzoylaconitine and paeoniflorin could significantly palliate the arthritic symptoms of CIA rats, and better therapeutic effects could be achieved if the two components were used in combination

Animals , Male , Rats , Arthritis, Experimental/chemically induced , Therapeutic Uses , Enzyme-Linked Immunosorbent Assay/methods , Dinoprostone/adverse effects , Interleukin-6/pharmacology , Interleukin-1/pharmacology , Interleukin-10/pharmacology , Matrix Metalloproteinases , Microscopy, Electron, Transmission/methods
An. bras. dermatol ; 96(3): 328-331, May-June 2021. graf
Article in English | LILACS | ID: biblio-1285053


Abstract Cutis rhomboidalis nuchae was assessed in a 65-year-old patient. Optical microscopy showed basophilic agglomerations in the reticular dermis with decreased elastic fibers. Trans- mission electron microscopy showed elongated, curved and fragmented structures, and in their interior the presence of electron-dense lumps was reduced and irregular, similar to modified elastic fibers, whereas the collagen fibers had a normal aspect. Scanning electron microscopy showed deposits between the bundles of collagen, resembling pebbles or stones. These findings demonstrate that, at one stage of the disease, the collagen remains normal and the alterations are seen in the elastic tissue.

Humans , Aged , Skin Diseases , Microscopy, Electron, Scanning , Dermis , Microscopy, Electron, Transmission , Elastic Tissue
Int. j. morphol ; 39(2): 371-377, abr. 2021. ilus
Article in English | LILACS-Express | LILACS | ID: biblio-1385372


SUMMARY: Diabetes mellitus increases the risk of developing chronic obstructive pulmonary disease (COPD). The small bronchiole is a prominent site of airflow obstruction that causes increased airway resistance in patients with the COPD. Therefore, the histological and ultrastructural changes in small bronchioles in streptozotocin (STZ)-induced chronic diabetes were determined. Twenty-four weeks after STZ induction, rats were sacrificed, and the right and left lungs were collected for examination by light and electron microscopy. The alterations to the small bronchioles were the same in both lungs of these diabetic rats. The bronchiolar epithelial cells, both ciliated and secretory club cells, showed pyknotic nuclei and damaged cytoplasmic organelles. Increased thickening of the bronchiolar wall occurred in diabetic rats due to smooth muscle layer thickening, inflammatory cell infiltration, and increased numbers of myofibroblasts with collagen deposition.These results indicated that chronic diabetes caused extreme damage to small bronchioles, which may lead to chronic small airway obstruction and ultimately increase the likelihood of COPD progression. This basic knowledge provides a better understanding of the progression of pathogenesis in the small airways of patients with prolonged diabetes.

RESUMEN: La diabetes mellitus aumenta el riesgo de desarrollar enfermedad pulmonar obstructiva crónica (EPOC). El bronquiolo es un sitio prominente de obstrucción del flujo de aire que causa una mayor resistencia de las vías respiratorias en pacientes con EPOC. Por lo tanto, se determinaron los cambios histológicos y ultraestructurales en los bronquiolos en la diabetes crónica inducida por estreptozotocina (STZ). 24 semanas después de la inducción de STZ, se sacrificaron las ratas y se analizaron los pulmones derecho e izquierdo por microscopía óptica y electrónica. Las alteraciones de los pequeños bronquiolos fueron las mismas en ambos pulmones de estas ratas diabéticas. Las células epiteliales bronquiolares, tanto ciliadas como secretoras, mostraban núcleos picnóticos y orgánelos citoplasmáticos dañados. Se produjo un aumento del engrosamiento de la pared bronquiolar en ratas diabéticas debido al engrosamiento de la capa de músculo liso, infiltración de células inflamatorias y un mayor número de miofibroblastos con colágeno. Estos resultados indicaron que la diabetes crónica causaba daño extremo a los pequeños bronquiolos, lo que puede conducir a una obstrucción crónica de las vías respiratorias pequeñas y además aumentar la probabilidad de progresión de la EPOC. Esta información proporcionará un mejor conocimiento de la patogénesis en las vías respiratorias pequeñas de los pacientes con diabetes prolongada.

Animals , Male , Rats , Bronchi/pathology , Diabetes Mellitus, Experimental/pathology , Bronchi/ultrastructure , Chronic Disease , Rats, Sprague-Dawley , Microscopy, Electron, Transmission
Article in English | WPRIM | ID: wpr-878442


