[Celiac disease--the chameleon among the food intolerances]. / Zöliakie. Das Chamäleon unter den Nahrungsmittelintoleranzen.

Celiac disease is an autoimmune disorder resulting from gluten intolerance and is based on a genetically predisposition. Symptoms occur upon exposure to prolamin from wheat, rye, barley and related grain. The pathogenesis of celiac disease has not yet been sufficiently elucidated but is being considered as an autoimmune process. At its core are the deamidation of prolamin fragments, the building of specific antibodies and the activation of cytotoxic T-cells. The immunological inflammatory process is accompanied by structural damages of the enterocytes (villous atrophy, colonization and crypt hyperplasia). The symptoms and their extent depend on the type of the celiac disease; classic and non-classic forms are being distinguished (atypical, oligosymptomatic, latent and silent celiac disease). Characteristics of the classic presentation are malabsorption syndrome and intestinal symptoms such as mushy diarrhea and abdominal distension. The diagnosis of celiac disease is based on four pillars: Anamnesis and clinical presentation, serological evidence of coeliac specific antibodies (IgA-t-TG; IgA-EmA), small intestine biopsy and improvement of symptoms after institution of a gluten-free diet. The basis of the therapy is a lifelong gluten-free diet, i. e. wheat, rye, barley, spelt, green-core, faro-wheat, kamuth and conventional oats as well as food items obtained therefrom. Small amounts of up to 50 mg gluten per day are usually tolerated by most patients; amounts of > or = 100 mg/day lead mostly to symptoms. Gluten-free foods contain < or = 20 ppm or 20 mg/kg (Sign: symbol of the 'crossed ear' or label 'gluten-free'). At the beginning of the therapy the fat and lactose intake may need to be reduced; also the supplementation of single micronutrients (fat-soluble vitamins, folic acid, B12, iron, and calcium) may be required. Alternative therapies are being developed but have not yet been clinically tested.

Immunologic effect and tolerability of intra-seasonal subcutaneous immunotherapy with an 8-day up-dosing schedule to 10,000 standardized quality-units: a double-blind, randomized, placebo-controlled trial.

BACKGROUND: International guidelines recommend that allergen-specific immunotherapy for pollen-induced rhinoconjunctivitis is initiated preseasonally. However, because subjects often present to physicians with allergy symptoms during the pollen season, "within-season" initiation of specific immunotherapy is of special interest. OBJECTIVE: We evaluated the immunomodulatory effects and tolerability of subcutaneous immunotherapy (SCIT) with Standardized Quality (SQ) 6-grass mix and rye allergen extract, using an 8-day intra-seasonal up-dosing schedule to 10,000 SQ-units (SQ-U). METHODS: In a 9-week, multicenter, randomized, double-blind, placebo-controlled trial, adults (mean age, 34.6 years; 99.3% whites) with grass pollen-induced rhinoconjunctivitis (mean disease duration, 15.1 years) were randomized 3:1 to receive SCIT or placebo. Treatment was initiated during the 2008 pollen season, with an 8-day up-dosing from 100 to 10,000 SQ-U (6 daily injections) followed by 2 maintenance injections of 10,000 SQ-U at intervals of 2 and 4 weeks, respectively. The primary end point examined immunologic effects, assessing the difference in IgE-blocking factor (serum components competing with IgE for allergen binding) between SCIT and placebo at week 9. Secondary/explorative end points included the difference in IgE-blocking factor, specific IgG(4), and specific IgE at various times. Tolerability (adverse events, local and systemic allergic reactions) of the up-dosing schedule was also evaluated. RESULTS: Of the 148 treated subjects 144 (SCIT n = 109; placebo n = 35) were analyzed for the primary parameter. Immunologic response (significantly higher increase in IgE-blocking factor and IgG(4) levels) occurred with SCIT versus placebo at week 9 (IgE-blocking factor, P = 0.0017; IgG(4,)P = 0.0215). Significant differences were observed as early as week 3. AEs were reported in 60.7% of SCIT- and 30.6% of placebo-treated subjects, with no treatment-related serious AEs. Local allergic reactions occurred in 46.4% of SCIT and 8.3% of placebo subjects (χ(2) test, P < 0.0001). No significant difference was observed between groups in the incidence of systemic reactions (7.1% SCIT vs 5.6% placebo; χ(2) test, P = 0.7413). CONCLUSIONS: This trial provides the first description of short (8-day) intra-seasonal up-dosing with SCIT, which induced immunologic effects after only 3 weeks, and was generally well tolerated, although it induced a marked increase in the rate of local reactions compared with placebo. ClinicalTrials.gov identifier NCT00807547; ALK trial ID SHX0562.

