Biomedical subjects

A Holm

Publications and source records attributed to A Holm.

At least 37 records · Page 2Linked to original sources

PubMed2001

Fluticasone propionate aqueous nasal spray reduces inflammatory cells in unchallenged allergic nasal mucosa: effects of single allergen challenge.

BACKGROUND: Topical corticosteroid therapy reduces symptoms and nasal mucosal inflammatory cells in patients with allergic rhinitis. Usually patients are advised to start their medication (1 week) before the beginning of the pollen season. The effect of pretreatment with a topical corticosteroid on unchallenged nasal mucosa is not well documented. OBJECTIVES: The purpose of this study was to investigate, in a double-blind, placebo-controlled study, the effect of 6 weeks' pretreatment with 200 microg twice daily fluticasone propionate on nasal symptoms and inflammatory cell numbers after nasal allergen provocation in patients with seasonal allergic rhinitis. METHODS: Nineteen patients with grass pollen-induced allergic rhinitis were treated for a 6-week period out of the grass pollen season. After completing the treatment period, patients were challenged with grass pollen. Nasal mucosal biopsy specimens were taken 5 times in every patient. In nasal mucosa changes in numbers of T cells, B cells, mast cells, eosinophils, macrophages, and Langerhans' cells were investigated. RESULTS: After 4 weeks of treatment but before allergen provocation, significantly fewer epithelial Langerhans' cells, macrophages, mast cells, T cells, and eosinophils were found in the fluticasone propionate group compared with those found in the placebo group. In the lamina propria significantly fewer Langerhans' cells and eosinophils were found in the fluticasone propionate group. Cell influx in nasal mucosa after allergen provocation was significantly inhibited in the fluticasone propionate group compared with that in the placebo group for epithelial Langerhans' cells, mast cells, macrophages, and T cells and for lamina propria eosinophils, mast cells, Langerhans' cells, macrophages, and T cells. CONCLUSIONS: Fluticasone propionate is effective in reducing early- and late-phase nasal symptoms. Topical corticosteroid treatment reduces inflammatory cells in unchallenged nasal mucosa.

Administration, Intranasal↗

PubMed2000

Efficient protection against Mycobacterium tuberculosis by vaccination with a single subdominant epitope from the ESAT-6 antigen.

We have investigated the vaccine potential of two peptides derived from the 6-kDa early secretory antigenic target (ESAT)-6 antigen in the mouse model of tuberculosis. The peptides were both strongly immunogenic in B6CBAF1 (H-2b,k) mice and primed recall responses of the same intensity after immunization. However, both tuberculosis infection and immunization with ESAT-6 resulted in responses focused towards ESAT-61-20. Multiple antigen peptide constructs as well as free peptides were emulsified with dimethyl dioctadecylammonium bromide/monophosphoryl lipid A/IL-2 and tested as experimental vaccines in an i.v. and aerosol model of tuberculosis in mice. The peptide were highly immunogenic and induced cellular responses of the same magnitude. However, only vaccines based on the subdominant ESAT-651-70 epitope promoted significant levels of protective immunity and the level of protection was equivalent to that achieved with ESAT-6 and BCG. These findings demonstrate the potential of peptide-based vaccines against tuberculosis and indicate that there is not direct correlation between the hierarchy of response to naturally processed peptides and their ability to induce protective immunity against Mycobacterium tuberculosis.

Amino Acid Sequence↗

PubMed2000

Longer peptide can be accommodated in the MHC class I binding site by a protrusion mechanism.

According to current consensus, CD8(+) T cell responses are focused upon short peptide sequences (8-11 amino acids) presented by MHC class I molecules. This size restriction is thought to operate mostly at the level of peptide-MHC class I interaction. Crystal structures have shown that the free N and C termini of a bound peptide interact through hydrogen bonding networks to conserved residues at either end of the class I binding site. Accordingly, it is thought that the termini are fixed and that only minor variations in peptide size are possible through a central bulging mechanism. We find that this consensus view is not always correct as some peptide-MHC class I interaction will accept significant extensions. Furthermore, our results indicate that in some cases protrusion, rather than bulging, may be the mechanism of extension. Depending upon the particular peptide-MHC combination in question, such extensions can occur at either the N or C terminus (but never both at the same time). Finally, we show that MHC and T cell in some cases can detect the identity of the extension, i.e. that extensions may be part of the specificity of the T cell immune response. We suggest that such extensions may play a physiological role.

Amino Acid Sequence↗

PubMed2000

Characterization of air-coupled ultrasound transducers in the frequency range 40 kHz-2 mHz using light diffraction tomography.

