Seroprotection against serogroup C meningococcal disease in adolescents in the United Kingdom: observational study.

OBJECTIVE: To determine the persistence of bactericidal antibody titres following immunisation with serogroup C meningococcal glycoconjugate vaccine at age 6-15 years in order to examine changes in persistence of antibodies with age. DESIGN: Observational study. SETTING: Secondary and tertiary educational institutions in the United Kingdom. PARTICIPANTS: Healthy adolescents aged 11-20 years previously immunised between 6 and 15 years of age with one of the three serogroup C meningococcal vaccines. INTERVENTION: Serum obtained by venepuncture. MAIN OUTCOME MEASURES: Percentage of participants with (rabbit complement) serum bactericidal antibody titres of at least 1:8; geometric mean titres of serogroup C meningococcal serum bactericidal antibody. RESULTS: Five years after immunisation, 84.1% (95% confidence interval 81.6% to 86.3%) of 987 participants had a bactericidal antibody titre of at least 1:8. Geometric mean titres of bactericidal antibody were significantly lower in 11-13 year olds (147, 95% confidence interval 115 to 188) than in 14-16 year olds (300, 237 to 380) and 17-20 year olds (360, 252 to 515) (P<0.0001 for both comparisons). Within these age bands, no significant difference in geometric mean titres of bactericidal antibody between recipients of the different serogroup C meningococcal vaccines was seen. More than 70% of participants had received a vaccine from one manufacturer; in this cohort, geometric mean titres were higher in those immunised at aged 10 years or above than in those immunised before the age of 10. CONCLUSIONS: Higher concentrations of bactericidal antibody are seen five years after immunisation with serogroup C meningococcal vaccine at age 10 years or above than in younger age groups, possibly owing to immunological maturation. This provides support for adolescent immunisation programmes to generate sustained protection against serogroup C meningococcal disease not only for the vaccine recipients but also, through the maintenance of herd immunity, for younger children.

Haplotypic diversity in human CEACAM genes: effects on susceptibility to meningococcal disease.

Adhesion between the opacity-associated adhesin (Opa) proteins of Neisseria meningitidis and human carcino-embryonic antigen cell adhesion molecule (CEACAM) proteins is an important stage in the pathogenesis of meningococcal disease, a globally important bacterial infection. Most disease is caused by a small number of meningococcal genotypes known as hyperinvasive lineages. As these are also carried asymptomatically, acquisition of them alone cannot explain why only some hosts develop meningococcal disease. Our aim was to determine whether genetic diversity in CEACAM is associated with susceptibility to meningococcal disease. Frequency distributions of alleles, genotypes and haplotypes were compared in four CEACAM genes in 384 case samples and 190 controls. Linkage disequilibrium among polymorphic sites, haplotype structures and relationships were also analysed. A number of polymorphisms were observed in CEACAM genes but the diversity of CEACAM1, to which most Opa proteins bind, was lower, and a small number of high-frequency haplotypes were detected. Dose-dependent associations of three CEACAM haplotypes with meningococcal disease were observed, with the effect of carrying these haplotypes amplified in homozygous individuals. Two haplotypes were protective while one haplotype in CEACAM6 was associated with a twofold increase in disease susceptibility. These data imply that human CEACAM may be one determinant of human susceptibility to meningococcal disease.

Development of an anti-Salmonella typhi Vi ELISA: assessment of immunocompetence in healthy donors.

