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L Wofsy

Publications and source records attributed to L Wofsy.

At least 19 recordsLinked to original sources
PubMed1986

Converting Sendai virus into a specific fusogen whose cell target can be selected.

Covalent intermolecular hybrids of Fab anti-hemagglutinin-neuraminidase (HN) monoclonal antibody and avidin were prepared and characterized. These conjugates were used to block and redirect the fusion activity of Sendai virus (SV). After incubation of SV with Fab anti-HN: avidin conjugate on ice for 1-2 h, the SV fused only those P815 or BW5147 cells which were labeled with biotin-modified anti-cell surface immunoglobulin. The levels of cell-cell fusion obtained were at least as high as those achieved with unmodified SV and unlabeled P815 or BW5147 cells. These results demonstrate that it is possible to block the normal agglutinating activity of the HN molecules of SV and to introduce a new cell recognition feature without negating the fusogenic potential of the virus. Such an approach may be useful in harnessing the fusion activity of SV to a targeted delivery system for microinjection of macromolecules into selected cell populations.

Agglutination↗
PubMed1984

Immunospecific vesicle targeting facilitates fusion with selected cell populations.

Antibody-directed targeting of vesicles to cells dramatically enhances polyethylene glycol-mediated fusion and microinjection. Sealed erythrocyte ghosts or liposomes, containing fluorescent bovine serum albumin, were targeted to murine spleen and thymus cells, and to lymphocyte and monocyte cell lines. In all cases, targeted cell populations showed substantial levels of microinjection, whereas populations treated with the fusogen in the absence of targeting were not significantly microinjected. Attachment of vesicles to selected cells was achieved by first labelling the cells with biotin-modified antibody and then treating them with avidin-coupled sealed ghosts or liposomes. Another approach to the promotion of selective fusion aims to alter the cell recognition properties of Sendai virus so that its fusogenic activity may be redirected to specific cellular targets. The agglutination and fusion of red cells by UV-inactivated Sendai virus were completely blocked by low concentrations of a Fab preparation of a monoclonal antibody against the viral haemagglutinin (HN) sites. Agglutination and fusion activity were restored in the presence of Fab-anti-HN by providing an alternative recognition system, namely, when the virus had been coupled with biotin and the red cells with avidin. Methods for facilitating microinjection by specifically directing vesicles to target cells may be particularly useful in overcoming barriers to the transfer of genes into lymphocytes by standard transfection techniques.

Animals↗
PubMed1983

Immunospecific vesicle targeting facilitates microinjection into lymphocytes.

Antibody-directed targeting of vesicles to cells dramatically enhances polyethylene glycol-mediated fusion and microinjection. Sealed erythrocyte ghosts, containing fluorescent bovine serum albumin, were targeted to murine spleen and thymus cells, and to lymphocyte, monocyte, and fibroblast cell lines. In all cases, targeted cell populations showed substantial levels of microinjection, whereas populations treated with the fusogen in the absence of targeting were not significantly microinjected. To achieve attachment of vesicles to selected cells, the cells were first labeled with biotin-modified antibody then treated with sealed ghosts prepared from avidin-coupled erythrocytes. This procedure should prove useful when the injection of specific cell populations is desired, or with cell types such as lymphocytes that are difficult to fuse, or when the use of limited reagents necessitates high injection efficiencies.

Animals↗
PubMed1981

Intramembrane particles and the organization of lymphocyte membrane proteins.

