Biomedical subjects

D E Briles

Publications and source records attributed to D E Briles.

At least 19 recordsLinked to original sources

PubMed1992

In vivo administration of bryostatin 1, a protein kinase C activator, decreases murine resistance to Salmonella typhimurium.

Bryostatin 1, a potent activator of protein kinase C, has antitumor activity against murine lymphoma, leukemia, and melanoma. In vitro, this compound stimulates the release of gamma-interferon, interleukins, and hematopoietic growth factors from accessory cells and activates both T- and B-cells. Bryostatin 1 is also able to stimulate neutrophils to undergo oxidative burst and degranulation. Because of the ability of this compound to stimulate the immune system, cause release of immune mediators, and activate neutrophils, we have examined its effect on bacterial infection by using the gram-negative bacterium Salmonella typhimurium in mice. We find that animals given injections i.v. of S. typhimurium have a shortened life span if they are also given injections i.p. of nonlethal doses of bryostatin 1. There is a dose-response relationship with 100 micrograms/kg bryostatin 1 having a greater effect on survival than 40 micrograms/kg. Below 40 micrograms/kg there are no effects on survival. Analysis of the first 4 h of Salmonella infection demonstrates that bryostatin 1 does not affect the blood clearance of the bacterium. However, by day 2 of infection greater numbers of bacteria are found in the livers and spleens of mice given injections of bryostatin 1. By day 5, 10-fold more S. typhimurium bacteria are found in the livers and spleens of mice receiving 40 micrograms/kg of bryostatin 1. To determine whether bryostatin 1 was affecting growth or causing the death of bacteria, we used a Salmonella carrying a plasmid which has a temperature-sensitive origin of replication and is unable to replicate when the bacteria are in mice. This experiment demonstrates that bryostatin 1 represses bacterial killing but does not affect bacterial growth. Bryostatin 1 given i.p. stimulates a transient syndrome of weight loss and diarrhea from which the mice recover and regain weight, suggesting that bryostatin 1 may release a number of important humoral mediators in vivo. The weight loss is exacerbated by Salmonella infection with mice receiving bryostatin 1 and S. typhimurium, in that they lose approximately 33% of body weight prior to death. Thus, at doses used to treat murine tumors, bryostatin 1 treatment does not affect the clearance of S. typhimurium from the blood but does decrease the killing of bacteria in the liver and spleen, leading to early animal death. Such potential effects of bryostatin 1 on the outcome of bacterial infections should be evaluated in ongoing human trials of this agent.

Animals↗

PubMed1992

A 'safe-site' for Salmonella typhimurium is within splenic polymorphonuclear cells.

Following oral or systemic infection with Salmonella typhimurium, the focus of infection is in the liver and spleen. The majority of Salmonella surviving in the liver and spleen by 4 h post infection are already in an environment where they are largely protected from subsequent killing. Previous studies have shown that the majority of surviving Salmonella are intracellular. In the present study we sought to determine the cell type containing most of the cell-associated Salmonella liberated from the spleen. We enriched for Salmonella-containing cells by Ficoll-Hypaque separation followed by fluorescence-activated cell sorting. Approximately 85% of the total intracellular Salmonella were found in Mac-1+/J-11d+ cell fractions of the Ficoll-Hypaque band and pellet. By microscopic examination of stained cells from the sorted cell populations, it was evident that virtually all of the Salmonella were in polymorphonuclear cells (PMN). The numbers of Salmonella observed microscopically were similar in numbers to Salmonella colony forming units detected by plating. Salmonella containing PMN in the Ficoll band generally contained a single bacterium, while those from the probably less healthy cells in the Ficoll pellet generally contained several Salmonella.

Animals↗

PubMed1992

Molecular localization of variable and conserved regions of pspA and identification of additional pspA homologous sequences in Streptococcus pneumoniae.

PspA is anchored to the surface of all pneumococci by the C-terminal end of the molecule. The N-terminal half of PspA is known to be serologically variable and to be able to elicit protective immune responses. Molecular analysis with DNA probes spanning different regions of pspA was carried out to identify homologous sequences among pneumococcal isolates. At high stringency, DNA probes derived from the 3'-half of pspA (encoding the C-terminal half of PspA) hybridized to all of 37 pneumococcal isolates tested, representing 20 capsular serotypes and 12 PspA serotypes. Most strains had two sequences highly homologous to this region of pspA. Using derivatives of strain Rx1, with insertion mutations in pspA, it was possible to identify the functional pspA sequence. At 50% stringency, the 3' pspA probes also detected lytA and additional sequences. lytA encodes autolysin and shares homology with the 3' portion of pspA. A probe derived from the 5'-half of pspA (encoding the N-terminal half of PspA) hybridized with only 75% of strains and generally detected only one of the two sequences recognized by the 3' probes. Thus, the 3'-half of pspA appears to contain more highly conserved sequences than the 5'-half of pspA and shares homology with several additional sequences, suggesting that the pneumococcus might make several proteins that interact with the surface by the same mechanism as PspA.

