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Biomedical subjects

A K Barlow

Publications and source records attributed to A K Barlow.

13 recordsLinked to original sources
PubMed1998

Subtle conformational changes induced in major histocompatibility complex class II molecules by binding peptides.

Intracellular trafficking of major histocompatibility complex (MHC) class II molecules is characterized by passage through specialized endocytic compartment(s) where antigenic peptides replace invariant chain fragments in the presence of the DM protein. These changes are accompanied by structural transitions of the MHC molecules that can be visualized by formation of compact SDS-resistant dimers, by changes in binding of mAbs, and by changes in T cell responses. We have observed that a mAb (25-9-17) that is capable of staining I-Ab on the surface of normal B cells failed to interact with I-Ab complexes with a peptide derived from the Ealpha chain of the I-E molecule but bound a similar covalent complex of I-Ab with the class II binding fragment (class II-associated invariant chain peptides) of the invariant chain. Moreover, 25-9-17 blocked activation of several I-Ab-reactive T cell hybridomas but failed to block others, suggesting that numerous I-Ab-peptide complexes acquire the 25-9-17(+) or 25-9-17(-) conformation. Alloreactive T cells were also able to discriminate peptide-dependent variants of MHC class II molecules. Thus, peptides impose subtle structural transitions upon MHC class II molecules that affect T cell recognition and may thus be critical for T cell selection and autiommunity.

Amino Acid Sequence↗
PubMed1998

Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins.

Abeta1-42 is a self-associating peptide whose neurotoxic derivatives are thought to play a role in Alzheimer's pathogenesis. Neurotoxicity of amyloid beta protein (Abeta) has been attributed to its fibrillar forms, but experiments presented here characterize neurotoxins that assemble when fibril formation is inhibited. These neurotoxins comprise small diffusible Abeta oligomers (referred to as ADDLs, for Abeta-derived diffusible ligands), which were found to kill mature neurons in organotypic central nervous system cultures at nanomolar concentrations. At cell surfaces, ADDLs bound to trypsin-sensitive sites and surface-derived tryptic peptides blocked binding and afforded neuroprotection. Germ-line knockout of Fyn, a protein tyrosine kinase linked to apoptosis and elevated in Alzheimer's disease, also was neuroprotective. Remarkably, neurological dysfunction evoked by ADDLs occurred well in advance of cellular degeneration. Without lag, and despite retention of evoked action potentials, ADDLs inhibited hippocampal long-term potentiation, indicating an immediate impact on signal transduction. We hypothesize that impaired synaptic plasticity and associated memory dysfunction during early stage Alzheimer's disease and severe cellular degeneration and dementia during end stage could be caused by the biphasic impact of Abeta-derived diffusible ligands acting upon particular neural signal transduction pathways.

Alzheimer Disease↗
PubMed1998

Exogenously provided peptides of a self-antigen can be processed into forms that are recognized by self-T cells.

Major histocompatibility complex (MHC) class II molecules can present peptides derived from two different sources. The predominant source of peptide in uninfected antigen presenting cells (APCs) is from self-proteins that are synthesized within the cell and traffic through the MHC class II compartment. The other source of antigen is endocytosed proteins, which includes both self- and foreign proteins. Foreign protein antigens generate adaptive immune responses, whereas self-peptides stabilize the MHC class II heterodimer on the cell surface, allowing positive and negative selection of thymocytes. Therefore, self-antigens play an important normal role in shaping the T cell receptor repertoire as well as a pathological role in autoimmunity. To determine whether processing and presentation of self-antigens by MHC class II molecules differs depending on whether the antigen is supplied through synthesis within the cell or by endocytosis, we used a T cell clone against an Ealpha peptide presented by I-Ab to show that processing through these two routes can differ. We also show that mice can be tolerant to the epitope formed through the endogenous route, but responsive to the epitope that can be formed through endocytosis. This suggests that negative selection occurs primarily against antigens that are synthesized within the APC, and that endocytosed self-antigens could serve as autoantigens. Finally, we also demonstrate that lipopolysaccharide-activated B cells are defective for uptake, processing, and presentation of this self-antigen, and that this correlates with the increased expression of the costimulatory molecules B7.1 and B7.2. This may provide a model for studying the onset of an autoimmune response.

