Comparison of polysaccharide glycoconjugates as candidate vaccines to combat Clostridiodes (Clostridium) difficile.

Two known Clostridiodes (Clostridium) difficile surface antigens, a lipoteichoic acid (LTA) and a polysaccharide (PS-II) were isolated and purified in order to prepare glycoconjugate vaccines to the carrier protein human serum albumin utilising a reductive amination strategy. Mice and rabbits were immunized with a prime and two boost strategy and the resulting sera were examined for their ability to recognise the purified homologous antigens and subsequently killed whole cells of C. difficile strains and other Clostridia species. Immunisation derived antisera from rabbits and mice, recognised all strains of C. difficile vegetative cells examined, with generally similar titers from animals that received the LTA or the PS-II conjugates. Sera raised to the LTA conjugates were able to recognise other Clostridia species C. butyricum, C. bifermentans and C. subterminale whereas sera raised to the PS-II conjugates were not. These LTA and PS-II sera recognised live cells in an immunofluorescence assay and were also able to recognise the spore form of the bacterium. This study has confirmed that the LTA and PS-II polysaccharides are both highly conserved surface polymers of C. difficile that are easily accessible to the immune system and as such may have potential as vaccine antigens or as targets for therapeutics to combat C. difficile infection.

Photonic wire biosensor microarray chip and instrumentation with application to serotyping of Escherichia coli isolates.

A complete photonic wire molecular biosensor microarray chip architecture and supporting instrumentation is described. Chip layouts with 16 and 128 independent sensors have been fabricated and tested, where each sensor can provide an independent molecular binding curve. Each sensor is 50 µm in diameter, and consists of a millimeter long silicon photonic wire waveguide folded into a spiral ring resonator. An array of 128 sensors occupies a 2 × 2 mm2 area on a 6 × 9 mm2 chip. Microfluidic sample delivery channels are fabricated monolithically on the chip. The size and layout of the sensor array is fully compatible with commercial spotting tools designed to independently functionalize fluorescence based biochips. The sensor chips are interrogated using an instrument that delivers sample fluid to the chip and is capable of acquiring up to 128 optical sensor outputs simultaneously and in real time. Coupling light from the sensor chip is accomplished through arrays of sub-wavelength surface grating couplers, and the signals are collected by a fixed two-dimensional detector array. The chip and instrument are designed so that connection of the fluid delivery system and optical alignment are automated, and can be completed in a few seconds with no active user input. This microarray system is used to demonstrate a multiplexed assay for serotyping E. coli bacteria using serospecific polyclonal antibody probe molecules.

Residential treatment for women with dependent children: one agency's approach.

The Salvation Army First Choice Program, located in Fort Worth, Texas, provides comprehensive-as well as gender-specific-treatment for addicted women while providing child care and therapeutic services for children. Specific program attributes (including therapeutic interventions, community linkages, and staffing patterns) are described, and the five-year evaluation initiative, designed to examine relationships between client characteristics, program participation, and client progress is outlined. Findings from initial analyses examining correlates of 90-day dropout suggest a complex interaction among specific problems a woman brings to treatment, her level of dysfunction at treatment entry, how much social support is available to her, and what services she receives.

Estrogen-induced activation of extracellular signal-regulated kinase signaling triggers dendritic resident mRNA translation.

Activated extracellular signal-regulated kinase (ERK) signaling mediated plasticity-related gene transcription has been proposed for one possible mechanism by which 17ß-estradiol (E2) enhances synaptic plasticity and memory. Because activated ERK also enhances plasticity-related mRNA translation in the dendrites of neurons, we sought to determine the effects of E2 on activation of ERK, phosphorylation of translation initiation factors, and dendritic mRNA translation in hippocampal neurons. Acute E2 application resulted in a rapid, transient increase in phosphorylation of translation initiation factors, ribosomal protein (S6) and eIF4E binding protein1 (4EBP1), in an activated ERK-dependent manner. Since phosphorylation of these translation factors enhance mRNA translation, we tested E2's effect on dendritic mRNA translation. Using a green fluorescent protein (GFP)-based dendritic mRNA translation reporter (reporter plasmid construct consisted of a GFP gene fused to the 3' untranslated region (UTR) from CAMKIIα, which contains dendritic resident mRNA targeting and mRNA translational regulatory elements) we showed that E2 treatment resulted in increased somatic and dendritic GFP mRNA translation in GFP-reporter transfected hippocampal neurons. Translation inhibitor anisomycin and ERK inhibitor U0126 blocked E2 effects. Taken together, our results provide a novel mechanism by which E2 may trigger local protein synthesis of α-CaMKII in the dendrites, which is necessary for modulation of synaptic plasticity.

