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Biomedical subjects

S Buus

Publications and source records attributed to S Buus.

At least 73 records · Page 4Linked to original sources
PubMed1995

Identification of a nonameric H-2Kk-restricted CD8+ cytotoxic T lymphocyte epitope on the Plasmodium falciparum circumsporozoite protein.

Class I-restricted CD8+ cytotoxic T lymphocytes (CTL) against the circumsporozoite protein (CSP) protect mice against the rodent malaria parasite, Plasmodium yoelii, and vaccines designed to produce protective CTL against the P. falciparum CSP (PfCSP) are under development. Humans and B10.BR (H-2k) mice have been shown to have CD8+ CTL activity against a 23-amino-acid region of the PfCSP (residues 368 to 390 from the PfCSP 7G8 sequence) that is too long to bind directly to class I major histocompatibility complex molecules. To identify within this 23-amino-acid peptide a shorter peptide that binds to an H-2k class I major histocompatibility molecule, a primarily CD8+ (97.8%) T-cell line (PfCSP TCL.1) was produced by immunizing B10.BR mice with recombinant vaccinia virus expressing the PfCSP and stimulating in vitro spleen cells from these immunized mice with L cells transfected with the PfCSP gene (LPF cells). PfCSP TCL.1 lysed LPF cells and L cells pulsed with peptide PfCSP 7G8 368-390. When 15 overlapping nonamer peptides spanning the 368 to 390 sequence were tested, only one peptide, PfCSP 7G8 375-383 (Y E N D I E K K I), which includes an H-2Kk-binding motif, E at amino acid residue 2, and I at residue 9, sensitized targets for lysis by PfCSP TCL.1. Furthermore, a 10(3)- to 10(4)-fold lower concentration of the nonamer than that of the 23-amino-acid peptide was required to sensitize target cells for lysis by PfCSP TCL.1. Presentation by H-2Kk was demonstrated by using 3T3 fibroblast cells transfected with the murine H-2Kk or H-2Dk genes, and only the H-2Kk transfectants were lysed by PfCSP TCL.1 after incubation with peptide PfCSP 7G8 375-383. Binding to H-2Kk was confirmed by competitive inhibition of binding of labelled peptides to affinity-purified Kk molecules. Substitution of the anchor amino acid residue, E, at position 2 with A dramatically reduced binding to Kk and eliminated the capacity of the peptide to sensitize target cells for killing. Variation of non-anchor residues did not markedly reduce binding to Kk but in some cases eliminated the capacity of the peptide to sensitize targets for cytolysis by PfCSP TCL.1, presumably by eliminating T-cell receptor-binding sites. These data suggest that similar studies with human T cells will be required for optimal development of peptide-based vaccines designed to produce protective class I-restricted CD8+ CTL against the PfCSP in humans.

3T3 Cells↗
PubMed1994

A quantitative assay to measure the interaction between immunogenic peptides and purified class I major histocompatibility complex molecules.

A direct and sensitive biochemical assay to measure the interaction in solution between peptides and affinity-purified major histocompatibility complex (MHC) class I molecules has been generated. Specific binding reflecting the known class I restriction of cytotoxic T cell responses was obtained. Adding an excess of beta 2-microglobulin (beta 2m) significantly increased the rate of peptide association, but it did not affect the rate of dissociation. Binding was complicated by a rapid and apparently irreversible loss of functional MHC class I at 37 degrees C which might limit the life span of empty MHC class I thereby preventing the inadvertent exchange of peptides at the target cell surface. All class I molecules tested bound peptides of the canonical octa- to nona-meric length. However, one class I molecule, Kk, also bound peptides, which were much longer suggesting that the preference of class I molecules for short epitopes is not absolute and may be caused by factors other than the peptide-MHC class I binding event itself.

Amino Acid Sequence↗
PubMed1994

Preformed purified peptide/major histocompatibility class I complexes are potent stimulators of class I-restricted T cell hybridomas.

