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.

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.

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.

ACTH-induced change of cAMP-dependent protein-kinase repartition in bovine adrenal cells.

Acute ACTH stimulation of isolated adrenal cells produced a modification of the subcellular distribution of cAMP-dependent protein kinase. Within 20 min, the protein-kinase ratio in all subcellular fractions, particularly in the 175 000 x g supernatant, was increased. Total protein-kinase activity, as well as the specific activity of both the homogenate and particulate enzymatic activities, was decreased while that of the 175 000 x g supernatant was increased. However, the increase of the soluble kinase activity represented only 29-46% of the lost particulate activity. On the other hand, under exchange conditions, the cAMP-binding capacity of all subcellular fractions was similar in control and ACTH-treated cells, except in the 175 000 x g pellet in which it was slightly decreased in ACTH-treated cells. These modifications were observed for supraphysiological (10(-8) M), as well as for physiological (10 (-11) M), concentrations of ACTH. These observations suggest that, after ACTH stimulation, a liberation of free catalytic sub-unit occurs from particulate into the soluble cell compartment, which shows an activation of particulate cAMP-dependent protein-kinase activity.

Distribution and characterization of cAMP-dependent protein kinase isoenzymes in bovine adrenal cells.

Subcellular localization and characterization of cAMP-kinase isoenzymes in fasciculata reticularis bovine adrenal cells has been investigated. Different subcellular fractions were purified on a Percoll gradient and characterized by marker enzymes. cAMP-kinase was located principally in cytosol and microsomes. In the low-speed particulate fractions cAMP-kinase was found associated mainly with plasma membrane but not with mitochondria. Characterization of isoenzyme patterns in subcellular fractions by conventional DEAE-cellulose chromatography and by anion-exchange HPLC gives essentially the same results. Isoenzyme I appears to be the main enzyme in cytosol whereas isoenzyme II predominates in solubilized microsome and plasma membrane enriched fraction. Photoaffinity labelling of chromatographic fractions demonstrated that HPLC separates both cAMP binding subunits. Photoaffinity labelling of the different subcellular fraction by 8-azido-[32P]cAMP confirmed the data obtained by anion-exchange chromatography. However, in microsomes this method revealed the presence of both isoenzymes and the preferential solubilization of isoenzyme II by Triton X-100. In summary, our results indicate a subcellular compartmentalization of cAMP-kinase in bovine adrenal cells with a preferential localization of isoenzyme I in cytosol and of isoenzyme II in membrane. However, the relation between the distribution and the role of each isoenzyme has so far not been documented.

[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.

[Anaphylactic shock to celery and sensitization to ragweed and mugwort. Crossed or concomitant allergy?]. / Choc anaphylactique au céleri et sensibilisation à l'ambroisie et à l'armoise. Allergie croisée ou allergie concomitante?

Two cases of anaphylactoid reaction from hypersensitivity to celery are reported. The two patients were also allergic to ragweed and mugwort pollen. The RAST inhibition test suggested slight cross-allergenicity between celery and mugwort pollen. The common allergenic factor could have been the alpha-methylene gamma-butyrolactone group.

[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.

[Rules concerning allergy to celery (and other Umbellifera)]. / Règles concernant l'allergie au céleri (et aux autres ombellifères).

1. Allergy to celery is associated with a pollinosis. Even if there are no overt symptoms of the pollinosis there are positive tests, including RAST. 2. The causative pollen is usually the most common of the region, for example in Sweden it is birch (Betula), whilst in the surroundings of Lyon in France ragweed (Ambrosia) and mugwort (Artemisia) and Compositae in general seem to play a particularly important role. 3. The allergy is strictly on-way, celery-pollen. If the reverse should occur the positive tests for celery are often found when allergy is caused by ragweed or mugwort. 4. Other Umbelliferae such as parsley and carrots produce the same effects. 5. The syndrome seems to implicate aromatic plants for culinary use. 6. The allergen has been shown to be heat-stable and this indicates that it is a small molecule.

[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.

[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.

[Correlation between the atmospheric level of antigen Amb-al (AgE) and the number of Ambrosia artemisiaefolia pollen grains in Lyon and neighboring regions]. / Etude de la corrélation entre le taux atmosphérique de l'antigène Amb-al (Ag E) et le nombre de grains de pollen d'Ambrosia artemisiae folia dans la région lyonnaise et les régions avoisinantes.

