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Objective: To investigate the possible role of Cryptococcus neoformans in HIV-1 pathogenesis., Design: An in vitro system was developed to study HIV-1 replication in freshly HIV-1-infected peripheral blood mononuclear cells (PBMC) incubated with whole azide-killed C. neoformans., Methods: Human PBMC or peripheral blood lymphocytes were infected with lymphocytotropic HIV-1 and incubated with azide-killed encapsulated or non-encapsulated C. neoformans for 10 days. Viral replication was followed by HIV-1 p24 enzyme-linked immunosorbent assay and median tissue culture infective dose determination. Tumour necrosis factor (TNF) release by PBMC, induced by C. neoformans, was measured. Anti-TNF monoclonal antibodies or pentoxifylline were used to inhibit TNF bioactivity., Results: Both encapsulated and non-encapsulated C. neoformans enhanced HIV-1 replication in PBMC but not in peripheral blood lymphocytes. C. neoformans induced TNF release by PBMC. Inhibition of TNF bioactivity did not block C. neoformans-enhanced HIV-1 replication in PBMC., Conclusions: C. neoformans can enhance HIV-1 replication in T cells only in the presence of monocytic cells. This enhancement is not dependent on encapsulation nor can it be attributed to TNF release.

Streptococcus pneumoniae type 23F capsular polysaccharide (PS23F) consitss of a repeating glycerol-phosphorylated branched tetrasaccharide. The immunogenicities of the following related antigens were investigated: (i) a synthetic trisaccharide comprising the backbone of one repeating unit, (ii) a synthetic tetrasaccharide comprising the complete repeating unit, and (iii) native PS23F (all three conjugated to keyhole limpet hemocyanin [KLH]) and (iv) formalin-killed S. pneumoniae 23F. All antigens except the trisaccharide-KLH conjugate induced relatively high anti-PS23F antibody levels in rabbits. The epitope specificity of such antibodies was then studied by means of an inhibition immunoassay. The alpha(1-->2)-linked L-rhamnose branch was shown to be immunodominant for immunoglobulin G (IgG) induced by tetrasaccharide-KLH, PS23F-KLH, and killed S. pneumoniae 23F: in most sera L-rhamnose totally inhibited the binding of IgG to PS23F. Thus, there appears to be no major difference in epitope specificity between IgG induced by tetrasaccharide-KLH and that induced by antigens containing the polymeric form of PS23F. Human anti-PS23F IgG (either vaccine induced or naturally acquired) had a different epitope specificity: none of the inhibitors used, including L-rhamnose and tetrasaccharide-KLH, exhibited substantial inhibition. These observations suggest that the epitope recognized by human IgG on PS23F is larger than the epitope recognized by rabbit IgG. Both human and rabbit antisera efficiently opsonized type 23F pneumococci, as measured in a phagocytosis assay using human polymorphonuclear leukocytes.

This study determines the sensitivity of human observers to 2D fronto-parallel angles. Angle discrimination thresholds vary as a function of base angle and stimulus configuration orientation. However, these variations can all be understood from the well-known meridional anisotropy for orientation discrimination of the orientations that define the angle. Specifically, observers do not show any special sensitivity to angles of 90 degrees and 180 degrees (straightness). Instead it is claimed that observers measure geometric angle by comparing the visual orientations that define the angle, although it is shown that they are not fully efficient in this comparison operation. An explicit visual reference angle does not improve discrimination thresholds (that is, observers can perfectly well supply one from memory), nor do observers need an explicit visual reference orientation in an orientation discrimination task.

We study the capacities of human observers to time order light sources that emit dynamic noise, identical for the different light sources, except for an adjustable delay. There is a range of temporal delays for which human observers are perfectly able to perform this task, using the direction of the motion percept that is evoked by the stimulus as a cue. An optimal delay between light sources at which the observers are most robust against any deterioration of the stimulus is defined. We claim that optimal delays (15-25 msec) correspond to the time delay of a putative Reichardt correlation mechanism in human motion vision. Contrary to the ability of human observers to sense temporal correlations in noise sequences, observers are totally unable to detect anticorrelation between noise sequences. This inability rules out motion opponency as a viable model for human front-end ("early") motion vision.

Overlapping synthetic disaccharide, trisaccharide and tetrasaccharide, derived from pneumococcal polysaccharide type 17F (PS17F), were coupled to keyhole limpet haemocyanin (KLH). The conjugates were tested in mice. The disaccharide-KLH and especially trisaccharide-KLH, in combination with Quil A, induced high titres of high-avidity anti-PS17F IgG. Both conjugates protected mice against challenge with Streptococcus pneumoniae 17F. Tetrasaccharide-KLH, although able to elicit anti-tetrasaccharide antibodies, induced a minimal non-protective anti-PS17F IgG response of low avidity. The tetrasaccharide-KLH conjugate, in contrast to the other conjugates, failed to bind rabbit anti-PS17F IgG.

