ABSTRACT
The third hypervirable (V3) domain of the HIV-1 envelope glycoprotein gp120 has been implicated in HIV pathogenesis via co-receptor usage of chemokine receptors CCR5 and CXCR4. As the protagonist cell populations in the asymptomatic phase of HIV-1 infection are infected macrophages and effector/memory (CD45RO+) CD4+ T cells that express CCR5, we established an in vitro model using human primary monocyte-derived macrophages and lymphocytes to investigate the role of V3 in affecting antigen presentation. We used staphylococcal enterotoxin A (SEA) as a superantigen at a low concentration of 1ng/ml, to activate naïve CD4+ T cells. Exposure of cells to SEA and lipoV3-liposomes increased the percentage of CD4+/CD45RO+/CCR5+ T cell population as compared to cells treated with SEA and plain liposomes. A consequent decrease of the percentage of CD4+/CD45RO+/CXCR4+ subset was observed. The V3-mediated activation was competitively inhibited by soluble V3-derived peptides with higher cationic charge. V3 enhanced also apoptosis as demonstrated by flow cytometry and intracellular calcium ion assays. These results reinforce the postulation that V3 alters the antigen presentation function itself, independent of specific antigens, thus leading to an enhanced activation-induced cell death (AICD) of responding T cells.
Subject(s)
Apoptosis/immunology , CD4-Positive T-Lymphocytes/immunology , HIV Envelope Protein gp120/immunology , HIV-1/immunology , Leukocyte Common Antigens/metabolism , Peptide Fragments/pharmacology , Apoptosis/drug effects , CD4-Positive T-Lymphocytes/drug effects , Calcium/metabolism , Enterotoxins/pharmacology , HIV Envelope Protein gp120/blood , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/pharmacology , Humans , Immunophenotyping , Leukocyte Common Antigens/immunology , Liposomes , Lymphocyte Activation/drug effects , Peptide Fragments/immunology , Receptors, CCR5/immunology , Receptors, CCR5/metabolism , Superantigens/pharmacologyABSTRACT
Fluorescence polarization immunoassay (FPIA) is a convenient homogeneous assay, the use of which is restricted in environmental analysis by low sensitivity and matrix effects. We selected the herbicides 2,4D and 2,4,5T to synthesize new L-lysine-based fluorescent tracers using solid-phase chemistry. In addition, three different immunogens of 2,4,5T were prepared for immunization and antibody production. The new tracers and antibodies were adapted to FPIA. Tracers with the hapten attached to the alpha-aminogroup of L-lysine and fluorescein to the e-amino group exhibited at least a 5-fold increased sensitivity when compared to the previously reported ethylenediamine-based tracer (2,4D-EDA-F). The isomeric structure (hapten attached to the e-amino and fluorescein to the alpha-amino group) appeared 7.6 times less sensitive, and all other alternative structures exhibited even lower sensitivities. This observation was confirmed against the monoclonal anti-2,4D antibody E2/G2 and polyclonal anti-2,4,5T antibodies. The affinity constant of 2,4D-EDA-F with E2/G2 was 8.1 times higher when compared with the new tracer, suggesting the more specific nature of the L-lysine-based tracer, the use of which leads to a more sensitive assay. This type of tracer could improve performance and lower substantially the detection limits of FPIAs.
