Protection against group B meningococcal disease. II. Infection and resulting immunity in a guinea pig model.

A guinea pig subcutaneous chamber model was used to evaluate the specificity of the immune response resulting from Neisseria meningitidis infection. Small numbers of meningococci easily infected the chambers. The infections persisted for 6-8 days with relatively high levels of organisms (10(5)-10(6)/milliliter) in the chambers, and were then rapidly eliminated and no organisms could be cultured beyond day 14. Clearance of infection correlated with appearance of circulating antibody. Antibody against both the protein serotype antigen and the capsular polysaccharide were induced as a result of meningococcal infection. The group-specific polysaccharide response peaked 2-3 wk after the animals were inoculated, while the type-specific protein response peaked at 5-6 wk. The animals were quite resistant to reinfection with either the homologous serogroup or serotype.

Protection against group B meningococcal disease. III. Immunogenicity of serotype 2 vaccines and specificity of protection in a guinea pig model.

Protein vaccines were prepared from the serotype antigen of group B Neisseria meningitidis strain M986. The detergents Triton X-100, Emulphogene BC-720, and deoxycholate were used to removed the toxic lipopolysaccharide (LPS) portion of the serotype antigen. The LPS was most preferentially solubilized by Emulphogene. Guinea pigs were immunized with one or two doses of vaccine given intramuscularly without adjuvants and the antibody response quantitated by an enzyme-linked immunosorbant assay. Immunization with graded doses of vaccine between 25 to 200 microgram protein indicated a wide range of effective dosage and that a two-dose immunization schedule was superior to a single immunization. The vaccines elicited peak mean serum antibody levels of approximately 30 microgram/ml with bactericidal titers of 1:1,600-1:6,400. The peak antibody levels occurred 5-6 wk after immunization and persisted above preimmune levels for several months. To evaluate the protective effects of immunization, stainless steel springs were implanted subcutaneously into the guinea pigs. The resulting chambers, in unimmunized animals, could be infected with less than 100 type 2 organisms. A single 25-50 microgram dose of vaccine protected 50% of animals from challenge by 5 X 10(5) type 2 meningococci, and as little as 1 microgram vaccine significantly reduced the severity of infection. A two-dose immunization schedule was best and provided nearly complete protection for at least 4 mo against type 2 strains of meningococcal groups B, C, and Y.

Vi capsular polysaccharide-protein conjugates for prevention of typhoid fever. Preparation, characterization, and immunogenicity in laboratory animals.

The Vi has proven to be a protective antigen in two double masked, controlled clinical trials in areas with high rates of typhoid fever (approximately 1% per annum). In both studies the protective efficacy of the Vi was approximately 70%. Approximately 75% of subjects in these areas responded with a fourfold or greater rise of serum Vi antibodies. In contrast, the Vi elicited a fourfold or greater rise in 95-100% of young adults in France and the United States. Methods were devised, therefore, to synthesize Vi-protein conjugates in order to both enhance the antibody response and confer T-dependent properties to the Vi (and theoretically increase its protective action in populations at high risk for typhoid fever). We settled on a method that used the heterobifunctional crosslinking reagent, N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP), to bind thiol derivatives of the Vi to proteins. This synthetic scheme was reproducible, provided high yields of Vi-protein conjugates, and was applicable to several medically relevant proteins such as diphtheria and tetanus toxoids. The resultant conjugates were more immunogenic in mice and juvenile Rhesus monkeys than the Vi alone. In contrast to the T-independent properties of the Vi, conjugates of this polysaccharide with several medically relevant proteins induced booster responses in mice and in juvenile Rhesus monkeys. Clinical studies with Vi-protein conjugates are planned. This scheme is also applicable to synthesize protein conjugates with other polysaccharides that have carboxyl functions.

31P nuclear magnetic resonance spectroscopic observation of the intracellular transformations of oncostatic cyclophosphamide metabolites.

31P NMR spectroscopy was used to directly monitor, for the first time, the intracellular chemistry of the ultimate active metabolite of cyclophosphamide, namely, phosphoramide mustard. These NMR studies utilized a human histiocytic lymphoma cell line (U937), embedded in agarose gel threads, and perfused with medium containing synthetically derived metabolites (4-hydroxycyclophosphamide, aldophosphamide, and phosphoramide mustard). Metabolites 2 or 3 or both readily crossed the cell membrane; in contrast, the membrane was relatively impermeable to 4. Intracellular concentrations of 4 could, therefore, be attributed primarily to the intracellular fragmentation of 3. Signals suggestive of either carboxyphosphamide or 4-ketophosphamide were not detected. Spectral data were used to calculate a rate constant of (5.4 +/- 0.3) X 10(-3) min-1 for the intracellular disappearance of 4 at 23 degrees C. The intracellular pH was determined to be 7.1 from the chemical shift of the internal inorganic phosphate signal.