Aegilops-Secale amphiploids: chromosome categorisation, pollen viability and identification of fungal disease resistance genes.

The aim of this study was to assess the potential breeding value of goatgrass-rye amphiploids, which we are using as a "bridge" in a transfer of Aegilops chromatin (containing, e.g. leaf rust resistance genes) into triticale. We analysed the chromosomal constitution (by genomic in situ hybridisation, GISH), fertility (by pollen viability tests) and the presence of leaf rust and eyespot resistance genes (by molecular and endopeptidase assays) in a collection of 6× and 4× amphiploids originating from crosses between five Aegilops species and Secale cereale. In the five hexaploid amphiploids Aegilops kotschyi × Secale cereale (genome UUSSRR), Ae. variabilis × S. cereale (UUSSRR), Ae. biuncialis × S. cereale (UUMMRR; two lines) and Ae. ovata × S. cereale (UUMMRR), 28 Aegilops chromosomes were recognised, while in the Ae. tauschii × S. cereale amphiploid (4×; DDRR), only 14 such chromosomes were identified. In the materials, the number of rye chromosomes varied from 14 to 16. In one line of Ae. ovata × S. cereale, the U-R translocation was found. Pollen viability varied from 24.4 to 75.4%. The leaf rust resistance genes Lr22, Lr39 and Lr41 were identified in Ae. tauschii and the 4× amphiploid Ae. tauschii × S. cereale. For the first time, the leaf rust resistance gene Lr37 was found in Ae. kotschyi, Ae. ovata, Ae. biuncialis and amphiploids derived from those parental species. No eyespot resistance gene Pch1 was found in the amphiploids.

The interaction between early life upper respiratory tract infection and birth during the pollen season on rye-sensitized hay fever and ryegrass sensitization--a birth cohort study.

Studies on early life viral respiratory infection and subsequent atopic disease in childhood have conflicting findings. Animal models show that viral respiratory infection in conjunction with allergen presentation can enhance sensitization. This prospective study assesses the influence of an upper respiratory tract infection (URI) in the first month of life and the season of birth on the development of hay fever and ryegrass allergen sensitization in childhood. From a Tasmanian cohort born during 1988 and 1989, a group of 498 children were followed up at 8 yr and another different group of 415 children were followed up at 16 yr. The ryegrass pollen season in Tasmania occurs in November and December. Forty-four (9.6%) children in Follow-up sample 1 and 47 (12.5%) children in Follow-up sample 2 were born in the pollen season. The parental report of an early upper respiratory tract infection (EURI) was documented prospectively by a home interview at 1 month of age (median age 5.1 wk). Sensitization to ryegrass and house dust mite (HDM) was determined at 8 yr of age by skin prick testing and at 16 yr by ImmunoCap. Ryegrass sensitized hay fever was defined as a positive response to a question on hay fever plus the presence of ryegrass allergy. For children tested at age 8 and born in the pollen season, a EURI by postnatal interview was associated with an increased risk of ryegrass sensitization (OR 5.80 95% CI 1.07, 31.31) but not for children with a EURI born outside the pollen season (OR 0.62 95% CI 0.35, 1.08). Similarly, EURI was significantly associated with early onset (< or = 8 yr) ryegrass sensitized hay fever for children born in the pollen season (AOR 4.78 95% CI 1.17, 19.47) but was not associated with early onset ryegrass sensitized hay fever for children born outside the pollen season (AOR 0.76 95% CI 0.43, 1.33). These findings suggest that early life viral URI interacts with ryegrass allergen exposure in the development of ryegrass allergen sensitization and ryegrass sensitized hay fever symptoms.

Allergy to kiwi in patients with baker's asthma: identification of potential cross-reactive allergens.

