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S Buus

Publications and source records attributed to S Buus.

At least 37 records · Page 2Linked to original sources
PubMed2000

Modeling the interactions of a peptide-major histocompatibility class I ligand with its receptors. II. Cross-reaction between a monoclonal antibody and two alpha beta T cell receptors.

The recombinant antibody, pSAN13.4.1, has a unique T cell like specificity; it binds an Influenza Hemagglutinin octapeptide (Ha255-262) in an MHC (H-2Kk)-restricted manner, and a detailed comparison of the fine specificity of pSAN13.4.1 with the fine specificity of two Ha255-262-specific, H-2Kk-restricted T cell hybridomas has supported this contention. A three-dimensional model of pSAN13.4.1 has been derived by homology modeling techniques. Subsequently, the structure of the pSAN13.4.1 antibody in complex with the antigenic Ha-Kk ligand was derived after a flexible and automated docking of the MHC-peptide pair into the Fab combining site. Interestingly, the most energetically favored binding mode shows numerous analogies to the recently determined recognition of class I MHC-peptide complexes by alpha beta T cell receptors (TCRs). The pSAN13.4.1 also binds diagonally across the MHC binding groove but is more deeply anchored to the peptide-MHC (pep/MHC) ligand than TCRs, notably through numerous interactions of its heavy chain. The present model accounts well for the experimentally determined binding affinity of a set of 144 single amino acid substituted Ha analogues and the observed shared specificity between the pSAN antibody and two different T cell receptors for the Ha-Kk antigenic ligand. Analogies and differences between Fab and TCR recognition are explained by dissecting the binding role of each chain of the immune receptors as well as the contribution of all peptide amino acids.

Amino Acid Sequence↗
PubMed2000

Modeling the interactions of a peptide-major histocompatibility class I ligand with its receptors. I. Recognition by two alpha beta T cell receptors.

A three-dimensional model of the complex between an Influenza Hemagglutinin peptide, Ha255-262, and its restricting element, the mouse major histocompatibility complex (MHC) class I molecule, Kk, was built by homology modeling and subsequently refined by simulated annealing and restrained molecular dynamics. Next, three-dimensional models of two different T cell receptors (TCRs) both specific for the Ha255-262/Kk complex were generated based on previously published TCR X-ray structures. Finally, guided by the recently published X-ray structures of ternary TCR/peptide/MHC-I complexes, the TCR models were successfully docked into the Ha255-262/Kk model. We have previously used a systematic and exhaustive panel of 144 single amino acid substituted analogs to analyze both MHC binding and T cell recognition of the parental viral peptide. This large body of experimental data was used to evaluate the models. They were found to account well for the experimentally obtained data, lending considerable support to the proposed models and suggesting a universal docking mode for alpha beta TCRs to MHC-peptide complexes. Such models may also be useful in guiding future rational experimentation.

Amino Acid Sequence↗
PubMed2000

Level discrimination of sinusoids as a function of duration and level for fixed-level, roving-level, and across-frequency conditions.

The ability of listeners to detect level differences between two sinusoidal stimuli in a two-interval forced-choice procedure was measured as a function of duration and level in three conditions: (1) the pedestal was fixed in level and the stimuli in the two intervals had the same frequency of either 1 or 2 kHz (fixed-level condition); (2) the pedestal was roved in level over a 20-dB range from trial to trial, but the stimuli still had the same frequency of either 1 or 2 kHz (roving-level condition); and (3) the pedestal was roved in level over a 20-dB range and the two stimuli differed in frequency, such that one was around 1 kHz while the other was around 2 kHz (across-frequency condition). In the fixed-level conditions, difference limens decreased (improved) with both increasing duration and level, as found in previous studies. In the roving-level conditions, difference limens increased and the dependence on duration and level decreased. Difference limens in the across-frequency conditions were generally highest and showed very little dependence on either stimulus duration or level. The results may be understood in terms of different internal noise components with additive variances: In the fixed-level conditions, sensation noise, which is dependent on stimulus attributes such as duration and level, is dominant. In more difficult conditions, where trace-memory and/or across-channel comparisons are required, a more central, stimulus-independent noise dominates.

