A purified inactivated Japanese encephalitis virus vaccine made in Vero cells.

A second generation, purified, inactivated vaccine (PIV) against Japanese encephalitis (JE) virus was produced and tested in mice where it was found to be highly immunogenic and protective. The JE-PIV was made from an attenuated strain of JE virus propagated in certified Vero cells, purified, and inactivated with formalin. Its manufacture followed current GMP guidelines for the production of biologicals. The manufacturing process was efficient in generating a high yield of virus, essentially free of contaminating host cell proteins and nucleic acids. The PIV was formulated with aluminum hydroxide and administered to mice by subcutaneous inoculation. Vaccinated animals developed high-titered JE virus neutralizing antibodies in a dose dependent fashion after two injections. The vaccine protected mice against morbidity and mortality after challenge with live, virulent, JE virus. Compared with the existing licensed mouse brain-derived vaccine, JE-Vax, the Vero cell-derived JE-PIV was more immunogenic and as effective as preventing encephalitis in mice. The JE-PIV is currently being tested for safety and immunogenicity in volunteers.

Changes in activity and mRNA for rat tryptophan hydroxylase and aromatic L-amino acid decarboxylase of brain serotonergic cell bodies and terminals following neonatal 5,7-dihydroxytryptamine.

In the present study, we examined time-dependent changes in activity, mRNA and immunoreactivity of the serotonin biosynthetic enzymes, tryptophan hydroxylase (TPH) and aromatic L-amino acid decarboxylase (AADC) in dorsal raphe nucleus (DRN), caudal brainstem and hypothalamus, following intracisternal injection of 5,7-dihydroxytryptamine (5,7-DHT) in neonatal rats. TPH activity in central serotonergic cell bodies and terminals was reduced to 20-30% of control levels at 1-8 weeks after neonatal, low-dose 5,7-DHT injection (24 micrograms free base). In contrast, AADC activity was either not changed or decreased to 40% of control levels, depending on the region. In situ hybridization and immunocytochemical staining indicated that 5,7-DHT caused a marked reduction in TPH and AADC message levels as well as the number of 5-HT and AADC-immunoreactive cells within the DRN as early as 1 week after 5,7-DHT. Even 15 weeks after drug administration recovery did not occur. This apparent neuronal loss was region-specific suggesting that some serotonergic neurons are more resistant to neonatal 5,7-DHT treatment than others. Taken together, these studies indicate that neonatal treatment with 5,7-DHT produces a marked and permanent (up to 15 weeks) reduction in the number of central serotonergic neurons.

Distribution of glucagonlike peptide I (GLP-I), glucagon, and glicentin in the rat brain: an immunocytochemical study.

Although glucagonlike immunoreactants (GLIs) are present in the central nervous system of several mammalian species, their structural relationship with pancreatic proglucagon is not defined, and their precise anatomical distribution has not been studied extensively. To obtain further information about the structure and biological significance of brain GLIs, the anatomical distribution of three different antigenic determinants of pancreatic proglucagon--glucagonlike peptide I (GLP-I), glucagon, and glicentin--was mapped in the brain of colchicine-treated rats by immunocytochemistry using the avidin-biotin-peroxidase method. Neuronal cell bodies immunoreactive with antisera specific for GLP-I, glucagon, and glicentin were found only in the caudal medulla oblongata. Within the caudal medulla immunostained cell bodies were found at levels from approximately 0.55 mm rostral to the obex to 0.45 mm caudal to the obex, and were located within the nucleus of the solitary tract (NTS) and the dorsal (MdD) and ventral (MdV) parts of the medullary reticular nucleus. The NTS contained three times more immunoreactive cell bodies than the MdD and MdV, and these cell bodies were located in the midline, medial, and lateral subnuclei of the caudal third of the NTS. Immunostaining of the same cell bodies in paired adjacent sections incubated with GLP-I and glucagon antisera or glucagon and glicentin antisera provided evidence for coexistence of the three antigens within the same neurons of the NTS. Nerve fibers and terminals immunoreactive with GLP-I, glucagon, and glicentin antisera were widely distributed throughout the rat brain and there was no discernible difference in the distribution of fibers and terminals immunoreactive with each of the three antisera. The highest densities of immunostained fibers and terminals were observed in the hypothalamus, thalamus, and septal regions, and the lowest in the cortex and hindbrain. The localization of neuronal cell bodies containing GLP-I, glucagon, and glicentin within the NTS and the MdD and MdV, and the extensive distribution of immunoreactive fibers and terminals throughout the rat brain suggest a role for these peptides in the integration of autonomic as well as central nervous system functions.

Bombesin-like immunoreactivity in the central nervous system of capsaicin-treated rats: a radioimmunoassay and immunohistochemical study.

The neuroanatomical distribution of bombesin-like immunoreactivity (BLI) in the rat central nervous system was investigated using radioimmunoassay and immunohistochemistry. Whereas cross-reactivity of the bombesin antiserum with substance P was problematic in the immunohistochemical experiments, no significant cross-reactivity with substance P was apparent in the radioimmunoassay. Results from the radioimmunoassay studies reveal particularly high concentrations of BLI in the hypothalamus, thalamus, medulla and spinal cord. Adult rats treated neonatally with capsaicin displayed significant depletions of somatostatin-like and substance P-like immunoreactivity and a small, statistically significant, reduction of BLI in the cervical spinal cord. Capsaicin treatment significantly reduced substance P-like immunoreactivity, but not somatostatin-like immunoreactivity, in the medulla and resulted in a small BLI depletion of borderline statistical significance in this brain region. Neonatally administered capsaicin treatment had no effect on the thalamic concentration of any of these three neuropeptides and neurotensin-like immunoreactivity was unchanged in all brain regions studied. These results suggest that the source of some of the BLI found in the spinal cord may be capsaicin-sensitive dorsal root ganglion cells.

[3H] Corticosterone binding in rat superior cervical ganglion.

Binding of [3H]corticosterone was examined in the soluble fraction (100,000 g) from superior cervical ganglia dissected from adrenalectomized and perfused rats. A large binding capacity (2100 fmol/mg protein) was found. However, no binding for [3H]-dexamethasone and [3H]triamcinolone was detected. The binding protein present in the ganglion preparation was characterized by DEAE-cellulose and DNA-cellulose chromatography and by isoelectric focusing. The corticosterone binding protein from the ganglia was found to be different from the cytoplasmic glucocorticoid receptor, but similar to plasma transcortin. A large portion (approximately 75%) of the transcortin-like [3H]corticosterone binding protein was located in the interstitial space between the sheath and the ganglionic cells. However, 25% of the binding protein was retained in the desheathed ganglion even after extensive washing with saline. Our findings indicate that cytoplasmic glucocorticoid receptors are not present in the superior cervical ganglion. It is possible that the transcortin-like protein associated with the desheathed ganglion may be intracellular, as other investigators have suggested similar intracellular presence of a transcortin-like protein in the liver, uterus, and pituitary.