Molecular evidence for the early history of living amphibians

PURPOSE: To evaluate the feasibility of introducing exogenous genes and phosphorothioate oligonucleotides into the anterior chamber tissues of rats and monkeys using the authors' fusogenic liposomes. METHODS: Hemagglutinating virus of Japan liposomes containing LacZ DNA-high-mobility group 1 complexes or fluorescein isothiocyanate (FITC)-labeled phosphorothioate oligonucleotides were prepared and injected into the anterior chambers of rats (3 microliters) and rhesus monkeys (30 microliters). The expression of LacZ DNA was visualized histochemically by beta-Galactosidase assay and was followed for as long as 60 days in rats and 30 days in monkeys. FITC-labeled phosphorothioate oligonucleotides were observed by fluorescence microscopy for as long as 14 days in rats and 7 days in monkeys. RESULTS: Injection of LacZ DNA-high-mobility group 1 complexes encapsulated in hemagglutinating virus of Japan liposomes resulted in blue staining in the trabecular meshwork and iris-ciliary body of rats and selectively in the trabecular meshwork of monkeys at the concentrations used. This LacZ expression lasted for as long as 14 days after injection in both animals. Phosphorothioate oligonucleotides (3 microM) also were introduced into the rat trabecular meshwork and iris-ciliary body and into the primate trabecular meshwork when encapsulated in hemagglutinating virus of Japan liposomes, although the injection of naked FITC-labeled phosphorothioate oligonucleotides at the same concentration resulted in little fluorescence in any anterior chamber tissue. CONCLUSIONS: This study shows that the use of hemagglutinating virus of Japan liposomes can transfer LacZ DNA and phosphorothioate oligonucleotides to adult rat and primate trabecular meshwork. This system may enable progress in glaucoma research and in the development of nonviral somatic gene therapy of the trabecular meshwork to treat glaucoma.     

In vivo labelling of the mouse brain 5-hydroxytryptamine1A receptor with the novel selective antagonist 3H-NAD-299

The in vivo labelling of 5-hydroxytryptamine (5-HT)1A receptors in the mouse brain was studied with the novel selective 5-HT1A receptor antagonist, NAD-299 ((R)-3-N,N-dicyclobutylamino-8-fluoro-3,4-dihydro-2H-1-benzopyran- 5-carboxamide hydrogen (2R,3R)-tartrate monohydrate). 3H-NAD-299 was injected in a tail vein and the radioactivity in various brain regions was determined. More than 90% of the radioactivity in hippocampus, 15 min after the injection, was intact NAD-299. At this time the amount of 3H-NAD-299 was highest in hippocampus followed by frontal cortex, mesencephalon, hypothalamus, striatum and cerebellum. The specific accumulation of radioactivity (after subtracting cerebellum values) in frontal cortex and hippocampus was maximal 10 to 30 min after the injection and had almost disappeared after 2 h. Saturation kinetics derived Bmax (pmol/g wet weight tissue) values of 19.6+/-2.0 in frontal cortex and 38.0+/-3.5 in hippocampus. The apparent Kd values expressed in nmol/kg 3H-NAD-299 injected, were 12.3+/-2.2 in frontal cortex and 20.3+/-3.1 in hippocampus. The 5-HT1A receptor antagonist, WAY-100,635 competitively inhibited the specific accumulation of 3H-NAD-299 and was about equipotent with unlabelled NAD-299 with ED50 values of 20-30 nmol/kg s.c. These compounds were about 10 times more potent than the 5-HT1A receptor antagonists, p-MPPI and NDL-249 and 100 times more potent than (S)-UH-301. 5-HT1A receptor agonists, e.g. 8-OH-DPAT and flesinoxan and partial agonists, e.g. pindolol, buspirone and ipsapirone had low potency in this in vivo assay. Spiperone and methiothepin inhibited the 3H-NAD-299 accumulation at 10 micromol/kg s.c. The alpha1-adrenoceptor antagonist, prazosin at 2 micromol/kg s.c. increased significantly the specific accumulation of 3H-NAD-299. Pretreatment of the mice with the non-selective, irreversible receptor antagonist, EEDQ produced a dose related long-lasting decrease in the accumulation of 3H-NAD-299. It is concluded that NAD-299 is a very suitable ligand for studies of 5-HT1A receptors in the brain in vivo.     