Adult↗
PubMed2000

Efficient across-frequency integration: evidence from psychometric functions.

Across-frequency integration of complex signals was investigated by measuring psychometric functions [log (d') versus signal level in dB SPL] for detection of brief and long signals presented in broadband noise. The signals were tones at 630, 1600, and 4000 Hz, and a nine-tone complex with components spaced at one-third-octave frequencies between 630 and 4000 Hz. The phase relationship of the components in the complex was varied such that adjacent components were in phase (at 0 degrees), 90, or 180 degrees out of phase. Signal durations (defined in terms of the number of cycles between the half-amplitude points of the Gaussian envelopes) of 4.7 and 150 cycles were tested. Results for six normal-hearing listeners showed that the slopes of the psychometric functions were steeper for the brief than for the long signals, and steeper for the tone complexes than for the tones, particularly for the brief signals. This suggests that the transformation from signal intensity to decision variable may be different for brief complex signals than for tonal signals and long complex signals. Thresholds obtained from the psychometric functions were in excellent agreement with those obtained with an adaptive procedure that employed three interleaved tracks. For the long signals, the threshold improvement for the tone complexes relative to a single tone was well described by a 5* log (n) integration rule. However, the threshold improvement for brief signals obeyed a more efficient integration rule of 7 to 8* log (n). A portion of this effect could be accounted for by the phase relationship of the tone complexes; thresholds for brief signals were lowest when the components were in phase at the envelope peak of the signal. This finding indicates that temporal synchrony across auditory channels may enhance detection of brief multi-tone complexes.

Adult↗
PubMed2000

Identifying cytotoxic T cell epitopes from genomic and proteomic information: "The human MHC project.".

Complete genomes of many species including pathogenic microorganisms are rapidly becoming available and with them the encoded proteins, or proteomes. Proteomes are extremely diverse and constitute unique imprints of the originating organisms allowing positive identification and accurate discrimination, even at the peptide level. It is not surprising that peptides are key targets of the immune system. It follows that proteomes can be translated into immunogens once it is known how the immune system generates and handles peptides. Recent advances have identified many of the basic principles involved. The single most selective event is that of peptide binding to MHC, making it particularly important to establish accurate descriptions and predictions of peptide binding for the most common MHC variants. These predictions should be integrated with those of other steps involved in antigen processing, as these become available. The ability to translate the accumulating primary sequence databases in terms of immune recognition should enable scientists and clinicians to analyze any protein of interest for the presence of potentially immunogenic epitopes. The computational tools to scan entire proteomes should also be developed, as this would enable a rational approach to vaccine development and immunotherapy. Thus, candidate vaccine epitopes might be predicted from the various microbial genome projects, tumor vaccine candidates from mRNA expression profiling of tumors ("transcriptomes") and auto-antigens from the human genome.

Antigen Presentation↗
PubMed1999

Induction of cytotoxic T-cell responses by gene gun DNA vaccination with minigenes encoding influenza A virus HA and NP CTL-epitopes.

Cytotoxic T-lymphocyte (CTL) response is an important component of anti-viral immunity. CTLs are specific to short peptides presented by MHC-I molecules and immunisation with the exact peptide sequence introduced in the cytosol is therefore a minimal approach, which potentially affords a high degree of controllability. We have examined the induction of murine CTL's by this approach using DNA plasmid minigene vaccines encoding known mouse K(k) minimal CTL epitopes (8 amino acids) from the influenza A virus hemagglutinin and nucleoprotein. We here report that such an approach is feasible and that wild type influenza virus flanking amino acid sequences can influence the CTL response but are not essential for optimal CTL induction. We also examined the effect of different new amino acid sequences flanking the CTL epitopes. In one version, two CTL epitopes were linked together as 'string of beads'. This did not improve CTL induction. In another version, one CTL epitope was inserted into a known T-helper protein (HBsAg). This did significantly augment the response probably due to immunological help from HBsAg Th epitopes. Finally, the CTL inducing minigene DNA vaccines were compared with Flu-induced CTL responses and tested for their protective effect against a lethal influenza A virus infection in mice and no effect was found. We conclude that a specific and highly directed CTL induction is possible by unlinked minigene DNA immunisation, but that CTL induction solely is not always sufficient to provide protection.