Bacterial Proteins↗

PubMed1992

Analysis of pneumococcal PspA microheterogeneity in SDS polyacrylamide gels and the association of PspA with the cell membrane.

Pneumococcal surface protein A (PspA) is a protection-eliciting surface protein found on all pneumococci. Although highly cross-reactive, it displays interstrain variation in its size and in the expression of individual antibody reactive epitopes. PspA was not released in significant amounts from pneumococcal membranes treated with sodium carbonate, but was solubilized with SDS. Thus, PspA is either an integral membrane protein or is attached to an integral membrane component. By SDS-PAGE and immunoblot analysis, we found two predominant molecular sizes of PspA in each strain examined. The smaller band was about the size expected from the inferred amino acid sequence of PspA and the larger band appeared to be a dimer of the monomer PspA. When higher concentrations of lysate were run on SDS gels, it was also possible to detect many additional high molecular weight components that reacted with antibodies to PspA. These multiple high molecular weight PspA bands were not due to the attachment of PspA to peptidoglycan or teichoic acids, did not appear to be composed of degraded PspA and most likely resulted from non-covalent polymerization or aggregation of PspA.

Antigens, Surface↗

PubMed1992

Geographic distribution of penicillin-resistant clones of Streptococcus pneumoniae: characterization by penicillin-binding protein profile, surface protein A typing, and multilocus enzyme analysis.

Examination of several hundred penicillin-resistant clinical isolates of Streptococcus pneumoniae has revealed extensive strain-to-strain variation in the number and molecular size of penicillin-binding proteins (PBPs). This polymorphism has been used to classify resistant isolates into groups (PBP families) that share distinct electrophoretic profiles. We describe herein properties of four such PBP families: two from Spain (and/or Ohio) and one each from Hungary and Alaska. We have discovered that representative isolates assigned to each PBP family also share capsular serotype, antibiotic resistance pattern, pneumococcal surface protein A type, and multilocus enzyme genotype. The results demonstrate independent clonal origin for strains assigned to each PBP family. Each resistant clone occurs with uniquely high incidence within specific geographic areas.

Alaska↗

PubMed1992

Evidence for a clonal origin of relative penicillin resistance among type 9L pneumococci in northwestern Canada.

The relationships among capsular type, protein type, and penicillin resistance for capsular group 9 Streptococcus pneumoniae collected in northwestern Canada between 1974 and 1987 were examined. The group 9 relatively penicillin-resistant (RPR) isolates were of the rare 9L capsular type. Of 47 penicillin-susceptible (PS) group 9 isolates that were typed for capsule, only 1 was 9L. Among 29 PS group 9 isolates that were protein typed, 9 protein types were observed. Of the 70 RPR isolates, 51 were protein type P23, 1 was P19, and 18 could not be typed (P0). Protein types P23 differed from P0 by a single epitope on pneumococcal surface protein A. These results suggest that the Canadian P23 and P0 capsular group 9 isolates are likely subclones of a primordial 9L RPR strain.

Antibodies, Monoclonal↗

PubMed1992

Strong association between capsular type and virulence for mice among human isolates of Streptococcus pneumoniae.

The relationship between capsular type and virulence for mice was examined with 69 fresh human isolates of Streptococcus pneumoniae. These isolates represented eight capsular types or groups. Serologic and molecular weight differences in PspA (pneumococcal surface protein A) indicated that the strains were clonally distinct. Mice were infected intravenously with washed bacteria of all 69 isolates in sterile salt solutions. Twenty-eight of the isolates were also injected intraperitoneally to permit comparisons between the intravenous and intraperitoneal routes. With a few exceptions, there was concordance between the ability of strains to cause fatal infections by the two routes. About 30% of the 69 isolates were virulent for mice. The abilities of the isolates to kill mice and the length of time between inoculation and death were strongly associated with capsular type. All type 4 isolates, 40% of type 3 isolates, and 60% of group 6 isolates were virulent for mice; type 1 isolates were marginally virulent; and all type or group 14, 19, and 23 isolates were avirulent. Times to death were generally longer for mice infected with group 6 or type 1 than for those infected with type 3 or 4 pneumococci. There was no relationship between clinical diagnosis or tissue source of the isolates and virulence for mice.

