ABSTRACT
BACKGROUND: Adjuvants are essential in the induction of immunity by vaccines and interact with receptors, including the Toll-like receptors (TLRs). Responsiveness of these receptors differs between and within populations, which impacts vaccine effectiveness. OBJECTIVE: Here we examine how the innate cytokine response towards TLR ligands differs between high and low socioeconomic status (SES) school-aged children from Makassar, Indonesia. METHODS: We stimulated whole blood from children, of which 27 attended a high SES school and 27 children a low SES school, with ligands for TLR-2/1, -2/6, -3, -4, -5, -7, -9 and measured pro- (TNF) and anti-inflammatory (IL-10) cytokines released. RESULTS: In the low SES there is an increased pro-inflammatory response after 24 h stimulation with TLR-2/1 ligand Pam3 and TLR-4 ligand LPS compared to the high SES. Comparison of the response to LPS after 24 h versus 72 h stimulation revealed that the pro-inflammatory response in the low SES after 24 h shifts to an anti-inflammatory response, whereas in the high SES the initial anti-inflammatory response shifts to a strong pro-inflammatory response after 72 h stimulation. CONCLUSION: We observed differences in the TLR-mediated innate immune response between children attending low and high SES schools, which can have important implications for vaccine development.
Subject(s)
Cytokines , Immunity, Innate , Socioeconomic Factors , Toll-Like Receptors , Child , Cytokines/immunology , Humans , Indonesia , Ligands , Toll-Like Receptors/immunologyABSTRACT
In the past decade several reports have claimed that peroxisomes play a critical role in the isoprenoid/cholesterol biosynthetic pathway based on the finding of a predominant peroxisomal localization of several of the enzymes involved. Other reports, however, do not support the peroxisomal localization of these enzymes. In this study we have studied the subcellular localization of one of the enzymes, human mevalonate pyrophosphate decarboxylase, by conventional subcellular fractionation and digitonin permeabilization studies, immunofluorescence microscopy, and immunoelectron microscopy. We found a cytosolic localization for both endogenous human mevalonate pyrophosphate decarboxylase (in human fibroblasts, liver, CV1 and HEK293 cells) and overexpressed mevalonate pyrophosphate decarboxylase (in human fibroblasts, HEK293 and CV1 cells) but no indication for a peroxisomal localization. Our results do not support a central role of peroxisomes in the isoprenoid/cholesterol biosynthetic pathway.
Subject(s)
Carboxy-Lyases/metabolism , Cytosol/enzymology , Homozygote , Peroxisomes/enzymology , Cell Fractionation , Cells, Cultured , Fibroblasts/enzymology , Fluorescent Antibody Technique , Humans , Hypercholesterolemia/genetics , Liver/enzymology , Microscopy, ImmunoelectronABSTRACT
In the past decade several reports have appeared which suggest that peroxisomes play a central role in isoprenoid/cholesterol biosynthesis. These suggestions were based primarily on the reported finding of several of the enzymes of the presqualene segment of the biosynthetic pathway in peroxisomes. More recently, however, conflicting results have been reported raising doubt about the postulated role of peroxisomes in isoprenoid biosynthesis, at least in humans. In this study we have studied the subcellular localisation of human mevalonate kinase (MK) using a variety of biochemical and microscopical techniques. These include conventional subcellular fractionation studies, digitonin permeabilisation studies, immunofluorescence microscopy and immunocytochemistry. We exclusively found a cytosolic localisation of both endogenous human MK (human fibroblasts, liver and HEK293 cells) and overexpressed human MK (human fibroblasts, HEK293 cells and CV1 cells). No indication of a peroxisomal localisation was obtained. Our results do not support a central role for peroxisomes in isoprenoid biosynthesis.
Subject(s)
Cytosol/enzymology , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Cell Line , Cell Membrane Permeability/drug effects , Cell Membrane Permeability/physiology , Cytosol/metabolism , Digitonin/pharmacology , Fibroblasts/cytology , Fibroblasts/enzymology , Humans , Kidney/cytology , Kidney/enzymology , Liver/enzymology , Liver/ultrastructure , Microscopy, Fluorescence , Peroxisomes/enzymology , Subcellular Fractions/enzymologyABSTRACT
In the past decade, a predominant peroxisomal localization has been reported for several enzymes functioning in the presqualene segment of the cholesterol/isoprenoid biosynthesis pathway. More recently, however, conflicting results have been reported raising doubts about the postulated role of peroxisomes in isoprenoid biosynthesis, at least in humans. In this study, we have determined the subcellular localization of human phosphomevalonate kinase using a variety of biochemical and microscopic techniques, including conventional subcellular fractionation studies, digitonin permeabilization studies, immunofluorescence, and immunoelectron microscopy. We found an exclusive cytosolic localization of both endogenously expressed human phosphomevalonate kinase (in human fibroblasts, human liver, and HEK293 cells) and overexpressed human phosphomevalonate kinase (in human fibroblasts, HEK293 cells, and CV1 cells). No indication of a peroxisomal localization was obtained. Our results do not support a central role of peroxisomes in isoprenoid biosynthesis.