Amino Acid Sequence↗
PubMed1999

Predicting binding affinities of protein ligands from three-dimensional models: application to peptide binding to class I major histocompatibility proteins.

A simple and fast free energy scoring function (Fresno) has been developed to predict the binding free energy of peptides to class I major histocompatibility (MHC) proteins. It differs from existing scoring functions mainly by the explicit treatment of ligand desolvation and of unfavorable protein-ligand contacts. Thus, it may be particularly useful in predicting binding affinities from three-dimensional models of protein-ligand complexes. The Fresno function was independently calibrated for two different training sets: (a) five HLA-A0201-peptide structures, which had been determined by X-ray crystallography, and (b) three-dimensional models of 37 H-2K(k)-peptide structures, which had been obtained by knowledge-based homology modeling. For both training sets, a good cross-validated fit to experimental binding free energies was obtained with predictive errors of 3-3.5 kJ/mol. As expected, lipophilic interactions were found to contribute the most to HLA-A0201-peptide interactions, whereas H-bonding predominates in H-2K(k) recognition. Both cross-validated models were afterward used to predict the binding affinity of a test set of 26 peptides to HLA-A0204 (an HLA allele closely related to HLA-A0201) and of a series of 16 peptides to H-2K(k). Predictions were more accurate for HLA-A2-binding peptides as the training set had been built from experimentally determined structures. The average error in predicting the binding free energy of the test peptides was 3.1 kJ/mol. For the homology model-derived equation, the average error in predicting the binding free energy of peptides to K(k) was significantly higher (5.4 kJ/mol) but still very acceptable. The present scoring function is thus able to predict with a good accuracy binding free energies from three-dimensional models, at the condition that the backbone coordinates of the MHC-bound peptide have first been determined with an accuracy of about 1-1.5 A. Furthermore, it may be easily recalibrated for any protein-ligand complex.

Crystallography, X-Ray↗
PubMed1999

Description and prediction of peptide-MHC binding: the 'human MHC project'.

MHC molecules are crucially involved in controlling the specific immune system. They are highly polymorphic receptors sampling peptides from the cellular environment and presenting these peptides for scrutiny by immune cells. Recent advances in combinatorial peptide chemistry have improved the description and prediction of peptide-MHC binding. It is envisioned that a complete mapping of human immune reactivities will be possible.

Binding Sites↗
PubMed1999

A single-chain fusion molecule consisting of peptide, major histocompatibility gene complex class I heavy chain and beta2-microglobulin can fold partially correctly, but binds peptide inefficiently.

The function of major histocompatibility complex class I (MHC-I) molecules is to sample peptides from the intracellular environment and present these peptides to CD8+ cytotoxic T lymphocytes (CTL). We have attempted to develop a general approach to produce large amounts of pure and active recombinant MHC-I molecules. A convenient source of MHC-I molecules would be a valuable tool in structural and biochemical analysis of MHC-I, and in experiments using MHC-I molecules to enable specific manipulations of experimental and physiological CTL responses. Here we describe the generation of a recombinant murine MHC-I molecule, which could be produced in large amounts in bacteria. The recombinant MHC-I protein was expressed as a single molecule (PepSc) consisting of the antigenic peptide linked to the MHC-I heavy chain and further linked to human beta2-microglobulin (hbeta2m). The PepSc molecule was denatured, extracted, purified and folded using a recently developed in vitro reiterative refolding strategy. This led to the formation of soluble, recombinant MHC-I molecules, which migrated as monomers of the expected size when submitted to non-reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Serological analysis revealed the presence of some, but not all, MHC-I-specific epitopes. Biochemically, PepSc could bind peptide, however, rather ineffectively. We suggest that a partially correctly refolded MHC-I has been obtained.

Antigen Presentation↗
PubMed1999

Temporal integration of loudness in listeners with hearing losses of primarily cochlear origin.