A panel of antigen-specific, major histocompatibility complex class I-restricted T cell hybridomas has been generated to examine the capacity of peptide/class I complexes to stimulate T cells at the molecular level. Peptide/class I complexes were generated in detergent solution, purified and quantitated. Latex particles were subsequently coated with known amounts of preformed complexes and used to stimulate the T cell hybridomas. Stimulation was specific, i.e. only the appropriate peptide/class I combination were stimulatory, and quite sensitive, i.e. as little as 300 complexes per bead could be detected by the T cells. Preformed complexes were about 500,000 times more potent than free peptide in terms of T cell stimulation, demonstrating the physiological relevancy of the biochemically generated complexes. Surprisingly, the majority (including the most sensitive of the hybridomas) had lost CD8 expression, suggesting that antigen-specific stimulation of class I-restricted T cell hybridomas, as assessed by IL-2 release, does not depend on CD8.

Amino Acid Sequence↗
PubMed1994

Binding of peptides to HLA-DQ molecules: peptide binding properties of the disease-associated HLA-DQ(alpha 1*0501, beta 1*0201) molecule.

Peptide binding to DQ molecules has not previously been described. Here we report a biochemical peptide-binding assay specific for the DQ2 [i.e. DQ(alpha 1*0501, beta 1*0201)] molecule. This molecule was chosen since it shows a strong association to diseases such as celiac disease and insulin-dependent diabetes mellitus. Initially we radiolabelled some selected peptides and tested them for binding to affinity-purified DQ2 molecules. One of the peptides, a Mycobacterium bovis (MB) 65 kDa 243-255Y peptide, displayed a good signal-to-noise ratio and was thus chosen as an indicator peptide in the DQ2 binding assay. The MB 65 kDa 243-255Y peptide bound to DQ2 in a strictly pH-dependent fashion, with optimal binding around pH 5 and only weak binding at pH 7.4. The association of the MB 65 kDa 243-255Y peptide to DQ2 was slow, but once formed, the peptide-HLA complexes were very stable. The binding of peptides to DQ2 was specific, as shown in inhibition experiments with a panel of 47 peptides, differing in length, sequence, and origin. The binding of peptides to DR3 was tested in a similar assay with a Mycobacterium tuberculosis 65 kDa 3-13 peptide as the binding indicator. DQ2 and DR3 molecules bound to different sets of peptides. However, the peptide binding to DQ2 and DR3 showed, in general, similar characteristics with respect to pH dependence and kinetic parameters, indicating that the overall rules for peptide binding to DQ molecules are the same as those previously shown for human DR and murine I-A and I-E molecules.

Amino Acid Sequence↗
PubMed1994

Direct binding of autoimmune disease related T cell epitopes to purified Lewis rat MHC class II molecules.

New strategies applied in the treatment of experimental autoimmune disease models involve blocking or modulation of MHC-peptide-TCR interactions either at the level of peptide-MHC interaction or, alternatively, at the level of T cell recognition. In order to identify useful competitor peptides one must be able to assess peptide-MHC interactions. Several well described autoimmune disease models exist in the Lewis rat and thus this particular rat strain provides a good model system to study the effect of competitor peptides. So far no information has been available on the peptide binding characteristics of the Lewis rat MHC class II RT1.B1 molecule. We have now developed a biochemical binding assay which enables competition studies in which the relative MHC binding affinity of a set of non-labelled peptides can be assessed while employing detection of biotinylated marker peptides by chemiluminescence. The assay is sensitive and specific. We have used this assay to determine the binding characteristics of several disease associated T cell determinants and their sequence analogues in the Lewis rat. Notably, most of the autoimmune disease associated peptide sequences tested were found to be intermediate to poor binders. Single amino acid substitutions at defined positions were sufficient to turn certain peptides into good binders. These results are relevant to the design of competitor peptides in the treatment of experimental autoimmune diseases.

Amino Acid Sequence↗
PubMed1994

The interaction between beta 2-microglobulin (beta 2m) and purified class-I major histocompatibility (MHC) antigen.

