Gibberellin-regulated proteins: Emergent allergens.

About 10 years ago, a protein family was shown for the first time to contain allergenic members, gibberellin-regulated protein (GRP). The first reported member was from peach, Pru p 7. One can hypothesize that it was not detected before because its physicochemical characteristics overlap with those of lipid transfer protein (LTP), a well-known allergen, or because the exposure to GRP increased due to an increase in the gibberellin phythormone level in plant food, either exogenous or endogenous. Like LTPs, GRPs are small cationic proteins with disulfide bridges, are resistant to heat and proteolytic cleavage, and are involved in the defense of the plant. Besides peach, GRP allergens have been described in Japanese apricot (Pru m 7), sweet cherry (Pru av 7), orange (Cit s 7), pomegranate (Pun g 7), bell pepper (Cap a 7), strawberry (Fra a GRP), and also in pollen with a restriction to Cupressaceae tree family (Cup s 7, Cry j 7, and Jun a 7). IgE cross-reactivities were described between GRPs, and the reported peach/cypress and citrus/cypress syndromes may therefore be explained because of these GRP cross-reactivities. GRPs are clinically relevant, and severe adverse reactions may sometimes occur in association with cofactors. More than 60% and up to 95% sequence identities are calculated between various allergenic GRPs, and three-dimensional models show a cleft in the molecule and predict at least three epitopic regions. The structure of the protein and its properties and the matrix effect in the original allergenic source should be unraveled to understand why, despite the ubiquity of the protein family in plants, only a few members are able to sensitize patients.

Identification of a polygalacturonase (Cup s 2) as the major CCD-bearing allergen in Cupressus sempervirens pollen.

As IgE glyco-epitopes, also referred to as cross-reactive carbohydrate determinants (CCDs), can share significant structural homologies between different plants, they are prone to extensive cross-reactivity among allergen pollen extracts. Here, cypress pollen allergens, especially a polygalacturonase (PG), were further characterized using double one-dimensional electrophoresis (D1-DE). The presence of specific IgE directed against CCDs was investigated by bromelain IgE inhibition and concanavalin A binding assays using sera of cypress pollen-sensitized patients. Our results showed that IgE reactivity to CCDs in Cupressus sempervirens pollen extracts is mainly related to bromelain-type epitopes of a newly identified cypress PG. This glycoprotein has been further characterized through an immunoproteomic approach and officially indexed as Cup s 2 by the WHO/IUIS allergen nomenclature. Cup s 2 could thus be associated with the increased prevalence of IgE reactivity to cypress pollen extracts because of CCD interference.

Complementarity between microarray and immunoblot for the comparative evaluation of IgE repertoire of French and Italian cypress pollen allergic patients.

Cypress pollen represents the primary cause of respiratory allergies in Mediterranean areas. Patients allergic to Cupressus sempervirens pollen (Cups) (CPA) can be discriminated on the basis of the immunoglobulin E (IgE) binding to a basic 14 kDa protein (BP14) or to high-molecular-weight (HMW) glycoproteins only. Specific IgE repertoires of two differentially exposed CPA cohorts, French and Italian, were investigated using an IgE microarray system (some known major allergens from several allergenic sources) and individual IgE immunoblotting (IB) of whole Cups pollen extract separated by SDS-PAGE (all allergens from one allergenic source: cypress pollen). The prevalence of sensitization to BP14 was higher in French (37 %) than in Italian patients (17 %) and major differences were observed in IgE reactivities to lipid transfer proteins (LTPs). Thirty percent of the Italian CPA (4 % in the French group) had specific IgE against the Parietaria pollen LTP, independently of IB subgroups. Regarding peach LTP sensitization, all Pru p 3+ Italian CPA (10 %) were in the HMW+ subgroup, while Pru p 3+ French CPA (20 %) were all included in the BP14+ subgroup. BP14 sensitization is likely a marker of Cups exposure and is, in French CPA, significantly correlated to Pru p 3 sensitization. The IgE immunoblot and microarray are complementary tools that highlight differences in the subtle sensitization profile between groups of patients in comparative studies.

