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Kenneth H Roux

Publications and source records attributed to Kenneth H Roux.

24 records · Page 2Linked to original sources
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↗