Techniques of genetic engineering applied to allergens have enabled the production of recombinant allergens. The validation of recombinant allergens implies that their immunological activity and their identity with natural allergens might be confirmed by in vitro and in vivo techniques carried out on a sufficiently large number of allergic subjects. Currently available results for the principal pneumoallergens are reported. Thus the work of validating recombinant allergen BeTv1 has been confirmed by in vitro tests and also by skin tests and nasal and bronchial provocation tests. The association of four recombinant allergens of phleole has enabled the detection in vitro of sensitisation to germinated pollens in 94.5% of patients. For mites the validity of group 2 recombinant allergens has been confirmed. A system enabling the expression of glycosylation of recombinant proteins was necessary to validate recombinant proteins in group 1 allergens. The recombinant allergen Blot5 is recognised as being effective in the detection of sensitization to Blomia tropicalis, a domestic allergen in sub tropical countries. The recombinant allergens Bla g 4 and Bla g 5 have been tested in vitro and in vivo and reactions were positive in nearly 50% of subjects sensitive to cockroaches. The recombinant Asp f 1 has been tested in subjects suffering from allergic bronchopulmonary aspergillosis and is positive in 60-85% of cases. Some studies are available for recombinant allergens of certain animal antigens (Equ c 1, Bos d 2). The consequences of clarifying recombinant allergens are then analysed: obtaining better standardised allergens for diagnostic tests, studying the spectrum of specificities of IgE induced by an allergen, the quantification of specific IgE, a better approach to mixed allergies with the help of recombinant allergens of the principal mixed allergens. Some recent progress has led to the production of modified recombinant allergens: the synthesis of recombinant polypeptides corresponding to T epitopes, the production of isoform recombinant allergens with reduced allergenic activity, the production of recombinant allergens of modified allergenic molecules by directed mutations and the production of recombinant fragments of allergenic molecules. The use of modified recombinant allergens is a way of permitting research which would, in the future, lead to new modalities of specific immunotherapy.

UNLABELLED:The study had the aim of establishing the incidence of sensitization to profilin (a panallergen found in pollens and foods of vegetal origin) in pollen-allergic patients. We evaluated the consequences of such sensitizations on the results of specific IgE, the positivity of skin tests and clinical signs.METHODS:94 consenting patients, allergic to pollens (trees and/or grasses and/or weeds) replied to a questionnaire and had skin tests to purified profilin and measurement of serum anti-profilin IgE.RESULTS:Two groups were defined: one group was sensitized to profilin (GSP), with positive skin test and anti profilin IgE of 31 patients, and a group non-sensitive to profilin (GNSP) (negative skin test and anti-profilin IgE) of 41 patients. Discordant results were found in 22 patients. Taking in account the two groups, sensitization to profilin was 43%. The two groups were homogenous for age, sex, ethnics and clinical signs. Food allergy was more frequent but not statistically different (p = 0.09) in the group GSP (51.6%) than the GNSP (31.7%), in particular allergy to fruits of the Rosaceae family. Pollen polysensitization (to three species, trees, weeds and grasses) was more frequent in the GSP group (64.5%) than the GNSP (12.5). Polysensitization to pollens and foods was also more frequent in the sensitized group (65.5%) than in the non-sensitized group (12.5%). In a sub-group with normal levels of total IgE pollen polysensitization was more frequent in patients who were sensitive to profilin. On biological investigation, sensitization to profilin influenced the result of anti-latex IgE and also the IgE to many vegetal allergens. These results show the value of seeking a sensitization to profilin in patients with pollinoses.

