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Biomedical subjects

G Dougan

Publications and source records attributed to G Dougan.

At least 145 records · Page 8Linked to original sources
PubMed1996

Intimin from enteropathogenic Escherichia coli restores murine virulence to a Citrobacter rodentium eaeA mutant: induction of an immunoglobulin A response to intimin and EspB.

The formation of attaching and effacing (A/E) lesions is central to the pathogenesis of enteropathogenic Escherichia coli (EPEC)-mediated disease in humans and Citrobacter rodentium (formerly C. freundii biotype 4280)-mediated transmissible colonic hyperplasia in mice. Closely related outer membrane proteins, known as intimins, are required for formation of the A/E lesion by both EPEC (Int(EPEC)) and C. rodentium (Int(CR)). A secreted protein, EspB (formally EaeB), is also necessary for A/E-lesion formation. Here we report that expression of a cloned Int(EPEC), encoded by plasmid pCVD438, restores murine virulence to an intimin-deficient mutant of C. rodentium DBS255. Replacement of Cys937 with Ala abolished the ability of the cloned EPEC intimin to complement the deletion mutation in DBS255. Ultrastructural examination of tissues from wild-type C. rodentium and DBS255(pCVD438)-infected mice revealed multiple A/E lesion on infected cells and loss of contact between enterocytes and basement membrane. Histological investigation showed that although both wild-type C. rodentium and DBS255(pCVD438) colonized the descending colon and induced colonic hyperplasia in orally infected 21-day-old mice, the latter strain adhered to epithelial cells located deeper within crypts. Nonetheless, infection with the wild-type strain was consistently more virulent, as indicated by a higher mortality rate. All the surviving mice, challenged with either wild-type C. rodentium or DBS255(pCVD438), developed a mucosal immunoglobulin A response to intimin and EspB. These results show that C. rodentium infection provides a relevant, simple, and economic model to investigate the role of EPEC proteins in the formation of A/E lesions in vivo and in intestinal disease.

Adhesins, Bacterial↗
PubMed1996

Induction of antigen-specific antibodies in vaginal secretions by using a nontoxic mutant of heat-labile enterotoxin as a mucosal adjuvant.

Immunization of the female reproductive tract is important for protection against sexually transmitted diseases and other pathogens of the reproductive tract. However, intravaginal immunization with soluble antigens generally does not induce high levels of secretory immunoglobulin A (IgA). We recently developed safe mucosal adjuvants by genetically detoxifying Escherichia coli heat-labile enterotoxin, a molecule with a strong mucosal adjuvant activity, and here we describe the use of the nontoxic mutant LTK63 to induce a response in the mouse vagina against ovalbumin (Ova). We compared intravaginal and intranasal routes of immunization for induction of systemic and vaginal responses against LTK63 and Ova. We found that LTK63 is a potent mucosal immunogen when given by either the intravaginal or intranasal route. It induces a strong systemic antibody response and IgG and long-lasting IgA in the vagina. The appearance of vaginal IgA is delayed in the intranasally immunized mice, but the levels of vaginal anti-LTK63 IgA after repeated immunizations are higher in the intranasally immunized mice than in the intravaginally immunized mice. LTK63 also acts as a mucosal adjuvant, inducing a serum response against Ova, when given by both the intravaginal and intranasal routes. However, vaginal IgA against Ova is stimulated more efficiently when LTK63 and antigen are given intranasally. In conclusion, our results demonstrate that LTK63 can be used as a mucosal adjuvant to induce antigen-specific antibodies in vaginal secretions and show that the intranasal route of immunization is the most effective for this purpose.

Animals↗
PubMed1996

Construction and characterization of a Yersinia enterocolitica O:8 high-temperature requirement (htrA) isogenic mutant.