OBJECTIVES@#This study aims to investigate the effects of ionizing radiation on the secretion of the paracellular pathway in rat submandibular glands (SMGs) and reveal the changes in the tight junction (TJ) protein claudin-4.@*METHODS@#A total of 24 Wistar rats were randomly divided into control and irradiation groups. The irradiation groups were further divided into 1, 4, and 12 weeks groups after irradiation. One-time 20 Gy irradiation was given to the SMG area on the experimental side of the irradiation group. At 1, 4, and 12 weeks after irradiation, the secretion of SMGs was measured using the Schirmer's test. The pathological changes in the gland tissues were observed under light microscopy after hematoxylin⁃eosin (HE) staining. The changes in the TJ ultrastructure were observed under transmission electron microscopy. The immunofluorescence staining and Western blot were used to detect the expression levels of muscarinic acetylcholine M3 receptor, aquaporin 5 (AQP5), and claudin-4 protein.@*RESULTS@#At 1, 4, and 12 weeks after irradiation, the secretion of SMGs in the irradiation group was significantly decreased and lower than that in the control group (@*CONCLUSIONS@#The changes in the TJ structure, the upregulation of the claudin-4 expression, and the damage in the paracellular pathway were involved in the hyposecretion of SMGs after irradiation.

Animals , Microscopy, Electron, Transmission , Radiation, Ionizing , Rats , Rats, Wistar , Submandibular Gland , Tight Junctions
São Paulo; s.n; s.n; 2021. 96 p. tab, graf, ilus.
Thesis in Portuguese | LILACS | ID: biblio-1416965


Os cubossomos são partículas nanoestruturadas em forma de bicamada lipídica, bicontínuas e altamente curvadas, as quais devem ser estabilizadas por um polímero não-iônico, neste caso o Pluronic® F-127. Podem ser compostos por alguns tipos de lipídios específicos que possuem a capacidade de se auto associar em estruturas cúbicas quando estão em excesso de água, como o fitantriol (PHY) e a monoleína (GMO). Devido a sua estrutura única, cubossomos possuem um grande potencial para serem considerados como sistemas drug delivery. Os sistemas drug delivery são amplamente utilizados na pesquisa farmacêutica e em contextos clínicos para aumentar a eficácia de compostos utilizados para diagnóstico e de fármacos. No caso da cinarizina (CNZ), fármaco já aprovado para o tratamento de náuseas, vômitos e vertigens causadas pela doença de Ménière, existem inúmeros efeitos colaterais associados a sua baixa solubilidade. Desta forma, a encapsulação em cubossomos se torna uma abordagem promissora para resolver os problemas de atividade farmacológica relacionados ao fármaco. Neste trabalho, realizamos uma caracterização biofísica da interação da CNZ em cubossomos (contendo PHY ou myverol, MYV, sendo este composto por 80% de GMO). As técnicas biofísicas utilizadas foram: espalhamento de raios-X em baixos ângulos (SAXS), espalhamento dinâmico de luz (DLS), microscopia eletrônica de transmissão (TEM), crio microscopia eletrônica de transmissão (Crio-TEM), análise de rastreamento de nanopartículas (NTA) e potencial zeta. A cromatografia líquida de alta eficiência (HPLC) foi realizada para verificar a porcentagem de eficiência de encapsulação (%EE) da CNZ nos cubossomos, enquanto que a citotoxicidade foi avaliada em eritrócitos através da análise da atividade hemolítica. Inicialmente, a influência de diferentes solventes (acetona, clorofórmio, etanol e octano) nas propriedades estruturais de cubossomos de PHY foi investigada, a fim de se minimizar os efeitos do solvente utilizados para a encapsulação da CNZ. Para amostras com acetona, descobriu-se que apenas altas concentrações tiveram influência na estrutura cristalográfica das nanopartículas, sendo o resultado foi totalmente reversível após 24h. O etanol fez com que o parâmetro de rede aumentasse de 10-15%. O clorofórmio e o octano tiveram efeitos diferentes sobre cubossomos de PHY em comparação com a acetona e o etanol; ambos induziram uma transição cúbico-hexagonal-micelar. Posteriormente, constatamos que as nanopartículas de PHY e MYV apresentaram diferentes estruturas cristalográficas, sendo elas Pn3m e Im3m, respectivamente. Devido a problemas com a baixa solubilidade de CNZ em PHY os estudos para esse lipídio foram suspensos. Nos testes para cubossomos de MYV ao incorporar a CNZ foi observado uma alteração da estrutura cúbica de Im3m para Pn3m e os valores dos parâmetros de rede se alteraram de acordo com a estrutura cristalina encontrada, porém os valores não apresentaram diferenças significativas de tamanho quando se trata da mesma estrutura, sugerindo que a CNZ não interferiu no parâmetro de rede. Os tamanhos das nanopartículas apresentaram uma população monodispersa com ~200 nm. DLS mostrou uma interferência da CNZ no tamanho dos cubossomos, variando de forma diretamente proporcional a concentração de CNZ na amostra, enquanto as técnicas de NTA e microscopia apresentaram nanopartículas de tamanhos bastante variados, mas independente da interferência da CNZ. A encapsulação de CNZ também foi dosada por HLPC em cubossomos de MYV, obtendo um limite superior de 0,6 mg/mL. A atividade citotóxica dos cubossomos foi testada em eritrócitos, revelando uma taxa de hemólise bastante inferior em cubossomos com CNZ em relação a cubossomos puros. Acreditamos que os cubossomos podem sim ser utilizados como sistemas carreadores de CNZ

