Micrograms Of Protein Research Articles

Specificity of human bactericidal antibodies against PorA P1.7,16 induced with a hexavalent meningococcal outer membrane vesicle vaccine

A set of isogenic strains was constructed from the meningococcal reference strain H44/76 (B:15:P1.7,16) which differed only in their outer membrane protein (OMP) compositions. First, three isogenic strains lacking the expression of either class 3 (PorB) or class 4 (RmpM) OMP or both were obtained. Second, three isogenic class 1 OMP loop-deficient strains of H44/76 lacking the predicted loop 1 or 4 or both of class 1 OMP (PorA) were obtained. Third, three isogenic class 1 OMP strains which differed by point mutations in the predicted loop 4 of subtype P1.16 were constructed. Strains were constructed through transformation with gene constructs made in Escherichia coli and their homologous recombination into the meningococcal chromosome. This study describes the contribution of one of the six class 1 OMPs, PorA P1.7,16, in the development of bactericidal antibodies after a single immunization of adult volunteers with 50 or 100 micrograms of protein within a hexavalent PorA outer membrane vesicle vaccine. PorA-, PorB-, and RpmM-deficient isogenic strains were used to define the human immune response against PorA. The loop-deficient isogenic strains were used to define the contribution of loops 1 and 4 of PorA in the development of bactericidal anti-PorA antibodies. The isogenic strains carrying a point mutation in loop 4 were used to study the cross-reactivity of the induced bactericidal antibodies against target strains showing microheterogeneity. The results indicate that a single immunization with the hexavalent PorA vaccine induced a dose-dependent bactericidal immune response, which is directed mainly against PorA. The epitope specificity of antibodies is directed mostly against loop 1, although loop 4 and as-yet-unidentified epitopes of PorA P1.7,16 are also involved.

Protoporphyrinogen oxidase: high affinity tetrahydrophthalimide radioligand for the inhibitor/herbicide-binding site in mouse liver mitochondria.

Protoporphyrinogen oxidase (protox), the last common enzyme in heme and chlorophyll biosynthesis, is the target of several classes of herbicides acting as inhibitors in both plants and mammals. N-(4-Chloro-2-fluoro-5-(propargyloxy)phenyl)-3,4,5,6-tetrahydro phthalimide (a potent protox inhibitor referred to as THP) was synthesized as a candidate radioligand ([3H]-THP) by selective catalytic reduction of 3,6-dihydrophthalic anhydride (DHPA) with tritium gas followed by condensation in 45% yield with 4-chloro-2-fluoro-5-(propargyloxy)aniline. Insertion of tritium at the 3 and 6 carbons of DHPA as well as the expected 4 and 5 carbons resulted in high specific activity [3H]THP (92 Ci/mmol). This radioligand undergoes rapid, specific, saturable, and reversible binding to the inhibitor/herbicide binding site of the protox component of cholate-solubilized mouse liver mitochondria with an apparent Kd of 0.41 nM and Bmax of 0.40 pmol/mg of protein. In the standard assay, mouse preparation (150 micrograms of protein) and [3H]THP (0.5 nM) are incubated in 500 microL of phosphate buffer at pH 7.2 for 15 min at 25 degrees C followed by addition of ammonium sulfate and filtration with glass fiber filters. The potencies of five nitrodiphenyl ethers and two other herbicides as inhibitors of [3H]THP binding correlate well with those for inhibition of protox activity (r2 = 0.97, n = 7), thus validating the binding assay as relevant to enzyme inhibition. It is also suitable to determine in vivo block as illustrated by an approximately 50% decrease in [3H]THP binding in liver mitochondria from mice treated ip with oxyfluorfen at 4 mg/kg. This is the first report of a binding assay for protox in mammals. The high affinity and specific activity of [3H]THP facilitate quantitation of protox and therefore research on a sensitive inhibition site for porphyrin biosynthesis.

Evidence for overlapping active sites for 17 alpha-ethynlestradiol and bilirubin in the human major bilirubin UDPglucuronosyltransferase.

