ABSTRACT
Animal models of X-linked juvenile retinoschisis (XLRS) are valuable tools for understanding basic biochemical function of retinoschisin (RS1) protein and to investigate outcomes of preclinical efficacy and toxicity studies. In order to work with an eye larger than mouse, we generated and characterized an Rs1h-/y knockout rat model created by removing exon 3. This rat model expresses no normal RS1 protein. The model shares features of an early onset and more severe phenotype of human XLRS. The morphologic pathology includes schisis cavities at postnatal day 15 (p15), photoreceptors that are misplaced into the subretinal space and OPL, and a reduction of photoreceptor cell numbers by p21. By 6 mo age only 1-3 rows of photoreceptors nuclei remain, and the inner/outer segment layers and the OPL shows major changes. Electroretinogram recordings show functional loss with considerable reduction of both the a-wave and b-wave by p28, indicating early age loss and dysfunction of photoreceptors. The ratio of b-/a-wave amplitudes indicates impaired synaptic transmission to bipolar cells in addition. Supplementing the Rs1h-/y exon3-del retina with normal human RS1 protein using AAV8-RS1 delivery improved the retinal structure. This Rs1h-/y rat model provides a further tool to explore underlying mechanisms of XLRS pathology and to evaluate therapeutic intervention for the XLRS condition.
Subject(s)
Cell Adhesion Molecules , Eye Proteins , Retinoschisis , Animals , Cell Adhesion Molecules/genetics , Dietary Supplements , Electroretinography , Exons/genetics , Eye Proteins/genetics , Eye Proteins/metabolism , Humans , Phenotype , Rats , Retina/metabolism , Retinoschisis/genetics , Retinoschisis/pathology , Retinoschisis/therapyABSTRACT
Nevirapine (NVP) therapy is associated with a high risk of serious liver injury and skin rash. Treatment of Brown Norway rats with NVP causes an immune-mediated skin rash. Even though NVP does not cause serious liver injury in wildtype animals, incubation of hepatocytes with NVP leads to the release of presumably danger-associated molecular pattern molecules (DAMPs), which activate macrophages. In this study, we examined the liver biopsies of Brown Norway rats treated with NVP to determine the histologic correlate to the release of DAMPs by hepatocytes. In vivo, debris from necrotic hepatocytes and endothelial cells were present in the liver sinusoids, a condition that can trigger an immune response. In addition to mitochondrial, hepatocytic, and endothelial damage, the drug induced large hepatocytic inclusions composed of lipid droplets surrounded by concentric whorls of smooth endoplasmic reticulum (SER) cisternae-lipid-SER (LSER) inclusions, which were deposited in the sinusoids. NVP is lipid soluble, and these LSER inclusions may be sinks of NVP or its metabolites. LSERs are deposited in the blood stream where they may be picked up by lymph nodes and contribute to initiation of an immune response leading to serious liver injury or skin rash. LSERs migration from liver to the blood stream may signify a novel mechanism of drug exocytosis.
Subject(s)
Anti-HIV Agents/toxicity , Endoplasmic Reticulum, Smooth/pathology , Lipid Droplets/pathology , Liver/drug effects , Nevirapine/toxicity , Animals , Drug Eruptions/etiology , Drug Eruptions/pathology , Endothelial Cells/pathology , Female , Hepatocytes/drug effects , Hepatocytes/pathology , Hepatocytes/ultrastructure , Inclusion Bodies/pathology , Liver/pathology , Liver/ultrastructure , Rats , Rats, Inbred BNABSTRACT
We have previously described the process by which mitochondria donate their membranes for the formation of autophagosomes, and in this study we show that the same process could be involved in drug sequestration and exocytosis resulting in multidrug-resistant cancerous cells. We examine the implications of mitochondrial vesicle formation of mitoautophagosomes (MAPS) in response to the cytotoxic drug MKT-077, which targets mortalin, in a drug-resistant breast carcinoma cell line overexpressing P-glycoprotein (P-gp). The breast cancer cell line MCF-7Adr is derived from MCF-7, but differs from its ancestral line in tolerance of MKT-077-induced mitochondrial toxicity. Our ultrastructural observations suggest that autophagy in the MCF-7Adr cells entails regional sequestration of MKT077 in multilamellar LC3-labeled MAPS, which then separate from their mitochondria, and fuse with or engulf each other. MAPS appeared to be migrating through the cytoplasm and fusing with the plasma membrane, thus carrying out exocytotic secretion. This mechanism, which seems ineffective in the ancestral cell line, provides a resistance mechanism for MKT-077 by enhancing the efflux process of the cells. After 8 hr of MKT-077 exposure, a fraction of the resistant cells appeared viable and contained larger number of smaller sized mitochondria. Mitoautophagosomes, therefore, provide a potentially novel model for multidrug resistance in cancerous cells and may contribute to the P-gp efflux process.