En 1998, la Communauté Européenne ainsi que chacun des pays membres, préparent les cadres législatifs de l'évaluation et de la gestion de la qualité de l'air ambiant. À l'heure de l'élaboration dans chaque région administrative de France, du « Plan Régional de la Qualité de l'Air , il semble indispensable d'attirer l'attention, avant la promulgation de ces plans, sur le fait que la pollution de l'air ne se limite pas à la seule pollution chimique, mais s'étend à la pollution biologique par certains pollens. En effet, certaines pollinoses ont des caractéristiques particulières qui devraient amener à considérer les pollens responsables comme de véritables polluants biologiques. En premier lieu, ces pollinoses affectent plus que d'autres, des sujets non prédisposés au développement des maladies allergiques. Ces pollens sont donc responsables de pathologies qui ne seraient pas intervenues en leur absence. En deuxième lieu, ces pollens sont produits par des plantes qui peuvent être soit éliminées soit contrôlées dans leur extension sans dommage pour l'homme. Les pollens à éliminer sont ceux de l'ambroisie dans la moyenne vallée du Rhône, les pollens à contrôler sont ceux des cyprès et thuyas dans le sud de la France. Dans ces deux cas, ils doivent être considérés comme des pollens polluants. La justification de la prise en compte de la pollution pollinique à côté de la pollution chimique vient également du fait que la pollution chimique semble renforcer le pouvoir pathogène des pollens.

Many cases of cross-allergy have been reported over recent decades. As a result of progress in the knowledge concerning the allergenic composition of allergic agents and the structure of allergens, it is now possible to attribute some of these cases to the development of cross-sensitizations, reflecting cross-reactivities between various allergens. A review of the immunochemical basis for cross-allergies can therefore be considered. In this context, laboratory methods such as immunodetection after transfer (<< Western blot >>) and assay of IgE specific for cross-allergens can facilitate the diagnosis of cross-allergies.

BACKGROUND:Eosinophil-derived neurotoxin/protein X (EDN/EPX), one of the cationic granule proteins released by polymorphonuclear eosinophils, can be detected in human urine.OBJECTIVE:We sought to evaluate whether the urinary release of EDN/EPX was dependent on the blood eosinophil cell count, the bronchoalveolar eosinophil cell count, or both and on the clinical diagnosis. We also attempted to determine the precise kinetics of decrease of EDN excretion and eosinophil counts after the onset of corticosteroid treatment.METHODS:Daily urinary release of EDN/EPX was measured by radioimmunoassay in 28 patients with high hypereosinophilia (group 1), 32 patients with moderate hypereosinophilia (group 2), 26 patients without hypereosinophilia at the time of the study but with a known pulmonary disease involving eosinophils (group 3), and 13 control patients (group 4).RESULTS:The urinary excretion of EDN/EPX was significantly higher in patients from groups 1 or 2 than in patients from groups 3 or 4. Particularly high levels of EDN/EPX excretion were observed in patients from groups 1 or 2 with chronic eosinophilic pneumonia (chronic eosinophilic pneumonia: 4.7 +/- 8.1 mg/day, control subjects: 0.39 +/- 0.33 mg/day, p < 0.001). Urinary excretion of EDN/EPX was significantly correlated with blood (r = 0.66, p < 0.001) and differential bronchoalveolar (r = 0.62, p = 0.04) eosinophil cell counts in patients from group 1 but not from the other groups. Corticosteroid treatment was followed by a significant decrease in EDN/EPX excretion. The kinetics of decrease in EDN/EPX were delayed as compared with the dramatic drop in peripheral eosinophil counts. Distinct kinetics between urinary EDN/EPX and eosinophil counts differentiated the recurrence of chronic eosinophilic pneumonia from an asthma attack in one patient.CONCLUSION:Measurement of urinary EDN/EPX excretion may be a useful indicator of eosinophil degranulation in vivo.

