Biomedical subjects

D C Jackson

Publications and source records attributed to D C Jackson.

At least 55 records · Page 3Linked to original sources

PubMed1996

The assembly and immunological properties of non-linear synthetic immunogens containing T-cell and B-cell determinants.

For the rational design of synthetic vaccines, a potential immunogen must contain the appropriate helper T-cell and B-cell determinants to elicit a strong and relevant immune response. In this study we describe a method for the assembly of antigenic determinants from influenza virus hemagglutinin onto a lysine-based support, resulting in dimeric and trimeric constructs bearing both T-cell and B-cell determinants. A panel of synthetic immunogens was constructed incorporating peptides representing: (i) the B-cell determinant TLKLATG and the T-cell determinant PKYVKQNTLKLA which overlaps this sequence in the heavy chain (HA1) of the hemagglutinin; and (ii) the same B-cell determinant with an alternate T-cell determinant ALNNRFQIKGVELKS from the light chain (HA2). With these peptides we were able to investigate the effects of altering the source of T-cell help, increasing the copy number of B-cell determinants as well as comparing the presentation of determinants in either linear tandem or branched geometries. In general, peptides incorporating the non-native helper T-cell determinant in a branched conformation were superior immunogens, eliciting higher titres of both peptide-specific and virus-specific antibody. Increasing the copy number of the B-cell determinant also proved to be an advantage in terms of increasing antibody titres. Other evidence was obtained indicating that presentation of determinants to T cells may be different for linear peptide constructs compared to branched immunogens bearing the same determinants.

Amino Acid Sequence↗

PubMed1996

Manipulation of the helper T cell response to influence antigenic competition occurring with a multivalent vaccine.

The reduction in antibody observed following inoculation with multiple heterologous Dichelobacter nodosus pili antigens is thought to be due to competition between antigen-specific B cells for a limited amount of T cell help. We demonstrate here that this competition is not further influenced by the expansion of cross-reactive antibody secreting cells at the expense of serogroup specific antibody secreting cells. The T cell determinants of pili recognized by sheep and BALB/c mice have been defined using 15 residue peptides. These T cell determinants include cross-reactive determinants in the conserved amino terminal region of the antigen. Here we investigate the effect of expanding the pili-specific T cell population by priming with pili derived T cell determinants. It was not possible to increase the antibody elicited in response to the multivalent vaccine by priming mice with either a synthetic peptide spanning a T cell determinant or with reduced and alkylated or heterologous serogroups of pili 4 weeks before inoculation with the multivalent vaccine. A strategy designed to increase the T cell population by inoculating animals with pili covalently coupled to an extrinsic T cell determinant was pursued.

Amino Acid Sequence↗

PubMed1996

Opsonic human antibodies from an endemic population specific for a conserved epitope on the M protein of group A streptococci.

This study demonstrates the presence of epitope-specific opsonic human antibodies in a population living in an area endemic for group A streptococci (GAS) infection. Antibodies recognizing a conserved C-terminal region epitope (p145, sequence in single letter amino acids: LRRDLDASREAKKQVEKALE) of the M protein of GAS were isolated from human patients by affinity chromatography and were shown to be of the immunoglobulin G1 (IgG1) and IgG3 subclasses. These antibodies could reduce the number of colonies of serotype 5 GAS in an in vitro opsonization assay by 71-92%, compared with an equal amount of IgG from control adult donors living in non-endemic areas and without antibodies to p145. Addition of the peptide, p145, completely inhibited this opsonization. Indirect immunofluorescence showed that p145-specific antibodies were capable of binding to the surface of M5 GAS whereas control IgG did not. Using chimeric peptides, which contain overlapping segments of p145, each 12 amino acids in length, inserted into a known helical peptide derived from the DNA binding protein of yeast, GCN4, we have been able to further define two minimal regions within p145, referred to as pJ2 and pJ7. These peptides, pJ2 and pJ7, were able to inhibit opsonization by p145 specific antibodies. Finally, we have observed an association between the age-related development of immunity to GAS and the acquisition of antibodies to the conserved epitope, p145, raising the possibility of using this epitope as a target in a prophylactic vaccine administered during early childhood.

Adolescent↗

PubMed1996

Lactate distribution and metabolism during and after anoxia in the turtle, Chrysemys picta bellii.

