Biomedical subjects

J D Clements

Publications and source records attributed to J D Clements.

At least 55 records · Page 3Linked to original sources

PubMed1997

Role of receptor binding in toxicity, immunogenicity, and adjuvanticity of Escherichia coli heat-labile enterotoxin.

The role of receptor binding in the toxicity, immunogenicity, and adjuvanticity of the heat-labile enterotoxin of Escherichia coli (LT) was examined by comparing native LT and LT(G33D), a B-subunit receptor binding mutant, with respect to the ability to bind to galactose and to GM1, toxicity on mouse Y-1 adrenal tumor cells, the ability to stimulate adenylate cyclase in Caco-2 cells, enterotoxicity in the patent mouse model, and oral immunogenicity and adjuvanticity. In contrast to native LT, LT(G33D) was unable to bind to the galactosyl moiety of Sepharose 4B or GM1 but did retain the lectin-like ability to bind to immobilized galactose on 6% agarose beads. LT(G33D) had no enterotoxicity in the patent mouse model but exhibited residual toxicity on mouse Y-1 adrenal tumor cells and had an ability equivalent to that of native LT to stimulate adenylate cyclase in Caco-2 cells (5,000 versus 6,900 pmol per mg of protein). In addition, LT(G33D) was unable to serve as an effective oral adjuvant for induction of immunoglobulin G or A directed against a coadministered antigen. Furthermore, LT(G33D) elicited negligible serum and mucosal antibody responses against itself. These data indicate that the toxicity, immunogenicity, and oral adjuvanticity of LT are dependent upon binding of the B subunit to ganglioside GM1.

Animals↗

PubMed1997

Intracellular Salmonella dublin induces substantial secretion of the 40-kilodalton subunit of interleukin-12 (IL-12) but minimal secretion of IL-12 as a 70-kilodalton protein in murine macrophages.

The induction by intracellular pathogens of interleukin-12 (IL-12) secretion is of particular importance since this cytokine has been shown to be necessary for optimal cell-mediated immune responses. Several recent investigations have suggested that cultured macrophages are a significant source of IL-12 following intracellular infection with pathogens such as Salmonella spp. In an effort to critically evaluate the magnitude of the IL-12 response in cultured macrophages following interaction with Salmonella dublin, enzyme-linked immunosorbent assays specific for the 40- and 70-kDa subunits of IL-12 (IL-12p40 and IL-12p70) and a sensitive bioassay for IL-12p70 were used. Using BALB/c macrophages, S. dublin at various challenge doses was a potent inducer of IL-12p40 secretion (>6,000 pg/10(7) macrophages). However when secretion of IL-12p70 was evaluated, S. dublin did not induce comparable IL-12p70 production (<80 pg/10(7) macrophages) at any time, despite varying the challenge dose of Salmonella. The limited ability of BALB/c (Ity(s)) macrophages to secrete IL-12p70 in response to Salmonella was not a strain-specific phenomenon since similar results were demonstrated for macrophages isolated from CBA/J (Ity(r)) and C3H/HeJ (lipopolysaccharide [LPS]-hyporesponsive) mice. While intracellular infection with Salmonella was not a potent stimulus for IL-12p70 secretion in these mouse strains, macrophages from these mice responded significantly to a stimulus of gamma interferon plus LPS. Taken together these results demonstrate a limited capacity for intracellular Salmonella to stimulate murine macrophage secretion of IL-12p70, despite being a significant stimulus for IL-12p40 secretion. Furthermore, our results suggest that Salmonella-induced IL-12p40 secretion by macrophages is not solely an LPS-mediated event.

Animals↗

PubMed1997

Long-term synaptic plasticity in the honeybee.

A monosynaptic response was recorded in vivo in the mushroom body of the bee brain, an important site for memory consolidation. Focal electrical stimulation of a major afferent input evoked an extracellular field potential that consisted of a presynaptic fiber volley and a postsynaptic response. We report a long-lasting potentiation of the synaptic response (2.6-fold increase; < or = 3.5 h). Potentiation of the response was induced by low-frequency stimulation (0.02-1.0 Hz), was input specific, and was maintained in the absence of stimulation. Paired-pulse facilitation of the response was converted to paired-pulse depression after potentiation, suggesting a presynaptic mechanism. This is the first demonstration of long-term synaptic plasticity in the insect brain.

