RESUMEN
With the incidence of liver disease on the rise globally, increasing numbers of patients are presenting with advanced hepatic fibrosis and significant mortality risk. The demand far outstrips possible transplantation capacities, and thus there is an intense drive to develop new pharmacological therapies that stall or reverse liver scarring. Recent late-stage failures of lead compounds have highlighted the challenges of resolving fibrosis, which has developed and stabilized over many years and varies in nature and composition from individual to individual. Hence, preclinical tools are being developed in both the hepatology and tissue engineering communities to elucidate the nature, composition, and cellular interactions of the hepatic extracellular niche in health and disease. In this protocol, we describe strategies for decellularizing cirrhotic and healthy human liver specimens and show how these can be used in simple functional assays to detect the impact on stellate cell function. Our simple, small-scale approach is translatable to diverse lab settings and generates cell-free materials which could be used for a variety of in vitro analyses as well as a scaffold for repopulating with key hepatic cell populations.
Asunto(s)
Hepatopatías , Hígado , Humanos , Hígado/fisiología , Cirrosis Hepática , Ingeniería de Tejidos/métodos , Matriz Extracelular , Andamios del TejidoRESUMEN
Infectious keratoconjunctivitis (IKC) is a common transmissible ocular disease in semi-domesticated Eurasian tundra reindeer (Rangifer tarandus tarandus). In large outbreaks, IKC may affect tens of animals in a herd, with the most severe cases often requiring euthanasia due to the destruction of the affected eyes and permanent blindness. An experimental inoculation with cervid herpesvirus 2 (CvHV2), alone or in combination with Moraxella bovoculi, demonstrated that CvHV2 has the ability to cause clinical signs of IKC in previously unexposed reindeer. Tissues collected from upper and lower eyelids, lacrimal gland and cornea, were processed for light and transmission electron microscopy. Histopathological analysis of the eyes inoculated with CvHV2 showed widespread and severe pathological findings. Mucosal tissues from these eyes showed fibrinous and purulent exudates, hyperemia, hemorrhages, necrosis, vascular thrombosis, vascular necrosis, infiltration of mononuclear cells and neutrophils, and lymphoid follicle reaction, which matches the described histopathology of IKC in reindeer. Characteristic alpha-herpesvirus particles matching the size and morphology of CvHV2 were identified by transmission electron microscopy in the conjunctival tissue. The quantification of viral particles by qPCR revealed high copy numbers of viral DNA in all CvHV2 inoculated eyes, but also in the non-inoculated eyes of the same animals. The histopathology of eye tissues obtained from the CvHV2 inoculated reindeer and the lack of inflammation from bacterial infection, together with the detection of CvHV2 DNA in swabs from the inoculated and non-inoculated eyes of the same animals, verified that CvHV2 was the primary cause of the observed histopathological changes.
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Ojo/virología , Infecciones por Herpesviridae/veterinaria , Queratoconjuntivitis Infecciosa/virología , Reno/virología , Varicellovirus/fisiología , Animales , ADN Viral/genética , Ojo/patología , Infecciones por Herpesviridae/patología , Infecciones por Herpesviridae/virología , Queratoconjuntivitis Infecciosa/patología , Varicellovirus/genéticaRESUMEN
The liver is constantly exposed to dietary antigens, viruses, and bacterial products with inflammatory potential. For decades cellular uptake of virus has been studied in connection with infection, while the few studies designed to look into clearance mechanisms focused mainly on the role of macrophages. In recent years, attention has been directed towards the liver sinusoidal endothelial cells (LSECs), which play a central role in liver innate immunity by their ability to scavenge pathogen- and damage-associated molecular patterns. Every day our bodies are exposed to billions of gut-derived pathogens which must be efficiently removed from the circulation to prevent inflammatory and/or immune reactions in other vascular beds. Here, we have used GFP-labelled Enterobacteria phage T4 (GFP-T4-phage) as a model virus to study the viral scavenging function and metabolism in LSECs. The uptake of GFP-T4-phages was followed in real-time using deconvolution microscopy, and LSEC identity confirmed by visualization of fenestrae using structured illumination microscopy. By combining these imaging modalities with quantitative uptake and inhibition studies of radiolabelled GFP-T4-phages, we demonstrate that the bacteriophages are effectively degraded in the lysosomal compartment. Due to their high ability to take up and degrade circulating bacteriophages the LSECs may act as a primary anti-viral defence mechanism.