We present data on the human sensitivity to temporal pulse modulations of target velocity. We measured threshold detection modulation amplitudes for pulse-shaped speed modulations, as a function of pulse duration and temporal frequency. At short pulse durations (up to 50 msec) and low modulation frequency (1 Hz), detection amplitudes are ruled by Bloch's law: the product of pulse duration and threshold modulation amplitude is a constant. This constant corresponds to a position modulation with an amplitude of 3 arc min in a coordinate frame that moves at the average speed (3 deg/sec) of the target. At longer pulse durations we find deviations from Bloch's law. Speed modulation thresholds are not critically dependent on target luminance contrast. These results are modeled by a modulation detection process in two stages. A functional description of the first stage is filtering of the true speed modulation signal by a second order low-pass filter with a characteristic time constant of 20-25 msec. The second (decision) stage is variance detection: modulations are detected when the variance of the filtered modulation function exceeds a certain threshold variance. The square-root threshold variance is estimated 8-10%. This two-parameter model accurately predicts the measured dependence of pulse modulation detection thresholds on pulse duration and pulse density.

We present data on the human sensitivity to optic acceleration, i.e. temporal modulations of the speed and direction of moving objects. Modulation thresholds are measured as a function of modulation frequency and speed for different periodical velocity vector modulation functions using a localized target. Evidence is presented that human detection of velocity vector modulations is not directly based on the acceleration signal (the temporal derivative of the velocity vector modulation). Instead, modulation detection is accurately described by a two-stage model: a low-pass temporal filter transformation of the true velocity vector modulation followed by a variance detection stage. A functional description of the first stage is a second order low-pass temporal filter having a characteristic time constant of 40 msec. In effect, the temporal low-pass filter is an integration of the velocity vector modulation within a temporal window of 100-140 msec. A non-trivial link of this low-pass filter stage to the temporal characteristics of standard motion detection mechanisms will be discussed. Velocity vector modulations are detected in the second-stage, whenever the variance of the filtered velocity vector exceeds a certain threshold variance in either the speed or direction dimension. The threshold standard deviations for this variance detection stage are estimated to be 17% for speed modulations and 9% for motion direction modulations.

Two monoclonal anti-idiotypic antibodies (ab2 mAb), designated 1.13A112 (IgG2a) and 1.13A321 (IgG1) and induced against Semliki Forest virus (SFV)-neutralizing mAb UM 1.13, were investigated with regard to their vaccine potential. 1.13A321 was coupled with glutaraldehyde to keyhole limpet haemocyanin (KLH) and mixed with the adjuvant Quil A. Then when injected subcutaneously into BALB/c mice, it evoked high levels of SFV-neutralizing anti-anti-idiotypic antibodies in serum. In contrast, 1.13A112 had to be indirectly cross-linked to KLH with anti-mouse immunoglobulin to induce a low neutralizing antibody response. Competition binding assay revealed that 1.13A112 and 1.13A321 were completely competitive. Furthermore, SFV neutralization by UM 1.13 and anti-anti-idiotypic (ab3) serum was blocked equally well by either ab2 mAb. These results indicate that ab1 (UM 1.13) and ab3 share at least one antigen-combining site-related idiotope. Induction of SFV-neutralizing antibodies is genetically restricted. Rabbit anti-anti-idiotypic sera against 1.13A321 and 1.13A112 contained no SFV-neutralizing activity. Moreover, in DBA/2, C57BL/6J, CAL-20, and CB-20 mice 1.13A321 did not develop SFV-neutralizing ab3 antibodies in contrast to BALB.K, 129, SWISS, and BAB-14 mice. CAL-20, CB-20, and BAB-14 mice are congenic strains with an inbred background of BALB/c. CB-20 mice derived both IgCH and IgVH from donor strain C57BL/Ka, while BAB-14 mice derived IgCH from C57BL/Ka mice but retained IgVH from BALB/c mice. Clearly, induction of SFV-neutralizing antibodies by 1.13A321 in BAB-14 mice is dependent on IgVH of BALB/c origin. The results suggest that 1.13A321 binds to a paratope-associated recurring idiotope and almost certainly does not bear the internal image of the discontinuous neutralization epitope recognized by mAb UM 1.13. The latter suggestion is sustained by the observation that 1.13A112 and 1.13A321 do not bind to cell receptors.

Noise correlation can easily occur in the densely connected systems observed in biological information processing. We study the consequences of noise correlation for a statistically optimal processing of noise-perturbed receptor array outputs. We find a critical importance of the noise correlation length as compared to the receptors' tuning width for both the structure and the performance of the ideal observer. We show the general consistency of our scheme with psychophysical discrimination thresholds obtained in human spatial vision.