To investigate how hearing loss of primarily cochlear origin affects the loudness of brief tones, loudness matches between 5- and 200-ms tones were obtained as a function of level for 15 listeners with cochlear impairments and for seven age-matched controls. Three frequencies, usually 0.5, 1, and 4 kHz, were tested in each listener using a two-interval, two--alternative forced--choice (2I, 2AFC) paradigm with a roving-level, up-down adaptive procedure. Results for the normal listeners generally were consistent with published data [e.g., Florentine et al., J. Acoust Soc. Am. 99, 1633-1644 (1996)]. The amount of temporal integration--defined as the level difference between equally loud short and long tones--varied nonmonotonically with level and was largest at moderate levels. No consistent effect of frequency was apparent. The impaired listeners varied widely, but most showed a clear effect of level on the amount of temporal integration. Overall, their results appear consistent with expectations based on knowledge of the general properties of their loudness-growth functions and the equal-loudness-ratio hypothesis, which states that the loudness ratio between equal-SPL long and brief tones is the same at all SPLs. The impaired listeners' amounts of temporal integration at high SPLs often were larger than normal, although it was reduced near threshold. When evaluated at equal SLs, the amount of temporal integration well above threshold usually was in the low end of the normal range. Two listeners with abrupt high-frequency hearing losses (slopes > 50 dB/octave) showed larger-than-normal maximal amounts of temporal integration (40 to 50 dB). This finding is consistent with the shallow loudness functions predicted by our excitation-pattern model for impaired listeners [Florentine et al., in Modeling Sensorineural Hearing Loss, edited by W. Jesteadt (Erlbaum, Mahwah, NJ, 1997), pp. 187-198]. Loudness functions derived from impaired listeners' temporal-integration functions indicate that restoration of loudness in listeners with cochlear hearing loss usually will require the same gain whether the sound is short or long.

Adult↗
PubMed1999

Temporal integration and multiple looks, revisited: weights as a function of time.

This study tests the hypothesis that temporal integration for detection of tone bursts with various durations can be explained by optimally combining multiple looks of brief signal segments whose contribution to detection increases over time. Detectability was measured for signals consisting of six consecutive 25-ms, 1-kHz tone pulses presented in a 50-Hz-wide masker or in maskers consisting of seven 50-Hz-wide noises, one critical band apart, with either coherent or incoherent envelopes. The level of each signal pulse varied randomly around masked threshold according to a Gaussian distribution. The slopes of conditional psychometric functions--plotted in terms of d'2 as a function of the squared signal-pulse intensity for pulses in a particular temporal position--yielded estimates of the contribution to detection provided by each pulse. Results for three normal listeners showed a small, but significant, effect of the temporal location of the pulse. Multiple-looks predictions of temporal-integration functions based on the measured weights and on measured psychometric functions were compared to measured temporal-integration functions. For the single-band and incoherent maskers, the predicted temporal-integration slopes were reasonably consistent with those measured, but for the coherent masker it was not. Whereas no current theory can explain the very steep temporal-integration functions obtained in the coherent masker, the present results are not inconsistent with the multiple-looks hypothesis as an explanation for the decrease in threshold with increasing duration of signals presented in random maskers.

Adult↗
PubMed1999

Psychometric functions for gap detection in a yes-no procedure.

To examine models of temporal resolution and to investigate the decision processes underlying the detection of a brief pause in a bandpass noise, psychometric functions for gap detection were measured at octave frequencies from 0.25 to 8 kHz. Three normal listeners were tested using a constant-stimulus procedure with a cued Yes-No paradigm. The Minimum Detectable Gap (MDG) estimated from the midpoint of the psychometric functions decreased systematically with increasing frequency. The slopes of the psychometric functions generally increased as the test frequency increased up to 2 kHz, but remained constant at the higher frequencies. Two models were investigated: an energy-detector model and a loudness-detector model. Both consisted of auditory filtering, a nonlinearity, and short-term integration. In the energy-detector model, the nonlinearity was a square law. In the loudness-detector model, it was a compressive power law. Using the usual Gaussian approximations, the energy-detector model fails at low frequencies because the probability distributions of short-term energy differ from Gaussian distributions. The probability distributions of short-term loudness closely follow Gaussian distributions. The loudness-detector model predicts the frequency dependence of the MDG quite accurately, except at 0.25 kHz. It also predicts psychometric functions that resemble the data at low frequencies, but the predicted slopes increase much less with frequency than the measured slopes. This result may indicate that the onset response to the trailing marker of the gap provides an important cue for detection of gaps with durations exceeding the MDG.

