Morphology and morphogenesis of SARS-CoV-2 in Vero-E6 cells

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.

Plesiomonas shigelloides exports a lethal cytotoxic-enterotoxin (LCE) by membrane vesicles

ABSTRACT Plesiomonas shigelloides isolated from water in Brazil was previously described as a hemorrhagic heat-labile cytotoxic-enterotoxin producer. We purified this toxin from culture supernatants using ion metallic affinity chromatography (IMAC) followed by molecular exclusion chromatography. The pure toxin presented molecular mass of 50 kDa and isoelectric point (pI) around 6.9 by 2D electrophoresis. When injected intravenously, the purified cytotoxic-enterotoxin induced also severe spasms followed by sudden death of mice. Hence, we entitled it as lethal cytotoxic-enterotoxin (LCE). The presence of membrane vesicles (MVs) on cell surfaces of P. shigelloides was observed by scan electron microscopy (SEM). From these MVs the LCE toxin was extracted and confirmed by biological and serological assays. These data suggest that P. shigelloides also exports this cytotoxic-enterotoxin by membrane vesicles, a different mechanism of delivering extra cellular virulence factors, so far not described in this bacterium.

Hydration status affects osteopontin expression in the rat kidney

Osteopontin (OPN) is a secretory protein that plays an important role in urinary stone formation. Hydration status is associated with the development of urolithiasis. This study was conducted to examine the effects of dehydration and hydration on OPN expression in the rat kidney. Animals were divided into three groups, control, dehydrated, and hydrated. Kidney tissues were processed for light and electron microscope immunocytochemistry, in situ hybridization, and immunoblot analysis. Dehydration induced a significant increase in OPN protein expression, whereas increased fluid intake induced a decrease in protein expression. Under control conditions, OPN protein and mRNA expression were only detected in the descending thin limb (DTL). Dehydration induced increased expression in the DTL and the development of detectable expression in the thick ascending limb (TAL). In contrast, OPN expression levels declined to less than the controls in the DTL after hydration, while no expression of either protein or mRNA was detectable in the TAL. Immunoelectron microscopy demonstrated that hydration status altered tubular ultrastructure and intracellular OPN expression in the Golgi apparatus and secretory cytoplasmic vesicles. These data confirm that changes in oral fluid intake can regulate renal tubular epithelial cell OPN expression.

Protective effect of bone marrow mesenchymal stem cell-derived microvesicles on glutamate injured PC12 cells / 中国实验血液学杂志

This study was aimed to investigate the protective effect of bone mesenchymal stem cell-derived microvesicles (BMMSC-MV) on glutamate injured PC12 cells so as to elucidate the mechanism of the neural damage repair. BMMSC were isolated and purified with density-gradient centrifugation method, BMMSC-MV were harvested from the supernatants of BMMSC by hypothermal ultracentrifugation method. The surface markers of BMMSC reacted against different antibodies were detected by flow cytometry. The morphology features of MV were observed under an electron microscope. Experiment was divided into three groups, one was a control group, and the other two were glutamate-injured group and co-culture group of BMMSC-MV and glutamate-damaged cells respectively. MTT test was used to evaluate the proliferative status of PC12 cells and the AnnexinV-FITC detecting kit and Hoechst33342 were used to detect the apoptosis of PC12 cells in different groups. The results showed that BMMSC isolated from rat bone marrow were highly positive for CD29, CD44 and negative for CD31, CD34 and CD45. The morphology of MV was round and the vesicles were homogenous in size. BMMSC-MV exhibited a protective effect on the excitotoxicity-injured PC12 cells, displaying increase of cell viability, decrease of Annexin-V/PI staining positive and nuclear condensed cells. It is concluded that BMMSC-MV can protect PC12 cells from glutamate-induced apoptosis, suggesting that BMMSC-MV may be a potential candidate for treatment of neurological diseases.This study provides the preliminary experimental and theoretical evidence for use of BMMSC-MV in treatment of neural excited damage.