The aim of this work was to show the applicability of light diffraction tomography on airborne ultrasound in the frequency range 40 kHz-2 MHz. Seven different air-coupled transducers were measured to show the method's performance regarding linearity, absolute pressure measurements, phase measurements, frequency response, S/N ratio and spatial resolution. A calibrated microphone and the pulse-echo method were used to evaluate the results. The absolute measurements agreed within the calibrated microphone's uncertainty range. Pulse waveforms and corresponding FFT diagrams show the method's higher bandwidth compared with the microphone. Further, the method offers non-perturbing measurements with high spatial resolution, which was especially advantageous for measurements close to the transducer surfaces. The S/N ratio was higher than or in the same range as that of the two comparison methods.

Air↗

PubMed2000

Ligand-presenting assembly: a method for C- and N-terminal antigen presentation.

Achiral dicarboxylic acids were coupled with 2 eq. of the free alpha-amino groups of two fully side-chain protected peptide chains while these were still attached to a synthesis resin. Cleavage from the resin with simultaneous side-chain deprotection afforded two assembled peptide chains with free C-terminals. Suitable functionalization of the achiral dicarboxylic acid alternatively permitted continued peptide synthesis in a C to N orientation leading to a final peptide assembly which, after cleavage from the resin, may have multiple N to C and C to N presentation of one or more epitopes.

Amino Acid Sequence↗

PubMed2000

Polypeptide binding properties of the chaperone calreticulin.

Calreticulin is a highly conserved eukaryotic ubiquitious protein located mainly in the endoplasmic reticulum. Two major characteristics of calreticulin are its chaperone activity and its lectin properties, but its precise function in intracellular protein and peptide processing remains to be elucidated. We have investigated the interactions of human calreticulin with denatured ovalbumin, proteolytic digests of ovalbumin, and different available peptides by solid phase assays, size-exclusion chromatography, capillary electrophoresis, and MS. The results show that calreticulin interacts better with unfolded ovalbumin than with native ovalbumin, that calreticulin strongly binds components in proteolytic digests of denatured ovalbumin, and that calreticulin interacts strongly with certain synthetic peptides.

Amino Acid Sequence↗

PubMed2000

Neurite outgrowth induced by a synthetic peptide ligand of neural cell adhesion molecule requires fibroblast growth factor receptor activation.

The neural cell adhesion molecule NCAM is involved in axonal outgrowth and target recognition in the developing nervous system. In vitro, NCAM-NCAM binding has been shown to induce neurite outgrowth, presumably through an activation of fibroblast growth factor receptors (FGFRs). We have recently identified a neuritogenic ligand, termed the C3 peptide, of the first immunoglobulin (lg) module of NCAM using a combinatorial library of synthetic peptides. Here we investigate whether stimulation of neurite outgrowth by this synthetic ligand of NCAM involves FGFRs. In primary cultures of cerebellar neurons from wild-type mice, the C3 peptide stimulated neurite outgrowth. This response was virtually absent in cultures of cerebellar neurons from transgenic mice expressing a dominant-negative form of the FGFR1. Likewise, in PC12E2 cells transiently expressing a dominant-negative form of the mouse FGFR1, induction of neurites by the C3 peptide was abrogated. These findings suggest that the neuritogenic effect of the C3 peptide requires the presence of functional FGFRs and support the hypothesis that FGFRs are essential in cell adhesion molecule-stimulated neurite outgrowth. The C3 peptide appears to stimulate neurite outgrowth by specifically activating an NCAM-FGFR-dependent signaling cascade and may therefore be of considerable interest as a tool for the determination of NCAM-dependent neurite outgrowth as well as a potential drug capable of promoting outgrowth and regeneration of NCAM-responsive axons.

3T3 Cells↗

PubMed2000

Creation of a selective antagonist and agonist of the rat VPAC(1) receptor using a combinatorial approach with vasoactive intestinal peptide 6-23 as template.