Pollen counts taken from the air of a ragweed-infested area such as those in the French department of the Rhone and the north of the Isere have two main interests, one immediate, the other long-term. The Cour's trap is located on the eastern outskirts of Lyon at the Lyon-Bron meteorological station. It is located on a plain, that is semi-urbanized with fairly low, widely-spaced dwellings. The filter rises 3 meters above a regularly-maintained meadow. The results are expressed as a number of grains per cubic meters of air and the data are rounded according to the rule of 4/5. The weekly data have been recorded for 14 years (French Association for Ragweed Study). In this study it is shown that ragweed has proliferated between the two periods analyzed, 1982-1989 and 1990-1995. The eradication campaign, launched in the Greater Lyon area in July 1995, for the first time since ragweed was brought into the area, has resulted in a decrease of the amount of pollen in the air. Four weeks weeding is insufficient, however, and it would seem better to extend the campaign, without using weedkillers, over six weeks, in order to avoid the later growth, as occurred in 1995. It is still necessary to measure pollen counts for ragweed in 1996 using the same method as previously. The suggestion made by some officials to measure pollen counts by other cheaper but less accurate means would not give a scientifically satisfactory comparison, because of the insufficient number of years of reference, studied on the same site, using this cheaper technique.

The application of genetic engineering techniques to the study of allergens has considerably developed over the last ten years. At the present time, about one hundred allergens representative of the main allergenic agents have been cloned and produced, mostly in the form of fusion proteins. This approach has allowed identification of allergens which had been purified by conventional methods and also characterization and identification of new allergens. The classification of allergens into families has therefore considerably advanced. The main consequence is a better understanding of sensitizations and cross-allergies. Banks of recombinant allergenic peptides also represent an easier approach to the study of B and T epitopes of allergens. Finally, several groups have validated the use of recombinant allergens in laboratory diagnosis and in skin tests.

OBJECTIVE: This study assesses the value of two recombinant birch allergens for diagnosis of patients sensitized to birch pollen with or without associated food allergy. METHODS: Fifty-one patients with positive skin test responses to Betulaceae and seven nonallergic control subjects were investigated; specific IgE antibodies were evaluated by specific immunoassay and blot immunodetection. RESULTS: Among 51 patients, 47 reacted to rBet v 1 and 10 to rBet v 2. Seven patients reacted to both recombinant allergens. In skin prick tests we found a correlation between the wheal produced by the commercial birch extract and the wheal produced by rBet v 1. Among 47 patients with positive test responses to rBet v 1, 83% had IgE binding to the Bet v 1 protein as determined by immunoblotting. Among 10 patients sensitized to rBet v 2, six had IgE binding to Bet v 2. Eleven patients with negative results, as determined by immunoblotting, had low levels of birch IgE in the sera (<10 kU/L) and low concentrations of IgE to rBet v 1 or rBet v 2 in ELISA. The nonallergic control subjects (n = 7) did not react to rBet v 1 or rBet v 2 in skin prick tests, nor did they have detectable amounts of specific IgE to rBet v 1 or rBet v 2. Histamine release tests confirmed sensitization to Bet v 1 in two patients with discordant results; for Bet v 2, one patient had positive results only at a high concentration, and one had results that remained negative. Thirty-four patients had birch pollinosis, and all reacted to rBet v 1. Patients who were monosensitized to birch never reacted to rBet v 2. Sensitization to rBet v 2 was only found in patients who reacted to other pollens (mainly grass). Twenty-nine patients demonstrated allergy to apples, cherries, or hazelnuts; and all reacted to rBet v 1. Among 11 patients with allergy to Umbelliferae, only three reacted to rBet v 2. CONCLUSIONS: Use of the two recombinant allergens (rBet v 1 and rBet v 2) always permits the diagnosis of birch sensitization. Sensitization to rBet v 1 is specific for birch and Rosaceae allergies, whereas sensitization to birch profilin, Bet v 2, is encountered in multisensitized subjects and is not always related to Umbelliferae allergy. (J ALLERGY CLIN IMMUNOL 1996;97:1100-9.)