The human major bilirubin UDP glucuronosyltransferase (transferase), HUG-Brl, and its mutants were expressed in the COS-1 cells using cDNA-based pSVL expression units to generate isoforms for the comparison of relative activities with 17 alpha-ethynlestradiol (17 alpha-EE) and bilirubin, its natural substrate. In comparison to bilirubin, 17 alpha-EE was a good substrate for HUG-Br1 under typical assay conditions of pH 7.2, confirming published studies [Ebner, T., et al. (1993) Mol. Pharmacol. 43, 649-654]. It was further shown that the estrogen derivative is 1.2-2-fold more effective as a substrate at pH 6.4 than at pH 7.2. The km for 17 alpha-EE was 40 microM under both pH conditions, while the Vmax values were 400 and 200 pmol per hour per 300 micrograms of protein at pH 6.4 and 7.2, respectively. The pattern of glucuronidation was similar for both bilirubin and 17 alpha-EE. Previously, a ratio of 2-3-fold more activity for bilirubin glucuronidation at pH 6.4 versus 7.6 was established, and km values of 2.5 microM at both pH conditions were determined [Ritter, J.K., et al. (1993) J. Biol. Chem. 268, 23573-23579]. In this study, the generation of 17 alpha-EE and bilirubin beta-glucuronides under both pH conditions was confirmed by the sensitivity of the products to beta-glucuronidase treatment. Concurrent glucuronidation reaction mixtures containing equal amounts of wild-type and mutant proteins demonstrated the following. P270G, V273D, and five different G276 mutants nearly or completely inactivated all glucuronidation at both pH levels. V273Q generated 81-94% of the normal activity for 17 alpha-EE and 42% of the normal activity for bilirubin turnover; H173R gave 37-60% of the normal turnover with both substrates, and V275I produced 15-24% of the normal level of glucuronide with both compounds. The most distinguishing amino acid tested was P176G which was approximately 50% normal for 17 alpha-EE at both pH conditions but was totally inactive for bilirubin. A second substitution, P285G, did not affect 17 alpha-EE turnover but was 50% normal for bilirubin. The parallel effects on the metabolism of both substrates by some mutants and the opposite results from two mutants are evidence for a common set of amino acids for their catalysis with the recruitment of additional amino acids to depend upon the substrate to be metabolized. Hence, amino acid substitutions in the protein are not necessarily universally inactivating.

Methodology for assaying iodide conductance in proteoliposomes: specific induction by thyroid membrane protein

A sensitive assay is developed to assess the existence of an iodide channel in a fraction of solubilized membrane proteins. This step is critical when considering various procedures for purification of this channel. Sodium cholate is used as a detergent as it does not denature the iodide channel. A simple and rapid method involving gel-filtration chromatography is used simultaneously to remove the detergent and to adjust the buffer composition, before protein insertion into liposomes. The presence of an iodide channel is investigated by measuring the iodide conductance of these proteoliposomes at 4 degrees C. An outward iodide gradient is set up across the proteoliposomal membrane by anion-exchange chromatography, allowing uptake of radiolabelled iodide. This uptake is conductive as it is abolished by valinomycin in the presence of potassium. It is specifically mediated by a thyroid plasma-membrane protein inserted into liposomes, as its denaturation before insertion totally abolished uptake. It was observed only within a well-defined fraction of thyroid membrane proteins collected by size-exclusion chromatography (molecular mass between 100 and 200 kDa). Furthermore, it was not observed with other membrane proteins such as ileal brush-border-membrane proteins or bacteriorhodopsin. Like many anion channels, this conductance was also inhibited by N-phenylanthranilic acid. Optimization of the assay is described, validating the measurement of conductive iodide uptake at 30 s by proteoliposomes reconstituted in a ratio of 10 micrograms of protein to 90 micrograms of lipid, with an outward iodide gradient (KI 15 mM inside and 1 microM outside). This assay provides a test of the biological activity of the iodide channel at each step of the purification; it can be applied to any anionic channel.

