Biomedical subjects

L de Haan

Publications and source records attributed to L de Haan.

29 records · Page 2Linked to original sources

PubMed1998

Mutational analysis of the role of ADP-ribosylation activity and GM1-binding activity in the adjuvant properties of the Escherichia coli heat-labile enterotoxin towards intranasally administered keyhole limpet hemocyanin.

The Escherichia coli heat-labile enterotoxin (LT) is known for its potent mucosal immunoadjuvant activity towards co-administered antigens. LT is composed of one A subunit, which has ADP-ribosylation activity, and a homopentameric B subunit, which has high affinity for the toxin receptor, ganglioside GM1. In previous studies, we have investigated the role of the LTA and LTB subunits in the adjuvanticity of LT towards influenza virus hemagglutinin (HA), administered intranasally to mice. We now studied the adjuvant properties of LT and LT variants towards keyhole limpet hemocyanin (KLH), which, in contrast to HA, does not bind specifically to mucosal surfaces. It is demonstrated that LT mutants without ADP-ribosylation activity, as well as LTB, retain mucosal immunoadjuvant activity when administered intranasally to mice in conjunction with KLH. As with influenza HA, adjuvanticity of LTB required GM1-binding activity, whereas GM1-binding was not essential for adjuvant activity of LT. Furthermore, we found that also recombinant LTA alone acts as a potent mucosal adjuvant, and that this adjuvanticity is independent of ADP-ribosylation activity. It is concluded that binding of the antigen to mucosal surfaces does not play an essential role in the immunostimulation by LT and LT variants, and that both recombinant LTA and LTB represent powerful nontoxic mucosal adjuvants.

Adenosine Diphosphate Ribose↗

PubMed1998

Mucosal immunoadjuvant activity of recombinant Escherichia coli heat-labile enterotoxin and its B subunit: induction of systemic IgG and secretory IgA responses in mice by intranasal immunization with influenza virus surface antigen.

The Escherichia coli heat-labile enterotoxin (LT) is a very potent mucosal immunogen. LT also has strong adjuvant activity towards coadministered unrelated antigens and is therefore of potential interest for development of mucosal vaccines. However, despite the great demand for such mucosal vaccines, the use of LT holotoxin as an adjuvant is essentially precluded by its toxicity. LT is composed of an A subunit, carrying the toxic ADP-ribosylation activity, and a pentamer of identical B subunits, which mediates binding to ganglioside GM1, the cellular receptor for the toxin. In this paper, we demonstrate that recombinant enzymatically inactive variants of LT, including the LTB pentamer by itself, retain the immunoadjuvant activity of LT holotoxin in a murine influenza model. Mice were immunized intranasally (i.n.) with influenza virus subunit antigen, consisting mostly of the isolated surface glycoprotein hemagglutinin (HA), supplemented with either recombinant LTB (rLTB), a nontoxic LT mutant (E112K, with a Glu112-->Lys substitution in the A subunit), or LT holotoxin, and the induction of systemic IgG and local S-IgA responses was evaluated by direct enzyme-linked immunosorbent assay (ELISA). Immunization with subunit antigen alone resulted in a poor systemic IgG response and no detectable S-IgA. However, supplementation of the antigen with E112K or rLTB resulted in a substantial stimulation of the serum IgG level and in induction of a strong S-IgA response in the nasal cavity. The adjuvant activity of E112K or rLTB under these conditions was essentially the same as that of the LT holotoxin. The present results demonstrate that nontoxic variants of LT, rLTB in particular, represent promising immunoadjuvants for potential application in an i.n. influenza virus subunit vaccine. Nontoxic LT variants may also be used in i.n. vaccine formulations directed against other mucosal pathogens. In this respect, it is of interest that LT(B)-stimulated antibody responses after i.n. immunization were also observed at distant mucosal sites, including the urogenital system. This, in principle, opens the possibility to develop i.n. vaccines against sexually transmitted infectious diseases.

Administration, Intranasal↗

PubMed1998

Role of GM1 binding in the mucosal immunogenicity and adjuvant activity of the Escherichia coli heat-labile enterotoxin and its B subunit.