Rabbits were immunized with immunotype L3,7,9 phosphoethanolamine (PEA) group containing oligosaccharide-tetanus toxoid conjugates both with and without the addition of the adjuvant Quil A. The epitope specificity of the antibodies present in these antisera was analysed in an immunotype L2 and L3,7,9 specific inhibition ELISA using the homologous and heterologous lipopolysaccharide, oligosaccharide and partial dephosphorylated oligosaccharide as inhibitors. Two groups of antisera could be identified. In one group of antisera, at least two antibody populations are present, namely directed against the PEA group containing determinants on immunotype L3,7,9 lipopolysaccharide and against immunotype L2 specific epitopes in which no PEA group is present. In the second group of antisera, one but probably more antibody populations are detected with a similar specificity towards the conserved epitopes of both immunotypes. In general, immunization with the conjugates only resulted in the induction of antibodies against the PEA group containing epitopes on the L3,7,9 lipopolysaccharide (80%). Antibodies directed against the conserved epitopes of both immunotypes are mainly evoked with the conjugates in combination with the adjuvant Quil A (80%). Although these results suggest that the epitope specificity of the antibodies induced depends on the use of Quil A, the influence of genetic factors cannot be excluded. At the moment it is not known whether the differences in epitope specificities are reflected in biological function of these antibodies. However, the induction of antibodies with clearly different epitope specificities after immunization of different rabbits with the same antigen stresses the importance of this kind of analysis when developing a vaccine based on oligosaccharide-protein conjugates.

This project investigated the effects of novel carriers and adjuvants on the isotype of murine immunoglobulin G (IgG) antibody to pneumococcal capsular polysaccharide type 14 (S14PS). S14PS conjugated to bovine serum albumin induced a weak antibody response which was 100% IgG1 following injection without adjuvant. The same polysaccharide conjugated to flagella of Salmonella typhi induced an antibody response which was 88% IgG3. S14PS-bovine serum albumin was injected with block copolymer L121 or Quil A in squalane-in-water emulsions. The copolymer L121 was at least as effective as Quil A or complete Freund adjuvant in inducing IgG antibodies. IgG1 was the dominant subclass for all. Addition of monophosphoryl lipid A, but not the threonyl derivative of muramyl dipeptide or nontoxic Rhodopseudomonas sphaeroides lipopolysaccharide, to copolymer L121 increased production of the IgG2a, IgG2b, and IgG3 subclasses. S14PS-flagella with copolymer L121 induced higher titers with a markedly altered isotype distribution: 13% IgG1, 52% IgG2a, 6% IgG2b, and 29% IgG3. Monophosphoryl lipid A added to L121 reduced IgG1 antibody to 5%, but increased IgG2a antibody to 14%, IgG2b antibody to 3%, and IgG3 antibody to 78%. These studies demonstrate that both the carrier and the adjuvant can influence the titer and isotype distribution of antipolysaccharide antibody responses.

Rabbit anti-idiotypic sera were prepared against Semliki Forest virus (SFV) neutralizing monoclonal antibody (MAb) UM 1.13 and mumps virus neutralizing MAb UM 10B. From these sera anti-idiotypic antibodies were purified by ammonium sulphate precipitation and subsequent affinity column chromatography. Anti-iso- and anti-allotypic antibodies were removed by binding to normal mouse serum immunoglobulins coupled to CNBr activated Sepharose. Peak protein fractions eluted from columns loaded with homologous MAb were used for anti-anti-idiotypic immunization of BALB/c mice to raise virus neutralizing anti-anti-idiotypic antibodies. Two intracutaneous immunizations, five weeks apart, with affinity purified rabbit polyclonal anti-idiotypic antibody (40 micrograms protein per animal) coupled to keyhole limpet hemocyanin and mixed with the adjuvant Quil A (50 microliters per animal) were sufficient to evoke neutralizing antibodies against either virus. Moreover the mice who developed SFV neutralizing serum antibodies upon anti-idiotypic immunization all survived an otherwise lethal challenge with virulent SFV.

We present an ambiguous motion paradigm that allows us to quantify the influence of aspects of form relevant to the perception of apparent motion. We report on the role of bar element orientation in motion paths. The effect of orientation differences between bar elements in a motion path is small with respect to the crucial role of the orientation of bar elements relative to motion direction. Motion perception between elements oriented along the motion direction dominates motion perception between elements oriented perpendicularly to motion direction. The perception of apparent motion is affected by bar length and width and is anisotropic.

A Streptococcus pneumoniae type 14-specific ELISA and ELISPOT assay have been developed based on the use of biotinylated type 14 capsular polysaccharide (S14PS-biotin). A major advantage of this application over other methods is the use of 10-100-fold less antigen than that reported in the literature for other similar assays. Moreover, the prepared biotinylated polysaccharides are very stable and it is possible to use the same procedures for other pneumococcal polysaccharide antigens (e.g., S6BPS) with no major changes necessary in the ELISA and ELISPOT protocols. Furthermore, a simple thin layer chromatography method has been developed as a method for quality control of the biotinylated polysaccharide. Immunization with the thymus-independent antigen S14PS resulted in the induction of IgM spot-forming cells (SFC) and antibodies while S14PS-protein conjugates induced a thymus-dependent response. The immune response to the conjugates was enhanced by the addition of the adjuvant Quil A resulting in high levels of both IgG SFC and antibodies at day 14 after immunization. The developed assays are reliable and reproducible tools for studying the humoral immune response against Streptococcus pneumoniae type 14 capsular polysaccharide derived antigens.

