Inhibition of anti-DNP antibody formation by high doses of DNP-polyacrylamide molecules; effects of hapten density and hapten valence

High-dose inhibition of anti-DNP antibody formation by a series of DNP-polyacrylamide molecules of varying hapten density and hapten valence was measured. It was found that a molecule's inhibitory ability correlated directly with its hapten density, but not with its hapten valence nor with its own ability at optimal dose to stimulate an anti-DNP response.     

Picogram quantities of anti-Ig antibodies coupled to dextran induce B cell proliferation

To investigate the properties which enable type 2 Ag, as exemplified by dextran and Ficoll, to stimulate high levels of antibody responses in the relative absence of T cells, we conjugated anti-IgD and anti-IgM mAb to both dextran and Ficoll and examined their B cell-activating properties. Such conjugated anti-Ig antibodies stimulated both early and later stages of B cell activation at picogram concentrations, which are at least 1000-fold lower than that required for B cell stimulation by unconjugated anti-Ig antibodies, and the level of proliferation they stimulated was on average 10-fold greater. Furthermore, concentrations of anti-Ig dextran (100 pg/ml) which modulated little sIgD from the B cell surface were strong inducers of enhanced B cell expression of MHC class II molecules. Conjugation of Fab fragments of anti-IgD or nonmitogenic anti-IgM mAb to dextran rendered them as mitogenic as dextran conjugated to strongly stimulatory anti-IgD or anti-IgM antibodies. The ability of dextran and Ficoll to serve as effective carrier molecules for anti-IgD was not related solely to their large m.w., because anti-IgD coupled to polymerized BSA (m.w. 1.5 X 10(6), was only 10- to 50-fold more potent than unconjugated anti-IgD antibodies at stimulating B cell DNA synthesis. These results suggest, therefore, that the unique ability of picogram concentrations of haptenated type 2 Ag to stimulate Ig secretion in the absence of T cells may be a function of their ability to promote effective cross-linking without resulting in the modulation of sIg. This would enable such Ag to mediate repetitive B cell signaling, a situation that cannot be achieved by unconjugated anti-Ig antibodies which result in modulation of sIg at their mitogenic concentrations. These compounds therefore may be employed to study B cell activation stimulated by sIg cross-linking at concentrations that may more closely reflect those which are achieved under physiologic conditions by type 2 Ag.     

Theoretical considerations of the role of antigen structure in B cell activation

Thymus-independent antigens generally are polymeric molecules with repeating arrays of antigenic determinants. Immunological studies of the activity of haptenated thymus-independent antigens have shown that small changes in hapten density can transform a polymeric antigen from nonimmunogenic to immunogenic, and from immunogenic to tolerogenic. In this paper we compute the equilibrium configuration of a linear flexible, haptenated polymer absorbed to a B cell surface, and correlate configurational features of the molecule with its immunological functioning. A polymeric molecule bound to a cell generally will not lie entirely on the surface; rather there will be sections that form loops extending into solution, separated by tightly bound sections, or trains. Trains link antibody receptors on the B cell surface in a fashion that restricts their mobility. Thus trains cause restrictive cross-linking. Our computations show that there is a critical hapten density below which the polymer does not bind to the surface. At hapten densities slightly above the critical density, the polymer binds weakly to the surface with a configuration dominated by a few, rather long loops. These loops cross-link receptors, but do so without bringing the cross-linked receptors into close proximity and without substantially restricting their motion. Long loops thus cause unrestrictive cross-linking. As the hapten density increases, the average loop length decreases and the average train length increases. Thus cross-linking becomes restrictive. In this density range, immune stimulation is observed. At high hapten densities long trains form, separated by few, very short loops and almost all receptors are cross-linked. Consequently cross-linking may be overly restrictive, freezing receptors into place and generating an abundance of cross-linking or other signals that induce a state of immunological tolerance.     