Hypoxia activates nitric oxide synthase and stimulates nitric oxide production in porcine coronary resistance arteriolar endothelial cells

Hypoxia significantly alters vascular tone in coronary resistance arterioles during prolonged ischemia, potentially through the modulation of endothelial cell metabolism as well as endothelial function. The objective of this study was to test the hypothesis that constitutive nitric oxide synthase (cNOS) is sensitive to oxygen tension and that hypoxia increases the activity of cNOS and nitric oxide production in the porcine coronary microcirculation. Monocultures of porcine coronary resistance arteriolar endothelial cells (RAEC) were isolated and proven to be endothelium based upon morphology, binding of acetylated LDL, and factor VIII antigen positivity. Cells were exposed to either hypoxia (pO2 = 10 mmHg) or normoxia (pO2 = 160 mmHg) for varying periods of time. Nitric oxide production was directly measured using a chemiluminescence method, while cNOS enzyme activity was assayed using a fibroblast-report cell method. cNOS protein was quantitated by Western blot analysis using the H32 monoclonal antibody to the endothelial cell constitutive isoform of NOS. Hypoxia significantly augmented A23187-stimulated nitric oxide production [23.77 (1.73) vs 14.94 (0.66) nmol . micrograms-1 protein, hypoxia vs. normoxia respectively, n = 8, P < 0.01]. Using the fibroblast reporter cell assay, cNOS activity was increased in RAEC after exposure to hypoxia for 30, 120 and 240 min [normoxia control: 0.16 (0.04) fmol . microgram-1 protein; hypoxia: 30 min = 1.00 (0.19), 120 min = 1.08 (0.04), 240 min = 1.26 (0.07) fmol . micrograms-1 protein (n = 6, p < 0.01)]. Western blots showed a single band at 135 kDa that was increased in homogenates of cells previously exposed to hypoxia. These experiments demonstrated that the regulation of cNOS is sensitive to oxygen tension. Hypoxia significantly activated constitutive nitric oxide synthase in coronary resistance arteriolar endothelial cells, and this was translated to an increased production of nitric oxide.

Delayed hypersensitivity to Trypanosoma cruzi antigen. I -- Experimental study in rabbits

Delayed-type skin hypersensitivity in rabbits infected with homologous or heterologous T. cruzi has been elicited upon injection of 50 micrograms of protein in the T12E antigen derived from a parasite clone. The specificity of this reaction was indicated by absence of skin reactivity in control rabbits that received same quantity of antigen. Also, the intradermal injection of the antigen in five occasions in control rabbits neither induced seroconversion nor shifted their skin sensitiveness. On the other hand, chagasic rabbits that underwent a series of five skin testings one week apart, did not alter the intensity of the skin reactivity, and their specific serum antibody titers remained similarly high. Of interest, the ECG patterns remained unchanged after a series of five skin testings in control and in chagasic rabbits. This study shows that the T12E antigen might be an useful tool for diagnosing T. cruzi infections, besides serving as an immunologic marker for morbidity in Chagas' disease.

Effect of thyrotropin-releasing hormone on microtubules in GH3 cells.

The effect of thyrotropin-releasing hormone (TRH) on the tubulin-microtubule system in GH3 cells was investigated. Prolactin (PRL) secretion by GH3 cells was stimulated by TRH at 1, 5, and 10 min of incubation. The polymerized tubulin levels increased from 0.4 units micrograms-1 protein in control cells to 0.6 units micrograms-1 protein in cells incubated with TRH for 1 min. Soluble tubulin levels increased from 0.8 units microgram-1 protein in control cells to 1.2 units micrograms-1 protein in cells exposed to TRH for 5 min. Tubulin levels were similar in control cells and cells treated with TRH for 10 min. These results suggest that TRH-stimulated PRL secretion is accompanied by a shift in the equilibrium between tubulin pools.

