Specific Disengagement of Cell-Bound Anti-Lam-1 (Anti-L-Selectin) Antibodies by Aurintricarboxylic Acid

Brief treatment of human peripheral blood lymphocytes with the potential anti-HIV compound aurintricarboxylic acid (ATA) prompts the selective release of already bound L-selectin-specific anti-leu8 and anti-LAM1-1 antibodies from the cells. Two other anti-LAM1 antibodies, anti-LAM1-3 and anti-LAM1-5 stay antigen-bound at the same time. interestingly, the ATA-sensitive anti-leu8 strongly competes with the ATA-resistant anti-LAM1-3 for binding. Photobleaching fluorescence resonance energy transfer (pFRET) measurements on flow-sorted cells suggests that these two antibodies compete for the same epitope, while anti-LAM1-5-FITC and anti-Leu8-PE bind to distinct sites, although they also compete for binding. Combining the data on competition, pFRET and ATA effect, we suggest that the ATA sensitive anti-leu8 and resistant anti-LAM1-3 bind to overlapping but non-identical epitopes. This remarkably specific effect may be exploited for designing anti-inflammatory drugs that modulate leukocyte adhesio

The L-Selectin (Leu8) Molecule Is Associated with the Tcr/Cd3 Receptor - Fluorescence Energy-Transfer Measurements on Live Cells

Several accessory molecules were shown to play important roles in T cell functions and be in close proximity to the T cell receptor (TcR/CD3). The L-selectin molecule (Leu8, LAM1-1, LECAM1) also plays an important role in lymphocyte homing and proliferation. We were interested in determining the proximity of this molecule to the TcR/CD3 complex on live peripheral human T cells. Using a fluorescence energy transfer method, designed to study individual cells, we could show that L-selectin is within 170 A of the TcR/CD3 complex. Monoclonal antibody directed against the LAM1-1 (Leu8) epitope of the L-selectin molecule suppressed the mitogenic activity of antibodies specific for various CD3 epitopes in vitro. Intracellular Ca2+ mobilization obtained with wt31 followed by cross-linking antibody or with anti-CD3 was not influenced by anti-leu8 antibody. Also antibody directed against the LAM1-1 epitope did not influence the binding of the mitogenic antibodies, as shown by fluorescence-based flow cytometry. Therefore, we suggest that binding of TcR/CD3 bound mitogenic antibodies to accessory cell Fc receptors may be hindered by antibodies bound to the close proximity L-selectin molecule

Cd4 Changes Conformation Upon Ligand-Binding

Aurintricarboxylic acid (ATA) has been shown to block the binding site for both HIV gp120 and mAb anti-Leu 3a on CD4. We have unexpectedly found that brief treatment with greater-than-or-equal-to 1 mug/ml ATA rapidly disengages another mAb, OKT4E, after it has been bound to CD4 on human PBL. OKT4E is specific for a discontinuous epitope overlapping the MHC class II-binding region in the N-terminal CD4 domain. Interestingly, among 10 other mAb tested, only anti-Leu 8, specific for a leukocyte homing receptor is also quickly released from the cells by ATA treatment. Disengagement of the OKT4E mAb is also seen on a CD4-positive cell line (HPB-ALL) and with recombinant soluble CD4 (sCD4) bound to immobilized OKT4E. In all of these cases, disengagement is prevented if OKT4E is cross-linked, or the Leu 3a site is blocked by the mAb, but not by gp120. Photobleaching fluorescence resonance energy transfer (pFRET) measurements suggest that OKT4E is released as an indirect consequence of ATA-evoked conformational changes of CD4. Similar changes were detected as a result of gp120 binding to PBL. These data raise the possibility of a novel type of immunomodulation: induced disengagement of a bound ligand from its A

Combination of Ascorbate and a-Tocopherol as a Preventive Therapy against Structural and Functional Defects of Erythrocytes in Visceral Leishmaniasis

The redox unbalance in erythrocytes has been found to contribute significantly in the development of anemia in visceral leishmaniasis (VL). The present study revealed enhanced production of reactive oxygen species (ROS) and gradual depletion of a-tocopherol and ascorbate in the erythrocytes of infected animals. The response of erythrocytes to chronic treatment with antioxidants was studied in hamsters during leishmanial infection. Treatment with a combination of a-tocopherol and ascorbate proved to be the most effective preventive for the proteolytic degradation of erythrocyte membrane. Erythrocytes from infected animals were thermally more sensitive compared to the control ones. Combination of both antioxidants was most successful in resisting heat induced structural defects in the cells. Cross-linking of membrane proteins subsequent to oxidative damage in the red cells was accompanied by the formation of high molecular weight protein band at the top of the resolving gel in the presence of the cross-linking agent dimethyladepimidate (DMA). Marked inhibition of cross-linking was observed with combination of both antioxidants. Treatment with a-tocopherol and ascorbate together could withstand osmotic lysis of erythrocytes in the infected animals very efficiently. Decreased hemoglobin (Hb) level was successfully replenished and was coupled with significant increase in the life span of red cells after treating the animals with both antioxidants. Results indicate better efficacy of the combination therapy with a-tocopherol and ascorbate in protecting the erythrocytes from structural and functional damages during leishmanial infection

Spectral line-shapes investigation with Pound-Drever-Hall-locked frequency-stabilized cavity ring-down spectroscopy

A review of recent experiments involving a newly developed Pound-Drever-Hall-locked frequency-stabilized cavity ring-down spectroscopy (PDH-locked FS-CRDS) system is presented. By comparison to standard FS-CRDS, the PDH lock of the probe laser to the ring-down cavity optimized coupling into the cavity, thus increasing the ring-down signal acquisition rate nearly 300-fold to 14 kHz and reducing the noise-equivalent absorption coefficient by more than an order of magnitude to 7 × 10−11 cm−1. We discuss how averaging approximately 1000 spectra yielded a signal-to-noise ratio of 220000. We also discuss how the spectrum frequency axis was linked to an optical frequency comb, thus enabling absolute frequency measurements of molecular optical transitions at sub-MHz levels. Applications of the spectrometer to molecular line-shape studies are also presented. For these investigations, we use semi-classical line-shape models that consider the influence of Dicke narrowing as well as the speed dependence of the pressure broadening and shifting to fit spectra. We show that the improved precision and spectrum fidelity of the spectrometer enable precise determinations of line-shape parameters. We also discuss the importance of line-shape analysis with regard to the development of new spectroscopic databases as well as in the optical determination of the Boltzmann constant