In vivo electroporation improves immune responses to DNA vaccination in sheep.

In vivo electroporation was utilised to enhance plasmid DNA expression in sheep muscle to improve the immune response to DNA vaccination. DNA encoding enhanced green fluorescence protein expressed at higher levels in sheep muscle following in vivo electroporation which caused minimal muscle damage. Groups of seven sheep were then given three intramuscular injections of plasmids encoding two Haemonchus contortus Ag, with and without electroporation at 0, 3 and 7 weeks. Humoral responses were enhanced in electroporated sheep. Four weeks after vaccination, all groups were injected subcutaneously with recombinant Ag formulated in Quil A. Induction of vaccine-specific immune memory was demonstrated in DNA-vaccinated sheep.

DNA injection in combination with electroporation: a novel method for vaccination of farmed ruminants.

Injection of plasmid DNA encoding antigens into rodents followed by electroporation improved the immune response when compared with injection without electroporation (Widera et al. J Immunol 2000;164:4635-40; Zucchelli et al. J Virol 2000;74:11598-607; Kadowaki et al. Vaccine 2000;18:2779-88). The present study describes the extension of this technology to farm animals, by injecting plasmid DNA encoding mycobacterial antigens (MPB70, Ag85B and Hsp65) into the muscles of goats and cattle using two different types of electrodes, both allowing DNA delivery at the site of electroporation. The animals were vaccinated under local anaesthesia without any observed immediate or long-term distress or discomfort, or any behavioural signs of muscle damage or pathological changes after the electroporation. DNA-injected and electroporated goats showed increased humoral response after the primary vaccination when compared with nonelectroporated animals. Improved T-cell responses following electroporation were observed in hsp65 DNA-vaccinated cattle. DNA injection with or without electroporation did not compromise the specificity of the tuberculin skin test. In conclusion, a protocol applying in vivo electroporation free of side effects to farmed ruminants was established. In addition, we show that DNA vaccination in combination with electroporation can improve the primary immune responses to the encoded antigens.

Photochemical transfection: a new technology for light-induced, site-directed gene delivery.

The development of methods for specific delivery of therapeutic genes into target tissues is an important issue for the further progress of in vivo gene therapy. In this article we report on a novel technology, named photochemical transfection, to use light to direct a precise delivery of therapeutic genes to a desired location. The technology makes use of photosensitizing compounds that localize mainly in the membranes of endosomes and lysosomes. On illumination these membrane structures will be destroyed, releasing endocytosed DNA into the cell cytosol. Using a green fluorescent protein gene as a model we show that illumination of photosensitizer-treated cells induces a substantial increase in the efficiency of transfection by DNA-poly-L-lysine complexes. Thus, in a human melanoma cell line the light treatment can increase the transfection efficiency more than 20-fold, reaching transfection levels of about 50% of the surviving cells. In this article various parameters of importance for the use of this technology are examined, and the potential use of the technology in gene therapy is discussed.

Phagosome dynamics and function.

Phagocytosis of microorganisms and other particles is mediated most efficiently by receptors such as Fc-receptors (FcR) and complement-receptors (C3R). Interaction between these receptors and ligands on the particle results in signal transduction events that lead to actin polymerisation and phagosome formation. The phagosome then undergoes a maturation process whereby it transforms into a phagolysosome. Phagosome maturation depends on interactions (fusion events) with early and late endosomes as well as with lysosomes. The fusion processes are regulated by small GTP-binding proteins and other proteins that are also involved in fusion processes in the endocytic pathway. Although most phagocytosed microorganisms are killed in the lysosome, some pathogens have developed survival strategies and are able to live in the harsh conditions in the phagolysosome or interfere with the maturation process and thereby evade destruction by acid hydrolases.

Role of endosomes in gene transfection mediated by photochemical internalisation (PCI).

BACKGROUND: Most non-viral gene therapy vectors deliver transgenes into cells through the endocytic pathway. Lack of escape from endocytic vesicles in many cases constitutes a major barrier for delivery of the functional gene. We have developed a new technology named photochemical internalisation (PCI) to achieve light-inducible cytosolic delivery of the transgene. The technology is based on a photochemical treatment employing photosensitisers localised in endocytic vesicles. In this work mechanisms involved in PCI-mediated transfection (photochemical transfection) were studied. METHODS: Human melanoma or colon carcinoma cells were pre-incubated with the photosensitiser aluminium phthalocyanine disulfonate (AlPcS2a) followed by treatment with plasmid encoding enhanced green fluorescent protein (EGFP) complexed with poly-L-lysine, N-(1-(2,3-dioleoxyloxy)propyl)-N,N,N,-trimethylammonium-methyl-sulfate (DOTAP) or polyethylenimine (PEI) and light exposure. The expression of the EGFP-gene was scored by fluorescence microscopy and flow cytometry. RESULTS: The photochemical treatment using light doses corresponding to D50 substantially improves the efficiency of transfection mediated by poly-L-lysine and PEI, but not by DOTAP. The treatment does not enhance the delivery of the plasmid complex across the plasma membrane, since the amount of internalised plasmid is similar for irradiated and non-irradiated cells. Light-inducible transfection occurs only under temperature conditions allowing endocytic uptake and is not improved by chloroquine or ammonium chloride, but is inhibited by bafilomycin A1 (agents that increase vesicular pH and interfere with the endocytic transport). CONCLUSIONS: Photochemical transfection occurs through endocytosis, followed by cytosolic release of the transfecting DNA from photochemically permeabilised endocytic vesicles. Release of plasmid from early endosomes seems to be of importance in photochemical transfection, although a role of later endocytic vesicles can, however, not be ruled out.