[Cypress pollen allergy]. / Allergie au pollen de cyprès.

Cypress belongs to the Cupressaceae family, which includes 140 species with non-deciduous foliage. The most important genera in allergic diseases are Cupressus sempervirens or Green cypress, Cupressus arizonica or Blue cypress, Juniperus oxycedrus, Juniperus communis and Thuya. Because J. oxycedrus pollinates in October, C. sempervirens in January and February, C. arizonica in February and March, J. communis in April, the symptomatic period is long-lasting. Because of global warming, the pollination period is tending to last longer and Cupressaceae species are becoming established further the north. In Mediterranean countries, cypress is by far the most important pollinating species, accounting for half of the total pollination. The major allergens belong to group 1. The other allergens from cypress and Juniper share 75 to 97 % structural homology with group 1 major allergens. The prevalence of cypress allergy in the general population ranges from 5 % to 13 %, according to exposure to the pollen. Among outpatients consulting an allergist, between 9 and 35 %, according to different studies, are sensitized to cypress pollen. Repeated cross-sectional studies performed at different time intervals have demonstrated a threefold increase in the percentage of cypress allergy. Risk factors include a genetic predisposition and/or a strong exposure to pollen, but air pollutants could play a synergistic role. The study of the natural history of cypress allergy allows the identification of a subgroup of patients who have no personal or family history of atopy, whose disease began later in life, with low total IgE and often monosensitization to cypress pollen. In these patients, the disease is allergic than rather atopic. In the clinical picture, rhinitis is the most prevalent symptom but conjunctivitis the most disabling. A cross-reactivity between cypress and peach allergy has been demonstrated. The pharmacological treatment of cypress allergy is not different from that for other allergies. Hyposensitization has been used, at first by injection, but nowadays mostly through the sublingual route, but clinical trials have included few patients. Avoidance can be implemented at the individual level but also at the community levels using alternative plants, low-pollinating cypresses or by trimming hedges prior to pollination.

Immunodominant semen proteins II: contribution of seminal proteins to female immune infertility.

Seminal fluid is a protective medium for sperm, but it also represents potential immunogenic structures for the female immune system. Anti-seminal antibodies may threaten early fertilization. The aim of our work is to detect and identify seminal proteins that are related to female isoimmunization. In this report, we quantified serum anti-seminal IgG antibodies. Seminal proteins were analysed by two-dimensional gel electrophoresis followed by immunoblotting. To identify IgG-binding proteins of interest, a proteomic approach was selected. The dominant seminal antigens were detected within the relative molecular mass ranging from 25 to 85 kDa and the isoelectric point from 5 to 7. The detected proteins were further identified as prostate-specific antigen, prostatic acid phosphatase, zinc-α-2-glycoprotein and zinc finger protein 778. Since these proteins were recognized by IgGs produced by infertile women and not by fertile women, we presume that major seminal antigens may play an important role in the pathogenesis of female immune infertility. Our study suggests the pattern of seminal proteins for further therapeutic attempts in the diagnosis of female immune infertility.

[Clinical evaluation of the new coat colour macchiato in a male Franches-Montagnes horse]. / Klinische und genetische Befunde bei einem männlichen Freiberger mit der neuen Fellfarbe Macchiato.

In April 2008 a Franches-Montagnes colt was born with an unusual coat colour phenotype which had never been observed in that population before. The foal showed extended white markings on body and legs, a white head and blue eyes. As both parents have an unremarkable bay coat colour phenotype, a de novo mutation was expected in the offspring and a candidate gene approach revealed a spontaneous mutation in the microphthalmia associated transcription factor gene (MITF). A detailed clinical examination in 2010 indicated an impaired hearing capacity. As in the American Paint Horse large white facial markings in combination with blue eyes are associated with deafness, the hearing capacity of the stallion was closer examined performing brainstem auditory-evoked responses (BAER). The BAER confirmed bilateral deafness in the Franches-Montagnes colt. It is assumed that the deafness is caused by a melanocyte deficiency caused by the MITF gene mutation. Unfortunately, due to castration of the horse, the causal association between the mutation in the MITF gene and clinical findings cannot be confirmed by experimental matings.