To determine the fate of lactate during and after prolonged anoxia, 14C-labeled lactate was injected into turtles after 2 h of a 6-h submergence at 20 degrees C. 14C activities of plasma and chamber water were tested at intervals during anoxia and also in expired air during 39 h of recovery. Partitioning of label in major body compartments [extracellular fluid (ECF), intracellular fluid (ICF), and shell] and 14C activity and glycogen in selected tissues (heart, liver, and muscle) were measured after anoxia (n = 7) and after recovery (n = 6). Shell 14C and [lactate] were extensively measured on six anoxic turtles. During anoxia all 14C remained in the animal indicating no urine production. At 6 h of anoxia 47% of recovered 14C, presumably still as lactate, was in the ECF, 27% in the ICF, and 30% in the shell. During recovery, plasma [lactate] fell from 35 to 5 meq, but surrounding water and expired air accounted for only 9 and 8%, respectively, of recovered label. The ICF portion grew to 41%, associated with a recovery in tissue glycogen. The shell still had 22% of total label. We conclude that, during recovery from anoxia, lactate is predominantly resynthesized to glycogen, and only a small fraction is directly oxidized. During anoxia, however, lactate is widely distributed in the body, and a surprisingly large and functionally significant fraction resides in the shell.

Animals↗

PubMed1996

1H NMR structural study of free and template-linked antigenic peptide representing the C-terminal region of the heavy chain of influenza virus hemagglutinin.

A 12 kDa template-assembled molecule, incorporating four oxime-linked synthetic peptides representing residues 306-328 of influenza virus hemagglutinin (HA), has been analysed by 1H NMR spectroscopy. The molecule (referred to as the 'tetraoxime') is of interest because it has been shown to elicit a better immune response than the free, monomeric peptide not only in the production of antibodies crossreactive with HA but also in its ability to elicit CD4+ T helper cells. We describe here an NMR structural analysis of (i) the unlinked template molecule and (ii) the free peptide and show that their conformations are not affected upon assembly of the tetraoxime. Our results suggest that the increased immune response observed for the tetraoxime may be due to its greater size and valency compared to that of the free peptide rather than being due to any induced structural effects.

Amino Acid Sequence↗

PubMed1995

Analysis of the requirements for class II-restricted T cell recognition of a single determinant reveals considerable diversity in the T cell response and degeneracy of peptide binding to I-Ed.

The amino acid sequences recognized by five I-E(d)-restricted and one E alpha A beta d-restricted murine T cell clones were determined. The clones had been raised to a synthetic peptide representing amino acids 305-328 of influenza virus hemagglutinin. It was found that although all of the T cell clones recognized a single 10-residue region of the peptide, 307KYVKQNTLKL316, different clones could recognize minimal ("core") determinants spanning 8, 9, or 10 of these amino acids. To see whether particular amino acids within the sequence 307-316 were universally important for T cell recognition, the six clones were assayed for their ability to tolerate single amino acid substitutions of the 10 residue peptide. In all, 190 analogues of the peptide in which each amino acid in the sequence was replaced, in turn, by each of the other 19 naturally occurring amino acids were tested. It was shown that 1) the six T cell clones had very different requirements for recognition of the peptide, 2) substitutions at every single position within the peptide could be shown to affect recognition in a T cell-specific manner, and 3) every single position within the peptide could be replaced by a large number of amino acids and still be recognized by at least one T cell clone. These results demonstrate the great diversity exhibited by the T cell repertoire in recognizing a 10-amino acid determinant, as well as the degeneracy of peptide binding to I-E(d).

Amino Acid Sequence↗

PubMed1995

Development of a cleavage-site-specific monoclonal antibody for detecting metalloproteinase-derived aggrecan fragments: detection of fragments in human synovial fluids.