BACKGROUND: Baker's asthma is a frequent IgE-mediated occupational disorder mainly provoked by inhalation of cereal flour. Allergy to kiwifruit has being increasingly reported in the past few years. No association between both allergic disorders has been described so far. METHODS: Twenty patients with occupational asthma caused by wheat flour inhalation were studied. Kiwi allergens Act d 1 and Act d 2 were purified by cation-exchange chromatography. Wheat, rye, and kiwi extracts, purified kiwi allergens, and model plant glycoproteins were analyzed by IgE immunodetection, enzyme-linked immunosorbent assay (ELISA), and inhibition ELISAs. RESULTS: Kiwifruit ingestion elicited oral allergy syndrome in 7 of the 20 patients (35%) with baker's asthma. Positive specific IgE and skin prick test responses to this fruit were found in all these kiwi allergic patients, and IgE to Act d 1 and Act d 2 was detected in 57% and 43%, respectively, of the corresponding sera. Actinidin Act d 1 and bromelain (harboring cross-reactive carbohydrate determinants) reached above 50% inhibition of the IgE binding to wheat and/or kiwi extracts. CONCLUSIONS: A potential association between respiratory allergy to cereal flour and allergy to kiwifruit has been disclosed. Cross-reactive carbohydrate determinants and thiol-proteaseshomologous to Act d 1 are responsible for wheat-kiwi crossreactivity in some patients.

Allergoid-specific T-cell reaction as a measure of the immunological response to specific immunotherapy (SIT) with a Th1-adjuvanted allergy vaccine.

BACKGROUND: Specific immunotherapy (SIT) is believed to modulate CD4+ T-helper cells. In order to improve safety, SIT vaccines are often formulated with allergoids (chemically modified allergens). Interaction between T-cells and allergoids is necessary to influence cellular cytokine expression. There have been few reports on identification the early cellular effects of SIT. METHOD: Patients allergic to grass and/or mugwort pollen (n= 21) were treated with a 4-shot allergy vaccine (Pollinex Quattro) containing appropriate allergoids (grass/rye and/or mugwort) adsorbed to L-tyrosine plus a Th1 adjuvant, monophosphoryl lipid A (MPL). Fourteen grass-allergic patients served as untreated controls. Using the peripheral blood mononuclear cells of these patients, an optimized lymphocyte transformation test (LTT) was employed to monitor the in vitro proliferative response of T-cells to an allergoid challenge (solubilised Pollinex Quattro) before the first and last injection and then 2 and 20 weeks after the final injection. Control challenges utilised preparations of a similar pollen vaccine without the adjuvant MPL and a tree pollen vaccine with and without MPL. RESULTS: The LTT showed increased LTT stimulation indices (SI) in 17/20 SIT patients when the solublised vaccine preparation was used as a challenge before the last injection and 2 weeks after, in comparison to pre-treatment levels. Twenty weeks after therapy, the SI decreased to baseline level. A vaccine challenge without MPL gave lower SI levels. A challenge of a clinically inappropriate tree allergoid vaccine gave no response, and a nontreated group also showed no response. CONCLUSION: Following a short-course SIT adjuvated with MPL, challenges of allergoids were shown to activate allergen-specific T cells in vitro. There was an additional stimulating effect when the challenge was in combination with MPL. There were no non-specific effects of MPL, shown by the tree allergoid/MPL control. The timing of the response was closely correlated to the treatment course; reactivity fell two weeks after the final injection and 20 weeks later it was at baseline level. Thus an immunological response to SIT was detected after very few injections. This methodology could provide a basis for monitoring the immediate progress of allergy vaccinations.

[Relation between specific IgE antibodies to grass pollens and to wheat, rye, soya in children with allergic disease]. / Wzpólzaleznosc mieczy obecnoscia swoistych przeciwcial klasy ige dla pylków traw, pszenicy, zyta i soi u dzieci z choroba alergiczna.

The aim of this study was to investigate the relationship between the hypersensitivity to grass pollens (gxl) and to wheat (f4), rye ff5) and soya (fl 4) in children with allergic disease. Specific IgE was determined by the FEIA CAP- System. Hypersensitivity was expressed in classes from 1 to 6. Specific IgE antibodies against grass (gxl) in 944 children with allergic disease, aged from 6 months to 18 years were detected. At the same time in 532 of these children - specific IgE to wheat and rye and in 122 children specific IgE to soya were also estimated. We detected very high correlation between the hypersensitivity to grass and the presence of specific IgE to wheat, rye (r=0.96, p<0.01) and soya (r=0.90, p<0.05).

Kiwi allergens and their cross-reactivity with birch, rye, timothy, and mugwort pollen.