Animals↗

PubMed1997

Evaluation of immunogenicity of an oral Salmonella vaccine expressing recombinant Plasmodium berghei merozoite surface protein-1.

A repetitive region of Plasmodium berghei merozoite surface protein-1 (PbMSP-1) was expressed as a fusion protein with either maltose binding protein or the B subunit of heat-labile enterotoxin from Escherichia coli. Vaccination of mice with the fusion proteins indicates that this region of PbMSP-1 is antigenic as evidenced by an antibody response. The fusion proteins were also expressed in Salmonella and mice were orally immunized with the recombinant Salmonella. Some of the vaccinated mice survived a challenge with P. berghei blood-stage parasites without developing parasitemia. All control mice became patent and succumbed to the challenge infection. This partial protection was also observed with purified recombinant protein and was independent of the adjuvant used. Mice immunized with recombinant Salmonella showed either extremely low or no antibody response to PbMSP-1, suggesting that cell-mediated immunity is important for the observed protection. These studies show that it is feasible to develop a cost effective oral vaccine against the blood stage of the malarial parasite.

Administration, Oral↗

PubMed1997

Reverse transcriptase-polymerase chain reaction amplification and partial sequence of T helper 1- and T helper 2-type lymphokine genes from the owl monkey (Aotus trivirgatus).

The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify selected lymphokine mRNAs from phytohemagglutinin-activated leukocytes of the owl monkey (Aotus trivirgatus). Interleukin-2 (IL-2), IL-4, IL-13, and interferon-gamma were selected as lymphokine mRNAs of interest, since expression of these cytokines helps define the type of T helper lymphocyte response (i.e., TH1 versus TH2). Because sequences for these lymphokine genes were not available for the owl monkey, multiple PCR primers for each lymphokine gene were designed based on published human sequences. Various PCR primer pairs were then used in the RT-PCR to determine the conditions for optimal amplification of each owl monkey cytokine mRNA. In addition, each PCR primer pair was compared for the ability to amplify lymphokine mRNAs from other primate species, including African green (Cercopithecus aethiops), squirrel (Saimiri sciureus), and rhesus (Macaca mulatta) monkeys. The specificity and sensitivity of optimal primer pair was also demonstrated by amplification of as little as 10 fg of each lymphokine gene in a background of 300 ng of irrelevant cDNA. Finally, partial sequences of owl monkey coding regions for IL-2, IL-13, and interferon-gamma were determined and compared for homology with their human counterparts. Together, these studies define specific and sensitive conditions for detection of lymphokine mRNA expression in the owl monkey and provide partial sequence information of the coding region for these lymphokines. This investigation should provide molecular probes to investigate the immune response against malaria and the effectiveness of malaria vaccines in the owl monkey that models this human disease.

Amino Acid Sequence↗

PubMed1996

Effects of mutating leucine to threonine in the M2 segment of alpha1 and beta1 subunits of GABAA alpha1beta1 receptors.

The conserved leucine residues at the 9' positions in the M2 segments of alpha1 (L264) and beta1 (L259) subunits of the human GABAA receptor were replaced with threonine. Normal or mutant alpha1 subunits were co-expressed with normal or mutant beta1 subunits in Sf9 cells using the baculovirus/Sf9 expression system. Cells in which one or both subunits were mutated had a higher "resting" chloride conductance than cells expressing wild-type alpha1beta1 receptors. This chloride conductance was blocked by 10 mM penicillin, a recognized blocker of GABAA channels, but not by bicuculline (100 microm) or picrotoxin (100 microm) which normally inhibit the chloride current activated by GABA: nor was it potentiated by pentobarbitone (100 microM). In cells expressing wild-type beta1 with mutated alpha1 subunits, an additional chloride current could be elicited by GABA but the rise time and decay were slower than for wild-type alpha1beta1 receptors. In cells expressing mutated beta1 subunits with wild-type or mutated alpha1 subunits (alphabeta(L9'T) and alpha(L9'T)beta(L9'T)), no response to GABA could be elicited: this was not due to an absence of GABAA receptors in the plasmalemma because the cells bound [3H]-muscimol. It was concluded that in GABAA channels containing the L9'T mutation in the beta1 subunit, GABA-binding does not cause opening of channels, and that the L9'T mutation in either or both subunits gives an open-channel state of the GABAA receptor in the absence of ligand.

