An ethnographic, controlled study of the use of a computer-based histology atlas during a laboratory course.

OBJECTIVE: To evaluate the use and effect of a computer-based histology atlas during required laboratory sessions in a medical school histology course. DESIGN: Ethnographic observation of students' interactions in a factorial, controlled setting. MEASUREMENTS: Ethnographer's observations; student and instructor self-report survey after each laboratory session with items rated from 1 (least) to 7 (best); microscope practicum scores at the end of the course. RESULTS: Between groups assigned the atlas and those not, the ethnographer found qualitative differences in the semantic categories used by students in communicating with each other and with the faculty. Differences were also found in the quality of the interactions and in the learning styles used with and without the computer present in the laboratory. The most interactive learning style was achieved when a pair of students shared a computer and a microscope. Practicum grades did not change with respect to historical controls. Students assigned the atlas, compared with those not assigned, reported higher overall satisfaction (a difference in score of 0.1, P = 0.003) and perceived their fellow students to be more helpful (a difference of 0.11, P = 0.035). They rated the usefulness of the microscope lower (a difference of 0.23, P<0.001). CONCLUSION: A computer-based histology atlas induces qualitative changes in the histology laboratory environment. Most students and faculty reacted positively. The authors did not measure the impact on learning, but they found that there are aspects of using the atlas that instructors must manipulate to make learning optimal. Ethnographic techniques can be helpful in delineating the context and defining what the interventions might be.

Low molecular weight antigen arrays delete high affinity memory B cells without affecting specific T-cell help.

An ongoing, T-cell dependent, secondary antibody response to an epitope can be suppressed in vivo by low molecular weight, soluble polymers, bearing multiple copies of the same epitope. This study illustrates that such suppressive T-cell independent antigen arrays target the epitope-specific, high affinity, memory B cells for long-term functional elimination. Splenocytes from hyperimmune unsuppressed donors, when adoptively transferred into irradiated recipients will readily reconstitute a secondary anti-hapten response after antigenic challenge. No such response was observed with splenocytes transferred from hyperimmune donors suppressed with antigen arrays. The extent of suppression depended on antigen array dose and duration of exposure in the donor animals. The suppressive antigen array carryover from the donors into the recipients was negligible and insufficient to account for the observed suppression. B cells from hyperimmune mice producing high affinity anti-fluorescein antibodies, generated by multiple fluoresceinated ovalbumin (FL-OVA) injections, were helped efficiently by T cells from hyperimmune donors, which were either unsuppressed or suppressed with antigen arrays. Accordingly, help from T cells, specific for the carrier protein remains intact after such suppression. Neither lipopolysaccharide (LPS), nor additional transferred carrier-primed T cells could reverse the unresponsiveness of adoptively transferred splenocytes from suppressed animals. Flow cytometry showed that the number of hapten-specific B cells was markedly reduced after suppression. Collectively, these data show that the long term elimination of an ongoing T-cell dependent antibody response by suppressive exogenous antigen arrays is due to the functional deletion of high affinity, antigen-specific B cells, even in the presence of adequate T-cell help. The long-term nature of such functional deletion strongly suggests physical deletion of the antigen-specific B cell population.

Durable elimination of high affinity, T cell-dependent antibodies by low molecular weight antigen arrays in vivo.

Ongoing Ab responses to a T cell-dependent Ag can be suppressed in hyperimmune animals by exogenous, multivalent Ag arrays. The pharmacologic basis for this suppression was studied by varying the molecular mass, ligand valence, and dose of Ag arrays, and then determining their efficacy, pharmacokinetics, and tissue distribution. Arrays ranging in molecular mass from 30 to 500 kDa caused initial clearance of specific serum Abs, but only the smaller arrays caused persistent suppression despite their relatively lower binding avidity and shorter retention in vivo. Suppression by the smaller arrays at lower doses was biphasic, implying two distinct modes of Ab elimination. High affinity IgG was eliminated preferentially, as shown by calibrated variable ligand-density ELISA. Suppressive arrays were localized discretely in the splenic germinal centers of hyperimmune animals. These results indicate that Ag array mass, ligand valence, and dose all play critical roles, and histologic compartmentalization may also be a pertinent parameter, in determining suppressive efficacy in vivo.