SummaryConsidering the high occurrence of profilin as an allergen in many plant species, the assumption was made that profilin might be an allergen in Hevea brasiliensis, a member of the latex producing Euphorbiaceae family. Using IgE‐binding inhibition by purified profilins we demonstrated that profilin is an IgE‐binding component in the cytosolic fraction of natural latex and, to a lower extent, in the rubber fraction. Thirty‐five out of 36 sera containing IgE to ragweed‐profilin reacted with profilin from latex, indicating structural homologies between profilins from latex and ragweed. A large percentage (59%) of these sera were found to be positive in CAP latex assay. The preincubation of these sera with purified ragweed profilin greatly inhibited the CAP latex. Because profilin is also present in banana extract, it is likely to be involved in cross‐sensitivity to banana and latex. In a group of 19 individuals allergic to latex only two had antiprofilin IgE antibodies. Profilin was barely detectable on glove extract immunoblots, whereas some sera from patients allergic to latex reacted with a 15 kDa allergen which was not profilin. Consequently, IgE antibodies to latex‐profilin is a questionable factor for sensitization of occupationally‐exposed patients; however, sensitization to profilin should be taken into account when interpreting the results of latex IgE antibody assays.

The expression of certain cell cycle regulatory proteins: cdk1, cdk2, cdk4, cyclin A, cyclin B, cyclin E, Bcl2 and PCNA was examined in peripheral blood lymphocytes (PBL) from 25 cases of chronic lymphocytic leukemias (CLL) in order to analyze a possible cell cycle involvement of CLL lymphocytes. For comparison, we also studied the expression of these proteins in: 23 samples of non-Hodgkin's lymphoma (NHL) tissue of different histological types, 10 samples of non-neoplastic lymphoid tissue (NLT), non-stimulated PBL (NS-PBL) and PHA-stimulated PBL (PHA-PBL) from three healthy donors. Samples were lysed and proteins were resolved on polyacrylamide gel followed by Western blot. The expression of cdk4 and cyclin E, both known to act in early cell cycle stage, was approximately on the same level in all groups of lymphoid pathology examined. In particular, we found that that 19 out of 24 CLL cases were cyclin E positive and all but one were cdk4 positive, ie they expressed these markers over twice the level of non-stimulated healthy PBL. The cdk1 expression was above the level seen in NS-PBL in 14 (56%) cases, but the average expression was significantly lower than in the other tissues examined, including low-grade lymphomas. Cdk2 expression was comparable in CLL and in low malignancy grade NHL, but weaker than in other NHL and in NLT. Cyclins A and B, normally observed in advanced cell cycle phases, were not seen in any CLL case. The presence of cdk4 and cyclin E in the blood cells of the majority of CLL cases studied, as well as cdk1 and cdk2 in some cases, indicate that the CLL cells are not quiescent, but are blocked in an early stage of the G1 cell cycle phase, and/or that the expression of these proteins is pathologically deregulated.

Using affinity chromatography on lactose‐agarose, five β‐galactoside binding lectins of 14 to 20 kDa were detected in the rat small intestinal mucosa. The prominant proteins of 17 and 19 kDa were purified to homogeneity by 2D‐electrophoresis. Direct N‐terminal sequencing of the 17 kDa protein and intrachain sequencing of the 19 kDa protein produced sequences which are part of the N‐terminal domain of the L‐36/galectin‐4. A rabbit polyclonal antibody was raised against the 19 kDa lectin, which specifically recognized the 17 and 19 kDa lectins and detected a related 36 kDa protein in human undifferentiated HT29 cells.

We report an anaphylactic reaction which occurred very shortly after ingestion of a fresh fig. The IgE-dependent mechanism was demonstrated on the basis of positivity of the prick test performed with fresh fig (Ficus carica) extract. In addition, we were able to detect specific IgE to the same extract in the serum. The patient did not demonstrate sensitization to other common allergens involved in respiratory and food allergies. However, detection of specific IgE to F. benjamina indicated a sensitization to weeping fig. The CAP F. benjamina was partially inhibited by preincubation of the serum with fig extract, suggesting that these two species of Ficus share some common allergens. In this context, the assumption can be made that weeping fig was responsible for the initial sensitization in this patient.

