[Incidence of sensitization to profilin in a population allergic to pollen: responsibility of profilin in pollen polysensitizations in patients with a normal level of total IgE]. / Incidence de la sensibilisation á profiline dans une population allergique aux pollens: responsabilité de la profiline dans des polysensibilisations polliniques chez des patients á taux normal d'IgE totales.

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.

[Recombinant allergens: diagnostic use and therapeutic perspectives]. / Allergènes recombinants: utilisation diagnostique et perspectives thérapeutiques.

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.

Urinary eosinophil-derived neurotoxin/protein X: a simple method for assessing eosinophil degranulation in vivo.

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.

Skin testing with recombinant allergens rBet v 1 and birch profilin, rBet v 2: diagnostic value for birch pollen and associated allergies.

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 (less than 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.

[Natural allergens and recombinant allergens]. / Allergènes naturels et allergènes recombinants.

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.

[Prevalence of ragweed hayfever in the south and east of the greater Lyon region in 1993]. / Prévalence de la pollinose à l'Ambroisie dans le Sud et l'Est de l'agglomération de Lyon en 1993.

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.

Expression of cell cycle regulatory proteins in chronic lymphocytic leukemias. Comparison with non-Hodgkin's lymphomas and non-neoplastic lymphoid tissue.

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.

First report of anaphylactic reaction after fig (Ficus carica) ingestion.

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.

Cdk1 is a marker of proliferation in human lymphoid cells.

To better understand the relationship between the proliferation of human lymphoid cells and the expression of cdk1, a catalytic subunit of the histone H1 kinase (H1K), 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 G1-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 G2/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 + G2/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.

Identification of profilin as an IgE-binding component in latex from Hevea brasiliensis: clinical implications.

Considering 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 anti-profilin 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.

Purification and characterization of the N-terminal domain of galectin-4 from rat small intestine.

Using affinity chromatography on lactose-agarose, five beta-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.

Identification of profilin as an actin-binding protein in higher plants.

Profilin is a low molecular weight protein involved in the organization of the mammalian and protozoan cytoskeleton as well as in signal transduction. In this study, profilin is identified as an actin-binding protein in higher plants which is present in monocot and dicot angiosperms. Birch pollen profilin and actin can be copurified as a complex, and purified recombinant birch profilin can be used as an affinity matrix to obtain birch pollen actin. The binding of 125I-labeled recombinant birch pollen profilin to plant and animal actins can be blocked by profilin-specific antibodies that react with different epitopes of birch profilin. One of the blocking antibodies was raised against the 25 COOH-terminal amino acids indicating the importance of this region in the profilactin complex formation.

The herpes simplex virus type 1 US11 gene product is a phosphorylated protein found to be non-specifically associated with both ribosomal subunits.

Microsequencing of a cyanogen bromide peptide obtained from a basic phosphoprotein co-sedimenting with purified ribosomes extracted from herpes simplex virus type 1-infected human epidermoid carcinoma 2 cells identified this protein as a product of the true late US11 gene. An antibody was raised against a recombinant fusion protein expressed in Escherichia coli from a plasmid carrying 75% of the US11 coding sequence including the carboxy terminus. This antibody was used to probe Western blots carried out under various conditions of one- and two-dimensional electrophoresis. The electrophoretic behaviour of the immunoreactive proteins offered further proof that they were indeed products of the US11 gene. This US11 protein, which has phosphates on multiple serine residues, is brought into the cell by the virion and found to be present within ribosome fractions early after infection. This association with ribosomes is non-specific and due to probable aggregation or oligomerization of this proline-rich basic protein allowing its co-sedimentation with ribosomes during the different subcellular fractionation steps used for the purification of ribosomal subunits.

Purification and characterization of an allergen from celery immunochemically related to an allergen present in several other plant species. Identification as a profilin.

The purification of a 15 kD allergen from celery was obtained by a four step procedure. Evidence for at least two isoallergenic forms was obtained after analysis by two-dimensional-electrophoresis. A rabbit polyclonal antibody raised against this purified allergen allowed us to confirm our precedent results on the occurrence of allergenically and molecular mass-related components in celery and birch and mugwort pollens. In addition such components were also present in numerous other species like Cynodon dactylon, Sorghum halopense, Poa pratensis, Ambrosia elatior and in apple and carrot. The 15 kD allergen was identified as profilin by use of a specific rabbit polyclonal antibody that recognized a recombinant birch profilin. In addition, the purified celery allergen binds IgE from sera of patients allergic to birch profilin. These results reinforce the concept of profilin as a panallergen responsible in some patients for cross-allergic manifestations to various and unrelated species of grasses, weeds, trees, vegetables and fruits.

[Biological and clinical prevalence of pollinosis caused by ragweeds of the upper valley of the Rhône corridor]. / Prévalence biologique et clinique de la pollinose due aux ambroisies dans la vallée supérieure du couloir rhodanien.

An epidemiological study of ragweed allergy was conducted on 646 employees belonging to 6 factories located in the Rhone valley south of the city of Lyon. Information on seasonal evocative clinical symptoms was obtained through a self-administered questionnaire. Biological prevalence was assessed by measuring anti-ragweed IgE specific antibodies. Measurements were performed by immunoenzymatic assay (W1 Phadezym RAST from Pharmacia). 34 (5,4%) subjects had evocative symptoms whereas 37 (5,9%) had increased specific IgE. Persons with the highest IgE levels were symptomatic. Concordance between symptoms and biology was 35% (12/34). Results indicate that sensitization level varies according to the location of the factory and people's residence, the risk to become allergic being of 10% in the most exposed population. This data emphasize the need to promote anti-ragweed eradication policy.

Profilins constitute a novel family of functional plant pan-allergens.

Type I allergy is a major health problem in industrialized countries where up to 15% of the population suffer from allergic symptoms (rhinitis, conjunctivitis, and asthma). Previously, we identified a cDNA clone that encoded a birch pollen allergen as profilin. Profilins constitute a ubiquitous family of proteins that control actin polymerization in eukaryotic cells; in particular, profilin participates in the acrosomal reaction of animal sperm cells. Although profilins had been unknown in plants so far, our finding led to the assumption that profilins might have similar functions in pollens during plant fertilization and therefore represent allergenic components in almost all pollens. We show that profilins are prominent allergens that can be isolated from tree pollens (Betula verrucosa, birch), from pollens of grasses (Phleum pratense, timothy grass), and weeds (Artemisia vulgaris, mugwort). About 20% of all pollen allergic patients tested (n = 65) displayed immunoglobulin E (IgE) reactivity to recombinant birch profilin that was expressed in pKK223-3. An IgE inhibition experiment performed with recombinant birch profilin and purified natural profilins from timothy grass and mugwort indicates common IgE epitopes. Moreover, all pollen profilins purified from these far distantly related plant species, and likewise the purified recombinant birch profilin, are able to elicit dose-dependent histamine release via high affinity Fc epsilon receptor of blood basophils from profilin allergic patients. The presence of profilin and possibly related proteins as crossreacting allergenic components in various plants therefore provides an explanation as to why certain allergic patients display type I allergic reactions with pollens and even food from distantly related plants. A functional pan-allergen, like profilin, available as purified recombinant protein, may be a useful diagnostic and probably therapeutic reagent.