Early interactions of Salmonella enterica serovar typhimurium with human small intestinal epithelial explants.

BACKGROUND: Salmonella enterica serovar typhimurium (S typhimurium) causes invasive gastroenteritis in humans, a disease involving significant penetration of the intestinal mucosa. However, few studies have been undertaken to investigate this interaction directly using differentiated human gut tissue. AIMS: To investigate the early interactions of an enteropathogenic strain of S typhimurium with human intestinal mucosa using human intestinal in vitro organ culture (IVOC). METHODS: Wild-type and mutant derivatives of S typhimurium TML were used to compare interactions with cultured human epithelial cells, bovine ligated loops, and human intestinal IVOC. RESULTS: S typhimurium TML was shown to attach to cultured Caco-2 brush border expressing cells and cause tissue damage and fluid accumulation in a ligated bovine loop model.S typhimurium TML bound predominantly to the mucus layer of human IVOC explants during the first four hours of IVOC incubation. From four to eight hours of IVOC incubation, small but characteristic foci of attaching and invading S typhimurium TML were detected as clusters of bacteria interacting with enterocytes, although there was no evidence for large scale invasion of explant tissues. Ruffling of enterocyte membranes associated with adherent Salmonella was visualised using electron microscopy. CONCLUSIONS: Human IVOC can be used as an alternative model for monitoring the interactions between S typhimurium and human intestinal epithelium, thus potentially offering insight into the early stages of human Salmonella induced gastroenteritis.

A vaccination strategy incorporating DNA priming and mucosal boosting using tetanus toxin fragment C (TetC).

Intramuscular (i.m.) immunisation of BALB/c mice with a DNA vaccine, pcDNA3/tetC, encoding fragment C (TetC) from tetanus toxin, stimulated production of TetC specific IgG2a antibodies in the serum and release of IFN-gamma from TetC stimulated splenocytes. A similar pattern of immune response was detected if pcDNA3/tetC primed mice were boosted i.m. with purified TetC protein or TetC and cholera toxin (included as an adjuvant). In contrast, control mice primed with the empty DNA vector pcDNA3 and boosted i.m. with TetC or TetC and CT, generated a dominant IgG1 specific anti-TetC response in the sera and low or undetectable levels of IFN-gamma from stimulated splenocytes. Thus, i.m. priming with a DNA vaccine modulated the subsequent immune response to the same antigen administered as a protein boost. Similar observations were made when DNA primed mice were boosted using the intranasal mucosal route of immunisation. Interestingly, although mice immunised with pcDNA3/tetC and boosted mucosally with TetC and CT produced anti-TetC IgA in mucosal secretions, the titres were reproducibly lower than those detected in mice immunised with the pcDNA3 vector alone. The immunomodulatory effect of pcDNA3/tetC appeared to be antigen specific as mucosal boosting with an unrelated antigen (pertactin) revealed no significant modulation in terms of the anti-pertactin immune response.

DNA topology and adaptation of Salmonella typhimurium to an intracellular environment.

The expression of genes coding for determinants of DNA topology in the facultative intracellular pathogen Salmonella typhimurium was studied during adaptation by the bacteria to the intracellular environment of J774A.1 macrophage-like cells. A reporter plasmid was used to monitor changes in DNA supercoiling during intracellular growth. Induction of the dps and spv genes, previously shown to be induced in the macrophage, was detected, as was expression of genes coding for DNA gyrase, integration host factor and the nucleoid-associated protein H-NS. The topA gene, coding for the DNA relaxing enzyme topoisomerase I, was not induced. Reporter plasmid data showed that bacterial DNA became relaxed following uptake of S. typhimurium cells by the macrophage. These data indicate that DNA topology in S. typhimurium undergoes significant changes during adaptation to the intracellular environment. A model describing how this process may operate is discussed.

The virulence plasmid of Salmonella typhimurium contains an autoregulated gene, rlgA, that codes for a resolvase-like DNA binding protein.