The high-temperature requirement (HtrA) family of stress response proteins are induced by different environmental stress conditions in a variety of bacteria and have been shown to contribute to the pathogenicity of some of these species. In this study, the htrA gene from Yersinia enterocolitica O:8 was amplified, cloned, and sequenced. Analysis of the deduced amino acid sequence predicted that the putative HtrA homolog contains a serine protease active site and a catalytic triad characteristic of trypsin-like serine proteases, structural features characteristic of previously described HtrA proteins. In order to evaluate the biological functions of Y. enterocolitica HtrA, an isogenic mutant was constructed by a reverse-genetics PCR-based approach. Characterization of the mutant provided evidence supporting a stress response function for the Y. enterocolitica htrA gene product. In contrast to the parent strain, the mutant showed increased sensitivity to killing by H2O2, O2- and temperature stress (50 degrees C). The mutant was avirulent in the murine yersiniosis injection model and offered partial protection to mice challenged with the parent strain. Further studies with the Y. enterocolitica htrA mutant should increase our knowledge of the host-pathogen interactions which occur during Yersinia infections.

Amino Acid Sequence↗
PubMed1996

Immune response in mice following immunization with DNA encoding fragment C of tetanus toxin.

Tetanus toxin is a potent neurotoxin synthesized by Clostridium tetani. Immunization with fragment C protein, the nontoxic C-terminal domain of tetanus toxin, will protect mice against lethal challenge with tetanus toxin. A synthetic gene encoding fragment C (tetC) had previously been shown to express high levels of fragment C in Saccharomyces cerevisiae. A plasmid, pcDNA3/tetC, which encodes the synthetic tetC gene expressed under the control of the human cytomegalovirus major intermediate-early promoter/enhancer region, was constructed. Expression of fragment C was observed in eukaryotic cells growing in vitro following transfection with pcDNA3/tetC. The immune response induced by intramuscular immunization with pure pcDNA3/tetC DNA was evaluated in a murine model. Anti-fragment C serum immunoglobulin and proliferative responses in splenocytes were observed in BALB/c mice following two immunizations with pcDNA3/tetC. The major immunoglobulin G subclass that recognized fragment C was immunoglobulin G2a, and the stimulated splenocytes secreted high levels of gamma interferon. Immunity to tetanus is dependent on the presence of neutralizing serum antibodies against tetanus toxin. Sufficient anti-fragment C serum immunoglobulins were induced by DNA-mediated immunization to protect mice against lethal challenge with tetanus toxin.

Animals↗
PubMed1996

Chimeric hepatitis B virus core particles as probes for studying peptide-integrin interactions.

An RGD-containing epitope from the foot-and-mouth disease virus (FMDV) VP1 protein was inserted into the e1 loop of the hepatitis B virus core (HBc) protein. This chimeric protein was expressed at high levels in Escherichia coli and spontaneously assembled into virus-like particles which could be readily purified. These fusion particles elicited high levels of both enzyme-linked immunosorbent assay- and FMDV-neutralizing antibodies in guinea pigs. The chimeric particles bound specifically to cultured eukaryotic cells. Mutant particles carrying the tripeptide sequence RGE in place of RGD and the use of a competitive peptide, GRGDS, confirmed the critical involvement of the RGD sequence in this binding. The chimeric particles also bound to purified integrins, and inhibition by chain-specific anti-integrin monoclonal antibodies implicated alpha 5 beta 1 as a candidate cell receptor for both the chimeric particle and FMDV. Some serotypes of FMDV bound to beta 1 integrins in solid- phase assays, and the chimeric particles competed with FMDV for binding to susceptible eukaryotic cells. Thus, HBc particles may provide a simple, general system for exploring the interactions of specific peptide sequences with cellular receptors.

Animals↗
PubMed1995

Tetanus toxin inhibits neuroexocytosis even when its Zn(2+)-dependent protease activity is removed.