Genetic diversity in an indigenous horse breed: implications for mating strategies and the control of future inbreeding.

The Franches-Montagnes is an indigenous Swiss horse breed, with approximately 2500 foalings per year. The stud book is closed, and no introgression from other horse breeds was conducted since 1998. Since 2006, breeding values for 43 different traits (conformation, performance and coat colour) are estimated with a best linear unbiased prediction (BLUP) multiple trait animal model. In this study, we evaluated the genetic diversity for the breeding population, considering the years from 2003 to 2008. Only horses with at least one progeny during that time span were included. Results were obtained based on pedigree information as well as from molecular markers. A series of software packages were screened to combine best the best linear unbiased prediction (BLUP) methodology with optimal genetic contribution theory. We looked for stallions with highest breeding values and lowest average relationship to the dam population. Breeding with such stallions is expected to lead to a selection gain, while lowering the future increase in inbreeding within the breed.

Patterning of polystyrene by scanning electrochemical microscopy. Biological applications to cell adhesion.

Polystyrene surfaces may be patterned by Ag(II), NO(3)(•), and OH(•) electrogenerated at the tip of a scanning electrochemical microscope. These electrogenerated reagents lead to local surface oxidation of the polymer. The most efficient surface treatment is obtained with Ag(II). The patterns are evidenced by XPS and IR and also by the surface wettability contrast between the hydrophobic virgin surface and the hydrophilic pattern. Such Ag(II) treatment of a polystyrene Petri dish generates discriminative surfaces able to promote or disfavor the adhesion of proteins and also the adhesion and growth of adherent cells. The process is also successfully applied to a cyclo-olefin copolymer and should be suitable to pattern any hydrogenated polymer.

Evaluation of ash pollen sensitization pattern using proteomic approach with individual sera from allergic patients.

BACKGROUND: In Europe, sensitization to ash pollen induces pollinosis with cross-reactivities with other pollen sources. The aim of the study was to identify the repertoire of ash pollen allergens and evaluate the extent of the diversity of the IgE response in ash allergic patients. METHODS: The IgE reactivities of 114 ash pollen- and eight grass pollen-sensitized patients were screened by 1D immunoblot (SDS-PAGE) against ash pollen extract. The IgE reactivities of 13 ash pollen- and two grass pollen-sensitized patients were then evaluated in 2D immunoblots. Some IgE- and non-IgE-reactive proteins were identified by mass spectrometry. RESULTS: In 1D analysis, 86% of sera showed binding to Fra e 1 (18-20 kDa), 23% to Fra e 2 (14 kDa), 3% to Fra e 3 (10 kDa) and 57% to High Molecular Weight allergens (HMW, >30 kDa). Individual analysis of 2D immunoblots showed several IgE-binding protein areas among which three were more often recognized: (i) Fra e 1 comprising, at least, 15 isoforms, (ii) a series of acidic spots (45 kDa), and (iii) Fra e 2, the ash profilin. HMW allergens could be resolved in four areas; two unidentified, one homologous to beta-galactosidase and the other to sugar transport proteins. A malate deshydrogenase and calmodulin were shown to be IgE-binding proteins and 10 non-IgE reactive proteins were identified. CONCLUSIONS: No direct correlation was evidenced between IgE profile and the degree of sensitization even though 2 spectrotypes could be distinguished. Our data contribute to a better delineation of ash pollen allergens and patterns of sensitization.

Seven novel KIT mutations in horses with white coat colour phenotypes.

