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.

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.

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.

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.

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.

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.

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.

MHC class II-dependent activation of CD4+ T cell hybridomas by human mast cells through superantigen presentation.

Human mast cells (MC) were examined for expression of MHC class II antigens and for their ability to activate CD4+ T cell hybridomas through presentation of superantigen (SAg). HMC-1, a leukemic immature MC line expressing class II Ags, was shown to efficiently present the staphylococcal enterotoxin B (SEB) SAg to responding T cell hybridoma on treatment with interferon-gamma (IFN-gamma), which up-regulated class II molecules. The study was then extended to human normal MC. Almost pure (>99%) cord blood-derived MC (CBMC) were shown to express class II Ags (HLA-DR and HLA-DQ) and CD80, which were up-regulated by IFN-gamma treatment and, to a lesser extent, by interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). CBMC directly activated CD4+ T cell hybridomas through presentation of SEB and TSST1 SAgs. The production of IL-2 required a cell-to-cell contact between T cells and CBMC and it was inhibited by anti-class II antibodies. Furthermore, an additional pretreatment of CBMC by IFN-gamma or GM-CSF or IL-4 had no effect on their presenting efficiency. This previously unknown function of human MC, i.e., MHC class II-dependent activation of CD4+ T cells, may be critical in subsequent cellular activation events because colocalization of mast and T cells is frequently observed at sites of antigen entry.

Immunochemical studies of polyspecific natural autoantibodies: charge, lipid reactivity, Fab'2 fragments activity and complement fixation.

Polyspecific natural autoantibodies (NAAb) are antibodies present in normal unimmunized animals and are able to react with very dissimilar antigens (Ag). To better delineate the characteristics of polyspecificity, we subjected monoclonal NAAb to four different immunochemical studies: (1) Two-dimensional gel electrophoresis performed on eight NAAb did not reveal any obvious relationship between charge and antigen specificities; (2) NAAb widely polyspecific on proteins and nucleic acid were reactive with lipids bearing either phosphate, sulfate or carboxyl polar groups; (3) pepsin digestion of polyspecific IgM NAAb yielded Fab'2 fragments which maintained their multireactivities, but exhibited a decrease in reactivity as compared to that seen with monospecific mAb (induced); (4) two different assays were used to analyse the complement fixation ability of IgM NAAb. While very weak or no complement fixation was observed with a classical complement fixation test (fluid phase), when a complement enzyme immunoassay was used where Ag is immobilized on a solid phase, polyspecific NAAb fixed reproducible and easily detectable amounts of complement.

Anti-histone autoantibodies react specifically with the B cell surface.

In an attempt to induce an immune response against Mls-1a antigens by immunizing C57B1/6 mouse (Mls-1b) with purified B cells from DBA/2 mouse (Mls-1a), we generated a panel of monoclonal antibodies from which the 5B9.6 mAb, taken as a representative antibody, was thoroughly investigated. This antibody specifically reacts with B cells from all mouse strains studied including C57Bl/6 mice as shown by FACS analysis of double-antibody labelled spleen cells. Using enzyme immunoassays and immunoprecipitation techniques, 5B9.6 mAb was found to be specific for histones. Amino acid sequence analysis of a peptide derived from a 5B9.6-immunoprecipitated polypeptide from DBA/2 B cells showed a 100% homology with a sequence within H2B histones. Furthermore, 5B9.6 mAb was able to interact with the cell surface of 7OZ/3 cell line, known as a typical pre-B cell line. The presence of histones can be modulated on the surface of 7OZ/3 cells since this antigen was upregulated after exposure of these cells to a cocktail of IL-1 and cAMP. Finally, 5B9.6 mAb was shown to interact with freshly isolated B cells from human peripheral blood.

Monoclonal autoantibodies against mouse red blood cells: a family of structurally restricted molecules.

