RESUMEN

OBJECTIVE: For treatment of medication-related osteonecrosis of the jaw, one proposed approach is the use of a topical agent to block entry of these medications in oral soft tissues. We tested the ability of phosphonoformic acid (PFA), an inhibitor of bisphosphonate entry through certain sodium-dependent phosphate contransporters (SLC20A1, 20A2, 34A1-3) as well as Dynasore, a macropinocytosis inhibitor, for their abilities to prevent zoledronate-induced (ZOL) death in human gingival fibroblasts (HGFs). METHODOLOGY: MTT assay dose-response curves were performed to determine non-cytotoxic levels of both PFA and Dynasore. In the presence of 50 µM ZOL, optimized PFA and Dynasore doses were tested for their ability to restore HGF viability. To determine SLC expression in HGFs, total HGF RNA was subjected to quantitative real-time RT-PCR. Confocal fluorescence microscopy was employed to see if Dynasore inhibited macropinocytotic HGF entry of AF647-ZOL. Endosomal acidification in the presence of Dynasore was measured by live cell imaging utilizing LysoSensor Green DND-189. As a further test of Dynasore's ability to interfere with ZOL-containing endosomal maturation, perinuclear localization of mature endosomes containing AF647-ZOL or TRITC-dextran as a control were assessed via confocal fluorescence microscopy with CellProfiler™ software analysis of the resulting photomicrographs. RESULTS: 0.5 mM PFA did not rescue HGFs from ZOL-induced viability loss at 72 hours while 10 and 30 µM geranylgeraniol did partially rescue. HGFs did not express the SLC transporters as compared to the expression in positive control tissues. 10 µM Dynasore completely prevented ZOL-induced viability loss. In the presence of Dynasore, AF647-ZOL and FITC-dextran co-localized in endosomes. Endosomal acidification was inhibited by Dynasore and perinuclear localization of both TRITC-dextran- and AF647-ZOL-containing endosomes was inhibited by 30 µM Dynasore. CONCLUSION: Dynasore prevents ZOL-induced viability loss in HGFs by partially interfering with macropinocytosis and by inhibiting the endosomal maturation pathway thought to be needed for ZOL delivery to the cytoplasm.

Asunto(s)

Supervivencia Celular , Difosfonatos , Endosomas , Fibroblastos , Encía , Hidrazonas , Imidazoles , Ácido Zoledrónico , Ácido Zoledrónico/farmacología , Humanos , Fibroblastos/efectos de los fármacos , Encía/efectos de los fármacos , Encía/citología , Difosfonatos/farmacología , Imidazoles/farmacología , Supervivencia Celular/efectos de los fármacos , Endosomas/efectos de los fármacos , Hidrazonas/farmacología , Células Cultivadas , Factores de Tiempo , Reacción en Cadena en Tiempo Real de la Polimerasa , Conservadores de la Densidad Ósea/farmacología , Reproducibilidad de los Resultados , Microscopía Confocal , Relación Dosis-Respuesta a Droga , Pinocitosis/efectos de los fármacos

RESUMEN

Macropinocytosis is a nonspecific endocytic process that may enhance cancer cell survival under nutrient-poor conditions. Ataxia-Telangiectasia mutated (ATM) is a tumor suppressor that has been previously shown to play a role in cellular metabolic reprogramming. We report that the suppression of ATM increases macropinocytosis to promote cancer cell survival in nutrient-poor conditions. Combined inhibition of ATM and macropinocytosis suppressed proliferation and induced cell death both in vitro and in vivo. Supplementation of ATM-inhibited cells with amino acids, branched-chain amino acids (BCAAs) in particular, abrogated macropinocytosis. Analysis of ATM-inhibited cells in vitro demonstrated increased BCAA uptake, and metabolomics of ascites and interstitial fluid from tumors indicated decreased BCAAs in the microenvironment of ATM-inhibited tumors. These data reveal a novel basis of ATM-mediated tumor suppression whereby loss of ATM stimulates protumorigenic uptake of nutrients in part via macropinocytosis to promote cancer cell survival and reveal a potential metabolic vulnerability of ATM-inhibited cells.

Asunto(s)

Proteínas de la Ataxia Telangiectasia Mutada , Neoplasias , Pinocitosis , Humanos , Adaptación Fisiológica , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Reprogramación Celular , Neoplasias/metabolismo , Microambiente Tumoral , Aminoácidos de Cadena Ramificada/metabolismo , Metabolómica , Animales , Ratones , Línea Celular Tumoral