White coat colour in horses is inherited as a monogenic autosomal dominant trait showing a variable expression of coat depigmentation. Mutations in the KIT gene have previously been shown to cause white coat colour phenotypes in pigs, mice and humans. We recently also demonstrated that four independent mutations in the equine KIT gene are responsible for the dominant white coat colour phenotype in various horse breeds. We have now analysed additional horse families segregating for white coat colour phenotypes and report seven new KIT mutations in independent Thoroughbred, Icelandic Horse, German Holstein, Quarter Horse and South German Draft Horse families. In four of the seven families, only one single white horse, presumably representing the founder for each of the four respective mutations, was available for genotyping. The newly reported mutations comprise two frameshift mutations (c.1126_1129delGAAC; c.2193delG), two missense mutations (c.856G>A; c.1789G>A) and three splice site mutations (c.338-1G>C; c.2222-1G>A; c.2684+1G>A). White phenotypes in horses show a remarkable allelic heterogeneity. In fact, a higher number of alleles are molecularly characterized at the equine KIT gene than for any other known gene in livestock species.

[Measurement of the attention time in the horse]. / Mesure de la durée d'attention chez le cheval.

A study carried out on 49 horses showed that it is possible to measure the attention time by operant conditioning. After teaching horses an instrumental task using a signal, we were then able to test their attention time by asking them to prolong it increasingly while setting success and failure criteria. Two tests were performed 3 weeks apart. The 2nd test was feasible without relearning, a proof of memory, and was repeatable, a proof of consistency in the attention time. A significant difference was observed between the 3 age groups. Young horses often performed very well during the 1st test but their attention dropped in the 2nd test while older horses were more stable with respect to attention and even increased it slightly. The study shows that there are individual differences but it was not possible to prove a significant influence of breed, gender and paternal influence. Consequently, learning appears to be one of the most interesting approaches for evaluating the attention of horses and for observing their behaviour.

[The influence of the socio-economic structure of the breeding farms of Franches-Montagnes horses on the conditions of husbandry]. / L'influence de la structure socio-economique des exploitations d'elevage de chevaux de race franches-montagnes sur les conditions de détention.

The socio-economic structure of the breeding farms of Franches-Montagnes horses (FM) in Switzerland is evaluated on the basis of an investigation carried out in 2002 by the Swiss FM breeding federation. Questionnaires were sent to 3500 of its members and the results include data from 968 breeding enterprises, housing a total of 3965 FM. The quality of the husbandry of FM varies according to factors such as the altitude and the geographical situation of the farms and studs. Socio-economic parameters, such as the role of FM in the business, their use (breeding, driving, riding) and the age and level of professional education of the owners may also have an effect on standards of husbandry. The results show that the owners for whom FM represent a source of income more frequently keep their horses in standing stalls, but give them more time to exercise at liberty than the horses belonging to amateur breeders. Younger and better educated breeders are more likely to house their animals in groups.

[Evaluation of the quality of husbandry of Franches-Montagnes horses in their breeding farms]. / Les conditions de détention des chevaux de race franches-montagnes dans leurs exploitations d'elevage.

The quality of husbandry of Franches-Montagnes horses (FM) in Switzerland is evaluated on the basis of an investigation carried out in 2002 by the Swiss FM breeding federation. Questionnaires were sent to 3500 of its members and the results include data from 968 breeding enterprises, housing a total of 3965 FM: 46.1% were breeding mares (61.0% with foal at foot), 26.5% young stock, 1.3% stallions and 26.0% non breeding stock (74.6% of which were pleasure horses and 25.4% working horses). 57.6% of the FM were housed in individual boxes with or without permanent outdoor access, 25.4% were hold in groups with or without permanent outdoor access, the remaining 17.0% were kept in standing stalls. 95.0% of the FM had at least visual contact with other equines and 99.2% had sufficient light in their stable. 88.1% were stabled on long stalk straw, while only 4.3% were bedded on other materials other than straw. The average time spent at pasture per horse and per week ranged from 96.5 +/- 51.6 hours in summer to 27.2 +/- 26.7 hours in winter. On average, a FM is used for 8.3 +/- 6.5 hours per week. Horses with an paddock at their disposal spend an average of 39.8 +/- 45.9 hours there per week.

Analysis of pedigree and conformation data to explain genetic variability of the horse breed Franches-Montagnes.