Roles of microvesicles in tumor progress and its clinical application / 浙江大学学报·医学版

Microvesicles transport special proteins, micro RNA and DNA segments, which provides new access to intercellular communication. Tumor-derived membrane microvesicles (TMV) are involved in the tumor progress by transporting tumor-derived proteins, delivering microRNA to surrounding normal cells to alter their phenotype and promoting reverse transcription to interfere gene stability and to create tumor microenvironment. TMV also play crucial roles in tumor angiogenesis and matrix degradation, which facilitates malignant cell metastasis. TMVs are also involved in escaping immunological surveillance by intensifying the function of suppressor T cell and inducing apoptosis of cytotoxic T cells. On the other hand, microvesicles carry tumor antigens and can be used for development of tumor vaccines; some new vaccines such as AEX and DEX are under early clinical trials. Circulating microRNA and DNA segments in body fluid can be a new potential biomarker for cancer diagnosis and prognosis. Purification of microvesicles needs to be further improved, which is important for identification of microvesicles and their subtypes.

Steatosis of indeterminate cause in a pediatric group: is it a primary mitochondrial hepatopathy? / Esteatose de causa não determinada em grupo pediátrico: hepatopatia mitocondrial primária?

CONTEXT AND OBJECTIVE: In children, hepatic steatosis may be related to inborn errors of metabolism (IEMs) or to non-alcoholic fatty liver disease (NAFLD). The aim of this study was to assess and characterize steatosis of indeterminate cause through morphological and morphometric analysis of liver tissue. DESIGN AND SETTING: Cross-sectional study at the Departments of Pathology of Faculdade de Ciências Médicas, Universidade Estadual de Campinas (FCM-Unicamp) and Faculdade de Medicina de Botucatu, Universidade Estadual Paulista (FMB-Unesp). METHODS: Eighteen consecutive liver biopsies obtained from 16 patients of ages ranging from 3 months to 12 years and nine months that were inserted in a database in the study period were analyzed using optical microscopy and transmission electron microscopy. Through electron microscopy, the mitochondrial density and mean mitochondrial surface area were determined in hepatocytes. Ten patients ranging in age from 1 to 14 years were used as a control group. RESULTS: "Pure" steatosis was detected, unaccompanied by fibrosis or any other histological alteration. Microvesicular steatosis predominated, with a significant increase in mean mitochondrial surface area. CONCLUSION: Microvesicular steatosis may be related to primary mitochondrial hepatopathy, especially due to reduction of β-oxidation or partial stagnation of oxidative phosphorylation. For these reasons, this form of steatosis (which should not be called "pure") is likely to represent an initial stage in the broad spectrum of NAFLD. We have drawn attention to cases of steatosis in the pediatric group, in which the microvesicular form predominates, since this may be associated with mitochondrial disorders.

CONTEXTO E OBJETIVO: Em crianças, a esteatose hepática pode se relacionar a erros inatos do metabolismo (EIMs) ou à doença hepática gordurosa não-alcoólica (DHGNA). O objetivo deste estudo foi avaliar e caracterizar esteatose de causa indeterminada por meio de análises morfológica e morfométrica em tecido hepático. TIPO DE ESTUDO E LOCAL: Estudo transversal nos Departamentos de Patologia da Faculdade de Ciências Médicas da Universidade Estadual de Campinas (FCM-Unicamp) e Faculdade de Medicina de Botucatu da Universidade Estadual Paulista (FMB-Unesp). MÉTODOS: Foram utilizadas 18 biópsias hepáticas consecutivas obtidas de 16 pacientes com idade variando de 3 meses a 12 anos e 9 meses, inseridas num banco de dados no período do estudo, que foram analisadas por microscopia óptica e eletrônica. Na microscopia eletrônica, foi realizada determinação da densidade mitocondrial e da área superficial média das mitocôndrias nos hepatócitos. Dez pacientes com idade variando de 1 a 14 anos foram usados como grupo controle. RESULTADOS: Foi detectada esteatose "pura", não acompanhada por fibrose ou outra alteração histológica. Foi verificado que, na predominância de esteatose microvesicular, houve aumento significativo da área mitocondrial média. CONCLUSÃO: A esteatose microvesicular pode estar relacionada à hepatopatia mitocondrial primária, principalmente devido à redução na β-oxidação ou parcial estagnação da fosforilação oxidativa. Por essas razões, esta forma de esteatose (que não pode ser chamada de "pura") possivelmente represente uma fase inicial no amplo espectro da DHGNA. Chamamos a atenção para casos de esteatose no grupo pediátrico com predomínio da forma microvesicular, uma vez que pode haver associação com desordens mitocondriais.