Forskolin carbamates: binding and activation studies with type I adenylyl cyclase.

Three series of analogs were regioselectively prepared from a protected forskolin precursor to afford 7-carbamoyl-7-desacetylforskolins (series 1), 6-carbamoyl-7-desacetylforskolins (series 2), and 6-carbamoylforskolins (series 3). The analogs were pharmacologically evaluated for binding (IC50) to and activation (EC50) of type I adenylyl cyclase in membranes from stably transfected Sf9 cell lines expressing a single adenylate cyclase subtype. The following ranges were determined for the IC50's and EC50's of each individual series: series 1, IC50 = 43-1600 nM, EC50 = 0.5-9.6 microM; series 2, IC50 = 65-680 nM, EC50 = 0.63-6.5 microM; series 3, IC50 = 21-271 nM, EC50 = 0.5-8.1 microM (forskolin IC50 = 41 nM and EC50 = 0.5 microM). Activation paralleled binding; however, some analogs exhibited poor binding and good activation whereas others demonstrated good binding but poor activation. Steric bulk tended to diminish binding and activation when at the 6- or 7-position, although bulk was accommodated at the 6-position if the 7-site was reacetylated. Acylation of the 7-position by the carbamoyl linker or acetyl was important for obtaining good binding and activation; however, the effect was more pronounced with binding. For both binding and activation, small, linear, lipophilic substituents (propyl, allyl, isopropyl) are well tolerated at the 7-position but less so in the 6-position, even when the 7-site is reacetylated. Planar aromatic moieties (phenyl and 2-pyridinyl) demonstrated moderate to good potency for binding and activation when located at either the 6- or 7-positions. There is an overall trend toward increasing potency for both binding and activation with polar substituents.

(Aminoalkyl)carbamates of forskolin: intermediates for the synthesis of functionalized derivatives of forskolin with different specificities for adenylyl cyclase and the glucose transporter.

(Aminoalkyl)carbamates of forskolin were synthesized at the 6- and 7-hydroxyl positions of forskolin with the length of the alkyl chain varying from ethyl to heptyl. Two of these derivatives, 7-[[(2-aminoethyl)amino]carbonyl]-7-desacetylforskolin (2) and 6-[[(2-aminoethyl)amino]carbonyl]forskolin (3), were used to synthesize iodinated derivatives of forskolin that bind with high affinity to adenylyl cyclase in bovine brain membranes and the glucose transporter in human erythrocyte membranes, respectively. Hydroxyphenyl derivatives of forskolin were prepared from the (aminoalkyl)carbamates and tested for their ability to bind to adenylyl cyclase in bovine brain membranes and the glucose transporter in human erythrocyte membranes. The 6-derivative (18) of forskolin had a Kd of 9 nM at adenylyl cyclase and was more potent than either the 7-derivatives or the 6-derivatives of 7-desacetylforskolin. The 7-derivatives were more potent at binding to the glucose transporter than forskolin. In contrast, the 6-derivatives had Kd's greater than 100 microM at the glucose transporter. Isothiocyanates and N-bromoacetyl derivatives were synthesized from 2 and 3 as potential alkylating agents for forskolin binding sites. The alkylating agents produced an irreversible loss of forskolin binding to adenylyl cyclase. In contrast, the alkylating agents bound reversibly to the glucose transporter.

Differential identification and localization of adenylyl cyclase and glucose transporter in brain using iodinated derivatives of forskolin.

Two radioiodinated derivatives of forskolin, [125I]6-IHPP-Fsk and [125I]7-IHPP-Fsk, were synthesized as specific ligands for adenylyl cyclase and glucose transporter, respectively. [125I]6-IHPP-Fsk bound to bovine brain homogenates with a Kd of 9 nM and binding was inhibited by forskolin but not 1,9-dideoxyforskolin, cytochalasin B, or D-glucose. [125I]7-IHPP-Fsk bound to bovine brain homogenates at two classes of binding sites with Kd's of 56 nM and 4.7 microM; cytochalasin B and D-glucose inhibited 75% of the high affinity binding while having no effect on the low affinity binding. [125I]6-IHPP-Fsk and [125I]7-IHPP-Fsk were used to localize adenylyl cyclase and glucose transporter in rat brain by receptor autoradiography. The pattern of binding obtained with [125I]6-IHPP-Fsk was similar to that observed using [3H]forskolin to detect adenylyl cyclase. In contrast, the pattern of binding obtained with [125I]7-IHPP-Fsk was similar to that observed by others using [3H]cytochalasin B to detect glucose transporter. These iodinated ligands are selective for adenylyl cyclase and glucose transporter and require significantly shorter exposure times to yield autoradiographs than tritiated ligands.