Monoclonal antibodies (MA) UM 21.1 and UM 21.2 protect mice against encephalomyocarditis virus (D-variant) induced diabetes and lethal disease. MA-mediated protection in vivo as well as neutralization in vitro could be specifically blocked by anti-idiotypic antibodies.

The effects of two synthetic adjuvants on the antibody response to sheep red blood cells (SRBC) as a thymus dependent (TD) antigen and to dinitrophenyl59-Ficoll as a thymus-independent (TI-1) antigen were investigated in mice. Both dimethyldioctadecylammonium bromide (DDA) and dextran sulfate (DXS) augmented the humoral response to SRBC but not to dinitrophenyl59-Ficoll if injected simultaneously with antigen. Dose-response curves of both antigen and adjuvant revealed that DXS compared to DDA is a more effective adjuvant for the induction of a humoral response to SRBC. Intraperitoneal injection of DDA or DXS evoked a sequence of distinct immune responsive states in mice, measured by the capacity to develop an anti-SRBC response. A short immune-potentiating period (less than 6 hr) is followed by a suppressive, second immune-potentiating state. The immune suppressive state lasted for a period of about 8 days and was restricted to TD-antigens. Suppression could be totally overridden by injection of DDA or DXS simultaneously with antigen, suggesting that the suppressive state was reversible. The kinetics of the observed alteration of the immune response by DDA and DXS were very similar. It is concluded that differences in the modulation of the immune response by DDA and DXS are limited to the initial state. Long-term effects like the induction of a succession of distinct immune responsive states, are more or less similar for both adjuvants. Possible mechanisms by which these immunomodulators interfere with the immune system are discussed.

The effects of the adjuvant dimethyl dioctadecyl ammonium bromide (DDA) on the immune responses to encephalomyocarditis (EMC) virus were studied in mice. The humoral response, as measured by appearance of neutralizing antibodies, was slightly enhanced in mice immunized by the intraperitoneal route. Intracutaneously, DDA almost did not affect the humoral response but resulted in distinct enhancement of delayed type hypersensitivity (DH), as measured by the footpad swelling test. DH to EMC virus was found to be antigen-specific and could be passively transferred to normal mice with peritoneal exudate cells from immunized mice. Dose-response curves for DH and humoral antibody responses to EMC virus were not concordant. Low doses induced DH on day 6 without measurable circulating antibodies; high doses gave good antibody responses but suboptimal DH reactions. Immunization conferred a state of resistance to infection with virulent EMC virus. Protection seemed more related to DH than to the prevalence of specific antibodies at the time of infection.

Intracutaneous immunization of BALB/c mice with inactivated Semliki Forest virus mixed with the cationic, surface active lipid dimethyl dioctadecyl ammoniumbromide produced a strong enhancement of delayed type hypersensitivity without detectable antibodies in serum. Furthermore these mice were more protected against intraperitoneal challenge than normal immunized mice. This protection might be explained by an enhancement of cellular immunity against Semliki Forest virus. Dimethyl dioctadecyl ammoniumbromide by itself has no effect on footpad swelling, mortality and mean survival time.

Mumps neutralizing monoclonal antibodies (MAs) were purified and labelled with horseradish peroxidase and used to detect virus-infected Vero cells, which were seeded as monolayers in wells of 96-well plates. This direct enzyme immunoassay (EIA) in cell culture proved to be a sensitive method for detection and titration of mumps virus and it may be useful for diagnostic purposes. The EIA is also suitable for the rapid determination of neutralizing antibodies. Neutralization of mumps virus by preincubation with either monoclonal or polyclonal antibodies was indicated by inhibition of the absorbance at 450 nm as measured with a multichannelled photometer. The EIA (duration 2 days) for determination of mumps neutralizing antibodies is an attractive alternative for the plaque reduction test (duration 6 days).

CBA/N mice harbor an X-linked B cell defect which is transmitted by CBA/N female mice to their hybrid male progeny. Hapten-specific plaque-forming cell (PFC) responses to haptenated proteins by B cell-defective CBA/N mice and CBA/N X C3H/HeN F1 male mice can be blockaded by concomitant exposure to polysaccharide agents bearing the same hapten. Various experimental approaches were explored in an attempt to study this phenomenon in a syngeneic cell transfer system. Unprimed donor spleen cells were unable to mount adequate PFC responses to dinitrophenylated-hemocyanin (DNP-KLH) in irradiated, syngeneic recipients. DNP-KLH-primed donor spleen cells produced strong PFC responses after challenge in irradiated recipients, and these secondary responses were subject to hapten-specific blockade by DNP-derivatized polysaccharide agents. Both direct and indirect PFC responses could be blocked. DNP conjugated to bovine serum albumin also produced hapten-specific blockade in this cell transfer system. Under some cell transfer conditions normal CBA/N X C3H/HeN F1 female spleen cells were just as susceptible to hapten-specific blockade as defective F1 male spleen cells.