RESUMEN

Clostridioides difficile is the main causative agent of hospital-acquired diarrhea and the potentially lethal disease, C. difficile infection. The cornerstone of the current therapy is the use of antibiotics, which is not fully effective. The molecular mechanisms, inflammatory conditions and host-immune responses that could benefit the persistence or elimination of C. difficile remain unclear. Macrophages perform different ways of endocytosis as part of their immune surveillance functions and platelets, classically known for their coagulatory role, are also important modulators of the immune system. The aim of this study was to evaluate the endocytosis of vegetative C. difficile by human macrophages and the involvement of platelets in this process. Our results showed that both macrophages and platelets interact with live and heat-killed C. difficile. Furthermore, platelets form complexes with human monocytes in healthy donor's fresh blood and the presence of C. difficile increased these cell-cell interactions. Using flow cytometry and confocal microscopy, we show that macrophages can internalize C. difficile and that platelets improve this uptake. By using inhibitors of different endocytic pathways, we demonstrate that macropinocytosis is the route of entry of C. difficile into the cell. Taken together, our findings are the first evidence for the internalization of vegetative non-toxigenic and hypervirulent C. difficile by human macrophages and highlight the role of platelets in innate immunity during C. difficile infection. Deciphering the crosstalk of C. difficile with immune cells could provide new tools for understanding the pathogenesis of C. difficile infection and for the development of host-directed therapies.

Asunto(s)

Clostridioides difficile , Humanos , Clostridioides , Plaquetas , Macrófagos , Pinocitosis

RESUMEN

Mycobacterium tuberculosis has developed diverse mechanisms to survive inside phagocytic cells, such as macrophages. Phagocytosis is a key process in eliminating invading pathogens; thus, M. tuberculosis efficiently disrupts phagosome maturation to ensure infection. However, inflammatory cytokines produced by macrophages in response to early M. tuberculosis infection are key to promoting bacterial clarification. IFN-γ enhances M. tuberculosis engulfment and destruction by reprogramming macrophages from phagocytosis to macropinocytosis. Here, we show that the transcription factor Krüppel-like factor 10 (Klf10) plays a positive role in M. tuberculosis survival and infection by negatively modulating IFN-γ levels. Naïve Klf10-deficient macrophages produce more IFN-γ upon stimulation than wild-type macrophages, thus enhancing bacterial uptake and bactericidal activity achieved by macropinocytosis. Moreover, Klf10⁻/ ⁻ macrophages showed cytoplasmic distribution of coronin 1 correlated with increased pseudopod count and length. In agreement with these observations, Klf10⁻/ ⁻ mice showed improved bacterial clearance from the lungs and increased viability. Altogether, our data indicate that Klf10 plays a critical role in M. tuberculosis survival by preventing macrophage reprogramming from phagocytosis to macropinocytosis by negatively regulating IFN-γ production upon macrophage infection.

Asunto(s)

Factores de Transcripción de Tipo Kruppel , Macrófagos , Mycobacterium tuberculosis , Tuberculosis , Animales , Factores de Transcripción de la Respuesta de Crecimiento Precoz , Interferón gamma , Factores de Transcripción de Tipo Kruppel/genética , Macrófagos/microbiología , Ratones , Fagocitosis , Pinocitosis

RESUMEN

The ciliate Tetrahymena thermophila can either synthesize tetrahymanol or when available, assimilate and modify sterols from its diet. This metabolic shift is mainly driven by transcriptional regulation of genes for tetrahymanol synthesis (TS) and sterol bioconversion (SB). The mechanistic details of sterol uptake, intracellular trafficking and the associated gene expression changes are unknown. By following cholesterol incorporation over time in a conditional phagocytosis-deficient mutant, we found that although phagocytosis is the main sterol intake route, a secondary endocytic pathway exists. Different expression patterns for TS and SB genes were associated with these entry mechanisms. Squalene synthase was down-regulated by a massive cholesterol intake only attainable by phagocytosis-proficient cells, whereas C22-sterol desaturase required ten times less cholesterol and was up-regulated in both wild-type and mutant cells. These patterns are suggestive of at least two different signaling pathways. Sterol trafficking beyond phagosomes and esterification was impaired by the NPC1 inhibitor U18666A. NPC1 is a protein that mediates cholesterol export from late endosomes/lysosomes in mammalian cells. U18666A also produced a delay in the transcriptional response to cholesterol, suggesting that the regulatory signals are triggered between lysosomes and the endoplasmic reticulum. These findings could hint at partial conservation of sterol homeostasis between eukaryote lineages.

Asunto(s)

Colesterol/metabolismo , Regulación de la Expresión Génica , Homeostasis , Fagocitosis , Pinocitosis , Proteínas Protozoarias/metabolismo , Esteroles/metabolismo , Tetrahymena thermophila/metabolismo , Animales , Transporte Biológico , Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Humanos , Proteínas Protozoarias/genética , Transducción de Señal , Tetrahymena thermophila/genética , Tetrahymena thermophila/crecimiento & desarrollo

