Retained T-Cell Reactivity of Rye Grass Pollen Extract following Cleavage with Cyanogen Bromide and Nitrothiocyanobenzoic Acid

To facilitate studies on establishing the nature of structure/function relationships of allergens, ryegrass pollen allergen, Lol p IV, was cleaved into smaller fragments by cyanogen bromide (CNBr) and the resulting peptides were further digested with trypsin. The resulting peptides were then fractionated by high performance liquid chromatography (HPLC) on a C-18 reverse phase column. The allergenic activity of the HPLC fractions was evaluated in terms of their ability to inhibit the binding of ¹²⁵l-Lol p IV to serum IgE antibodies of a grass-allergic patient. Many of these fractions inhibited the binding between the native allergen and IgE antibodies in a dose-dependent manner. The inhibitions were specific, i.e., the fractions did not inhibit the binding between ¹²⁵l-Lol p I (a group-I ryegrass pollen allergen) and the IgE antibodies present in the allergic human serum. The possibility that the allergenic peptide fractions were contaminated by the native undegraded allergen, which might have accounted for the observed inhibition, was ruled out by the fact that the native allergen could not be detected by SDS-PAGE and the elution profiles of allergenically active peptides did not coincide with that of native allergen. One of the allergenic sites recognized by monoclonal antibody (Mab) 90, i.e., site A, was located in HPLC fractions 90–100 while another allergenic site B (recognized by Mab 12) appeared to be lost following the sequential digestion of Lol p IV with CNBr and trypsin.

Section: Discussionsupporting

confidence: 56%

Allergenic Fragments of Ryegrass (Lolium perenne) Pollen Allergen Lol p IV

Jaggi¹,

Ekramoddoullah²,

Kisil³

1989

“…In addition other modified allergens have been described that have extensively reduced IgE-reactivities and, poor IgG-inducing pro perties, but yet have various retained T-cell-stimulating capacities in experimental animals. For example, fragments of RE produced by treatment with cya nogen bromide and thiocyanonitrobenzoic acid were shown to be able to stimulate the production of T cells which could respond to accessory-cell-processed RE [8]. Conjugates of the biologically active peptide f met-leu-phe and allergens had similar properties to the fragments but in addition were shown to induce delayed hypersensitivity in guinea pigs [7], Chymotryptically cleaved fragments of rye extract also induced the development of unmodified allergenspecific T cells which could collaborate to produce DNP-specific antibody from B cells following in vivo transfer and challenge with dinitrophenylated grass pollen extract [9].…”

Section: Discussionmentioning

confidence: 99%

“…Modified RE samples were those described in a number of pre vious publications (table I): polysarcosine-RE [6]; f met-leuphe-RE [7]; chemically (cyanogen bromide and thiocyanonitrobenzoic acid) fragmented RE [8], and enzymatically (chymotrypsin) fragmented RE [9].…”

Section: Modified Allergensmentioning

confidence: 99%

“…Although this form of treatment is generally considered to be beneficial, the local and systemic anaphylactic side effects of the therapy can be serious [2,3]. Considerable effort, therefore, has been expended in a search for allergen forms with re duced allergenicity but maintained efficacy [4], Modified allergens that have mainly T-cell-stimulating properties retained have been proposed as candidates for safer therapy [5], such as conjugates of polysarcosine [6] or f met-leu-phe [7] and grass pollen extract, and both chemically [8] and enzymatically [9] produced cleavage products of allergens. Experimen tal animal systems are often used for evaluation of these potential-therapeutic agents [10] but a more rele vant system using human lymphocytes would be of advantage.…”

Section: Introductionmentioning

confidence: 99%

“…[8] reduced reduced Enzymatically much much maintained maintained maintained ND no fragmented RE [9] reduced reduced ND = Not done.…”

Section: Introductionmentioning

confidence: 99%

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Demonstration of Human, Rye Grass Pollen Extract-Specific, Helper and Suppressor Stimulating Activity of Modified Allergens by Effects on in vitro Hapten-Specific Antibody Production

Wheeler

Johansson²

1989

Self Cite

An in vivo system is described in which penicilloyl antibody was produced from peripheral leucocytes of a grass pollen-sensitive patient who had received penicillin therapy, by challenge of the cells with penicilloyl-grass pollen extract conjugate. Incubation of these leucocytes with a number of modified preparations of grass pollen extract with various T-cell-stimulating properties was shown to affect penicilloyl antibody production. Both chymotryptically fragmented rye grass pollen extract and a conjugate of f met-leu-phe and rye grass pollen extract enhanced penicilloyl-specific antibody similarly to the enhancement induced by unmodified extract, though at high concentration some suppression was seen. A conjugate of polysarcosine and rye grass pollen extract, previously shown to cause antibody suppression in mice, was similarly suppressive for penicilloyl-specific antibody. The system therefore shows potential for the evaluation of the effects of modified allergen treatment on antibody levels via T-cell mechanisms.

Immunochemical Characterization of Antigenic Fragments of Olea europaea and Phleum pratense

Fernández-Caldas¹,

Romano²,

Gallego³

et al. 2001

Background: The modulation of T cell responses by natural peptides is of potential use in the treatment of allergic diseases and reduces the risk of IgE-mediated reactions. The objectives of this study were to evaluate the allergenicity of depigmented, pepsin-digested fragments of Oela europaea and Phleum pratense extracts and their capacity to induce T cell proliferation. Methods: Lyophilized O. europaea and P. pratense extracts were depigmented following standardized methods, digested with pepsin and fractionated by ultrafiltration; a fraction containing components in the molecular weight range of 1–10 kD was selected. The blood of 20 allergic individuals was used to conduct T cell proliferation studies. Results: Pepsin-digested fragments of both pollen extracts lost more than 95% of their IgE-binding capacity by ELISA inhibition. Greater proliferation indexes than their respective native extracts were obtained in 55% of O. europaea and in 95% of P. pratense allergic patients. Conclusions: This study demonstrates that pepsin-digested allergenic fragments of depigmented extracts can be produced in a standardized, reproducible manner. These fragments are significantly less allergenic and retain, or even increase, their capacity to induce T cell proliferations.