An experimental system was developed in which the majority of all lymphocyte cell-surface proteins, regardless of antigenic specificity, could be cross-linked and redistributed in the membrane to determine whether this would induce a corresponding redistribution of intramembrane particles (IMP). Mouse spleen cells were treated with P-diazoniumphenyl- beta-D-lactoside (lac) to modify all exposed cell-surface proteins. Extensive azo- coupling was achieved without significantly reducing cell viability or compromising cellular function in mitogen- or antigen-stimulated cultures. When the lac-modified cell- surface proteins were capped with a sandwich of rabbit antilactoside antibody and fluorescein-goat anti-rabbit Ig, freeze-fracture preparations obtained from these cells revealed no obvious redistribution of IMP on the majority of fracture faces. However, detailed analysis showed a statistically significant 35 percent decrease (P less than 0.01) in average IMP density in the E face of the lac-capped spleen cells compared with control cells, whereas a few E-face micrographs showed intense IMP aggregation. In contrast, there was no significant alteration of P-face IMP densities or distribution. Apparently, the majority of E-face IMP and virtually all P-face IMP densities or distribution. Apparently, the majority of E-face IMP and virtually all P-face IMP do not present accessible antigenic sites on the lymphocyte surface and do not associate in a stable manner with surface protein antigens. This finding suggests that IMP, as observed in freeze-fracture analysis, may not comprise a representative reflection of lymphocyte transmembrane protein molecules and complexes because other evidence establishes: (a) that at least some common lymphocyte surface antigens are indeed exposed portions of transmembrane proteins and (b) that the aggregation of molecules of any surface antigen results in altered organization of contractile proteins at the cytoplasmic face of the membrane.

Animals↗
PubMed1979

Hapten-sandwich labeling. IV. Improved procedures and non-cross-reacting hapten reagents for double-labeling cell surface antigens.

New procedures are presented for preparation of hapten-antibody conjugates with the bifunctional amidinating reagent methyl-p-hydroxybenzimidate (HB). Conjugates with improved solubility are effective for hapten-sandwich labeling of cell surface antigens with high sensitivity and specificity. Several non-cross-reacting hapten-antihapten antibody systems are described which are well-suited for simultaneous labeling of different surface antigens.

Animals↗
PubMed1979

Hapten-antibody conjugates as probes of the lymphocyte surface.

Various forms and applications of the hapten-sandwich method for labeling cell surface antigens are reviewed. This is a two-layer antibody technique: the first antibody that binds antigen has been chemically modified so that a number of hapten groups are attached; the second antibody is directed against the hapten and has been coupled to a marker for amplified detection. This methodology is particularly suited to circumstances where high sensitivity and specificity are required for labeling alloantigens or for discriminating between antigens in double-labeling procedures. It is applicable to fluorescence visualization, electron microscopy, and radioisotopic labeling. It lends itself also to procedures for specific separation of differing lymphoid cell populations, and has been adapted to studies of cell activation in the immune response.

Animals↗
PubMed1978

Role of membrane receptors in the induction of an in vitro secondary anti-hapten response. I. differentiation of B memory cells to plasma cells is independent of antigen-immunoglobulin receptor interaction.

In these experiments, we show that the interaction of antigen and B cell surface immunoglobulin is not essential for the generation of an IgG in vitro response to the hapten p-azophenyl-lactoside (lac). In our experimental system, keyhole limpet hemocyanin (KLH) was first selectively attached either to H-2, Ia or Ig receptors of lac-primed B cells by a hapten sandwich technique or to Fc receptors by complexes of azophenyl arsonate (ars)-coupled KLH and anti-ars. The labeled cells were then cultured with KLH-specific T cells for 5 days in the absence of antigen. Under all conditions of attachment we observed a significant anti-lac IgG response. We have demonstrated an absolute requirement for KLH-specific helper T cells. The results thus indicate that T helper cells are by themselves, regardless of the B cell antigen that serves to effect bridging, sufficient to activate B memory cells. We could find no evidence to support either a matrix theory or a two-signal hypothesis as currently proposed.

Animals↗
PubMed1977

No direct association between Ia antigens and Fc receptors on the B cell membrane.

