ABSTRACT
The disaccharide trehalose is increasingly being used as a very efficient stabilizer of cells, membranes and macromolecules during cryo- and lyoconservation. Although extracellular trehalose can reduce cryo- and lyodamage to mammalian cells, the sugar is required on both sides of the plasma membrane for maximum protection efficiency. In the present study, mouse myeloma cells were loaded with the disaccharide by means of reversible electropermeabilization in isotonic trehalose-substituted medium, which contained 290 mM trehalose as the major solute. By using the membrane-impermeable fluorescent dye propidium iodide as the reporter molecule, optimum electropulsing conditions were found, at which most permeabilized cells survived and recovered (i.e., resealed) their original membrane integrity within a few minutes after electric treatment. Microscopic examination during the resealing phase revealed that electropulsed cells shrank gradually to about 60% of their original volume. The kinetics of the dye uptake and the volumetric response of cells to electropulsing were analyzed using a theoretical model that relates the observed cell volume changes to the solute transport across the transiently permeabilized cell membrane. From the best fit of the model to the experimental data, the intracellular trehalose concentration in electropulsed cells was estimated to be about 100 mM. This loading efficiency compares favorably to other methods currently used for intracellular trehalose delivery. The results presented here point toward application of the electropermeabilization technique for loading cells with membrane-impermeable bioprotectants, with far-reaching implications for cryo- and lyopreservation of rare and valuable mammalian cells and tissues.
Subject(s)
Electric Stimulation , Electrochemistry/methods , Electroporation/methods , Multiple Myeloma/pathology , Preservation, Biological/methods , Trehalose/administration & dosage , Animals , Cell Line , Cell Membrane Permeability , Cell Size , Cell Survival , Flow Cytometry/methods , Mice , Multiple Myeloma/physiopathology , Sensitivity and SpecificityABSTRACT
To monitor early and late events of immune system activation after primary and secondary flavivirus infection, 17 healthy persons were vaccinated with the standard 17D vaccine virus strain of yellow fever (YF). Twelve of these persons had not received YF vaccine previously and 5 had been vaccinated once at least 10 years before. Viremia and various parameters of humoral and cellular immune activation were followed daily for 7 days and weekly thereafter. Viremia was detected by reverse transcriptase-polymerase chain reaction in all 12 first-time vaccinees beginning from the second to the sixth day after vaccination; most tested positive between the fourth and sixth day. Infectious 17D virus was detected using a plaque forming assay in the serum of 7 of the 12 first-time vaccinees. As first parameters of immune activation, neopterin and beta2-microglobulin markedly increased between day 2 and day 6 postvaccination. In parallel to the viremia, circulating CD8+ T-cells significantly increased, with peak levels at day 5 after primary vaccination, indicating an activation of the cellular immune system. Neither viremia nor significant changes of these activation markers were observed in the five revaccinated persons. Neutralizing antibodies directed against the 17D vaccine strain developed in all persons within 2 weeks after vaccination. No correlation was found between the extent of viremia and the titer of neutralizing antibodies. Revaccination was followed by a minor and transient increase of neutralizing antibodies. High titers of neutralizing antibodies persisted for at least 10 years after primary vaccination.
Subject(s)
Antibodies, Viral/blood , Vaccines, Attenuated/immunology , Viral Vaccines/immunology , Viremia/immunology , Yellow Fever/prevention & control , Yellow fever virus/immunology , Adolescent , Adult , Antibodies, Viral/biosynthesis , Antibody Formation , Flow Cytometry , Fluorescent Antibody Technique , Humans , Middle Aged , Neopterin/blood , Neutralization Tests , Reverse Transcriptase Polymerase Chain Reaction , Vaccination , Vaccines, Attenuated/adverse effects , Viral Plaque Assay , Viral Vaccines/adverse effects , Viremia/etiology , Yellow Fever/immunologyABSTRACT
A radioligand receptor assay (RRA) for TSH and TSH-binding-inhibiting antibodies (TBIAb) using solubilized receptor is presented. Introducing charcoal for separation of bound and free hormone has made the assay easier to handle. The detection limit for TSH is 30 microU/ml as measured by RIA. TBIAb was found in the sera of 19 of 26 hyperthyroid patients. Only 16 sera were positive when the RRA using membrane fragments was applied.