The function of MHC class-I molecules is to sample peptides from the intracellular environment and present them to CD8+ cytotoxic T lymphocytes. To understand the molecular details of the assembly (and disassembly) of peptide-beta 2m-class-I complexes a biochemical peptide-class-I binding assay has been generated recently and this paper reports on a similar assay for the interaction between beta 2m and class I. As a model system human beta 2m binding to mouse class I was used. The assay is strictly biochemical using purified reagents which interact in solution and complex formation is determined by size separation. It is specific and highly sensitive. The observed affinity of the interaction, KD, is close to 0.4 nM. The rate of association at 37 degrees C is very fast (the ka is around 5 x 10(4)/M/s) whereas the dissociation is slow (the kd is around 8 x 10(-6)/s); the ratio of dissociation to association yields a calculated KD close to the observed value. At 37 degrees C almost all of the purified class I participates in binding of the exogenously offered beta 2m showing that a considerable exchange of the endogenous beta 2m occurs. Finally, it was demonstrated that exogenous beta 2m enhances binding to MHC class-I of short perfectly-matching peptides as well as longer peptides.

Amino Acid Sequence↗
PubMed1994

Antibodies directed against monomorphic and evolutionary conserved self epitopes may be generated in 'knock-out' mice. Development of monoclonal antibodies directed against monomorphic MHC class I determinants.

Beta-2 microglobulin (beta 2m) gene 'knock-out' mice (C1D) were primed with purified H-2Kb and H-2Db molecules and spleen cells from immunized mice were used to generate monoclonal antibody secreting B-cell hybridomas. Approximately 0.2% of the Ig-secreting primary microcultures contained H-2b binding antibodies. Three stable anti-MHC class I (MHC-I) antibody secreting hybridoma clones were established and subcloned. All three MoAbs precipitated radiolabelled H-2 molecules as analysed by SDS PAGE, and all three MoAbs stained H-2b, H-2d, as well as H-2k cells by FACS analysis. The MoAbs stained to two beta 2m loss mutant cell lines, C4.4-25- and R1E, suggesting that some MHC-I heavy chain is exported to the cell surface even in the absence of endogenous beta 2m. Staining of murine cell lines kept under serum-free culture conditions was strongly influenced by the addition of bovine or human serum as a source of exogenous beta 2m suggesting that xenogeneic beta 2m affects the conformation of class I molecules. Furthermore, all three MoAbs strongly stained the peptide transporter deficient cell line, RMA-S, when cultured at 26 degrees C, however, staining was reduced five-fold when RMA-S cells were cultured at 37 degrees C. In total, these observations suggest that the MoAbs recognize conformational, presumably beta 2m and peptide dependent, self epitopes on MHC-class I. One of the three MoAbs stained rat blood mononuclear blood cells (BMC), all three MoAbs stained hamster BMC, whereas two of the MoAbs stained human cells. These data suggest that the MoAbs recognize determinants which are conserved between species. All three antibodies strongly inhibited the development of CTLs generated in an allogeneic one-way MLC, provided that the MoAbs were present during the first 24 h of culture. It is concluded that MoAbs reacting with monomorphic self epitopes may be generated using animals deleted of the gene of interest. The implications may be far reaching since such MoAbs potentially identify evolutionary conserved and physiologically important epitopes.

Animals↗
PubMed1994

Auditory facilitation: procedural or sensory effect?