Differential spatial displacement discrimination thresholds were determined for stimuli consisting of blobs with Gaussian spatial and temporal contrast envelopes. The stimuli were presented at detection threshold luminance contrast. The tasks were similar to the two-point discrimination acuity task and the three-dot alignment hyperacuity task. Thresholds were determined as a function of eccentricity along the horizontal meridian of the visual field (from 45 degrees nasal to 65 degrees temporal). The spatial spread or blur parameter of the blobs was adopted as a scale parameter. The results show that the performance of the visual system in differential spatial displacement discrimination tasks becomes progressively more homogeneous for a progressive increase in the blur parameter of the stimuli. Scaling (i) the three-blob alignment results with estimates of the cortical magnification factor and (ii) the two-blob separation discrimination results with their corresponding neural blur parameter shows an impressive isotropy and blur scale-invariance for the mechanisms mediating differential spatial displacement discrimination across the visual field. These results are interpreted in terms of a scaled sampling lattice model of the visual system, in combination with an automatic scale-selection mechanism.

The reliability of the determination of antibody avidity in polyclonal sera by indirect sandwich ELISA was studied. Binding of IgM and IgG (sub)classes in unpurified serum to Streptococcus pneumoniae type 3 capsular polysaccharide, which was coated onto ELISA plates, was inhibited with different inhibitors. The inhibitor concn at which 50% inhibition of antibody binding to the ELISA coat was achieved, was used as a measure for antibody avidity. As this 50% inhibition value is dependent upon the dilution of the serum and thus upon the initial amount of free antibody, it is necessary to define (a narrow range of) final ELISA absorbance values to which the dilutions of non-inhibited sera have to be adjusted. The shapes of the serum dilution curves have a good correlation with the numerical 50% inhibition values of the antibody avidity. The inhibition ELISA is suitable to compare the avidity values of the different antibody isotypes, but two remarks should be made: (1) antibody heterogeneity should be considered to influence the results and prevent the accurate measurement of absolute numerical avidity values. Because in the ELISA system merely antibody "activity" is measured, comparison of the efficacy of vaccines by means of the 50% inhibition (avidity) value of various antibody (sub)classes can still be performed in a reliable way; (2) results of the determination of the 50% inhibition values of the different antibody (sub)classes showed them to be dependent on the molecular ratio between antibody (sub)class levels. More aspects of the determination should be taken into account, like shapes of simple dilution curves, influences of various inhibitor concns in the diluent and whole (extended) inhibition curves.

Synergistic effects of two synthetic adjuvants, dimethyldioctadecylammonium bromide (DDA) and dextran sulfate (DXS) on the humoral response to sheep red blood cells (SRBC) were investigated. Mice received intraperitoneal (ip) injections of adjuvant and antigen simultaneously. The number of plaque-forming cells (PFC) in the spleen were determined 5 days later and circulating anti-SRBC antibodies were measured till 16 weeks after immunization. Although combinations of DDA and DXS were very effective in enhancing the PFC response to both moderate (2 X 10(7] and low (2 X 10(6] doses of SRBC, synergy between the adjuvants was only observed at the low dose of SRBC. Optimal augmentation of the primary response to the low antigen dose was evoked by the combination of the highest dose tested of either adjuvant (1 mumol DDA and 1 nmol DXS) resulting in a 560-fold increase of the number of PFC in the spleen as compared to controls. Even combinations of relatively small amounts of both adjuvants were very effective in augmenting the response to SRBC. Mice receiving half the amounts of both adjuvants with 2 X 10(6) SRBC displayed increased numbers of PFC in the spleen at Day 5 as well as increased titers of total anti-SRBC antibodies at Week 1 and Week 2 and 2-mercaptoethanol-resistant antibodies from Week 4 till Week 16 as compared to the calculated sum of responses in mice which received either DDA (0.05 mumol per mouse) or DXS (0.05 nmol per mouse). The mechanism behind the synergy between these adjuvants is discussed and the possibility of discerning adjuvants on their modes of action is suggested.