Cubosomes are nanostructured particles in the form of a lipid bilayer, bicontinuous and highly curved, which must be stabilized by a non-ionic polymer, in this case Pluronic® F-127. They can be composed of some types of specific lipids that have the ability to self-associate in cubic structures when they are in excess of water, such as phytantriol (PHY) and monolein (GMO). Due to their unique structure, cubosomes have a great potential to be considered as drug delivery systems. Drug delivery systems are widely used in pharmaceutical research and clinical settings to increase the efficacy of compounds used for diagnostics and drugs. In the case of cinnarizine (CNZ), a drug already approved for the treatment of nausea, vomiting and vertigo caused by Ménière's disease, there are numerous side effects associated with its low solubility. Thus, cubosomal encapsulation becomes a promising approach to solve drug-related problems of pharmacological activity. In this work, we performed a biophysical characterization of the CNZ interaction in cubosomes (containing PHY or myverol, MYV, which is composed of 80% GMO). The biophysical techniques used were: low angle X-ray scattering (SAXS), dynamic light scattering (DLS), transmission electron microscopy (TEM), cryo transmission electron microscopy (Crio-TEM), nanoparticle tracking analysis (NTA) and zeta potential. High performance liquid chromatography (HPLC) was performed to verify the percentage of encapsulation efficiency (%EE) of CNZ in cubosomes, while cytotoxicity was evaluated in erythrocytes by analyzing the hemolytic activity. Initially, the influence of different solvents (acetone, chloroform, ethanol and octane) on the structural properties of PHY cubosomes was investigated in order to minimize the effects of the solvent used for the encapsulation of CNZ. For samples with acetone, it was found that only high concentrations had an influence on the crystallographic structure of the nanoparticles, with the result being fully reversible after 24h. Ethanol caused the network parameter to increase by 10-15%. Chloroform and octane had different effects on PHY cubosomes compared to acetone and ethanol; both induced a cubic-hexagonal-micellar transition. Later, we found that PHY and MYV nanoparticles presented different crystallographic structures, being Pn3m and Im3m, respectively. Due to problems with the low solubility of CNZ in PHY, studies for this lipid were suspended. In the tests for MYV cubosomes when incorporating CNZ, a change in the cubic structure from Im3m to Pn3m was observed and t he lattice parameters changed according to the crystal structure found, but the differences observed were not significant when it comes to the same structure, suggesting that the CNZ did not interfere with the network parameter. The nanoparticle sizes showed a monodisperse population with ~200 nm. DLS showed an interference of CNZ in the size of the cubosomes, varying directly proportionally to the concentration of CNZ in the sample, while NTA and microscopy techniques showed nanoparticles of widely varying sizes, but independent of CNZ interference. CNZ encapsulation was also dosed by HLPC in MYV cubosomes, obtaining an upper limit of 0.6 mg/ml. The cytotoxic activity of cubosomes was tested in erythrocytes, revealing a much lower rate of hemolysis in cubosomes with CNZ compared to pure cubosomes. We believe that cubosomes can indeed be used as CNZ carrier systems

Cinnarizine/analysis , Efficiency , Acetone/agonists , Chromatography, High Pressure Liquid/methods , Chromatography, Liquid/methods , Microscopy, Electron, Transmission/instrumentation , Nanoparticles/adverse effects , Dynamic Light Scattering/instrumentation , Pharmaceutical Research , Lipid Bilayers/pharmacology , Meniere Disease/pathology
Rev. Soc. Bras. Med. Trop ; 54: e08502021, 2021. graf
Article in English | LILACS | ID: biblio-1288094


Abstract INTRODUCTION: Electron microscopy (EM) is a rapid and effective tool that can be used to create images of a whole spectrum of virus-host interactions and, as such, has long been used in the discovery and description of viral mechanisms. METHODS: Electron microscopy was used to evaluate the pulmonary pathologies of postmortem lung sections from three patients who died from infection with SARS-associated coronavirus 2 (SARS-CoV-2), a new member of the Coronaviridae family. RESULTS: Diffuse alveolar damage (DAD) was predominant in all three patients. The early exudative stage was characterized principally by edema and extravasation of red blood cells into the alveolar space with injury to the alveolar epithelial cells; this was followed by detachment, apoptosis, and necrosis of type I and II pneumocytes. The capillaries exhibited congestion, exposure of the basement membrane from denuded endothelial cells, platelet adhesion, fibrin thrombi, and rupture of the capillary walls. The proliferative stage was characterized by pronounced proliferation of type II alveolar pneumocytes and multinucleated giant cells. The cytopathic effect of SARS-CoV-2 was observed both in degenerated type II pneumocytes and freely circulating in the alveoli, with components from virions, macrophages, lymphocytes, and cellular debris. CONCLUSIONS: Viral particles consistent with the characteristics of SARS-CoV-2 were observed mainly in degenerated pneumocytes, in the endothelium, or freely circulating in the alveoli. In the final stage of illness, the alveolar spaces were replaced by fibrosis.