Allergenicity and antigenicity of chicken egg ovomucoid (Gal d III) compared with ovalbumin (Gal d I) in children with egg allergy and in mice.

When attempting to generate mouse monoclonal antibodies to hen's egg ovalbumin, injection of commercially purified ovalbumin resulted in monoclonal antibodies, which when assayed against commercially purified ovalbumin (Gal d I) or ovomucoid (Gal d III), appeared to be specific to both. With the use of high-performance liquid chromatography (HPLC)-repurified ovalbumin and ovomucoid in assay procedures, monoclonal antibodies generated by commercially purified ovalbumin were found to be specific for ovomucoid only. To clarify this phenomenon, mice were serially injected with commercially purified ovalbumin or HPLC-repurified ovalbumin. It was found that most of the antibody response to commercially purified ovalbumin was directed against the minor (< 1%) ovomucoid contaminant and that HPLC-repurified ovalbumin failed to produce antibodies to ovomucoid. Commercially purified ovomucoid resulted in only minimal amounts of antibodies to ovalbumin. Thus when commercially purified ovalbumin is used both for immunization and immunoassay, most of the antibodies produced are actually against the small amount of ovomucoid contaminant, and not ovalbumin. To determine whether ovomucoid is the major antigenic and allergenic egg white protein in human beings, one group of 18 children with egg allergy were skin prick tested with half-log dilutions of egg white extract and diethylaminoethyl cellulose (DEAE)-repurified ovomucoid, ovalbumin, and lysozyme. Ovomucoid mean wheal diameters were significantly greater than wheal diameters in response to ovalbumin, lysozyme, and egg white extract at the three most concentrated of five dilutions tested: 0.01, 0.03, and 0.1 mg/ml (p < 0.01). Serum ovomucoid-specific IgE and IgG antibody concentrations to DEAE-repurified ovomucoid were significantly greater than that to DEAE-repurified ovalbumin (p < 0.05). In a second study, 10 patients with egg allergy and persistent egg hypersensitivity were compared with 11 patients with egg allergy in whom clinical tolerance to egg developed. IgE antibodies to repurified ovomucoid were significantly greater in patients with persistent egg hypersensitivity compared with patients in whom clinical tolerance developed at the time of both initial and follow-up food challenges. In contrast, there were no significant differences in IgE antibody concentrations to repurified ovalbumin in either group at any time. These results suggest that ovomucoid is the immunodominant protein fraction in egg white and that the use of commercially purified ovalbumin has led to an overestimation of the dominance of ovalbumin as a major egg allergen and antigen in human beings.

A comparison of the immunogenicity of a pair of enantiomeric proteins.

The immunogenicity of a folded, all D-amino acid protein, rubredoxin, has been compared with that for the corresponding L-protein enantiomer. Following multiple administrations with alum adjuvant, the L-protein induced a strong, specific IgG antibody response, whereas the D-protein did not. This relative lack of responsiveness to the D-protein cannot be attributed to rapid excretion, since it is retained at least 4 times longer than the natural L-protein. These observations provide the first direct evidence that a folded D-amino acid protein has low immunogenicity and is long lived in vivo. Proteins with such properties may be useful as molecular platforms in a variety of chemical and pharmacological applications.

Inhibition or activation of human T cell receptor transfectants is controlled by defined, soluble antigen arrays.