Escherichia coli (E. coli) heat-labile toxin (LT) is a potent mucosal immunogen and immunoadjuvant towards co-administered antigens. LT is composed of one copy of the A subunit, which has ADP-ribosylation activity, and a homopentamer of B subunits, which has affinity for the toxin receptor, the ganglioside GM1. Both the ADP-ribosylation activity of LTA and GM1 binding of LTB have been proposed to be involved in immune stimulation. We investigated the roles of these activities in the immunogenicity of recombinant LT or LTB upon intranasal immunization of mice using LT/LTB mutants, lacking either ADP-ribosylation activity, GM1-binding affinity, or both. Likewise, the adjuvant properties of these LT/LTB variants towards influenza virus subunit antigen were investigated. With respect to the immunogenicity of LT and LTB, we found that GM1-binding activity is essential for effective induction of anti-LTB antibodies. On the other hand, an LT mutant lacking ADP-ribosylation activity retained the immunogenic properties of the native toxin, indicating that ADP ribosylation is not critically involved. Whereas adjuvanticity of LTB was found to be directly related to GM1-binding activity, adjuvanticity of LT was found to be independent of GM1-binding affinity. Moreover, a mutant lacking both GM1-binding and ADP-ribosylation activity, also retained adjuvanticity. These results demonstrate that neither ADP-ribosylation activity nor GM1 binding are essential for adjuvanticity of LT, and suggest an ADP-ribosylation-independent adjuvant effect of the A subunit.

Adenosine Diphosphate Ribose↗

PubMed1998

The role of ADP-ribosylation and G(M1)-binding activity in the mucosal immunogenicity and adjuvanticity of the Escherichia coli heat-labile enterotoxin and Vibrio cholerae cholera toxin.

The mucosal route of vaccination has attracted a great deal of attention recently. Not only is mucosal application of vaccines, for example, orally or intranasally, particularly convenient, it also offers the possibility to induce locally produced and secreted S-IgA antibodies in addition to systemic IgG antibodies. These IgA antibodies are known to play a key role in protection against pathogens that invade the host through mucosal surfaces. Induction of such responses is not readily achieved by currently used vaccination strategies, which generally involve intramuscular or subcutaneous injection with inactivated pathogens or antigens thereof. For the induction of a mucosal immune response, the vaccine needs to be applied locally. However, local vaccination with non-replicating antigens is usually ineffective and may result in tolerance unless a mucosal immunoadjuvant is included. The most potent mucosal immunoadjuvants known to date are probably cholera toxin (CT) and the closely related Escherichia coli heat-labile enterotoxin (LT). Although CT and LT have become standard adjuvants for experimental mucosal vaccines, the intrinsic toxicity has thus far precluded their use as adjuvants for human vaccine formulations. In the present review, the mucosal immunogenic and adjuvant properties of LT and CT are described, with special emphasis on the functional role of the individual subunits on their immune-stimulatory properties.

Adenosine Diphosphate Ribose↗

PubMed1998

Early detection and intervention in schizophrenia.

During the course of schizophrenia, symptoms tend to increase at the highest rate during the first 5 years of the disease. Moreover, 10% of suicides by schizophrenic patients occur within the first 10 years of schizophrenia being diagnosed. These facts emphasize the importance of early intervention to improve the course of the disease before further deterioration. The use of psychosocial interventions and drug management programmes, in addition to maintenance antipsychotic medication, reduces the risk of psychotic relapse. Continuity of care from inpatient to outpatient treatment also significantly improves outcome, largely as a result of better drug compliance. It appears, however, that the addition of a behavioural family intervention alone to a standard programme offers little additional benefit. The benefits of intervention programmes last only as long as the programme, and patients should continue with such intensive treatment strategies for at least the duration of the critical phase. Under these circumstances, very mild psychotic complaints may be recognized at an early stage so that treatment can begin even earlier, further increasing the chance of an optimal long-term outcome. Further studies of early intervention and relapse prevention are required to support these findings.

Antipsychotic Agents↗

PubMed1996

Mucosal immunogenicity of the Escherichia coli heat-labile enterotoxin: role of the A subunit.