In order to study kiwi allergens and examine their cross-reactivity to birch, rye, timothy, and mugwort pollen, immunoblot and enzyme immunoassay (EIA) inhibition tests were performed with self-prepared kiwi extract. For the investigations, the sera of 22 kiwi-allergic patients were used, which were characterized by radioallergosorbent (RAST) measurements for kiwi, birch pollen, and apple with commercial allergen disks. The RAST values for kiwi were compared with those obtained by self-prepared kiwi extract disks. In the RAST, the allergen potency of this extract was found to be very similar to that of the commercial extracts. This extract was able to bind immunoglobulin E from kiwi-allergic patients in the immunoblots and EIA. Immunoblot results revealed a broad spectrum of IgE specificities; 12 allergens were identified within the range of 15 to 94 kDa, 10 of which cross-reacted with birch, timothy, rye, and mugwort pollen, while two (25 and 30 kDa) were not inhibited homologously or by pollen. EIA inhibition additionally revealed kiwi-specific allergens. Three proteins of the kiwi extract (25, 30 and 43 kDa) were considered to contain a carbohydrate moiety. Profilin seems to be relevant in cross-reactivity of kiwi allergens.

Cloning and sequencing of Lol pI, the major allergenic protein of rye-grass pollen.

We have isolated a full length cDNA clone encoding the major glycoprotein allergen Lol pI. The clone was selected using a combination of immunological screening of a cDNA expression library and PCR amplification of Lol pI-specific transcripts. Lol pI expressed in bacteria as a fusion protein shows recognition by specific IgE antibodies present in sera of grass pollen-allergic subjects. Northern analysis has shown that the Lol pI transcripts are expressed only in pollen of rye-grass. Molecular cloning of Lol pI provides a molecular genetic approach to study the structure-function relationship of allergens.

IgE-binding proteins from pine (Pinus radiata D. Don) pollen: evidence for cross-reactivity with ryegrass (Lolium perenne).

A complex mixture of pine (Pinus radiata D. Don) pollen proteins are rapidly released into aqueous solutions. IgE-binding proteins have been identified in these extracts using combined SDS-PAGE immunoblotting techniques. These IgE-binding proteins were detected using atopic patient and commercial pooled human sera known to be high in ryegrass-specific IgE. Enzyme-immunoassay inhibition studies revealed that leached P. radiata pollen proteins could partially inhibit serum IgE binding to ryegrass RAST discs thus providing preliminary evidence for allergen cross-reactivity between these two unrelated species.

Mapping of Lol p I allergenic epitopes by using murine monoclonal antibodies.

Murine monoclonal antibodies (MAbs) against three non-overlapping epitopes of Lol p I allergen were previously produced and subsequently used for purification of the allergen. In the present study, these MAbs were further characterized, and the biological activity of the purified allergen assessed. The three MAbs were of the IgG isotype and carried a kappa light chain. Their affinity constants were in the range of 7.4-15.1 x 10(-9) mol/l. Purified Lol p I kept its biological activity, as shown by its ability to induce histamine release by basophils of Lol p I-sensitive patients. The profiles of histamine release induced by either Lol p I or crude Lolium perenne extracts were comparable. This observation suggests that human IgE bound to basophils are polyspecific which has been confirmed by immunoblot and inhibition assay. Our data indicated also that Lol p I possesses a major allergenic epitope recognized by all human serum IgE tested. This epitope seems to be partially shared by those recognized by the three MAbs. Finally, preincubation of Lol p I with either one of the Mabs did not affect significantly the basophil-histamine release induced by the purified allergen. This suggests that Lol p I possesses allergenic sites other than the one shared by MAbs and IgE Abs.

Immunologic and clinical evaluation of a 12-month course of specific immunotherapy.

Allergen specific immunoglobulins (IgE, IgG, IgG1, and IgG4) were evaluated during the course of a 12-month specific immunotherapy (ITS) in 27 allergic subjects: 12 Dermatophagoides pteronyssinus (DP)-, 9 Parietaria officinalis (W19)-, six perennial and timothy rye grass (G5 and G6)-sensitive cases. Further, the modifications of the specific Ig levels were compared with the significant improvement in symptoms and drug consumption observed after 12 months of ITS. IgE levels significantly decreased after 6 and 12 months, while IgG1 significantly increased in the early phases of ITS (3rd month), and IgG4 significantly and progressively increased during the course of ITS. No modification was observed in specific total IgG. Regression analysis test among the various Ig levels revealed a strict correlation only between IgE and IgG4 (high levels of IgE before ITS correlated with high levels of IgG4 after 12 months of ITS). No correlation was observed between total Ig changes and improvement of clinical status or drug consumption.

