Belén de Andrés

Ole e I: Epitope Mapping, Cross-Reactivity with Other Oleaceae Pollens and Ultrastructural Localization

Ole e I is the major allergen derived from olive tree pollen (Olea europaea) and it is composed of two polypeptides with molecular weights (MWs) of 18 and 20 kD. A panel of six monoclonal antibodies (mAbs) has been prepared and used to map antigenic determinants on this molecule. Four epitope determinants have been identified on Ole e I. Using the purified mAbs produced against Ole e I, we have analyzed the common epitope determinants in olive (O. europaea) and different Oleaceae pollens: ash (Fraxinus excelsior); privet (Ligustrum vulgare); lilac (Syringa vulgaris), and forsythia (Forsythia suspensa). ELISA showed three reactivity groups depending on the recognition of monoclonal antibodies: (1) olive and ash; (2) olive, ash, privet and lilac; and (3) olive, ash, privet, lilac and forsythia. Immunoblotting studies on Oleaceae pollen extracts with these mAbs showed a very similar cross-reactivity pattern. The 18- and 20-kD MW proteins were present in each pollen, except in the case of forsythia. In this case the reactivity pattern was associated with 50- to 55-kD protein bands. This band was recognized by a pool of sera from olive-allergic patients. Finally, ultrastructural localization of Ole e I antigen was performed on the mature olive pollen grain. Ole e I was located in association with dilated endoplasmic reticulum cisternae. Pollen grain walls, nuclei and cytoplasmic organelles were totally devoid of the allergen.

A population of c-Kitlow(CD45/TER119)– hepatic cell progenitors of 11-day postcoitus mouse embryo liver reconstitutes cell-depleted liver organoids

Embryo liver morphogenesis takes place after gastrulation and starts with a ventral foregut evagination that reacts to factor signaling from both cardiac mesoderm and septum transversum mesenchyme. Current knowledge of the progenitor stem cell populations involved in this early embryo liver development is scarce. We describe here a population of 11-day postcoitus c-Kit(low)(CD45/TER119)- liver progenitors that selectively expressed hepatospecific genes and proteins in vivo, was self-maintained in vitro by long-term proliferation, and simultaneously differentiated into functional hepatocytes and bile duct cells. Purified c-Kit(low)(CD45/TER119)- liver cells cocultured with cell-depleted fetal liver fragments engrafted and repopulated the hepatic cell compartments of the latter organoids, suggesting that they may include the embryonic stem cells responsible for liver development.

DR7 and DQ2 are positively associated with immunoglobulin-E response to the main antigen of olive pollen (Ole e I) in allergic patients

We have studied the relationship between HLA class II haplotypes and alleles, and the IgE antibody response to a highly purified allergen, Ole e I, in allergic patients. Ole e I, is the major antigen from the pollen of olive tree that grows mainly in the Mediterranean. Genomic DNA typing was performed in 40 unrelated patients with seasonal allergic pollenosis who had specific IgE antibodies against Ole e I, detected by double-antibody radioimmunoassay. HLA-DRB and -DQB loci were analyzed by PCR-SSO and RFLP. Phenotypic frequencies were compared with those of 179 healthy unrelated individuals. Significant increases in the phenotypic frequencies of DR7 (pf = 67.5% vs 31.3% in the control population, pc = 0.0023) and DQ2 (pf = 90.0% vs 48.0%, pc = 0.0003) were found, indicating an association between DRB1*0701/2, DQB1*0201 alleles and the IgE antibody response to Ole e I. This is the first time that the HLA-DQ gene has been associated with a positive allergic response.

A Role for DNA Polymerase μ in the Emerging DJH Rearrangements of the Postgastrulation Mouse Embryo

ABSTRACT The molecular complexes involved in the nonhomologous end-joining process that resolves recombination-activating gene (RAG)-induced double-strand breaks and results in V(D)J gene rearrangements vary during mammalian ontogeny. In the mouse, the first immunoglobulin gene rearrangements emerge during midgestation periods, but their repertoires have not been analyzed in detail. We decided to study the postgastrulation DJH joints and compare them with those present in later life. The embryo DJH joints differed from those observed in perinatal life by the presence of short stretches of nontemplated (N) nucleotides. Whereas most adult N nucleotides are introduced by terminal deoxynucleotidyl transferase (TdT), the embryo N nucleotides were due to the activity of the homologous DNA polymerase μ (Polμ), which was widely expressed in the early ontogeny, as shown by analysis of Polμ−/− embryos. Based on its DNA-dependent polymerization ability, which TdT lacks, Polμ also filled in small sequence gaps at the coding ends and contributed to the ligation of highly processed ends, frequently found in the embryo, by pairing to internal microhomology sites. These findings show that Polμ participates in the repair of early-embryo, RAG-induced double-strand breaks and subsequently may contribute to preserve the genomic stability and cellular homeostasis of lymphohematopoietic precursors during development.

Immunological basis of toxic oil syndrome (TOS)

The toxic oil syndrome (TOS), a multisystemic disease, that occurred in Spain in 1981, was caused by the ingestion of rapeseed oil denatured with 2% aniline. Due to the clinical course of the disease, immunopathological mechanisms have been suspected but a direct connection was never demonstrated. To analyse this possibility, we determined several immunological parameters in the sera of patients with TOS and without the disease, using a case-control design: total immunoglobulins, IgG and IgE antibodies against different toxic agents (oleylanilide, aniline, linoleyl-anilide, and 3-phenylaminopropane-1-2-diol), autoantibodies, cytokines (IL-4, IL-6, TNF, GM-CSF) and soluble receptors (sCD23 and sIL-2R). We detected high levels of sIL-2R in TOS patients compared to controls (P < 0.0001). A higher levels of sCD23 and IgE were also found. In addition, the response to oleyl-anilide of peripheral blood lymphocytes from TOS patients was studied and a significant proliferative response in 30% of TOS patients versus 5% controls was observed. Our data support the implication of the immune system in the acute phase of TOS, with a possible activation of T-cells and release of cytokines, that could explain some of the clinical findings in this phase of the disease.

