Search for correlates of protective immunity conferred by anthrax vaccine.

Vaccination by anthrax protective antigen (PA)-based vaccines requires multiple immunization, underlying the need to develop more efficacious vaccines or alternative vaccination regimens. In spite of the vast use of PA-based vaccines, the definition of a marker for protective immunity is still lacking. Here we describe studies designed to help define such markers. To this end we have immunized guinea pigs by different methods and monitored the immune response and the corresponding extent of protection against a lethal challenge with anthrax spores. Active immunization was performed by a single injection using one of two methods: (i) vaccination with decreasing amounts of PA and (ii) vaccination with constant amounts of PA that had been thermally inactivated for increasing periods. In both studies a direct correlation between survival and neutralizing-antibody titer was found (r(2) = 0.92 and 0.95, respectively). Most significantly, in the two protocols a similar neutralizing-antibody titer range provided 50% protection. Furthermore, in a complementary study involving passive transfer of PA hyperimmune sera to naive animals, a similar correlation between neutralizing-antibody titers and protection was found. In all three immunization studies, neutralization titers of at least 300 were sufficient to confer protection against a dose of 40 50% lethal doses (LD(50)) of virulent anthrax spores of the Vollum strain. Such consistency in the correlation of protective immunity with anti-PA antibody titers was not observed for antibody titers determined by an enzyme-linked immunosorbent assay. Taken together, these results clearly demonstrate that neutralizing antibodies to PA constitute a major component of the protective immunity against anthrax and suggest that this parameter could be used as a surrogate marker for protection.

A vasoactive intestinal peptide antagonist inhibits the growth of glioblastoma cells.

The effects of a vasoactive intestinal peptide (VIP) receptor antagonist (VIPhyb) on human glioblastoma cells were characterized. Pituitary adenylate cyclase activating polypeptide (125I-PACAP-27) bound with high affinity to U87, U118, and U373 cells. Specific 125I-PACAP-27 binding to U87 cells was inhibited, with high affinity, by PACAP but not VIP or VIPhyb (IC50 = 10, 1500, and 500 nM, respectively). By reverse transcriptase-polymerase chain reaction (RT-PCR), a major 305 bp band was observed indicative of PAC1 receptors. PACAP-27 caused cAMP elevation and the increase in cAMP caused by PACAP-27, was inhibited by the VIPhyb. Also, PACAP-27 caused cytosolic Ca2+ elevation in Fura-2AM loaded U87 cells and the VIPhyb inhibited this increase. Using the MTT growth assay, the VIPhyb was shown to inhibit glioblastoma growth in a concentration-dependent manner. Using a clonogenic assay in vitro, 10 microM VIPhyb significantly inhibited proliferation of U87, U118, and U373 cells. In vivo, 0.4 microg/kg VIPhyb inhibited U87 xenograft proliferation in nude mice. These results suggest that the VIPhyb antagonizes PAC1 receptors on glioblastoma cells and inhibits their proliferation.

Attenuated nontoxinogenic and nonencapsulated recombinant Bacillus anthracis spore vaccines protect against anthrax.

Several highly attenuated spore-forming nontoxinogenic and nonencapsulated Bacillus anthracis vaccines differing in levels of expression of recombinant protective antigen (rPA) were constructed. Biochemical analyses (including electrospray mass spectroscopy and N terminus amino acid sequencing) as well as biological and immunological tests demonstrated that the rPA retains the characteristics of native PA. A single immunization of guinea pigs with 5 x 10(7) spores of one of these recombinant strains, MASC-10, expressing high levels of rPA (>/=100 microgram/ml) from a constitutive heterologous promoter induced high titers of neutralizing anti-PA antibodies. This immune response was long lasting (at least 12 months) and provided protection against a lethal challenge of virulent (Vollum) anthrax spores. The recombinant B. anthracis spore vaccine appears to be more efficacious than the vegetative cell vaccine. Furthermore, while results clearly suggest a direct correlation between the level of expression of PA and the potency of the vaccine, they also suggest that some B. anthracis spore-associated antigen(s) may contribute in a significant manner to protective immunity.