We have used combinatorial chemistry with amino acid mixtures (X) at positions 6 to 23 in vasoactive intestinal peptide (VIP) to optimize binding affinity and selectivity to the rat VPAC(1) receptor. The most efficient amino acid replacement was a substitution of alanine at position 18 to diphenylalanine (Dip), increasing the displacement efficiency of (125)I-VIP by 370-fold. The [Dip(18)]VIP(6-23) was subsequently used to find a second replacement, employing the same approach. Tyrosine at position 9 was selected and the resulting [Tyr(9),Dip(18)]VIP(6-23) analog has a K(i) value of 90 nM. This analog was unable to stimulate cAMP production at 10(-6) M but was able to inhibit VIP-induced cAMP stimulation (K(b) = 79 nM). The K(i) values of [Tyr(9),Dip(18)]VIP(6-23) using the rat VPAC(2) and PAC(1) receptors were 3,000 nM and >10,000 nM, respectively. Thus, [Tyr(9),Dip(18)]VIP(6-23) is a selective VPAC(1) receptor antagonist. The C-terminally extended form, [Tyr(9),Dip(18)]VIP(6-28), displays improved antagonistic properties having a K(i) and K(b) values of 18 nM and 16 nM, respectively. On the contrary, the fully extended form, [Tyr(9),Dip(18)]VIP(1-28), was a potent agonist with improved binding affinity (K(i) = 0.11 nM) and ability to stimulate cAMP (EC(50) = 0.23 nM) compared with VIP (K(i) = 1.7 nM, EC(50) = 1.12 nM). Furthermore, the specificity of this agonist to the VPAC(1) receptor was high, the K(i) values for the VPAC(2) and PAC(1) receptors were 53 nM and 3,100 nM, respectively. Seven other analogs with the [Tyr(9),Dip(18)] replacement combined with previously published VIP modifications have been synthesized and described in this work.

Amino Acid Substitution↗

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

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

Mechanical manipulation of bone and cartilage cells with 'optical tweezers'.

The single beam optical gradient trap (optical tweezers) uses a single beam of laser light to non-invasively manipulate microscopic particles. Optical tweezers exerting a force of approximately 7 pN were applied to single bone and cartilage derived cells in culture and changes in intracellular calcium levels were observed using Fluo-3 labelling. Human derived osteoblasts responded to optical tweezers with an immediate increase in [Ca2+]i that was inhibited by the addition of a calcium channel blocker nifedipine. Force applied to different regions of cells resulted in a variable response. [Ca2+]i elevation in response to load was lower in rat femur derived osteoblasts, and not apparent in primary chondrocytes and the osteocytic cell line (MLO Y4).

Calcium↗

PubMed1999

Mechanical manipulation of polymorphonuclear leukocyte plasma membranes with optical tweezers causes influx of extracellular calcium through membrane channels.

Optical tweezers are used mechanically to manipulate the plasma membrane of polymophonuclear leukocytes attached to the bottom of a glass manipulation chamber. The laser trapping beam is dragged across the membrane of cells in calcium-containing and calcium-depleted extracellular medium. This treatment causes a significant rise in the intracellular calcium concentration compared with controls, in cells in calcium-containing medium (239.8 +/- 49.0% against 75.4 +/- 16.4%, respectively), but not in cells in calcium-depleted medium (69.1 +/- 9.6% against 83.4 +/- 18.5%, respectively), indicating that the calcium rise is caused by an influx of calcium from the environment. The rise in calcium concentration is blocked (23.5 +/- 7.1% against 17.1 +/- 4.1%, respectively) by the addition of lansoprazole, indicating that the influx is not due to unspecific membrane damage caused by the mechanical manipulation of the cell. It can therefore be concluded that mechanical manipulation of the neutrophil membrane, in the piconewton force range exerted by the optical tweezer, does not damage the plasma membrane but stimulates a mechanically inducible, membrane channel-mediated influx of extracellular calcium.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed1999

High resolution light diffraction tomography: nearfield measurements of 10 MHz continuous wave ultrasound

Light diffraction tomography is an ultrasound measurement method that offers possibilities to make high spatial resolution measurements. The aim of this study was to investigate the spatial resolution of light diffraction tomography by characterizing the complex pressure and phase patterns 0.5-1 mm from the surface of a 5 mm diameter 10 MHz ultrasound transducer. Three different transducers were measured with a detection width of 5 microm, sample distances of 20-40 microm and 50-100 projections/180 degrees. They were excited with a 10 Vpp CW-signal. To verify the results the optical measurements were compared with 75 microm diameter hydrophone measurements and with theoretical calculations. The light diffraction tomography results show very good agreement with the hydrophone measurements and pressure peaks separated 160 microm were resolved. Comparison with the theoretical calculation shows that small distortions, caused by defects in the matching layer or in the piezo-electric disc, disturb the symmetric ring-pattern characteristic of an ideal piston source.

Journal Article↗

PubMed1999

Identification of a neuritogenic ligand of the neural cell adhesion molecule using a combinatorial library of synthetic peptides.