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Bacteriófago T4/patogenicidad , Hígado/citología , Hígado/virología , Animales , Bacteriófago T4/genética , Bacteriófago T4/metabolismo , Células Cultivadas , Endocitosis , Células Endoteliales/metabolismo , Células Endoteliales/virología , Proteínas Fluorescentes Verdes/genética , Interacciones Huésped-Patógeno/fisiología , Lisosomas/virología , Masculino , Microorganismos Modificados Genéticamente , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Ratas Sprague-DawleyRESUMEN
The environmental temperature has profound effects on biological systems of marine aquatic organisms and plays a critical role in species distribution and abundance. Particularly during the warmer seasons, variations in habitat temperature may introduce episodes of stressful temperatures which the organisms must adapt to and compensate for to maintain physiological homeostasis. The marine environment is changing and predicted raises in water temperatures will affect numerous marine species. Translocation of pathogens follow migration of species and alternations in physical environmental parameters may have influence upon the virulence of pathogens, as well as the hosts immune responses. While pathogenicity of many true pathogens is expected to increase following climate induced temperature stress, the impact from environmental stressors on the occurrence and severity of opportunistic infections is unknown. Here we describe how thermal stress in the cold-water species Atlantic cod influenced the fish immune responses against an opportunistic intracellular bacterium. Following experimental infection with Brucella pinnipedialis at normal water temperature (6°C) and sub-optimal temperature (15°C), cod cleared the intracellular bacteria more rapidly at the highest temperature. The overall immune response was faster and of higher amplitude at 15°C, however, a significant number of cod died at this temperature despite efficient clearance of infection. An increased growth rate not affected by infection was observed at 15°C, confirming multiple energy demanding processes taking place. Serum chemistry suggested that general homeostasis was influenced by both infection and increased water temperature, highlighting the cumulative stress responses (allostatic load) generated by simultaneous stressors. Our results suggest a trade-off between resistance and tolerance to survive infection at sub-optimal temperatures and raise questions concerning the impact of increased water temperatures on the energetic costs of immune system activation in aquatic ectotherms.
RESUMEN
Triethylene glycol dimethacrylate (TEGDMA) is commonly used in polymer resin-based dental materials. This study investigated the molecular mechanisms of TEGDMA toxicity by identifying its time- and dose-dependent effects on the proteome of human THP-1 monocytes. The effects of different concentrations (0.07-5 mM) and exposure times (0-72 h) of TEGDMA on cell viability, proliferation, and morphology were determined using a real-time viability assay, automated cell counting, and electron microscopy, and laid the fundament for choice of exposure scenarios in the proteomic experiments. Solvents were not used, as TEGDMA is soluble in cell culture medium (determined by photon correlation spectroscopy). Cells were metabolically labeled [using the stable isotope labeled amino acids in cell culture (SILAC) strategy], and exposed to 0, 0.3 or 2.5 mM TEGDMA for 6 or 16 h before liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. Regulated proteins were analyzed in the STRING database. Cells exposed to 0.3 mM TEGDMA showed increased viability and time-dependent upregulation of proteins associated with stress/oxidative stress, autophagy, and cytoprotective functions. Cells exposed to 2.5 mM TEGDMA showed diminished viability and a protein expression profile associated with oxidative stress, DNA damage, mitochondrial dysfunction, and cell cycle inhibition. Altered expression of immune genes was observed in both groups. The study provides novel knowledge about TEGDMA toxicity at the proteomic level. Of note, even low doses of TEGDMA induced a substantial cellular response.
