Biomedical subjects

L E Brown

Publications and source records attributed to L E Brown.

At least 55 records · Page 3Linked to original sources

PubMed1993

Modulation of CD4+ T-cell recognition of influenza hemagglutinin by carbohydrate side chains located outside a T-cell determinant.

Two distinct T-cell clones raised to the H3 subtype influenza virus A/Memphis/71 fail to proliferate in response to other H3 viruses that contain the amino acid substitution Asp63-->Asn within the heavy (HA1) chain of the hemagglutinin (HA). These nonstimulatory viruses nevertheless have an intact sequence corresponding to the determinants recognized by the clones. The substitution at residue 63 results in the creation of a potential glycosylation site which occurs outside the boundaries of the T-cell determinants. A synthetic peptide encompassing the two determinants and extended to include residue Asn63 stimulated levels of proliferation of the clones similar to those obtained with a peptide containing Asp63. These results indicate that the presence of Asn rather than Asp does not in itself affect T-cell recognition. Partial enzymatic deglycosylation of isolated HA possessing the glycosylation site restored the ability of the T-cell clones to proliferate. More extensive deglycosylation of HA1 had a differential effect on the clones, allowing one to proliferate but not the other. These results illustrate that even when attached to residues outside a determinant, the carbohydrate of a glycoprotein antigen can influence T-cell recognition. In this way, changes in glycosylation patterns of HA from different isolates of the virus may influence the efficacy of preexisting T-cell immunity.

Amino Acid Sequence↗

PubMed1993

Conservation of determinants for class II-restricted T cells within site E of influenza virus hemagglutinin and factors influencing their expression.

The determinants recognized by helper T cells specific for the site E region of H3 subtype influenza virus hemagglutinin (HA) have been defined by examining the reactivity of T-cell clones with sets of overlapping peptides of various lengths covering the site. Two overlapping sequences, TLIDALLG and LIDALLGDP, were identified as the minimal determinants for four of five representative clones. These sequences are located within a loop of the molecule closed by a disulfide bond and presumably require cleavage of this bond for interaction with the class II major histocompatibility molecule. In contrast, the determinant recognized by the fifth clone was dependent on the presence of an intact disulfide bond for its expression and could not be represented by a synthetic peptide homolog of the linear sequence. Both TLIDALLG and LIDALLGDP are conserved within all field strains of the H3 subtype. Nevertheless, recognition of these sequences by the T-cell clones is affected by the glycosylation pattern of the hemagglutinin and by residues lying outside the minimal determinant. Three distinct clones directed towards the sequence LIDALLGDP were remarkably similar in their pattern of response to a set of synthetic analogs of the determinant, suggesting that residues of the T-cell receptor other than those contacting the minimal determinant may be responsible for the different specificities observed for these clones with different field strains of virus.

Amino Acid Sequence↗

PubMed1993

Immunization of BALB/c mice with a monoclonal anti-DNA antibody induces an anti-idiotypic antibody reactive with a cell-surface DNA binding protein.

DNA binds to cell-surface proteins on human and murine leukocytes and induces secretion of the cytokine interleukin 6 (IL-6). Cell-surface DNA binding molecules have been shown to serve as target antigens for the production of autoantibodies in patients with systemic lupus erythematosus (SLE), and in lupus-prone mice. Recent studies have demonstrated that a subset of anti-anti-DNA antibodies, isolated from patients with SLE, are idiotypically related to antibodies reactive with a cell-surface DNA binding molecule. We now report that immunization of normal mice with a murine monoclonal anti-DNA antibody induces an anti-idiotypic response which has reactivity with a cell-surface DNA binding molecule. An anti-idiotypic anti-DNA monoclonal antibody (LB17) was isolated from the spleen of an immunized mouse. This monoclonal antibody blocked the binding of DNA to murine splenocytes and mimicked the functional effect of DNA by stimulating the secretion of IL-6. These experiments provide further evidence for an idiotypic connectivity between antibodies to cell-surface DNA binding proteins and anti-DNA antibodies. It is hypothesized that this idiotypic system is part of the network of natural autoantibodies and that its perturbation may give rise to pathogenic antibodies.

