Assessment of the immunoglobulin E-mediated immune response to milk-specific proteins in allergic patients using microarrays.

BACKGROUND: Cow's milk allergy (CMA) is one of the most widespread human allergies, especially in young children. Although CMA is intensively studied, little is known about the recognition patterns of milk allergens in allergic patients, and the determination these patterns is a prerequisite for the development of efficient diagnostic and prognostic tools. Several factors present difficulties for such a determination, because (i) milk contains a large number of potential allergens; (ii) the majority of these allergens consist of complex suspensions rather than solutions; (iii) the major allergens, such as caseins, cannot be highly purified in large amounts; and (iv) most of the time, very small amount of young patients' sera are readily available. METHODS: To overcome these difficulties, we developed a sensitive microarray assay that, in combination with near-infrared fluorescence detection, was used to study the immune response to milk and purified native milk proteins. RESULTS: This new assay allowed us to assess the binding ability of IgE to milk allergens from a large number of young patients using reduced amounts of clinical material. The data show that bovine lactoferrin can be classed as a strong milk allergen. We confirmed that bovine caseins are the main allergens in milk and that alpha(S1)-casein is more allergenic than alpha(S2)-, beta- and kappa-caseins, which were recognized with almost a similar frequency by the sera of patients. CONCLUSION: Microarray methods, in combination with near-infrared fluorescence detection, can be useful for the in vitro diagnosis of food allergies.

New GPCRs from a human lingual cDNA library.

Sweet and bitter taste perception involve G protein coupled receptors (GPCRs) present at the taste receptor cell surface. It is likely that various mechanisms are active and various families of GPCRs are involved in the perception of these tastes. The expression of GPCRs in human tongue was studied using degenerated primers corresponding to transmembrane domains 2 or 3 (for 5' primer), 6 or 7 (for 3' primer) of olfactory-like receptors in reverse transcription-polymerase chain reaction experiments. It was demonstrated that four previously identified, eight new olfactory-like receptor genes, three previously known and eight new olfactory-like receptor pseudogenes, mostly located on chromosome 11, are expressed in adult tongue and/or in fetal tongue. Previously identified genes include HGMP071, HTPCR06, TPCR120 and TPCR85 whose cDNAs were originally isolated from male germinal cells. New genes were named JCG1, JCG2, JCG3, JCG4, JCG5, JCG6, JCG9 and JCG10. HGMP071, HTPCR06, TPCR120, JCG3 and JCG5 are also expressed in the epithelium of adult tongue, whereas all these genes are expressed in fetal tongue. Although functional studies are needed before definitive conclusions are made, the obtained results imply that lingual olfactory-like receptors could be involved in taste perception.

Esterified milk proteins inhibit DNA replication in vitro.

DNA replication was studied in vitro in the presence of native and esterified milk proteins [alpha-lactalbumin (ALA), beta-lactoglobulin (BLG) and beta-casein (BCN)]. Addition of unmodified proteins to the PCR medium did not change the result of the reaction seen by electrophoresis, even at excessive ratios of basic amino acids in proteins:phosphate groups in DNA as high as 100:1. Addition of esterified proteins greatly reduced the intensity of the bands corresponding to the newly synthesized DNA, at ratios as low as 1:1 and 5:1 in case of methylated-BLG and methylated-ALA, respectively. The inhibitory effect of esterified proteins was directly proportional to their extent of esterification and strongly related to their DNA-binding capacity. Generally, inhibition of PCR with esterified proteins was similar to what can be observed with histones. However, stronger inhibition was observed with highly esterified proteins when using a higher ratio of basic:acid residues (1:1) when compared with 0.5:1 ratio in case of histones. Highly esterified BCN did not exert any inhibitory effect because of its relatively lower pI when compared with that of other esterified milk proteins and due to its lower positive net charge at the pH used for PCR. During a second PCR run, only the addition of new DNA template was able to reinitiate the reaction, giving rise to new synthesized DNA. Addition of Taq DNA polymerase did not enhance DNA synthesis, showing that inhibition was performed only by binding of DNA template and not by the inhibition of the polymerase.