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Monocitos/efectos de los fármacos , Polietilenglicoles/toxicidad , Ácidos Polimetacrílicos/toxicidad , Proteoma , Células THP-1/efectos de los fármacos , Técnicas de Cultivo de Célula , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cromatografía Liquida , Daño del ADN , Materiales Dentales , Relación Dosis-Respuesta a Droga , Humanos , Ensayo de Materiales , Mitocondrias/efectos de los fármacos , Estrés Oxidativo , Especies Reactivas de Oxígeno , Solventes , Espectrometría de Masas en TándemRESUMEN
General comprehension of terms and confounding factors associated with in vitro experiments can maximize the potential of in vitro testing of substances. In this systematic review, we present an overview of the terms and methods used to determine low-dose effects of matrix constituents in polymer resin-based dental materials in cell-culture studies and discuss the findings in light of how they may influence the comprehension and interpretation of results. Articles published between 1996 and 2015 were identified by searches in the Scopus, Web of Science, MEDLINE, PubMed, and Embase databases using keywords associated with low-dose effects, polymer resin-based materials, in vitro parameters, and dental materials. Twenty-nine articles were included. Subtoxic (n = 11), sublethal (n = 10), and nontoxic (n = 6) were the terms most commonly used to describe the low-dose effects of methacrylates. However, definition of terms varied. Most (82%) studies employed only one method to define the exposure scenario, and no agreement was seen between studies on the use of solvents. Prophylactic use of antibiotics was widespread, and mycoplasma screening was not reported. In conclusion, cell-culture conditions and tests used to define exposure scenarios have changed little in the last decades, despite development in recommendations. Nomenclature alignment is needed for a better understanding of possible biohazards of methacrylates.
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Técnicas de Cultivo de Célula , Materiales Dentales , Metacrilatos , Humanos , PolímerosRESUMEN
Previously, we have proposed mupirocin-in-liposomes-in-hydrogel delivery system as advanced delivery system with the potential in treatment of burns. In the current studies, we evaluated the system for its cytotoxicity, ability to prevent biofilm formation, act on the mature biofilms, and finally determined its potential as wound treatment in in vivo mice burn model. The system was found to be nontoxic against HaCaT cells, that is, keratinocytes. It was safe for use and exhibited antibiofilm activity against S. aureus biofilms, although the activity was more significant against planktonic bacteria and prior to biofilm formation than against mature biofilms as shown in the resazurin and the crystal violet assays. An in vivo mice burn model was used to evaluate the biological potential of the system and the healing of burns observed over 28 days. The in vivo data suggest that the delivery system enhances wound healing and is equally potent as the marketed product of mupirocin. Histological examination showed no difference in the quality of the healed scar tissue, whereas the healing time for the new delivery system was shorter as compared to the marketed product. Further animal studies and development of more sophisticated in vivo model are needed for complete evaluation.
Asunto(s)
Quemaduras/tratamiento farmacológico , Mupirocina/administración & dosificación , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacos , Animales , Antibacterianos/administración & dosificación , Antibacterianos/química , Biopelículas/efectos de los fármacos , Quemaduras/microbiología , Quemaduras/patología , Modelos Animales de Enfermedad , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/administración & dosificación , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Liposomas/administración & dosificación , Liposomas/química , Ratones , Mupirocina/química , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/patología , Cicatrización de HeridasRESUMEN
Marine mammal Brucella spp. have been isolated from pinnipeds (B. pinnipedialis) and cetaceans (B. ceti) from around the world. Although the zoonotic potential of marine mammal brucellae is largely unknown, reports of human disease exist. There are few studies of the mechanisms of bacterial intracellular invasion and multiplication involving the marine mammal Brucella spp. We examined the infective capacity of two genetically different B. pinnipedialis strains (reference strain; NTCT 12890 and a hooded seal isolate; B17) by measuring the ability of the bacteria to enter and replicate in cultured phagocytes and epithelial cells. Human macrophage-like cells (THP-1), two murine macrophage cell lines (RAW264.7 and J774A.1), and a human malignant epithelial cell line (HeLa S3) were challenged with bacteria in a gentamicin protection assay. Our results show that B. pinnipedialis is internalized, but is then gradually eliminated during the next 72-96 hours. Confocal microscopy revealed that intracellular B. pinnipedialis hooded seal strain colocalized with lysosomal compartments at 1.5 and 24 hours after infection. Intracellular presence of B. pinnipedialis hooded seal strain was verified by transmission electron microscopy. By using a cholesterol-scavenging lipid inhibitor, entrance of B. pinnipedialis hooded seal strain in human macrophages was significantly reduced by 65.8 % (± 17.3), suggesting involvement of lipid-rafts in intracellular entry. Murine macrophages invaded by B. pinnipedialis do not release nitric oxide (NO) and intracellular bacterial presence does not induce cell death. In summary, B. pinnipedialis hooded seal strain can enter human and murine macrophages, as well as human epithelial cells. Intracellular entry of B. pinnipedialis hooded seal strain involves, but seems not to be limited to, lipid-rafts in human macrophages. Brucella pinnipedialis does not multiply or survive for prolonged periods intracellulary.