Les protéines du latex constituent un mélange polyallergénique dont la composition est variable en fonction de paramètres botaniques, techniques et immunologiques. Malgré des résultats parfois discordants, les très nombreuses études portant sur l'identification des allergènes montrent le rôle de l'hévéine et de son précurseur la prohévéine, du facteur d'élongation du latex et des β glucosidases. L'existence d'allergènes croisés avec d'autres végétaux de familles botaniques parfois très éloignées, en particulier les fruits, rend compte de la conservation interespèces de motifs protéiques très voisins. Parmi ces allergènes croisés, le lysozyme paraît être un panallergène végétal, appartenant au groupe des protéines de stress.Natural rubber latex is a complex polyallergenic mixture containing numerous proteins with variable concentrations according to botanical, technical and immunological patterns. Despite apparently discordant results, many studies have indicated the role of hevein and its precursor prohevein, rubber elongation factor and β glucosidases. Another point concerns the existence of cross-reacting antigens; among phytogenetically different species, especially fruits, some antigens are very widespread. Lysozyme has been identified as a cross-allergen in natural rubber latex and many plant species. This defense-related protein could be a plant panallergen.

Since forty years, many allergens from different species responsible for allergies, have been purified and sometimes identified using classical methods of protein chemistry. For the first time in 1988, molecular biology technologies were applied to allergens, namely to a major allergen of the mite, Dermatophagoides pteronyssinus. During the recent years many other allergens have been produced as recombinant proteins expressed in bacteria or yeast leading to the growing family of recombinant allergens. This talk presents a general view on the allergen cloning procedure and gives an account on future applications of recombinant allergens in both fields of fundamental and practical allergy.

To better understand the relationship between the proliferation of human lymphoid cells and the expression of cdk1, a catalytic subunit of the histone HI kinase (HIK), we examined its mRNA and protein content in 3 B‐cell lines: Ramos, Reh‐6 and IARC 963. Cells were elutriated according to their position in the cell cycle. Cell fractions were analyzed for cdk1 mRNA and protein cellular content by Northern blot and immunoblot, respectively, as well as for H1K activity. Both mRNA and protein amounts and H1K activity varied according to cell cycle phase, the lowest values being observed in G 1 ‐enriched fractions. For comparison, elutriated fractions were also tested for the expression of cdk2 and cdk4 proteins. Both showed some variations among fractions, but they were less clear than those of cdk1. We also tested 29 samples of lymphoid neoplastic and non‐neoplastic tissues for proliferative activity (percentage of S and G 2 /M cells estimated by flow cytometry) and expression of cdk1, cdk2 and cdk4 proteins. We found a significant correlation between the percentage of cells in S or S + G 2 /M phases and cdk1 protein content but not cdk2 or cdk4 content. We conclude that cdk1 expression in human lymphoid cells varies during the cell cycle at both mRNA and protein levels. © 1995 Wiley‐Liss, Inc .

In December 1993, AFEDA conducted an epidemiological survey in order to establish the prevalence of ragweed-induced pollinosis and to evaluate how well the public was informed about this danger. A survey by telephone was made, taking a random sample of the population drawn by lot from telephone directories in the Rhône district. This involved about 300,000 homes. Those in which at least one person was under 50 were questioned. The questionnaire included a section aimed at everyone, and another section aimed at homes where at least one person suffered from pollinosis in August and/or September, in Lyon. 1,800 homes were selected at random, 905 persons responded: 22.4% were off target, 77.6% matched up to the chosen target. Of these, 32.9% were aware of Ragweed and 31.9% knew of the dangers. The 702 homes targeted made a total of 2,060 persons, 59.6% of which live in town and 40.4% in suburban areas; 51.6% are women and 48.4% men. 53 people, i.e. 2.57%, presented at least one of the following symptoms in 1993: rhinitis 86.8%, conjunctivitis 69.8%, itching of the pharynx and/or ears 47.2%, tracheitis and/or asthma 41.5%. 77.4% received medication. In 1993, the prevalence of ragweed-induced symptoms is a certainty for 1.8% of the population, if we evaluate only those patients presenting these symptoms every year. This prevalence therefore does not take account of new cases nor of newcomers to the district. Moreover, we have probably under-estimated this prevalence, owing to the torrential rain that fell in Lyon in late August 1993. This made a break in the pollen curve, a phenomenon that has never yet been observed in 13 years of recording pollen calendars.