Protective immunity to progressive tumors can be induced by antigen presented on regressor tumors

Immunization of animals with 1591-RE tumor cells, a highly immunogenic UV-induced epithelia cell tumor from C3H/HeN mice, that were haptenated with trinitrophenol (TNP) leads to protective immunity against a challenge of TNP-haptenated 3152-PRO tumor cells, a progressive highly malignant. MCA-induced fibrosarcoma from syngeneic mice. Animals that rejected TNP-1591-RE and subsequently TNP-3152-PRO tumor cells showed increased tumor-specific resistance to another challenge of 3152-PRO tumor cells, even when these fibrosarcoma cells had not been haptenated with TNP. Induction of protection required the presence of TNP-hapten groups on both 1591-RE and 3152-PRO during the initial immunization, and could be induced by immunization with other haptenated syngeneic highly immunogenic regressor tumor lines. In addition, TNP-haptenated progressor variants of the 1591-RE were ineffective in generating protection, suggesting that the immunogenicity of the haptenated tumor used for the initial immunization was a determining factor in whether or not protective immunity against the highly malignant tumor was later generated. Protection required at least two T cell types: a Lyt-1-2+ T cells, and a Lyt-1+2- T cell that also expressed I-J determinants and was Vicia villosa lectin adherent, suggesting it was not a classical helper T cell. These results suggest that presentation of a hapten by highly immunogenic tumor cells can lead to enhanced protective immunity to poorly immunogenic noncross-reactive tumors that co-express the same hapten, and rejection of these haptenated poorly immunogenic tumors leads to enhanced protection against a subsequent challenge of the same, but not noncross-reactive progressor tumors.     

Intracellular interference with antigen presentation

Murine peritoneal macrophages were cultured with heat-killed Listeria monocytogenes organisms and then with the protein hen egg white lysozyme. Hen egg lysozyme is well known to need intracellular processing for presentation to T cells. The presentation to T cells of lysozyme was affected despite no reduction in the amount taken up or catabolized by the macrophage. This interference with Ag presentation was not found if the macrophages were cultured with lysozyme before the Listeria pulse. The interference with Ag presentation induced by Listeria was found for a second Ag (conalbumin). Uptake of Listeria did not affect the presentation of the lysozyme peptide 46-61, indicating that MHC class II molecules were available at the macrophage surface. Other materials that are retained in the macrophages affected presentation of lysozyme but not of the processed peptide. These included SRBC, dextran, sucrose, cellobiose, polyvinyl pyrrolidone, sodium dextran sulfate, Ficoll, and polyethylene glycol. Except for SRBC, which were not tested, the remaining molecules did not interfere with presentation of 46-61 by formaldehyde-fixed macrophages, an indication that they did not affect the peptide interaction with class II molecules. Finally, uptake of latex beads did not affect presentation of lysozyme. We conclude that retention in the macrophage of a variety of soluble or particulate molecules can interfere with the intracellular events that result in the creation of an immunogenic determinant. This interference is independent of the catabolism of the Ag or of the availability of class II molecules to bind peptides.     

The interaction of nominal antigen with T cell antigen receptors. I. Specific binding of multivalent nominal antigen to cytolytic T cell clones