RESUMEN

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan parasite. Toxoplasma can invade and multiply inside any nucleated cell of a wide range of homeothermic hosts. The canonical process of internalization involves several steps: an initial recognition of the host cell surface and a sequential secretion of proteins from micronemes followed by rhoptries that assemble a macromolecular complex constituting a specialized and transient moving junction. The parasite is then internalized via an endocytic process with the establishment of a parasitophorous vacuole (PV), that does not fuse with lysosomes, where the parasites survive and multiply. This process of host cell invasion is usually referred to active penetration. Using different cell types and inhibitors of distinct endocytic pathways, we show that treatment of host cells with compounds that interfere with clathrin-mediated endocytosis (hypertonic sucrose medium, chlorpromazine hydrochloride, and pitstop 2 inhibited the internalization of tachyzoites). In addition, treatments that interfere with macropinocytosis, such as incubation with amiloride or IPA-3, increased parasite attachment to the host cell surface but significantly blocked parasite internalization. Immunofluorescence microscopy showed that markers of macropinocytosis, such as the Rab5 effector rabankyrin 5 and Pak1, are associated with parasite-containing cytoplasmic vacuoles. These results indicate that entrance of T. gondii into mammalian cells can take place both by the well-characterized interaction of parasite and host cell endocytic machinery and other processes, such as the clathrin-mediated endocytosis, and macropinocytosis.

Asunto(s)

Toxoplasma , Toxoplasmosis , Animales , Endocitosis , Interacciones Huésped-Parásitos , Pinocitosis , Vacuolas

RESUMEN

There is an urgent need for safe, efficacious, affordable, and field-adapted drugs for the treatment of cutaneous leishmaniasis, which newly affects around 1.5 million people worldwide annually. Chitosan, a biodegradable cationic polysaccharide, has previously been reported to have antimicrobial, antileishmanial, and immunostimulatory activities. We investigated the in vitro activity of chitosan and several of its derivatives and showed that the pH of the culture medium plays a critical role in antileishmanial activity of chitosan against both extracellular promastigotes and intracellular amastigotes of Leishmania major and Leishmania mexicana Chitosan and its derivatives were approximately 7 to 20 times more active at pH 6.5 than at pH 7.5, with high-molecular-weight chitosan being the most potent. High-molecular-weight chitosan stimulated the production of nitric oxide and reactive oxygen species by uninfected and Leishmania-infected macrophages in a time- and dose-dependent manner at pH 6.5. Despite the in vitro activation of bone marrow macrophages by chitosan to produce nitric oxide and reactive oxygen species, we showed that the antileishmanial activity of chitosan was not mediated by these metabolites. Finally, we showed that rhodamine-labeled chitosan is taken up by pinocytosis and accumulates in the parasitophorous vacuole of Leishmania-infected macrophages.

Asunto(s)

Antiprotozoarios/farmacología , Quitosano/farmacología , Leishmania major/efectos de los fármacos , Leishmania mexicana/efectos de los fármacos , Estadios del Ciclo de Vida/efectos de los fármacos , Anfotericina B/farmacología , Animales , Quitosano/análogos & derivados , Medios de Cultivo/química , Medios de Cultivo/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Concentración de Iones de Hidrógeno , Leishmania major/inmunología , Leishmania major/metabolismo , Leishmania mexicana/inmunología , Leishmania mexicana/metabolismo , Estadios del Ciclo de Vida/fisiología , Macrófagos/efectos de los fármacos , Macrófagos/parasitología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/parasitología , Ratones , Ratones Endogámicos BALB C , Peso Molecular , Óxido Nítrico/metabolismo , Pruebas de Sensibilidad Parasitaria , Pinocitosis/efectos de los fármacos , Cultivo Primario de Células , Especies Reactivas de Oxígeno/metabolismo , Células THP-1 , Factor de Necrosis Tumoral alfa/biosíntesis

RESUMEN

Shiga toxin-producing Escherichia coli (STEC) strains are responsible for multiple clinical syndromes, including hemolytic uremic syndrome (HUS). E. coli O157:H7 is the most prevalent serotype associated with HUS and produces a variety of virulence factors being Stx2 the responsible of the most HUS severe cases. After intestinal colonization by STEC, Stx2 is released into the intestinal lumen, translocated to the circulatory system and then binds to its receptor, globotriaosylceramide (Gb3), in target cells. Thus, Stx2 passage through the colonic epithelial barrier is a key step in order to produce disease, being its mechanisms still poorly understood. We have previously reported that STEC interaction with the human colonic mucosa enhanced Stx2 production. In the present work, we have demonstrated that infection with O157:H7Δstx2, a mutant unable to produce Stx2, enhanced either Stx2 cytotoxicity on an intestinal cell line (HCT-8), or translocation across HCT-8 monolayers. Moreover, we found that translocation was enhanced by both paracellular and transcellular pathways. Using specific endocytosis inhibitors, we have further demonstrated that the main mechanisms implicated on Stx2 endocytosis and translocation, either when O157:H7Δstx2 was present or not, were Gb3-dependent, but dynamin-independent. On the other hand, dynamin dependent endocytosis and macropinocytosis became more relevant only when O157:H7Δstx2 infection was present. Overall, this study highlights the effects of STEC infection on the intestinal epithelial cell host and the mechanisms underlying Stx2 endocytosis, cytotoxic activity and translocation, in the aim of finding new tools toward a therapeutic approach.