Insulin-like growth factor receptors and their ligands in gonads of a hermaphroditic species, the gilthead seabream (Sparus aurata): expression and cellular localization.

Expression of insulin-like growth factor (IGF)-I, IGF-II, and IGF type I receptor (IGF-1R) genes was studied in gonads at different developmental stages of the protandrous hermaphroditic species the gilthead seabream (Sparus aurata) by reverse transcription-polymerase chain reaction and Northern blot analysis. Both IGF-I and IGF-II mRNA levels were highest in bisexual gonads and decreased during gonadal development. Regardless of the stage of gametogenesis, IGF-II mRNA levels exceeded those of IGF-I. Transcripts for IGF-1R RNA were detected in gonads at all stages studied. A major transcript of 11 kb was found in gonads and in gill arch and brain, but it was not found in liver and muscle. Distribution of the two types of IGF-1R and IGF-I in gonads was studied by immunohistochemistry. Immunoreactive IGF-I was found in the granulosa and theca cells of follicles at different vitellogenic stages and in oocytes at the chromatin-nucleolus and perinucleolus stage. In the testis, immunoreactive IGF-I was found in somatic cells of the cyst wall, interstitial cells, and spermatogonia A. In addition, IGF-1R was detected in the membrane of previtellogenic oocytes and in the theca and granulosa cells of vitellogenic and late vitellogenic follicles. In the testis, a positive reaction was identified in spermatogonia A and spermatids for the germ cells and in somatic cells of the cyst walls and interstitial cells. Local expression and production of IGFs and their receptors in fish gonads support a role for the IGF system in fish gonadal physiology.

VPAC1 receptors and lung cancer.

VIP/PACAP are autocrine growth factors for lung cancer. VIP and/or PACAP mRNA is present in most lung cancer cell lines examined. Although mRNA for VPAC2-R is not common, VPAC1-R and PAC1-R mRNA is present in many lung cancer cell lines. 125I-VIP binds with high affinity to lung cancer cells and specific 125I-VIP binding is inhibited with high affinity by (Lys15, Arg16, Leu27)VIP1-7 GRF8-27, the VPAC1-R specific agonist, but not by Ro25-1553(18), the VPAC2-R specific agonist. VIP elevates cAMP and increases c-fos gene expression. The increase in cAMP and c-fos mRNA caused by VIP is inhibited by SN(VH). (SH)VH inhibited the proliferation of NCIH1299 cells in the MTT assay, which is based on cytotoxicity. In a recent cell line screen, (SN)VH inhibited the growth of 51 of 56 cancer cell lines including leukemia, lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, breast cancer, and prostate cancer (T. Moody, unpublished). It remains to be determined if (SN)VH will be useful for treatment of a wide variety of cancers.

Sulfur mustard toxicity in macrophages: effect of dexamethasone.

Cells from the murine macrophage-like cell line J774A.1 (J774) and cultures of primary alveolar macrophages (PAM) obtained from guinea pigs were exposed to sulfur mustard (HD, 50-200 microM) and treated with dexamethasone (2.5 microM) 10 min after HD exposure. Cell cultures were studied at 3 and 24 h after exposure by the cleavage of Thiazolyl blue reaction (MTT) reaction and crystal violet staining (viability assays), by morphological observation and by [3H]thymidine incorporation. Exposure of J774 cells to HD caused a dose-dependent decrease in viability that was evident at 24 h. Although no significant change in viability was observed at 3-4 h after HD exposure, a dose-dependent decrease in [3H]thymidine incorporation was observed. Treatment with dexamethasone caused a dose-dependent decrease in viability. However, the combined exposure to HD and dexamethasone had a synergistic effect on the decrease of cell viability. This synergistic effect is not due to a change in DNA synthesis rate because [3H]thymidine incorporation was not affected by dexamethasone. In PAM cultures, HD caused some 'activating' effect on [3H]thymidine incorporation and an increase in cell number at the lower dose (100 microM) but this was less at 200 microM. Both effects were reduced by dexamethasone treatment. We conclude that macrophages derived from different sources exhibit a different responsiveness to immunomodulators (HD and dexamethasone) and that dexamethasone can reduce the 'inflammatory' effect of HD in PAM.