Photochemical internalization: a novel technology for delivery of macromolecules into cytosol.

The therapeutic usefulness of macromolecules, such as in gene therapy, is often limited by an inefficient transfer of the macromolecule to the cytosol and a lack of tissue-specific targeting. The possibility of photochemically releasing macromolecules from endosomes and lysosomes into the cytosol was examined. Endocytosed macromolecules and photosensitizer were exposed to light and intracellular localization and the expression of macomolecules in the cytosol was analyzed. This novel technology, named photochemical internalization (PCI), was found to efficiently deliver type I ribosome-inactivating proteins, horseradish peroxidase, a p21ras-derived peptide, and a plasmid encoding green fluorescent protein into cytosol in a light-dependent manner. The results presented here show that PCI can induce efficient light-directed delivery of macromolecules into the cytosol, indicating that PCI may have a variety of useful applications for site-specific drug delivery, e.g., in gene therapy, vaccination, and cancer treatment.

Retinyl esters are hydrolyzed in early endosomes of J774 macrophages.

The aim of the current study was to identify the subcellular compartment(s) responsible for the hydrolysis of chylomicron remnant-retinyl esters, in J774.1 cells. The cells were incubated with medium containing chylomicron remnant [(3)H]retinyl ester. Subcellular fractionation was used to separate early endosomes from late endosomes and lysosomes. About 26% and 80% of the total [(3)H]retinyl esters taken up by the J774 cells were hydrolyzed after 10 min and 60 min of chase, respectively. In the early endosomes, there was a 4-fold increase of radioactivity (nearly all radioactivity associated with retinyl esters) during the first 10 min of chase. The radioactivity in early endosomes was reduced by 43% from 10 min to 60 min and remained stable from 60 to 180 min of chase. From 10 to 60 min the amount of retinol in early endosomes increased from 44% to 82%, indicating an efficient hydrolysis of retinyl esters. Less than 10% and 5% of the total cell-associated radioactivity was found in the late endosomes and lysosomes during the entire chase period. In the chase medium, 84% of the total amount of retinoid released during 180 min was present already after 10 min. The percentage of retinol in the medium increased from 25% to 82% during incubation from 10 to 180 min. These data suggest that retinyl esters are endocytosed together with the chylomicron remnant particle and hydrolyzed in the early endosomes in this cell model.-Hagen, E., A. M. Myhre, T. E. Tjelle, T. Berg, and K. R. Norum. Retinyl esters are hydrolyzed in early endosomes of J774 macrophages.

In vitro fusion of phagosomes with different endocytic organelles from J774 macrophages.

We describe novel biochemical and electron microscopy assays to investigate in vitro fusion of latex bead phagosomes with three different endocytic organelle fractions from J774 macrophages. After formation, early phagosomes fuse avidly with early and late endosomes and for a longer period of time with lysosomes, but they subsequently become fusion-incompetent. The fusion of early, but not late, phagosomes with all three endocytic fractions could be significantly stimulated by Rab5. In contrast to other cell types investigated, this Rab is uniquely enriched on both early and late endosomes in J774 macrophages. Moreover, exogenous Rab5 stimulates homotypic fusion between both sets of organelles. This was shown by a quantitative electron microscopy fusion assay that can directly assay fusion between any combination of morphologically defined organelles. By the same approach, we discovered an unexpected Rab5-stimulatable fusion between early and late endosomes in J774, but not in BHK cells. Thus, in J774 cells both Rab5 and the endocytic pathway seem to have evolved additional functions not yet seen in nonphagocytic cells.

Degradation of phagosomal components in late endocytic organelles.