Asunto(s)

Endocitosis , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Escherichia coli O157/fisiología , Mucosa Intestinal/inmunología , Mucosa Intestinal/microbiología , Toxina Shiga II/metabolismo , Línea Celular , Supervivencia Celular , Células Cultivadas , Interacciones Huésped-Patógeno/inmunología , Humanos , Mucosa Intestinal/metabolismo , Pinocitosis , Transporte de Proteínas , Toxina Shiga II/genética , Transducción de Señal , Factores de Virulencia

RESUMEN

We have previously shown that infectious pancreatic necrosis virus (IPNV) enters the embryo cell line CHSE-214 by macropinocytosis. In this study, we have extended our investigation into SHK-1 cells, a macrophage-like cell line derived from the head kidney of Atlantic salmon, the most economically important host of IPNV. We show that IPNV infection stimulated fluid uptake in SHK-1 cells above the constitutive macropinocytosis level. In addition, upon infection of SHK-1 cells, IPNV produced several changes in actin dynamics, such as protrusions and ruffles, which are important features of macropinocytosis. We also observed that the Na+/H+ pump inhibitor EIPA blocked IPNV infection. On the other hand, IPNV entry was independent of clathrin, a possibility that could not be ruled out in CHSE 214 cells. In order to determine the possible role of accessory factors on the macropinocytic process, we tested several inhibitors that affect components of transduction pathways. While pharmacological intervention of PKI3, PAK-1 and Rac1 did not affect IPNV infection, inhibition of Ras and Rho GTPases as well as Cdc42 resulted in a partial decrease in IPNV infection. Further studies will be required to determine the signalling pathway involved in the macropinocytosis-mediated entry of IPNV into its target cells.

Asunto(s)

Virus de la Necrosis Pancreática Infecciosa/fisiología , Macrófagos/virología , Pinocitosis , Salmón/virología , Internalización del Virus , Actinas/metabolismo , Animales , Infecciones por Birnaviridae/virología , Línea Celular , Enfermedades de los Peces/virología , Riñón Cefálico/virología , Macrófagos/citología

RESUMEN

Protein kinase CK2 is a highly conserved and constitutively active Ser/Thr-kinase that phosphorylates a large number of substrates, resulting in increased cell proliferation and survival. A known target of CK2 is Akt, a player in the PI3K/Akt/mTORC1 signaling pathway, which is aberrantly activated in 32% of colorectal cancer (CRC) patients. On the other hand, mTORC1 plays an important role in the regulation of protein synthesis, cell growth, and autophagy. Some studies suggest that CK2 regulates mTORC1 in several cancers. The most recently developed CK2 inhibitor, silmitasertib (formerly CX-4945), has been tested in phase I/II trials for cholangiocarcinoma and multiple myeloma. This drug has been shown to induce autophagy and enhance apoptosis in pancreatic cancer cells and to promote apoptosis in non-small cell lung cancer cells. Nevertheless, it has not been tested in studies for CRC patients. We show in this work that inhibition of CK2 with silmitasertib decreases in vitro tumorigenesis of CRC cells in response to G2/M arrest, which correlates with mTORC1 inhibition and formation of large cytoplasmic vacuoles. Notably, molecular markers indicate that these vacuoles derive from massive macropinocytosis. Altogether, these findings suggest that an aberrantly elevated expression/activity of CK2 may play a key role in CRC, promoting cell viability and proliferation in untreated cells, however, its inhibition with silmitasertib promotes methuosis-like cell death associated to massive catastrophic vacuolization, accounting for decreased tumorigenicity at later times. These characteristics of silmitasertib support a potential therapeutic use in CRC patients and probably other CK2-dependent cancers.

Asunto(s)

Muerte Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Naftiridinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Vacuolas/patología , Carcinogénesis/efectos de los fármacos , Quinasa de la Caseína II/antagonistas & inhibidores , Puntos de Control del Ciclo Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias Colorrectales/patología , Células HCT116 , Células HT29 , Humanos , Fenazinas , Pinocitosis/efectos de los fármacos , Transfección

RESUMEN

It is well established that B cells play an important role during infections beyond antibody production. B cells produce cytokines and are APCs for T cells. Recently, it has become clear that several pathogenic bacterial genera, such as Salmonella, Brucella, Mycobacterium, Listeria, Francisella, Moraxella, and Helicobacter, have evolved mechanisms such as micropinocytosis induction, inflammasome down-regulation, inhibitory molecule expression, apoptosis induction, and anti-inflammatory cytokine secretion to manipulate B cell functions influencing immune responses. In this review, we summarize our current understanding of B cells as targets of bacterial infection and the mechanisms by which B cells become a niche for bacterial survival and replication away from extracellular immune responses such as complement and antibodies.