Subject(s)
Autophagosomes/ultrastructure , Breast Neoplasms/ultrastructure , Drug Resistance, Neoplasm/physiology , Mitochondria/ultrastructure , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Autophagosomes/drug effects , Autophagosomes/pathology , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Drug Resistance, Multiple/drug effects , Drug Resistance, Multiple/physiology , Drug Resistance, Neoplasm/drug effects , Female , Humans , Microscopy, Electron, Transmission , Mitochondria/drug effects , Mitochondria/pathology , Pyridines/pharmacology , Thiazoles/pharmacologyABSTRACT
Apoptosis, autophagosomes, and lysosomes are lacking in the retinal pigment epithelium (RPE) of age-related macular degeneration (AMD) eyes. Necrosis, not apoptosis, appeared to be the prominent type of cell death in RPE, which led to the accumulation of cell debris within and on both sides of Bruch's membrane. The endothelium of the choriocapillaris had an altered planar cell polarity which encompassed the disappearance of fenestrations, the thickening of cytoplasm, and anterior nuclear dislocation. There were no significant differences in RPE and choroidal aberrations between macular and temporal regions. Loss of endothelial polarity could be at the crux of AMD initiation and progression.
Subject(s)
Bruch Membrane/ultrastructure , Cell Polarity/physiology , Macular Degeneration/etiology , Aging , Choroid/cytology , Endothelial Cells/ultrastructure , Humans , Macular Degeneration/therapy , Retinal Pigment Epithelium/ultrastructureABSTRACT
Human immunodeficiency virus and antiretroviral therapy (ART) together can be far more detrimental to liver cells than either of the two unaided. However, ultrastructural aspects of the synergistic effects of HIV and ART have been understudied. In a patient cohort receiving ART, this study characterizes ultrastructurally sinusoidal degeneration, hepatocytic aberrations, mitochondrial dysfunction, accumulation of bulky lipid droplets (steatosis), and occlusion of sinusoidal lumina. Mitochondrial dysfunction causes the accumulation of acetyl-CoA which leads to insulin upregulation and resistance, lipid synthesis, and steatosis. Lipid droplets deposited in the sinusoids could be the source of the blood's lipid profile alterations in HIV patients on ART.
Subject(s)
Anti-HIV Agents/adverse effects , Chemical and Drug Induced Liver Injury/pathology , Endothelium/drug effects , HIV Infections/pathology , Hepatocytes/drug effects , Endothelium/pathology , Endothelium/ultrastructure , Female , HIV Infections/drug therapy , Hepatocytes/pathology , Hepatocytes/ultrastructure , Humans , Male , Microscopy, Electron, Transmission , Middle AgedABSTRACT
In order to maintain visual sensitivity at all light levels, the vertebrate eye possesses a mechanism to regenerate the visual pigment chromophore 11-cis retinal in the dark enzymatically, unlike in all other taxa, which rely on photoisomerization. This mechanism is termed the visual cycle and is localized to the retinal pigment epithelium (RPE), a support layer of the neural retina. Speculation has long revolved around whether more primitive chordates, such as tunicates and cephalochordates, anticipated this feature. The two key enzymes of the visual cycle are RPE65, the visual cycle all-trans retinyl ester isomerohydrolase, and lecithin:retinol acyltransferase (LRAT), which generates RPE65's substrate. We hypothesized that the origin of the vertebrate visual cycle is directly connected to an ancestral carotenoid oxygenase acquiring a new retinyl ester isomerohydrolase function. Our phylogenetic analyses of the RPE65/BCMO and N1pC/P60 (LRAT) superfamilies show that neither RPE65 nor LRAT orthologs occur in tunicates (Ciona) or cephalochordates (Branchiostoma), but occur in Petromyzon marinus (Sea Lamprey), a jawless vertebrate. The closest homologs to RPE65 in Ciona and Branchiostoma lacked predicted functionally diverged residues found in all authentic RPE65s, but lamprey RPE65 contained all of them. We cloned RPE65 and LRATb cDNAs from lamprey RPE and demonstrated appropriate enzymatic activities. We show that Ciona ß-carotene monooxygenase a (BCMOa) (previously annotated as an RPE65) has carotenoid oxygenase cleavage activity but not RPE65 activity. We verified the presence of RPE65 in lamprey RPE by immunofluorescence microscopy, immunoblot and mass spectrometry. On the basis of these data we conclude that the crucial transition from the typical carotenoid double bond cleavage functionality (BCMO) to the isomerohydrolase functionality (RPE65), coupled with the origin of LRAT, occurred subsequent to divergence of the more primitive chordates (tunicates, etc.) in the last common ancestor of the jawless and jawed vertebrates.