Animals↗
PubMed1995

Direct physical interaction involving CD40 ligand on T cells and CD40 on B cells is required to propagate MMTV.

The propagation of mouse mammary tumor virus (MMTV) has been analyzed in mice defective for expression of CD40 ligand (CD40L). Mice with endogenous viral superantigen (SAG) delete T cells with cognate V beta independent of CD40L expression. Nevertheless, CD40L-mice do not show deletion of cognate T cells after being exposed to infectious MMTV and have greatly diminished viral replication. The response of CD40L- T cells to SAG in vitro is also impaired, but can be reconstituted by adding B cells activated by recombinant CD40L to express costimulatory molecules. Thus, direct CD40L-dependent B cell activation appears to be a critical step in the life cycle of MMTV. The initial step in SAG-dependent T cell activation, and hence the MMTV life cycle, may be mediated by non-B cells, because splenocytes from B cell-deficient SAG-transgenic mice are able to activate cognate T cells.

Animals↗
PubMed1992

Anti-CD2 monoclonal antibodies prevent spontaneous and adoptive transfer of diabetes in the BB/Wor rat.

We studied the effects of anti-CD2 monoclonal antibodies (MAb) on spontaneous and induced autoimmune diabetes mellitus in diabetes-prone (DP) and diabetes-resistant (DR) BB/Wor rats. In DP rats, all anti-CD2 MAb prevented spontaneous diabetes and the adoptive transfer of diabetes with Con-A--stimulated acute diabetic spleen cells; OX34 prevented Poly I:C induced accelerated onset of diabetes and the adoptive transfer of diabetes with Con-A--stimulated RT6.1+ T cell depleted DR splenocytes. In DP rats, all anti-CD2 MAb except OX53 depleted CD4+ T cells, without depleting natural killer cells or CD8+ T cells. OX34 injected DR rats were profoundly depleted of CD4+ T cells without evidence of decreased CD8+ T cells, but were not protected against the induction of diabetes by RT6.1+ T-cell depletion and Poly I:C injections. We conclude that anti-CD2 MAbs protect against BB/Wor autoimmune diabetes by depleting CD4+ T cells, preventing the activation of effector cells, or by blocking CD2/ligand interaction between effector and target cells.

Animals↗
PubMed1991

Point mutation in meningococcal por A gene associated with increased endemic disease.

The por A gene, which encodes expression of meningococcal class 1 outer membrane protein, responsible for antigenic subtype specificity, has been cloned and sequenced in an isolate of Neisseria meningitidis (B:15:P1.7,16) from a patient in the Gloucester area with meningococcal meningitis. Comparison of the sequence with that of the equivalent gene from the P1.7,16 reference strain reveals a point mutation which generates a single aminoacid change in the epitope responsible for P1.16 specificity. Monoclonal antibodies with P1.16 specificity do not react with synthetic peptides that correspond to the altered epitope, and do not promote complement-mediated bactericidal killing of the isolate. Analysis of other strains shows widespread distribution of infections due to B:15:P1.7,16 meningococci with the altered epitope (P1.16b) in England and Wales.

Antibodies, Monoclonal↗
PubMed1990

Deduced amino acid sequences of class 1 protein (PorA) from three strains of Neisseria meningitidis. Synthetic peptides define the epitopes responsible for serosubtype specificity.

The previously determined nucleotide sequence of the porA gene, encoding the class 1 outer membrane protein of meningococcal strain MC50, has been used to clone and sequence the porA gene from two further strains with differing serosubtype specificities. Comparison of the predicted amino acid sequences of the three class 1 proteins revealed considerable structural homology with major variation confined to two discrete regions (VR1 and VR2). The high degree of structural homology between the sequences gave predicted secondary structures that were almost identical, with the variable domains located in hydrophilic regions that are likely to be surface located and hence accessible to antibody binding. The predicted amino acid sequences have been used to define the epitopes recognized by mAbs with serosubtype specificity. A series of overlapping decapeptides spanning each of the class 1 protein sequences have been synthesized on solid-phase supports and probed with mAbs. Antibodies with P1.16 and P1.15 subtype specificity reacted with sequences in the VR2 domain, while antibodies with P1.7 subtype specificity reacted with sequences in the VR1 domain. Further peptides have been constructed to define the minimum epitopes recognized by each antibody. Thus we have been able to define linear peptides on each class 1 protein molecule that are responsible for subtype specificity and that represent targets for a protective immune response.