Asunto(s)

Linfocitos B/inmunología , Infecciones Bacterianas/microbiología , Bacterias Gramnegativas/inmunología , Bacterias Grampositivas/inmunología , Evasión Inmune , Animales , Anticuerpos Antibacterianos/biosíntesis , Apoptosis/inmunología , Linfocitos B/microbiología , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/patología , Citocinas/biosíntesis , Citocinas/inmunología , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Gramnegativas/patogenicidad , Bacterias Grampositivas/crecimiento & desarrollo , Bacterias Grampositivas/patogenicidad , Humanos , Inflamasomas/inmunología , Viabilidad Microbiana/inmunología , Pinocitosis/inmunología

RESUMEN

Burkholderia cenocepacia is an opportunistic pathogen that infects individuals with cystic fibrosis, chronic granulomatous disease, and other immunocompromised states. B. cenocepacia survives in macrophages in membrane-bound vacuoles; however, the mechanism by which B. cenocepacia gains entry into macrophages remains unknown. After macrophage internalization, survival of B. cenocepacia within a bacteria-containing membrane vacuole (BcCV) is associated with its ability to arrest the maturation of the BcCV. In this study, we show that B. cenocepacia induces localized membrane ruffling, macropinocytosis, and macropinosomes-like compartments upon contact with the macrophage. The Type 3 Secretion System (T3SS) of B. cenocepacia contributes to macrophage entry and macropinosome-like compartment formation. These data demonstrate the ability of Burkholderia to enter macrophages through the induction of macropinocytosis.

Asunto(s)

Burkholderia cenocepacia/fisiología , Macrófagos/microbiología , Pinocitosis , Animales , Sistemas de Secreción Bacterianos , Femenino , Macrófagos/ultraestructura , Ratones Endogámicos BALB C

RESUMEN

Infectious pancreatic necrosis virus (IPNV) is a non-enveloped virus belonging to the Birnaviridae family. IPNV produces an acute disease in salmon fingerlings, with high mortality rates and persistent infection in survivors. Although there are reports of IPNV binding to various cells, the viral receptor and entry pathways remain unknown. The aim of this study was to determine the endocytic pathway that allows for IPNV entry. We observed that IPNV stimulated fluid uptake and virus particles co-localysed with the uptake marker dextran in intracellular compartments, suggesting a role for macropinocytosis in viral entry. Consistent with this idea, viral infection was significantly reduced when the Na+/H+ exchanger NHE1 was inhibited with 5-(N-Ethyl-N-isopropyl) amiloride (EIPA). Neither chlorpromazine nor filipin complex I affected IPNV infection. To examine the role of macropinocytosis regulators, additional inhibitors were tested. Inhibitors of the EGFR pathway and the effectors Pak1, Rac1 and PKC reduced viral infection. Together, our results indicate that IPNV is mainly internalized into CHSE-214 cells by macropinocytosis.

Asunto(s)

Virus de la Necrosis Pancreática Infecciosa/fisiología , Pinocitosis , Internalización del Virus , Animales , Infecciones por Birnaviridae/virología , Caveolinas/metabolismo , Línea Celular , Clorpromazina/farmacología , Dinaminas/metabolismo , Endocitosis , Enfermedades de los Peces/tratamiento farmacológico , Enfermedades de los Peces/virología , Microdominios de Membrana , Pinocitosis/efectos de los fármacos , Salmón/virología , Internalización del Virus/efectos de los fármacos

RESUMEN

BACKGROUND: Poly-lactic acid nanoparticles (PLA-NP) are a type of polymeric NP, frequently used as nanomedicines, which have advantages over metallic NP such as the ability to maintain therapeutic drug levels for sustained periods of time. Despite PLA-NP being considered biocompatible, data concerning alterations in cellular physiology are scarce. METHODS: We conducted an extensive evaluation of PLA-NP biocompatibility in human lung epithelial A549 cells using high throughput screening and more complex methodologies. These included measurements of cytotoxicity, cell viability, immunomodulatory potential, and effects upon the cells' proteome. We used non- and green-fluorescent PLA-NP with 63 and 66 nm diameters, respectively. Cells were exposed with concentrations of 2, 20, 100 and 200 µg/mL, for 24, 48 and 72 h, in most experiments. Moreover, possible endocytic mechanisms of internalization of PLA-NP were investigated, such as those involving caveolae, lipid rafts, macropinocytosis and clathrin-coated pits. RESULTS: Cell viability and proliferation were not altered in response to PLA-NP. Multiplex analysis of secreted mediators revealed a low-level reduction of IL-12p70 and vascular epidermal growth factor (VEGF) in response to PLA-NP, while all other mediators assessed were unaffected. However, changes to the cells' proteome were observed in response to PLA-NP, and, additionally, the cellular stress marker miR155 was found to reduce. In dual exposures of staurosporine (STS) with PLA-NP, PLA-NP enhanced susceptibility to STS-induced cell death. Finally, PLA-NP were rapidly internalized in association with clathrin-coated pits, and, to a lesser extent, with lipid rafts. CONCLUSIONS: These data demonstrate that PLA-NP are internalized and, in general, tolerated by A549 cells, with no cytotoxicity and no secretion of pro-inflammatory mediators. However, PLA-NP exposure may induce modification of biological functions of A549 cells, which should be considered when designing drug delivery systems. Moreover, the pathways of PLA-NP internalization we detected could contribute to the improvement of selective uptake strategies.