Brazil , SARS-CoV-2 , Endothelial Cells , Microscopy, Electron, Transmission , COVID-19 , Lung
Braz. j. med. biol. res ; 54(6): e10722, 2021. graf
Article in English | LILACS | ID: biblio-1285669


Continuous industrial productivity and modern societies have resulted in excess artificial light. The altered circadian rhythm causes many diseases. During intrauterine life, the mother's maternal melatonin rhythm has a major role in influencing organ development. The aim of this study was to investigate the effect of maternal exposure to constant light on the structure and ultrastructure of neonatal skin. Twenty pregnant New Zealand rabbits were divided into two groups (n=10 each): control group (12-h light/dark) and constant light group (24-h light). Plasma maternal melatonin and corticosterone during pregnancy were determined. At the end of the experiment, the dorsal skin of the neonates of both groups was collected and prepared for histological, morphometric, and transmission electron microscopic study. Histological and morphometric results of skin of neonates from the constant light group revealed statistically significantly reduced epidermal thickness, decreased number of hair follicle, increased surface area of collagen, and decreased proliferating cell nuclear antigen (PCNA) positive cells. Ultrastructural examination showed wide intercellular spaces and disrupted desmosomal junctions in the epidermis. Earlier stages of hair follicles were also observed with indented shrunken nuclei, vacuolization, and swollen mitochondria. Dermal fibroblasts with dilated cisternae of rough endoplasmic reticulum containing electron-dense material were detected. Maternal melatonin was significantly reduced in the constant light group while maternal corticosterone showed no significant difference between groups. Therefore, normal maternal circadian rhythm is a key factor for the integrity of neonatal skin structure.

Animals , Female , Pregnancy , Rabbits , Skin , Melatonin , Circadian Rhythm , Microscopy, Electron, Transmission , Epidermis
Mem. Inst. Oswaldo Cruz ; 116: e200443, 2021. tab, graf
Article in English | LILACS | ID: biblio-1154874


BACKGROUND The coronaviruses (CoVs) called the attention of the world for causing outbreaks of severe acute respiratory syndrome (SARS-CoV), in Asia in 2002-03, and respiratory disease in the Middle East (MERS-CoV), in 2012. In December 2019, yet again a new coronavirus (SARS-CoV-2) first identified in Wuhan, China, was associated with a severe respiratory infection, known today as COVID-19. This new virus quickly spread throughout China and 30 additional countries. As result, the World Health Organization (WHO) elevated the status of the COVID-19 outbreak from emergency of international concern to pandemic on March 11, 2020. The impact of COVID-19 on public health and economy fueled a worldwide race to approve therapeutic and prophylactic agents, but so far, there are no specific antiviral drugs or vaccines available. In current scenario, the development of in vitro systems for viral mass production and for testing antiviral and vaccine candidates proves to be an urgent matter. OBJECTIVE The objective of this paper is study the biology of SARS-CoV-2 in Vero-E6 cells at the ultrastructural level. METHODS In this study, we documented, by transmission electron microscopy and real-time reverse transcription polymerase chain reaction (RT-PCR), the infection of Vero-E6 cells with SARS-CoV-2 samples isolated from Brazilian patients. FINDINGS The infected cells presented cytopathic effects and SARS-CoV-2 particles were observed attached to the cell surface and inside cytoplasmic vesicles. The entry of the virus into cells occurred through the endocytic pathway or by fusion of the viral envelope with the cell membrane. Assembled nucleocapsids were verified inside rough endoplasmic reticulum cisterns (RER). Viral maturation seemed to occur by budding of viral particles from the RER into smooth membrane vesicles. MAIN CONCLUSIONS Therefore, the susceptibility of Vero-E6 cells to SARS-CoV-2 infection and the viral pathway inside the cells were demonstrated by ultrastructural analysis.

Humans , Animals , Vero Cells/virology , Cytoplasmic Vesicles/virology , Cytopathogenic Effect, Viral , SARS-CoV-2/physiology , Chlorocebus aethiops , Nucleocapsid , Reverse Transcriptase Polymerase Chain Reaction , Microscopy, Electron, Transmission , Endocytosis , Endoplasmic Reticulum/virology , Virus Internalization , Real-Time Polymerase Chain Reaction
Braz. arch. biol. technol ; 64: e21190387, 2021. tab, graf
Article in English | LILACS | ID: biblio-1153290


HIGHLIGHTS Production of lipid nanoemulsions (<100 nm) of industrial interest with low energy demand. The antioxidant properties of babassu oil have been improved and the nanoemulsions are not cytotoxic. Babassu oil is a food and medicinal product. The nanoemulsion is strategic for the developed of new antioxidants phytotherapeutics.

