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Biomedical subjects

Kenneth H Roux

Publications and source records attributed to Kenneth H Roux.

12 recordsLinked to original sources
PubMed2003

Antibody domain exchange is an immunological solution to carbohydrate cluster recognition.

Human antibody 2G12 neutralizes a broad range of human immunodeficiency virus type 1 (HIV-1) isolates by binding an unusually dense cluster of carbohydrate moieties on the "silent" face of the gp120 envelope glycoprotein. Crystal structures of Fab 2G12 and its complexes with the disaccharide Manalpha1-2Man and with the oligosaccharide Man9GlcNAc2 revealed that two Fabs assemble into an interlocked VH domain-swapped dimer. Further biochemical, biophysical, and mutagenesis data strongly support a Fab-dimerized antibody as the prevalent form that recognizes gp120. The extraordinary configuration of this antibody provides an extended surface, with newly described binding sites, for multivalent interaction with a conserved cluster of oligomannose type sugars on the surface of gp120. The unique interdigitation of Fab domains within an antibody uncovers a previously unappreciated mechanism for high-affinity recognition of carbohydrate or other repeating epitopes on cell or microbial surfaces.

Amino Acid Sequence↗
PubMed2003

A sensitive sandwich ELISA for the detection of trace amounts of cashew (Anacardium occidentale L.) nut in foods.

Trace amounts of cashew nut protein can provoke severe allergic reactions in sensitive patients. Consequently, commercial food processors and regulatory agencies must be vigilant to prevent cashew nut cross-contamination among foods and ensure proper labeling. Toward this end, we have developed a sandwich enzyme-linked immunosorbent (ELISA) to detect the predominant cashew protein fraction (anacardein or cashew major protein, CMP) that can be extracted in aqueous buffer from food matrixes. Protein G-purified goat antiwhole cashew extract IgG and rabbit anti-CMP IgG were used as capture and secondary antibodies, respectively. Immunoadsorption against several nut and seed proteins significantly minimized the inherent cross-reactivity of these reagents. Food samples spiked with cashew flour and CMP were extracted and tested in a sandwich ELISA where standard curves were based on reactivity with CMP. The assay was optimized to detect as little as 20 ng/mL (0.02 ppm) of CMP and was successfully used to quantify CMP, and thus cashew, in various food matrixes.

Allergens↗
PubMed2003

Tree nut allergy.

Tree nuts are clinically associated with severe immunoglobulin E-mediated systemic allergic reactions independent of pollen allergy and with reactions that are usually confined to the oral mucosa in patients with immunoglobulin E directed toward cross-reacting pollen allergens. The latter reactions can progress to severe and life-threatening episodes in some patients. Many patients with severe tree nut allergy are co-sensitized to peanut. Clinical studies on cross-reactivity between the tree nuts are few in number, but based on reports to date, avoidance of the other tree nuts once sensitivity is diagnosed appears prudent unless specific challenges are performed to ensure clinical tolerance. Even then, great care must be taken to avoid cross-contamination. As with other severe food allergies, a recurrent problem in clinical management is the failure of physicians to prescribe self-injectable epinephrine to patients who are at risk of anaphylaxis.

Clinical Trials as Topic↗
PubMed2003

Tree nut allergens.

Allergic reactions to tree nuts can be serious and life threatening. Considerable research has been conducted in recent years in an attempt to characterize those allergens that are most responsible for allergy sensitization and triggering. Both native and recombinant nut allergens have been identified and characterized and, for some, the IgE-reactive epitopes described. Some allergens, such as lipid transfer proteins, profilins, and members of the Bet v 1-related family, represent minor constituents in tree nuts. These allergens are frequently cross-reactive with other food and pollen homologues, and are considered panallergens. Others, such as legumins, vicilins, and 2S albumins, represent major seed storage protein constituents of the nuts. The allergenic tree nuts discussed in this review include those most commonly responsible for allergic reactions such as hazelnut, walnut, cashew, and almond as well as those less frequently associated with allergies including pecan, chestnut, Brazil nut, pine nut, macadamia nut, pistachio, coconut, Nangai nut, and acorn.

Allergens↗
PubMed2002

Characterization of the soluble allergenic proteins of cashew nut (Anacardium occidentale L.).