Th2-type granuloma development in acute murine schistosomiasis is only partly dependent on CD4+ T cells as the source of IL-4

Schistosome granulomas produce IL‐4, important for Th2 granuloma expression. We defined the origins of IL‐4 within these granulomas and the role of IL‐4‐producing CD4+ T cells in Th2 granuloma development. Dispersed granuloma cells spontaneously produced IL‐4 independently of T cells, whereas IL‐5 production was T cell dependent. Granuloma IL‐4 mRNA localized to the non‐T cells and IL‐5 to T cells. Granuloma CD4+ T and NK cells, but not B cells produced IL‐4 and IL‐5 in vitro. B cell–/– mice generated Th2 granulomas that produced IL‐4 and IL‐5 normally. Granuloma eosinophils expressed no IL‐4 or IL‐5 mRNA. Granulomas in WWv mast cell‐deficient mice lacked mast cells. The dispersed granuloma cells from WWv mice released IL‐4 only after T cell stimulation, suggesting that mast cells influenced the constitutive component of IL‐4 production. Rag‐1 animals (T/B/NK T cell deficient) given schistosomiasis after reconstitution with splenocytes from naive mice produced Th2 granulomas. Mice reconstituted to create selective CD4+ T cell IL‐4 knockout animals developed eosinophilic granulomas that made IL‐4. Thus, granulomas contain several cell types that produce IL‐4. Mast cells are not needed to form Th2 granulomas, but influence IL‐4 release. Th2 granuloma development in schistosomiasis is only partly dependent on IL‐4‐producing CD4+ T cells.

Study of apoptosis in human lymphocytes by toxic substances implicated in toxic oil syndrome.

Toxic Oil Syndrome is a multisystemic disease that occurred in epidemic proportions in Spain in 1981 caused by the ingestion of rapeseed oil denatured with aniline. Several data implicate T cells in the pathogenesis of the disease. We evaluated the mechanisms of cytotoxicity in human lymphocytes of TOS-related products: aniline, 3-(N-phenylamino)-1,2-propanediol and its mono- and di-oleyl esters and eosinophilia myalgia-related product such as 3-(phenylamino)-L-alanine, which is chemically similar to 3-(N-phenylamino)-1,2-propanediol, and has been found in manufactured L-tryptophan. Our results show that only di-oleyl ester of 3-(N-phenylamino)-1,2-propanediol induces apoptosis in human lymphocytes, in a concentration and time-dependent way, confirmed by morphology, expression of phosphatidylserine in membrane and analysis of DNA degradation.

A regulatory role for Fcγ receptors (CD16 and CD32) in hematopoiesis

Abstract Progenitor cells of the T- and B-lineages in mice express (CD32) and FcγRIII (CD16) but as the developing lymphocytes begin to express clonal antigen receptors, CD16 and CD32 are downregulated in T-cells, and CD16 is downregulated in B-cells. Considering that counter-receptors for FcγR occur on thymic and bone marrow stromal cells, the possibility exists that FcγR might participate in some aspect of T- and B-lineage development prior to the stage of antigen receptor expression. Previous studies provided evidence that FcγR can influence murine T-lineage development. In the present studies we found that anti-FcγRII/III mAb accelerated B-lineage development in bone marrow cultures from normal mice, but not in cultures from CD16-/- or CD32-/- mice. Similar results were observed when FACS-purified B-progenitor cells were co-cultured with BMS2, a bone marrow stromal cell line. Fresh bone marrow from CD32-/- mice contained about two-fold more B-lineage cells compared to bone marrow from normal or CD16-/- mice. These studies indicate that the FcγR on B-lineage progenitor cells can influence their further development and add to a growing body of evidence that implicates FcγR as regulatory elements in hematopoiesis.

B Cell Strategies of Ag Recognition in a Stratified Immune System

From the old good times in which pioneering immunologists were simply specialised in either T or B lymphocytes, to the current lymphoid complexities that powerful quantitative technologies have revealed, the adaptive immune system has become a highly complicated subject of study. Also, as a part of the hematopoietic system, the lymphoid tissue is constituted by cells that undergo great turnover rates, with continuous renewal during an individual’s lifespan, extending from immature, bone marrow-derived precursors up to the final exhaustion of highly-differentiated cells. To exert their functions, B and T lymphocytes first need to recognise small molecular epitopes, through their highly diverse clonotypic receptors, with different degrees of specificity (the result of combinatorial gene rearrangements, with inexact joining ends). But lymphocytes also bear an evolutionary history that “informs” them about those more or less efficient receptor choices that previously influenced the survival of the species. Those cellular adjustments that were previously successful have remained preserved and are therefore preferentially exploited by the immune system (IS). Subsequently, immune strategies to “see” the antigenic world (self or foreign, harmless or dangerous) are balanced between an unrestricted openness to novelty (which has been named the promethean character of the IS [1]) and the evolutionary preservation of cellular activities and receptors that have already been tested and demonstrated their utility. In the following pages, we try to provide a few insights regarding when and where those functional polarisations are at work, with a special focus on B lymphocytes. We will leave out the differentiation steps and genetic programmes leading to the production of mature B lymphocytes, except in those selected cases when they may provide relevant information.