Ontogeny of the insulin-like growth factor system (IGF-I, IGF-II, and IGF-1R) in gilthead seabream (Sparus aurata): expression and cellular localization.

There is evidence for the presence of an insulin-like growth factor (IGF) system during fish development. The pattern of gene expression of IGF-I, IGF-II, and their cognate receptors during early development of gilthead seabream (Sparus aurata) was studied by reverse transcription-polymerase chain reaction (RT-PCR). Transcripts for IGF-I, IGF-II, and IGF-1R were detected throughout development in unfertilized eggs, embryos, and larvae, suggesting that these mRNAs are products of both the maternal and the embryonic genomes. Analysis of IGF-1R mRNA in various adult tissues using RT-PCR revealed expression in all tissues studied, with the highest levels in gill cartilage, skin, kidney, heart, pyloric caeca, and brain. The distribution of the two types of IGF-1R and IGF-I in gilthead seabream larvae was studied by immunohistochemistry and found to be tissue-specific and age-dependent. IGF-I and its receptors are widely distributed and appear in various tissues of seabream larvae. IGF-I immunoreactivity was highest in skeletal muscle and pancreas. The general distribution of the two types of IGF receptors in larval tissues appeared similar except for the muscle and the corpus cerebelli, in which IGF-1R was detected only by SpIR6 antisera. Both IGF-I and IGF-II may thus play a role during early development of teleosts, as in other vertebrates.

PACAP(6-38) is a PACAP receptor antagonist for breast cancer cells.

The effects of pituitary adenylate cyclase activating polypeptide (PACAP) analogs were investigated using breast cancer cells. 125I-PACAP-27 bound with high affinity (Kd = 5 nM) to T47D cells (Bmax = 29,000 per cell). Specific 125I-PACAP-27 binding was inhibited half maximally by PACAP-27, PACAP-38, PACAP(6-38) and PACAP(28-38) with IC50) values of 8, 17, 750 and >3000 nM, respectively. By RT-PCR, PACAP receptor mRNA was present in MCF-7 and T47D cell lines. Polyclonal antibodies to a PACAP receptor fragment (A-8-C) were elicited. The antibodies were affinity purified, recognized a 60-kDa protein by western blot, and stained malignant cells in breast cancer biopsy specimens by immunohistochemistry. PACAP-27 elevated the cAMP in T47D cells and the increase in cAMP caused by PACAP was inhibited by PACAP(6-38). PACAP-27 stimulated c-fos mRNA in T47D cells and the increase in c-fos gene expression caused by PACAP was reversed by PACAP(6-38). PACAP(6-38) inhibited colony formation using a soft agar assay and inhibited breast cancer xenograft growth in nude mice. These data suggest that PACAP(6-38) functions as a breast cancer PACAP receptor antagonist.

Activity-dependent neurotrophic factor: a potent regulator of embryonic growth and development.

Activity-dependent neurotrophic factor is a potent, neuroprotective molecule released from astroglia following stimulation by vasoactive intestinal peptide and, at least in part, accounts for the neuroprotective actions of vasoactive intestinal peptide. As well as enhancing neuronal survival, vasoactive intestinal peptide is known to regulate embryonic growth during the early postimplantation period of development. The current study was designed to assess activity-dependent neurotrophic factor's role in the growth-regulatory properties of vasoactive intestinal peptide. Treatment of whole cultured day-9 mouse embryos with activity-dependent neurotrophic factor (10(-13) M) resulted in a growth of 3.1 somites, compared with 1.6 somites in control embryos after a 4 h incubation period. Significant increases were also seen in cross-sectional area, protein and DNA content and bromodeoxyuridine incorporation. Activity-dependent neurotrophic factor-treated embryos were morphologically indistinguishable from control embryos of the same size. Anti-activity-dependent neurotrophic factor ascites significantly inhibited growth. In addition, co-treatment of embryos with anti-activity-dependent neurotrophic factor ascites inhibited vasoactive intestinal peptide-stimulated growth. Although anti-vasoactive intestinal peptide treatment inhibited growth, it did not inhibit activity-dependent neurotrophic factor-induced growth. These data indicate that an activity-dependent neurotrophic factor-like substance is an endogenous and potent growth-promoting factor in the early postimplantation embryo and that vasoactive intestinal peptide-regulated growth of embryos occurs, at least in part, through the action of activity-dependent neurotrophic factor.