This paper describes the minimal requirements to protect mice adoptively against challenge with virulent Semliki Forest virus (SFV). Early immune serum, from donor mice infected with an avirulent strain of SFV, contained mainly neutralizing IgM immunoglobulins. More of these antibodies (1.80 PND50 vs 0.06 PND50) were needed to protect recipient mice against intraperitoneal challenge (10 LD50 of SFV) than against subcutaneous challenge (7 LD50). Adoptive transfer experiments indicated that a minimum of 3 X 10(7) six-day immune spleen cells were also able to protect recipient mice against intraperitoneal challenge with 10 LD50 of SFV. Treatment of these donor cells with cytotoxic antisera and complement revealed both T- and B-lymphocytes were required for optimum adoptive immunity. Surviving recipients of either immune serum or immune spleen cells developed significantly less neutralizing antibodies than control mice. The lower antibody titres in protected mice might be related to either immune serum or immune spleen cell mediated restriction of virus replication; meaning a reduced antigenic stimulus in these mice compared to control mice.

This paper describes the immunogenicity of haptenated liposomes in a number of inbred mouse strains. The tripeptide-enlarged hapten, dinitrophenyl-beta-alanylglycylglycine (J) was conjugated to phosphatidyl-ethanolamine (PE) and incorporated into liposomal model membranes. The magnitude of the response to this thymus-independent (TI) antigen was measured by the appearance of direct plaque-forming cells in the spleen. The response to J-PE-liposomes in the different mouse strains varied sufficiently to suggest the existence of high, intermediate and low responders. On the basis of this preliminary survey no correlation was found between the immunogenicity of J-PE-liposomes and H-2 haplotype. The positive responses obtained in CBA/N mice appear to classify J-PE-liposomes as a TI-1 antigen. Further characterization of this response was carried out in CBA/N X C3H/HeN F1 male and female mice. The magnitude of the response was dependent on dose and epitope density of the hapten-PE derivative. The response could be blocked in F1 male mice by prior immunization with the TI-2 antigen J53-Ficoll. This suggests that J-PE-liposomes have no intrinsic mitogenic properties. Non-haptenated liposomes could not be used as an adjuvant to restore the response in F1 male mice to haptenated Ficoll (TI-2) preparations.

Virus infected monolayers, in wells of 96-well plates, could be used as antigen in competition binding assays to identify epitopes on respectively Semliki Forest virus, encephalomyocarditis virus and mumps virus.

CBA/N mice have an X-linked B cell defect which prevents them from responding to nonmitogenic thymic independent (TI-2) antigens such as dinitrophenylated DNP-Ficoll (1,2). The F1 male progeny of CBA/N female mice express the same defect. Spleen cell suspensions from such defective mice (CBA/N X C3H/HeN F1 males) could not respond to DNP-Ficoll following in vitro immunization and subsequent transfer into irradiated, syngeneic, F1 male recipients as expected. In contrast, normal CBA/N X C3H/HeN F1 female spleen cells could respond and effect a "rescue"; they mounted strong plaque-forming cell responses 7 days after in vitro exposure to DNP-Ficoll and subsequent transfer into irradiated F1 male recipients. Defective F1 male spleen cells, however, could bind significant quantities of 125I-DNP-Ficoll after in vitro exposure. Extensive washing of these spleen cells could not reverse this binding. Such DNP-Ficoll-exposed and washed F1 male spleen cells could, after transfer, aid normal untreated F1 female cells in their rescue function. The defective F1 male spleen cells could convey immunogenic quantities of DNP-Ficoll to the "rescuing" F1 female cells. Mitomycin treatment of F1 male cells did not interfere with their conveyor function. Goat anti-mouse mu serum impeded the passive antigen conveyor function of defective F1 male cells as did prior exposure to high concentrations of free DNP hapten. Our data support the view that the B cell defect of CBA/N X C3H/HeN F1 male mice does not relate to antigen binding, but rather to an inability to be effectively triggered by certain cell-bound polymeric antigens.

The capacity of immunoadjuvants to enhance serum amyloid P component (SAP) levels and to modulate the humoral immune response to sheep red blood cells in a number of different mouse strains was investigated. Although the synthetic adjuvants dimethyldioctadecylammonium bromide, dextran sulphate and bacterial-derived lipopolysaccharide did not enhance SAP levels in some of the mouse strains tested, these strains responded normally to the immunomodulating effects of the adjuvants. We conclude that increased SAP levels and modulation of immune responses are induced via at least partially different pathways. For these reasons, it is impossible to screen drugs for potential adjuvant activity by only measuring SAP levels in mice.