In this report, we describe an experimental strategy for analyzing the interaction of nominal antigen with antigen-specific T cell clones. Our approach was based on the notion that low affinity interactions between nominal antigen and T cell antigen receptors might be detected by using a highly multivalent form of the antigen in which a large number of identical, appropriately spaced epitopes are attached to a polymer backbone. Antigens of this kind should be capable of multivalent binding to receptors on the T cell, resulting in a marked enhancement of the overall avidity of the interaction. To examine this possibility, we established a series of murine cytolytic T cell (Tc) clones specific for the readily detectable hapten fluorescein isothiocyanate (FL). These clones lysed FL-conjugated target cells in an antigen-specific fashion and also showed specificity for target cell MHC gene products. The interaction of these clones with the nominal antigen FL was assessed by flow cytometry, using a series of water-soluble FL-conjugated polymers varying in polymer backbone and FL isomer. High m.w. (600 to 2000 Kd) polymers of acrylamide, dextran, or Ficoll conjugated with 300 to 800 FL groups/molecule bound specifically to anti-FL Tc clones. There was little binding to syngeneic spleen cells, thymocytes, noncytolytic T cell clones, or T cell clones specific for other haptens such as NIP. Polymer concentrations in the 1 to 10 micrograms/ml range produced readily detectable binding within minutes at 20 degrees C, and the binding approached plateau levels at polymer concentrations of between 100 and 300 micrograms/ml. Studies with closely related FL isomers showed that the same antigen fine specificity was operative in both lysis of FL-conjugated target cells and in binding of FL-conjugated polymers. The functional significance of the observed binding was assessed by measuring the effect of FL-conjugated polymers on lymphokine secretion by the clones. High m.w. FL-conjugated polymers caused a dose-dependent increase in the production of macrophage activation factor (MAF) by anti-FL Tc clones, but did not increase MAF production by an NIP-specific clone. In contrast, concanavalin A induced MAF production by both FL-specific and NIP-specific clones. Thus, the observed binding is both specific and functionally significant. These results suggest that soluble nominal antigen, in an appropriately multivalent form, can bind specifically to antigen receptors on Tc clones.     

The effect of neutral polymers on glutamate dehydrogenase activity of mouse liver mitochondria upon administration of endotoxin

It is shown that neutral polymers administered intraperitoneally to intact animals considerably affect glutamate dehydrogenase activity in the liver cell mitochondria as well as in the supernatant. Of the tested polymers, only polyvinyl methylacetamide and dextran inhibit a decrease in the level of mitochondrial enzyme activity which develops with administration of endotoxin. Polyvinyl pyrrolidone with molecular weight of 100 kDa, polyvinyl methylacetamide, dextran and polyvinyl caprolactam prevent an increase of glutamate dehydrogenase activity in the supernatant in case of endotoxin administration to animals. It is possibly a result of the effect of the mitochondrial structure stabilization by the above polymers. A physiological effect of polyvinyl pyrrolidone revealed as an effect on the activity level of mitochondrial glutamate dehydrogenase and in the supernatant after endotoxin administration to animals, depends on the molecular weight of the polymer.     

Suppression of reaginic antibody formation. III. Relationship between immunogenecity and tolerogenicity of hapten-carrier conjugates.

From the study of the effect of epitope density on the immunogenicity of haptenated ovalbumin (DNP-OA) it was concluded that the lightly haptenated conjugate, DNP0-5-OA, induced, on the one hand, only low titers of anti-DNP hemagglutinating antibody and no reaginic antibodies to the hapten and, on the other, high reaginic and high hemagglutinating antibody responses to the carrier. The conjugate with a slightly higher degree of haptenation, i.e., DNP2.3-OA, induced both reaginic and hemagglutinating antibodies to both the hapten and the carrier. By contrast, the heavily haptenated conjugate, DNP20-OA, elicited reaginic and hemagglutinating antibodies only against the hapten but not against the carrier. Specific suppression of anti-hapten reaginic antibody formation had been achieved by treatment of mice with a tolerogen consisting of the hapten (DNP) conjugated covalently to isologous gamma globulins (MgammaG). The epitope density of the DNPx-MgammaG conjugates was shown to play a dominant role in determining whether or not the conjugate was tolerogenic. Thus, lightly haptenated conjugates (DNP0.5-MgammaG, DNP1.3-MgammaG or DNP1.9-MgammaG) were not tolerogenic, moderately haptenated conjugates (DNP4.2-MgammaG, DNP8-MgammaG, and DNP 14-MgammaG) were tolerogenic, and heavily haptenated conjugates (DNP32-MgammaG and DNP53-MgammaG) were immunogenic, being capable of priming the recipients for the DNP hapten. Further evidence for the nonimmunogenicity of DNP 8-MgammaG conjugate was inferred from its rate of clearance in tolerized and normal mice. Thus, the half-life of 125I-labeled DNP8-MgammaG in circulation was not significantly different for normal and tolerized mice; it was 3.7 and 3.5 days, respectively, which is within the range of data reported for clearance of normal MgammaG. These results suggest that DNP8-MgammaG was catabolized at a rate similar to that of nonconjugated, isologous MgammaG. Moreover, there was no significant difference in the localization of DNP8-MgammaG in identical difference in the localization of DNP8-MgammaG in identical organs (spleen, thymus, kidney, and liver) of normal and tolerized mice. All the multivalent DNPx-MgammaG conjugates were shown to be able to elicit passive cutaneous anaphylaxis (PCA) reaction on i.v. challenge of rats which had been pre-sensitized i.d. with anti-DNP reaginic antibodies.     