Abstract Background: Babassu oil is an extract from a Brazilian native coconut (Orbignya phalerata Martius) and is used both as a food and a medicinal product. Methods: we produced two babassu oil nanoemulsions and evaluated them regarding their nanoscopic stability, antioxidant activity and cytotoxicity.The nanoemulsions were characterized by Dynamic Light Scattering, and their stability was investigated for 120 days. The antioxidant activity was assessed by Spectroscopy Electron Paramagnetic Resonance, and the cytotoxicity was assessed by a colorimetric method (MTT) with the NIH/3T3 cell lineage. Results: the results showed nanoemulsions with average hydrodynamic diameter lower than 100 nm (p(0.001).and a polydispersity index of less than 0.3 (p(0.001), indicating monodisperse systems and good stability at room temperature. The exposure of nanoemulsions at varying pH revealed that the isoelectric point was at 3.0, and the images obtained by Transmission Electron Microscopy showed spherical droplets with a size 27 nm. The antioxidant activity showed that the babassu nanoemulsions exposed to free radicals had a better response when compared to the oil free samples. The cell viability assays showed low toxicity of the formulation with viability over 92% (p(0.05). Conclusion: babassu oil nanoformulations showed low polydispersity and kinetic stability with effective antioxidant action. Therefore, they can be promising for application in the food industry or as antioxidant phytotherapeutics.

Palm Oil/chemistry , Nanotechnology , Antioxidants , Plant Extracts/chemistry , Food Industry , Cytotoxins , Microscopy, Electron, Transmission , Emulsions , Nanocomposites
Rev. bras. parasitol. vet ; 30(1): e017020, 2021. tab, graf
Article in English | LILACS | ID: biblio-1156227


Abstract Autophagy plays an important role in maintaining cell homeostasis through degradation of denatured proteins and other biological macromolecules. In recent years, many researchers focus on mechanism of autophagy in apicomplexan parasites, but little was known about this process in avian coccidia. In our present study. The cloning, sequencing and characterization of autophagy-related gene (Etatg8) were investigated by quantitative real-time PCR (RT-qPCR), western blotting (WB), indirect immunofluorescence assays (IFAs) and transmission electron microscopy (TEM), respectively. The results have shown 375-bp ORF of Etatg8, encoding a protein of 124 amino acids in E. tenella, the protein structure and properties are similar to other apicomplexan parasites. RT-qPCR revealed Etatg8 gene expression during four developmental stages in E. tenella, but their transcriptional levels were significantly higher at the unsporulated oocysts stage. WB and IFA showed that EtATG8 was lipidated to bind the autophagosome membrane under starvation or rapamycin conditions, and aggregated in the cytoplasm of sporozoites and merozoites, however, the process of autophagosome membrane production can be inhibited by 3-methyladenine. In conclusion, we found that E. tenella has a conserved autophagy mechanism like other apicomplexan parasites, and EtATG8 can be used as a marker for future research on autophagy targeting avian coccidia.

Resumo A autofagia desempenha um papel importante na manutenção da homeostase celular através da degradação de proteínas desnaturadas e outras macromoléculas biológicas. Nos últimos anos, muitos pesquisadores se concentraram no mecanismo da autofagia em parasitas apicomplexos, mas pouco se sabe sobre esse processo na coccidia aviária. No presente estudo, a clonagem, sequenciamento e caracterização de gene relacionado à autofagia Etatg8 foram investigados pela PCR quantitativa em tempo real (RT-qPCR), mancha ocidental (WB), ensaios indiretos de imunofluorescência (IFAs) e microscopia eletrônica de transmissão (TEM), respectivamente. Os resultados mostraram que o gene Etatg8 de E. tenella possui uma ORF de 375 bp, codificando uma proteína de 124 aminoácidos com estrutura e propriedades semelhantes à de outros apicomplexos. RT-qPCR revelou que Etatg8 é expresso durante os quatro estágios de desenvolvimento de E. tenella. Entretanto, seus níveis transcricionais foram significativamente mais elevados na fase de oocisto não esporulados. Os ensaios de manchas ocidental (WB) e de imunofluorescência (IFA) mostraram que a proteína EtATG8 foi lipidada para ligar-se à membrana do autofagossomo sob condições de deficiência nutritiva (em presença de rapamicina) e se agregar no citoplasma de esporozoítas e merozoítas. No entanto, o processo de produção de membrana do autofagossomo pode ser inibido por um inibidor de autofagia (3-meetiladeninatiladenina, 3-MA). Em conclusão, foi demonstrado que E. tenella tem um mecanismo de autofagia conservado, semelhante ao de outros parasitas apicomplexos, e que EtATG8 pode ser usado como um marcador para futuras pesquisas sobre autofagia direcionada à coccidiose aviária.