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.

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.

Allergenic fragments of ryegrass (Lolium perenne) pollen allergen Lol p IV.

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 125I-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 125I-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.

Characterization of anti-idiotypic antibodies and their use as probes for determination of shared idiotopes expressed on murine and human IgE anti-rye I antibodies.

This study describes the production and characterization of rabbit anti-idiotypic antibodies (anti-ID Abs) against three idiotypes of three mAbs with different specificities. The anti-ID Abs were rendered idiotype specific by appropriate adsorption. Binding of labelled mAb to homologous anti-ID Ab bound to a polystyrene matrix was completely inhibited when the same mAb was added. In contrast, addition of other mAbs sharing the same isotype and the same light chain but with different specificity did not affect the binding reaction. Each anti-ID Ab inhibited completely and selectively the reaction between the allergen and the homologous mAb idiotype. Labelled rye I binding to a given polystyrene-bound mAb idiotype was completely blocked if the relevant anti-ID Ab was used as an inhibitor. Murine polyclonal anti-rye I antisera inhibited the reaction between all three mAbs and the antigens, as well as the reaction between all three mAb idiotypes and their homologous anti-ID Abs. On another hand, goat polyclonal anti-rye I antisera only inhibited the reaction between the mAbs and the antigens. These results suggest that the anti-ID Abs produced are directed against idiotopes located within the paratopes and such idiotopes are shared by murine monoclonal and polyclonal Abs. Human rye I-specific IgE and murine anti-rye I mAbs could share common idiotopes, since human IgE binding to the antigen was inhibited by the anti-ID Abs. These observations imply structural similarity in the V gene coding for the variable region of the antibody of two different species.

Analysis of rye pollen (Secale cereale) allergens using patients' IgE, immunoprint, Western Blot and monoclonal antibodies.

Rye pollen (Secale cereale) was investigated by Immunoprint and Western Blot analysis. Using Immunoprint, more than 30 proteins could be shown to be allergens in terms of their IgE binding, and 9 were determined as major allergens. In Western Blot 17 allergens were discovered, 3 of them major allergens. 4 monoclonal antibodies raised against a pollen extract of 6 grasses showed cross-reactivity to rye pollen, thus indicating common epitopes. 1 of 3 monoclonal antibodies produced against an extract of rye pollen was only reacting to this pollen. Looking at the overall binding pattern of the 7 monoclonal antibodies, they showed partial identity to patients' IgE in Immunoprint and Western Blot. The possibility of defining epitopes using preparative isoelectric focusing and monoclonal antibodies is discussed.

Induction of allergen-specific T cells by conjugates of N-formyl-methionyl-leucyl-phenylalanine and rye grass pollen extract.

A conjugate of the biologically active peptide N-formyl-methionyl-leucyl-phenylalanine and rye grass pollen extract (F-MLP/rye), previously shown to react with rye grass pollen extract-specific T cells, induced the formation of allergen-specific T cells in mice. Lymph node cells prepared from mice immunized with either native extract or F-MLP/rye gave an enhanced response to unmodified rye grass pollen allergens in vitro. Syngeneic spleen macrophages were able to present the unmodified allergens to T cells obtained from both groups of mice causing their proliferation in vitro. Conjugation of the peptide into the extract brought about an extensive reduction in its reactivity with grass pollen-specific human IgE, and a loss of its ability to induce specific IgG antibody in guinea-pigs. A state of delayed hypersensitivity specific for rye grass pollen extract was produced in guinea-pigs by immunization with either the F-MLP/rye or unmodified extract. It is concluded that conjugates such as F-MLP/rye or other T' allergoids could be used as probes to investigate whether changes in T cell activity are important in immunotherapy.

Allergenicity and cross-reactivity of rye grass pollen extracts revealed by monoclonal antibodies.

Using monoclonal antibodies the immunogenic and allergenic characteristics of rye Group I were redefined by SDS-PAGE analysis and immunoblotting. The purified rye Group I from NIH possesses a major component of approximately 34000 Da against which most of the sera from grass-sensitive patients and none from non-atopic volunteers contain specific IgE antibodies. Monoclonal antibodies to rye Group I were raised and used to purify the antigen and to verify the cross-reactivity between grass extracts. The 3 monoclonal antibodies studied recognized different components of timothy grass and 2 of them recognized kentucky june grass but none recognized components of ragweed extracts.