Animals↗

PubMed1992

Mouse antibody to phosphocholine can protect mice from infection with mouse-virulent human isolates of Streptococcus pneumoniae.

Previous studies have demonstrated that mouse antibodies to phosphocholine (PC) can protect mice against fatal infection caused by several, but not all, mouse-virulent laboratory strains of Streptococcus pneumoniae. Because the pneumococcal strains used in previous studies had been mouse passed and were propagated for many years outside of humans, it was not known whether antibody to PC would be able to protect mice against S. pneumoniae freshly isolated from humans. In the present study, we examined the ability of an immunoglobulin G (IgG) monoclonal antibody (MAb) to PC to protect against infections in mice caused by 14 pneumococcal strains of capsular types 3, 4, 6A, and 6B. Nine of these strains were selected as the most virulent strains for mice from a group of 69 fresh clinical isolates. Five were mouse-passed laboratory strains. Mouse IgG3 MAb to PC was able to exhibit protective effects (survival or increased time to death) against infection with virtually all of the strains injected intravenously and against infection with 70% of the strains injected intraperitoneally. The protective effects of antibody to PC appeared to be partially dependent on capsular type. MAb to PC was most effective against capsular type 3 strains and least effective against type 4 strains. With type 3 and type 4 strains, MAb to PC could frequently protect against larger numbers of CFU injected intravenously than intraperitoneally. For capsular type 6A and 6B strains the reverse was true.

Animals↗

PubMed1992

Structural properties and evolutionary relationships of PspA, a surface protein of Streptococcus pneumoniae, as revealed by sequence analysis.

Analysis of the sequence for the gene encoding PspA (pneumococcal surface protein A) of Streptococcus pneumoniae revealed the presence of four distinct domains in the mature protein. The structure of the N-terminal half of PspA was highly consistent with that of an alpha-helical coiled-coil protein. The alpha-helical domain was followed by a proline-rich domain (with two regions in which 18 of 43 and 5 of 11 of the residues are prolines) and a repeat domain consisting of 10 highly conserved 20-amino-acid repeats. A fourth domain consisting of a hydrophobic region too short to serve as a membrane anchor and a poorly charged region followed the repeats and preceded the translation stop codon. The C-terminal region of PspA did not possess features conserved among numerous other surface proteins, suggesting that PspA is attached to the cell by a mechanism unique among known surface proteins of gram-positive bacteria. The repeat domain of PspA was found to have significant homology with C-terminal repeat regions of proteins from Streptococcus mutans, Streptococcus downei, Clostridium difficile, and S. pneumoniae. Comparisons of these regions with respect to functions and homologies suggested that, through evolution, the repeat regions may have lost or gained a mechanism for attachment to the bacterial cell.

Amino Acid Sequence↗

PubMed1992

Truncated forms of PspA that are secreted from Streptococcus pneumoniae and their use in functional studies and cloning of the pspA gene.

Insertion-duplication mutagenesis was used to generate mutants of Streptococcus pneumoniae that produced truncated forms of PspA (pneumococcal surface protein A). The truncated products, representing from 20 to 80% of the complete PspA molecule, were all secreted from the cell and could be detected in unconcentrated culture medium. Analysis of the truncated molecules showed that the antigenic variability known to be associated with PspA is located in the alpha-helical N-terminal half of the molecule. This region was also found to contain immunogenic and protection-eliciting epitopes and to define the maximum region of the molecule that is likely to be surface exposed. The apparent molecular weight variability seen for PspA molecules of different S. pneumoniae strains was localized to both the N- and C-terminal halves of the protein. Attachment of PspA to S. pneumoniae was found to require regions located carboxy to the fifth repeat unit in the C-terminal end of the molecule. From the insertion-duplication mutants, the complete pspA gene was cloned and expressed in Escherichia coli. Differences in apparent molecular weight were observed when the same cloned product was expressed in E. coli and S. pneumoniae, suggesting that PspA is modified differently in the two hosts.

Antibodies, Monoclonal↗

PubMed1991

The Salmonella typhimurium locus mviA regulates virulence in Itys but not Ityr mice: functional mviA results in avirulence; mutant (nonfunctional) mviA results in virulence.