Amino Acid Sequence↗
PubMed1990

Stable expression of meningococcal class 1 protein in an antigenically reactive form in outer membranes of Escherichia coli.

The entire gene encoding the class 1 outer membrane protein of Neisseria meningitidis is located on a 2.2kb fragment, obtained on digestion of chromosomal DNA with Xbal. This Xbal fragment from strain MC50 (subtype P1-16), which had previously been cloned in bacteriophage M13, has been transferred to the plasmid vector pMTL20. The resulting plasmid (pPORA100) was propagated in Escherichia coli (JM109) and cell lysates were subjected to SDS-PAGE. Western blotting with anti-class 1 protein antibodies revealed constitutive expression of a protein of 41 kD, corresponding to the class 1 protein of the parent meningococcal strain, which was absent in the E. coli control. Fractionation of E. coli cells carrying the recombinant plasmid revealed that the protein was exclusively located in the outer membrane, and N-terminal amino acid analysis of the expressed protein revealed that normal processing of the signal peptide had occurred. Immuno-gold electron microscopy showed that the protective epitope recognized by a P1-16 subtype-specific monoclonal antibody was exposed in an antigenically reactive form on the surface of E. coli cells carrying plasmid pPORA100. In contrast, expression in E. coli of a second plasmid (pPORA104) lacking the coding sequence for the first 15 amino acids of the signal peptide resulted in accumulation of recombinant class 1 protein only in the cytoplasm of the cells. Thus the presence of the meningococcal signal sequence ensures expression of this meningococcal porin protein in an antigenically native conformation in outer membranes of E. coli, while its absence results in expression of a soluble protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

The class 1 outer membrane protein of Neisseria meningitidis: gene sequence and structural and immunological similarities to gonococcal porins.

The class 1 protein is a major protein of the outer membrane of Neisseria meningitidis, and an important immunodeterminant in humans. The complete nucleotide sequence for the structural gene of a class 1 protein has been determined. The sequence predicts a protein of 374 amino acids, preceded by a typical signal peptide of 19 residues. The hydropathy profile of the predicted protein sequence resembles that of the Escherichia coli and gonococcal porins. The predicted protein sequence of the class 1 protein exhibits considerable structural similarity to the gonococcal porins PIA and PIB. Western blot studies also reveal immunologically conserved domains between the class 1 protein, PIA and PIB. A restriction fragment from the class 1 gene hybridizes to gonococcal genomic fragments in Southern blots. In addition to the class 1 gene coding region there is a large open reading frame on the opposite strand.

Amino Acid Sequence↗
PubMed1987

Molecular cloning and expression of Neisseria meningitidis class 1 outer membrane protein in Escherichia coli K-12.

A genomic library of meningococcal DNA from a clinical isolate of Neisseria meningitidis was constructed in the expression vector lambda gt11. Outer membrane complex was prepared from the same strain and used to immunize rabbits to raise polyclonal anti-outer membrane complex serum. The amplified library was probed with this polyclonal serum, and seven expressing recombinants were isolated; further investigations indicated these to be identical. The expressed meningococcal gene in these recombinants was fused to vector beta-galactosidase and shown to encode epitopes present on the 42-kilodalton class 1 outer membrane protein. Estimation of the size of the recombinant fusion protein suggests that up to 40 kilodaltons of protein-coding sequence is present. The lambda gt11 recombinant contains a 3.4-kilobase DNA insert, which has been recloned into a plasmid and characterized by restriction endonuclease analysis. A restriction fragment from the insert, representing the protein-coding region hybridizes to a single 2.2-kilobase XbaI fragment from the homologous strain and to similar-sized XbaI fragments in other strains of meningococci, expressing antigenically distinct class 1 proteins.

Antigens, Bacterial↗