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Brucella/patogenicidad , Células Epiteliales/microbiología , Macrófagos/microbiología , Phocidae/microbiología , Zoonosis/microbiología , Animales , Brucella/fisiología , Línea Celular , Gentamicinas , Humanos , Inmunohistoquímica , Ratones , Microscopía Confocal , Microscopía Electrónica de TransmisiónRESUMEN
Cervid herpesvirus 2 (CvHV2) has been isolated from reindeer (Rangifer tarandus tarandus), and serological data indicate that in reindeer this virus is endemic in Fennoscandia, Alaska, Canada, and Greenland. CvHV2 has been described as a cause of subclinical genital infections in reindeer, but little information on primary infections exists. In this study, six seronegative and presumably pregnant reindeer were allocated to one of two groups. Two animals were inoculated with CvHV2 intratracheally, and two animals intravaginally, with one control animal in each group receiving sterile water. Mild hyperthermia and serous discharges from the vagina and nose were observed. No abortions were recorded, but one calf died shortly after birth. Inoculated animals seroconverted and had neutralizing antibodies after days 7 to 10 postinfection. CvHV2 was detected by PCR in nasal and vaginal swabs from animals in both groups but could be isolated only from nasal swabs in the respiratory group and from vaginal swabs in the genital group. CvHV2 was detected by PCR in various organs and tissues postmortem. In control animals, the virus could not be isolated in spite of PCR-positive nasal and vaginal swab samples and some degree of positive immunostaining. One of the animals that were inoculated intratracheally developed a hemorrhagic, necrotizing bronchopneumonia, which was CvHV2 positive by PCR and immunohistochemistry. We conclude that CvHV2 can cause systemic infection, that both genital and respiratory inoculations can lead to virus shedding, and that the virus can infect the fetus in utero.
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Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Infecciones por Herpesviridae/veterinaria , Complicaciones Infecciosas del Embarazo/veterinaria , Reno/virología , Varicellovirus/inmunología , Animales , Femenino , Feto/inmunología , Feto/virología , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Pulmón/inmunología , Pulmón/patología , Pulmón/virología , Embarazo , Complicaciones Infecciosas del Embarazo/inmunología , Complicaciones Infecciosas del Embarazo/virología , Reno/inmunología , Útero/inmunología , Útero/patología , Útero/virología , Vagina/inmunología , Vagina/virología , Esparcimiento de Virus/inmunologíaRESUMEN
Morphological changes in the hepatic sinusoid with old age are increasingly recognized. These include thickening and defenestration of the liver sinusoidal endothelial cell, sporadic deposition of collagen and basal lamina in the extracellular space of Disse, and increased numbers of fat engorged, nonactivated stellate cells. In addition, there is endothelial up-regulation of von Willebrand factor and ICAM-1 with reduced expression of caveolin-1. These changes have been termed age-related pseudocapillarization. The effects of old age on Kupffer cells are inconsistent, but impaired responsiveness is likely. There are functional implications of these aging changes in the hepatic sinusoid. There is reduced sinusoidal perfusion, which will impair the hepatic clearance of highly extracted substrates. Blood clearance of a variety of waste macromolecules takes place in liver sinusoidal endothelial cells (LSECs). Previous studies indicated either that aging had no effect, or reduced the endocytic capacity of LSECs. However, a recent study in mice showed reduced endocytosis in pericentral regions of the liver lobules. Reduced endocytosis may increase systemic exposure to potential harmful waste macromolecules such as advanced glycation end products Loss of fenestrations leads to impaired transfer of lipoproteins from blood to hepatocytes. This provides a mechanism for impaired chylomicron remnant clearance and postprandial hyperlipidemia associated with old age. Given the extensive range of substrates metabolized by the liver, age-related changes in the hepatic sinusoid and microcirculation have important systemic implications for aging and age-related diseases.