We present evidence that direct T cell receptor (TCR) occupancy by antigen can either activate or inhibit T cells, depending upon whether or not a threshold number of local TCRs are crosslinked by multivalent arrays of the antigen. Variants of Jurkat cells were previously transfected with TCR alpha and beta chains that bind fluorescein, yielding FL-TCR+ human T cells. The transfectants are activated upon binding soluble multivalent antigen arrays at concentrations well below those required for monovalent interactions. This activation, measured by calcium fluxes and interleukin 2 (IL-2) production, indicates the superior binding avidity of multivalent ligands. Smaller, less multivalent arrays do not activate the cells, but antagonize larger arrays, demonstrating that antigen can bind TCR as either agonist or antagonist. The balance between activation and inhibition depends upon antigen array size, ligand valence, and concentration, indicating that a threshold extent of receptor crosslinking, and not individual perturbations of single TCR, is required for activation by antigen. Approximately 100 stimulatory arrays specifically bind per FL-TCR+ cell at concentrations where IL-2 production is half-maximal.

Rational design of conjugate vaccines.

Whereas bacterial polysaccharides, classified as T-cell-independent antigens, elicit protective antibodies in adults, booster injections fail to produce an augmented response or promote antibody class switching. Because T-cell-dependent antigens, typically proteins, both produce boosted antibody levels and promote antibody class switching, it has been considered highly desirable to attempt to convert the T-cell-independent polysaccharide antigens into T-cell-dependent antigens, particularly for use in high-risk groups. A number of clinical trials now report the efficacy of conjugate vaccines in inducing the production of antibody in response to a number of previously poorly immunogenic--mainly T-cell-independent--antigens. In addition to conjugate vaccines containing bacterial polysaccharides, vaccines containing relevant peptides from a variety of pathogens are also being formulated and investigated. Questions remain, however, regarding their synthesis, use, and efficacy. The best ages for vaccine administration and selection of the optimal protein carrier are still under investigation, as are questions regarding the use of adjuvants, which can greatly affect the vaccine's potency. Spacing and size of epitope and size and composition of the final structure also must be considered; the importance of molecular size and aggregation of antigen in increasing immunogenicity have been well documented. These questions must be addressed for the much-needed development of conjugate vaccines against some common infections worldwide, including malaria, bacterial meningitis, and infections from Pseudomonas aeruginosa and Neisseria gonorrhoeae because of increasing susceptibility to these infections and resistance of the pathogens to chemotherapeutic agents and/or antibiotics.

Profound specific suppression by antigen of persistent IgM, IgG, and IgE antibody production.

Ongoing, high-titer T-cell-dependent immune responses in adult mice, consisting of IgM, IgG, and IgE anti-fluorescein antibodies, can be specifically and substantially reduced (90-99%) when the mice are injected with appropriate doses of fluoresceinated dextran of defined molecular weight and hapten valence. This suppressive form of the antigen is nontoxic and specific, as responses to other antigens are unaffected. The suppression is long lasting and reduces high-affinity antibodies most markedly. Moreover, plasma cell secretion of specific antibody is virtually eliminated. This demonstrates that the reduction in antibody titer is not simply due to masking of serum antibody by the suppressive polymer. The results are discussed with reference to proposed models of B-cell and T-cell tolerance. Extension of these findings to disease-related immunogens may yield effective antigen-specific treatments of human allergy and autoimmune diseases.

Major histocompatibility complex independent T cell receptor-antigen interaction: functional analysis using fluorescein derivatives.

We have isolated T cell receptor (TCR) cDNAs from fluorescein (FL)-specific human T cell clones (alpha FL beta FL), and transferred them to TCR beta- Jurkat cells in order to study direct FL-binding to the TCR. Using either FL-conjugated polymers (FL-polymer) or FL-substituted Sepharose beads, we are able to demonstrate the direct binding of antigen to the T cell surface, and the functional activation of the T cell transfectants. We present evidence against the involvement of major histocompatibility complex (MHC) molecules or antigen presentation in the interaction of FL with the alpha FL beta FL transfectants. Additionally, we have examined the effect of ring substitutions on the FL molecule as well as specific alterations of substituents attached to the 5' position, and we have found that all of them interfere with the functional recognition of the alpha FL beta FL TCR. These experiments demonstrate that TCRs like antibodies have intrinsic affinities for antigen, even without the involvement of MHC molecules.