Animals , Autophagy/physiology , Bird Diseases/parasitology , Chickens/parasitology , Eimeria tenella/physiology , Coccidiosis/veterinary , Autophagy-Related Protein 8 Family/chemistry , Autophagy/genetics , Bird Diseases/prevention & control , Genetic Markers/physiology , China , Polymerase Chain Reaction , Eimeria tenella/genetics , Cloning, Molecular/methods , Coccidiosis/prevention & control , Oocysts/isolation & purification , Oocysts/physiology , Sporozoites/isolation & purification , Sporozoites/physiology , Microscopy, Electron, Transmission , Merozoites/isolation & purification , Merozoites/physiology , Autophagy-Related Protein 8 Family/genetics
Int. j. morphol ; 38(6): 1525-1527, Dec. 2020. graf
Article in English | LILACS | ID: biblio-1134471


SUMMARY: The study reported the influence of the high and acute dose of Letrozole on the testis morphology in paca (Cuniculus paca), an aromatase inhibitor that reduces the endogenous estrogen, the essential hormone for spermatogenesis. Morphological changes were observed in seminiferous epithelium with germ cells with apoptotic characteristics and presence of vacuoles and nuclei in pycnose.

RESUMEN: El objetivo de este estudio fue analizar la influencia de una dosis alta de Letrozol en la morfología de los testículos de la paca (Cuniculus paca), un inhibidor de la aromatasa que reduce el estrógeno endógeno, la hormona esencial para la espermatogénesis. Se observaron cambios morfológicos en el epitelio seminífero con células germinales con características apoptóticas y la presencia de vacuolas y núcleos en picnosis.

Animals , Male , Testis/drug effects , Aromatase Inhibitors/administration & dosage , Cuniculidae , Letrozole/administration & dosage , Seminiferous Epithelium/drug effects , Spermatogenesis/drug effects , Immunohistochemistry , Orchiectomy , Microscopy, Electron, Transmission , Germ Cells/drug effects
Int. j. morphol ; 38(6): 1606-1613, Dec. 2020. tab, graf
Article in English | LILACS | ID: biblio-1134486


SUMMARY: Disturbances of sensory and motor nerve conduction velocity in the spinal cord as well as degenerated myelin sheaths are observed in diabetic patients and animal models. Indeed, oligodendrocytes (OLs), which are important neuroglial cells, generate myelin in the central nervous system. Spinal enlargement, including cervical and lumbar enlargements, innervates all limbs. Thus, the purposes of this study were to examine and compare the ultrastructural alterations of OLs in spinal enlargements of streptozotocin (STZ)- induced diabetic rats and controls. Thirteen male Sprague-Dawley rats were induced with STZ in citrate buffer and six control rats were injected with the same buffer solution. All rats were sacrificed after inductions at four (short-term DM) and twenty-four weeks (long-term DM). The selected spinal enlargements were processed for transmission electron microscopy. The OL alterations in both the cervical and lumbar enlargements were apparently the same. In short-term DM, the nuclei of OLs became swelled with chromatin clumping. Cytoplasmic organelles were moderately damaged. In long-term DM, OLs contained shrinkage nuclei with thick heterochromatin clumping. Severely degenerated mitochondria with disrupted cristae and broken membranes were observed. Moreover, distended and fragmented rough endoplasmic reticulum were observed, and large clear areas were present in the cytoplasm. Additionally, the loosening, splitting, and destruction of myelin lamellae were found. This study can provide important preliminary information about the alteration of OLs in the spinal cords of diabetic patients, which might be involve in the impairments of sensory and motor conduction velocities in these individuals.

RESUMEN: En pacientes diabéticos y modelos animales se observan alteraciones de la velocidad de conducción nerviosa sensorial y motora en la médula espinal, así como vainas de mielina degeneradas. De hecho, los oligodendrocitos (OL), que son importantes células neurogliales, generan mielina en el sistema nervioso central. La intumescencia espinal, a nivel cervical y lumbar, inerva los miembros. Por lo tanto, los propósitos de este estudio fueron examinar y comparar las alteraciones ultraestructurales de los OL en la intumescencia espinal de ratas diabéticas inducidas por estreptozotocina (STZ) y controles. Se indujeron trece ratas macho Sprague-Dawley con STZ en tampón citrato y se inyectaron seis ratas de control con la misma solución tampón. Todas las ratas se sacrificaron después de la inducción a las cuatro (DM a corto plazo) y a las veinticuatro semanas (DM a largo plazo). Las ampliaciones de la columna seleccionadas se procesaron para microscopía electrónica de transmisión. Las alteraciones de OL en las intumescencias cervical y lumbar eran aparentemente las mismas. En la DM a corto plazo, los núcleos de los OL se hincharon con la acumulación de cromatina. Los orgánulos citoplasmáticos sufrieron daños moderados. En la DM a largo plazo, los OL contenían núcleos de contracción con aglutinación de heterocromatina gruesa. Se observaron mitocondrias severamente degeneradas con crestas y membranas rotas. Además, se observó un retículo endoplásmico rugoso distendido y fragmentado, y estaban presentes grandes áreas claras en el citoplasma. Además, se encontraron el aflojamiento, la división y la destrucción de las laminillas de mielina. Este estudio puede proporcionar información preliminar importante sobre la alteración de los OL en la médula espinal de los pacientes diabéticos, que podría estar involucrada en las alteraciones de las velocidades de conducción sensorial y motora en estos individuos.