A few studies have reported that, under certain conditions, thresholds following exposure to a sound may be lower than those measured without prior stimulation. For example, Rubin [J. Acoust. Soc. Am. 32, 670-681 (1960)] found a 7-dB negative shift in threshold, for a 20-ms tone presented 160 ms after termination of a low-level cue tone. This "auditory facilitation" was obtained with an up-and-down psychophysical method. In the present study, auditory facilitation was reexamined using an adaptive, two-interval, two-alternative-forced-choice (2I, 2AFC) procedure. Thresholds were measured for 20-ms tones at 1 and 5 kHz, preceded by a 15-dB SL 80-ms cue tone of the same frequency. In addition, thresholds were measured with the cue absent. The results show that thresholds can be lower in the presence of the cue than in its absence, but the effect averages 0.5 dB or less and seems to be independent of cue-signal delay between 80 and 640 ms. Because this finding contrasts with the 7-dB facilitation, which Rubin obtained using an up-and-down method, part of his experiment was replicated and the results were essentially the same as his. The failure to find a pronounced facilitation effect with a 2I, 2AFC procedure indicates that the poststimulatory lowering of threshold found by Rubin primarily resulted from biases and criterion shifts.

Auditory Perception↗
PubMed1994

Discrimination of envelope frequency in one spectral region in the presence of modulation in another.

This paper examines how discrimination of envelope frequency in one spectral region is affected by the presence of either correlated or uncorrelated modulation in another spectral region. Envelope-frequency discrimination thresholds, delta fe's, for a 60-dB SPL two-tone complex centered at 2 kHz were measured in the presence or absence of an auxiliary two-tone complex at another frequency. When present, the auxiliary complex had either the same envelope as the standard signal or an envelope that was uncorrelated with both the standard and variable signal. The results show that listeners can take advantage of correlation between simultaneous envelopes in different frequency regions to improve discrimination of a small change in envelope frequency, whereas the presence of uncorrelated envelopes causes interference with discrimination. Except when the auxiliary complex and the signal are close together in frequency, the discrimination advantage afforded by the correlated auxiliaries appears to reflect across-channel envelope processing. The discrimination interference caused by the uncorrelated auxiliaries, on the other hand, may reflect primarily within-channel interaction such as peripheral masking, although a small amount of across-channel masking also is apparent.

Adult↗
PubMed1993

Intensity perception. XIV. Intensity discrimination in listeners with sensorineural hearing loss.

Intensity discrimination of pulsed tones (also called level discrimination) was measured as a function of level in 13 listeners with sensorineural hearing impairment of primarily cochlear origin, one listener with a vestibular schwannoma, and six listeners with normal hearing. Measurements were also made in normal ears presented with masking noise spectrally shaped to produce audiograms similar to those of the cochlearly impaired listeners. For unilateral impairments, tests were made at the same frequency in the normal and impaired ears. For bilateral-sloping impairments, tests were made at different frequencies in the same ear. The normal listeners showed results similar to other data in the literature. The listener with a vestibular schwannoma showed greatly reduced intensity resolution, except at a few levels. For listeners with recruiting sensorineural impairments, the results are discussed according to the configuration of the impairment and are compared across configurations at equal SPL, equal SL, and equal loudness level. Listeners with increasing hearing losses at frequencies above the test frequency generally showed impaired resolution, especially at high levels, and less deviation from Weber's law than normal listeners. Listeners with decreasing hearing loss at frequencies above the test frequency showed nearly normal intensity-resolution functions. Whereas these trends are generally present, there are also large differences among individuals. Results obtained from normal listeners who were tested in the presence of masking noise indicate that elevated thresholds and reduced dynamic range account for some, but not all, of the effects of recruiting sensorineural impairment on intensity resolution.

Acoustic Stimulation↗
PubMed1992

MHC molecules protect T cell epitopes against proteolytic destruction.

There is a subtle duality in the role of proteolytic enzymes in Ag processing. They are required to fragment protein Ag ingested by APC. However, prolonged exposure to proteolytic enzymes may lead to a complete degradation of the Ag, leaving nothing for the T cell system to recognize. What ensures that some of the Ag is salvaged? Using a cell-free system we demonstrate that an Ag fragment, once bound to a MHC class II molecule, is effectively protected against proteolytic destruction by cathepsin B and pronase E. The bound fragment, however, can be modified by aminopeptidase N. We suggest that MHC class II molecules play an important regulatory role in the physiologic processing of Ag.