Animals↗

PubMed1992

Nucleosomes and DNA bind to specific cell-surface molecules on murine cells and induce cytokine production.

The molecular basis for the cellular interaction of DNA and nucleosomes and the physiological consequences of this binding were examined. Both DNA and nucleosomes were demonstrated to bind specifically to the surface of human peripheral blood mononuclear cells and the murine T cell line S49. Western blots of S49 cell membranes, using probes of biotin-labeled DNA and nucleosomes, showed reactivity at 29 and 69 kDa. Functionally, the interaction of DNA and nucleosomes with murine spleen cells stimulated the release of significant amounts of IL-6 activity. There is evidence that nucleosomes, a product of apoptosis, are the major component of circulating DNA found in the plasma of patients with systemic lupus erythematosus (SLE). The interaction of nucleosomes with cell-surface DNA binding molecules may have physiological relevance to some of the immune aberrations observed in patients with SLE.

Animals↗

PubMed1992

DNA binding to mouse cells is mediated by cell-surface molecules: the role of these DNA-binding molecules as target antigens in murine lupus.

Autoimmunity to a 28-29-kDa cell-surface DNA-binding molecule has previously been described in patients with systemic lupus erythematosus and related autoimmune diseases. This report describes experiments that implicate a similar antigen-antibody system in the evolution of autoimmunity in lupus-prone mice. DNA binding to murine spleen cells was found to be a saturable phenomenon that was inhibited by excess cold DNA and trypsinization. The role of autoimmunity to murine cell-surface DNA-binding molecules in lupus-prone mice (MRL lpr/lpr, MRL +/+, BXSB) was compared to normal mice (BALB/c, C3H.SW) by means of an assay that measured the inhibition of cell-surface DNA binding. Only sera from lupus strains had inhibitory activity and this component was shown to be an IgM autoantibody. Furthermore, we isolated a spontaneously occurring IgM monoclonal antibody from the spleen of an MRL/lpr mouse, which inhibited DNA binding to mouse cells. Time-course studies indicated that young female MRL/lpr mice lacked detectable activity against cell-surface DNA-binding molecules; however, by 8-10 weeks maximal inhibitory activity was observed. This response occurred prior to the development of significant antinuclear antibody activity. With the appearance of overt disease and anti-DNA antibodies, inhibition of DNA-binding activity became undetectable. These findings mirror previous studies on autoimmunity to a cell-surface DNA-binding molecule on human leucocytes, but have the added advantage of permitting the study of the temporal evolution of this inhibitory activity in relation to disease expression.

Animals↗

PubMed1991

Rationale and design of a secondary prevention trial of lowering normal plasma cholesterol levels after acute myocardial infarction: the Cholesterol and Recurrent Events trial (CARE)

Recent clinical trials of primary and secondary prevention of cardiovascular disease have demonstrated that lowering plasma cholesterol decreases the incidence of coronary heart disease in patients with elevated plasma cholesterol. However, it is not known whether patients with established coronary artery disease and normal plasma cholesterol can be benefited. Several previous prevention trials reviewed in this report found that patients who had plasma cholesterol levels at baseline in the upper portion of the eligibility range (e.g., greater than 240 mg/dl) received greater benefit from hypolipidemic diet or drug therapy than patients who had lower plasma cholesterol levels at baseline. The recent availability of drugs that are more potent and less prone to cause adverse reactions than previous regimens permits this important question to be addressed. The Cholesterol and Recurrent Events trial is testing whether pravastatin, a hydroxymethylglutaryl coenzyme A reductase inhibitor, will decrease the sum of fatal coronary heart disease and nonfatal myocardial infarction (MI) in patients who have recovered from a MI and who have normal total cholesterol levels. Fatal cardiovascular disease and total mortality are important secondary end points. The trial is enrolling 4,000 men and women from 80 centers throughout North America, age 21 to 75 years, who have survived MI for 3 to 20 months, who have plasma total cholesterol less than 240 mg/dl (6.2 mmol/liter) and low-density cholesterol of 115 to 174 mg/dl (3.0 to 4.5 mmol/liter), and who are representative of the general population of patients with MI. Patients are randomized to either active or inactive drug therapy. Active therapy consists of pravastatin, 40 mg/day, designed to achieve an average decrease in low-density lipoprotein cholesterol of approximately 30%, and an increase in high-density lipoprotein of 5%. The average duration of follow-up will be greater than or equal to 5 years. To protect against a lower than expected rate of recurrent events, the trial will be continued until a predetermined fixed number of coronary heart disease events occurs in the entire cohort so that the original sensitivity of the trial will be maintained.