We have developed a monoclonal antibody AF-28 that specifically recognizes a neo-epitope on polypeptides with N-terminal FFGVG ... sequences. This sequence is found at the N-terminus of aggrecan fragments that have been digested with matrix metalloproteinases (MMPs). By immunoblotting, monoclonal antibody AF-28 specifically detected G2 fragments derived from an aggrecan G1-G2 substrate digested with stromelysin, collagenase, gelatinase and matrilysin, but failed to detect G2 fragments obtained from elastase, trypsin or cathepsin B digests. Undigested G1-G2 was not detected. In addition, AF-28 antibody detected fragments derived from whole aggrecan and this detection did not require prior treatment with chondroitinase or keratanase. Competition experiments confirmed that peptides containing internal ... FFGVG ... sequences were not detected by the antibody, while native MMP-digested aggrecan fragments and a synthetic 32-mer peptide with FFGVG ... N-termini were equally competitive on a molar basis. An FFGVG 5-mer, and an FGVGGEEDI9-mer which lacked the N-terminal phenylalanine residue, were 50 times and 230 times respectively less competitive than the FFGVG ... 32-mer. Two fragments from the interglobular domain, F342-F373 and F342-D441, that are predicted products of G1-G2 digestion by neutrophil collagenase but have not previously been detected, could be detected with AF-28. The epitope recognized by AF-28 was also detected in human synovial fluids by Western blot analysis. A broad band of 100-200 kDa was detected in some patients and a dominant band of 40-60 kDa was found in two patients. The size of this small fragment corresponds with that seen for the porcine F342-E373 product and may represent the natural physiological product of aggrecan cleaved in vivo at both the MMP site (... DIPEN341 decreases F342FGVG ...) and the aggrecanase site (... ITEGE373 decreases A374RGSVI ...).

Aggrecans↗

PubMed1995

The effect of prolonged anoxia at 3 degrees C on tissue high energy phosphates and phosphodiesters in turtles: a 31P-NMR study.

Selected tissues (skeletal muscle, heart ventrical, and liver), sampled from turtles (Chrysemys picta bellii) at 3 degrees C either under normoxic conditions or after 12 weeks of anoxic submergence were quantitatively analysed for intracellular pH and phosphorus metabolites using 31P-NMR. Plasma was tested for osmolality and for the concentrations of lactate, calcium, and magnesium to confirm anoxic stress. We hypothesized that, in the anoxic animals, tissue ATP levels would be maintained and that the increased osmolality of the body fluids of anoxic turtles would be accounted for by a corresponding increase in the concentrations of phosphodiesters. The responses observed differed among the three tissues. In muscle, ATP was unchanged by anoxia but phosphocreatine was reduced by 80%; in heart, both ATP and phosphocreatine fell by 35-40%. The reduction in phosphocreatine in heart tissue at 3 degrees C was similar to that observed in isolated, perfused working hearts from turtles maintained at 20 degrees C but no decrease in ATP occurred in the latter tissues. In liver, although analyses of several specimens were confounded by line-broadening, neither ATP nor phosphocreatine was detectable in anoxic samples. Phosphosdiesters were detected in amounts sufficient to account for 30% of normoxic cell osmotic concentration in heart and 11% and 12% in liver and muscle, respectively. The phosphodiester levels did not change in anoxia. Heart ventricular phosphodiester levels in turtles at 3 degrees C were significantly higher than those determined for whole hearts from turtles at 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1995

Synthetic peptides representing sequences within gp41 of HIV as immunogens for murine T- and B-cell responses.

Within the gp41 glycoprotein of the human immunodeficiency virus type 1 (HIV-1) there is a relatively conserved region which appears accessible to the immune system during the course of HIV infection and is recognised by antibody from virtually all patients with AIDS. This region has also been shown to function as a target for human T cells. We have examined synthetic peptides spanning this sequence, between residues 572 and 604, with a view to evaluating their potential as immunogens. Peptides 572GIKQLQARILAVERYLKDQQ591 and 579RILAVERYLKDQQLLGGIWGCSGK601 were good immunogens in two different strains of mice while peptide 576LQARILAVERYLKDQQ591 was an inferior immunogen, and peptide 593LGIWGCSGKLIC604 was non-immunogenic unless coupled to a carrier protein. For both antibody and T cell responses it was apparent that sequences that could function as determinants within one peptide could not do so in the context of a different peptide immunogen. It follows that by judicious choice of immunogen sequence it may be possible to direct the immune response towards a desired fine specificity. Unwanted responses by CD4+ T cells isolated from certain peptide-primed animals were also observed. These T cells showed an unusual reactivity in that they were incapable of recognising their determinant AVERYLKDQQ if it was extended at the C-terminal end with the native sequence and as such would not be expected to recognise the native molecule unless processing created the identical C-terminus.

Amino Acid Sequence↗

PubMed1995

A synthetic peptide-based polyoxime vaccine construct of high purity and activity.

An artificial protein containing four copies of a peptide comprising the C-terminal 23 residues of influenza virus hemagglutinin was constructed using oxime chemistry and compared with two tetrameric multiple antigenic peptide (MAP) constructions of the same peptide displayed either radially or linearly which were made by conventional techniques. The tetra-oxime was much more homogeneous yielding a single peak on reversed phase HLPC and the correct mass spectrum. In addition, the tetra-oxime was found to be recognized by anti-peptide antibodies, to stimulate at low concentrations a T-cell clone and also to elicit in mice high titres of antibodies which were able to recognize native virus. The modular polyoxime approach, which permits artificial proteins to be assembled rapidly, in high yield and in high purity, is expected to lead to an increase in the use of artificial proteins in vaccine technology.