The virulence plasmid of Salmonella typhimurium contains a gene, rlgA, that shows strong homology to several reported resolvase-like proteins. This gene maps 5 kb upstream of spv locus, the major virulence determinant on the plasmid. Regulation of rlgA was studied using a lacZ transcriptional reporter fusion. The rlgA gene was found to be repressed at the level of transcription by its own product and to be expressed maximally in the late exponential phase of growth. The transcription start site of the rlgA gene was determined and the RlgA binding site was mapped and found to overlap with the transcription initiation signals. A derivative of the virulence plasmid was constructed with a knockout mutation in rlgA. This mutation did not alter the stability of the virulence plasmid nor did it affect the ability of S. typhimurium to cause systemic disease in mice.

Salmonella enterica serovar typhimurium surA mutants are attenuated and effective live oral vaccines.

A previously described attenuated TnphoA mutant (BRD441) of Salmonella enterica serovar Typhimurium C5 (I. Miller, D. Maskell, C. Hormaeche, K. Johnson, D. Pickard, and G. Dougan, Infect. Immun. 57:2758-2763, 1989) was characterized, and the transposon was shown to be inserted in surA, a gene which encodes a peptidylprolyl-cis, trans-isomerase. A defined surA deletion mutation was introduced into S. enterica serovar Typhimurium C5 and the mutant strain, named S. enterica serovar Typhimurium BRD1115, was extensively characterized both in vitro and in vivo. S. enterica serovar Typhimurium BRD1115 was found to be defective in the ability to adhere to and invade eukaryotic cells. Furthermore, S. enterica serovar Typhimurium BRD1115 was attenuated by at least 3 log units when administered orally or intravenously to BALB/c mice. Complementation of the mutation with a plasmid carrying the intact surA gene almost completely restored the virulence of BRD1115. In addition, S. enterica serovar Typhimurium BRD1115 demonstrated potential as a vaccine candidate, since mice immunized with BRD1115 were protected against subsequent challenge with S. enterica serovar Typhimurium C5. S. enterica serovar Typhimurium BRD1115 also showed potential as a vehicle for the effective delivery of heterologous antigens, such as the nontoxic, protective fragment C domain of tetanus toxin, to the murine immune system.

Use of the stationary phase inducible promoters, spv and dps, to drive heterologous antigen expression in Salmonella vaccine strains.

We have investigated the ability of the growth phase regulated promoters dps and spv, to drive expression of heterologous antigens in Salmonella vaccine strains. Reporter plasmids were constructed which directed beta-galactosidase expression from dps (pDpslacZ) or spv (pSpvlacZ) and these were introduced independently into the Salmonella typhimurium vaccine strain SL3261 (aroA(-)). beta-galactosidase expression was induced 20-fold and 100-fold when broth cultures of SL3261 (pDpslacZ) or SL3261 (pSpvlacZ) respectively, entered the stationary phase of growth. Within macrophages, beta-galactosidase expression was induced 3.5-fold with SL3261 (pDpslacZ) and 7-fold with SL3261 (pSpvlacZ). The spv and dps promoters were used to drive independent expression of the C fragment domain of tetanus toxin (TetC) from plasmids harboured in S. typhimurium SL3261. Levels of anti-TetC antibodies were significantly higher in the sera of BALB/c mice perorally inoculated with SL3261 (pSpvtetC) or SL3261 (pDpstetC) compared to unvaccinated controls. This suggests that these promoter systems may be used to drive foreign antigen expression in live oral Salmonella vaccines.

At least four percent of the Salmonella typhimurium genome is required for fatal infection of mice.

Salmonella typhimurium infection of mice is an established model system for studying typhoid fever in humans. Using this model, we identified S. typhimurium genes which are absolutely required to cause fatal murine infection by testing independently derived transposon insertion mutants for loss of virulence in vivo. Of the 330 mutants tested intraperitoneally and the 197 mutants tested intragastrically, 12 mutants with 50% lethal doses greater than 1, 000 times that of the parental strain were identified. These attenuated mutants were characterized by in vitro assays which correlate with known virulence functions. In addition, the corresponding transposon insertions were mapped within the S. typhimurium genome and the nucleotide sequence of the transposon-flanking DNA was obtained. Salmonella spp. and related bacteria were probed with flanking DNA for the presence of these genes. All 12 attenuated mutants had insertions in known genes, although the attenuating effects of only two of these were previously described. Furthermore, the proportion of attenuated mutants obtained in this study suggests that mutations in about 4% of the Salmonella genome lead to 1,000-fold or greater attenuation in the mouse typhoid model of infection. Most of these genes appear to be required during the early stages of a natural infection.