Animals , Male , Rats , Spinal Cord/pathology , Oligodendroglia/pathology , Diabetes Mellitus, Experimental/pathology , Spinal Cord/ultrastructure , Central Nervous System , Oligodendroglia/ultrastructure , Rats, Sprague-Dawley , Microscopy, Electron, Transmission , Myelin Sheath
Int. j. morphol ; 38(3): 585-591, June 2020. graf
Article in English | LILACS | ID: biblio-1098291


Acetaminophen (also called paracetamol, or APAP) induced nephrotoxicity is reported after accidental or intentional ingestion of an overdose of the drug. Renal tubular ultrastructural alterations induced by APAP overdose associated with the induction of biomarkers of kidney injury have not been investigated before. Also, we investigated whether the combined polyphenolic anti-inflammatory and antioxidants agents, resveratrol (RES) and quercetin (QUR) can protect against APAP-induced acute kidney injury. The model group of rats received a single dose of APAP (2 g/kg), whereas the protective group of rats was pre-treated for 7 days with combined doses of RES (30 mg/kg) and QUR (50 mg/kg) before being given a single dose of APAP. All rats were then sacrificed one day post APAP ingestion. Harvested kidney tissues were prepared for transmission electron microscopy (TEM) staining and blood samples were assayed for urea, creatinine, and biomarkers of inflammation and oxidative stress. TEM images and blood chemistry analysis showed that APAP overdose induced kidney damage as demonstrated by substantial alterations to the proximal convoluted tubule ultrastructure, and a significant (p<0.05) increase in urea, creatinine, tumor necrosis factor-alpha (TNF-a), and malondialdehyde (MDA) blood levels, which were protected by RES+QUR. These findings indicate that APAP induces alterations to the renal tubular ultrastructure, which is inhibited by resveratrol plus quercetin, which also decreases blood levels of kidney injury biomarkers.

El objetivo de este trabajo fue estudiar la nefrotoxicidad inducida por acetaminofeno (también llamado paracetamol o APAP) después de la ingestión accidental o intencional de una sobredosis de la droga. Las alteraciones ultraestructurales tubulares renales inducidas por sobredosis de APAP asociadas con la inducción de biomarcadores de daño renal no se han investigado. Además, estudiamos si los agentes combinados antiinflamatorios y antioxidantes polifenólicos, el resveratrol (RES) y la quercetina (QUR) pueden proteger contra la lesión renal aguda inducida por APAP. El grupo modelo de ratas recibió una dosis única de APAP (2 g / kg), mientras que el grupo protector de ratas se trató previamente durante 7 días con dosis combinadas de RES (30 mg / kg) y QUR (50 mg / kg) antes de recibir una dosis única de APAP. Todas las ratas se sacrificaron un día después de la ingestión de APAP. Los tejidos renales fueron preparados para el análisis a través de la microscopía electrónica de transmisión (MET). En las muestras de sangre se determinaron la urea, creatinina y los biomarcadores de inflamación y estrés oxidativo. Las imágenes MET y el análisis químico de la sangre mostraron que la sobredosis de APAP inducía daño renal, como lo demuestran las alteraciones sustanciales en la ultraestructura del túbulo contorneado proximal, y además, de un aumento significativo (p <0,05) de la urea, creatinina, factor de necrosis tumoral alfa y niveles sanguíneos de malondialdehído, protegidos por RES + QUR. Estos hallazgos indican que APAP induce alteraciones en la ultraestructura tubular renal, inhibida por el resveratrol más quercetina, que también disminuye los niveles sanguíneos de biomarcadores de daño renal.

Animals , Rats , Quercetin/administration & dosage , Resveratrol/administration & dosage , Kidney Tubules/drug effects , Acetaminophen/toxicity , Quercetin/pharmacology , Urea/blood , Rats, Sprague-Dawley , Creatinine/blood , Microscopy, Electron, Transmission , Disease Models, Animal , Drug Overdose , Resveratrol/pharmacology , Kidney Tubules/pathology , Anti-Inflammatory Agents/administration & dosage , Antioxidants/administration & dosage
Int. j. morphol ; 38(3): 602-610, June 2020. graf
Article in English | LILACS | ID: biblio-1098294


