ABSTRACT
PURPOSE: The complement system is involved in the pathogenesis of age-related macular degeneration (AMD). Because activated microglia are also associated with AMD, we studied the relationship between complement anaphylatoxin receptors and microglial recruitment. METHODS: We assessed the effect of anaphylatoxin C3a receptor (C3aR) and C5a receptor (C5aR) knockout (KO) on light damage-induced migration of microglia/macrophages into the mouse outer retina via immunofluorescence and real-time quantitative PCR. RESULTS: We found that the mRNA levels of C3, C5, C3aR, C5aR, and two activators of the complement alternative pathway, Cfb and Cfd, were all upregulated after light exposure. Retinal Iba1-positive microglia/macrophages express receptors for C3a and C5a. Light damage increased the number of retinal Iba1-positive cells and the mRNA levels of Iba1. Compared with the wild-type (WT) mice, these increases were attenuated in the C5aR KO mice but not in the C3aR KO mice. CONCLUSIONS: C5aR but not C3aR promoted the recruitment of microglia/macrophages. These divergent properties of complement anaphylatoxins in the light damage model provide a rationale for testing the differential effects of these receptors in additional retinal and neurodegeneration models.
Subject(s)
Cell Movement/radiation effects , Gene Knockout Techniques , Light/adverse effects , Macrophages/physiology , Microglia/physiology , Receptor, Anaphylatoxin C5a/genetics , Retinal Degeneration/pathology , Animals , Calcium-Binding Proteins/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Microfilament Proteins/metabolism , RNA, Messenger/genetics , Radiation Injuries, Experimental/etiology , Radiation Injuries, Experimental/pathology , Real-Time Polymerase Chain Reaction , Receptors, G-Protein-Coupled/genetics , Retina/radiation effects , Retinal Degeneration/etiologyABSTRACT
Aminolevulinic acid (ALA)-mediated protoporphyrin IX (PpIX) production is being explored for tumor fluorescence imaging and photodynamic therapy (PDT). As a prodrug, ALA is converted in heme biosynthesis pathway to PpIX with fluorescent and photosensitizing properties. To better understand the role of heme biosynthesis enzymes in ALA-mediated PpIX fluorescence and PDT efficacy, we used lentiviral shRNA to silence the expression of porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD) and ferrochelatase (FECH) in SkBr3 human breast cancer cells. PBGS and PBGD are the first two cytosolic enzymes involved in PpIX biosynthesis, and FECH is the enzyme responsible for converting PpIX to heme. PpIX fluorescence was examined by flow cytometry and confocal fluorescence microscopy. Cytotoxicity was assessed after ALA-mediated PDT. Silencing PBGS or PBGD significantly reduced ALA-stimulated PpIX fluorescence, whereas silencing FECH elevated basal and ALA-stimulated PpIX fluorescence. However, compared with vector control cells, the ratio of ALA-stimulated fluorescence to basal fluorescence without ALA was significantly reduced in all knockdown cell lines. PBGS or PBGD knockdown cells exhibited significant resistance to ALA-PDT, while increased sensitivity to ALA-PDT was found in FECH knockdown cells. These results demonstrate the importance of PBGS, PBGD and FECH in ALA-mediated PpIX fluorescence and PDT efficacy.
Subject(s)
Aminolevulinic Acid/metabolism , Gene Silencing , Heme/biosynthesis , Photochemotherapy , Protoporphyrins/metabolism , Cell Line, Tumor , HumansABSTRACT
Desde os anos 1980, em resposta aos desafios trazidos pelas doenças raras, alguns países desenvolvidos vêm estabelecendo quadros regulatórios. Os países em desenvolvimento devem fazer o mesmo? Este artigo argumenta que a limitação de recursos de um país em desenvolvimento, embora seja um fator importante para se considerar cuidadosamente a distribuição dos gastos com saúde, não pode ser uma desculpa para negar a necessidade de uma regulamentação. O trabalho apresenta argumentos legais e políticos para o estabelecimento de tal regulação nos países em desenvolvimento. Também relata os recentes esforços legislativos para o cuidado das doenças raras por parte das autoridades locais chinesas, explica as razões para a adoção de uma nova definição de doenças raras e elabora os segmentos necessários dentro de um contexto integrado, racional e adequado à China para o cuidado dessas enfermidades.