The candidacidal activity (CA) of peritoneal cells (PC) in vitro was used as a measure of nonspecific microbicidal activity of phagocytes after intraperitoneal injection of mice with different adjuvants. Dilutions of PC were incubated with constant numbers of C. parapsilosis in a 96-well culture plate. The PC number causing 50% reduction of yeast colonies formed after 48 hr at 37 degrees C was called 1 CA50 unit. CA was expressed in CA50 units per 10(6) PC. Optimal reduction of the number of viable candida cells in vitro was established within 1.5 hr while 50% reduction was reached after 0.5 hr. In this test CA was, within limits, independent of the number of viable candida cells added per well (22 to 152 yeast cells), of the concentration of fetal calf serum (1-20%) and of the presence of heat-labile serum components. The CA of PC of individual mice was measured 6, 24, and 96 hr after injection of an adjuvant. In most instances optimal CA was observed 6 hr after administration of adjuvant and varied from 3.7 (methylamine) to 50 (Corynebacterium parvum strain 4982) units. With respect to the titer and duration of CA, the adjuvants were arranged in the following order of increasing efficacy: methylamine, heparin, polyol L 121, suramin, dextran sulfate, polyol L 101, dimethyldioctadecylammonium bromide, Liquoid, heat-killed Listeria monocytogenes, formalin-killed C. parvum strain 10387, and strain 4982. The CA induced by the latter strain persisted at least till 96 hr after injection. The induction of CA was accompanied by recruitment of polymorphonuclear cells. The contribution of distinct phagocytic effector cells to CA and the correlation between modulation of the specific and nonspecific immunity are discussed.

Regulation of the immune response by macrophages was studied with cellular resistance to Listeria monocytogenes as parameter. The use of agents which suppress macrophage activity during the induction-phase of immunity enabled the induction of protective immunity with killed listeria. Fractionation of the cell content of listeria yielded an RNA'se sensitive fraction which in a dose of 300 ng and in combination with the cationic surfactant dimethyl dioctadecyl ammonium bromide induced protective immunity against listeria.

After subcutaneous infection of mice with Semliki Forest virus, a delayed-type hypersensitivity (DH) could be demonstrated by footpad swelling. Pretreatment with cyclophosphamide resulted in enhanced DH if neutralizing antibodies were undetectable in serum.

Eleven monoclonal antibodies (MAs) reacted strongly in an enzyme immunoassay with virulent Semliki Forest virus (SFV) replicating in L cell monolayers. Three MAs showed a considerably diminished reaction with an avirulent strain of SFV both in enzyme immunoassays and plaque reduction tests.

Liposomes haptenated with tripeptide-enlarged dinitrophenyl (DNP) are known to act as thymus-independent antigens which induce a strong IgM response and only limited amounts of circulating IgG. When haptenated liposomes are used in vaccination studies, it is of practical importance to improve the immunogenicity of these complexes. Therefore, an evaluation was made of the potency of various substances to modulate the immune response in such a way that the total antibody production is increased, including a relative great increase of 2-mercaptoethanol (2-ME)-resistant antibodies and immunological memory is induced. The following substances were used: glycophorin A (GP-A), sialogangliosides (monosialo-, disialo- and trisialoganglioside), 6-0-stearoyl-MDP (MDP-SA) and lipid A (lip A). Lip A incorporated into liposomes was the only substance inducing considerable increases of both total and 2-ME-resistant haemagglutination (HA) titre after immunization. Depending on the dose tested, the sialic-acid-containing protein GP-A had a small and varying influence on the serum antibody response. Sialogangliosides transiently decreased in a dose-dependent manner the total antibody titre in serum. In contrast to lip A, the lipophilic bacterial adjuvant MDP-SA did not influence HA titres significantly. The number of plaque-forming cells (PFC) in the spleen was enhanced considerably after both primary and secondary immunization with liposomes containing lip A. The other substances tested induced only minor differences of the number of PFC. To some extent, lip A induced immunological memory. In conclusion, it can be stated that of the agents tested, only lip A is a potent and consistent stimulator of the humoral immune response to liposomes haptenated with DNP groups.(ABSTRACT TRUNCATED AT 250 WORDS)

A sensitive ELISA has been developed to study immune responses in mice against Streptococcus pneumoniae type 3 capsular polysaccharide (S3PS) and hexasaccharide (HS)-protein conjugates derived therefrom. An advantage of the described system is that the same microtiter plates can be used for both ELISA and ELISPOT tests with a standardized washing procedure and diluent composition. S3PS induced predominantly IgM antibodies and minute amounts of IgG as measured by ELISA in serum. This was accompanied by large numbers (greater than 14000) of IgM spot-forming cells in the spleen. A shift towards IgG production was achieved by addition of lipid A. HS-protein conjugates induced predominantly IgG antibodies after booster immunization(s). Furthermore these conjugates induced large numbers (greater than 40000) of IgG spot-forming cells (SFC) in the spleen. ELISA and ELISPOT assays on microtiter plates are both reliable and highly reproducible assays for the evaluation of immune responses to S. pneumoniae antigens.

The two highly competitive, monoclonal antibodies (MAs), UM 5.1 and UM 8.1, recognize on the E2 glycoprotein of Semliki Forest virus different determinants as indicated by the absence of cross-reactive anti-idiotypic antibodies in rabbit immune sera induced against these highly neutralizing MAs.