Safety, tolerability and immunogenicity of concomitant injections in separate locations of M-M-R II, VARIVAX and TETRAMUNE in healthy children vs. concomitant injections of M-M-R II and TETRAMUNE followed six weeks later by VARIVAX

OBJECTIVES AND STUDY DESIGN: The primary objectives of this study were to compare immunologic responses, antibody persistence, safety and varicella breakthrough rates when VARIVAX (varicella vaccine) is given at the same time as M-M-R II (measles, mumps, rubella vaccine) and TETRAMUNE (conjugate Haemophilus influenzae type b, diphtheria, tetanus and whole cell pertussis vaccine) at separate injection sites (Group A) vs. VARIVAX given 6 weeks after M-M-R II and TETRAMUNE (Group B). Six hundred nine healthy children, 12 to 23 months of age, were randomized to one of two treatment (immunization) groups (Group A and Group B). Blood for antibody titers was drawn on the day of immunization, 6 weeks after each injection and 1 year later. Local and systemic adverse reactions were recorded. Exposure and cases of varicella were documented through a 1-year follow-up period. RESULTS: Measles, mumps and rubella seroconversion rates and geometric mean titers (GMTs) were similar for both treatment groups. Varicella seroconversion rates were also similar between groups. However, varicella GMTs and percent with a varicella-protective level [> or =5.0 glycoprotein (gp) enzyme-linked immunosorbent assay (ELISA) units] did not meet the prespecified criteria for similarity were lower for Group A (GMT 10.5; 82.8% > or =5.0 gp ELISA units) than for Group B (GMT 14.5; 91.2% > or =5.0 gp ELISA units). The GMTs between groups for other antibodies were similar. At the 1-year follow-up antibody titers were comparable in both groups and breakthrough varicella cases appeared generally similar. There were fewer local adverse events (AEs) at the VARIVAX injection sites (9.8% and 2.9%, Group A and B, respectively) than at the TETRAMUNE sites (27.9% and 24.0%). Systemic AEs were not statistically different when M-M-R II was administered alone (8.6%) or concomitantly with VARIVAX (8.9%). When VARIVAX was given alone AEs were 1.8%. The rate of fever > or =102 degrees F after M-M-R II and TETRAMUNE administered together was 10.7% on Days 0 to 3 and 23.7% on Days 7 to 21. When VARIVAX was administered alone, the rate of fever was 5.4% on Days 0 to 3 (P = 0.018) and 10.8% on Days 7 to 21 (P<0.001). CONCLUSION: Because the varicella titers were comparable and varicella breakthrough rates generally similar at 1 year in both groups, we expect that the concomitant administration of VARIVAX with M-M-R II and TETRAMUNE has clinical effectiveness similar to that with VARIVAX 6 weeks after the administration of these other two vaccines. VARIVAX appears to be less reactogenic than M-M-R II and TETRAMUNE.     

The Australian brushtail possum (Trichosurus vulpecula) gonadotrophin alpha-subunit: analysis of cDNA sequence and pattern of expression

Substantial evidence has accumulated to suggest that in the near future implementation of the veto-cell-suppressor concept in the treatment of kidney allograft recipients might lead to the establishment of life-long specific allograft tolerance in the absence of further immunosuppressive therapy. Veto suppression prevents the generation of antigen-specific T-helper and cytotoxic T lymphocytes in vitro provided that the T-lymphocyte precursors specifically recognize antigenic peptides associated with the major histocompatibility complex molecules class II and class I, respectively, expressed on the surface of the veto-active cell. Data from a large number of experimental and clinical studies strongly indicate that veto-active cells function in vivo and are capable of preventing allograft rejection. Thus, donor-cell-mediated veto activity is the most likely explanation for the well-known graft tolerizing effect of pretransplant donor blood transfusions in kidney graft recipients. A prerequisite for a veto-active environment in vivo is the establishment of lymphoid microchimerism, in which veto-active donor and recipient cells mutually downregulate potential alloaggression.     

Capitated risk-bearing managed care systems could improve end-of-life care

A surface protein antigen (PAc) of Streptococcus mutans, in particular the A-region of this PAc molecule, has been noted as a possible target in research for an effective dental caries vaccine. To identify the antigenic peptide binding to major histocompatibility complex (MHC) class II (HLA-DR) molecules in the A-region, we prepared a panel of overlapping synthetic peptides in the second unit of the A-region, and established that a simple enzyme-linked immunosorbent assay (ELISA) binding assay could be achieved by incubating the DR-crude. Binding to DR molecules of these peptides from nine donors was investigated by using the ELISA binding assay. It was revealed that the PAc(316-334) peptide bound more strongly to the HLA-DR molecule in seven out of nine subjects. In particular, DR8 (DRB1*0802), DR5 (DRB1*1101) and DR6 (DRB1*1402 and *1405), which bound strongly to PAc(316-334) peptide, were identified. Moreover, we synthesized glycine-substituted peptide analogues of the peptide and examined the binding motif of the binding region. As a result, the multiple binding motif in DR8, DR5 and DR6 was found in L-RV-K-A. It is suggested that a peptide vaccine for dental caries that is more effective for humans, with fewer adverse side-effects, could be designed by combining the multiple binding motif with the B-cell epitope to produce only the inhibiting antibody against dental caries. The peptide could therefore be useful for peptide vaccine development in the general human population.