The allergens associated with cashew food allergy have not been well-characterized. We sought to identify the major allergens in cashew nut by performing IgE immunoblots to dissociated and reduced or nonreduced cashew protein extracts, followed by sequencing of the peptides of interest. Sera from 15 subjects with life-threatening reactions to cashews and 8 subjects who tolerate cashews but have life-threatening reactions to other tree nuts were compared. An aqueous cashew protein extract containing albumin/globulin was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subjected to IgE immunoblotting using patient sera. Selected IgE reactive bands were subjected to N-terminal amino acid sequencing. Each of the 15 sera from cashew-allergic subjects showed IgE binding to the cashew protein extract. The dominant IgE-binding antigens in the reduced preparations included peptides in the 31-35 kD range, consistent with the large subunits of the major storage 13S globulin (legumin-like protein). Low-molecular-weight polypeptides of the 2S albumin family, with similarity to the major walnut allergen Jug r 1, also bound IgE. The sera from eight patients who tolerate cashew but displayed allergies to other tree nuts showed only minimal or no IgE binding to cashew. Cashew food allergy is associated with the presence of IgE directed against the major seed storage proteins in cashew, including the 13S globulin (legumin group) and 2S albumins, both of which represent major allergen classes in several plant seeds. Thus, the legumin-group proteins and 2S albumins are again identified as major food allergens, which will help further research into seed protein allergenicity.

Adult↗
PubMed2002

Biochemical characterization of amandin, the major storage protein in almond (Prunus dulcis L.).

The almond major storage protein, amandin, was prepared by column chromatography (amandin-1), cryoprecipitation (amandin-2), and isoelectric precipitation (amandin-3) methods. Amandin is a legumin type protein characterized by a sedimentation value of 14S. Amandin is composed of two major types of polypeptides with estimated molecular weights of 42-46 and 20-22 kDa linked via disulfide bonds. Several additional minor polypeptides were also present in amandin. Amandin is a storage protein with an estimated molecular weight of 427,300 +/- 47,600 Da (n = 7) and a Stokes radius of 65.88 +/- 3.21 A (n = 7). Amandin is not a glycoprotein. Amandin-1, amandin-2, and amandin-3 are antigenically related and have similar biochemical properties. Amandin-3 is more negatively charged than either amandin-1 or amandin-2. Methionine is the first essential limiting amino acid in amandin followed by lysine and threonine.

Amino Acid Sequence↗
PubMed2002

Idiotypic-anti-idiotypic complexes and their in vivo metabolism.

BACKGROUND: Different strategies can be used to improve the tumor:non-tumor ratio of radiolabeled antibodies in immunotargeting. One approach is to use secondary antibodies to clear out redundant, circulating primary antibodies. In the current study, the in vitro complex formation and in vivo clearing capabilities and metabolism of the monoclonal antibody TS1 and its monoclonal anti-idiotype, alphaTS1, were studied. METHODS: Complex formation studies were performed using polyacrylamide gel electrophoresis (PAGE), gel permeation chromatography, and electron microscopy. The clearance and metabolism of the complexes were studied in nude mice. RESULTS: PAGE and gel permeation chromatography showed that more than 70% of the antibodies formed complexes. The electron microscopy studies revealed that the complexes formed between TS1 and alphaTS1 are mainly ring-shaped (66.6-73.4%), comprising 4 to > 8 antibodies. These rings consist of equal numbers of idiotype and anti-idiotype. The most commonly observed complexes were tetrameric rings (26.8-40.5%), hexameric rings (10.7-11.9%), and rings containing more than eight monoclonal antibodies (6.6-14-4%). The in vivo study illustrated that within 24 hours 80% of the total nuclide content had been degraded and excreted via the urine, compared with 25% for similarly treated mice that did not receive any anti-idiotype. CONCLUSIONS: Interestingly, the electron microscopy study demonstrated that dimers were rare (0.4-1.2%), probably reflecting a location of epitopes incompatible with tight, sterically constrained dimeric interactions; insufficient flexibility of the immunoglobulin G1 subtype hinge regions; or both. The anti-idiotypic clearing mechanisms proved efficient in nude mice. In vivo metabolic studies indicate that the accumulation and degradation of TS1/alphaTS1 immune complexes, to a large extent, take place in the liver, where a substantial amount was detected as soon as 1 hour after anti-idiotype injection.

Animals↗
PubMed2002

Variation in the ordered structure of complexes between CD154 and anti-CD154 monoclonal antibodies.

The cell surface co-stimulatory protein CD154 (CD40L) is a target for monoclonal antibody (mAb) inhibitors of T-cell mediated immune diseases. This protein, like most other members of the TNF ligand family, forms homotrimeric complexes on the cell surface and in solution, with a three-fold axis of symmetry. We find that several different anti-CD154 monoclonal antibodies form distinctive complexes with soluble CD154. These soluble complexes have been analyzed using size exclusion chromatography, static and dynamic light scattering, and electron microscopy and shown to consist of caged structures of various geometries. The cell surface complexes have been analyzed by confocal microscopy and, depending on the mAb, remain as small, separate complexes or form large aggregates. The formation of these complexes in solution is likely to have an impact on measures of affinity, while the cell surface complexes could affect binding potency and provoke other biological effects.

Antibodies, Monoclonal↗
PubMed2002

Linear IgE epitope mapping of the English walnut (Juglans regia) major food allergen, Jug r 1.