Location of epitopes on Campylobacter jejuni flagella.

Flagella were isolated from strains of Campylobacter jejuni belonging to different heat-labile serogroups and from a strain of Campylobacter fetus, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the flagellin molecular weights (Mr) were approximately 62,000. The flagellins were cleaved by hydrolysis with cyanogen bromide, and sodium dodecyl sulfate-urea peptide gel electrophoresis showed that the C. jejuni flagellins were structurally similar, and differed from C. fetus flagellin. Immunochemical analysis by Western blotting, enzyme-linked immunosorbent assay, immune electron microscopy, and immunoprecipitation with polyclonal and monoclonal antibodies revealed the presence of both internal and surface-exposed epitopes. The internal epitopes were antigenically cross-reactive and linear, and in the case of C. jejuni flagellin were located on cyanogen bromide peptides of apparent Mr 22,400 and 11,000. Antigenically cross-reactive epitopes were also present on an Mr 43,000 cyanogen bromide peptide of C. fetus flagellin. The Mr 22,400 peptide of C. jejuni VC74 flagellin also carried closely positioned internal linear epitopes for two monoclonal antibodies. One epitope was strain specific, while the other was shared by some but not all Campylobacter flagellins. The flagella of C. jejuni VC74 also displayed both surface-exposed antigenically cross-reactive and surface-exposed serospecific epitopes. Both linear and conformational epitopes contributed to the serospecificity of C. jejuni VC74 flagella, and a linear serospecific epitope was located on a cyanogen bromide peptide of apparent Mr 4,000.

Molecular identification of surface protein antigens of Campylobacter jejuni.

The technique of immunoblotting was used to identify the surface protein antigens of Campylobacter jejuni. Polyclonal antisera were raised in rabbits to formalinized cells of a typical human fecal isolate, C. jejuni VC74. Surface components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fractions analyzed included whole cell lysates, sarcosinate-extracted outer membranes, released outer membrane blebs (fragments), isolated flagella, 0.2 M glycine-hydrochloride (pH 2.2) extract, saline extract, and material released by osmotic shocking. The ability of the antisera to recognize corresponding antigens on other strains of thermophilic campylobacters and Campylobacter fetus was also determined. The results demonstrated that heat-labile antigenic specificity was conferred on C. jejuni VC74 by an outer membrane protein with an approximate molecular weight of 92,500. Both the major outer membrane protein and the flagella were immunogenic but did not confer either strain or species serospecificity on the strains tested. Another major antigen on thermophilic campylobacter cells was a surface protein with an approximate molecular weight of 31,000. This common antigen was preferentially removed by glycine extraction but was not detectable in outer membrane prepared by sarcosinate extraction.

Structural and antigenic heterogeneity of lipopolysaccharides of Campylobacter jejuni and Campylobacter coli.

The techniques of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, silver staining, and immunoblotting were used to analyze the lipopolysaccharide (LPS) structure of 20 strains of Campylobacter jejuni and 4 strains of Campylobacter coli belonging to more than 22 thermostable serotypes. The LPSs of all strains examined were shown to be of a low-molecular-weight type, and these low-molecular-weight LPSs conferred heat-stable serospecificity. High-molecular-weight banding observed with both in vivo LPS in proteinase K digests of whole cell lysates and purified LPS was shown to be due to the ready ability of Campylobacter lipopolysaccharide to form aggregates rather than to the presence of O polysaccharide chains. Purified LPSs from two strains of C. jejuni were also subjected to gross chemical analysis. The high-lipid A to low-neutral sugar ratio of both LPSs was typical of LPSs lacking O polysaccharide chains.

Outer membrane characteristics of Campylobacter jejuni.