Glucose-6-phosphatase activity in islets from ob/ob and lean mice and the effect of dexamethasone

In previous studies we demonstrated a much greater rate of glucose cycling (glucose—>glucose-6-P—>glucose) in islets from ob/ob mice than from lean litter mates. Cycling was further augmented by dexamethasone treatment. To determine whether these findings could be accounted for by increased islet glucose-6-phosphatase activity, we have now measured that enzyme's activity in permeabilized and sonicated islets and in islet microsomes. Activity in permeabilized islets from ob/ob mice was 19 times more than from lean litter mates (17.7 +/- 2.9 vs. 0.9 +/- 0.2 pmol/islet/min). Activity was 6 times higher when calculated per microgram of protein or microgram of DNA. Treatment of ob/ob mice with dexamethasone (25 micrograms/daily for 3 days) increased activity 2- to 3-fold. Activities were about twice as much in sonicated as permeabilized islets. There was no difference between glucose-6-phosphatase activity in microsomes prepared from islets of ob/ob and from lean mice, and the activity was relatively low. Thus, permeabilized islets can be used to determine glucose-6-phosphatase activity and study its regulation. The higher glucose cycling in islets of ob/ob mice and its stimulation by dexamethasone can be attributed to increased glucose-6-phosphatase activity.

Quantification of apolipoproteins B-100, B-48, and E in human triglyceride-rich lipoproteins.

We have developed a procedure to quantify apolipoprotein (apo) B-100, apoB-48, and apoE in human triglyceride-rich lipoproteins. This procedure permits delipidation of small amounts of triglyceride-rich lipoproteins without appreciable losses, and quantification of these apolipoproteins in samples containing as little as 10 micrograms of protein. Delipidated triglyceride-rich lipoproteins are subjected to sodium dodecyl sulfate polyacrylamide slab gel electrophoresis, and the mass of apolipoproteins is estimated after densitometric scanning and volume integration of Coomassie blue-stained bands. The chromogenicities of apoB-100 and apoB-48 are virtually identical, and twofold lower than that of apoE. The standard curve for each apolipoprotein follows a power function over a wide protein range, permitting quantification of as little as 0.2 microgram of apoB-48 and as much as 30 micrograms of apoB-100 from a single application of triglyceride-rich lipoproteins to the gels. This method is suitable for routine use in studies of the intestinal and hepatic contributions to triglyceride-rich lipoproteins and their responses to postprandial lipemia.

Highly sensitive and specific gas chromatographic-tandem mass spectrometric method for the determination of trace amounts of antipyrine metabolites in biological material.

A highly sensitive and specific gas chromatographic-tandem mass spectrometric method was developed for the determination of the antipyrine (INN: phenazone) metabolites, norantipyrine, 4-hydroxyantipyrine and 3-hydroxy-methylantipyrine, in biological material. Deuterated analogues of the metabolites were used as internal standards. The method has a limit of quantitation of 5 ng per sample for the determination of norantipyrine, 4-hydroxy-antipyrine and 3-hyddroxymethylantipyrine with coefficients of variation of 19.4, 14.6 and 20.7%, respectively. Precision and accuracy are good over the whole range measured (5-500 ng/sample) with a coefficient of variation, respectively error of determination < or = 20%. Due to its high sensitivity the method can be used to study the formation of these metabolites in microsomal preparations containing only 100 micrograms of protein.

Targeting gene expression to the vascular wall in transgenic mice using the murine preproendothelin-1 promoter.

To develop a system for overexpressing genes in the vascular wall, we created transgenic mice using the reporter gene luciferase and the murine preproendothelin-1 promoter. In vitro analysis suggested that the murine 5'-flanking region contained endothelial-specific elements in a 5.9-kb fragment. Five transgenic mice colonies established from independent founders all exhibited the highest level of luciferase activity in the aorta with up to 8,540 light units per microgram of protein. Immunohistochemistry with anti-luciferase antisera revealed high levels of expression in the endothelial cells of both large and small arteries and lower levels of expression in veins and capillaries. Significant expression was also seen in arterial smooth muscle cells and in select epithelial surfaces which is consistent with the known distribution of endothelin-1 in mammals. The further demonstrate the targeting capability of this system, we overexpressed the lipid-peroxidating enzyme, human 15-lipoxygenase, in the vessel wall of transgenic mice. As with luciferase, expression of active enzyme and immunohistochemical localization in vascular cells were documented in transgenic animals. Hence, this new system can be used to direct expression of molecules to the vascular wall for the purpose of examining the biological significance of either overexpression or inhibition of select proteins.