Protection by extracellular glutathione against sulfur mustard induced toxicity in vitro.

1. The present study characterizes the role of extracellularly added glutathione in protection against sulfur mustard (HD) toxicity in a macrophage monocyte cell line J774. 2. Toxic effects of HD depend on dose and duration of exposure with an ED50 of 50 and 75 microM for dividing and confluent cells respectively. 3. Exposure to HD, 100-200 microM caused approximately 15% decrease in the cellular glutathione (GSH) content 2 h after exposure, pretreatment with GSH, 0.2-10 mM, elevated cellular GSH approximately x 1.5. 4. GSH pretreatment increased cell viability after HD 2-3-fold. Similar protective effects of GSH treatment were found in a human epidermoid carcinoma cell line (KB). 5. Protection by post treatment with GSH was apparent even 60 min post HD exposure. 6. No protection was afforded when the intracellular GSH concentration was elevated prior to exposure and the extracellular GSH had been washed out. However, GSH depleted cells were more sensitive to HD than normal cells, and were also protected by addition of GSH to the growth medium, although the intracellular GSH content remained low. 7. We conclude that it is essential for the GSH to be present extracellularly in order to protect cells from HD toxicity. 8. Our findings have therapeutic implications in particular for the protection of lungs after inhalation exposure to HD vapor.

Antiserum to activity-dependent neurotrophic factor produces neuronal cell death in CNS cultures: immunological and biological specificity.

Activity-dependent neurotrophic factor (ADNF) is a glia-derived protein that is neuroprotective at femtomolar concentrations. ADNF is released from astroglia after treatment with 0.1 nM vasoactive intestinal peptide (VIP). To further assess the biological role of ADNF, antiserum was produced following sequential injections of purified ADNF into mice. Anti-ADNF ascites fluid (1:10,000) decreased neuronal survival by 45-55% in comparison to untreated cultures or those treated with control ascites. The neuronal death after anti-ADNF treatment was observed in cultures derived from the spinal cord, hippocampus or cerebral cortex at similar IC50's. Using a terminal deoxynucleotidyl transferase in situ assay to estimate apoptosis in cerebral cortical cultures, anti-ADNF was shown to produce a 70% increase in the number of labeled cells in comparison to controls. In spinal cord cultures, anti-ADNF treatment produced a 20% decrease in choline acetyltransferase activity in comparison to controls. Neuronal cell death produced by the antiserum to ADNF was prevented in cultures co-treated with purified ADNF or ADNF-15, an active peptide derived from the parent ADNF. In vitro binding between the anti-ADNF and ADNF-15 was demonstrated with size exclusion chromatography. Comparative studies with other growth factors (insulin-like growth factor-1, platelet-derived growth factor, nerve growth factor, epidermal growth factor, ciliary neurotrophic growth factor, and neurotrophin-3) demonstrated that only ADNF prevented neuronal cell death associated with electrical blockade. These investigations indicated that an ADNF-like substance was present in cultures derived from multiple locations in the central nervous system and that ADNF-15 exhibited both neuroprotection and immunogenicity. ADNF appears to be both a regulator of activity-dependent neuronal survival and a neuroprotectant.

Breast cancer growth is inhibited by vasoactive intestinal peptide (VIP) hybrid, a synthetic VIP receptor antagonist.