The virulent Salmonella typhimurium strain WB600 carries the mviA allele of the gene mouse virulence A. As shown here, the virulent phenotype of WB600 is the result of a nonfunctional mviA gene. As compared to the functional allele mviA+, mviA increases virulence in Itys mice, but not in Ityr mice. A specific BglII site, mviA4185, between osmZ and galU, located at approximately 35 min on the salmonella chromosome, was within mviA. Insertion of an antibiotic cassette in the mviA4185 site of mviA+ or the homologous mviA4093 site of mviA DNA resulted in virulence when either cassette was recombined into the chromosome. When mviA and mviA+ were both expressed in the same strain with one carried in the chromosome and the other on a plasmid, avirulence was dominant. Replacement of the mviA allele of strain WB600 using P22 transductions of linked antibiotic cassettes cloned into the chromosome of virulent S. typhimurium strains (SR-11, TML, SL1344, C5, ATCC14028, W118-2, and WB600) showed that all but WB600 contained the avirulent mviA+ allele. Southern hybridizations provided no evidence for a second mviA allele anywhere in the genome of the six non-WB600 strains.

Alleles↗

PubMed1991

A 'safe-site' for Salmonella typhimurium is within splenic cells during the early phase of infection in mice.

Salmonella typhimurium infection in mice is focused on the spleen and liver, and prolonged infection can lead to sepsis and death. After intravenous infection with a moderate dose of S. typhimurium, the few bacteria that survive in the spleen and liver grow in a 'safe-site' where they are protected from immune destruction. In this study, we demonstrated that the lack of killing of resident salmonella in the spleen and liver was not because the salmonella were transformed within the host and became resistant to killing, or because the infected mice lost the ability to kill salmonella. We showed that the salmonella were within an intracellular 'safe-site' that protected them from killing. Brief treatment of salmonella-infected mice with gentamicin reduced the numbers of salmonella in the blood but had no effect on the numbers in the liver and spleen, suggesting an intracellular location of the salmonella. After dissociation of spleen cells from recently infected mice, 60% of the salmonella remained cell associated. These cell-associated salmonella, unlike cell-free salmonella, were resistant to killing by gentamicin. The cell-associated salmonella were rendered susceptible to gentamicin after sonication, providing confirmation of their previous intracellular location.

Animals↗

PubMed1991

A hemA mutation renders Salmonella typhimurium avirulent in mice, yet capable of eliciting protection against intravenous infection with S. typhimurium.

The hemA mutation reduces the virulence of Salmonella typhimurium for mice by at least 10(7)-fold, as measured by change in LD50. The hemA mutation does not appear to affect killing of salmonella in mice. The salmonella with the hemA mutation persist in the spleen and liver for 2 to 3 weeks following intravenous injection. The most likely effect of the hemA mutation is to block, or retard, growth of S. typhimurium in an aerobic in vivo environment. Intravenous vaccination of susceptible ltys mice with hemA salmonella was able to elicit about 4 logs of protection against invasive infection with wild-type S. typhimurium 78 days after vaccination, at a time when the vaccine strain was no longer detectable in the spleen and liver.

Aldehyde Oxidoreductases↗

PubMed1991

Epidemiologic studies of group 9 pneumococci in terms of protein type and 9N versus 9V capsular type.

The variability among Streptococcus pneumoniae isolates of capsular group 9 was analyzed with a panel of six monoclonal antibodies to pneumococcal proteins. These antibodies differentiated the 104 group 9 isolates into 18 protein types and a group (PO) not typable with the antibody panel was used. Capsular type was determined for 87 isolates: 70% were capsular type 9V, 28% 9N, and 2% 9A. In terms of protein type, the 9V isolates were four times as diverse as the 9N isolates. Significant associations were observed between the protein type, geographic origin, and year of isolation. Statistically significant associations were also observed between different manifestations of pneumococcal disease and protein type of the capsular type 9V isolates. Thus, even within capsular types, pneumococci can be highly diverse; pneumococcal protein types may be useful in epidemiologic studies to distinguish related strains in the environment.

Adolescent↗

PubMed1991

PspA, a surface protein of Streptococcus pneumoniae, is capable of eliciting protection against pneumococci of more than one capsular type.