Outer membranes were isolated from type strains and wild-type isolates of Campylobacter jejuni and Campylobacter coli by sodium lauryl sarcosinate extraction, and the polypeptide complement and lipopolysaccharide (LPS) content were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein profiles exhibited by membranes from both species were quite similar, but could be distinguished from the type strain of the genus, C. fetus subsp. fetus CIP5396. The sodium dodecyl sulfate electrophoretograms of C. jejuni and C. coli were dominated by a major polypeptide band. In the reference strain C. jejuni VC74, this polypeptide had an apparent molecular weight of 45,000, was heat modifiable, and was shown to be transmembrane by virtue of its peptidoglycan association and surface exposure. Two other proteins with approximate molecular weights of 37,000 and 73,000 were also surface exposed on C. jejuni VC74 and represented potential surface antigens. The LPS of C. jejuni and C. coli was of low molecular weight, suggesting that serotypic differences due to LPS were based on different carbohydrate compositions of core LPS. In contrast, the LPS of C. fetus CIP5396 exhibited O antigen polysaccharide chains of intermediate chain length. Fragments of outer membranes released during growth of C. jejuni VC74 displayed a polypeptide profile which differed from that of sarcosinate-extracted outer membranes. Radiolabeling demonstrated that the proteins exposed on the surface of this released membrane differed from those exposed on the cell surface and would likely contribute to the antigenic complexity of C. jejuni.

Isolation and characterization of Campylobacter flagellins.

Sequential acid pH dissociation, differential ultracentrifugation, and neutral pH reassociation were used to partially purify serotypically distinct flagella from three strains of Campylobacter jejuni and the two antigenic phases of flagella of Campylobacter coli VC167. Each C. jejuni flagellin and C. coli VC167 antigenic phase 1 flagellin were purified to homogeneity by reverse-phase high-performance liquid chromatography with a C8 Spheri-10 column. C. coli VC167 antigenic phase 2 was purified to homogeneity by ion-exchange chromatography with a Mono-Q column. Amino acid compositional analysis put the C. jejuni flagellin molecular weight in the range 63,200 to 63,800 and the C. coli antigenic phase 1 and 2 flagellins at 61,500 and 59,500, respectively. The amino acid compositions of the C. jejuni were similar to each other and to the C. coli VC167 antigenic phase 1 and phase 2 flagellins. One-dimensional peptide mapping of the C. jejuni flagellins by partial digestion with trypsin or chymotrypsin confirmed the structural similarities of the C. jejuni flagellins and the C. coli VC167 antigenic phase 1 flagellin and showed that C. coli VC167 antigenic phase 2 flagellin was structurally distinct from the phase 1 flagellin. The antigenic phase 2 flagellin was especially sensitive to digestion by chymotrypsin. Amino-terminal sequence analysis showed that the 20 N-terminal amino acids of the Campylobacter flagellins were highly conserved. The Campylobacter flagellins also shared limited sequence homology with the N-terminal sequences reported for Salmonella and Bacillus flagellins.

Demonstration of a flagellar antigen shared by a diverse group of spiral-shaped bacteria that colonize intestinal mucus.

Western blot analysis showed that there is little immunological cross-reactivity of the human pathogens Campylobacter jejuni and Campylobacter coli with Campylobacter pyloridis, except for a very strong cross-reaction between the flagellins. This same antigenic cross-reaction was found with two isolates of gram-negative spiral microaerophilic bacteria that are known to colonize the intestinal mucosa of rodents, but not with the flagellins of a number of other motile bacteria. It is proposed that this shared flagellin antigen may be important structurally and functionally.

Genes for tryptophan biosynthesis in the halophilic archaebacterium Haloferax volcanii: the trpDFEG cluster.

Tryptophan auxotrophs of the archaebacterium Haloferax volcanii define a cluster of overlapping genes homologous to eubacterial-eukaryotic trpD, -F, -E, and -G, linked in that order and each preceded by a possible ribosome binding site. Residues involved in feedback inhibition of eubacterial anthranilate synthetases are conserved.

Genomic rearrangements associated with antigenic variation in Campylobacter coli.

Campylobacter coli and Campylobacter jejuni share a limited number of highly conserved DNA sequences with members of the family Enterobacteriaceae. One of these sequences was cloned from C. coli VC167, and the region of homology to the enteric sequences was determined to be confined to a 700-base-pair region. The DNA represented in this clone undergoes a programmed, reversible rearrangement in VC167 that is associated with flagellar antigenic variation.

Protein kinase C modulation of recombinant NMDA receptor currents: roles for the C-terminal C1 exon and calcium ions.