The Escherichia coli heat-labile enterotoxin (LT) is a potent mucosal immunogen, inducing high secretory as well as systemic antibody responses upon oral or intranasal (i.n.) administration. In addition, LT has the capacity to act as an adjuvant in antibody responses against coadministered other antigens. To investigate the role of the individual subunits of LT in the mucosal immunogenicity and adjuvanticity of LT, the LT holotoxin and the non-toxic B subunit (LTB) were cloned separately and purified from overproducing E. coli cultures. Mice were immunized i.n. with the recombinant LT, LTB and combinations of the two and the induction of LTB-specific serum IgG and IgA as well as mucosal S-IgA was monitored. Intranasal administration of 2 micrograms LTB by itself induced a moderate systemic and a low mucosal antibody response, the latter being restricted to the site of immunization. However, addition of a trace amount (50 ng) of LT holotoxin to LTB strongly stimulated both serum antibody and mucosal S-IgA responses to LTB: the antibody levels induced by 2 micrograms LTB supplemented with 50 ng LT were similar to those seen after immunization with 2.9 micrograms of the LT holotoxin alone (representing an amount of 2 micrograms LTB). Furthermore, immunization with LT-supplemented LTB or with LT holotoxin alone, but not immunization with LTB alone, induced an S-IgA response in distant mucosal tissues including the lung, intestine and the urogenital system. Nicking of the LTA chain with trypsin did not enhance the immunogenicity of LT. These results indicate that, although the LTA chain plays an important role in the mucosal immunogenicity of LT including priming of the common mucosal immune system, extremely low amounts of the LT holotoxin suffice for the induction of high antibody responses to LTB, the trace LT and LTB acting in a synergistic fashion.

Administration, Intranasal↗

PubMed1996

Protein engineering studies of A-chain loop 47-56 of Escherichia coli heat-labile enterotoxin point to a prominent role of this loop for cytotoxicity.

Heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, is a close relative of cholera toxin (CT). These two toxins share approximately 80% sequence identity, and consists of one 240-residue A chain and five 103-residue B subunits. The B pentamer is responsible for GM1 receptor recognition, whereas the A subunit carries out an ADP-ribosylation of an arginine residue in the G protein, Gs alpha, in the epithelial target cell. This paper explores the importance of specific amino acids in loop 47-56 of the A subunit. This loop was observed to be highly mobile in the inactive R7K mutant of the A subunit. The position of the loop in wild-type protein is such that it might require considerable reorganization during substrate binding and is likely to have a crucial role in substrate binding. Five single-site substitutions have been made in the LT-A subunit 47-56 loop to investigate its possible role in the enzymatic activity and toxicity of LT and CT. The wild-type residues Thr-50 and Val-53 were replaced either by a glycine or by a proline. The glycine substitutions were intended to increase the mobility of this active-site loop, and the proline substitutions were intended to decrease the mobility of this same loop by restricting the accessible conformational space. Under the hypothesis that mobility of the loop is important for catalysis, the glycine-substitution mutants T50G and V53G would be expected to exhibit activity equal to or greater than that of the wild-type A subunit, while the proline substitution mutants T50P and T53P would be less active. Cytotoxicity assays showed, however, that all four of these mutants were considerably less active than wild-type LT. These results lend support for assignment of a prominent role to loop 47-56 in catalysis by LT and CT.

Animals↗

PubMed1996

Mutants of the Escherichia coli heat-labile enterotoxin with reduced ADP-ribosylation activity or no activity retain the immunogenic properties of the native holotoxin.

The Escherichia coli heat-labile enterotoxin (LT) is a potent inducer of mucosal immune responses. In a previous study (L. DeHaan, W. R. Verweij, M. Holtrop, E. Agsteribbe, and J. Wilschut, Vaccine 14:620-626, 1996), we have shown that efficient induction of an LTB-specific mucosal immune response by LT requires the presence of the LTA chain, suggesting a possible role of the enzymatic activity of LTA in the induction of these responses. In the present study, we generated LT mutants with altered ADP-ribosylation activities and evaluated their immunogenicity upon intranasal administration to mice. The results demonstrate that the mucosal immunogenicity of LT is not dependent on its ADP-ribosylation activity.

Adenosine Diphosphate↗

PubMed1994

Site-directed mutagenesis of proposed active-site residues of penicillin-binding protein 5 from Escherichia coli.