Tumour-derived exosomes and their roles in cancer / 中南大学学报(医学版)

Exosomes are nanometer sized membrane vesicles, released in the extracellular milieu following the fusion of the external membrane of multivesicular body (MVB) with plasma membrane. They perform a certain function in immune regulation. Exosomes have been shown to be released by cells of hematopoietic and non-hematopoietic origin. Tumour-derived exosomes (TEX) exist in the supernatant of tumour cells, plasma and malignant effusions of tumour patients. They contain native candidate tumour associated antigen and are capable of transferring antigens to T lymphocytes, therefore efficiently promoting cytotoxic T lymphocyte (CTL) activation and producing antitumor immunity. However, recent evidence shows that tumor exosomes may induce immunologic tolerance and even activate immunosuppression which makes tumour escape from the immune surveillance of the host immune system. In addition, tumor exosomes may mediate a growth-promoting effect on tumor cells. These discrepancies are almost certainly due to differences in the phenotype of the exosomes.

Contribution of matrix vesicles and alkaline phosphatase to ectopic bone formation

Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 ± 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PPi), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PPi as substrates. While the enzymatic efficiency (k cat/Km) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat/Km for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.

Dehydration Increases Osteopontin Expression in Rat Kidney / 대한해부학회지

Osteopontin (OPN), is a secreted phosphoprotein that is expressed in the normal kidney. However, little is known about the role of OPN in the kidney. The purpose of this study was to establish the effect of dehydration on renal OPN expression. Dehydrated rats had free access to normal rat chow, but were deprived of water for 3 days. Kidney tissues were preserved by in vivo perfusion with paraformaldehyde-lysine-periodate (PLP) and processed for immunocytochemistry and in situ hybridization. Serum sodium concentration and urine osmolality were increased in dehydrated rats. Both OPN mRNA and protein were expressed restrictively in the descending thin limb (DTL) and papillary surface epithelium (PSE) in control kidneys. In dehydrated kidneys, there was an increase in OPN mRNA and protein expression in the thick ascending limb (TAL) as well as DTL and PSE. Electron microscopy revealed that OPN immunoreactivity in the DTL and TAL cells was located in the Golgi apparatus and in small cytoplasmic vesicles. These results demonstrate that dehydration status increases the expression of OPN in renal tubules and stimulates the secretion into the urine.

Mechanisms of leukocyte lipid body formation and function in inflammation

An area of increasingly interest for the understanding of cell signaling are the spatio-temporal aspects of the different enzymes involved in lipid mediator generation (eicosanoid-forming enzymes, phospholipases and their regulatory kinases and phosphatases) and pools of lipid precursors. The compartmentalization of signaling components within discrete and dynamic sites in the cell is critical for specificity and efficiency of enzymatic reactions of phosphorilation, enzyme activation and function. We hypothesized that lipid bodies - inducible non-membrane bound cytoplasmic lipid domains - function as specialized intracellular sites of compartmentalization of signaling with major roles in lipid mediator formation within leukocytes engaged in inflammatory process. Over the past years substantial progresses have been made demonstrating that all enzymes involved in eicosanoid synthesis localize at lipid bodies and lipid bodies are distinct sites for eicosanoid generation. Here we will review our current knowledge on the mechanisms of formation and functions of lipid bodies pertinent to inflammation.

Características de nuevos cultivos celulares derivados de tejidos embrionarios de Aedes aegypti (Diptera: Culicidae) / Characteristics of new cell cultures derived from embryonic tissues of Aedes aegypti (Diptera: Culicidae)