Baculoviridae↗

PubMed1996

Transmitter timecourse in the synaptic cleft: its role in central synaptic function.

The speed of clearance of transmitter from the cleft influences many aspects of synaptic function, including the timecourse of the postsynaptic response and the peak postsynaptic receptor occupancy. The timecourse of transmitter clearance can be estimated either by detailed theoretical modelling, or from the attenuation of synaptic transmission produced by a low-affinity competitive antagonist. These approaches have been applied to several classes of central synapse, and results obtained are in broad agreement. The average concentration of transmitter peaks in the range 1-5 mM, and clearance is biphasic, with time constants of approximately 100 microseconds and 2 ms. The pulse of transmitter, although very brief, can prolong the timecourse of the fastest AMPA synaptic currents, and is sufficient to saturate postsynaptic GABA, glycine or NMDA receptors.

Animals↗

PubMed1996

In vivo treatment with anti-interleukin-13 antibodies significantly reduces the humoral immune response against an oral immunogen in mice.

Interleukin-13 (IL-13) is a cytokine which significantly enhances the proliferation and differentiation of B lymphocytes. We therefore evaluated its role in the formation of a humoral immune response in vivo. Upon oral immunization with the B subunit of Escherichia coli heat-labile enterotoxin (LT-B), rapid up-regulation of IL-13 mRNA expression in the mesenteric lymph nodes of LT-B intubated mice occurred. This result suggested that IL-13 might be involved in the formation of a mucosal antibody response against LT-B if this cytokine was in fact secreted. To test this possibility, the coding region for murine IL-13 was cloned into the pFLAG-1 expression vector. Recombinant murine IL-13 was purified from bacterial lysates and used as an immunogen to produce polyclonal anti-IL-13 antibodies. Groups of BALB/c mice treated in vivo with anti-IL-13 antibody 2 days before and on the day of oral immunization with LT-B had significantly reduced intestinal IgA and serum IgG and IgA anti-LT-B antibody responses when compared to mice treated with control antibody. Furthermore, groups of mice primed with LT-B and then treated with anti-IL-13 antibody prior to oral immunization with a second dose of LT-B also had significantly reduced intestinal IgA and serum IgG and IgA anti-LT-B antibody titres compared to controls. In vitro LT-B restimulation experiments using splenic mononuclear leucocytes isolated from LT-B primed mice treated with anti-IL-13 antibody demonstrated decreased expression of IL-4 and IL-13 mRNA and decreased IL-4 secretion when compared to controls. Together these results demonstrate an important role for IL-13 in the formation of a humoral immune response at mucosal surfaces.

Administration, Oral↗

PubMed1996

Mechanisms for mucosal immunogenicity and adjuvancy of Escherichia coli labile enterotoxin.

Escherichia coli labile toxin (LT) was assessed as mucosal immunogen and as adjuvant for tetanus toxoid (TT) in mice. After oral administration of LT, C57BL/6 (H-2b) and BALB/c(H-2d) mice were high mucosal and serum antibody responders, while C3H/HeN (H-2k) mice were low responders. High responders exhibited mainly serum IgG (including IgG1, IgG2a, and IgG2b), as well as IgM and IgA, while mucosal responses were IgA. Analysis of LT-B-specific CD4+ T helper (Th) cells from Peyer's patches (PP) or from spleen revealed a mixed Th1 (interferon-gamma) and Th2 (interleukin-4 and -5) cell pattern. Oral LT given with TT induced TT-specific response patterns identical to LT-B. Analysis of mRNA from TT-specific PP CD4+ Th cells also revealed a mixed Th1- and Th2- type response. Thus, antibody response profiles induced by LT are regulated by both CD4+ Th1 and Th2 cell types.