Alignment of the amino acid sequence of penicillin-binding protein 5 (PBP5) with the sequences of other members of the family of active-site-serine penicillin-interacting enzymes predicted the residues playing a role in the catalytic mechanism of PBP5. Apart from the active-site (Ser44), Lys47, Ser110-Gly-Asn, Asp175 and Lys213-Thr-Gly were identified as the residues making up the conserved boxes of this protein family. To determine the role of these residues, they were replaced using site-directed mutagenesis. The mutant proteins were assayed for their penicillin-binding capacity and DD-carboxypeptidase activity. The Ser44Cys and the Ser44Gly mutants showed a complete loss of both penicillin-binding capacity and DD-carboxypeptidase activity. The Lys47Arg mutant also lost its DD-carboxypeptidase activity but was able to bind and hydrolyse penicillin, albeit at a considerably reduced rate. Mutants in the Ser110-Gly-Asn fingerprint were affected in both acylation and deacylation upon reaction with penicillin and lost their DD-carboxypeptidase activity with the exception of Asn112Ser and Asn112Thr. The Asp175Asn mutant showed wild-type penicillin-binding but a complete loss of DD-carboxypeptidase activity. Mutants of Lys213 lost both penicillin-binding and DD-carboxypeptidase activity except for Lys213His, which still bound penicillin with a k+2/K' of 0.2% of the wild-type value. Mutation of His216 and Thr217 also had a strong effect on DD-carboxypeptidase activity. Thr217Ser and Thr217Ala showed augmented hydrolysis rates for the penicillin acyl-enzyme. This study reveals the residues in the conserved fingerprints to be very important for both DD-carboxypeptidase activity and penicillin-binding, and confirms them to play crucial roles in catalysis.

Acylation↗

PubMed1993

Domain organization of penicillin-binding protein 5 from Escherichia coli analysed by C-terminal truncation.

The structural organization of penicillin-binding protein (PBP) 5 was investigated by C-terminal truncation. Compared with other low-M(r) penicillin-interacting proteins, PBP5 carries a C-terminal extension of about 100 amino acids. The sites for introduction of stop codons were chosen on the basis of the established three-dimensional structure of the Streptomyces albus G beta-lactamase [Dideberg, Charlier, Wéry, Dehottay, Dusart, Erpicum, Frère and Ghuysen (1987) Biochem. J. 245, 911-913] and comparative hydrophobic cluster analysis [Gaboriaud, Bissery, Bencheritt and Mornon (1987) FEBS Lett. 224, 149-155]. Two stop codons were introduced at positions Ile-354 or Val-348 to construct an optimized soluble form of PBP5 for crystallization purposes. The newly constructed soluble and enzymically active form (PBP5s353) was isolated by dye-affinity chromatography and gave rise to small crystals. Another two stop codons were introduced at positions Arg-261 or Ala-276 to determine the minimal enzymically active 'core protein'. The truncated form (PBP5s275), missing the entire C-terminal extension, showed unaltered penicillin-binding characteristics and a catalytic-centre activity 40% that of PBP5s353 + 9 using bisacetyl-L-Lys-D-Ala-D-Ala as substrate. This protein, however was more susceptible to proteolytic degradation, which might indicate a role of the C-terminal portion in stabilizing the protein.

Amino Acid Sequence↗

PubMed1992

Possible role of Escherichia coli penicillin-binding protein 6 in stabilization of stationary-phase peptidoglycan.

Plasmids for high-level expression of penicillin-binding protein 6 (PBP6) were constructed, giving rise to overproduction of PBP6 under the control of the lambda pR promoter in either the periplasmic or the cytoplasmic space. In contrast to penicillin-binding protein 5 (PBP5), the presence of high amounts of PBP6 in the periplasm as well as in the cytoplasm did not result in growth as spherical cells or in lysis. Deletion of the C-terminal membrane anchor of PBP6 resulted in a soluble form of the protein (PBP6s350). Electron micrographs of thin sections of cells overexpressing both native membrane-bound and soluble PBP6 in the periplasm revealed a polar retraction of the cytoplasmic membrane. Cytoplasmic overexpression of native PBP6 gave rise to the formation of membrane vesicles, whereas the soluble PBP6 formed inclusion bodies in the cytoplasm. Both the membrane-bound and the soluble forms of PBP6 were purified to homogeneity by using the immobilized dye Procion rubine MX-B. Purified preparations of PBP6 and PBP6s350 formed a 14[C]penicillin-protein complex at a 1:1 stoichiometry. The half-lives of the complexes were 8.5 and 6 min, respectively. In contrast to PBP5, no DD-carboxypeptidase activity could be detected for PBP6 by using bisacetyl-L-Lys-D-Ala-D-Ala and several other substrates. These findings led us to conclude that PBP6 has a biological function clearly distinct from that of PBP5 and to suggest a role for PBP6 in the stabilization of the peptidoglycan during stationary phase.

Amino Acid Sequence↗