Minimal requirements for in vitro activity of chemically defined synthetic antigens as immunogenic carrier molecules

A primary antibody response to 2,4-dinitrophenyl (Dnp)-oligolysines of defined chain length was induced in vitro. A molecular size of at least eight lysine residues was critical for the induction of antibody response to the hapten, but the location of the hapten on the carrier did not seem to play a significant role in this regard. Shorter peptides were nonimmunogenic, but did not paralyze the response to other, noncrossreacting antigens. The electric charge of the carrier molecule was found to have an effect on immunogenicity in vitro. A synthetic copolymer with a positive charge was immunogenic, whereas a similar molecule carrying a negative charge was inactive. On the other hand, changing the negative charge of carriers such as polyglutamic acid was not sufficient to render them immunogenic. Furthermore, neutralization of the negative charge of the surface of the spleen tissue by preincubation with positive polymers did not enhance the response to conjugates of the hapten with negatively charged carriers. These observations are interpreted on the basis of higher affinity of positively charged molecules for the negatively charged cell surface. Accordingly, the specific binding of the antigen to the cell surface is made more stable, and this ensures stimulation.     

Suppressive effect of bacterial polysaccharides on BAFF system is responsible for their poor immunogenicity

Capsular polysaccharides of encapsulated bacteria are weakly immunogenic T cell-independent type 2 (TI-2) Ags. Recent findings suggest that BAFF system molecules have a critical role in the development of Ab responses against TI-2 Ags. In this study, we investigated the effect of bacterial polysaccharides on B cell responses to BAFF and a proliferation-inducing ligand (APRIL). We determined that B cells exposed to meningococcal type C polysaccharide (MCPS) or group B Streptococcus serotype V (GBS-V) were unresponsive to BAFF- and APRIL-induced Ig secretion. Moreover, MCPS and GBS-V strongly downregulated transmembrane activator and calcium-modulator and cyclophilin ligand interactor, the BAFF and APRIL receptor that is responsible for Ab development against TI-2 Ags. Interestingly, (4-hydroxy-3-nitrophenyl)acetyl-Ficoll (NP-Ficoll), a prototype TI-2 Ag, did not manifest a suppressive effect on B cells. Paradoxically, whereas GBS-V and MCPS inhibited IFN-γ-induced BAFF production from dendritic cells, NP-Ficoll strongly increased BAFF secretion. TLR 9 agonist CpG deoxyoligonucleotide (ODN) was able to reverse the MCPS-mediated transmembrane activator and calcium-modulator and cyclophilin ligand interactor suppression but could not rescue the Ig secretion in BAFF- or APRIL-stimulated B cells. In support of these in vitro observations, it was observed that CpG ODN could help augment the Ab response against NP in mice immunized with a CpG ODN-containing NP-Ficoll vaccine but exhibited only marginal adjuvant activity for MCPS vaccine. Collectively, these results suggest a mechanism for the weak immunogenicity of bacterial polysaccharides and explain the previously observed differences between bacterial polysaccharide and NP-Ficoll immunogenicity.     