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Envejecimiento/patología , Hígado/patología , Envejecimiento/inmunología , Envejecimiento/fisiología , Animales , Molde por Corrosión , Endocitosis , Células Endoteliales/inmunología , Células Endoteliales/patología , Células Endoteliales/fisiología , Humanos , Macrófagos del Hígado/patología , Macrófagos del Hígado/fisiología , Hígado/irrigación sanguínea , Hígado/inmunología , Hígado/fisiología , Circulación Hepática/fisiología , Microcirculación/inmunología , Microcirculación/patología , Microcirculación/fisiología , Microscopía ElectrónicaRESUMEN
BACKGROUND: In a project to determine the causes of winter mortality in reindeer in Finnmark County, northern Norway, the most frequent diagnosis turned out to be complete emaciation, despite several of the reindeer having been given silage for up to 4 weeks before they died. The present paper describes autopsy results and other findings in these animals. METHODS: Autopsies were made of 32 reindeer carcasses, and 28 of these were diagnosed as completely emaciated based on lack of visible fat and serous atrophy of subepicardial and bone marrow fat. Other investigations of the carcasses included histology, bacteriology, parasitology (counting of macro parasites and faecal egg counting), analysis of vitamin E and selenium in liver, chemical and botanical analysis of rumen content, analysis of lipid content in femur bone marrow and estimation of muscle atrophy by use of a muscle index. RESULTS: Main findings were: Low carcass weight, severe muscle atrophy, hemosiderosis in liver and spleen, subcutaneous oedema (18%) and effusions to body cavities (18%). Two types of lipofuscin granula were identified in the liver: One type occurred in liver endothelial cells of all carcasses, while the other type occurred in hepatocytes, and prevailed in adult animals. Abomasal haemorrhages, consistent with previously described stress lesions, was present in 68% of the carcasses. Diarrhoea occurred in 2 cases, and loose faecal consistency was associated with silage feeding. Rumen content was low in crude protein. Grass dominated rumen content in silage-fed carcasses, while reindeer on natural pastures had mainly woody plants, mosses and litter in rumen. Stem dominated the grass fraction in rumens with high grass content, indicating ruminal indigestion as a cause of emaciation in silage fed animals. Some cases had heavy infestation of parasites such as warble fly larvae (Hypoderma tarandi), throat bot larvae (Cephenemyiae trompe) and lung nematodes. CONCLUSION: Lack of appropriate amounts and/or appropriate quality of feed has been the main cause of emaciation, though heavy infestation of parasites may have contributed to the emaciation in some cases.
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Alimentación Animal/efectos adversos , Fenómenos Fisiológicos Nutricionales de los Animales/fisiología , Emaciación/veterinaria , Reno , Inanición/veterinaria , Alimentación Animal/normas , Animales , Causas de Muerte , Emaciación/etiología , Femenino , Masculino , Noruega , Inanición/etiología , Inanición/mortalidad , Inanición/patologíaRESUMEN
The liver sinusoidal endothelial cell (LSEC) influences the transfer of substrates between the sinusoidal blood and hepatocytes and has a major role in endocytosis; therefore, changes in the LSEC have significant implications for hepatic function. There are major morphological changes in the LSEC in old age called pseudocapillarization. These changes include increased LSEC thickness and reduced numbers of pores in the LSEC, which are called fenestrations. Pseudocapillarization has been found in old humans, rats, mice, and nonhuman primates. In addition, old age is associated with impaired LSEC endocytosis and increased leukocyte adhesion, which contributes to reduced hepatic perfusion. Given that fenestrations in the endothelium allow passage of some lipoproteins, including chylomicron remnants, age-related reduction in fenestrations impairs hepatic lipoprotein metabolism. In old rats, caloric restriction was associated with complete preservation of LSEC morphology and fenestrations. In conclusion, pseudocapillarization of the LSEC is a newly discovered aging change that, through its effects on lipoproteins, contributes to the association between old age, dyslipidemia, and vascular disease.