Amino Acid Sequence↗
PubMed1992

pH dependence of the interaction between immunogenic peptides and MHC class II molecules. Evidence for an acidic intracellular compartment being the organelle of interaction.

The pH dependence of the interaction between immunogenic peptide and MHC class II was studied both in a direct biochemical binding assay and in a functional Ag presentation assay. The two approaches yielded similar results. All of the peptides tested bound optimally to their relevant MHC class II restriction element at around pH 4.5. Indeed, several of the peptides did not bind at neutral pH. These results demonstrate that Ag under physiologic conditions meet MHC class II in a quite acidic environment. The very acidic pH optimal for peptide-MHC class II interaction is only found intracellularly and most notably in the endosome-lysosome compartment in which Ag processing is thought to occur. Thus, Ag processing and interaction with MHC class II molecules can potentially happen in the very same compartment. This yet undefined acidic compartment would have to contain proteolytic enzymes and MHC class II molecules.

Amino Acid Sequence↗
PubMed1992

Complete dissection of the Hb(64-76) determinant using T helper 1, T helper 2 clones, and T cell hybridomas.

We have generated cloned Th1 cells, Th2 cells, and T cell hybridomas specific for the single immunogenic peptide from the beta-chain of murine hemoglobin (Hb(64-76)). The availability of these various types of T cells provided us an unique opportunity to examine and dissect the T cell response to an immunogenic peptide. A panel of altered Hb peptides was made by replacing each amino acid in the Hb peptide (positions 64-76) with a conservative amino acid substitution or an alanine. Although none of the eleven T cell clones and hybridomas tested exhibited the same pattern of reactivity to the substituted Hb peptides, some general features were identified for all T cell responses. The primary T cell contact residue of Hb(64-76) was shown to be asparagine 72. For every Hb(64-76) specific T cell, no activation was observed using a peptide containing the conservative substitution of a glutamine for the asparagine at position 72. The flanking glutamic acid at position 73 was also required for a proliferative response for all of the Th1 and Th2 clones. The Th subtypes were not grossly unique in their responses to the substituted Hb peptides, but exhibited minor differences in fine specificity with the Th1 cells identifying more critical amino acids then did the Th2 cells. For the Th1 cells and also the T cell hybridomas, the phenylalanine at position 71 was critical for a T cell response. Analysis of peptide affinity for IEk molecules indicated that position 71 played a role in peptide binding to MHC. Secondary T cell contact residues, which were important for many but not all of the T cells, were identified at positions 69, 70, and 76. Overall T cell responses were minimally affected by changes in the amino acid residues at positions 64-68, 74, and 75. We have also demonstrated that cloned Th1 cells, Th2 cells and T hybridomas can be generated against the same Hb(64-76) determinant.

Amino Acid Sequence↗
PubMed1991

Effective attenuation of signals in noise under focused attention.

When attending to a tone at a given frequency, listeners are most sensitive to that tone and others within a restricted band of frequencies surrounding it. This region of enhanced sensitivity defines the attention band that was measured in two experiments using a modified version of the probe-signal method of Greenberg and Larkin [J. Acoust. Soc. Am. 44, 1513-1523 (1968)]. Experiment 1 showed that at five center frequencies, from 0.25 to 4.0 kHz, the shape of the attention band resembles that of the auditory filter as inferred from notched-noise masking experiments by other investigators. The width of the attention band is close to the critical band at higher frequencies, but only half as wide at 0.25 and 0.5 kHz. Experiment 2 produced psychometric functions for unattended probe tones at least 0.23 kHz away from a fully attended, 1-kHz target tone. From these functions, the effective attenuation, measured as the threshold difference between the 1-kHz target and the probes, was estimated to be 7 dB; the amount of attenuation appeared to be about the same regardless of how far the probe frequency was from the attended band. One interpretation of these results is that bands centered on the unattended tones contribute to the decision process with some small but measurable weight and are not entirely ignored.

Acoustics↗