Enhancement by monoclonal anti-Lyb-2 antibody of antigen-specific B lymphocyte expansion stimulated by TNP-Ficoll and T lymphocyte-derived factors

By using antigen-specific populations of B cells (TNP-ABC) we have demonstrated that the type-2 antigen TNP-Ficoll was capable of initiating B cell proliferation only in the presence of T cell-derived factors. Monoclonal-anti-Lyb-2.1 antibody acted synergistically with a T cell-derived supernatant, as well as with B cell-stimulating factor (BSF-1) to enhance the level of B cell expansion obtained in this in vitro system. This effect of anti-Lyb-2.1 mAB was observed at each day of the antigen-driven B cell expansion and was seen only with B cells purified from strains expressing the Lyb-2.1 allele. The epitope density of hapten on the Ficoll plays a critical role in this process, because Ficoll that is haptenated with low density of hapten was not found to be stimulatory. These results suggest that the Lyb-2 surface molecule influences the antigen-driven B cell growth that is stimulated by type 2 antigens and BSF-1.     

Artificial peptide and carbohydrate antigens. Immobilization of haptens and adjuvant (MDP) on polyacrylamide

To study the influence of the polyacrylamide carrier on immunogenic properties of the peptide and oligosaccharide haptens, we have prepared artificial antigens by conjugation of a synthetic hexapeptide (homologous to the fragment 95-100 of the murine H-2Db antigen heavy chain) or of an oligosaccharide (antigenic determinant of human blood groops, Lea) with polyacrylamide. In some cases the conjugates containing also a synthetic glycopeptide adjuvant, N-acetylmuramoyl-L-alanyl-D-isoglutamine (MDP), were used. Antisera against haptens were obtained by immunization of BALB/c mice with corresponding conjugates. By the enzyme-linked immunosorbent assay it was shown that these antisera had a high binding titer (up to 10 000) to corresponding hapten, and MDP immobilized on the same carrier as hapten possessed a considerable immunostimulating activity. Thus, usefulness of polyacrylamide for preparation of immunogenic artificial molecules carrying peptide and oligosaccharide haptens was demonstrated.     

Affinity separation of proteins in aqueous three-phase systems

Aqueous polymer three-phase systems composed of dextran-Ficoll-polyethylene glycol-water have been used for affinity partition of proteins. The upper, middle, and lower phases are rich in polyethylene glycol, Ficoll, and dextran, respectively. Affinity partition was performed using the reactive dyes Cibacron Blue F36-A and Remazol Yellow GCL which are known as specific ligands for albumin and prealbumin from human serum. When the ligands were bound alternatively to polyethylene glycol, Ficoll, or dextran the target proteins were directed toward the upper, middle, or lower phase, respectively. In the presence of two ligands immobilized to two different polymers the distribution of two proteins could be steered to different phases at the same time. Serum albumin and prealbumin could be separated by using Cibacron Blue-Ficoll and Remazol Yellow-dextran or Cibacron Blue-polyethylene glycol and Remazol Yellow-dextran as polymer ligands.     

Adjuvant effects of nonionic block polymer surfactants on liposome-induced humoral immune response

The ability of several surface-active agents to stimulate the humoral immune response in mice against haptenated liposomes was tested. The surfactants were block copolymers of hydrophilic polyoxyethylene (POE) and hydrophobic polyoxypropylene (POP) that differed in m.w., percentage of POE, and mode of linkage of POP to POE. The liposomes were haptenated with tripeptide-enlarged dinitrophenyl coupled to phosphatidylethanolamine, which was incorporated into the liposomal membrane. Additional injection of mice with surfactant stimulated serum hemagglutination titers and splenic plaque-forming cell (PFC) numbers to varying extents. Block polymers with POP chains flanking a POE center, as well as polymers with POE chains flanking a POP center, displayed high adjuvant activity. These block polymers stimulated the antibody response in a dose-dependent manner. They stimulated the antibody response with both high and low antigen doses. Furthermore, the addition of one of these adjuvants (25R1) reduced the amount of carrier lipid required in the liposome in order to obtain an optimal antibody response. The surfactants, which displayed high adjuvant activity, did not interfere with liposome stability as measured with a liposome lysis assay. Moreover, in vitro preincubation of liposomes with a block polymer did not affect their immunogenicity. Optimal adjuvant activity was observed when both adjuvant and liposomes were administered by the same route. Simultaneous injection of both components, however, is not a prerequisite. Conclusively, it can be stated that nonionic block polymer surfactants are potent adjuvants for stimulation of the antibody response against haptenated liposomes.     

