Development of a cell-free binding assay for rat ICAM-1/LFA-1 interactions using a novel anti-rat LFA-1 monoclonal antibody and comparison with a cell-based assay.

The importance of the interaction between lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) in the progression of inflammatory responses in vivo has been demonstrated mainly in rats. The present study was undertaken to develop binding assays suitable for measuring the rat ICAM-1/LFA-1 interaction in vitro. We first examined binding of rat T lymphoma FTL43 cells, which express LFA-1, to immobilized rat ICAM-1. Although FTL43 cells bound avidly to immobilized ICAM-1 and the binding was abolished with anti-LFA-1 monoclonal antibodies (mAbs), the binding was not completely inhibited by most anti-ICAM-1 mAbs. We next purified rat LFA-1 from FTL43 cells and constructed a cell-free binding assay. By using a newly developed anti-rat LFA-1 mAb RL14/9, which does not inhibit ICAM-1/LFA-1 interactions, binding of purified rat LFA-1 to immobilized ICAM-1 was successfully detected, whereas only a low signal to noise ratio was observed when binding of ICAM-1 to immobilized LFA-1 was examined. Moreover, we found that simultaneous addition of purified LFA-1 and biotinylated RL14/9 to ICAM-1-coated wells resulted in more sensitive detection of rat ICAM-1/LFA-1 binding. The binding was completely blocked with both anti-LFA-1 and anti-ICAM-1 mAbs and was much more sensitive to inhibition by the ICAM-1-IgG chimera, as compared with the cell-based assay. These results indicate that the cell-free binding assay provides a rapid and sensitive method for screening rat ICAM-1/LFA-1 antagonists, whose therapeutic effect on inflammatory diseases can further be evaluated in vivo.

Human T lymphocyte populations which bind to P- or E-selectin are enriched with cells expressing core 2 O-glycans.

The present study was undertaken to investigate whether core 2 O-glycans are involved in binding of resting human T lymphocytes to P- or E-selectin and in recruitment of these cells to inflammatory sites. Freshly isolated human peripheral blood T lymphocytes were incubated with P- or E-selectin-coated dishes, and expression of core 2 O-glycans by the adherent and nonadherent cells was examined using the anti-1D4 mAb, which specifically recognizes human CD43 modified with core 2 O-glycans. The results indicated that both the P-selectin/adherent and E-selectin/adherent populations were significantly enriched with ID4+ cells, as compared with the initial population. An enrichment of ID4+ cells in the P- and E-selectin/adherent populations was observed in both CD4 and CD8 T cell subsets and even in the CD45RO+ memory CD4 T-cell subset. However, the anti-1D4 mAb did not inhibit binding of human T lymphocytes to P- or E-selectin, indicating that the 1D4 antigen itself is not directly involved in selectin binding. We also found that the percentage of ID4+ cells in synovial fluid T lymphocytes of rheumatoid arthritis patients was significantly increased as compared with normal peripheral blood T lymphocytes. Taken together, our results support the notion that core 2 O-glycans, which are located apart from the ID4 antigen, are involved in binding of human resting T lymphocytes to both P- and E-selectin, and these interactions may contribute to preferential recruitment of human memory CD4 T lymphocytes to inflammatory sites, including the synovium of rheumatoid arthritis patients.

Core 2-containing O-glycans on CD43 are preferentially expressed in the memory subset of human CD4 T cells.

Human CD4 T cells can be divided into two functionally distinct subsets: a CD45RO+ memory subset and a CD45RA+ naive subset. In an attempt to identify novel cell surface molecules on these cells, we have developed a mAb, anti-1D4. The antigen defined by anti-1D4 was preferentially expressed on the memory subset of freshly isolated peripheral CD4 T cells and 1D4+ CD4 T cells functionally corresponded to memory T cells. Retrovirus-mediated expression cloning revealed that the 1 D4 antigen is human CD43. Transfection of CHO-leu cells, which stably express human CD43, with core 2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT) conferred expression of the 1D4 antigen and mRNA of C2GnT was detected by RT-PCR only in 1D4+ T cells but not in 1D4- T cells, implying that the 1 D4 antigen is composed of core 2-containing O-glycans on CD43. Reactivity with anti-1 D4 was completely abolished when cells were treated with neuraminidase, while them remained weak binding of anti-T305, a previously described mAb which also reacts with CD43 modified with core 2-containing O-glycans. Moreover, anti-1D4 markedly reacted with NIH-3T3 cells expressing human CD43 and low levels of endogenous C2GnT, whereas anti-T305 reacted slightly. These results indicate that the 1D4 antigen is distinct from the epitope defined by anti-T305 and anti-1D4 is a more sensitive probe to detect core 2-containing O-glycans than anti-T305. Taken together, our results indicate that core 2-containing O-glycans, whose expression can easily be detected with anti-1D4, are preferentially expressed in the CD45RO+ memory subset of CD4 T cells.

Characterization of glycosylphosphatidylinositol (GPI)-anchored NCAM on mouse skeletal muscle cell line C2C12: the structure of the GPI glycan and release during myogenesis.