We have developed a solid-phase enzyme-linked immunosorbent assay (ELISA) to study the vaccination responses to Vi capsular polysaccharide of Salmonella typhi (S. typhi Vi) vaccine. Purified S. typhi Vi polysaccharide was biotinylated and bound to streptavidin coated microtitre plates. Reproducibility was determined across a range of IgG antibody levels: mean interassay coefficients of variation (CVs) were <11.9% for non-vaccinated sera with low levels and <11.1% for sera with very high levels of anti-S. typhi Vi IgG. Specificity was assessed by inhibition studies using salmonella antigen. We have developed the ELISA based on normal adult serum responses to test immunization with S. typhi Vi vaccine. We also report here anti-S. typhi Vi IgG levels in a group of healthy preschool children. In non-vaccinated adult sera (n = 104), the median value of anti-S. typhi Vi IgG, expressed in S. typhi Vi arbitrary units (AU/ml), was 5.3 AU/ml and in non-vaccinated sera from children (n = 44) the median value was 1.4 AU/ml. The data from immunization of healthy volunteers (n = 23) show that geometric mean levels of anti-S. typhi Vi IgG were significantly higher (P < 0.0001) for post-vaccination subjects (39.2 AU/ml) compared to paired prevaccination (3.9 AU/ml) values. A total of 21/23 vaccine recipients had <8 AU/ml S. typhi Vi IgG in their sera prior to vaccination and of these 20/21 (95%) exhibited threefold increases and 14/21 (67%) fourfold increases in their S. typhi Vi IgG following vaccination. Based on the data in this study, we propose a threefold increase in anti-S. typhi Vi IgG post-vaccination to be considered a positive vaccination response. The ability to demonstrate clearly an antibody rise in response to immunization with S. typhi Vi capsular polysaccharide vaccine suggests that this is likely to be a useful vaccine for the assessment of B cell function in patients with suspected immune deficiency.

Influence of physical and chemical characteristics of diesel fuels and exhaust emissions on biological effects of particle extracts: a multivariate statistical analysis of ten diesel fuels.

The emission of diesel exhaust particulates is associated with potentially severe biological effects, e.g., cancer. The aim of the present study was to apply multivariate statistical methods to identify factors that affect the biological potency of these exhausts. Ten diesel fuels were analyzed regarding physical and chemical characteristics. Particulate exhaust emissions were sampled after combustion of these fuels on two makes of heavy duty diesel engines. Particle extracts were chemically analyzed and tested for mutagenicity in the Ames test. Also, the potency of the extracts to competitively inhibit the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the Ah receptor was assessed. Relationships between fuel characteristics and biological effects of the extracts were studied, using partial least squares regression (PLS). The most influential chemical fuel parameters included the contents of sulfur, certain polycyclic aromatic compounds (PAC), and naphthenes. Density and flash point were positively correlated with genotoxic potency. Cetane number and upper distillation curve points were negatively correlated with both mutagenicity and Ah receptor affinity. Between 61% and 70% of the biological response data could be explained by the measured chemical and physical factors of the fuels. By PLS modeling of extract data versus the biological response data, 66% of the genotoxicity could be explained, by 41% of the chemical variation. The most important variables, associated with both mutagenicity and Ah receptor affinity, included 1-nitropyrene, particle bound nitrate, indeno[1,2,3-cd]pyrene, and emitted mass of particles. S9-requiring mutagenicity was highly correlated with certain PAC, whereas S9-independent mutagenicity was better correlated with nitrates and 1-nitropyrene. The emission of sulfates also showed a correlation both with the emission of particles and with the biological effects. The results indicate that fuels with biologically less hazardous potentials should have high cetane number and contain less PAC and sulfur. The results also indicate that engine factors affect the formation and emission of nitrated PAC.

Fatty acid activation of peroxisome proliferator-activated receptor (PPAR).

Peroxisome proliferators such as clofibric acid, nafenopin, and WY-14,643 have been shown to activate peroxisome proliferator-activated receptor (PPAR), a member of the steroid nuclear receptor superfamily. We have cloned the cDNA from rat that is homologous to that from mouse, which encodes a 97% similar protein. To search for physiologically occurring activators, we established a transcriptional transactivation assay by stably expressing in CHO cells a chimera of rat PPAR and the human glucocorticoid receptor that activates expression of the placental alkaline phosphatase reporter gene under the control of the mouse mammary tumor virus promoter. 150 microM concentrations of arachidonic or linoleic acid but not of dehydroepiandrosterone, cholesterol, or 25-hydroxy-cholesterol, activated the receptor chimera. In addition, saturated fatty acids induced the reporter gene. Shortening the chain length to n = 6 or introduction of an omega-terminal carboxylic group abolished the activation potential of the fatty acid. To test whether a common PPAR binding metabolite might be formed from free fatty acids we tested the effects of differentially beta-oxidizable fatty acids and inhibitors of fatty acid metabolism. The peroxisomal proliferation-inducing, non-beta-oxidizable, tetradecylthioacetic acid activated PPAR to the same extent as the strong peroxisomal proliferator WY-14,643, whereas the homologous beta-oxidizable tetradecylthiopropionic acid was only as potent as a non-substituted fatty acid. Cyclooxygenase inhibitors, radical scavengers or cytochrome P450 inhibitors did not affect activation of PPAR. In conclusion, beta-oxidation is apparently not required for the formation of the PPAR-activating molecule and this moiety might be a fatty acid, its ester with CoA, or a further derivative of the activated fatty acid prior to beta-oxidation of the acyl-CoA ester.