Asunto(s)

Materiales Biocompatibles/química , Caveolas/metabolismo , Células Epiteliales/efectos de los fármacos , Microdominios de Membrana , Nanopartículas/química , Poliésteres/química , Células A549 , Supervivencia Celular , Clatrina/química , Sistemas de Liberación de Medicamentos , Células Epiteliales/citología , Humanos , Interleucina-12/metabolismo , MicroARNs/metabolismo , Tamaño de la Partícula , Pinocitosis , Proteoma , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

The proposed order Megavirales comprises the nucleocytoplasmic large DNA viruses (NCLDV), infecting a wide range of hosts. Over time, they co-evolved with different host cells, developing various strategies to penetrate them. Mimiviruses and other giant viruses enter cells through phagocytosis, while Marseillevirus and other large viruses explore endocytosis and macropinocytosis. These differing strategies might reflect the evolution of those viruses. Various scenarios have been proposed for the origin and evolution of these viruses, presenting one of the most enigmatic issues to surround these microorganisms. In this context, we believe that giant viruses evolved independently by massive gene/size gain, exploring the phagocytic pathway of entry into amoebas. In response to gigantism, hosts developed mechanisms to evade these parasites.

Asunto(s)

Acanthamoeba/virología , Virus Gigantes/crecimiento & desarrollo , Virus Gigantes/metabolismo , Interacciones Huésped-Patógeno/fisiología , Pinocitosis/fisiología , Internalización del Virus , ADN Viral/genética , Evolución Molecular , Mimiviridae/metabolismo

RESUMEN

In situ tissue engineering within a stroke cavity is gradually emerging as a novel therapeutic paradigm. Considering the varied lesion topology within each subject, the placement and distribution of cells within the lesion cavity is challenging. The use of multiple cell types to reconstruct damaged tissue illustrates the complexity of the process, but also highlights the challenges to provide a non-invasive assessment. The distribution of implanted cells within the lesion cavity and crucially the contribution of neural stem cells and endothelial cells to morphogenesis could be visualized simultaneously using two paramagnetic chemical exchange saturation transfer (paraCEST) agents. The development of sophisticated imaging methods is essential to guide delivery of the building blocks for in situ tissue engineering, but will also be essential to understand the dynamics of cellular interactions leading to the formation of de novo tissue.

Asunto(s)

Imagen por Resonancia Magnética , Accidente Cerebrovascular/patología , Ingeniería de Tejidos , Animales , Rastreo Celular/métodos , Tratamiento Basado en Trasplante de Células y Tejidos , Modelos Animales de Enfermedad , Células Endoteliales/citología , Células Endoteliales/metabolismo , Células Endoteliales/trasplante , Procesamiento de Imagen Asistido por Computador , Imagen por Resonancia Magnética/métodos , Masculino , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Pinocitosis , Ratas , Coloración y Etiquetado , Trasplante de Células Madre , Accidente Cerebrovascular/diagnóstico , Accidente Cerebrovascular/terapia , Ingeniería de Tejidos/métodos

RESUMEN

Here, we investigated the role of EhVps32 protein (a member of the endosomal-sorting complex required for transport) in endocytosis of Entamoeba histolytica, a professional phagocyte. Confocal microscopy, TEM and cell fractionation revealed EhVps32 in cytoplasmic vesicles and also located adjacent to the plasma membrane. Between 5 to 30 min of phagocytosis, EhVps32 was detected on some erythrocytes-containing phagosomes of acidic nature, and at 60 min it returned to cytoplasmic vesicles and also appeared adjacent to the plasma membrane. TEM images revealed it in membranous structures in the vicinity of ingested erythrocytes. EhVps32, EhADH (an ALIX family member), Gal/GalNac lectin and actin co-localized in the phagocytic cup and in some erythrocytes-containing phagosomes, but EhVps32 was scarcely detected in late phagosomes. During dextran uptake, EhVps32, EhADH and Gal/GalNac lectin, but not actin, co-localized in pinosomes. EhVps32 recombinant protein formed oligomers composed by rings and filaments. Antibodies against EhVps32 monomers stained cytoplasmic vesicles but not erythrocytes-containing phagosomes, suggesting that in vivo oligomers are formed on phagosome membranes. The involvement of EhVps32 in phagocytosis was further study in pNeoEhvps32-HA-transfected trophozoites, which augmented almost twice their rate of erythrophagocytosis as well as the membranous concentric arrays built by filaments, spirals and tunnel-like structures. Some of these structures apparently connected phagosomes with the phagocytic cup. In concordance, the EhVps32-silenced G3 trophozoites ingested 80% less erythrocytes than the G3 strain. Our results suggest that EhVps32 participates in E. histolytica phagocytosis and pinocytosis. It forms oligomers on erythrocytes-containing phagosomes, probably as a part of the scission machinery involved in membrane invagination and intraluminal vesicles formation.