The capping of Ia antigens does not induce redistribution of Fc receptors (FcR) on B lymphocytes. This rules out the possibility of a unidirectional association between Ia and FcR such as has been reported to link Ig and FcR. Ia-capping was achieved with hapten-sandwich antibodies devoid of Fc regions: hapten-conjugated Fab anti-Ia followed by (Fab')2 anti-hapten antibody. Three different immune complex systems were used to label FcR. With fluorescent double labeling, Ia and FcR were readily distinguished. The independent labeling and surface mobility of Ia and FcR are considered in connection with reports of the inhibition of FcR by anti-Ia antibodies.

Animals↗
PubMed1976

Immunospecific labeling of mouse lymphocytes in the scanning electron microscope.

Bone marrow-derived (B) and thymus-derived (T) Balb/c mouse lymphocytes were identified in the scanning electron microscope (SEM) by the immunospecific attachment of one of several kinds of large-molecular-weight markers distinguishable in SEM. These markers (tobacco mosaic virus, keyhole limpet hemocyanin, bushy stunt virus, and bacteriophage T4) could be modified with hapten groups and linked with anti-hapten antibody, in an indirect (sandwich) scheme, to hapten-modified anti-cell-surface antibody bound to the cell surface. Hapten-modified antibodies to B cell antigens (goat anti-mouse-immunoglobulin) or to T cell antigens (rabbit anti-mouse brain) were employed to identify these two lymphoid cell types in unfractionated spleen, mesenteric lymph node, bone marrow, and thymus cell populations. The topography of B cells was always indistinguishable from that of T cells. No surface features were found to be unique to either cell type. In suspension, the majority of B and T cells had one or no microvilli regardless of the tissue source of the labeled cells. Cells in suspension that had microvilli (usually 10% of the total cell population) were always unlabeled. However, after cell contact with a glass surface, approximately half of both the B and T cell population had a villous topography.

Animals↗
PubMed1976

Hapten-sandwich labeling. III. Bifunctional reagents for immunospecific labeling of cell surface antigens.

Bifunctional reagents have been prepared which permit extensive coupling of haptens to antibodies with retention of antibody-binding capacity. This modification procedure increases the sensitivity with which cell-surface antigens can be labeled for fluorescent or electron microscopy by the hapten-sandwich method to a level comparable to or greater than may usually be achieved by conventional indirect techniques. With two different haptens, and amplifying with anti-hapten antibodies bearing fluorescent or other markers, different antigens can be distinguished readily on a single cell or on separate cells. Mouse alloantigens detected only with difficulty by conventional fluorescence can be discerned clearly and with high specificity. The reagent, either methyl hydroxybenzimidate (HB) or methyl 3,5-dihydroxybenzimidate (DHB), is first reacted with a diazonium phenyl hapten. Since the resulting azo-derivative retains the imido ester function, the reaction solution can be used directly to amidinate antibodies. Both HB and DHB are easily prepared and may be stored for use as needed. With these reagents, the hapten-sandwich procedure may be applied to proteins other than immunoglobulins, e.g., hormones and mitogens. In addition to labeling antigens for fluorescent or electron microscopy, haptenamidinated antibodies or other ligands may be used in conjunction with appropriately modified antihapten amplifiers for a variety of purposes in cell biology, such as radioisotope studies, selective cell killing or suppression, and isolation of membrane antigens.

Animals↗
PubMed1975

Hapten-sandwich labeling. II. Immunospecific attachment of cell surface markers suitable for scanning electron microscopy.

A hapten-sandwich procedure has been used for immunospecific labeling of cell surface antigens with markers visible by scanning electron microscopy. Antihapten antibody was used to link hapten-modified tobacco mosaic virus, bushy stunt virus, or hemocyanin to hapten-modified human erythrocytes. The antihapten antibody bridge was also used to link the hapten-virus marker to hapten-modified antibodies against mammary tumor virus on mouse mammary tumor cells, or against immunoglobulin receptors on mouse splenic lymphocytes. In all cases, labeling was highly specific. With this technique, it is possible to (a) compare morphological features of cells bearing differing cell surface antigens, and (b) examine the arrangement of specific antigenic sites on a cell surface or their distribution relative to membrane structures such as microvilli.

Animals↗