Introducción. Los cultivos celulares de insectos son una metodología útil en estudios biomédicos y tecnológicos.Objetivo. El propósito principal del presente trabajo fue obtener y caracterizar cultivos celulares derivados de tejidos embrionarios de Aedes aegypti. Materiales y métodos. Se emplearon huevos embrionados para los explantes de tejidos en los medios de cultivos MM/VP12 y L-15/Grace, con suplemento de 20 por ciento de suero fetal bovino y una mezcla al 1 por ciento de antibiótico y antimicótico, con un rango de pH entre 6,8 y 7,0. Los cultivos se incubaron a una temperatura de 28oC sin atmósfera de CO2. Resultados. El crecimiento celular se obtuvo en el medio L-15/Grace, 3 semanas después de haber sido sembrados los tejidos embrionarios; sin embargo, se necesitaron 6 meses para la formación de la monocapa confluente. Desde agosto de 2003 hasta junio de 2004, se habían realizado 28 subcultivos. Las células se caracterizaron morfológicamente; predominaron las formas epitelioides en subcultivos de pases altos. También se reconocieron las particularidades morfométricas del cariotipo y, además, se determinaron los perfiles isoenzimáticos y moleculares de los cultivos celulares, los cuales se compararon con muestras de adultos de la especie tomadas de la misma colonia y con líneas celulares derivadas de otros insectos.Discusión y conclusiones. Estas células representan, potencialmente, un importante sistema in vitro en investigaciones básicas y aplicadas

Expression of Osteopontin in Chronic Potassium Depletion-induced Renal Injury / 대한해부학회지

Osteopontin (OPN), a potent chemoattractant for the monocyte/macrophage infiltration, is highly upregulated in the renal tubular epithelium during various pathologic conditions associated with tubulointerstitial injury. The purpose of this study was to establish the distribution and localization of OPN in the tubulointerstitial injury induced by chronic potassium (K+) deprivation in the rat kidney. Sprague-Dawley rats were fed either a normal or a K+ -deficient diet for 2 weeks. Kidney tissues were preserved by in vivo perfusion with paraformaldehyde-lysine-periodate (PLP) and processed for light and electron microscope immunocytochemistry using monoclonal antibodies to OPN. K+ -depleted kidneys showed tubulointerstitial injury with renal hypertrophy, monocyte/macrophage infiltration, interstitial fibrosis, and OPN overexpression. OPN immunoreactivity was observed only in the descending thin limb (DTL) and the papillary surface epithelium (PSE) in the control kidney. However, the OPN labeling was observed not only in DTL and PSE but also in the thick ascending limb (TAL) in the K+ -depleted kidney. Electron microscopy revealed that OPN induced in the TAL cells was not located in the basal plasma membrane, but in the Golgi apparatus and in subapical cytoplasmic vesicles. There was no OPN expression in the epithelium of the collecting ducts, which showed marked morphological damages with mononuclear cell infiltration. These results demonstrate that chronic K+ -deprivation causes renal injury with the increased OPN expression in tubular epithelial cells. However, the localization of the induced OPN suggests other roles rather than a chemoattractant function in this renal injury model.

V2 receptor-mediated regulation of aquaporin-2 by oxytocin in the kidney / 대한내과학회지

BACKGROUND: Oxytocin is a nonapeptide hormone secreted from posterior pituitary gland and has a very similar structure to vasopressin. The aquaporin-2 (AQP2) water channel is predominantly expressed in the kidney and plays a key role in regulation of water permeability of mammalian collecting duct, exerted by both short-term and long-term vasopressin action. We speculated that oxytocin may be involved in some part of vasopressin-independent urinary concentrating mechanism by regulating AQP2 trafficking in the kidney. METHODS: This study was undertaken to investigate whether and how the acute stimulation of oxytocin induces changes in AQP2 localization in the kidney. Immunohistochemistry and semiquantitative immunoblotting of AQP2 were carried out from Sprague-Dawley rat kidneys after a single intraperitoneal injection of oxytocin with or without pretreatment of a vasopressin-2 receptor (V2R) antagonist. RESULTS: Urinary cAMP excretion was increased by oxytocin administration. Immuno- histochemistry of inner medullary collecting duct (IMCD) revealed that AQP2 was shifted from diffuse cytoplasmic localization in controls to the apical and basolateral membrane domains in oxytocin-treated rats. This pattern of AQP2 redistribution was noted in connecting tubule, cortical collecting duct and outer medullary collecting duct as in IMCD, although the tendency to basolateral localization was somewhat less. Semiquantitative immunoblotting of membrane fractions of whole kidney homogenates was also used to assess redistribution of AQP2. The band density ratio of the plasma membrane-rich fraction over cytoplasmic vesicle-rich fraction was higher in oxytocin-treated rats than in controls (3.64+/-0.60 vs. 1.09+/-0.14, P<0.05). Regarding the receptor pathway of oxytocin action in the kidney, we found that pretreatment with a V2R antagonist (OPC-31260) blocked redistribution of AQP2 which was induced by oxytocin. CONCLUSION: In conclusion, oxytocin induces a V2R-mediated redistribution of AQP2-containing cytoplasmic vesicles to both apical and basolateral plasma membrane domains in rat kidney. Oxytocin may be one of the factors that accounts for vasopressin-independent AQP2 targeting in the kidney.