Adjuvants, Immunologic↗

PubMed1996

Differential production of interleukin-12 mRNA by murine macrophages in response to viable or killed Salmonella spp.

The use of attenuated Salmonella spp. as live oral vaccine carriers fo r foreign antigens has been extensively studied. We have shown that appropriately prepared nonviable organisms are as effective as viable organisms in eliciting humoral immune responses against a foreign antigen delivered by these vectors. It is not clear how strain viability affects the development of a cell-mediated immune response. In the present study, we demonstrate that BALB/c mice orally immunized with viable attenuated Salmonella spp. were protected against subsequent challenge while animals immunized with killed organisms were not. Protection was correlated with increased production of interleukin-12 (IL-12) p40 mRNA in the Peyer's patches within hours of oral administration. Peritoneal macrophages from lipopolysaccharide (LPS)-responsive and LPS-unresponsive mice were also examined for production of IL-12 p40 mRNA following exposure to the viable or killed attenuated Salmonella carrier. There was dramatic upregulation of IL-12 p40 mRNA following exposure of macrophages to either viable or killed organisms. By 4 h postexposure, viable organisms had induced a 27-fold increase in IL-12 p40 mRNA levels while killed organisms had induced a 9-fold increase in IL-12 p40 mRNA levels. This was observed in macrophages isolated from both LPS-responsive and unresponsive mice. The higher levels of IL-12 induced by viable Salmonella spp. may result in the development of a Th1 response and cell mediated immunity, while the lower levels of IL-12 induced by killed Salmonella spp. may not be sufficient to promote a Th1 response.

Animals↗

PubMed1996

Epitope maps of the Escherichia coli heat-labile toxin B subunit for development of a synthetic oral vaccine.

Linear B- and T-cell epitopes spanning all 103 amino acids of the Escherichia coli heat-labile toxin B subunit (LT-B) were assessed in mice orally immunized with native LT or with recombinant Salmonella enteritidis expressing LT-B. Oral administration of native LT induced mucosal immunoglobulin A (IgA) antibodies reactive with an epitope at residues 85 to 91, while IgA induced by recombinant Salmonella LT-B reacted with an epitope at residues 36 to 44. Serum IgG anti-LT-B antibodies from mice orally immunized with either LT or with recombinant Salmonella LT-B were directed to both epitopes. A single T-cell epitope spanning residues 34 to 42 was identified by T-cell proliferative and cytokine responses. When a 20-mer peptide (residues 26 to 45) with B- and T-cell epitopes was given orally to BALB/c (H-2(d)) and B10 congenic (I-A(d), I-A(b), and I-A(k)) mice, significant fecal IgA and serum IgG anti-LT-B antibodies were induced. The peptide also induced LT-B-specific T-cell proliferative responses in these mice. Orally administered LT-B peptide (residues 26 to 45) induced a cytokine profile indicative of both T helper 1- and 2-type cells. The remarkable immunogenicity of this 20-mer peptide makes it a candidate for a vaccine to protect against enterotoxigenic E. coli.

Administration, Oral↗

PubMed1996

Endogenous and exogenous interleukin-12 augment the protective immune response in mice orally challenged with Salmonella dublin.

Following oral challenge with Salmonella dublin, we observed significant increases in interleukin-12 (IL-12) protein expression in the mesenteric lymph nodes. The importance of this endogenous cytokine production in the immune response against S. dublin was demonstrated by in vivo depletion of IL-12 with an anti-IL-12 monoclonal antibody prior to oral S. dublin challenge. Mice pretreated with anti-IL-12 antibody had increased salmonellosis and reduced survival times compared with mice receiving control antibody. Furthermore, administration of exogenous murine recombinant IL-12 dramatically increased survival times of mice challenged orally with S. dublin. Together, these results demonstrate that endogenous and exogenous IL-12 significantly augment the mucosal immune response against the intracellular pathogen S. dublin.

Administration, Oral↗

PubMed1995

Oral immunization with a recombinant bacterial antigen produced in transgenic plants.