Subject(s)
Retinoids/chemistry , Retinoids/metabolism , Vision, Ocular/physiology , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Sequence , Animals , Catalysis , HEK293 Cells , Humans , Models, Molecular , Molecular Sequence Data , Multigene Family , Phylogeny , Protein Conformation , Retinal Pigment Epithelium/metabolism , Retinaldehyde/biosynthesis , Sequence Alignment , Vertebrates/genetics , Vertebrates/metabolism , beta-Carotene 15,15'-Monooxygenase/metabolism , cis-trans-Isomerases/chemistry , cis-trans-Isomerases/geneticsABSTRACT
Cone photoreceptors are the primary initiator of visual transduction in the human retina. Dysfunction or death of rod photoreceptors precedes cone loss in many retinal and macular degenerative diseases, suggesting a rod-dependent trophic support for cone survival. Rod differentiation and homeostasis are dependent on the basic motif leucine zipper transcription factor neural retina leucine zipper (NRL). The loss of Nrl (Nrl(-/-)) in mice results in a retina with predominantly S-opsin-containing cones that exhibit molecular and functional characteristics of wild-type cones. Here, we report that Nrl(-/-) retina undergoes a rapid but transient period of degeneration in early adulthood, with cone apoptosis, retinal detachment, alterations in retinal vessel structure, and activation and translocation of retinal microglia. However, cone degeneration stabilizes by 4 months of age, resulting in a thinner but intact outer nuclear layer with residual cones expressing S- and M-opsins and a preserved photopic electroretinogram. At this stage, microglia translocate back to the inner retina and reacquire a quiescent morphology. Gene profiling analysis during the period of transient degeneration reveals misregulation of genes related to stress response and inflammation, implying their involvement in cone death. The Nrl(-/-) mouse illustrates the long-term viability of cones in the absence of rods and retinal pigment epithelium defects in a rodless retina. We propose that Nrl(-/-) retina may serve as a model for elucidating mechanisms of cone homeostasis and degeneration that would be relevant to understanding diseases of the cone-dominant human macula.
Subject(s)
Apoptosis/genetics , Basic-Leucine Zipper Transcription Factors/genetics , Eye Proteins/genetics , Retina/abnormalities , Retina/metabolism , Retinal Cone Photoreceptor Cells/metabolism , Retinal Degeneration/physiopathology , Animals , Basic-Leucine Zipper Transcription Factors/deficiency , Disease Models, Animal , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Retinal Cone Photoreceptor Cells/pathology , Retinal Degeneration/genetics , Retinal Degeneration/pathology , Retinal Detachment/genetics , Retinal Detachment/pathology , Retinal Detachment/physiopathologyABSTRACT
El objetivo del presente trabajo, es dar a conocer conceptos clásicos y actuales del proceso terapéutico integrado, describir sus fases desde la óptica de diversos autores. Partiendo de la importancia de una correcta historia clínica del paciente, destacando el trabajo en equipo que sistematiza un protocolo de trabajo con un enfoque interdiciplinario. Se evalúa la implicancia del paciente en un tratamiento y el perfil profesional que debe reunir el odontólogo dedicado a esta área odontológica. El propósito de esta revisión bibliográfica, es convertirse en un punto de referencia para la lectura de graduados y estudiantes de odontología que desean incursionar dentro de la disciplina de clínica integrada, porque una buena planificación constituye la piedra angular que da lugar al proceso rehabilitador. Finalmente el profesional, tendrá una idea aproximada de la correcta elaboración de un plan salud, pudiendo educar a la situación individual de cada paciente y podrá acceder a un modelo de presupuesto odontológico donde se registra el trabajo a realizar y el costo en forma clara y ordenada.