Franches-Montagnes is the only native horse breed in Switzerland, therefore special efforts should be made for ensuring its survival. The objectives of this study were to characterize the structure of this population as well as genetic variability with pedigree data, conformation traits and molecular markers. Studies were focused to clarify if this population is composed of a heavy- and a light-type subpopulation. Extended pedigree records of 3-year-old stallions (n = 68) and mares (n = 108) were available. Evaluations of body conformation traits as well as pedigree data and molecular markers did not support the two-subpopulation hypothesis. The generation interval ranged from 7.8 to 9.3 years. The complete generation equivalent was high (>12). The number of effective ancestors varied between 18.9 and 20.1, whereof 50% of the genetic variability was attributed to seven of them. Genetic contribution of Warmblood horses ranged from 36% to 42% and that of Coldblood horses from 4% to 6%. The average inbreeding coefficient reached 6%. Inbreeding effective population size was 114.5 when the average increase of the inbreeding coefficient per year since 1910 was taken. Our results suggest that bottleneck situations occurred because of selection of a small number of sire lines. Promotion of planned matings between parents that are less related is recommended in order to avoid a reduction of the genetic diversity.

Genetic diversity among horse populations with a special focus on the Franches-Montagnes breed.

Genetic characterization helps to assure breed integrity and to assign individuals to defined populations. The objective of this study was to characterize genetic diversity in six horse breeds and to analyse the population structure of the Franches-Montagnes breed, especially with regard to the degree of introgression with Warmblood. A total of 402 alleles from 50 microsatellite loci were used. The average number of alleles per locus was significantly lower in Thoroughbreds and Arabians. Average heterozygosities between breeds ranged from 0.61 to 0.72. The overall average of the coefficient of gene differentiation because of breed differences was 0.100, with a range of 0.036-0.263. No significant correlation was found between this parameter and the number of alleles per locus. An increase in the number of homozygous loci with increasing inbreeding could not be shown for the Franches-Montagnes horses. The proportion of shared alleles, combined with the neighbour-joining method, defined clusters for Icelandic Horse, Comtois, Arabians and Franches-Montagnes. A more disparate clustering could be seen for European Warmbloods and Thoroughbreds, presumably from frequent grading-up of Warmbloods with Thoroughbreds. Grading-up effects were also observed when Bayesian and Monte Carlo resampling approaches were used for individual assignment to a given population. Individual breed assignments to defined reference populations will be very difficult when introgression has occurred. The Bayesian approach within the Franches-Montagnes breed differentiated individuals with varied proportions of Warmblood.

[Molecular genetic studies of the "Einsiedler" horse population]. / Molekulargenetische Untersuchungen beim Einsiedler-Pferd.

In this study it was investigated whether the "Einsiedler" warmblood horse, a historically old horse population from central Switzerland (Abbey of Einsiedeln), is distinguishable from micellaneous horse breeds, using molecular genetic techniques. The breeding history of Einsiedler horses is characterised by systematic line breeding through the dams. Therefore, two Einsiedler dam lines (N = 28), going back to the middle of the 19th century according to pedigree entries, were the focus of the survey. Random samples of diverse warmblood horse populations, but also samples from more distinct types of horse breeds, served as comparison populations (N = 52). Variation in the mitochondrial genome appeared to be only partially informative to demarcate the studied horses, as horses of distinct breeds may share identical mtDNA sequence fragments. Both dam lines revealed haplotypes commonly found in Iberian horse breeds. This is to take as an indication on the genetic origin of Einsiedler horses. Furthermore, the Klima dam line held a homologous mtDNA sequence fragment with E. ferus przewalskii. Therefore, this seems to be a phylogenetically old haplotype. The analysis of microsatellite loci revealed that horses from the two Einsiedler dam lines were in fact distinguishable from more distinct types of horses, but not from closely related European warmblood horse breeds and English thoroughbred.

Lipid transfer protein 1 is a possible allergen in Arabidopsis thaliana.