Adult↗

PubMed1991

Probing the idiotype/anti-idiotype antibody interaction with a set of synthetic peptide homologues.

Anti-idiotypic (anti-Id) antibodies were raised against two murine monoclonal antibodies (mAb 1/1 and mAb 2/1) which recognise two distinct and well-characterised epitopes on a 24-residue synthetic peptide representing part of the haemagglutinin (HA) of influenza virus. A monoclonal anti-Id antibody, specific for mAb 2/1, could bind to mAb 2/1 when the paratope of the latter was occupied with peptide, indicating that this anti-Id antibody is directed to a framework idiotope. In contrast, an anti-Id mAb derived from mAb 1/1-immunised mice was inhibited in its binding to Id by the parent peptide and also by the heptapeptide NVPEKQT which constitutes the epitope recognised by mAb 1/1. The small size of this synthetic peptide eliminates the possibility of significant steric inhibition in the system, and establishes that this mAb is a true paratope-directed anti-Id antibody. The interaction of this anti-Id mAb with the paratope of mAb 1/1 in the presence of a set of peptide homologues of the epitope was also examined. A peptide as short as 5 residues, which contains two of the three irreplaceable residues of the epitope, could inhibit binding between the two mAbs.

Amino Acid Sequence↗

PubMed1991

Extension of a minimal T cell determinant allows relaxation of the requirement for particular residues within the determinant.

The determinant recognized by a class II restricted helper T cell clone raised against a peptide corresponding to the C-terminal 24 residues of the heavy chain of influenza virus hemagglutinin (HA) was examined in detail. The sequence 309VKQNTLKL316 was identified as the minimal determinant for T cell activation but its stimulatory capacity was augmented by extension at either end. Sets of peptide analogs, in which each residue within the minimal determinant was replaced in turn by every one of the other naturally occurring amino acids, revealed either an absolute requirement for the native residue or a very limited degree of replaceability, at seven of the eight positions. Only the N-terminal residue 309V could be replaced with almost any other amino acid without loss of reactivity; in fact, substitution at this position with residues containing bulky side groups enhanced the response. The reactivity of the clone with analogs of the longer peptide 307KYVKQNTLKL316, which induces maximal levels of stimulation, revealed a very different pattern of replaceability for certain residues; in particular, the requirement for a lysine at position 310 was no longer apparent. This study presents a complete analysis of the importance of each individual residue to the integrity of a T cell determinant and provides evidence that the critical requirement for a particular amino acid at a given location may be overridden by N-terminal extension of the minimal determinant. These findings indicate that, within different homologs of the native sequence, particular residues may assume quite different roles.

Amino Acid Sequence↗

PubMed1991

Introduction of exogenous DNA into Chlamydomonas reinhardtii by electroporation.

The fate of exogenous DNA introduced into Chlamydomonas reinhardtii by electroporation was analyzed. With single and double electrical pulses, plasmids as large as 14 kb were introduced into cells with and without intact cell walls. Within hours after introduction, exogenous plasmid DNA was associated with nuclei isolated from cells; several weeks after introduction, exogenous DNA was stably integrated into the Chlamydomonas genome. These studies establish electroporation as a method for introducing DNA, and potentially other molecules, into C. reinhardtii.

Chlamydomonas↗

PubMed1990

An analysis of the properties of monoclonal antibodies directed to epitopes on influenza virus hemagglutinin.