BACKGROUND: Peanut and tree nut allergies can be life-threatening, and they appear to be growing in prevalence. Jug r 1, a 2S albumin seed storage protein, was previously characterized as a major English walnut food allergen. OBJECTIVE: We sought to identify the linear IgE-binding epitopes of Jug r 1 and to determine which, if any, amino acids are necessary for this binding to occur. METHODS: Pools of sera from walnut-allergic patients and overlapping peptides synthesized on an activated cellulose membrane were used to screen for IgE-binding epitopes. Mutational analysis of the immunodominant epitope was carried out through single and multisite amino acid substitutions. Inhibition assays were performed through use of affinity-purified IgE, soluble forms of the epitope peptide, and the recombinant 2S albumin, rJug r 1. RESULTS: One immunodominant linear epitope was identified. Amino acid mutations to the epitope demonstrated that the residues RGEE, at positions 36 through 39, were minimally required for IgE binding. Probing of this epitope with sera from each of 20 patients revealed 15 of the sera to be positive. Binding of patients' IgE to the epitope was inhibited with a soluble form of the peptide; however, soluble peptide did not completely inhibit the binding of IgE to the intact rJug r 1. CONCLUSION: One major linear IgE-reactive epitope and its critical core amino acid residues have been identified. Mutation of any of these core amino acids resulted in loss of IgE binding to the epitope, and this points toward the feasibility of reducing allergenicity in genetically modified walnuts. However, strong evidence for the existence of conformational epitopes was also obtained.

Allergens↗
PubMed2002

Ana o 1, a cashew (Anacardium occidental) allergen of the vicilin seed storage protein family.

BACKGROUND: The allergens responsible for cashew food allergy have not been well characterized. OBJECTIVES: We initiated a study to clone cDNAs encoding cashew food allergens. METHODS: A cashew cDNA library was screened with human serum for IgE-reactive clones and rabbit IgG anti-cashew extract antisera. Reactive clones were sequenced and expressed, and linear epitopes were identified by means of solid-phase overlapping peptide analysis. Immunoblot inhibition was used to identify the native peptide in cashew extract. RESULTS: Four closely related clones reactive with both human and rabbit antisera were sequenced. Sequence analysis showed that these encode members of the vicilin/sucrose-binding protein family of plant seed storage proteins. Screening of the recombinant protein with sera from 20 patients with cashew allergy and 8 cashew-tolerant patients with allergies to other tree nuts showed that 50% and 25% of sera from patients with cashew allergy and cashew-tolerant subjects, respectively, bound the recombinant protein. The corresponding native allergen protein, designated Ana o 1, was located at approximately 50 kd. Epitope mapping revealed 11 linear IgE-binding epitopes, of which 3 appear to be immunodominant. None of the epitopes were shared in common with those of the peanut vicilin allergen Ara h 1. CONCLUSION: Ana o 1, a vicilin-like protein, is a major food allergen in cashews. Cashew and peanut vicilins do not share linear epitopes.

Allergens↗
PubMed2002

Oligomeric and conformational properties of a proteolytically mature, disulfide-stabilized human immunodeficiency virus type 1 gp140 envelope glycoprotein.

We describe the further properties of a protein, designated SOS gp140, wherein the association of the gp120 and gp41 subunits of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein is stabilized by an intersubunit disulfide bond. HIV-1(JR-FL) SOS gp140, proteolytically uncleaved gp140 (gp140(UNC)), and gp120 were expressed in stably transfected Chinese hamster ovary cells and analyzed for antigenic and structural properties before and after purification. Compared with gp140(UNC), SOS gp140 reacted more strongly in surface plasmon resonance and radioimmunoprecipitation assays with the neutralizing monoclonal antibodies (MAbs) 2G12 (anti-gp120), 2F5 (anti-gp41), and 17b (to a CD4-induced epitope that overlaps the CCR5-binding site). In contrast, gp140(UNC) displayed the greater reactivity with nonneutralizing anti-gp120 and anti-gp41 MAbs. Immunoelectron microscopy studies suggested a model for SOS gp140 wherein the gp41 ectodomain (gp41(ECTO)) occludes the "nonneutralizing" face of gp120, consistent with the antigenic properties of this protein. We also report the application of Blue Native polyacrylamide gel electrophoresis (BN-PAGE), a high-resolution molecular sizing method, to the study of viral envelope proteins. BN-PAGE and other biophysical studies demonstrated that SOS gp140 was monomeric, whereas gp140(UNC) comprised a mixture of noncovalently associated and disulfide-linked dimers, trimers, and tetramers. The oligomeric and conformational properties of SOS gp140 and gp140(UNC) were largely unaffected by purification. An uncleaved gp140 protein containing the SOS cysteine mutations (SOS gp140(UNC)) was also oligomeric. Surprisingly, variable-loop-deleted SOS gp140 proteins were expressed (although not yet purified) as cleaved, noncovalently associated oligomers that were significantly more stable than the full-length protein. Overall, our findings have relevance for rational vaccine design.

Animals↗