Modification, optimization and simplification of the spot ELISA technique for the enumeration of cells secreting anti-hapten antibodies.

We describe here a modification of the spot ELISA assay for the detection and enumeration of antibody-secreting cells. The modification increases sensitivity, convenience and simplicity while optimizing parameters of time, temperature and reagents. Laboratory reagents and 96 well microtiter plates commonly used for the standard ELISA assay are adapted for use in the spot ELISA assay. Special emphasis is placed on the detection of anti-hapten IgM or IgG production by splenocytes in response to stimulation by a range of doses of antigen. In addition, two procedures are described whereby the spot ELISA assay is altered to yield a measurable optical density: (1) the blue precipitate obtained in the spot ELISA assay is dissolved and the optical density of the resultant blue color is measured, and (2) a standard ELISA substrate is substituted for the spot ELISA substrate to yield a colored solution rather than a precipitate. Both of these are shown to correlate with spot numbers and can therefore supplement or substitute for the enumeration of spots (produced by antibody-secreting cells), thus speeding and mechanizing the assay.

Inhibition of antibody formation by receptor cross-linking: the molecular characteristics of inhibitory haptenated polymers.

Previous studies from this laboratory have reported on the characteristics of a molecule which make it inhibitory. These findings were based on studies using the T cell-independent haptenated polymer, 2,4-dinitrophenyl-polyacrylamide (DNP-PA). The present study was undertaken to determine whether the same molecular properties which defined the inhibitory potentials of DNP-PA were characteristic of other haptenated polymers as well. The molecules studied here consisted of a series of five diverse fluoresceinated polymers with varying molecular mass and hapten valence. In agreement with the previous findings on DNP-PA, definable molecular forms of fluoresceinated dextrans. Ficolls, polyacrylamides, carboxymethyl celluloses and polyvinyl alcohols were each found to be capable of inhibiting the anti-fluorescein response to immunogenic forms of haptenated polymers of either the same or different carrier chemistry. This inhibitory ability was relatively independent of the chemical composition and conformation of the carrier polymer. These studies allow some logical generalizations to be made as to which quantitative molecular properties of haptenated type 2 T cell-independent antigens determine whether they will be stimulatory or inhibitory of an anti-hapten immune response.

The immunogenicity of soluble haptenated polymers is determined by molecular mass and hapten valence.

T cell-independent Ag are believed to stimulate antibody formation in the relative absence of Ag processing and T cell help. Previous studies on the type 2 T cell independent (TI-2) Ag DNP-polyacrylamide, have shown that when one systematically varies the molecular mass and hapten valence, the immunogenic potential of this type of molecule depends on definable molecular characteristics. It was found that to be immunogenic, these molecules had to exceed a threshold molecular mass of 100,000 Da and a threshold hapten valence of 20. The present study was undertaken to determine whether such findings could be generalized to other molecules belonging to the TI-2 class of Ag. The molecular characteristics of five chemically different fluoresceinated (FL)-polymers were systematically varied, and their ability to stimulate an IgM antihapten immune response was measured. The polymers used as carriers were carefully size-fractionated and consisted of one natural polymer (dextran), one modified natural polymer (carboxymethyl cellulose), and three synthetic polymers (Ficoll, polyvinyl alcohol, and polyacrylamide). The carriers varied in physical structure from the highly cross-linked Ficoll, to the somewhat branched dextran, to the linear polyacrylamide, carboxymethyl cellulose, and polyvinyl alcohol. Polymers were haptenated with FL and size-fractionated so as to yield a panel of molecules with varying molecular mass, hapten valence, and hapten density. Anti-FL IgM response to these haptenated polymers was measured in vivo after i.p. injection of the FL-polymer in saline, and measured in vitro after culture with unfractionated spleen cells from naive mice. In agreement with the previous studies on DNP-polyacrylamide, it was found that to be immunogenic, each of the FL-polymers had to exceed a comparable threshold value of molecular mass and of hapten valence. Optimal immunogenicity occurred when the FL-polymers had values of mass and hapten density lying within a predictable range. Immunogenicity decreased when these optimal parameters were substantially increased or decreased. We conclude that the immunogenicity of soluble haptenated polymers depends on predictable physical molecular characteristics, and is relatively independent of the chemical composition and conformation of the carrier polymer.