Protein kinase C (PKC) positively modulates NMDA receptor (NMDAR) currents. In contrast to previous reports, this study determines the importance of individual exons in the mechanism underlying the potentiation process by examining the complete set of eight naturally occurring splice variants expressed in Xenopus oocytes both as homomers and as heteromeric NR1/NR2A or NR1/NR2B complexes. After PKC stimulation, homomeric currents demonstrated a high level of potentiation ( approximately 500% of untreated baseline currents) that reduced to a lower level ( approximately 300% of baseline) in variants containing the first C-terminal exon (C1). An ANOVA showed that only C1 and no other exon or interaction of exons determined the degree of NMDAR current modulation by PKC. When recordings were performed in solutions in which barium replaces calcium, only the lower form of potentiation was observed, regardless of the splice variant exon composition. This suggested an important role for calcium in the PKC modulation of homomeric NMDA splice variant currents in which the C1 exon also participates. The effectiveness of the C1 exon to reduce the higher form of potentiation is modulated by heteromeric assemblies with NR2A heteromers yielding smaller levels of potentiation and a larger C1 exon effect compared with NR2B heteromers. The heteromers demonstrated the higher form of potentiation even in the absence of calcium. Furthermore, calcium had different effects in the potentiation of the heteromers depending on the NR2 subunit. This study refines the region of the NR1 subunit involved in a modulation crucial to the function of NMDA receptors and provides evidence that the NR2A and NR2B subunits realize this modulation differentially.

Evidence for posttranslational modification and gene duplication of Campylobacter flagellin.

A gene encoding a flagellin protein of Campylobacter coli VC167 has been cloned and sequenced. The gene was identified in a pBR322 library by hybridization to a synthetic oligonucleotide probe corresponding to amino acids 4 to 9 of the N-terminal sequence obtained by direct chemical analysis (S. M. Logan, L. A. Harris, and T. J. Trust, J. Bacteriol. 169:5072-5077, 1987). The DNA was sequenced and shown to contain an open reading frame encoding a protein with a molecular weight of 58,945 and a length of 572 amino acids. The deduced amino acid sequence was identical to the published N-terminal amino acid sequence of VC167 flagellin and to four internal regions whose partial sequences were obtained by direct chemical analysis of two tryptic and two cyanogen bromide peptides of VC167 flagellin. The C. coli flagellin protein contains posttranslationally modified serine residues, most of which occur within a region containing two 9-amino-acid repeating peptides separated by 34 unique amino acids. Comparisons with the sequences of flagellins from other bacterial species revealed conserved residues at the amino- and carboxy-terminal regions. Hybridization data suggest the presence of a second flagellin copy located adjacent to the first on the VC167 chromosome.

Predictors of program completion for women in residential substance abuse treatment.

Although there is increasing emphasis on providing drug treatment programs for women that address their specific needs (including parenting and childcare), some women still fail to complete treatment. Because of the limited information about the barriers involved, this study examines pretreatment characteristics as predictors of program completion for 87 women who were pregnant or who entered residential treatment with their children. By using a multivariate prediction model, three significant predictors of treatment completion were identified: education level, recent arrests, and peer deviance. Women who completed program requirements were more likely to have a high school degree or equivalent, no arrests in the 6 months before admission, and friends who were less deviant. These findings support the need for specialized education and services that address social deviancy of pregnant and/or parenting women. Other predictors that approached significance and deserve further study include marital status, number of children in treatment, child welfare involvement, cocaine use, and psychological depression.

Elevated levels of Legionella pneumophila stress protein Hsp60 early in infection of human monocytes and L929 cells correlate with virulence.

Legionella pneumophila 2064 was selectively radiolabelled in mouse L929 cells and human monocytes to identify proteins expressed early in the course of infection. Polypeptide profiles (sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography) of adherent or intracellular bacteria indicated that a 60-kDa stress protein (Hsp60) was preferentially synthesized. Hsp60 synthesis was not induced by medium alone. The synthesis of many polypeptides, including OmpS (major outer membrane protein), diminished over the 1-h period postinfection. However, by 17 h postinfection OmpS and Hsp60 were the dominant proteins synthesized by 2064. To establish whether induction of Hsp60 was a correlate of virulence, an isogenic avirulent strain (2064M) of 2064 was isolated following selection on a nonpermissive medium. 2064M did not exhibit a stress response when adherent or intracellular in L929 cells or in human monocytes and failed to abrogate phagosome-lysosome fusion. When grown in vitro, 2064M exhibited no deficiencies in the heat shock response and its polypeptide profile resembled that of 2064. Immunogold electron microscopy was used to localize Hsp60 in L. pneumophila-infected L929 cells. There was an increase in the number of gold particles associated with phagosomes for phagosomes harboring single 2064 bacteria compared with those harboring 2064M. Moreover, by 1 h postinfection, a sixfold increase in the number of gold spheres associated with the membranes of phagosomes was observed for phagosomes harboring 2064 compared with those harboring 2064M. These studies indicate that virulent, but not NaCl-tolerant avirulent, strains of L. pneumophila respond to host-cell-associated environmental signals early in the course of infection. This response includes increased synthesis and possibly extracellular secretion of Hsp60 concomitant with repression of the expression of other genes, including ompS.

