Evaluation of the DNA stability of forensic markers used in betel-quid chewers' oral swab samples and oral cancerous specimens: implications for forensic application.

Chewed betel-quid (BQ) residues are often considered vital biological evidence at crime scenes, since the human DNA extracted from the residues is actually from buccal epithelial cells and can be associated with suspects. BQ-chewing is also a risk factor for oral diseases and/or cancers. Archived medical oral-specimens can be used to identify specific individuals under adverse conditions, although STR markers are known to be unstable in various tumor tissues. This study evaluates the DNA stability of forensic marker systems in BQ-chewers' oral epithelial cells, and in archived clinical specimens of oral cancer patients. The genotypes of oral and paired peripheral blood samples in 200 subjects were compared, using the commercialized typing systems of HLA-DQA1, PM (including LDLR, GYPA, HBGG, D7S8, and GC loci), and AmpFlSTR markers (including 9 STR loci and the Amelogenin gene). The 100 healthy BQ-chewers had consistent oral swab and paired blood sample genotypes analyzed withboth DQA1/PM and STR marker systems. In the 100 oral cancer patients, one discordant result at D7S8 was found in the 600DQA1/PM-marker loci, and 25 allelic alterations with expansion or contraction were detected in the 900 STR loci. The findings herein suggest that when cancerous specimens were tested, the HLA-DQA1/PM system with point polymorphism appears more reliable than the STR system with length polymorphism. Our results also indicate that healthy BQ-chewers' oral cotton swabs containing buccal epithelial cells are useful for forensic purposes using the HLA-DQA1, PM, and STR marker systems.

The T cell response to Art v 1, the major mugwort pollen allergen, is dominated by one epitope.

Mugwort (Artemisia vulgaris) pollen allergens represent the main cause of pollinosis in late summer in Europe. At least 95% of sera from mugwort pollen-allergic patients contain IgE against a highly glycosylated 24- to 28-kDa glycoprotein. Recently, this major allergen, termed Art v 1, was characterized, cloned in Escherichia coli, and produced in recombinant form. In the present study we characterized and compared the T cell responses to natural (nArt v 1) and recombinant Art v 1 (rArt v 1). In vitro T cell responses to nArt v 1 and rArt v 1 were studied in PBMC, T cell lines (TCL), and T cell clones (TCC) established from PBMC of mugwort-allergic patients. Stimulation of PBMC or allergen-specific TCL with either nArt v 1 or rArt v 1 resulted in comparable proliferative T cell responses. Eighty-five percent of the TCC reactive with rArt v 1 cross-reacted with the natural protein. The majority of the CD4(+)CD8(-)TCR alphabeta(+) Art v 1-specific TCC, obtained from 10 different donors, belonged to the Th2 phenotype. Epitope mapping of TCL and TCC using overlapping peptides revealed a single immunodominant T cell epitope recognized by 81% of the patients. Inhibition experiments demonstrated that the presentation of this peptide is restricted by HLA-DR molecules. In conclusion, the T cell response to Art v 1 is characterized by one strong immunodominant epitope and evidently differs from the T cell responses to other common pollen allergens known to contain multiple T cell epitopes. Therefore, mugwort allergy may be an ideal candidate for a peptide-based immunotherapy approach.

Parietaria judaica-specific T-cell clones from atopic patients: heterogeneity in restriction, V beta usage, and cytokine profile.

The pollen of Parietaria spp. is one of the most clinically relevant sources of allergens in the Mediterranean area. CD4+ T-lymphocyte clones specific for Parietaria allergens were isolated from peripheral blood of atopic donors, and their phenotype, HLA restriction, V beta usage, and cytokine profile were determined. All the T-cell clones expressed the alpha/beta T-cell receptor and were induced to express CD40 ligand after activation with phorbol-myristate-acetate plus ionomycin. When the proliferative response to three chromatographic fractions of the extract was analyzed, distinct reactivity patterns were found. Interestingly, most of the clones responded to the fraction that was the most enriched for the major allergen Par j 1. The clones were either HLA-DR- or HLA-DQ-restricted and did not show any preferential usage of T-cell receptor V beta segments. Five of the 17 clones tested produced only IL-4 and no interferon-gamma, thus displaying a TH2 phenotype. The other clones displayed a TH0 phenotype in that they produced both IL-4 and interferon-gamma. These results show that in atopic patients T-cell response against Parietaria judaica allergen involves different T-cell subsets in terms of restriction, V beta usage, and cytokine profile.

[Immunological study of the HLA class II antigen associated with birch pollen allergy].

Pollinosis is known as a hereditary disease. Recently, association of HLA antigens and pollinosis caused by several pollen allergens has been reported. Furthermore, HLA antigens are considered to have a very important role in the development of pollinosis. We performed an HLA population study and tested the lymphocyte proliferative response (LPR) to birch pollen allergens with birch pollen allergic patients and healthy control subjects. The results of the HLA population study indicated that HLA-DR9 and HLA-DQw3 correlated with the development of birth pollinosis (relative risk [R. R] = 6.37 for DR9 and R. R = 7.92 for DQw3). DNA typing revealed that serotype DQw3 in patients were DQw9 specific. Two sources of birch pollen allergens, betula pendula and betula platyla var. japonica, were used in LPR. Lymphocytes from the patients, but not from the healthy control subjects, proliferated in response to these two allergens. Anti-HLA-DR antibody, but not anti-HLA-DQ antibodies, inhibited the patients' LPR. These results suggest that the HLA-DR molecules are responsible to present the pollen specific antigen to T lymphocytes inducing LPR in the patients. Furthermore, in some of the healthy control subjects, anti HLA-DQ antibodies enhanced this LPR almost to the same level as that of the patients'. This result indicates that HLA-DQ molecules might be associated with the suppression of T cell proliferation.

Gamma interferon induces different keratinocyte cellular patterns of expression of HLA-DR and DQ and intercellular adhesion molecule-I (ICAM-I) antigens.

With indirect immunofluorescence techniques we demonstrated that recombinant gamma-interferon induced the expression of the class II antigens HLA-DR and HLA-DQ as well as intercellular adhesion molecule-I (ICAM-I) on normal, cultured human keratinocytes grown in low-calcium, serum-free medium. Each antigen displayed a distinctive cellular staining pattern. HLA-DR was strongly localized to perinuclear zones with intense cell surface expression; HLA-DQ displayed a perinuclear accentuation, but with minimal cell surface staining, and ICAM-I was strongly expressed in a diffuse cytoplasmic pattern with intense cell surface expression. Keratinocytes grown in medium supplemented with 10% fetal calf serum underwent differentiation, with a diminished expression of all three antigens as compared to those grown in low-calcium, serum-free medium. These results confirm that gamma interferon can differentially regulate HLA-DR and HLA-DQ expression; that there are probably different biochemical metabolic pathways by which these three molecules are expressed on keratinocytes, and that the expression is also a function of the degree of keratinocyte differentiation. The strong cell surface expression of ICAM-I is suggested to be of major importance as the recognition molecule, by which T cells bind to gamma interferon exposed keratinocytes, and suggests an integral role for this molecule in epidermal lymphocyte trafficking.