Fatty acid activation of the peroxisome proliferator activated receptor, a member of the nuclear receptor gene superfamily.

The peroxisome proliferator activated receptor is a member of the steroid receptor gene superfamily, sharing amino acid sequence homology with other receptors and also showing similarities at the level of gene structure. This receptor is activated both by xenobiotic compounds that induce peroxisome proliferation and by fatty acids at physiological concentrations. Upon activation the receptor mediates transcription of responsive genes through binding to peroxisome proliferator response elements. These genes include those encoding peroxisomal enzymes and members of the cytochrome P450 family of drug metabolizing enzymes. It is therefore possible that the peroxisome proliferator activated receptor may play a crucial role in regulating lipid homeostasis.

Role of brain cytochrome P450 in regulation of the level of anesthetic steroids in the brain.

The role of brain cytochrome P450 (P450) in regulating the levels of the potent anesthetic steroid 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-OH-DHP) has been investigated. By analogy with the elimination of androgen from its target tissues, we present evidence that it is 3 beta-hydroxy-5 alpha-pregnan-20-one (3 beta-OH-DHP) and not 3 alpha-OH-DHP that represents the major pathway for the formation of more polar metabolites and thus the elimination of the 5 alpha-reduced metabolites of progesterone from target tissues. No polar metabolites were formed when 3 alpha-OH-DHP was incubated with microsomal fractions prepared from rat brain, but 3 beta-OH-DHP was hydroxylated at the 6 alpha- and 7 alpha-positions. These 3 beta-diols were not formed to any detectable extent in the liver or kidney but were formed in prostate, pituitary, brain, and breast. The highest catalytic activity, 512 nmol of products formed/g of tissue/hr, was found in the prostate. The corresponding rates in the pituitary, brain, and breast were 71.9, 28.1, and 6.7 nmol/g/hr, respectively. These hydroxylations were confirmed to be P450-catalyzed reactions by solubilization of the P450 from prostate, brain, and breast microsomes and reconstitution of the catalytic activity with NADPH-P450 reductase (EC 1.6.2.4) and lipid. Because 5 alpha-androstane-3 beta,17 beta-diol (3 beta-Adiol) has been shown to be a good substrate for prostate and brain P450, competition experiments were performed to determine whether the same form of P450 is involved in the elimination of 3 beta-Adiol and 3 beta-OH-DHP in the brain. These two substrates competed with each other for metabolism in microsomal fractions and in reconstitution experiments with P450 extracted from the brain or prostate. To test the hypothesis that the hydroxylation of 3 beta-OH-DHP represents a pathway for regulation of the level of 3 alpha-OH-DHP in the brain, the effect of inhibition of the hydroxylation of 3 beta-OH-DHP on the duration of 3 alpha-OH-DHP-induced anesthesia was examined. The nonanesthetic steroid 3 beta-Adiol was used as a competitive inhibitor of the metabolism of 3 beta-OH-DHP. The duration of anesthesia upon intravenous administration of 3 alpha-OH-DHP was increased by 33% when 3 beta-Adiol was coadministered.(ABSTRACT TRUNCATED AT 400 WORDS)

A systematic analytical chemistry/cell assay approach to isolate activators of orphan nuclear receptors from biological extracts: characterization of peroxisome proliferator-activated receptor activators in plasma.