Clinical and functional investigation of 10 missense mutations and a novel frameshift insertion mutation of the gene for copper-zinc superoxide dismutase in UK families with amyotrophic lateral sclerosis.

Mutations of the gene SOD-1, which encodes the enzyme copper-zinc superoxide dismutase, occur in patients with a familial form of amyotrophic lateral sclerosis (ALS). We investigated 71 families with more than one individual affected by ALS for clinical features and SOD-1 mutations. Mutations were identified in 14 families, indicating the presence of SOD-1 mutations in around 20% of this population. There were 10 different heterozygote missense point mutations in eight different codons, and a novel two-base frameshift insertion (132insTT), which leads to substitution of aspartic acid for glutamic acid at codon 132, and a premature stop codon at 133, with predicted truncation of the protein. SOD enzyme activity was reduced to around 50% of normal in individuals with SOD-1 mutations, and may be a useful predictor for the presence of these mutations. A predilection for disease onset in the lower limbs appears to be a distinguishing feature of familial ALS with SOD-1 mutations, and accords with findings in transgenic mouse models. In general, the finding of an SOD-1 mutation does not accurately predict a prognosis or disease severity.

The pef fimbrial operon of Salmonella typhimurium mediates adhesion to murine small intestine and is necessary for fluid accumulation in the infant mouse.

We investigated the role of the pef operon, containing the genes for plasmid-encoded (PE) fimbriae of Salmonella typhimurium, in adhesion to the murine small intestine. In an organ culture model, a mutant of S. typhimurium carrying a tetracycline resistance cassette inserted in pefC was found to be associated in lower numbers with murine small intestine than the wild-type. Similarly, heterologous expression of PE fimbriae in Escherichia coli increased the bacterial numbers recovered from the intestine in the organ culture model. Adhesion to villous intestine mediated by PE fimbriae was further demonstrated by binding of an E. coli strain expressing PE fimbriae to thin sections of mouse small intestine. The contribution of pef-mediated adhesion on fluid accumulation was investigated in infant mice. Intragastric injection of S. typhimurium 14028 and SR-11 caused fluid accumulation in infant mice. In contrast, pefC mutants of S. typhimurium 14028 and SR-11 were negative in the infant mouse assay. Introduction of a plasmid containing pefBACD and orf5, the first five genes of the pef operon, into the pefC mutant complemented for fluid accumulation in the infant mouse assay. However, heterologous expression of PE fimbriae in E. coli did not result in fluid accumulation in the infant mouse, suggesting that factors other than fimbriae are involved in causing fluid accumulation.

A cytolysin encoded by Salmonella is required for survival within macrophages.

A Salmonella gene encoding a cytolysin has been identified by screening for hemolysis on blood agar. DNA sequence analyses together with genetic mapping in Salmonella suggest that it is unrelated to other toxins or hemolysins. The gene (slyA) is present in every strain of Salmonella examined, in Shigella, and in enteroinvasive Escherichia coli but not in other Enterobacteriaceae. SlyA (salmolysin) purified from a derivative of the original clone has hemolytic and cytolytic activity and has a molecular weight predicted by the DNA sequence. The median lethal dose and infection kinetics in mice suggest that the toxin is required for virulence and facilitates Salmonella survival within mouse peritoneal macrophages.

Getting there. Update on recommendations by the Commission on Education of the Deaf.

Major progress on the recommendations of the Commission on Education of the Deaf was made by the Education of the Deaf Act Amendments of 1992 (PL 102-421); the Rehabilitation Act Amendments of 1992 (PL 102-569); and a U.S. Department of Education "Notice of Policy Guidance" clarifying its views on the commission's recommendations about elementary and secondary education. The changes, taken together, brought implementation of commission recommendations almost to closure. They also represented significant steps forward on behalf of deaf children, youth, and adults, setting the stage for further progress.

Alterations of the LPS determine virulence of Neisseria gonorrhoeae in guinea-pig subcutaneous chambers.