Inorganic Polyphosphate in Mammalian Cells and Tissues

Inorganic polyphosphate (polyP), a linear polymer of hundreds of orthophosphate (Pi) residues linked by high-energy, phosphoanhydride bonds, has been identified and measured in a variety of mammalian cell lines and tissues by unambiguous enzyme methods. Subpicomole amounts of polyP (0.5 pmol/100 micrograms of protein) were determined by its conversion to ATP by Escherichia coli polyphosphate kinase and, alternatively, to Pi by Saccharomyces cerevisiae exopolyphosphatase. Levels of 25 to 120 microM (in terms of Pi residues), in chains 50 to 800 residues long, were found in rodent tissues (brain, heart, kidneys, liver, and lungs) and in subcellular fractions (nuclei, mitochondria, plasma membranes, and microsomes). PolyP in brain was predominantly near 800 residues and found at similar levels pre- and postnatally. Conversion of Pi into polyP by cell lines of fibroblasts, T-cells, kidney, and adrenal cells attained levels in excess of 10 pmol per mg of cell protein per h. Synthesis of polyP from Pi in the medium bypasses intracellular Pi and ATP pools suggesting the direct involvement of membrane component(s). In confluent PC12 (adrenal pheochromocytoma) cells, polyP turnover was virtually complete in an hour, whereas in fibroblasts there was little turnover in four hours. The ubiquity of polyP and variations in its size, location, and metabolism are indicative of a multiplicity of functions for this polymer in mammalian systems.

Enzyme-Linked Immunosorbent Assay for Detection of Serum Antibody to Blastocystis hominis in Symptomatic Infections

An enzyme-linked immunosorbent assay was devised in order to search for antibodies against Blastocystis hominis in infected humans. Reaction proteins were obtained from washed, axenic B. hominis cells, as sonicate. Sonicate was diluted to provide 17 and 34 micrograms of protein per well. Dilutions of patients' sera were applied, followed by phosphatase-conjugated goat anti-human serum and phosphatase substrate. Color was measured at 405 microns wavelength. Immunoglobulin G antibodies to high titers were found. Of 30 sera tested from 28 patients, 3 were negative at the 1/50 threshold dilution, 8 were positive at 1/50, 3 at 1/100, 2 at 1/200, 3 at 1/400, 6 at 1/800, and 5 at 1/1,600. Normal sera (42 blood bank sera) were all negative at 1/50. Each serum was subjected to multiple testing. Duplicate tests were included for each run, and runs were made from 4 to 6 for each serum. Blastocystis hominis is increasingly recognized to be a cause of human enteric disease, with symptoms often like those in giardiasis. Demonstration of strong antibody response is consistent with this view.

Bacterial adsorption to fetuin and mucin pellicle.

The ability of Actinomyces viscosus (A. viscosus) WVU 627 to attach to hydroxyapatite (HA) beads treated with either bovine fetuin or bovine mucin was studied. All preparations used were blocked with bovine serum albumin prior to incubation with [3H] thymidine-labeled A.viscosus cells. The quantity of fetuin or mucin adsorbed on the HA beads was determined by use of [3H] fetuin or [3H] mucin. Approximately 6 micrograms protein of [3H] fetuin and 20 micrograms of protein of [3H] mucin adsorbed to 5 mg of HA beads at saturation, respectively. The presence of fetuin or mucin on HA beads promoted attachment of the organism. However, HA beads treated with human whole saliva as a positive control promoted A.viscosus attachment more effectively than HA beads treated with these glycoproteins. Attachment of two additional strains (B 236 and B 25) of A. viscosus to HA beads was also promoted by these glycoproteins. The number of A.viscosus cells which attached to fetuin-treated HA beads was dose-dependent, except for strain B 25. For all three A.viscosus strains tested, attachment to the experimental pellicle with mucin was dose-dependent. These findings suggest that the use of these newly developed bacterial adhesion assay systems may be effective for elucidating bacterial adhesion mechanisms.

Cultured cerebellar granule cells, but not astrocytes, produce an ester of ganglioside GD1b, presumably GD1b monolactone, from exogenous GD1b.