Adapter molecule Grb2-associated binder 1 is specifically expressed in marginal zone B cells and negatively regulates thymus-independent antigen-2 responses

Grb2-associated binder 1 (Gab1) is a member of the Gab/daughter of sevenless family of adapter molecules involved in the signal transduction pathways of a variety of growth factors, cytokines, and Ag receptors. To know the role for Gab1 in hematopoiesis and immune responses in vivo, we analyzed radiation chimeras reconstituted with fetal liver (FL) cells of Gab1(-/-) mice, because Gab1(-/-) mice are lethal to embryos. Transfer of Gab1(-/-) FL cells of 14.5 days post-coitum rescued lethally irradiated mice, indicating that Gab1 is not essential for hematopoiesis. Although mature T and B cell subsets developed normally in the peripheral lymphoid organs, reduction of pre-B cells and increase of myeloid cells in the Gab1(-/-) FL chimeras suggested the regulatory roles for Gab1 in hematopoiesis. The chimera showed augmented IgM and IgG1 production to thymus-independent (TI)-2 Ag, although they showed normal responses for thymus-dependent and TI-1 Ags, indicating its negative role specific to TI-2 response. Gab1(-/-) splenic B cells stimulated with anti-delta-dextran plus IL-4 plus IL-5 showed augmented IgM and IgG1 production in vitro that was corrected by the retrovirus-mediated transfection of the wild-type Gab1 gene, clearly demonstrating the cell-autonomous, negative role of Gab1. Furthermore, we showed that the negative role of Gab1 required its Src homology 2-containing tyrosine phosphatase-2 binding sites. Cell fractionation analysis revealed that nonfollicular B cells were responsible for the augmented Ab production in vitro. Consistent with these results, the Gab1 gene was expressed in marginal zone B cells but not follicular B cells. These results indicated that Gab1 is a unique negative regulator specific for TI-2 responses.     

Effects of dextran molecular weight on red blood cell aggregation

The reversible aggregation of human red blood cells (RBC) by proteins or polymers continues to be of biologic and biophysical interest, yet the mechanistic details governing the process are still being explored. Although a depletion model with osmotic attractive forces due to polymer depletion near the RBC surface has been proposed for aggregation by the neutral polyglucose dextran, its applicability at high molecular mass has not been established. In this study, RBC aggregation was measured over a wide range of dextran molecular mass (70 kDa to 28 MDa) at concentrations ≤2 g/dL. Our results indicate that aggregation does not monotonically increase with polymer size; instead, it demonstrates an optimum dextran molecular mass around 200-500 kDa. We used a model for depletion-mediated RBC aggregation to calculate the expected depletion energies. This model was found to be consistent with the experimental results and thus provides new insight into polymer-RBC interactions.     

Role of solvent properties of aqueous media in macromolecular crowding effects

Analysis of the macromolecular crowding effects in polymer solutions show that the excluded volume effect is not the only factor affecting the behavior of biomolecules in a crowded environment. The observed inconsistencies are commonly explained by the so-called soft interactions, such as electrostatic, hydrophobic, and van der Waals interactions, between the crowding agent and the protein, in addition to the hard nonspecific steric interactions. We suggest that the changes in the solvent properties of aqueous media induced by the crowding agents may be the root of these “soft” interactions. To check this hypothesis, the solvatochromic comparison method was used to determine the solvent dipolarity/polarizability, hydrogen-bond donor acidity, and hydrogen-bond acceptor basicity of aqueous solutions of different polymers (dextran, poly(ethylene glycol), Ficoll, Ucon, and polyvinylpyrrolidone) with the polymer concentration up to 40% typically used as crowding agents. Polymer-induced changes in these features were found to be polymer type and concentration specific, and, in case of polyethylene glycol (PEG), molecular mass specific. Similarly sized polymers PEG and Ucon producing different changes in the solvent properties of water in their solutions induced morphologically different α-synuclein aggregates. It is shown that the crowding effects of some polymers on protein refolding and stability reported in the literature can be quantitatively described in terms of the established solvent features of the media in these polymers solutions. These results indicate that the crowding agents do induce changes in solvent properties of aqueous media in crowded environment. Therefore, these changes should be taken into account for crowding effect analysis.     