Breast cancer vasoactive intestinal peptide (VIP) receptors were characterized. Using in vitro autoradiographic techniques, 125I-labeled VIP bound with high affinity to breast biopsy sections. 125I-labeled VIP bound specifically to give breast cancer cell lines examined using receptor-binding techniques. Specific 125I-labeled VIP binding to MDA-MB-231 cells was inhibited with high affinity by VIP and pituitary adenylate cyclase-activating polypeptide (IC50, = 2 nM) and with moderate affinity by the VIP hybrid (IC50 = 0.5 microM). VIP elevated the cAMP in a dose-dependent manner, and VIP hybrid (10 microM) inhibited the increase in cAMP caused by VIP. Using Northern blot analysis, VIP (10 nM) stimulated c-fos and c-myc mRNA, and the increase caused by VIP was reversed by the VIP hybrid. The VIP hybrid inhibited breast cancer growth in vitro and in vivo using nude mice bearing breast cancer xenografts. These data suggest that the VIP hybrid is a breast cancer VIP receptor antagonist.

BW 1023U90: a new GRP receptor antagonist for small-cell lung cancer cells.

Gastrin-releasing peptide (GRP) receptor antagonists were synthesized and their ability to interact with small-cell lung cancer (SCLC) cells determined. [125I] BW1023U90, bound with high affinity (Kd = 2 nM) to a single class of sites (Bmax = 55 fmol/mg protein) using SCLC cell line NCI-H345. [125I] BW1023U90 binding was time dependent and reversible even at 37 degrees C as the ligand was minimally internalized. Specific [125I] BW1023U90 binding was inhibited with high affinity by GRP as well as bombesin (BB) but not neuromedin B (NMB). BW1023U90 inhibited the ability of BB to elevate cytosolic Ca2+ and increase the growth of SCLC cells. A BW1023U90 analogue, BW2258U89 (10 micrograms/day, SC) slowed SCLC xenograft format on in nude mice and [125I] BW 1023U90 localized to SCLC tumors 1 h after injection into nude mice. BW2258U89 (4% by weight) was placed in microspheres and slowly released over a 3-week period in nude mice bearing SCLC xenografts. The microspheres containing BW2258U89 strongly inhibited SCLC growth in vivo. A radioimmunoassay was developed for the GRP receptor antagonists and the rabbit antiserum cross-reacted totally with BW2258U89 or BW1023U90. BW2258U89 immunoreactivity (5 nM) was detected in the plasma of nude mice containing the microspheres after 1 week. These data suggest that GRP receptor antagonists bind to receptors on SCLC tumors.

Production and secretion of high levels of recombinant human acetylcholinesterase in cultured cell lines: microheterogeneity of the catalytic subunit.

To allow for structural analysis of the human acetylcholinesterase (hAChE) subunit, a series of eukaryotic vectors was designed for efficient expression. Several eukaryotic multicistronic expression vectors were tested in various mammalian cell lines. All expression vectors contained the selectable neo gene under control of a weak promoter, while the hAChE cDNA was under control of the cytomegalovirus (CMV) immediate-early or Rous sarcoma virus long terminal repeat (RSV LTR) or simian virus 40 (SV40) early promoters. Optimal production and secretion of recombinant hAChE (rehAChE) was achieved in the embryonal kidney 293 cell line transfected either with the RSV-hAChE or with CMV-hAChE expression vectors. Clones expressing and secreting as much as 5-25 pg of enzyme per cell per 24 h were obtained without resorting to coamplification techniques or continuous maintenance of cells under selective pressure. The purified (specific activity of 6000 units per mg protein) homodimer and tetramer enzyme molecules displayed typical AChE biochemical properties: a Km value of 120 microM for acetylthiocholine; a kcat value of 3.9 x 10(5)/min, and selective by AChE-specific inhibitors. Catalytic subunit dimers (130 kDa) exhibit differential N-glycosylation patterns, and upon reduction resolve into 67- and 70-kDa monomeric subunits. These two forms appear as a single discrete 62-kDa band following deglycosylation by N-glycanase. The N-terminal amino acid sequence analysis of the purified mature enzyme suggests the existence of two alternative cleavage sites for the removal of the signal peptide, in which the 'mature' position 1 is either Ala31 or Gly33. Both of these positions conform with the consensus signal peptide recognition sequences and demonstrate bidirected processing of signal peptides on a native molecule.