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Envejecimiento/patología , Dislipidemias/fisiopatología , Endotelio Vascular/fisiopatología , Hígado/irrigación sanguínea , Envejecimiento/sangre , Animales , Dislipidemias/sangre , Dislipidemias/patología , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Humanos , Hígado/metabolismo , Hígado/patologíaRESUMEN
Aging of the liver is associated with impaired metabolism of drugs, adverse drug interactions, and susceptibility to toxins. Since reduced hepatic blood flow is suspected to contribute this impairment, we examined age-related alterations in hepatic microcirculation. Livers of C57Bl/6 mice were examined at 0.8 (pre-pubertal), 3 (young adult), 14 (middle-aged), and 27 (senescent) months of age using in vivo and electron microscopic methods. The results demonstrated a 14% reduction in the numbers of perfused sinusoids between 0.8 and 27 month mice associated with 35% reduction in sinusoidal blood flow. This was accompanied by an inflammatory response evidenced by a fivefold increase in leukocyte adhesion in 27 month mice, up-regulated expression of ICAM-1, and increases in intrahepatic macrophages. Sinusoidal diameter decreased 6-10%. Liver sinusoidal endothelial cell (LSEC) dysfunction was seen as early as 14 months when there was a threefold increase in the numbers of swollen LSEC. The endocytotic capacity of LSEC also was found to be reduced in older animals. The sinusoidal endothelium in 27 month old mice exhibited pseudocapillarization. In conclusion, the results suggest that leukocyte accumulation in the sinusoids and narrowing of sinusoidal lumens due to pseudocapillarization and dysfunction of LSEC reduce sinusoidal blood flow in aged livers.
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Envejecimiento/fisiología , Circulación Hepática/fisiología , Envejecimiento/patología , Animales , Adhesión Celular , Endocitosis , Células Endoteliales/patología , Células Endoteliales/fisiología , Molécula 1 de Adhesión Intercelular/metabolismo , Leucocitos/patología , Hígado/irrigación sanguínea , Hígado/patología , Hígado/fisiología , Macrófagos/patología , Macrófagos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Microcirculación/fisiología , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Receptores Depuradores/metabolismoRESUMEN
UNLABELLED: The purpose of this study was to identify the receptor responsible for endocytosis of denatured collagen from blood. The major site of clearance of this material (at least 0.5 g/day in humans) is a receptor on liver sinusoidal endothelial cells (LSECs). We have now identified an 180-kDa endocytic receptor on LSECs, peptide mass fingerprinting of which revealed it to be the mannose receptor. Challenge of mannose-receptor knockout mice and their cultured LSECs revealed significantly reduced blood clearance and a complete absence of LSEC endocytosis of denatured collagen. Organ analysis of wild-type versus knockout mice after injection of denatured collagen revealed significantly reduced liver uptake in the knockout mice. Clearance/endocytosis of ligands for other receptors in these animals was as that for wild-type mice, and denatured collagen uptake in wild-type mice was not affected by other ligands of the mannose receptor, namely mannose and mannan. Furthermore, unlike that of mannose and mannan, endocytosis of denatured collagen by the mannose receptor is calcium independent. This suggests that the binding site for denatured collagen is distinct from that for mannose/mannan. Mannose receptors on LSECs appear to have less affinity for circulating triple helical type I collagen. CONCLUSION: The mannose receptor is the main candidate for being the endocytic denatured collagen receptor on LSECs.
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Colágeno/metabolismo , Células Endoteliales/metabolismo , Cirrosis Hepática/metabolismo , Hígado/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Animales , Colágeno/química , Endocitosis/fisiología , Hígado/citología , Masculino , Glicoproteínas de Membrana/aislamiento & purificación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Desnaturalización Proteica , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptores de Superficie Celular/aislamiento & purificación , Sus scrofaRESUMEN
OBJECTIVE: To evaluate the possibility and mechanism of the turnover of the negatively charged macromolecule by retinal pigment epithelial (RPE) cells. METHODS: Cultured porcine RPE eye cups were incubated with fluorescence labeled formaldehyde treated serum albumin (F-FSA), a classical ligand for scavenger receptors. The endocytosis of F-FSA by RPE cells under different conditions was evaluated systematically by fluorescent microscopy and electron microscopy. RESULTS: The amount of F-FSA ingested by RPE cells depends on the incubation time and the concentration of ligand. Similar ligands compete for the binding site of RPE cells. Small vesicles containing F-FSA are noticed in the cytosol of RPE cells as demonstrated by electron microscopy. CONCLUSION: The RPE cells can effectively ingest negatively charged macromolecules, possibly by scavenger receptor mediated endocytosis.