Adult↗
PubMed1999

Complete protection against lethal Toxoplasma gondii infection in mice immunized with a plasmid encoding the SAG1 gene.

Infection with the protozoan parasite Toxoplasma gondii is transmitted to humans from infected animals by tissue cysts and oocysts excreted by cats. Immunization with inactivated parasites or recombinant proteins has at best shown partial protection. We constructed a plasmid expressing the SAG1 surface antigen of T. gondii, p1tPASAG1, and showed that animals immunized with the plasmid produce anti-SAG1 antibodies which recognize the native SAG1. Mice immunized with p1tPASAG1 showed 80 to 100% protection against challenge with the non-cyst-producing, virulent RH isolate, compared to an 80% mortality in mice immunized with empty plasmid, which is the greatest efficacy of any vaccine against T. gondii produced so far. The SAG1 molecule was analyzed for potential cytotoxic T-lymphocyte (CTL) epitopes, and four peptides with the best fit were synthesized. The ability of the peptides to stimulate gamma interferon production by CD8(+) T cells from p1tPASAG1-immunized mice was tested in an ELISPOT assay, and one new CTL epitope was identified. Adoptive transfer of CD8(+) T cells from p1tPASAG1-immunized to naïve mice showed partial protection. In conclusion, DNA vaccination with p1tPASAG1 gave effective protection in mice against T. gondii infection and the protection could be adoptively transferred by purified CD8(+) T cells.

Adoptive Transfer↗
PubMed1998

Improved humoral and cellular immune responses against the gp120 V3 loop of HIV-1 following genetic immunization with a chimeric DNA vaccine encoding the V3 inserted into the hepatitis B surface antigen.

The gp120-derived V3 loop of HIV-1 is involved in co-receptor interaction, it guides cell tropism, and contains an epitope for antibody neutralization. Thus, HIV-1 V3 is an attractive vaccine candidate. The V3 of the MN strain (MN V3) contains both B- and T-cell epitopes, including a known mouse H-2d-restricted cytotoxic T lymphocyte (CTL) epitope. In an attempt to improve the immunogenicity of V3 in DNA vaccines, a plasmid expressing MN V3 as a fusion protein with the highly immunogenic middle (pre-S2 + S) surface antigen of hepatitis B virus (HBsAg) was constructed. Epidermal inoculation by gene gun was used for genetic immunization in a mouse model. Antibody and CTL responses to MN V3 and HBsAg were measured and compared with the immune responses obtained after vaccination with plasmids encoding the complete HIV-1 MN gp160 and HBsAg (pre-S2 + S), respectively. DNA vaccination with the HIV MN gp160 envelope plasmid induced a slow and low titred anti-MN V3 antibody response at 12 weeks post-inoculation (p.i.) and a late appearing (7 weeks), weak and variable CTL response. In contrast, DNA vaccination with the HBsAg-encoding plasmid induced a rapid and high titred anti-HBsAg antibody response and a uniform strong anti-HBs CTL response already 1 week p.i. in all mice. DNA vaccination with the chimeric MN V3/HBsAg plasmid elicited humoral responses against both viruses within 3-6 weeks which peaked at 6-12 weeks and remained stable for at least 25 weeks. In addition, specific CTL responses were induced in all mice against both MN V3 and HBsAg already within the first 3 weeks, lasting at least 11 weeks. Thus, HBsAg acts as a 'genetic vaccine adjuvant' augmenting and accelerating the cellular and humoral immune response against the inserted MN V3 loop. Such chimeric HIV-HBsAg plasmid constructs may be useful in DNA immunizations as a 'carrier' of protein regions or minimal epitopes which are less exposed or poorly immunogenic.