The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration, and learning. In this study, we identified a synthetic peptide-ligand of the NCAM Ig1 module by combinatorial chemistry and showed it could modulate NCAM-mediated cell adhesion and signal transduction with high potency. In cultures of dissociated neurons, this peptide, termed C3, stimulated neurite outgrowth by activating a signaling pathway identical to that activated by homophilic NCAM binding. A similar effect was shown for the NCAM Ig2 module, the endogenous ligand of NCAM Ig1. By nuclear magnetic resonance spectroscopy, the C3 binding site in the NCAM Ig1 module was mapped and shown to be different from the binding site of the NCAM Ig2 module. The C3 peptide may prove useful as a lead in development of therapies for neurodegenerative disorders, and the C3 binding site of NCAM Ig1 may represent a target for discovery of nonpeptide drugs.

Amino Acid Sequence↗

PubMed1999

Epitope mapping of Aleutian mink disease parvovirus virion protein VP1 and 2.

Six overlapping fragments of the Aleutian Mink Disease parvoVirus (AMDV) virion protein VP1 and 2 (VP1/2) gene were inserted into the expression vector pMAL-c2. Four of the clones carried large overlapping fragments covering the entire VP1/2 gene. The remaining two clones covered specifically chosen regions within the VP1/2 gene. Using a Western blotting detection system, sera from AMDV-infected mink were tested against the recombinant polypeptides. These studies showed reactions primarily directed against the two AMDV polypeptides ranging from amino acids 297 to 518. Weaker reactions against other regions of the VP1/2 were also observed. The small fusion protein designed to cover the presumed AMDV VP1/2 loop 4 was purified by affinity chromatography and used to develop solid-phase immunoassays. Twelve small synthetic peptides were constructed and used as inhibitors. A peptide covering amino acids S428 to T448 was shown to block the reactivity of a pool of positive mink sera, indicating the presence of one dominant linear epitope.

Aleutian Mink Disease↗

PubMed1999

Human T cell responses to the ESAT-6 antigen from Mycobacterium tuberculosis.

Human T cell responses to ESAT-6 and eight synthetic overlapping peptides were investigated in tuberculosis (TB) patients and control subjects from regions of high and low endemicity for TB. ESAT-6 was recognized by 65% of all tuberculin purified protein derivative-responsive TB patients, whereas only 2 of 29 bacille Calmette-Guérin-vaccinated Danish healthy donors recognized this molecule. In Ethiopia, a high frequency (58%) of healthy contacts of TB patients recognized ESAT-6. All of the peptides were recognized by some donors, indicating that the molecule holds multiple epitopes. Danish and Ethiopian patients differed in the fine specificity of their peptide responses. Recognition of the C-terminal region (aa 72-95) was predominant in Danish patients, whereas recognition of aa 42-75 was predominant in Ethiopia. The relationship of these differences to the distribution of HLA types in the two populations is discussed. This study demonstrates that ESAT-6 is frequently recognized during early infection and holds potential as a component of a future TB-specific diagnostic reagent.

Antigens, Bacterial↗

PubMed1999

The outcomes of an inpatient treatment program for geriatric patients with dementia and dysfunctional behaviors.

This study evaluated outcomes of an inpatient program designed to reduce severe agitated behavior in geriatric patients with dementia who could not be successfully treated on an outpatient basis. An individualized treatment plan was created for each patient (N = 250) that involved pharmacological and nonpharmacological interventions with behavioral, environmental, and psychological components. Assessment of behavioral, cognitive, and functional status was conducted for each patient on admission to the program and at discharge. Significant improvements on these assessments were observed. We conclude that the longitudinal, multidisciplinary approach used in this study was effective in significantly reducing intrusive and dangerous behaviors while preserving or enhancing patients' cognitive and functional abilities.

Aged↗

PubMed1998

Selection of peptides that bind to plasminogen activator inhibitor 1 (PAI-1) using random peptide phage-display libraries.

Large random hexa- and decapenta-peptide libraries were constructed and displayed on the surface of the filamentous phagemid pComb8. Panning of the hexa-peptide library on immobilized plasminogen activator inhibitor 1 (PAI-1) specifically selected a minor fraction of concatemers, indicating that binding to PAI-1 requires an extended amino acid sequence. Accordingly, the decapenta-peptide library exclusively yielded PAI-1 binding peptides of 15 amino acid residues. None of these phage-bound peptides prevented the interaction between PAI-1 and its target serine protease urokinase (u-PA). To isolate peptides that block the interaction between PAI-1 and u-PA, phages bound to immobilized PAI-1 were eluted by incubation with u-PA. Remarkably, this procedure resulted in elution of a unique phage type that harbors a concatemer of decapentamers, consisting of 49 amino acid residues with no obvious similarity to the primary sequence of PAI-1 or u-PA.

Amino Acid Sequence↗