Monoclonal antibodies against pneumococcal surface protein A (PspA) have been shown to protect mice from fatal pneumococcal infection. PspA is highly variable serologically, raising the possibility that PspA from one strain might not be able to elicit protective responses against strains which possess serologically different PspA. We have prepared a lambda gt11 library of pneumococcal genomic DNA and identified a clone expressing PspA. The recombinant PspA in this phage lysate elicited protection against pneumococcal infections with three strains of two different capsular serotypes. This finding demonstrated that PspA could elicit a protective response in the absence of other pneumococcal antigens. The observed protection was probably antibody mediated because it could be passively transferred with immune sera. Lambda lysates producing pneumococcal proteins other than PspA failed to elicit protection against fatal pneumococcal infection.

Animals↗

PubMed1991

Mouse hepatitis virus strain UAB infection enhances resistance to Salmonella typhimurium in mice by inducing suppression of bacterial growth.

We have previously shown that intranasal infection of mice with mouse hepatitis virus (MHV) strain UAB (MHV-UAB) increases their resistance to Salmonella typhimurium injected intravenously 6 days later. To study how salmonella resistance was induced, BALB/cAnNCr mice were infected with salmonella strains carrying specific genetic alterations. One set of studies compared the effect of MHV infection on subsequent salmonella infections with AroA- (avirulent) and Aro+ (virulent) salmonellae. Unlike its effect on Aro+ salmonellae, MHV failed to reduce the number of AroA- salmonellae recovered from mice. Because AroA- S. typhimurium shows almost no growth in vivo, this failure indicated that the effect of MHV on salmonella resistance required growth of the infecting salmonellae. In other studies, the effect of MHV infection on both growth and killing were monitored simultaneously in mice with growing salmonellae carrying a single copy of the temperature-sensitive pHSG422 plasmid, which is unable to replicate in vivo. MHV infection reduced salmonella growth but caused no increase in salmonella killing. MHV infection of mice given wild-type salmonellae also resulted in no increase in salmonella killing 4 h after salmonella challenge. These studies demonstrate that MHV-UAB infection increases host resistance to salmonellae by enhancing suppression of bacterial growth instead of by increasing the amount of salmonella killing.

Animals↗

PubMed1991

A 43-kilodalton pneumococcal surface protein, PspA: isolation, protective abilities, and structural analysis of the amino-terminal sequence.

PspA is an antigenically variable surface protein of Streptococcus pneumoniae that appears to be essential for full pneumococcal virulence. In addition, monoclonal antibodies to PspA protect mice against infection with specific strains of pneumococci virulent for mice. In this study, we have isolated the 43-kDa N-terminal half of the native 84-kDa PspA and determined the sequence of the first 45 amino acids. This sequence, the first obtained for a pneumococcal surface protein, is consistent with that of an amphiphatic coiled-coil alpha helix with a 7-residue periodicity common to fibrous proteins such as tropomyosin and streptococcal M protein. The 7-residue periodicity begins with residue 8 and extends throughout the remaining sequence for nearly 11 turns of the helix. Mice immunized with this purified PspA segment were protected from fatal pneumococcal challenge, thus demonstrating that those PspA epitopes eliciting protection were present in the N-terminal half of the molecule.

Amino Acid Sequence↗

PubMed1991

Antigen-independent selection of T15 idiotype during B-cell ontogeny in mice.

Precursors of B cells capable of responding to a T-independent form of phosphorylcholine (PC) in splenic focus assays were detected in the spleens of neonatal mice as early as 4 days after birth. The earliest anti-PC B cells were T15-. T15+ foci-forming B cells were first detected 6 days after birth and expanded rapidly to constitute greater than 80% of the total PC-specific foci by day 10. Injection of heat-killed S. pneumoniae (R36A) into neonatal mice resulted in priming of the antibody response to PC, with an idiotype profile reflecting that of precursors of foci-forming B cells at the time of antigen administration. Priming of 2-day-old mice with 2 x 10(6) and 2 x 10(7) R36A induced a five- and ten-fold increase in the antibody response to phosphorylcholine 6 to 8 weeks later. However, only 10 to 15% of the serum antibodies expressed the normally dominant T15 idiotype. Doses below 2 x 10(5) R36A showed no detectable priming activity. PC-specific hybridomas derived from mice injected with 2 x 10(7) R36A 2 days after birth lacked the idiotypic and molecular characteristics typical of T15+ antibodies. Antibodies to phosphorylcholine, raised by immunization of 6-week-old mice are normally protective against pneumococcal infection. However, serum antibodies from mice treated with R36A 2 days after birth and responding to phosphorylcholine following challenge with R36A at 6 weeks of age failed to protect against deliberate infection with virulent S. pneumoniae. These observations imply that the antigen phosphorylcholine does not play a role in the selective expansion and dominant expression of the T15 idiotype.

Animals↗