Mutagenesis of human acetylcholinesterase. Identification of residues involved in catalytic activity and in polypeptide folding.

Evidence for the involvement of Ser-203, His-447, and Glu-334 in the catalytic triad of human acetylcholinesterase was provided by substitution of these amino acids by alanine residues. Of 20 amino acid positions mutated so far in human acetylcholinesterase (AChE), these three were unique in abolishing detectable enzymatic activity (less than 0.0003 of wild type), yet allowing proper production, folding, and secretion. This is the first biochemical evidence for the involvement of a glutamate in a hydrolase triad (Schrag, J.D., Li, Y., Wu, M., and Cygler, M. (1991) Nature 351, 761-764), supporting the x-ray crystal structure data of the Torpedo californica acetylcholinesterase (Sussman, J.L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L. and Silman, I. (1991) Science 253, 872-879). Attempts to convert the AChE triad into a Cys-His-Glu or Ser-His-Asp configuration by site-directed mutagenesis did not yield effective AChE activity. Another type of substitution, that of Asp-74 by Gly or Asn, generated an active enzyme with increased resistance to succinylcholine and dibucaine; thus mimicking in an AChE molecule the phenotype of the atypical butyrylcholinesterase natural variant (D70G mutation). Mutations of other carboxylic residues Glu-84, Asp-95, Asp-333, and Asp-349, all conserved among cholinesterases, did not result in detectable alteration in the recombinant AChE, although polypeptide productivity of the D95N mutant was considerably lower. In contrast, complete absence of secreted human AChE polypeptide was observed when Asp-175 or Asp-404 were substituted by Asn. These two aspartates are conserved in the entire cholinesterase/thyroglobulin family and appear to play a role in generating and/or maintaining the folded state of the polypeptide. The x-ray structure of the Torpedo acetylcholinesterase supports this assumption by revealing the participation of these residues in salt bridges between neighboring secondary structure elements.

Divergent envelope E2 alphavirus sequences spanning amino acids 297 to 352 induce in mice virus-specific protective immunity and antibodies with complement-mediated cytolytic activity.

We have proposed a general algorithm for identification of potential immunoprotective domains (cassettes) on the envelope E2 polypeptide of alphaviruses (H. Grosfeld, B. Velan, M. Leitner, S. Cohen, S. Lustig, B.E. Lachmi, and A. Shafferman, J. Virol. 63:3416-3422, 1989). To assess the generality of our approach, we compared analogous E2 cassettes from Sindbis virus (SIN) and Semliki Forest virus (SFV), two alphaviruses which are philogenetically very remote. The antigenically distinct SFV E2 and SIN E2 cassettes exhibit comparable immunological characteristics. Most significantly, the SIN E2 LMN cassette cluster (E2 amino acids 297 to 352 fused to beta-galactosidase), like the analogous SFV E2 LMN cassettes, elicited high titers of antivirus antibodies in mice and proved to be highly effective in protection against lethal challenge. Mice immunized with SIN E2 LMN were completely protected against intracerebral challenge of 10 to 100 50% lethal doses of different neurovirulent SIN strains. Anti-SIN LMN antibodies, like anti-SFV LMN antibodies, lacked in vitro neutralizing activity, yet both exerted protection against homologous challenge upon transfer to mice. The two antibody preparations exhibited virus-specific complement-mediated cytolysis of cells infected with the homologous but not heterologous virus. These results suggest a possible mechanism for virus-specific E2 LMN-induced protection and demonstrate the generality of our methodology for deciphering immunogenic and protective domains in alphavirus systems. Results suggest also that the E2 LMN sequence of any given alphavirus should be considered as a component of a synthetic vaccine against that specific virus.

The effect of elimination of intersubunit disulfide bonds on the activity, assembly, and secretion of recombinant human acetylcholinesterase. Expression of acetylcholinesterase Cys-580----Ala mutant.