Due to the great change in the morphology of squamate vomeronasal organ (VNO), the histomorphology characteristics of VNO in Scincella tsinlingensis were studied by light and electronic microscopy. The results indicated that the VNO of S. tsinlingensis was located at the base of nasal cavity and consisted of a mushroom body situated anteroventrally and a sensory epithelium (SE) situated dorsocaudally. SE was composed of supporting cells, receptor cells and basal cells, and the supporting cells contained secretory granules near the surface membrane. Most of receptor cells were irregular in shape with long cytoplasmic extensions and characterized by microtubules, vesicles, and mitochondria. The basal cells with long cytoplasmic extensions were also irregular in shape and appeared a greater electron density than others. The thick nerve bundles were found on the dorsomedial area of VNO, and the surface of mushroom body was non-sensory epithelium consisting of ciliated and basal cells, without goblet cells. Epithelial cells were arranged in irregular, with many cilia and microvilli distributed on its free surface. Cells on the basal layer were irregularly circular in shape and arranged sparsely. Taken together, the results indicated that the fine structure of VNO in S. tsinlingensis was similar to other species from scincomorphs.

Debido al gran cambio en la morfología del órgano vomeronasal (OVN), se estudiaron las características histomorfológicas en la Scincella tsinlingensis por microscopías de luz y electrónica. Los resultados indicaron que el OVN de S. tsinlingensis se localizaba en la base de la cavidad nasal y consistía en un cuerpo como hongo situado anteroventralmente y un epitelio sensorial (ES) situado dorso caudamente. El ES estaba compuesto de células de soporte, células receptoras y células basales, y las células de soporte contenían gránulos secretores cerca de la membrana superficial. En gran parte de la mayoría de las células receptoras se observó una forma irregular con largas extensiones citoplasmáticas, caracterizadas por microtúbulos, vesículas y mitocondrias. Las células basales con extensiones citoplasmáticas también tenían forma irregular y algunas parecían tener una mayor densidad de electrones. Los haces gruesos nerviosos se encontraron en el área dorsomedial del OVN, la superficie del cuerpo de estaba compuesto de epitelio no sensorial y consistía de células ciliadas y basales, sin células caliciformes. Las células epiteliales estaban dispuestas de manera irregular, con muchos cilios y microvellosidades distribuidas en su superficie libre. Las células en la capa basal eran escasas y de forma circular irregular. Tomados en conjunto, los resultados indicaron que la estructura fina del OVN en S. tsinlingensis era similar a otras especies de scincomorpha.

Animals , Vomeronasal Organ/anatomy & histology , Lizards/anatomy & histology , Microscopy, Electron, Scanning , Vomeronasal Organ/ultrastructure , Microscopy, Electron, Transmission
Arq. bras. oftalmol ; 83(2): 146-148, Mar.-Apr. 2020. graf
Article in English | LILACS | ID: biblio-1088972


ABSTRACT Lisch corneal dystrophy is a rare corneal disease characterized by the distinctive feature of highly vacuolated cells. Although this feature is important, the nature of these vacuoles within corneal cells remains unknown. Here, we sought to analyze corneal cells from a patient diagnosed with Lisch dystrophy to characterize the vacuoles within these cells. Analyses using histopathology examination, confocal microscopy, and transmission electron microscopy were all consistent with previous descriptions of Lisch cells. Importantly, the vacuoles within these cells appeared to be autophagosomes and autolysosomes, and could be stained with an anti-microtubule-associated protein 1A/1B-light chain 3 (LC3) antibody. Taken together, these findings indicate that the vacuoles we observed within superficial corneal cells of a patient with Lisch corneal dystrophy constituted autophagosomes and autolysosomes; this finding has not been previously reported and suggests a need for further analyses to define the role of autophagy in this ocular disease.

RESUMO A distrofia corneana de Lisch é uma doença rara, caracterizada principalmente pela presença de células altamente vacuoladas. Embora esta característica seja importante, a natureza desses vacúolos dentro das células da córnea permanece des conhecida. Aqui, procuramos analisar as células da córnea de um paciente diagnosticado com distrofia de Lisch para caracte rizar os vacúolos dentro dessas células. Análises utilizando exame histopatológico, microscopia confocal e microscopia eletrônica de transmissão foram todas consistentes com descrições previas de células de Lisch. Importante, os vacúolos dentro dessas células pareciam ser autofagossomos e autolisossomos, e po deriam ser corados com um anticorpo proteico 1A/1B-cadeia leve 3 (LC3) da proteína anti-microtúbulo associado a microtúbulos. Em conjunto, esses achados indicam que os vacúolos observados nas células superficiais da córnea de um paciente com distrofia corneana de Lisch constituíram autofagossomos e autolisossomos. Esse achado não foi relatado anteriormente e sugere a necessidade de mais análises para definir o papel da autofagia nessa doença ocular.

Humans , Female , Adult , Vacuoles/pathology , Corneal Dystrophies, Hereditary/pathology , Autophagosomes/pathology , Corneal Dystrophies, Hereditary/diagnostic imaging , Microscopy, Confocal/methods , Corneal Opacity/pathology , Corneal Opacity/diagnostic imaging , Tomography, Optical Coherence/methods , Microscopy, Electron, Transmission/methods , Microautophagy