Phagosomes are formed when phagocytic cells ingest particles such as bacteria, viruses or synthetic beads of different kinds. The environment within the phagosome gradually changes to generate degradative conditions. These changes require multiple interactions between the maturing phagosomes and the endocytic and the biosynthetic pathway. The phagosomes probably communicate with endocytic organelles by a transient fusion event, often referred to as the 'kiss-and-run' hypothesis. We have studied the role of endocytic organelles in the phagocytic pathway of J774 cells, a mouse macrophage cell line. We have used magnetic Dynabeads coated with 125ITC-IgG and 125ITC-OVA as phagocytic probes and were able to isolate the phagosomal fraction by means of a magnet. To separate lysosomes from other organelles in the endocytic pathway we allowed the cells to endocytose a pulse of colloidal gold particles complexed with ovalbumin. By combining this density shift technique with subcellular fractionation of a postnuclear supernatant in Percoll gradients we could isolate three endocytic fractions corresponding to early endosomes (the light Percoll fraction), late endosomes (the dense Percoll fraction) and lysosomes (the gold fraction). We observed that the proteins linked to the ingested beads are initially cleaved in the phagosomes. This cleavage is inhibited by leupeptin, a thiol-protease inhibitor, and requires an acidic environment. However, efficient communication between the phagosomes and the endocytic pathway leads to the transfer of dissociated phagocytosed peptides of different sizes to late endosomes and lysosomes for further processing. Consequently, the late endosomes and the lysosomes may be involved in the degradation of phagocytosed compounds.

Isolation and characterization of early endosomes, late endosomes and terminal lysosomes: their role in protein degradation.

Although endosomal proteolysis has been reported (e.g. for peptide hormones and lysosomal enzymes), lysosomes are believed to be the main site of degradation in the endocytic pathway. We have studied the separate roles of lysosomes and prelysosomal endocytic organelles in the degradation of ovalbumin in J774 cells. The ovalbumin was labelled with 125I-tyramine cellobiose (125I-TC-ova). The labelled degradation products formed from this probe are trapped at the site of formation. To separate lysosomes efficiently from prelysosomal endocytic organelles we allowed the cells to endocytose a pulse of colloidal gold particles complexed with ovalbumin. By combining this density shift technique with subcellular fractionation of a postnuclear supernatant in Percoll gradients we could isolate three fractions that were sequentially involved in the endocytic pathway: a light Percoll fraction, a dense Percoll fraction and a gold fraction. The light Percoll fraction contained early endosomes since it was transferrin positive and received endocytic markers such as ovalbumin and horseradish peroxidase (HRP) early (< 5 minutes) after internalization. The dense Percoll fraction was transferrin negative, rab7 positive and received endocytic markers after 10-15 minutes of internalization. The gold-filled fraction was negative for both transferrin and rab7 but highly enriched in the lysosomal enzyme beta-hexosaminidase and was therefore defined as a lysosome. To study the role of endosomes and lysosomes in the degradation of endocytosed material we allowed the cells to take up (via the mannose receptor) 125I-TC-ova. It was found that the main degradation of 125I-TC-ova (measured as acid soluble radioactivity trapped in the organelle) took place in the late endosomes (and not in the lysosomes containing the bulk of the lysosomal enzymes). Our data therefore suggest that the late endosomes operate as an early lysosomal compartment. The terminal lysosomes may serve as storage bodies for acid hydrolases that may be called upon when needed (for instance during phagocytosis).

Binding and intracellular fate of beta-very low density lipoprotein in isolated rat liver parenchymal cells.

Binding of rabbit 125I-tyramine-cellobiose-beta-very low density lipoprotein (125ITC-beta-VLDL) was saturable both in suspended and cultured rat liver parenchymal cells, and in isolated rat liver membrane fractions. The specific binding had KD values ranging from 8 to 10 micrograms/ml. At 37 degrees C beta-VLDL was internalized and degraded in parenchymal cells in culture, but only negligible degradation was measured in suspended cells. In cultured cells the degradation was inhibited by lysosomal and microtubular inhibitors, these agents had no effects in suspended cells. Studies of release suggested internalization in suspended cells and subcellular fractionation experiments confirmed that internalized 125ITC-beta-VLDL reached the lysosomal compartment in cultured cells, but not in suspended cells. Since lactosylated (lac-) beta-VLDL was taken up and degraded efficiently by suspended cells, these cells are not in general unable to transport large lipoprotein particles to the lysosomes. We believe that beta-VLDL does not dissociate from the receptor in the endosomes and therefore is retroendocytosed to the plasma membrane. In isolated parenchymal cells the beta-VLDL binding site is supposedly different from the methylamine-activated-alpha 2-macroglobulin (ma-alpha 2M) binding site, since; 1) Excess beta-VLDL did not reduce ma-alpha 2M binding; 2) In suspended parenchymal cells ma-alpha 2M was readily degraded, whereas beta-VLDL was not degraded and 3) The binding of beta-VLDL was not Ca+(+)-dependent.(ABSTRACT TRUNCATED AT 250 WORDS)