Tolerogen-mediated suppression of the immune response. Suppressor cells as passive transfer agents.

The anti-dinitro-phenyl (Dnp)IgM antibody response in mice was inhibited by administration of either a non-immunogenic form of Dnp-polyacrylamide (Dnp-Pa) or an excess amount of an immunogenic form of Dnp-Pa. Spleen cells, alive or heat-killed, from mice tolerized in vivo by either method, inhibited the anti-Dnp response of naive spleen cells co-cultured in vitro with antigen. Conversely, donor cells tolerized in vivo by a high dose of immunogenic Dnp-Pa, when titrated into a naive cell culture which contained no antigen, produced a stimulatory dose-response curve. Both the dose-dependent inhibition and stimulation correlated strongly with the amount of 125I-labelled Dnp-Pa carried over by tolerized spleen cells into the naive cell culture system. Because the doses and experimental procedures used were comparable to those commonly used for suppressor cell generation and assay, it is suggested that antigen-specific suppressor cells may produce their effects through passive transfer of antigen and/or tolerogen.

Direct evidence for the existence of nominal antigen binding sites on T cell surface Ti alpha-beta heterodimers of MHC-restricted T cell clones.

The binding of nominal antigen to Ti alpha-beta heterodimers on MHC-restricted human T cell clones specific for fluorescein-5-isothiocyanate (FL) was detected by flow cytometry and affinity chromatography. The FL-Ti interaction is of physiologic significance, since T cell activation is induced by cross-linked arrays of FL in the absence of the specific MHC recognition. High antigen valence is required to achieve stable binding to cells and subsequent activation, which is consistent with estimated Ti-FL association constants of less than 3 X 10(5) l/mol. In addition to providing direct evidence that the Ti alpha-beta heterodimer is the receptor for antigen, these data suggest that nominal antigen binding sites exist on the Ti molecules of at least some MHC-restricted clones.

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.

Antigen valence determines the binding of nominal antigen to cytolytic T cell clones.

We have shown that cytotoxic T cell clones specific for the nominal antigen FL will bind high molecular weight (600,000 to 2,000,000) polyacrylamide and Ficoll polymers conjugated with 200-600 FL groups per molecule. Low molecular weight polymers (40,000) with the same epitope density did not give stable binding. A high molecular weight polymer with a lower epitope density also failed to bind. Taken together, these results suggest that a substantial degree of multivalence is a necessary factor in the stable binding of nominal antigen to T cell clones.

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.

Studies on the immunogenicity and tolerogenicity of T-independent antigens.

The T-cell independent immunological responses of naive mice to a series of size-fractionated dinitrophenylpolyacrylamide molecules were measured. These responses were correlated in vitro and in vivo with hapten number per molecule and hapten density per unit length. We found that to be immunogenic, a T-independent molecule must contain a threshold number (approximately 20) of appropriately spaced haptens or epitopes. We infer that this condition is necessary for simultaneous B-cell receptor binding and clustering. Molecules with less than this number of haptens are not immunogenic at any dose. Some nonimmunogenic molecules are capable of inhibiting the response to the immunogenic ones; this capability increases with increasing hapten density. In order for a molecule to accommodate 20 or more appropriately spaced haptens, it must be above a threshold size. Increasing hapten density in a molecule at or above threshold size and hapten number increases its immunogenicity. Increasing hapten density in a molecule below threshold size increases its tolerogenicity. Conclusions concerning immunogenicity and tolerogenicity of T-independent antigens may not be warranted unless the molecular weight and hapten or epitope number per molecule are well characterized.