Tetanus toxin (TeTX) is a dichain protein that blocks neuroexocytosis, an action attributed previously to Zn(2+)-dependent proteolysis of synaptobrevin (Sbr) by its light chain (LC). Herein, its cleavage of Sbr in rat cerebrocortical synaptosomes was shown to be minimized by captopril, an inhibitor of certain metalloendoproteases, whereas this agent only marginally antagonized the inhibition of noradrenaline release, implicating a second action of the toxin. This hypothesis was proven by preparing three mutants (H233A, E234A, H237A) of the LC lacking the ability to cleave Sbr and reconstituting them with native heavy chain. The resultant dichains were found to block synaptosomal transmitter release, albeit with lower potency than that made from wild type LC; as expected, captopril attenuated only the inhibition caused by the protease-active wild type toxin. Moreover, these protease-inactive toxins or their LCs blocked evoked quantal release of transmitter when micro-injected inside Aplysia neurons. TeTX was known to stimulate in vitro a Ca(2+)-dependent transglutaminase (TGase) (Facchiano, F., and Luini, A. (1992) J. Biol. Chem. 267, 13267-13271), an affect found here to be reduced by an inhibitor of this enzyme, monodansylcadaverine. Accordingly, treatment of synaptosomes with the latter antagonized the inhibition of noradrenaline release by TeTX while not affecting Sbr cleavage. This drug also attenuated the inhibitory action of all the mutants. Hence, it is concluded that TeTX inhibits neurotransmitter release by proteolysis of Sbr and a protease-independent activation of a neuronal TGase.

Animals↗
PubMed1995

Mutants of Escherichia coli heat-labile toxin lacking ADP-ribosyltransferase activity act as nontoxic, mucosal adjuvants.

A nontoxic mutant (LTK7) of the Escherichia coli heat-labile enterotoxin (LT) lacking ADP-ribosylating activity but retaining holotoxin formation was constructed. By using site-directed mutagenesis, the arginine at position 7 of the A subunit was replaced with lysine. This molecule, which was nontoxic in several assays, was able to bind to eukaryotic cells and acted as a mucosal adjuvant for co-administered proteins; BALB/c mice immunized intranasally with LTK7 and ovalbumin developed high levels of serum and local antibodies to ovalbumin and toxin. In addition, mice immunized intranasally with fragment C of tetanus toxin and LTK7 were protected against lethal challenge with tetanus toxin. Thus nontoxic mutants of heat-labile toxin can act as effective intranasal mucosal adjuvants.

Adjuvants, Immunologic↗
PubMed1995

Expression of LacZ from the htrA, nirB and groE promoters in a Salmonella vaccine strain: influence of growth in mammalian cells.

Attenuated Salmonella strains are currently being evaluated as live vectors for the delivery of heterologous antigens to the mammalian mucosal and systemic immune systems. An approach to improving the stability of heterologous antigen expression during vaccination is to drive expression of the foreign protein from promoters, e.g. nirB, that become activated when Salmonella enter the host. Salmonella strains were constructed that harboured similar multicopy plasmids encoding the lacZ gene. In each strain, lacZ expression was driven from either the nirB, htrA or groE promoters. Expression of LacZ increased in all vaccine strains as they were shifted from conditions of low to high temperature. In addition, expression of lacZ driven from the htrA and nirB promoters significantly increased when the Salmonella entered eukaryotic cells, including macrophages. Expression of lacZ from the groE promoter was significantly elevated in macrophages but not in cells derived from epithelia. These promoters may be useful for optimising heterologous antigen expression within immune cells of the host.

Antigens↗
PubMed1995

Effect of deoxymannojirimycin and Brefeldin A on Yersinia pseudotuberculosis invasin--eukaryotic cell interaction.

The cell surface receptors of the Yersinia pseudotuberculosis invasin protein are the alpha 3-6 beta 1 integrins. Invasin and the extracellular matrix protein fibronectin bind to the same or closely located sites on alpha 5 beta 1 integrin, although invasin bind with a much greater affinity. Invasin-integrin interaction promotes bacterial penetration into eukaryotic cells. Binding of fibronectin to its integrin receptor seems to be dependent on terminal oligosaccharides processing. In this paper, we have examined the effect of 1-deoxymannojirimycin (dMNJ), an inhibitor of Golgi alpha-mannosidases involved in processing of N-glycan precursors, and of Brefeldin A (BFA), a natural product of fungi which has profound effects on the structure and function of the Golgi apparatus, on invasin-cell interaction. We found that unlike fibronectin, the interaction of invasin with cells was resistance to dMNJ. However, preincubating cells with BFA caused a dose dependent inhibition of invasin-mediated cell entry, while cell invasion by Salmonella typhimurium was not affected.