Site-directed mutagenesis was used to study the cysteine residue involved in the assembly of human acetylcholinesterase (HuAChE) catalytic subunits. Substitution of the cysteine at position 580 by alanine resulted in impairment of interchain disulfide bridge formation; the mutagenized enzyme (C580A) was secreted from recombinant cells in the monomeric form and failed to assemble into dimers. The mutant monomeric HuAChE did not differ from the native oligomeric enzyme neither in rate of catalysis nor in affinity to acetylthiocholine. Mutant monomers were also shown to retain the acetylcholinesterase characteristic sensitivity to high substrate concentrations. The mutation did not seem to affect the efficiencies of either synthesis or secretion of recombinant HuAChE polypeptides, as was demonstrated in cell lines derived from human embryonic kidney (293 cells) as well as from a human neuroblastoma (SK-N-SH). Furthermore, the mutation did not lead to an increase in accumulation of intracellular HuAChE polypeptides, suggesting that export of acetylcholinesterase from cells may not be coupled to subunit assembly.

Recombinant human acetylcholinesterase is secreted from transiently transfected 293 cells as a soluble globular enzyme.

1. Coding sequences for the human acetylcholinesterase (HuAChE; EC 3.1.1.7) hydrophilic subunit were subcloned in an expression plasmid vector under the control of cytomegalovirus IE gene enhancer-promoter. The human embryonic kidney cell line 293, transiently transfected with this vector, expressed catalytically active acetylcholinesterase. 2. The recombinant gene product exhibits biochemical traits similar to native "true" acetylcholinesterase as manifested by characteristic substrate inhibition, a Km of 117 microM toward acetylthiocholine, and a high sensitivity to the specific acetylcholinesterase inhibitor BW284C51. 3. The transiently transfected 293 cells (100 mm dish) produce in 24 hr active enzyme capable of hydrolyzing 1500 nmol acetylthiocholine per min. Eighty percent of the enzymatic activity appears in the cell growth medium as soluble acetylcholinesterase; most of the cell associated activity is confined to the cytosolic fraction requiring neither detergent nor high salt for its solubilization. 4. The active secreted recombinant enzyme appears in the monomeric, dimeric, and tetrameric globular hydrophilic molecular forms. 5. In conclusion, the catalytic subunit expressed from the hydrophilic AChE cDNA species has the inherent potential to be secreted in the soluble globular form and to generate polymorphism through self-association.

A VIP antagonist distinguishes VIP receptors on spinal cord cells and lymphocytes.

Vasoactive intestinal peptide (VIP) is a neuropeptide which also interacts with cells of the immune system. The paucity of specific VIP receptor antagonists has hampered studies of possible receptor heterogeneity and of VIP function. To aid in achieving these goals, a new VIP antagonist, a hybrid between neurotensin and VIP, has been synthesized. This peptide interacted with VIP receptors on spinal cord cells with an affinity 10-fold greater than VIP itself. In contrast, 1000-fold higher concentrations of the antagonist were required to displace labeled VIP from its receptor on lymphoid cells as compared to VIP itself, suggesting VIP receptor heterogeneity between immune and spinal cord cells.

Sodium dodecylsulphate induces a breach in the blood-brain barrier and enables a West Nile virus variant to penetrate into mouse brain.

A novel and convenient assay was used to determine the effect of recombinant Interleukin-2 (IL-2) on the function of the blood-brain barrier (BBB). The assay is based on a variant of the West Nile virus, WN-25, which had lost its neuroinvasiveness but not its neurovirulence. WN-25, when injected intravenously, can cause the death of mice only if the function of the BBB is impaired. Sodium dodecylsulphate (SDS), a component in IL-2 excipient, was found to cause a short term breach in the BBB, enabling the penetration of viruses into the brain. Minimal amounts (30 ng/mouse) can induce a breach of about 10 min, which allows 0.1% of the injected virus to enter the brain. These findings demonstrate the possible use of SDS as a mean for intentional introduction of drugs into the brain, however they also call attention to the danger of using detergents as additives for drugs given intravenously.