Nuclear localisation of wild type and mutant galectin-3 in transfected cells.

Galectin-3, a member of a family of carbohydrate-binding proteins, is present generally in the cytoplasm of cells. However, galectin 3 can also be located in nuclei under certain conditions although it lacks any known nuclear localisation signal and the mechanism by which the protein is sequestered in nuclei is unknown. Here we describe that Cos-7 cells or rabbit smooth muscle Rb-1 cells transfected with cDNA encoding hamster galectin-3 sequester the protein in nuclei whereas untransfected BHK cells expressing the endogenous hamster lectin or transfected BHK cells over-expressing the protein, do not. Confocal immunofluorescence microscopy of Cos-7 cells or rabbit smooth muscle Rb-1 cells transfected with cDNAs encoding mutants of hamster galectin-3 containing N-terminal or internal deletions shows that nuclear localisation does not require the first 103 amino acid residues of the protein. Further deletion of residues 104-110 dramatically prevents sequestration in nuclei. However, the sequence A104PTGALT110 by itself is not obligatory for nuclear localisation and can be substituted by other unrelated sequences. A truncated galectin-3 protein, that is blocked in nuclear expression, retains carbohydrate-binding activity, making less likely the possibility that severe N-terminal truncations of galectin-3 induce mis-folding leading to aggregation and cytoplasmic sequestration and an incidental effect on nuclear trafficking. These studies indicate that nuclear import and retention of galectin-3 is a property of the CRD domain and is independent of N-terminal domains that others have shown to contain binding domains for various nuclear components.

Galectin-3 gene (LGALS3) expression in experimental atherosclerosis and cultured smooth muscle cells.

The galectin-3 gene (LGALS3) encodes a beta-galactose binding lectin. LGALS3 expression is associated with neoplastic transformation and with differentiation of monocytes to macrophages. Factors involved in migration, proliferation, adhesion and differentiation of vascular smooth muscle cells (SMC) play a major role during atherosclerosis development. Expression of the galectin-3 gene was not detected in quiescent SMC but was activated in aortas of hypercholesterolemic rabbits, in aortas of rats after balloon injury and in cultured SMC. These results suggest that galectin-3 production is involved in the developmental process of atherogenesis.

Modulation of the expression of the rabbit galectin-3 gene by p53 and c-Ha-ras proteins and PMA.

Galectin-3 is a galactose-binding lectin that has been found in several mammalian tissues. Galectin-3 gene is expressed in a wide range of normal and tumoral cells. In the case of myeloid cells, its expression correlates with the differentiation of monocytes to macrophages. In the case of cancer cell lines, its expression correlates with tumorigenicity and metastatic potential. The regulation of the expression of this gene is still largely unknown. The rabbit galectin-3 gene has been isolated and characterized. Its structure revealed an organization similar to that of the murine galectin-3 gene. The genomic sequences located upstream from its 5' end, upon insertion upstream from a promoter-free reporter gene, exhibited a strong promoter activity. This activity was upregulated upon treatment of transfected smooth muscle cells with phorbol 12-myristate 13-acetate (PMA) as well as upon transfection with a EJ/ras encoding plasmid. Conversely, it was downmodulated upon transfection with wild-type p53 but not with mutated p53. The regulatory sequences involved in the positive regulation of the gene were located upon serial deletion experiments.

Cloning of the cDNA encoding rabbit galectin-3.

The complete coding sequence of the rabbit galectin-3-encoding cDNA (LGALS3) has been cloned in a single step by using RT-PCR and specific human LGALS3 cDNA primers. The putative protein contains three domains with different degrees of homology to other known LGALS3. The homology is high in the C-terminal moiety corresponding to the carbohydrate-binding domain and is relatively low in the N-terminal moiety.