The binding subunit of Escherichia coli heat-labile enterotoxin (LT-B) is a highly active oral immunogen. Transgenic tobacco and potato plants were made with the use of genes encoding LT-B or an LT-B fusion protein with a microsomal retention sequence. The plants expressed the foreign peptides, both of which formed oligomers that bound the natural ligand. Mice immunized by gavage produced serum and gut mucosal anti-LT-B immunoglobulins that neutralized the enterotoxin in cell protection assays. Feeding mice fresh transgenic potato tubers also caused oral immunization.

Administration, Oral↗

PubMed1995

Mucosal T cells induce systemic anergy for oral tolerance.

Heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli is known to possess strong immunoregulatory potential in terms of inhibition of the induction of oral tolerance and adjuvanticity in oral immunization. We found that oral administration of an immunogenic peptide of LT [LT-B(26-45); spanning the residues 26-45 of LT-B] induced systemic unresponsiveness in BALB/c mice resulting in diminished serum IgG responses. It was also shown that the spleen (SP) CD4+ T cells of tolerized mice failed to proliferate, whereas the Peyer's patches (PP) CD4+ T cells responded to the peptide. RT-PCR revealed that the SP CD4+ T cells did not generate IL-2 mRNA, while the PP CD4+ T cells expressed significant levels of IFN-gamma, IL-2, IL-4, and TGF-beta mRNA. Adoptive transfer of LT-B-specific intraepithelial lymphocytes to the tolerant mice abrogated the tolerance. In the reversed mice, LT-B(26-45)-stimulated SP CD4+ T cells expressed significant levels of IFN-gamma, IL-2, IL-4, and IL-6 mRNA. These results indicate that PP CD4+ T cells induce oral tolerance due to systemic T cell anergy.

Amino Acid Sequence↗

PubMed1995

In vivo induction of interleukin-12 mRNA expression after oral immunization with Salmonella dublin or the B subunit of Escherichia coli heat-labile enterotoxin.

Mice orally immunized with Salmonella dublin EL23, a nonreverting, aromatic-dependent, histidine-requiring mutant transformed with a plasmid which carries a gene that codes for production of the B subunit of the heat-labile toxin (LT-B) of enterotoxigenic Escherichia coli, or with purified LT-B alone were compared for their ability to initiate expression of interleukin-12 (IL-12) mRNAs at mucosal sites. At 6 or 20 h following oral immunization, the Peyer's patches and mesenteric lymph nodes were removed, and polyadenylated mRNA was prepared from each tissue. Constitutive expression of an mRNA encoding the p35 subunit of IL-12 was observed in control as well as immunized mice. Conversely, expression of an mRNA encoding the p40 subunit of IL-12 was not detected in control animals but was dramatically upregulated in immunized mice. By using semiquantitative reverse transcription-PCR (RT-PCR) followed by competitive RT-PCR, differences in the magnitude of IL-12 p40 mRNA expression were quantified. Six hours after oral immunization with the Salmonella construct, mice had 12.1- and 8.4-fold increases in expression of IL-12 p40 mRNA in the Peyer's patches and mesenteric lymph nodes, respectively, compared with control mice receiving only saline. By 20 h, the pattern of increased mRNA expression was reversed, showing 2.5- and 17.6-fold increases in the Peyer's patches and mesenteric lymph nodes, respectively. Oral immunization with LT-B alone also stimulated IL-12 p40 mRNA expression, but to a lesser extent. The constitutive expression of IL-12 p35 mRNA at these mucosal sites coupled with a rapid and dramatic induction of IL-12 p40 mRNA following immunization with wild-type or attenuated strains of S. dublin is consistent with other investigations which support a role for IL-12 in modulating cell-mediated immune responses against intracellular pathogens.

Administration, Oral↗

PubMed1995

Dissociation of Escherichia coli heat-labile enterotoxin adjuvanticity from ADP-ribosyltransferase activity.