Amino Acid Sequence↗

PubMed1995

Immunological parameters associated with antigenic competition in a multivalent footrot vaccine.

A murine model for antigenic competition with multivalent D. nodosus pili vaccine has been established that parallels the phenomenon observed in sheep where levels of antibody, specific for any particular serogroup of pili, are significantly lower following vaccination in the presence of multiple serogroups of pili than with that serogroup alone. This competition was observed in both high and low responder strains of mice and was not dependent on the multiplicity of the antigens in the multivalent vaccine but could be observed with a large excess of a single heterologous serogroup. Competition was manifest by a reduction in the number of serogroup-specific antibody secreting cells elicited in response to vaccination. The antibody response to a single serogroup of pili reached a plateau at high doses and it was at these doses that antigenic competition was most pronounced, under conditions where both B- and T-cell responses were limiting. The limit in T-cell responsiveness was not imposed at the level of presentation of antigen. Pili-specific T cells were largely cross-reactive for different serogroups, and under conditions of limiting T-cell stimulation within a lymph node the available T cells would have to be shared between B cells specific for each serogroup of pili, which may in turn result in the decrease of serogroup-specific antibody induced following inoculation with the multivalent vaccine.

Animals↗

PubMed1995

Effects of input pressure on in vitro turtle heart during anoxia and acidosis: a 31P-NMR study.

In vitro working hearts of the turtle, Chrysemys picta bellii, paced at 30 beats/min, were studied over a range of input pressures in the following sequence of perfusion conditions: control normoxia, control anoxia, lactacidotic normoxia, and lactacidotic anoxia. Two such series of experiments were performed. In series 1 (n = 12), ventricular pressure (PV) and cardiac output were measured, and power output and dPV/dt were calculated. In series 2 (n = 5), intracellular phosphorus metabolites and intracellular pH (pHi) were also measured using 31P-nuclear magnetic resonance (31P-NMR) spectroscopy. In series 1 all mechanical variables increased with input pressure in generally similar fashion, except during anoxic acidosis, during which mechanical performance was depressed and was increased less or not at all by input pressure. Creatine phosphate (CP) and pHi fell significantly in anoxia and anoxic acidosis, but neither these variables, ATP, CP/ATP, nor, presumably, ADP changed as a function of input pressure with any perfusate despite often large increments in mechanical output. We conclude that anoxia and acidosis act synergistically to depress cardiac function in turtle hearts. Also, the insensitivity of NMR variables to changes in input pressure and cardiodynamics suggests that changes in these variables are unimportant for controlling energy turnover in this preparation.

Acidosis↗

PubMed1995

1H NMR studies of peptide fragments from the N-terminus of chicken and human transthyretin.

Two synthetic peptides corresponding to N-terminal fragments of human and chicken transthyretin have been synthesized and their structures examined in solution using 1H NMR spectroscopy. Complete sequence-specific assignments obtained for the two peptides are reported together with coupling constant and nuclear Overhauser data. The peptides were found to adopt random-coil conformations in aqueous solution. This is consistent with findings from X-ray structures of the native human transthyretin where the N-terminal region could not be defined, presumably because of conformational disorder.

Amino Acid Sequence↗

PubMed1995

Identification and synthesis of altered peptides modulating T cell recognition of a synthetic peptide antigen.

In studies of T cell responses to synthetic peptides we have observed agonist and antagonist activities associated with contaminants identified within the parent synthesis. The synthesis of two candidate analogues implied by a peptide contaminant formed during the synthesis of La 51-58 (IMIKFNRL) has been carried out. The peptide contaminant was 17-18 Da smaller than the parent peptide consistent with a modified asparagine residue at position 6 and so we synthesised both an aspartimide and a nitrile analogue, representing cyclisation or dehydration of the asparagine residue. The candidate aspartimide and nitrile analogues both bound empty MHC class I molecules to form allo determinants recognised by monoclonal antibodies. These results demonstrate that altered synthetic peptides can bind class I MHC molecules and prompt caution in the use of synthetic peptides as a source of immunising antigen.

Amino Acid Sequence↗

PubMed1995

Antigenic and immunogenic properties of synthetic peptide-based T-cell determinant polymers.