The mouse myoblast cell line C2C12 constitutively expressed 160-kDa transmembrane NCAM isoform and 135-kDa GPI-anchored isoform before differentiation. During differentiation into multinucleated myotubes, the cells newly expressed 150-kDa GPI-anchored isoform and the level of 135-kDa GPI-anchored isoform increased. Structural analysis of the GPI glycan of NCAM, which was purified from C2C12 myotubes after metabolic labeling with [3H]inositol, was performed by sequential exoglycosidase digestion and Wistaria floribunda agglutinin-agarose column chromatography. The core GPI glycan structure, Man alpha 1-2Man alpha-Man alpha-GlcNH2-myoInositol, was conserved and variations were observed in additional mannose and N-acetylgalactosamine residues. Structural analysis of the GPI glycans of the two GPI-anchored isoforms, GPI-NCAM 135 and GPI-NCAM 150, showed the enhanced attachment of the N-acetylgalactosamine residue to the GPI glycan core of GPI-NCAM 150. These GPI-anchored NCAM isoforms were released from C2C12 cells during the myoblast differentiation. Release of GPI-anchored NCAMs was observed when C2C12 cells were cultured in a serum-free medium, and inositol but not inositol phosphate was detected after nitrous acid deamination of the released NCAM. These results suggest that the GPI-anchored NCAM was released from the cell surface by the action of an endogeneous phospholipase D.

Couple counselling and HIV testing in Uganda

The objective was to assess provision of counseling and HIV testing for couples as an HIV prevention strategy. HIV counseling and testing (C/T) for couples has been offered at the AIDS Information Centre in Uganda since 1990 with support from USAID. When clients come as a couple; and if they consent to their partner learning their sero-status; clients receive pre-and post-test counseling and results together. Data have been routinely collected on all clients since 1992. Intensive interviews have been conducted with a selected number of couples to assess their reasons for coming; and their response to discordant results. Results showed that over time; there has been an increase in clients coming as couples; from 9in 1992 to 12in 1993 to 19in 1994. In 1994; 77of all clients were HIV-; among couples 84were both HIV-. Discordant results were found in 12.6of couples and 3.6of couples were both HIV+. Receiving discordant results is highly distressing to couples and requires special skills from the counsellors. Demand for HIB C/T before marriage and among couples in new relationships is increasing significantly in Uganda. In 1994; 16of AIC clients who came as couples learned they were either discordant or were both HIV+. This is a critical opportunity for intensive HIV prevention counseling. Providing a setting for couples to receive HIV C/T together is an important strategy for HIV prevention; especially in high prevalence settings such as Uganda

Anthropology and AIDS: the cultural context of sexual risk behavior among urban Baganda women in Kampala, Uganda.

One hundred and thirty Baganda women (65 HIV antibody positive and 65 HIV antibody negative), recruited from the Makerere University-Case Western Reserve University Collaborative Pediatric follow-up clinic in Kampala, Uganda were interviewed about cultural rules and norms for sexual behavior and HIV-specific risk behaviors. Interviews were analyzed for themes related to sexual risk, cultural rules regarding sex, and individual sexual practices. Statistical relationships were tested using chi 2 and t-test statistics. The mean age of the women was 21 years (range 15-30). Despite sexual norms prohibiting sex for women outside marriage, subjects reported that there are certain circumstances when a woman may take other partners, including economic need, desire for greater sexual satisfaction, or revenge on a husband with other partners. Cases were more likely to state that women may have outside partners for economic reasons (P < 0.05) and that women have outside partners for sexual satisfaction (P < 0.01). Women interviewed for this study are complying with Ugandan AIDS control messages to 'zerograze' and 'stick to one partner'. Fear of AIDS remains high, however, because women fear that their partners have not responded to risk reduction messages. Of those women stating fear of AIDS, 57% of cases and 62% of controls based their fear on their perceptions of their partners' activities. Therefore, women feel that they remain at risk of infection despite their own behavior change. We find that, while the potential for risk reduction is high for these women, cultural norms permitting males to have multiple partners limit a woman's ability to control her risk reduction. Important conclusions are: (1) a focus on women's behavior alone is not sufficient as both partners must respond to risk reduction messages; (2) knowledge about AIDS is not sufficient to achieve change in sexual behavior because sexual behavior is linked to economics, gender relations, and other complex socio-cultural factors; and (3) a study of Baganda male sexual values and behavior is urgently needed.

Couple counselling and HIV testing in Uganda

The objective was to assess provision of counseling and HIV testing for couples as an HIV prevention strategy. HIV counseling and testing (C/T) for couples has been offered at the AIDS Information Centre in Uganda since 1990 with support from USAID. When clients come as a couple; and if they consent to their partner learning their sero-status; clients receive pre-and post-test counseling and results together. Data have been routinely collected on all clients since 1992. Intensive interviews have been conducted with a selected number of couples to assess their reasons for coming; and their response to discordant results. Results showed that over time; there has been an increase in clients coming as couples; from 9in 1992 to 12in 1993 to 19in 1994. In 1994; 77of all clients were HIV-; among couples 84were both HIV-. Discordant results were found in 12.6of couples and 3.6of couples were both HIV+. Receiving discordant results is highly distressing to couples and requires special skills from the counsellors. Demand for HIB C/T before marriage and among couples in new relationships is increasing significantly in Uganda. In 1994; 16of AIC clients who came as couples learned they were either discordant or were both HIV+. This is a critical opportunity for intensive HIV prevention counseling. Providing a setting for couples to receive HIV C/T together is an improtant strategy for HIV prevention; especially in high prevalence settingssuch as Uganda