Using a novel combination of analytical chemical and molecular biological techniques, lipophilic components of human plasma separated according to their physico-chemical properties were screened for their ability to activate the rat peroxisome proliferator-activated receptor (rPPAR). Activation of an rPPAR/glucocorticoid receptor chimera stably expressed in CHO cells by fractions in the initial screening guided further subfractionation. Characterization of an active subfraction by gas chromatography alone and in combination with mass spectrometry (GC-MS), indicated the presence of free fatty acids. Individual active components in this mixture were isolated by a final fractionation using high performance liquid chromatography (HPLC). GC-MS analyses of HPLC fractions able to activate the chimeric receptor identified palmitic acid, oleic acid, linoleic acid, and arachidonic acid as endogenous activators of rPPAR. No other activators were identified. This approach is able to specifically extract and identify endogenous activators of PPAR from a complex biological extract and as such may be valuable in the identification of activators of other orphan receptors in the steroid hormone receptor superfamily.

Neurosteroids: 3 alpha-hydroxy-5 alpha-pregnan-20-one and its precursors in the brain, plasma, and steroidogenic glands of male and female rats.

A RIA procedure for measuring progesterone (PROG), 5 alpha-pregnane-3,20-dione (5 alpha-DH PROG), and 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 alpha-TH PROG) has been developed and validated by GLC/mass spectrometry. Measurements were made in intact and adrenalectomized (ADX) male rats, in cyclic, pregnant, spayed, and spayed-ADX females, and in both males and spayed females injected with PROG. The predominant contribution of the ovary to the concentrations of 3 alpha,5 alpha-TH PROG in plasma and brain, was indicated by its larger levels in females, in particular during pregnancy, and by its presence in ovarian tissue and disappearance after ovariectomy. An additional adrenal origin in both males and females was shown. Neither PROG nor 5 alpha-DH PROG disappeared from brain, contrary to plasma, after combined adrenalectomy and gonadectomy, thus suggesting that PROG might be synthetized de novo in brain. However, the concentrations of 3 alpha,5 alpha-TH PROG in plasma and brain of female rats were positively correlated with the concentrations of PROG in plasma, indicating that plasma PROG was the major precursor of 3 alpha,5 alpha-TH PROG. The direct formation of 3 alpha,5 alpha-TH PROG from PROG in brain was strongly suggested by the increased 3 alpha,5 alpha-TH PROG/PROG ratios in brain vs. plasma, when measured in control females, and after injection of PROG to both males and OVX females. It was previously reported that 3 alpha,5 alpha-TH PROG is a sedative/anxiolytic steroid, as a result of its binding to gamma-aminobutyric acid (GABA)A receptors and allosteric potentiation of GABAcergic neurotransmission. Its concentrations in brain reach indeed the neuroactive range in cyclic and pregnant females, and are compatible with a physiological role of this neurosteroid.

Effect of altered acid-base balance and of various agonists on levels of renal glutamate dehydrogenase mRNA.

Rat kidney contains 3.5-kb and 2.8-kb mRNAs that encode for glutamate dehydrogenase (GDH). The levels of both mRNAs are increased gradually after onset of chronic metabolic acidosis and reach a maximum induction of 2.5-fold after 7 days. In contrast, during recovery from chronic acidosis, the levels of the GDH mRNAs are returned to normal within 1 day. The development of an acute metabolic acidosis causes a more rapid induction of GDH mRNA. This increase occurs after a 7-h lag and plateaus after 18 h at a level that is threefold greater than normal. A very similar profile was observed after the transfer of LLC-PK-F+ cells from normal medium to an acidic medium containing 10 mM bicarbonate and adjusted to pH 6.9. However, the transfer of cells from acidic to normal medium caused an immediate and rapid [half-life (t) = 1 h] decrease in GDH mRNA. The apparent half-lives of GDH mRNA were measured by treating cells grown in normal (t = 4 h) and acidic media (t = 12 h) with actinomycin D. Thus, increased stability may account for the induction of GDH mRNA that occurs during growth in response to acidosis. The levels of GDH mRNA are independently affected by changes in medium pH or bicarbonate concentration. The levels of GDH mRNA are also increased by treating cells with adenosine 3',5'-cyclic monophosphate, epinephrine, triiodothyronine, or retinoic acid, whereas treatment with angiotensin II, vasopressin, phorbol 12-myristate 13-acetate, or cycloheximide did not produce an increase. The inductive effect of dexamethasone, which is observed in vivo, is not reproduced in the LLC-PK-F+ cells.