Cancer immunotherapy using dendritic cell-derived exosomes

Dendritic cells (DCs) are the most potent antigen presenting cells and the only ones capable of inducing primary cytotoxic immune responses. We found that DCs secrete a population of membrane vesicles, called exosomes. Exosomes are 60-80 nm vesicles of endocytic origin. The protein composition of exosomes was subjected to a systematic proteomic analysis. Besides MHC and co-stimulatory molecules, exosomes bear several adhesion proteins, most likely involved in their specific subjected to targeting. We also found that exosomes accumulate several cytosolic factors, probably involved in their endosomal biogenesis. Like DCs, exosomes induced immune responses in vivo. Indeed, a single injection of DC-derived exosomes sensitized with tumor peptides induced potent anti tumor immune responses in mice and the eradication of established tumors. Tumor-specific cytotoxic T lymphocytes were found in the spleen of exosome-treated mice, and the anti tumor effect of exosomes was sensitive to in vivo depletion of CD8+ T cells. These results show that exosomes induce potent anti tumor effects in vivo, and strongly support the implementation of human DC-derived exosomes for cancer immunotherapy.

Light and electron microscopic study of liver glycogen heterogeneity

Seven ad-libidum fed rats and seven overnight fasted rats where used in this experiment. Their livers where subjected to enzymatic glycogen determination, PAS staining and E.M. examination. High glycogen content was detected in the livers of fed rats. The livers showed discrete glycogen particles in contact with vesicles of SER in cetrilobuar hepatocytes and glycogen lacks surrounded with few vesicles in periportal areas. Liver of fasted animals showed low glycogen level and marked reduction of glycogen content with extensive SER proliferation especially in centrilobular hepatocytes. The study provides evidence of the regional variation in ultrastructure and glycogen distribution in fasted and fed animals. The study reflects more light about the dynamic action of SER in relation to both hepatocytic glycogen deposition and depletion according to the nutritional status. The variation in strength and distribution of PAS staining of hepatocytes of fed rats could be clarified in light of their ultrastrucre and lobular location. A close relationship was observed between peroxisomes and both glycogen particles and SER suggesting a role of peroxisomes in glycogen synthesis. We recommend that the study of the ultrastructure of the liver should be always correlated to the nutritional status of the animals, a precise determination of liver glycogen level for each particular spacemen and accurate localization of the hepatocytes in relation to their lobular patern

Morphologic Changes of the Urinary Bladder Mucosa Induced by E. coli / 대한비뇨기과학회지

This study was carried out to investigate pathogenesis of E. coli-induced cystitis by light and electron microscopy with immunocytochemistry. A group of ICR mice was intravesically given 0.3ml of E. coli DIE 174 lysate( 1000000000/ml). Another group was given E. coli lysate after acid solution(pH 4.5) washing for 30 minutes. The urinary bladders were observed at 2 hours and 24 hours after E.coli lysate irrigation. On light microscopy, submucosal edema, congestion, and perivascular neutrophilic infiltration occurred 2 hours after E. coli lysate irrigation. The congestion and acute inflammation were more pronounced 24 hours after. In cases of acid washing and E. coli lysate irrigation, mucosal acute inflammatory infiltration with edema and congestion was marked at early stage. Necrosis of transitional cells developed with time. On immunostain for E. coli, surface of transitional cells was stained 2 hours after E. coli lysate irrigation. At 24 hours after irrigation, intercellular spaces of transitional cells were stained. In cases of acid washing and E. coli lysate irrigation, entire mucosa was stained at early stage. Electron microscopically, transitional cells revealed increase of cytoplasmic processes, enlargement of cytoplasmic vesicles, and widening of intercellular spaces. There were large amounts of various shapes of lysosomes in neutrophils and monocytes infiltrated in the mucosa in acid washing groups. According to the above results, it is concluded that E. coli lysate may easily penetrate into the bladder mucosa and that the mucosal mucopolysaccharide plays as a barrier against bacterial invasion.