Studies on replacing most of the penetrating cryoprotectant by polymers for embryo cryopreservation.

Solutions used for vitrification or rapid cooling of embryos usually contain high concentrations of penetrating cryoprotectants. At these concentrations embryos can tolerate the penetrating cryoprotectants for only short periods of time without damage. This study designed and tested cryoprotectant solutions that combined high polymer concentrations with low penetrating cryoprotectant concentrations. Mouse 2-cell embryo development was not compromised by up to 15-min exposure to 30 wt% solutions of the polymers Ficoll 70,000 MW or dextran 69,000 MW at room temperature. However, our batches of polyvinylpyrrolidone (PVP) 10,000 and PVP 40,000 were embryo-toxic even after extensive dialysis against Milli-Q water. As both Ficoll and dextran contribute to a solution's physical vitrification properties, we formulated vitrifying solutions containing only 11 to 27 wt% ethylene glycol (EG) by including 34 to 49 wt% polymers (27 wt% EG + 34 wt% Ficoll, 27 wt% EG + 34 wt% dextran, 16 wt% EG + 39 wt% Ficoll, or 11 wt% EG + 49 wt% Ficoll, in phosphate-buffered saline (PBS)). Novel solutions were designed for 0.25 ml straw as a viscous matrix for encapsulation of embryos. These yielded high rates of development of 2-cell mouse embryos after rapid cooling and warming (> or = 96% expanded blastocysts in vitro and > or = 62% viable fetuses as assessed on day 15 of gestation in vivo) in all tested solutions. All control 2-cell embryos formed expanded blastocysts in vitro and 78% formed fetuses in vivo. Comparable results were obtained with both 4-cell and 8- to 16-cell mouse embryos. The lower toxicity of Ficoll and dextran may explain why these new solutions gave better results than had previously been reported for solutions containing 7.5% PVP and low concentrations of EG (2 M).     

Beneficial Effects of Additional Adjuvants on the Immune Response to Haptenated Liposomes

Abstract

The humoral immune response to haptenated liposomes is well documented. This review summarizes our efforts in this field of research. The immunogenicity of haptens, small oligosaccharides and peptides linked to phosphatidyl ethanolamine are studied in mice. Special attention is given to the immunomodulating properties of lipid A, its derivatives and the synthetic adjuvants non-ionic block polymers and ditnethyldioctadecylammonium bromide on the outcome of the response to these haptenated liposomes.     

Effect of priming with a thymus-independent antigen on susceptibility to B-cell tolerance.

The effects of priming on the susceptibility of B-cell subsets to tolerance induction have been tested in a model system in which anti-immunoglobulin (anti-Ig) has been employed as a surrogate for tolerogen. T-cell-depleted B cells were primed in vitro with fluorescein or trinitrophenylated Ficoll (a thymus-independent (TI) antigen) and then exposed overnight to anti-Ig to attempt to induce B-cell anergy. Primed cells were relatively resistant to this tolerance protocol and resistance was hapten specific. The dose response and kinetics suggested that this process was not due to receptor blockade or modulation, but was an active process. Moreover, this priming for resistance to tolerance was reproduced in vivo upon intraperitoneal treatment with haptenated Ficoll. Such in vivo priming for tolerance resistance was long-lasting and did not occur with a thymus-dependent priming protocol with fluoresceinated hemocyanin. These results are discussed in terms of TI priming to drive B cells into cycle and express novel functional and phenotypic properties.