1-Deoxynojirimycin↗
PubMed1995

A method to screen T lymphocyte epitopes after oral immunisation of humans: application to cholera toxin B subunit.

The response to oral immunisation of humans with classical biotype cholera toxin B subunit was studied to identify immunodominant T lymphocyte determinants. The in vitro proliferative response to pools of 12-mer peptides and larger peptides used individually was analysed by a novel statistical approach, and identified an immunodominant region in residues 70-79 in immunised subjects, when either pools or individual peptides were employed. In contrast, a patient infected with El Tor biotype had a dominant response to residues 40-60. The statistical software employed in this study may enable efficient screening of antigens for immunodominant T lymphocyte determinants when blood precursor frequencies are low following immunisation, and may therefore be of special relevance to mucosal vaccines.

Administration, Oral↗
PubMed1995

Mucosal and systemic immunogenicity of a recombinant, non-ADP-ribosylating pertussis toxin: effects of formaldehyde treatment.

The effect of formaldehyde treatment on the mucosal and systemic immunogenicity of the genetically detoxified pertussis toxin (PT-9K/129G) was investigated. Groups of BALB/c were immunized intranasally (i.n.) or subcutaneously (s.c.) with untreated, lightly formaldehyde treated (LFT) or heavily formaldehyde treated (HFT) recombinant pertussis toxin (PT) mutant, PT-9K/129G. Intranasal immunization with native PT-9K/ 129G induced significant levels of anti-toxin antibodies in serum and IgA anti-toxin responses in nasal and lung lavages of these mice. Similar local and systemic responses were observed following intransal immunization with LFT toxin. However, i.n. immunization with HFT toxin failed to induce a local IgA response and elicited a much diminished anti-toxin response in the serum. In contrast, the total antibody response following s.c. immunization was not significantly affected. In addition, i.n. immunization with native PT-9K/129G induced low but detectable levels of toxin neutralizing antibodies in the serum. These results show that native PT-9K/129G protein acts as a mucosal immunogen in mice and that this activity is greatly diminished by HFT of the protein.

Administration, Intranasal↗
PubMed1995

The development of oral vaccines against parasitic diseases utilizing live attenuated Salmonella.

Genetically defined, live attenuated Salmonella vaccines are proving useful both as oral vaccines against salmonellosis and for the development of multivalent vaccines based on the expression of heterologous antigens in such strains. Several candidate attenuated S. typhi strains are at present being evaluated as new single dose oral typhoid vaccines in human volunteers. The emergence of such a vaccine will facilitate the development of multivalent vaccines for humans. Many antigens from different infectious organisms have been expressed in attenuated Salmonella. A focus of this work has been on developing vaccines against parasitic diseases. This review will summarize the efforts that have been made in this area.

Administration, Oral↗
PubMed1995

Molecular characterization of a carboxy-terminal eukaryotic-cell-binding domain of intimin from enteropathogenic Escherichia coli.

A eukaryotic cell-binding domain from the intimin (Int) polypeptide of enteropathogenic Escherichia coli O127 (EPEC) was investigated. Derivatives of the carboxy-terminal 280-amino-acid domains of Int (Int-EPEC280) and the Int homolog invasin (Inv) from Yersinia pseudotuberculosis (InvYP280) were fused to the E. coli maltose-binding protein (MBP), expressed, and purified. The smallest MBP-IntEPEC fusion protein that efficiently mediated binding to HEp-2 cells, monitored by using purified fusion proteins in fluorescence activated cell sorter analysis or by using fluorescent Covaspheres coated with purified fusions, contained the carboxy-terminal 150 amino acids of Int. Replacement of Cys-937 with Ser (IntEPEC280CS) destroyed the cell-binding activity of IntEPEC280. Covaspheres coated with MBP-IntEPEC280 were associated with HEp-2 cell microvilli but failed to induce actin accumulation underneath bound particles or cell spreading on coated plastic surfaces. MBP-IntEPEC280, but not MBP, MBP-IntEPEC280CS, or MBP-InvYP280, inhibited EPEC entry into HEp-2 cells.