The effect of the adjuvant dimethyl dioctadecyl ammonium bromide (DDA) on the induction of cellular immunity in guinea pigs was studied. DDA, a surface-active lipid, was mixed with the antigen bovine serum albumin (BSA) or with a conjugate of BSA and dinitrophenol (DNP22--BSA) and injected into the footpads of guinea pigs. At varying intervals skin tests were performed to test the immediate and delayed hypersensitivity (DH) reactions. Optimal DH reactions to BSA were observed from 3 to 6 weeks after immunization with BSA in DDA. The hapten-specific response to DNP22--BSA had an optimum at 3 weeks and was highly specific for the homologous antigen. Histological examinations of skin test sites confirmed that the reaction was rather of the tuberculin type than of the cutaneous basophil hypersensitivity type. When guinea pigs were immunized with DNP22--BSA in Freund's complete adjuvant (FCA) a long-lasting DH to both carrier and hapten groups developed but the DH was always complicated by an Arthus reaction due to antibodies to the DNP hapten. In conclusion, DDA is superior to FCA as adjuvant for the induction of a state of pure DH in guinea pigs.

Modulation of delayed-type hypersensitivity reaction (DTH) in mice by synthetic adjuvants and the mode of their action were investigated. Intracutaneous injection of azobenzenearsonate coupled to phosphatidylethanolamine (A-PE) without adjuvant did not induce DTH. Administration of A-PE with the quaternary amines dimethyldioctadecylammonium bromide (DDA) or N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)propane diamine (CP-20,961) induced a strong response. Other surfactants, dextran sulfate (DXS) and dextran were not effective. In combination with 200 nmol DDA the optimal dose of antigen was 5 nmol A-PE, while at higher antigen doses DTH was diminished. Responses on combination of two adjuvants and A-PE revealed that DXS counteracted the stimulatory effects of both DDA and CP-20,961. In vitro, DDA formed insoluble complexes with 14C-A-PE and at optimal antigen concentration more than 90% of the antigen was bound to the adjuvant. The percentage of 14C-A-PE bound to 200 nmol DDA decreased with increasing doses of 14C-A-PE. Addition of DXS to the mixture of 14C-A-PE and DDA reduced the percentage of 14C-A-PE bound to DDA. Dose-response curves demonstrated a close relationship between the inhibitory effects of DXS on the DTH and the A-PE/DDA complex formation. Nonsulfated dextran affected neither the DTH nor the formation of complexes in vitro. In conclusion, cellular adjuvanticity of DDA for the lipophilic antigen A-PE is probably the result of formation of insoluble complexes with the antigen. Free A-PE suppresses the cellular response to A-PE/DDA complexes. The adjuvant DXS inhibits DTH by reducing the amount of immunogenic A-PE/DDA complexes and thus increasing the amount of free, immunosuppressive A-PE.

Multiplication of encephalomyocarditis virus (EMCV) in human HEp-2 cells, and its suppression by interferon (IFN), was demonstrated by direct enzyme immunoassay (EIA) in cell culture. EMCV was detected in glutaraldehyde fixed HEp-2 cell monolayers, in wells of 96-well plates, with a horse radish peroxidase (HRPO) labelled EMCV specific monoclonal antibody. Multiplication of EMCV (multiplicity of infection: 50) was indicated by a steep rise of absorbance values measured against infected monolayers starting as early as 5 h after infection and reaching relatively high values at 6 and 7 h. The rise in absorbance values did not occur after preincubation of the HEp-2 cells with either Newcastle disease virus-induced IFN, recombinant gamma IFN or recombinant alfa-2a IFN. Absorbance values were inversely dependent on the amount of IFN used. Therefore the EIA was suitable for rapid titration of IFN. The titres of recombinant gamma and alfa-2a IFN determined with EIA proved to be similar to those given by the manufacturers. The described bioassay of human IFN is objective, rapid and easy to perform and suitable for large scale experiments.

Mapping of T-cell epitopes on the structural proteins of Semliki Forest virus (SFV) was performed by measuring the ability of cloned SFV protein fragments to induce delayed-type hypersensitivity (DTH). The cloned SFV protein fragments were expressed as hybrid proteins with cro-beta-galactosidase in Escherichia coli from constructed recombinant plasmids. DTH reactions were measured, as footpad swelling, in BALB/c mice after immunization with whole, UV-inactivated SFV and challenge with the hybrid proteins, and vice versa, using the adjuvant dimethyl dioctadecyl ammonium bromide to enhance DTH. Only two of the tested hybrid proteins induced DTH, and these DTH reactions were equally strong. The largest DTH-inducing hybrid protein contained the N-terminal 350 amino acids of E2 and part of E3, the smallest contained only the region from amino acid residues 115 to 151 of the E2 membrane protein without any other SFV protein parts. It was concluded that the segment between amino acid residues 115 and 151 of the E2 membrane protein of SFV was responsible for the observed DTH, and thus, contains a T-cell epitope. Sequence homology with known T-cell epitopes on other proteins makes it likely that the DTH-inducing T-cell epitope is located from amino acid residues 120 to 128 of E2.