Role of two flagellin genes in Campylobacter motility.

Campylobacter coli VC167 T2 has two flagellin genes, flaA and flaB, which share 91.9% sequence identity. The flaA gene is transcribed from a o-28 promoter, and the flaB gene from a o-54 promoter. Gene replacement mutagenesis techniques were used to generate flaA+ flaB and flaA flaB+ mutants. Both gene products are capable of assembling independently into functional filaments. A flagellar filament composed exclusively of the flaA gene product is indistinguishable in length from that of the wild type and shows a slight reduction in motility. The flagellar filament composed exclusively of the flaB gene product is severely truncated in length and greatly reduced in motility. Thus, while both flagellins are not necessary for motility, both products are required for a fully active flagellar filament. Although the wild-type flagellar filament is a heteropolymer of the flaA and flaB gene products, immunogold electron microscopy suggests that flaB epitopes are poorly surface exposed along the length of the wild-type filament.

Isolation and biochemical and molecular analyses of a species-specific protein antigen from the gastric pathogen Helicobacter pylori.

A protein of Mr 26,000 which was present in large quantities in extracts of cells of Helicobacter pylori was purified to homogeneity by ammonium sulfate precipitation followed by gel filtration and reversed-phase chromatography or anion-exchange chromatography. The protein appeared to be associated with the soluble fraction of the cell, and antibodies raised against the protein were reactive with whole-cell lysates of a variety of H. pylori strains in a simple immunodot blot assay. This reaction was species specific. Protein sequence determination of the amino terminus and internal cyanogen bromide fragments and amino acid composition analysis were performed. An oligonucleotide derived from these data was used to clone a fragment encoding most of the coding sequence. Expression in Escherichia coli was dependent on vector promoters. The DNA sequence of the fragment was determined. DNA probes derived from the cloned fragment hybridized to genomic DNA of all H. pylori strains tested, but not to DNAs of Helicobacter mustelae, Wolinella succinogenes, various Campylobacter species, and a panel of gram-negative enteric bacteria. The apparent uniqueness of this protein may be exploited for the development of species-specific diagnostics for this gastric pathogen.

Antigenic variation of Campylobacter flagella.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of flagella dissociated from strains of Campylobacter coli and Campylobacter jejuni belonging to the heat-labile serogroup LIO 8 showed that some strains were capable of producing flagellin subunits of two different molecular weights (MrS), 59,500 and 61,500. Immunoelectron microscopy of cultures of the type strain of this serogroup, C. coli VC167, showed the presence of two flagellum filaments of different antigenic specificity. Epitopes on the surface of one of these flagella bound antibodies in LIO 8 typing antiserum, and Western blotting (immunoblotting) and immunoprecipitation showed that the flagellum was composed of flagellin of Mr 61,500. The other flagellum antigenic type did not bind LIO 8 antibodies but did possess serospecific epitopes which bound a second polyclonal antiserum, LAH2. This second antigenic flagellum type was composed of the Mr 59,500 flagellin. Cells producing either of the flagellum antigenic types serotyped as LIO 8, indicating that flagella composed of the Mr 61,500 flagellin do not carry the serological determinants for this serogroup. The ability of C. coli VC167 to produce these flagella of different subunit MrS was shown to represent a bidirectional antigenic variation. When measured in culture medium, the phase 1-to-phase 2 transition occurred at a rate of approximately 2.0 x 10(-5) per cell per generation, and the phase 2-to-phase 1 transition occurred at a rate of 1.2 x 10(-6) per cell per generation.

Flagellin from Listeria monocytogenes is glycosylated with beta-O-linked N-acetylglucosamine.

Glycan staining of purified flagellin from Listeria monocytogenes serotypes 1/2a, 1/2b, 1/2c, and 4b suggested that the flagellin protein from this organism is glycosylated. Mass spectrometry analysis demonstrated that the flagellin protein of L. monocytogenes is posttranslationally modified with O-linked N-acetylglucosamine (GlcNAc) at up to six sites/monomer. The sites of glycosylation are all located in the central, surface-exposed region of the protein monomer. Immunoblotting with a monoclonal antibody specific for beta-O-linked GlcNAc confirmed that the linkage was in the beta configuration, this residue being a posttranslational modification commonly observed in eukaryote nuclear and cytoplasmic proteins.