Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.     

The structure of tight junctions in the tracheal epithelium may not correlate with permeability

Summary To test the hypothesis that cigarette smoke produces changes in the morphology of tight junctions guinea pigs were exposed to cigarette smoke or air in a previously standardized fashion (Simani et al. 1974). Permeability is greatest one half hour following exposure to cigarette smoke (Hulbert et al. 1981). The animals were sacrificed at that time. The tracheal epithelium was studied using both thin-section and freeze-fracture techniques. A quantitative analysis of the organization and integrity of junctional complexes was performed for each animal. Organization was assessed by measuring and comparing areas delimited by PF fibers and EF furrows. PF fiber integrity was assessed by measuring uninterrupted lengths of fibers and furrows from freeze-fracture replicas. This assessment did not demonstrate a change in tight-junction morphology following exposure to cigarette smoke.     

ANALYSIS OF SPATIAL DISTRIBUTIONS OF CELLULAR MOLECULES DURING MECHANICAL STRESSING OF CELL SURFACE RECEPTORS USING CONFOCAL MICROSCOPY

Confocal laser scanning microscopy represents a suitable technique to study the localization of cellular components in three dimension. The authors used this technique to analyse cellular events related to mechanical stimulation of integrin receptors on the cell surface. By performing optical sections the distribution of integrin receptors on the apical surface of an osteoblastic cell was determined. Concerning intracellular compartimentalization of signal transduction events, it was demonstrated that mechanical stimulation of integrins induced their linkage to the cytoskeleton. Cytoskeletally associated proteins like vinculin and talin accumulated in the vicinity of the site where the mechanical stress was applied to integrins on the cell surface. Optical sections revealed that clustering of these proteins proceeded to the base of the cell with gradually decreasing extent. In summary, it was demonstrated that the local distribution of cellular components is an important factor in mechanically induced signal transduction.     

Extended shelf life of enzyme-based biosensors using a novel stabilization system

The storage stability of amperometric enzyme electrodes has been enhanced by a combination of a soluble, positively charged polymer, diethylaminoethyl (DEAE)-dextran, and a sugar alcohol, lactitol. Two different types of alcohol biosensor have been produced using the enzyme alcohol oxidase, isolated from the methylotrophic yeast Hansenula polymorpha. The first employs enzyme entrapment between two membranes with direct hydrogen peroxide amperometry at +0·65 V. The second was based on the mediated, coupled reaction with horseradish peroxidase and N-methyl phenazimiumtetracyanoquinonedimethane (NMP-TCNQ) on a graphite electrode. In both cases, addition of the stabilizers promoted a considerable increase in the storage stability of the enzyme component, as indicated by an increase in the shelf life of desiccated biosensors under conditions of thermal stress at 37°C. In addition, an L-glutamate biosensor constructed from NMP-TCNQ-modified graphite electrodes and L-glutamate oxidase also exhibited an increase in shelf life when stored, desiccated in the presence of stabilizers.     

Human insulin autoantibody fine specificity and H and L chain use

Fine specificity and H and L chain isotypes of insulin autoantibodies in sera from 11 subjects were examined. None of these 11 subjects was treated with exogenous insulin. Two patterns of fine specificity were found. In one, the autoantibodies were specific for human insulin, with a requirement for threonine at B30. The conservative substitution in pork insulin (threonine to alanine) abrogated IgG binding by these sera. Insulin autoantibodies in other sera cross-reacted with beef, pork, and human insulin; not requiring threonine at B30. Reciprocal competitive inhibition experiments showed that epitopes recognized by the human specific insulin autoantibodies were exclusively on the B chain, whereas the cross-reactive sera contain autoantibodies that recognize both the B chain and combinatorial (A and B chain) epitopes. The fine specificity of cross-reactive insulin autoantibodies are thus similar to insulin antibodies from insulin-treated subjects. When IgG subclasses and L chains of insulin autoantibodies were examined, however, restricted C region usage was found. The hierarchy was IgG3 greater than G1 greater than G2 greater than G4; with one subclass dominant in each serum, although others were used. L chain use was similarly restricted. There was no correlation between isotype and fine specificity or between H and L chain type. It is concluded that heterogeneity of insulin autoantibodies is restricted. The response is probably more oligo- or pauciclonal than insulin antibody from insulin-treated subjects.     

The phosphoenol-pyruvate branchpoint in adult Hymenolepis diminuta (Cestoda): a study of pyruvate kinase and phosphoenol-pyruvate carboxykinase.

The properties of pyruvate kinase (PK) and phosphoenol pyruvate carboxykinase (PEP CK), two enzymes that determine the preferrential accumulation of either succinate or lactate as endproducts of carbohydrate metabolism, are described in adult Hymenolepis diminuta. PK activity at Vmax and Km levels of PEP was unaffected by ATP, alanine, FDP4, OR H+ ions, but was inhibited by 50% at 6.3 mM L-lactate and 30 mM HCO3. The addition of 30 mM HCO3 increased the Km(PEP) by 6-fold but did not alter the Vmax. The inhibition of PK by HCO3 cannot be explained entirely by an effect of ionic strength, but probably represents a specific modulator-enzyme interaction. Under similar conditions PEP CK was maximally activated. Although L-lactate inhibited PEP CK (Ki(lac) = 1.8 mM), this effector may play a minor role in regulation of PEP flux. These results implicate the poise of the HCO3-:CO2 system as a major determiner of endproduct accumulation in H. diminuta.