Monoclonal antibodies (MAbs) specific for the hemagglutinin (HA) of the H3 subtype of influenza A virus were grouped according to their inability to bind to particular MAb-selected neutralization escape mutants of the virus having an amino acid substitution in one of the five postulated antigenic sites on the molecule. Additional residues critical to the binding of the MAbs were deduced from their patterns of reactivity with a panel of field strains and receptor mutants of the H3 subtype. The relationship of these residues to the actual epitopes recognized by the MAbs was inferred from their location on the three-dimensional structure of the HA molecule. In this way it was generally possible to identify a number of residues that are critical to the integrity of the epitope recognized by each of the MAbs examined. It was found that: (1) Several of these epitopes appear to be discontinuous and some may depend on residues contributed by more than one monomer. For example, residue 205, in the interface between monomers of the HA, was found to affect the integrity of the epitopes for several MAbs, possibly by stabilizing the conformation of residues around the receptor-binding pocket and/or in site B on the adjacent monomer. The activity of these particular MAbs was greatly decreased if the virus was exposed to pH 5. (2) All the MAbs tested neutralized viral infectivity and inhibited hemagglutination, although the single MAb directed to site C, which is the most distant from the receptor-binding site, was the least efficient. (3) Hemagglutination inhibition, and particularly neutralization tests, were more discriminating than ELISA in discerning subtle differences between the corresponding epitopes recognized by MAbs on different field strains. (4) Efficiency of neutralization of infectivity did not correlate consistently with hemagglutination inhibiting efficiency; MAbs postulated to bind to epitopes close to the receptor-binding pocket were very efficient at inhibiting hemagglutination, whereas neutralization efficiency tended to be more influenced by the affinity of binding of the MAb. (5) A MAb binding to any particular epitope could affect the binding of a second MAb directed to an epitope within the same or even a different antigenic site. The observed effect was most commonly inhibition of binding, which was not always reciprocal; enhancement of binding was also observed with certain combinations of MAbs. The relative affinity of the MAbs, in addition to steric constraints, were shown to be important factors in the ability to compete for interaction with HA.

Amino Acid Sequence↗

PubMed1990

The stoichiometry of binding between monoclonal antibody molecules and the hemagglutinin of influenza virus.

The number of neutralizing monoclonal IgG molecules that can bind to a single trimeric molecule of influenza viral hemagglutinin (HA) was calculated by estimating the molecular weight of the immune complexes formed under conditions of antibody excess and was found to be dependent upon the antigenic site to which the MAb is directed. Whereas three antibody molecules directed to site A or site E are able to bind simultaneously to a single trimer of HA, generally only one molecule directed to site B (the "tip") or site B/D ("tip/interface") can be accommodated. Using mixtures of MAbs, more IgG molecules can be accommodated, but steric hindrance limits simultaneous binding of different MAbs directed to the same antigenic site or even to neighboring sites. At limiting antibody concentration, some MAbs can form much larger aggregates in which several HA molecules are crosslinked by antibody. However, the fact that certain MAbs do not crosslink HA molecules in this way indicates that MAbs directed to different epitopes within the same general antigenic site differ significantly in their geometry of binding.

Antibodies, Monoclonal↗

PubMed1990

Novel multivalent effects of pyrazinoylguanidine in patients with azotemia.

In patients with azotemia, urea excretion, urea clearance, and urea/creatinine clearance ratio were increased by pyrazinoylguanidine in a dose-related manner. Urine volume and excretion of sodium greater than chloride greater than potassium tended to increase during administration of pyrazinoylguanidine. Systemic arterial pressure declined while pyrazinoylguanidine was given at 300 or 600 mg b.i.d. for 3 days. At both doses pyrazinoylguanidine reduced plasma renin activity during the first 2 hours. Between days 1 and 3 only the high dose of pyrazinoylguanidine decreased plasma renin activity and plasma aldosterone levels. These findings with pyrazinoylguanidine are consistent with those of secretion of urea in human subjects across the renal tubules and indicate that this process is susceptible to pharmacologic alteration, even in the presence of severe renal insufficiency.