The virulence of lipopolysaccharide (LPS) variants of Neisseria gonorrhoeae strain Gc40 was studied in vivo using the guinea-pig subcutaneous chamber model. Survival of variants D1, D2, D4 and D5 was assessed by viable counts made on chamber fluid at various times after inoculation. Chemotactic effect was measured by counts of white cells in the chambers. Differential cell counts and assessments of the location of the gonococci were made on Giemsa-stained smears of chamber fluid. Sensitivity of the variants to normal guinea-pig serum was determined by in vitro bactericidal assays. D1 and D5 had relatively high Mr LPS which was shed in the medium, were serum resistant, produced intense infections and were mainly extracellular. Large number of damaged white cells were present. D2 and D4, had low Mr LPS which was poorly shed in the medium, were serum sensitive and produced low grade infections. D2 was the least infective and was seen mainly inside neutrophils. Collectively the data indicates that the type of LPS on the gonococcal surface and possibly the amount of shed LPS strongly influence the fate of gonococci in vivo, in an environment in which antibodies, complement and phagocytic cells are freely available. This may be decisive at some stages of the human infection.

National survey on telephone services and products. The views of deaf and hard-of-hearing people.

A national study of 128 deaf and hard-of-hearing adults aged 18 to 70 years found that they are willing to pay as much as $15 per month for new telephone-based information services, in part because they are frustrated with what they regard as inadequate access to the nation's telecommunications network. The services they most want are "enhanced 911," fire, police, and other life and safety services. A majority wants the telecommunications network equipped with speech recognition capabilities as soon as the state-of-the-art permits, so that they can gain full access to network-based services. These findings suggest that changes in the national telecommunications policy might enhance the lives of deaf and hard-of-hearing adults. Policy changes might also permit them, as well as younger deaf Americans, to benefit more from the recently enacted Americans with Disabilities Act and to benefit more from other rights and services promoting access to work, education, and independent living.

Yersinia enterocolitica aroA mutants as carriers of the B subunit of the Escherichia coli heat-labile enterotoxin to the murine immune system.

Plasmid p5F which directs the expression of the Escherichia coli heat-labile enterotoxin B subunit (LT-B) from the ptac promoter was introduced into the attenuated Yersinia enterocolitica O:8 aroA mutant strain YAM.1. YAM.1 (p5F) expressed high levels of cell-associated and secreted LT-B in a stable fashion when grown on normal laboratory medium. The strain was used as a live oral vaccine in BALB/c mice and vaccinated mice developed high levels of gut-associated and systemic antibodies to both LT-B and the lipopolysaccharide (LPS) of the vaccine strain. Anti-LT-B and anti-LPS responses in the sera were predominantly of the IgG class whereas gut-associated antibodies were predominantly IgA. ELISPOT assays carried out on selected tissues prepared from vaccinated mice showed significant numbers of cells synthesising IgG and IgA antibodies to LT-B. These results show that Y. enterocolitica aroA mutants can be used effectively as carriers of heterologous antigens to the murine immune system.

Disabled and elderly people in the First, Second and Third Worlds.

While disabilities occur in all societies, their causes and effects differ in the First (developed), Second (communist) and Third (developing) 'worlds'. Of the 500 million persons with disabilities, about 80% live in the Third World, and United Nations projections suggest that the proportion soon will be 90%. Two values, integration and normalization, may guide development of national policies in all three worlds. In addition, two design principles, accessibility and adaptability, may prove useful. It may also be that information-age technology will assist in disability prevention and rehabilitation, as well as in employment of individuals with disabilities. First World economics facilitate application of technology to meet special needs, Second World economies impede that, and Third World economies often cannot support it. The challenge for policy makers is to find ways of bringing together integration, normalization, accessibility and adaptability so as to help nations to fashion cost-effective solutions to their disability-related problems.

Virulence, persistence, and immunogenicity of Yersinia enterocolitica O:8 aroA mutants.

The virulent Yersinia enterocolitica strain 8081 killed BALB/c mice within 5 days of oral infection with a 50% lethal dose of log10 7.1, whereas an aroA mutant of 8081, YAM.1, and the plasmidless variant 8081c failed to kill mice. Unlike 8081, YAM.1 and 8081c did not persist or grow in the Peyer's patches, mesenteric lymph nodes, livers, or spleens of mice. Mice immunized orally with single doses of live YAM.1 were poorly protected against a lethal 8081 challenge, whereas mice immunized with three doses of YAM.1 were moderately well protected.