Granule cells and astrocytes from rat cerebellum were fed in culture with 2 microM ganglioside [Gal-3H]GD1b and then analysed for the presence of carboxyl esters of that ganglioside. Before extraction and purification of gangliosides, cells were treated with NaBH4 under conditions that would allow complete reductive cleavage of carboxyl ester linkages, [Gal-3H]GD1b monolactone and dilactone being used as reference esters of GD1b. These conditions, established by adding harvested cells (250 micrograms of protein) with 0.01-2 nmol of standard [Gal-3H]GD1b monolactone or dilactone and [Gal-3H]GD1b-1ol or -2ol formed respectively, consisted of an NaBH4/cell protein ratio of 2:1 (w/w). Cerebellar granule cells, but not astrocytes, were able to produce a radioactive compound which was identified as GD1b-1ol. The formation of this compound increased with pulse (up to 4 h) and chase (up to 3 h) time after a 2 h pulse and also occurred when ganglioside endocytosis was blocked. It can be concluded that cerebellar granule cells are able to convert ganglioside GD1b into a carboxyl ester form, presumably GD1b monolactone. The natural occurrence of the same GD1b carboxyl ester in cerebellar granule cells was also demonstrated.

Levels of platelet calmodulin for the prediction of progression and severity of adolescent idiopathic scoliosis.

Calmodulin is a calcium-binding receptor protein that regulates the contractile protein systems of skeletal muscle and platelets. The levels of platelet calmodulin were measured in twenty-seven adolescents to determine whether there was a relationship between these levels and the progression or the severity of idiopathic scoliosis. The study included seventeen patients who had idiopathic scoliosis of varying severity and patterns and a control group consisting of ten age and sex-matched subjects: eight patients who were being managed for non-scoliosis-related problems and two normal volunteers. Platelets were isolated from the venous blood of all adolescents. The platelets were homogenized and centrifuged, and the calmodulin-containing supernatant was isolated. The level of calmodulin was then measured with use of a radioimmunoassay that employs competitive binding between native, unlabeled calmodulin and 125I-labeled calmodulin. The results showed that the level of platelet calmodulin in the patients who had a progressive curve (more than 10 degrees of progression in the previous twelve months) (3.83 nanograms per microgram of protein) was significantly higher than the level in the patients who had a stable curve (less than 5 degrees of progression in the previous twelve months) (0.60 nanogram per microgram of protein) (p < 0.01); the levels in the stable group and the control group (0.69 nanogram per microgram of protein) were similar. The level of platelet calmodulin appeared to be an independent and possibly more acute predictor of progression of the curve than the Risser sign alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Porphyromonas gingivalis fimbriae stimulate bone resorption in vitro.

Our previous study demonstrated that Porphyromonas gingivalis fimbriae induce the expression of interleukin-1, a potent bone-resorbing cytokine, in macrophages. This demonstration suggested to use the possibility that the fimbriae may stimulate bone resorption via the generation of an inflammatory cytokine(s). The present study was performed to test this suggestion. The bone-resorbing activity was evaluated by measuring the area of resorption lacunae on bone slices incubated with calvarial bone cells taken from 14-day-old mouse embryos. Fimbriae at 0.5 micrograms of protein per ml stimulated the bone-resorbing activity significantly, and the effect was dose and treatment time dependent. Since it is well known that interleukin-1 and granulocyte macrophage colony-stimulating factor induce differentiation of osteoclast lineage cells, we examined the involvement of these cytokines in fimbria-stimulated bone resorption. Fimbria-stimulated bone resorption was abolished significantly by antisera against both cytokines. We observed by Northern (RNA) blot assay that both cytokine genes were markedly expressed in the fimbria-treated calvarial bone cells. Our present data demonstrate that P. gingivalis fimbriae stimulate bone resorption in vitro.