AIDS Vaccines↗
PubMed1998

A peptide-binding assay for the disease-associated HLA-DQ8 molecule.

The study of peptide binding to HLA class II molecules has mostly concentrated on DR molecules. Since many autoimmune diseases show a primary association to particular DQ molecules rather than DR molecules, it is also important to study the peptide-binding properties of DQ molecules. Here we report a biochemical peptide-binding assay for the type I diabetes-associated DQ8, i.e. DQ (alpha1*0301, beta1*0302), molecule. Affinity-purified DQ8 molecules were tested in peptide-binding assays using a radiolabelled influenza haemagglutinin (Ha) peptide encompassing positions 255-271(Y) as an indicator peptide. The Ha 255-271(Y) peptide bound to DQ8 in a pH-dependent fashion showing optimal binding around pH 5. The association kinetics were relatively slow and the resulting complexes were heat labile. The specificity of peptide binding to DQ8 was investigated in competitive inhibition experiments with a panel of 43 peptides of different lengths and sequences. The DQ8 molecules showed a different pattern of peptide binding compared to a previously studied DQ2 molecule. Peptides derived from thyroid peroxidase, HLA-DQ(alpha1*0301), HLA-DQ(alpha1*0302), retinol receptor and p21ras were among the high-affinity binders, whereas peptides derived from myelin basic protein were among the low-affinity binders. The sequence of the high-affinity peptides conformed with a previously published peptide-binding motif of DQ8.

Amino Acid Sequence↗
PubMed1998

The P9 pocket of HLA-DQ2 (non-Aspbeta57) has no particular preference for negatively charged anchor residues found in other type 1 diabetes-predisposing non-Aspbeta57 MHC class II molecules.

Susceptibility and resistance to type 1 diabetes are associated with MHC class II alleles that carry non-Asp and Asp at residue 57 of their beta chain respectively. The effect of Asp or non-Aspbeta57 may relate to a differential ability of distinct class II molecules to bind specific immuno-pathogenic peptides. Recent studies in man and mouse have revealed that some type 1 diabetes-predisposing non-Aspbeta57 class II molecules (i.e. DQ8, DR4Dw15 and I-Ag7) preferentially bind peptides with a negatively charged anchor residue at P9. It has been suggested that this is a common feature of type 1 diabetes-predisposing class II molecules. The molecular explanation for such a phenomenon could be that class II beta chains with Aspbeta57 form a salt bridge between Aspbeta57 and a conserved Arg of the a chain, whereas in non-Aspbeta57 molecules the Arg is unopposed and free to interact with negatively charged P9 peptide anchor residues. We have investigated the specificity of the P9 pocket of the type 1 diabetes-associated DQ2 molecule and in particular examined for charge effects at this anchor position. Different approaches were undertaken. We analyzed binding of a high-affinity binding ligand and P9-substituted variants of this peptide, and we analyzed the binding of a set of synthetic random peptide libraries. The binding analyses were performed with wild-type DQ2 and a mutated DQ2 with Ala at beta57 substituted with Asp. Our results indicate that the wild-type DQ2 (non-Aspbeta57) prefers large hydrophobic residues at P9 and that there is no particular preference for binding peptides with negatively charged residues at this position. The specificity of the P9 pocket in the mutated DQ molecule is altered, indicating that the beta57 residue contributes to determining the specificity of the P9 pocket. Our data do not lend support to the hypothesis that all non-Asp beta57 class II molecules predispose to development of disease by binding peptides with negatively charged P9 anchor residues.

Alanine↗
PubMed1998

On the behavioral characteristics of loud-music listening.

To provide insight into the behavioral characteristics of people who listen excessively to loud music, the 32-item Northeastern Excessive Music Listening Survey was developed and administered to 90 subjects. Results indicate that 8 of the 90 subjects scored within a range that would suggest the presence of a maladaptive pattern of music-listening behavior similar to that exhibited by substance abusers. Implications for further research and models of treatment are discussed.

Adult↗