Asunto(s)

Entamoeba histolytica/metabolismo , Fagocitosis/fisiología , Pinocitosis/fisiología , Proteínas Protozoarias/metabolismo , Western Blotting , Eritrocitos/parasitología , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoprecipitación , Microscopía Confocal , Microscopía Electrónica , Vacuolas/metabolismo

RESUMEN

A utilização da transgenia com a proteína fluorescente verde (GFP) como marcador de células de origem fetal nas placentas de clones bovinos servirá de modelo inédito para estudo morfofisiológico e imunológico da interação materno-fetal, visto que possibilitará o seu mapeamento, diferenciando as células fetais das maternas. Tal modelo terá aplicação direta, principalmente porque estes são animais que apresentam problemas em relação ao seu desenvolvimento. Com o auxílio deste modelo, pretende-se verificar o transporte de substâncias entre a mãe e o feto via endocitose, pela imunolocalização das proteínas chamadas de caveolinas. Para tanto foram utilizados 06 bovinos clonados e 30 bovinos de inseminação artificial (IA) com idade até 90 dias de gestação, os quais tiveram seu desenvolvimento interrompido mediante abate humanitário das receptoras e ovariosalpingohisterectomia, com posterior recuperação do útero gestante. Foram coletados os placentônios e o cório. Uma parte das amostras foi recortada e fixada, por imersão, em solução de parafolmaldeído a 4% ou formoldeído a 10% em tampão fosfato de sódio (PBS) a 0,1M pH 7.4, solução de Zamboni (4% de paraformoldeído, 15% de ácido pícrico, em tampão fosfato de sódio a 0,1M pH 7.4), metacarn (60% de metanol, 30% de clorofórmio, e 10% de ácido acético glacial), para verificação da morfologia e realização de imuno-histoquímica para as proteínas caveolinas -1 e -2 (CAV -1 e CAV-2)...

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The transgenic application of green fluorescent protein (GFP) as fetal cell marker on cattle cloned placenta could provide an exclusive model for studying the morphologic and immunologic maternal-fetal interactions, providing information about its mapping, distinguishing the fetal from maternal cells. This model will have direct application, mainly because these animals present problems during its development. With this model's support, we intend to verify the substances transport between mother and fetus during endocytosis, through the immunolocalization of protein named caveolae. For these, we used 06 cloned bovine and 30 cattle samples of artificial insemination (AI) with 90 days of pregnancy, which had been their development interrupted by humanitarian slaughter of the recipient and recovery of the pregnant uterus. We collected the placentome and the chorion. A part of the samples was cut and fixed, by immersion, on a solution containing 4% of parafomaldehyde or 10% of formaldehyde on a sodium phosphate buffer (PBS), at 0,1M pH 7.4, Zamboni solution (4% of paraformaldehyde, 15% of picric acid, on sodium phosphate buffer 0,1M pH 7.4), metacarn (60% of metanol, 30% of chloroform, and 10% glacial acetic acid), for morphologic and immunohistochemistry verification for caveolinas proteins -1 and -2 (CAV -1 and CAV- 2). The caveolins -1 were found in fetal and maternal villi, but its strongest staining was observed in the endometrial stroma. The caveolins -2 had positive staining in trophoblast and chorioallantoic membrane, and specifically in giant trophoblastic binucleated cell. Therefore the results were compared between cloned cattle and from AI or natural mating, for assisting on detection of the reason of many placental alterations, embryonic losses, spontaneous abortion, post-natal mortality and large offspring syndrome on laboratory-manipulated animals. The result suggests that the proteins caveolins -1 and -2 (CAV-1 and CAV-2)...

Asunto(s)

Animales , Femenino , Embarazo , Lactante , Bovinos , Animales Modificados Genéticamente/embriología , Caveolas/ultraestructura , Caveolinas/genética , Clonación de Organismos/veterinaria , Apoptosis , Aumento de la Célula , Endocitosis , Técnica del Anticuerpo Fluorescente/veterinaria , Metabolismo de los Lípidos , Pinocitosis , Vellosidades Coriónicas/fisiología