Presentation of T-cell determinants to the immune system in multimeric form has clear advantages and the production of synthetic peptide-based polymers using the solubilisable KS resin described by Goddard et al. [1] provides a method of assembling such polymers and also offers the means for making heteropolymers. The present study investigates the potential of polymeric synthetic peptide constructs in eliciting proliferative T-cell responses to determinants of the influenza virus hemagglutinin. The induction of vigorous CD4+ T-cell immunity was achieved with a polymeric construct containing two different T-cell determinants. The data presented here also highlight the fact that distancing the determinant from the support backbone with appropriate amino acid residues is an important consideration for the success of these polymeric immunogens. This approach may be readily applied in other systems where induction of helper T-cell responses are required.

Amino Acid Sequence↗

PubMed1994

Conserved determinants for CD4+ T cells within the light chain of the H3 hemagglutinin molecule of influenza virus.

Helper T-cell clones were isolated from BALB/c mice that had been inoculated with purified light chain (HA2) from H3 subtype influenza virus hemagglutinin (HA). The clones were divided into two distinct groups based on their ability to proliferate in response to bromelain-derived HA (BHA) and the light chain derived from it (BHA2), both of which lack the C-terminal 46 amino acid residues of the HA2 chain. The first group contained two I-Ad restricted clones that proliferated in response to BHA and BHA2 and were found to recognize the determinant 96AELLVALEN104. The remaining seven clones were I-Ed restricted, required intact HA2 for proliferation, and responded to synthetic peptides containing the sequence 170RFQIKGVEL178 which spans the bromelain cleavage site. Although all T-cell clones proliferated in response to a wide range of different H3 virus strains, they showed no cross-reactivity with viruses of the H1 or H2 subtype. The T-cell clones from each group were able to provide help to virus-primed B cells allowing them to produce anti-HA antibody in vitro.

Amino Acid Sequence↗

PubMed1994

Glycosylation of a synthetic peptide representing a T-cell determinant of influenza virus hemagglutinin results in loss of recognition by CD4+ T-cell clones.

Synthetic glycopeptides were used to study possible mechanisms for the reduction observed in the response of influenza virus-specific CD4+ T-cells to strains of virus in which amino acid substitution in the hemagglutinin has led to attachment of a carbohydrate side chain. The peptide NCTLIDALLGDPH stimulates vigorous proliferation of hemagglutinin-specific T-cell clones F1-36 and F1-40 but addition of a heptasaccharide, which approaches the size of natural carbohydrate antennae, eliminated the stimulatory capacity of the peptide. This occurs even though the site of carbohydrate attachment at the N-terminal asparagine lies outside the T-cell determinants encompassed by this sequence. A glycopeptide with only two sugar units was stimulatory for F1-36 but not F1-40, suggesting that peptides with a carbohydrate side chain are able to bind to MHC molecules but that approach of the T-cell receptor of certain clones to the glycopeptide-MHC complex is hindered. Loss of T-cell recognition following attachment of a long carbohydrate side-chain to T-cell determinants is not a general finding because attachment of six carbohydrate units to the peptide, NKYVKQNTLKLA, had little or no effect on the stimulation of a T-cell clone specific for this sequence.

Amino Acid Sequence↗

PubMed1994

Analysis of antibody-antigen and antibody-anti-(idiotypic antibody) cross-reactivity using synthetic peptide probes.

The extent, nature and structural basis of immunological cross-reactivity of an anti-synthetic peptide monoclonal antibody (MAb) with the parent antigen (influenza virus haemagglutinin) from which the peptide was derived, and with a paratope-directed anti-idiotypic (anti-Id) antibody was investigated. Use of synthetic homologs and analogs of the peptide indicated that the anti-peptide MAb utilizes a common binding site to complex with peptide, haemagglutinin (HA) and anti-Id antibody, and the affinity constants for the binding of the anti-peptide MAb to peptide and to the anti-Id MAb were found to differ only by three fold. Determination of the amino acid sequence of the heavy chain variable domain (VH) of the anti-Id MAb did not reveal any obvious sequence homology with the peptide. Consideration of the spatial arrangement of residues, however, disclosed a region within the framework of the anti-Id VH with similarity to the epitope recognized by the anti-peptide MAb. This region, formed from antiparallel chains, contains amino acid residues arranged in a conformation similar to that assumed by amino acid residues comprising the epitope within the intact HA.

Amino Acid Sequence↗