A method for characterization of endogenous ligands to orphan receptors belonging to the steroid hormone receptor superfamily--isolation of progesterone from pregnancy plasma using progesterone receptor ligand-binding domain.

An analytical method is described whereby progesterone is isolated from pregnancy plasma on the basis of the high affinity and specificity of the progesterone receptor for its ligand. Partially purified progesterone receptor ligand-binding domain, expressed as a protein A fusion protein in Escherichia coli, is incubated with a neutral steroid fraction obtained by extraction and ion-exchange chromatography of human late-pregnancy plasma. The incubated sample is passed through two Lipidex 1000 (lipophilic gel) beds. The first, at 4 degrees C, separates the specific ligand-fusion protein complex from nonspecifically bound and unbound compounds, and the second, at 40 degrees C, separates the specific ligand from the protein. Elution of the second bed with methanol yields a fraction containing specific ligand that can be characterized by gas chromatography-mass spectrometry. This methodology may be valuable for identification of endogenous ligands to orphan receptors of the steroid hormone receptor superfamily.

Sources of plant sterol contaminants encountered in low level steroid analysis.

During development of an analytical method to characterize ligands to new members of the steroid hormone receptor superfamily, a persistant contaminant profile was observed during gas chromatographic analysis of reagent blanks. Mass spectrometric analysis identified three of the contaminant peaks as cholesterol and the plant sterols stigmasterol and sitosterol. Laboratory articles made of natural rubber, i.e. pipette fillers and latex gloves, were found to be the source of these and other compounds in the reagent blank profile.

Enterococcus seriolicida sp. nov., a fish pathogen.

The properties and taxonomic position of bacterial strains isolated from diseased specimens of cultured yellowtail and eels were examined. The isolates were gram-positive, short-chain-forming, catalase-negative, facultatively anaerobic cocci. Growth at 10 and 45 degrees C in 6.5% NaCl (pH 9.6) with 40% bile and in 0.1% methylene blue-milk were both positive. The isolates could be distinguished from other species of the genus Enterococcus by several biochemical characteristics and by Lancefield's group antigen. Guanine-plus-cytosine content of DNA was 44 mol% as determined by the thermal melting temperature. The value for DNA-DNA hybridization was sufficiently low to warrant distinguishing this species from reported. Enterococcus species. The name Enterococcus seriolicida is proposed. The type strain is YT-3 (=ATCC 49156).

A new value for mol percent guanine + cytosine of DNA for the salmonid fish pathogen Renibacterium salmoninarum.

The mol% G + C of DNA extracted from seven different isolates of Renibacterium salmoninarum was determined. Organisms studied were from selected geographical areas (U.S.A., Canada, England and France) and were isolated from five different species of salmonid fish. The mol% G + C was determined to be 55.5, higher than the currently reported value of 53.

Regional localization of the human glutaminase (GLS) and interleukin-9 (IL9) genes by in situ hybridization.

Phosphate-activated glutaminase is found in mammalian small intestine, brain, and kidney, but not in liver. The enzyme initiates the catabolism of glutamine as the principal respiratory fuel in the small intestine, may synthesize the neurotransmitter glutamate in the brain, and functions in the kidney to help maintain systemic pH homeostasis. Interleukin-9 (IL9) is a relatively new cytokine that supports the growth of helper T-cell clones, mast cells, and megakaryoblastic leukemia cells. cDNA clones have recently been obtained for each of these genes. The human loci for phosphate-activated glutaminase (GLS) and IL9 have previously been mapped to chromosomes 2 and 5, respectively, by analysis of somatic cell hybrid DNAs. By using chromosomal in situ hybridization, we have regionally mapped GLS to 2q32----q34 and IL9 to 5q31----q35.

Multiple system degeneration with glutamate dehydrogenase deficiency: pathology and biochemistry.

The neuropathological findings in a patient with antemortem diagnosis of olivopontocerebellar atrophy (OPCA) and reduced leucocytic glutamate dehydrogenase (GDH) activity included cerebellar cortical degeneration, most marked in the superior vermis, mild atrophy of the pons and the inferior olivary nucleus, marked reduction of anterior horn cells at all levels and gliosis in both lateral columns. GDH activities and their thermolability in "soluble" and "particulate" fractions in the cerebral cortex, cerebellar hemisphere and vermis were not significantly different from the values in two control brains. GDH mRNA in the patient's brain was not altered in size or amount.