BACKGROUND: The Arabidopsis thaliana genome was recently fully sequenced, and this plant is now considered as the most useful model to study the effects of genetic engineering. The aim of the present study was to identify A. thaliana IgE-binding molecules and to localize their genes in order to evaluate the potential effect of gene insertion on the expression of IgE-binding molecules. METHODS: A. thaliana flower proteins were separated by two-dimensional gel electrophoresis and transferred onto a nitrocellulose sheet. The nitrocellulose sheet was successively incubated with human sera known to contain IgE that binds to rapeseed proteins, alkaline phosphatase-conjugated goat anti-human IgE and 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium. One allergen was further identified by N-terminal amino acid microsequencing. RESULTS: The results showed that some individuals possessed IgE that recognized numerous proteins with high molecular masses and various isoelectric points. This binding pattern strongly suggests that the epitopes recognized by these IgE could be, at least partly, sugar residues. Otherwise, out of the 10 sera that possessed IgE to Arabidopsis flower proteins, one serum strongly recognized a unique basic protein with an apparent molecular mass of around 14 kD. This protein was identified by amino acid microsequencing as the lipid transfer protein 1 (LTP1). CONCLUSION: We have demonstrated that A. Thaliana LTP1 is IgE reactive. The gene encoding this protein is located on chromosome 2, but it has been described that family 1 of A. Thaliana LTPs constitutes a multigenic family with genes located on various chromosomes.

Polygalacturonase (pectinase), a new oilseed rape allergen.

BACKGROUND: Type I hypersensitivity to rapeseed pollen allergens was described as the result of a cross-sensitization with various pollens that could constitute an aggravating factor in birch or grass pollen allergies. Recently, a few rapeseed pollen allergens were described. The aim of the present work was to identify new rapeseed pollen allergens by using two-dimensional gel analysis, microsequencing, and mass spectrometry. METHODS: Water extractable proteins from oilseed rape pollen or stamen were separated by two-dimensional gel electrophoresis. The proteins were then electroblotted onto a nitrocellulose (NC) sheet. The NC sheets were successively incubated with (1) individual human sera pre-selected for their immunoglobulin E (IgE) reactivity to rapeseed pollen proteins, (2) alkaline phosphatase (AP)-conjugated goat anti-human IgE and (3) AP substrate. The allergens localized by this method were then identified by microsequencing and MALDI-TOF mass spectrometry analysis. RESULTS: Of the 18 sera studied, five recognized a wide multispot zone with a molecular mass around 43 kD and pIs between 6.5 and 8.5. The results obtained with two representative sera are shown. From this zone, two isoforms of the polygalacturonase enzyme were identified by microsequencing. Confirmation was obtained through MALDI-TOF mass spectrometry analysis. CONCLUSION: The present results allow the identification of a new rapeseed allergen that can be the main allergen for some patients.

[Horse breeding: genetic tests for the coat colors chestnut, bay and black. Results from a preliminary study in the Swiss Freiberger horse breed]. / Pferdezucht: Genetische Tests für die Fellfarben Fuchs, Braun und Schwarz. Ergebnisse einer ersten Untersuchung in der Schweizer Freibergerpferderasse.

Coat color played an important role during domestication and formation of breeds. Livestock breeders often had special preferences for particular color phenotypes because they believed them to be associated with performance or fitness traits. Socio-cultural reasons might have had an influence on color selection as well. Recently genetic tests on DNA level got available to genotype in any individual horse for basic horse coat colors (chestnut, bay, black). In particular, hidden carriers of the recessive chestnut and black allele are recognizable with these tests. A sample of 162 Franches-Montagnes horses from Switzerland was genotyped for the alleles for chestnut and black. The analysis of allele frequencies revealed a high prevalence of the chestnut allele and a low frequency of the black allele in this population. Rare colors are in demand on the market. The statistical analysis of 1369 offspring from five stallions indicate, that darker shades of basic color phenotypes (dark chestnut, dark bay) follow a recessive mode of inheritance in the Franches-Montagnes horse breed.