Adult↗

PubMed1989

Class II-restricted T-cell clones to a synthetic peptide of influenza virus hemagglutinin differ in their fine specificities and in the ability to respond to virus.

Fifteen T-cell clones were derived from BALB/c or DBA/2 mice immunized with a synthetic peptide corresponding to the C-terminal 24 residues (residues 305 to 328) of the HA1 chain of H3 subtype influenza virus hemagglutinin. All of the clones proliferated when the peptide was presented in association with I-Ed. By using shorter homologs, it was shown that the T-cell response was focused predominantly on the region at the N-terminal end of the peptide encompassed by residues 306 to 319. Individual clones recognizing this region differed in their absolute requirements for residues at the extremities of the site and also in their patterns of efficiency of recognition of shorter homologs. One particular clone defined another site of T-cell recognition within residues 314 to 328. The response of the clones to peptide analogs identified certain residues within the sites that were critical for recognition, with the substitution Gln-311----Ser having a differential effect on clones responding to the N-terminal site. Only one of the clones responded well to influenza virus itself. This clone also required relatively low concentrations of the parent peptide for optimum stimulation and was suppressed by higher concentrations. The data demonstrate striking heterogeneity in the T-cell response even to a short synthetic peptide, with different T-cell clones recognizing slightly different but overlapping areas of the molecule.

Amino Acid Sequence↗

PubMed1989

Neutralizing monoclonal antibodies against three serotypes of porcine rotavirus.

Using three serotypes (four strains) of cultivable porcine rotavirus as immunizing antigens, 10 neutralizing monoclonal antibodies were characterized. One VP4-specific monoclonal antibody directed against porcine rotavirus BEN-144 (serotype G4) neutralized human rotavirus strain ST-3 in addition to the homologous porcine virus. All nine VP7-specific monoclonal antibodies were highly specific for viruses of the same serotype as the immunizing rotavirus strain. One exception was the VP7-specific monoclonal antibody C3/1, which neutralized both serotype G3 and G5 rotaviruses. However, this monoclonal antibody did not neutralize the porcine rotavirus AT/76, also of serotype G3, nor mutants of SA-11 virus (serotype G3) which were selected with monoclonal antibody A10/N3 and are known to have mutations affecting the C antigenic region.

Animals↗

PubMed1988

Epitopes of an influenza viral peptide recognized by antibody at single amino acid resolution.

Antibodies raised against the synthetic peptide corresponding to the carboxy-terminal 24 amino acids (305-328) of the heavy chain of the hemagglutinin molecule of influenza virus A/X-31 (H3) bind this peptide at three antigenic sites. These sites were identified by assaying binding of polyclonal BALB/c mouse antipeptide sera to the complete set of all possible di-, tri, tetra-, penta-, hexa-, hepta-, and octapeptides homologous with the 24-residue sequence. Individual epitopes were defined and essential residues identified by testing the binding of monoclonal antibodies to sets of peptide analogues in which every one of the homologous residues was replaced in turn by each of the 19 alternative genetically coded amino acids. The immunodominant epitope was shown to be a linear sequence of five amino acids, 314LKLAT318. Replacement of any one of these residues with any other amino acid resulted in loss of antibody binding, indicating that all five are essential to the interaction and that they are probably contact residues. Another antigenic site contains at least two overlapping epitopes: polyclonal sera recognize predominantly an epitope or epitopes encompassed by the linear sequence 320MRNVPEKQT328, whereas the epitope defined by a particular monoclonal antibody comprises the seven amino acids 322NVPEKQT328, of which N322, E325, and Q327 were implicated as contact residues.

Amino Acid Sequence↗

PubMed1988

Distinct epitopes recognized by I-Ad-restricted T-cell clones within antigenic site E on influenza virus hemagglutinin.