Accurate quantitation of the atmospheric levels of pollens and other allergenic particulate is laborious and may not always correlate with the concentration of allergen within the particulate. This study examined the atmospheric contamination by short ragweed (Ambrosia artemisiae folia) over 7 weekly periods during ragweed pollination in 6 localities of France centered around Lyon. The correlation between data obtained from the conventional microscopic numeration of pollen grain and and the quantitation of the allergen Amb-al level by radioimmunoassay was investigated. For the total of 41 paired analyses of the actual numbers of ragweed pollen grains and computed numbers of pollen grains from the Amb-al levels there was an excellent overall correlation (r = 0.90, p 0.001) thus the radioimmunoassay of Amb-al represent a simple and relevant alternative to microscopic numeration of pollen grains.

[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.

Guanyl cyclase activity of human blood lymphocytes.

An enzyme, guanyl cyclase, which catalyzes formation of CYCLIC 3',5'-GMP from 5'-GTP, has been identified in human peripheral lymphocytes. The activity in lymphocyte homogenate is 14 pmol (min 10-7 lymphocytes). No activity is detected in red blood cells, and the amount found in platelets is very low. The properties of this enzyme are very close to those reported for other guanyl cyclases studied in other tissues: namely, its intracellular localization, its requirement for cation Mn-2+, its inhibition by Hg-2+, Zn-2+ and by nucleotides especially 5'-ATP. No change in enzyme activity occurs when phytohemagglutinin P is added to disrupted lymphocytes. However, when the mitogen is incubated with intact cells, guanyl cyclase activity increases in a few minutes.

Guanylate cyclase activity of human lymphocytes from peripheral blood, thymus, and tonsils. A comparative study.

The subcellular repartition and the distinctive properties of guanylate cyclase (EC 4.6.1.2) vary according to the lymphocyte population studied and according to the presence of detergent. Guanylate cyclase of non-adherent peripheral lymphocytes and of thymus lymphocytes is recovered by more than 90% in the soluble fraction of the homogenate. Kinetics according to the substrate (5'-GTP-Mn2+) is Michaelian, the Ca2+ ion acts as an activator, especially in the case of blood lymphocytes, and the detergent has no effect on the enzyme activity. On the other hand, the guanylate cyclase of tonsil lymphocytes reside in the particulate fraction. It has non-Michaelian kinetics for the substrate, a strong stimulating effect of detergent, and an inhibitory effect of Ca2+. A comparison of the enzymatic activities of unseparated and of non-adherent tonsil lymphocytes obtained from the same donor points to a correlation between their T and B properties: predominant soluble activity in the T population and particulate guanyl cyclase activity in the B subset.

[Inhibition of DNA biosynthesis in human lymphocytes by alpha 1-antitrypsin]. / Inhibition de la biosynthèse du DNA chez les lymphocytes humains par l'effet de l'alpha1-antitrypsine.

Incorporation of tritiated thymidine into human lymphocytes from tonsils is markedly inhibited by purified alpha1-antitrypsin-preparations. This inhibitory effect is observed in lymphocytes stimulated by a mitogen factor (phytohemagglutinin) as in non stimulated lymphocytes.

A study of allergens in celery with cross-sensitivity to mugwort and birch pollens.

Sixty-one sera with positive RAST to mugwort pollen (Artemisiae vulgaris) were submitted to RASTs for birch pollen (Betula verrucosa) and celery (Apium graveolens). In 36 cases RAST results were positive for celery. In addition, 23 sera presented specific IgE to birch pollen. The binding of specific IgE to individual allergens in celery, mugwort pollen and birch pollen was studied by the immunoblotting technique. This involved electrophoretic separation of allergenic extracts, electrotransfer of proteins onto nitrocellulose sheets and sensitive immunoenzymatic detection. Eighteen sera had specific IgE binding to two celery components of molecular weight around 15 kD. All these sera also detected a 15 kD allergen in mugwort and two allergens in birch of 14 kD and 16 kD molecular weight. The sera that did not detect the 15 kD bands in celery failed to react with both the 15 kD mugwort component and the 14 and 16 kD birch components. Specific cross-inhibitions of the detection of these allergens on immunoblots were obtained by pre-incubation of the sera with crude extract of the three species. These results strongly suggest that such allergens display some structural identity and that they could be at the origin of some cases of crossed hypersensitivity to celery, mugwort pollen and birch pollen.

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.