31P- and 13C-n.m.r.-spectral and chemical characterization of the end-group and repeating-unit components of oligosaccharides derived by acid hydrolysis of Haemophilus influenzae type b capsular polysaccharide.

Haemophilus influenzae type b capsular polysaccharide [repeating unit, leads to 3)-beta-D-Ribf-(1 leads to 1)-D-Ribol-5-(PO2H leads to] was partially hydrolyzed with HCl to give oligosaccharides that were isolated by size-exclusion chromatography, and then characterized by 31P- and 13C-n.m.r.-spectral and chemical methods, in order to determine the end-group composition and, hence, the number-average chain-length (L). The ratio (approximately 17:8) of monophosphate end-groups to D-ribofuranose end-groups revealed the relative rates of hydrolysis of the phosphoric diester linkage and the glycosidic linkage in the repeating-unit structure. Cleavage of the phosphoric diester linkage was approximately 92% regioselective, as indicated by the approximately 12:1 ratio of D-ribofuranose monophosphate end-groups to D-ribitol monophosphate end-groups. The n.m.r. spectra of the oligosaccharide repeating-unit provided evidence for partial stereomutation (approximately 3-8%) that involved rearrangement of the D-ribofuranose phosphoric diester linkage and anomerization at C-1 of D-ribofuranose. Variously sized oligosaccharides (L = 4, 7, and 12), that had D-ribofuranose end-groups reacted with bovine serum albumin that had an average of approximately 9 adipyl hydrazide functionalities, to give, within experimental error, quantitative yields of the corresponding hydrazone-linked, oligosaccharide-protein conjugates.

Effect of clonidine, quipazine, and LY 53857 on the prolactin-suppressant action of bromocriptine in rats.

The alpha 2-adrenergic agonist clonidine hydrochloride, the serotonin agonist quipazine maleate, and the serotonin (5-HT2) antagonist LY 53857 were tested alone and in various combinations for their capabilities to increase mean serum prolactin (MSP) concentrations in rats given the synthetic ergot alkaloid CB-154 (2-bromo-alpha-ergocriptine), a known prolactin suppressor. The LY 53857 and the combination of clonidine, quipazine, and LY 53857 significantly decreased MSP concentrations. Quipazine given alone (10 mg/kg of body weight) was best able to increase MSP concentration and has potential to antagonize prolactin-depressant effects of ergot alkaloids.

Effect of yohimbine hydrochloride on serum prolactin concentration in the rat: possible antagonist for fescue toxicosis.

Yohimbine hydrochloride is an indole alkaloid which blocks alpha 2-adrenergic and dopamine receptors and stimulates serotonergic receptors. Yohimbine was selected for testing as a possible antagonist in fescue toxicosis. Reduced body weight gains in cattle with chronic fescue toxicosis may be due to ergot alkaloids produced by fungi which infect the fescue grass. Ergot alkaloids stimulate dopamine receptors, antagonize serotonin, and lower serum prolactin concentrations. It was hypothesized that yohimbine may reverse or counteract the effects of the toxic fescue. Investigation was made of the treatment effects of multiple doses of yohimbine given in rats by intraperitoneal and oral routes. Given intraperitoneally once a day for 8 days, yohimbine hydrochloride increased serum prolactin concentrations. When given orally in feed for 7 days, the drug decreased the serum prolactin concentration. The effects of yohimbine on prolactin concentrations were dependent on the dosages and routes of administration. The inability of yohimbine, when given orally, to increase serum prolactin levels decreased its potential usefulness for prolonged treatment of fescue toxicosis.

Reexamination of the protective role of the capsular polysaccharide (Vi antigen) of Salmonella typhi.

The role of the Vi antigen, the capsular polysaccharide of Salmonella typhi, in the pathogenesis of and immunity to typhoid fever remains the subject of controversy. Vi-positive S. typhi resist phagocytosis and the action of serum complement, both of which actions are initiated by antibodies to Vi antigen. Both the laboratory potency in mice and the clinical effectiveness of whole-cell vaccines were related to their content of immunogenic Vi antigen. A Vi polysaccharide used for immunizing humans against experimental challenge with S. typhi failed to prevent typhoid fever; experimental conditions used to prepare this ineffective Vi antigen were shown to denature it and to reduce its immunogenicity. Assay of serum antibodies to Vi antigen with purified Vi antigen is a reliable method for diagnosis of typhoid fever and asymptomatic carriage of S. typhi. Vi polysaccharides prepared by modern techniques passed the requirements for meningococcal polysaccharide vaccines and had approximately 13 times the protective activity in the mouse potency assay as did the US Standard 6A whole-cell typhoid vaccine.