Improved purification and biologic activities of staphylococcal toxic shock syndrome toxin 1

An improved method for producing highly purified toxic shock syndrome toxin 1 (TSST-1) by preparative isoelectric focusing in a Bio-Rad Rotofor cell and then chromatofocusing is described. Purification to homogeneity was confirmed by silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 50 micrograms of protein was loaded), by immunoblotting with polyclonal rabbit antiserum raised against the crude culture supernatant used for purification, and by autoradiography after iodination and SDS-PAGE. Biologic activity was demonstrated by mitogenicity and cytokine induction (tumor necrosis factor alpha [TNF-alpha], interleukin 1-beta [IL-1 beta], and IL-6) of human peripheral blood mononuclear cells (PBMCs) and by lethality in New Zealand White rabbits following subcutaneous infusion. In contrast to commercial TSST-1 preparations, our TSST-1 preparation required the presence of both monocytes and T cells for the induction of TNF-alpha and IL-1 beta from human PBMCs. A 46-kDa contaminating protein in the commercial TSST-1 preparation, identified as staphylococcal lipase, was likely responsible for the induction of TNF-alpha and IL-1 beta from human monocytes in the absence of T cells, a biologic activity falsely attributed to purified TSST-1. Our improved purification procedure for TSST-1 provides a high yield and is both more rapid and less labor intensive than previously reported methods. Furthermore, our studies clearly demonstrate the need for stringent methods of purity assessment of TSST-1 preparations before ascribing to them their potent biologic activities.

Immunogenicity of Purified F Glycoprotein of Respiratory Syncytial Virus: Clinical and Immune Responses to Subsequent Natural Infection in Children

Purified F glycoprotein from respiratory syncytial virus (RSV, subgroup A antigenic type) was evaluated in 18- to 36-month-old children as a vaccine. Children had been previously infected with RSV during natural outbreaks of the virus. Single injections of 5, 20, or 50 micrograms of protein resulted in greater than eightfold increases in ELISA and neutralizing antibodies. Second doses of vaccine did not result in further boosts in antibody. Neutralizing antibodies increased not only to the A2 and Long strains (subgroup A strains) but also to strain 18537 (subgroup B). Four of 11 vaccinees became naturally infected during the subsequent RSV outbreak, suggesting that the vaccine was not effective in preventing recurrent RSV infections. Severe illnesses did not occur, indicating that there was not an increase of severity of infection following vaccine in seropositive children. Only 1 of 8 vaccinees tested had fourfold increases in nasal wash IgA to RSV after immunization. Vaccine strategies to stimulate secretory antibodies as well as circulating neutralizing antibodies to RSV need to be developed.

Immunization against Porcine Cysticercosis in an Endemic Area in Mexico: a Field and Laboratory Study

An antigenic extract from Taenia solium metacestodes was evaluated for immunogenicity in pig populations from a large area of endemic porcine cysticercosis in the State of Guerrero, Mexico. A total of 3,295 pigs from 18 villages were immunized with a single dose of 250 micrograms of protein administered intramuscularly. Systematic immunization was also performed on pigs (1,076 immunizations) from two of the villages with the highest percentages of cysticercosis. A year after immunization, porcine cysticercosis decreased from 4.8% and 5.4% to 0%. Immunity against the T. solium metacestode was estimated in vitro by measurements of 3H-thymidine uptake and inhibition of leukocyte migration. Peripheral blood lymphocytes from immunized cysticercotic (pigs that had cysticercosis prior to immunization), cysticercotic immunized (pigs that acquired cysticercosis after immunization), and normal control pigs incorporated 3H-thymidine better than lymphocytes from cysticercotic pigs when stimulated with concanavalin A. A significant inhibition in the leukocyte migration inhibition test was also found in leukocytes from immunized cysticercotic pigs (P < 0.01). Histopathologic studies revealed granuloma formation surrounding the metacestodes of the immunized cysticercotic and cysticercotic immunized pigs. These metacestodes exhibited several stages of destruction. Large numbers of eosinophils were frequently observed in a close association with the degeneration and destruction of parasites. Metacestodes in control cysticercotic pigs were intact and surrounded by a minor inflammatory reaction. Finally, the rate of in vitro evagination of scolices was high in metacestodes obtained from cysticercotic pigs and low or absent in those from immunized pigs (P < 0.01).