Characterization of human cDNA and genomic clones for glial fibrillary acidic protein.

Both a partial cDNA clone and a complete genomic clone have been isolated for human gfa, the gene encoding the major component of astrocyte intermediate filaments, glial fibrillary acidic protein (GFAP). The nucleotide sequence of the entire coding region and 102 bp of the 5' flanking DNA was determined. The mRNA start site was identified by primer extension and probe protection experiments, and a novel in vitro transcription and translation procedure was then used to establish that the first ATG in the mRNA initiates GFAP synthesis. The predicted amino-terminal sequence for human GFAP differs greatly from that previously deduced for mouse GFAP from its gene sequence, despite otherwise high homology. This discrepancy was resolved by determining that the published mouse genomic sequence has an incorrect additional base. The corrected sequence produces strong homology between human and mouse GFAP in their predicted amino acid sequences, and suggests that human and mouse GFAP initiate at homologous positions. The beginning sequence deduced here for both proteins is matched closely by that previously obtained for porcine GFAP by direct sequencing of its amino-terminal end. This supports the protein initiation sites proposed, and also indicates that GFAP is not processed at its amino-terminal end.

Neural control of glutamine synthetase activity in rat skeletal muscles.

The mechanism of glutamine synthetase induction in rat skeletal muscle after denervation or limb immobilization was investigated. Adult male rats were subjected to midthigh section of the sciatic nerve. At 1, 2, and 5 h and 1, 2, and 7 days after denervation, rats were killed and denervated, and contralateral control soleus and plantaris muscles were excised, weighted, homogenized, and assayed for glutamine synthetase. Glutamine synthetase activity increased approximately twofold 1 h after denervation in both muscles. By 7 days postdenervation enzyme activity had increased to three times the control level in plantaris muscle and to four times the control level in soleus muscle. Increased enzyme activity after nerve section was associated with increased maximum velocity with no change in apparent Michaelis constant. Immunotitration with an antiglutamine synthetase antibody suggested that denervation caused an increase in the number of glutamine synthetase molecules in muscle. However, Northern-blot analysis revealed no increase in the steady-state level of glutamine synthetase mRNA after denervation. A mixing experiment failed to yield evidence for the presence of a soluble factor involved in regulating the activity of glutamine synthetase in denervated muscle. A combination of denervation and dexamethasone injections resulted in additive increases in glutamine synthetase. Thus the mechanism underlying increased glutamine synthetase after denervation appears to be posttranscriptional and is distinct from that of the glucocorticoid-mediated glutamine synthetase induction previously described by us.

Effect of diabetes on glutamine synthetase expression in rat skeletal muscles.

The regulation of glutamine synthetase expression in muscles from normal and diabetic (streptozotocin-treated) rats was studied. Muscle and body weights were markedly reduced in diabetic animals. Glutamine synthetase activity was significantly (2-fold) elevated 7 days after induction of diabetes. Increased enzyme activity persisted for at least 14 days after induction of diabetes, and it was apparent in both slow (soleus) and fast (plantaris) muscles. The diabetes-induced increase in enzyme activity was reflected in an increased steady-state level of glutamine synthetase mRNA. The increases in glutamine synthetase activity and mRNA level in muscle from diabetic rats were reversed by insulin administration. Increased expression of glutamine synthetase may be important for accelerated glutamine production by muscle from diabetic rats.

Toward a molecular etiology of Alzheimer's disease.

Recent progress in the biochemical characterization of Alzheimer's disease (AD) neuropathology has led to the proposal of three hypotheses for the molecular etiology of AD. One focuses on calcium-activated neutral proteases or calpains (Nixon, 1989). Another focuses on protein phosphorylation (Saitoh & Iimoto, 1989). A third is centered on altered phospholipid metabolism (Pettegrew, 1989). Interestingly, all three hypotheses are mutually compatible, involving closely interlocking biochemical systems. Disturbances in any one of these systems might result in the same type of neuropathology, consistent with suggestions that AD could have multiple etiologies. Future investigations of the function and interrelation of these systems in the central nervous system in general and at the synaptic junction in particular are likely to have significant bearing on our understanding of AD.