Adhesins, Bacterial↗
PubMed1995

Biological and genetic characterization of TnphoA mutants of Salmonella typhimurium TML in the context of gastroenteritis.

TnphoA transposon insertion mutants of phoN-negative derivatives of Salmonella typhimurium TML (of human gastroenteritic origin) were selected by growing mutagenized recipient bacteria under a variety of growth conditions. Ninety-seven individual mutants, which expressed alkaline phosphatase, were collected and tested for their ability to invade HEp-2 cells. Seven smooth mutants had a reduced ability to invade HEp-2 cells, and three smooth mutants were consistently more invasive than their corresponding parental strains. One rough mutant was of similar invasiveness and two were of reduced invasiveness when compared with that of parental strains. The seven smooth hypoinvasive mutants, the three smooth hyperinvasive mutants, and the three rough mutant strains were tested for their abilities to invade ileal enterocytes by the rabbit ileal invasion assay described previously (3). All smooth mutants exhibited parental levels of invasiveness. The rough mutants were hypoinvasive in the rabbit ileal invasion assay. The HEp-2 system is therefore not a good predictor of behavior in gut tissue in this model. DNA sequences flanking the transposon were determined for five mutants which were hypoinvasive in the HEp-2 cell assay. The mutations were found to be insertions in two previously identified invasion genes, invG and invH, and in a gene not normally associated with invasion, pagC. These observations lead one to be cautious in the interpretation of the biological significance of data obtained from invasion of tissue culture monolayers when extrapolated to gut tissue.

Alkaline Phosphatase↗
PubMed1995

A mutant pertussis toxin molecule that lacks ADP-ribosyltransferase activity, PT-9K/129G, is an effective mucosal adjuvant for intranasally delivered proteins.

We examined the capacity of a genetically detoxified derivative of pertussis toxin (PTX), PT-9K/129G, to act as a mucosal adjuvant for an intranasally (i.n.) administered tetanus vaccine. Groups of mice were immunized i.n. with the nontoxic C-terminal 50-kDa portion of tetanus toxin (fragment C [Frg C]) either alone or mixed with PT-9K/129G, PTX, or cholera toxin (CT) or were immunized subcutaneously (s.c.) with an equivalent amount of Frg C adsorbed to alhydrogel. In response to a single immunization, mice receiving Frg C plus PT-9K/129G or CT i.n. and parenterally immunized mice developed high-titer (> 20,000) anti-Frg C antibodies, whereas mice immunized i.n. with Frg C plus PTX or with Frg C alone seroconverted only after being boosted. The serum anti-Frg C response was dominated by immunoglobulin G1 (IgG1) in mice immunized with Frg C plus PT-9K/129G, with Frg C plus PTX, or s.c. In contrast, IgG1, IgG2a, and IgG2b contributed almost equally to the Frg C response when CT was the adjuvant. Anti-Frg C IgE was detected only in the sera of mice immunized i.n. with Frg C plus PTX and immunized s.c. with Frg C plus alhydrogel. High levels of IgA antibodies were present in nasal lavage fluid from mice immunized i.n. with Frg C plus PT-9K/129G, PTX, or CT but not in that from mice given Frg C alone i.n. or parenterally. The mucosal adjuvanticity of PT-9K/129G was manifested in inbred as well as outbred mice. A single i.n. dose of Frg C plus either PT-9K/129G or PTX (with high specific activity) was sufficient to protect all immunized mice from tetanus toxin challenge, in contrast to the case for mice that received Frg C alone i.n. We conclude that the pertussis toxin analog PT-9K/129G, which is devoid of ADP-ribosyltransferase activity, is a potent mucosal adjuvant for vaccines delivered via the respiratory tract.

Adjuvants, Immunologic↗