Cultured mouse peritoneal cells from unstimulated mice developed plaque-forming activity against isologous bromelain-treated erythrocytes. Several IgM monoclonal autoantibodies obtained by fusion of peritoneal cells from NZB or CBA origin with BALB/c myeloma cells were purified by affinity chromatography on trimethyl ammonium (TMA) column on the basis of their cross-reactivity with TMA, phosphorylcholine (PC) or choline haptens. Binding affinity for PC hapten was of the order of 10(3) M-1. Idiotypic studies with a polyclonal rabbit anti-idiotypic reagent revealed strong cross-reactions with all hybridoma autoantibodies thus far tested. In addition, the rabbit anti-idiotypic serum detected idiotypes or cross-reactive idiotypes in the sera of NZB and CBA as well as BALB/c mice. N-terminal amino acid sequence analyses of three hybridoma autoantibodies from NZB mice and one from CBA mice were carried out. The sequences of the first 32 residues of the four heavy chains showed that three were identical while one had one amino acid interchange; they belong to the VHIII-subgroup. The light chains were identical in the first 35 residues with the exception of a substitution at position 3 in two light chains and are members of the VK-9-subgroup. These results entirely support the idiotypic data. These monoclonal autoantibodies from NZB and CBA mice although isolated and eluted from PC-related haptens do not have any apparent structural nor idiotypic relationship to PC-specific antibodies. Idiotypic and V-region N-terminal sequence data suggest that these autoantibodies constitute a highly restricted family of molecules likely to be encoded by unique germ-line genes which may be expressed as such or as somatic variants in different mouse strains.

[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.

Targeted transfection of the IL-3 gene into primary human hematopoietic progenitor cells through the c-kit receptor.

We recently showed that an antibody-mediated gene transfer procedure termed antifection can be used for targeted gene delivery into lymphoid cells in vitro and in vivo. We here report that antifection also is effective for targeted gene transfer to immature hematopoietic cells. A human IL3-expressing plasmid was chemically linked to an anti-human CD117 antibody. Delivery of the IL3 plasmid into IL-3-dependent myeloid TF-1 cells (bearing the CD117 antigen) was specific and resulted in the transient proliferation of the targeted cells in the absence of exogenous IL-3. Transfection of primary human CD34+ hematopoietic stem/progenitor cells led to transient production of IL-3 and transient proliferation of the target cells. Interestingly, by using a semisolid progenitor cell assay, we found that transfected primary CD34+ cells were able to generate normal numbers of cell colonies in the absence of exogenous IL-3. Polymerase chain reaction analysis confirmed the presence and expression of the IL-3 transgene in the progenitor-derived colonies. In conclusion, our data show that CD117 is a suitable cell surface target to specifically transfer gene by antifection into primary CD34+ cells and that delivery of IL-3 gene in these cells resulted in the expression of a functional IL-3 able to support cell growth in absence of exogenous cytokine. Thus, antifection may provide new therapeutic modality relying on the transient production of appropriate growth factors acting via autocrine and/or paracrine mechanisms.

Antibody-mediated endocytosis of G250 tumor-associated antigen allows targeted gene transfer to human renal cell carcinoma in vitro.

Specific gene transfer into targeted tumor cells remains a critical issue for the development of systemic gene therapy protocols. With this end in view, we have tested the possibility of selectively directing genes to tumor cells through the recognition of tumor-associated antigens (TAA). This was approached in vitro on four human renal cell carcinoma (RCC) lines by means of the highly specific mouse G250 monoclonal antibody (mAb) chemically conjugated to a plasmid DNA conveying a reporter activity. This mAb directed to a TAA that is present on 95% of primary RCCs and on 60% of metastatic human RCCs was extensively characterized, including during clinical trials. Epifluorescence microscopy analysis indicated that upon specific binding to G250 TAA, G250 mAb alone or conjugated to plasmid DNA was internalized by an active endocytic process and colocalized with the transferrin concentrated in the late recycling perinuclear compartment. We also observed that both unconjugated G250 mAb or G250 mAb conjugated to plasmid DNA remained in the perinuclear region of the cells for > or = 20 hours and were not rapidly translocated to lysosomes or recycled to the plasma membrane. In contrast, unconjugated plasmid DNA was not internalized. After transfection of G250 TAA-positive RCC lines with G250 mAb conjugated to a plasmid cDNA encoding mouse interleukin-2, a significant and sustained production of mouse interleukin-2 protein was detected from days 5-15 and was abrogated by inhibiting the internalization process. Altogether, our data showed that endocytosis of G250 TAA should be the basis of gene transfer to RCC, suggesting that targeting of TAA capable of internalization may be the basis of new approaches for designing alternative cancer gene therapy procedures.