A total of 14 I-Ad-restricted helper T-cell clones specific for the hemagglutinin (HA) molecule of influenza virus were isolated from spleens of BALB/c or (BALB/c X C57BL/10)F1 mice immunized with the H3 subtype influenza virus A/Memphis/71 (Mem 71) and from lymph nodes of BALB/c mice primed with purified HA. The specificity of these T-cell clones was assessed in proliferation assays by reactivity with naturally occurring strains of viruses that arose by antigenic drift and contain known amino acid sequence changes in HA and with a panel of monoclonal antibody (MAb)-selected mutants of Mem 71 with single amino acid substitutions in HA. The HA genes of those mutant viruses that failed to stimulate one or more of the T-cell clones were sequenced. The clones could be allocated to at least four groups, each group having a distinct pattern of reactivity with the panel of natural field strains. The epitopes recognized by the four groups of clones were found, by reactivity with MAb-selected mutants, to be in very close proximity to one another and probably overlapping. All of the distinct epitopes recognized by the T-cell clones were adversely affected by a single amino acid substitution, either at residue 60 or at residue 63 in the HA1 polypeptide chain, within the region known from antibody-binding studies as site E. Some, but not all, of the epitopes may be influenced by the addition of a carbohydrate side chain to the HA of a particular MAb-selected mutant and certain field strains containing an Asp----Asn substitution at residue 63. Site E is therefore a major site of H-2d helper T-cell recognition on the H3 HA.

Amino Acid Sequence↗

PubMed1988

Genetic control and fine specificity of the immune response to a synthetic peptide of influenza virus hemagglutinin.

The immune response to a synthetic peptide, H3 HA1(305-328), representing the C'-terminal 24 amino acid residues of the HA1 chain of the hemagglutinin of the H3 subtype of influenza virus is controlled by genes in the I region of the major histocompatibility complex. Mice of the H-2d haplotype are high responders and produce antibody for several months after a single injection of peptide without carrier. Mice of the H-2b, H-2k, and H-2q haplotypes are low antibody responders. Investigation of recombinant and congenic mouse strains revealed that high responsiveness requires the genes that encode the I-Ed molecule. Immunoassays, involving direct binding to analogs of this peptide and inhibition by both these analogs and synthetic epitopes, were used to analyze the specificity of the polyclonal response. In BALB/c mice, the primary antibody response is directed principally against the antigenic site 314-LKLAT-318, whereas the secondary response after a boost is predominantly directed to a distinct site, 320-MRNVPEKQT-328. The T-cell response to the peptide H3 HA1(305-328), as measured by antigen-induced proliferation of primed T cells in vitro, is also I-Ed restricted in high-responder H-2d mice and is directed against an antigenic site that does not require the four C-terminal residues unique to the H3 influenza subtype. A different epitope appears to be recognized by T cells from CBA (H-2k) mice, which proliferate to a moderate extent on exposure to the peptide but, nevertheless, do not provide help for an antibody response.

Animals↗

PubMed1987

Characterization of subtype-specific and cross-reactive helper-T-cell clones recognizing influenza virus hemagglutinin.

The specificity and function of two T-cell clones derived from A/Memphis/1/71 (H3) influenza virus (Mem 71)-immune BALB/c spleen cells have been compared. One clone, X-31 clone 1, was subtype specific, proliferating in response to influenza strains of the H3 subtype only. The other, Jap clone 3, cross-reacted in proliferation assays with heterologous subtypes of influenza A, but not type B. Both clones recognized the HA1 chain of the hemagglutinin (HA) molecule and their proliferation in response to detergent-disrupted virus could be specifically inhibited by monoclonal antibodies to the HA. The T-cell clones were of the L3T4+ phenotype. Both recognized antigen in association with I-Ed, as indicated by studies with H-2 recombinant strains of mice and by blocking with monoclonal anti-I-E antibody. In vivo, both clones elicited a delayed-type hypersensitivity (DTH) reaction when inoculated into mouse footpads together with virus, X-31 clone 1 again displaying subtype specificity and Jap clone 3 being cross-reactive. The clones were also able to provide factor-mediated help in vitro to virus-primed B cells in an anti-HA antibody response. The cross-reactive T-cell clone provided help not only for B cells primed with influenza A subtype H3 and responding to H3 virus in culture, but also for H2 virus-primed B cells making anti-H2 antibody.

Animals↗