The heat-labile enterotoxin (LT) of Escherichia coli is immunologically and physiochemically related to cholera enterotoxin. A number of studies have been performed to determine the relationship of the ADP-ribosylating enzymatic activity of these enterotoxins to toxicity and adjuvanticity. These studies have generally examined the effect of abolishing the ADP-ribosyltransferase activity of A1 by a variety of chemical or genetic manipulations. In every case, loss of enzymatic activity was associated with loss of biological activity and also with the ability of the molecules to function as oral adjuvants. Consequently, we explored an alternate approach to detoxification of LT without altering its adjuvanticity. Specifically, we generated a novel mutant form of LT by genetic modification of the proteolytically sensitive residues that join the A1 and A2 components of the A subunit. This mutant contains a single amino acid substitution within the disulfide subtended region joining A1 and A2. This mutant toxin, designated LT(R192G), is not sensitive to proteolytic activation, has negligible activity on mouse Y-1 adrenal tumor cells, and is devoid of ADP-ribosyltransferase activity. Nonetheless, LT(R192G) retains the ability to function as a mucosal adjuvant, increasing the serum immunoglobulin G (IgG) and mucosal IgA responses to coadministered antigen (OVA) beyond that achieved with administration of that antigen alone. Further, LT(R192G) prevented the induction of tolerance to coadministered antigen and did not induce tolerance against itself, as demonstrated by the presence of significant serum anti-LT IgG and mucosal anti-LT IgA antibodies in immunized mice.

Adjuvants, Immunologic↗

PubMed1994

Influence of strain viability and antigen dose on the use of attenuated mutants of Salmonella as vaccine carriers.

It is now accepted that oral killed typhoid vaccines are not effective at inducing protective anti-typhoid immunity. It is not known whether oral killed Salmonella can function as an effective carrier of other antigens to the immune system. In order to test this hypothesis, we immunized groups of mice with viable and non-viable preparations of aroA Salmonella dublin strain EL23 which codes for production of the binding subunit of the heat-labile enterotoxin of Escherichia coli (LT-B). Animals immunized orally with viable EL23 developed serum and mucosal anti-LT-B responses consistent with our previous findings. Significantly, mice immunized orally with ultraviolet-killed EL23 developed serum and mucosal antibody responses equivalent to those which developed in animals orally immunized with the same number of viable EL23. We extended these observations to include a number of methods of killing the organisms which may also preserve the ability of these strains to function as carriers. Our findings indicate that viability is not a requirement for use of a Salmonella strain as an immunological carrier. Moreover, our evidence indicates that bacteraemia and persistence in tissues are not necessary for oral priming, and therefore it may be best to dissociate the question of what makes the best live oral anti-typhoid vaccine from the question of what makes a good carrier of heterologous antigens.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed1994

Laboratory and preliminary clinical characterization of Vi capsular polysaccharide-protein conjugate vaccines.

To improve its immunogenicity for children and adults and to make it suitable for routine immunization of infants against typhoid fever, the capsular polysaccharide of Salmonella typhi (Vi) was bound to the B subunit of the heat-labile toxin (LT-B) of Escherichia coli or the recombinant exoprotein A (rEPA) of Pseudomonas aeruginosa. The conjugates elicited higher levels of antibodies (micrograms per milliliter of serum) in mice and in guinea pigs than did Vi and, unlike Vi alone, elicited booster antibody responses in both species. In adult volunteers, Vi-LT-B and Vi-rEPA, respectively, elicited higher levels of antibodies than Vi alone after the first injection (4.74 versus 1.77 and 4.91 versus 1.77; P < 0.005) and 26 weeks later (2.32 and 2.69 versus 0.54; P < 0.04); a second injection of the conjugates did not elicit a booster response of Vi antibodies. None of the 51 vaccinees had fever or significant local reactions. Vi-rEPA elicited slightly higher levels of Vi antibodies than did Vi-LT-B at all intervals after injection, but these differences were not significant. Each conjugate elicited antibodies to its carrier protein. The antibody responses elicited in adults by Vi bound to LT-B and rEPA are similar to those of other polysaccharide-protein conjugates. These conjugates promise to be an improved Vi vaccine. Studies of Vi conjugates with adults and infants in areas where typhoid is endemic are planned.

Adolescent↗