RESUMEN

A utilização da transgenia com a proteína fluorescente verde (GFP) como marcador de células de origem fetal nas placentas de clones bovinos servirá de modelo inédito para estudo morfofisiológico e imunológico da interação materno-fetal, visto que possibilitará o seu mapeamento, diferenciando as células fetais das maternas. Tal modelo terá aplicação direta, principalmente porque estes são animais que apresentam problemas em relação ao seu desenvolvimento. Com o auxílio deste modelo, pretende-se verificar o transporte de substâncias entre a mãe e o feto via endocitose, pela imunolocalização das proteínas chamadas de caveolinas. Para tanto foram utilizados 06 bovinos clonados e 30 bovinos de inseminação artificial (IA) com idade até 90 dias de gestação, os quais tiveram seu desenvolvimento interrompido mediante abate humanitário das receptoras e ovariosalpingohisterectomia, com posterior recuperação do útero gestante. Foram coletados os placentônios e o cório. Uma parte das amostras foi recortada e fixada, por imersão, em solução de parafolmaldeído a 4% ou formoldeído a 10% em tampão fosfato de sódio (PBS) a 0,1M pH 7.4, solução de Zamboni (4% de paraformoldeído, 15% de ácido pícrico, em tampão fosfato de sódio a 0,1M pH 7.4), metacarn (60% de metanol, 30% de clorofórmio, e 10% de ácido acético glacial), para verificação da morfologia e realização de imuno-histoquímica para as proteínas caveolinas -1 e -2 (CAV -1 e CAV-2). As caveolinas -1 foram localizadas nos vilos fetais e maternos, mas sua marcação mais forte foi observada no estroma endometrial. As caveolinas -2 tiveram marcação positiva no trofoblasto e membrana córioalantoide, e, especificamente em célula trofoblástica gigante binucleada. Sendo assim, os resultados mostram que a proteína CAV-1 teve uma maior expressão em relação ...(AU)

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The transgenic application of green fluorescent protein (GFP) as fetal cell marker on cattle cloned placenta could provide an exclusive model for studying the morphologic and immunologic maternal-fetal interactions, providing information about its mapping, distinguishing the fetal from maternal cells. This model will have direct application, mainly because these animals present problems during its development. With this model's support, we intend to verify the substances transport between mother and fetus during endocytosis, through the immunolocalization of protein named caveolae. For these, we used 06 cloned bovine and 30 cattle samples of artificial insemination (AI) with 90 days of pregnancy, which had been their development interrupted by humanitarian slaughter of the recipient and recovery of the pregnant uterus. We collected the placentome and the chorion. A part of the samples was cut and fixed, by immersion, on a solution containing 4% of parafomaldehyde or 10% of formaldehyde on a sodium phosphate buffer (PBS), at 0,1M pH 7.4, Zamboni solution (4% of paraformaldehyde, 15% of picric acid, on sodium phosphate buffer 0,1M pH 7.4), metacarn (60% of metanol, 30% of chloroform, and 10% glacial acetic acid), for morphologic and immunohistochemistry verification for caveolinas proteins -1 and -2 (CAV -1 and CAV- 2). The caveolins -1 were found in fetal and maternal villi, but its strongest staining was observed in the endometrial stroma. The caveolins -2 had positive staining in trophoblast and chorioallantoic membrane, and specifically in giant trophoblastic binucleated cell. Therefore the results were compared between cloned cattle and from AI or natural mating, for assisting on detection of the reason of many placental alterations, embryonic losses, spontaneous abortion, post-natal mortality and large offspring syndrome on laboratory-manipulated animals. The result suggests that the proteins caveolins -1 and -2 (CAV-1 and CAV-2) are part of the caveolae...(AU)

Asunto(s)

Animales , Femenino , Embarazo , Lactante , Bovinos , Caveolinas/genética , Clonación de Organismos/veterinaria , Animales Modificados Genéticamente/embriología , Caveolas/ultraestructura , Técnica del Anticuerpo Fluorescente/veterinaria , Endocitosis , Pinocitosis , Vellosidades Coriónicas/fisiología , Apoptosis , Aumento de la Célula , Metabolismo de los Lípidos

RESUMEN

Infectious bursal disease virus (IBDV) internalization is sparsely known in terms of molecular components of the pathway involved. To describe the cell biological features of IBDV endocytosis, we employed perturbants of endocytic pathways such as pharmacological inhibitors and overexpression of dominant-negative mutants. Internalization analysis was performed quantifying infected cells by immunofluorescence and Western blot detection of the viral protein VP3 at 12 h post-infection reinforced by the analysis of the capsid protein VP2 localization after virus uptake at 1 h post-infection. We compared IBDV infection to the internalization of well-established ligands with defined endocytic pathways: transferrin, cholera-toxin subunit B and dextran. To describe virus endocytosis at the morphological level, we performed ultrastructural studies of viral internalization kinetics in control and actin dynamics-blocked cells. Our results indicate that IBDV endocytic internalization was clathrin- and dynamin-independent, and that IBDV uses macropinocytosis as the primary entry mechanism. After uptake, virus traffics to early endosomes and requires exposure to the low endocytic pH as well as a functional endocytic pathway to complete its replication cycle. Moreover, our results indicate that the GTPase Rab5 is crucial for IBDV entry supporting the participation of the early endosomal pathway in IBDV internalization and infection of susceptible cells.

Asunto(s)

Endosomas/virología , Virus de la Enfermedad Infecciosa de la Bolsa/fisiología , Pinocitosis , Internalización del Virus , Proteínas de Unión al GTP rab5/metabolismo , Animales , Transporte Biológico , Aves , Western Blotting , Línea Celular , Microscopía Fluorescente , Factores de Tiempo , Proteínas Estructurales Virales/análisis