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Biomedical subjects

P Samuelson

Publications and source records attributed to P Samuelson.

At least 19 recordsLinked to original sources
PubMed2002

Depletion of Vbeta5.2/5.3 T cells with a humanized antibody in patients with multiple sclerosis.

A potentially pathogenic expansion of T cells expressing T cell receptor (TCR) Vbeta5.2/5.3 has been demonstrated in patients with multiple sclerosis (MS). A humanized antibody (ATM-027) directed against these T cells has been developed to further investigate the role of this subpopulation of T cells in MS. The pharmacokinetics/dynamics and safety of ATM-027 (0.3-300 mg intravenously over 30 min) were investigated in 14 patients with MS. The effect of treatment on cytokine expression and autoreactivity to peptides of myelin basic protein (MBP) was also studied. ATM-027 was well tolerated and raised no safety concerns. Clearance of the antibody was low and elimination half-life was approximately 3 weeks. The majority of the target Vbeta5.2/5.3 expressing T cells were depleted for at least 18 months. The small remaining fraction of target cells showed a marked decrease in their TCR expression, which was recovered within 8 months. The numbers of peripheral blood mononuclear cells (PBMCs) with spontaneous expression of IFN-gamma was decreased at 72 h and 8 weeks after treatment, whilst no clear effects on TNF-alpha, IL-4, IL-10, TGF-beta expression were observed. There was also a significant decrease in the number of PBMCs producing IFN-gamma in response to MBP peptide 80-102. We conclude that long-term depletion of T cells expressing defined Vbeta subgroups in MS patients is feasible using selective immunotherapy. The selective depletion of Vbeta5.2/5.3 expressing T cells in this study resulted in a decrease in potentially disease promoting anti-MBP reactivity and pro-inflammatory cytokine production.

Adolescent↗
PubMed2001

Anticircumvention rules: threat to science.

Scientists who study encryption or computer security or otherwise reverse engineer technical measures, who make tools enabling them to do this work, and who report the results of their research face new risks of legal liability because of recently adopted rules prohibiting the circumvention of technical measures and manufacture or distribution of circumvention tools. Because all data in digital form can be technically protected, the impact of these rules goes far beyond encryption and computer security research. The scientific community must recognize the harms these rules pose and provide guidance about how to improve the anticircumvention rules.

Computer Security↗
PubMed2001

Directed immobilization of recombinant staphylococci on cotton fibers by functional display of a fungal cellulose-binding domain.

The immobilization of recombinant staphylococci onto cellulose fibers through surface display of a fungal cellulose-binding domain (CBD) was investigated. Chimeric proteins containing the CBD from Trichoderma reesei cellulase Cel6A were found to be correctly targeted to the cell wall of Staphylococcus carnosus cells, since full-length proteins could be extracted and affinity-purified. Furthermore, surface accessibility of the CBD was verified using a monoclonal antibody and functionality in terms of cellulose-binding was demonstrated in two different assays in which recombinant staphylococci were found to efficiently bind to cotton fibers. The implications of this strategy of directed immobilization for the generation of whole-cell microbial tools for different applications will be discussed.

Cell Wall↗
PubMed2001

Surface display on gram positive bacteria.

Heterologous surface display on Gram-positive bacteria was first described almost a decade ago and has since then developed into an active research area. Gram-positive bacterial surface display has today found a range of applications, in immunology, microbiology and biotechnology. Live bacterial vaccine delivery vehicles are being developed through the surface display of selected foreign antigens on the bacterial surfaces. In this field, "second generation" vaccine delivery vehicles are at present being generated by the addition of mucosal targeting signals through co-display of adhesins, in order to achieve targeting of the live bacteria to immunoreactive sites to thereby increase immune responses. Engineered Gram-positive bacteria are further being evaluated as novel microbial biocatalysts with heterologous enzymes immobilized as surface exposed on the bacterial cell surface. A discussion has started whether bacteria can find use as new types of whole-cell diagnostic devices since single-chain antibodies and other variants of tailor-made binding proteins can be displayed on bacteria. Bacteria with increased binding capacity for certain metal ions can be created and potential environmental or biosensor applications for such recombinant bacteria as biosorbents are being discussed. This article explains the basis of Gram-positive bacterial surface display, and discusses current uses and possible future trends of this emerging technology.

Animals↗
PubMed2000

Partial protection to respiratory syncytial virus (RSV) elicited in mice by intranasal immunization using live staphylococci with surface-displayed RSV-peptides.

A live bacterial vaccine-delivery system based on the food-grade bacterium Staphylococcus carnosus was used for delivery of peptides from the G glycoprotein of human respiratory syncytial virus, subtype A (RSV-A). Three peptides, corresponding to the G protein amino acids, 144-159 (denoted G5), 190-203 (G9) and 171-188 (G4 S), the latter with four cysteine residues substituted for serines, were expressed by recombinant means as surface-exposed on three different bacteria, and their surface accessibility on the bacteria was verified by fluorescence-activated cell sorting (FACS). Intranasal immunization of mice with the live recombinant staphylococci elicited significant anti-peptide as well as anti-virus serum IgG responses of balanced IgG1/IgG2a isotype profiles, and upon viral challenge with 10(5) tissue culture infectious doses(50) (TCID(50)), lung protection was demonstrated for approximately half of the mice in the G9 and G4 S immunization groups. To our knowledge, this is the first study in which protective immunity to a viral pathogen has been evoked using food-grade bacteria as vaccine-delivery vehicles.

Administration, Intranasal↗
PubMed2000

Staphylococcal surface display and its applications.

Novel surface proteins can be introduced onto the bacterial cell surface by recombinant means. Here, we describe the development of such display systems for two food-grade bacteria, Staphylococcus carnosus and Staphylococcus xylosus, and present how such engineered bacteria can be used in different applications. A study will be described in which such staphylococci were employed as vaccine delivery vehicles to elicit protective antibody responses to respiratory syncytial virus (RSV). The use of surface-engineered staphylococci as novel microbial biocatalysts, as a new type of whole-cell diagnostic devices or for adsorption of metal ions with potential environmental or biosensor applications, will also be discussed.

Animals↗
PubMed2000

Staphylococcal surface display of metal-binding polyhistidyl peptides.

Recombinant Staphylococcus xylosus and Staphylococcus carnosus strains were generated with surface-exposed chimeric proteins containing polyhistidyl peptides designed for binding to divalent metal ions. Surface accessibility of the chimeric surface proteins was demonstrated and the chimeric surface proteins were found to be functional in terms of metal binding, since the recombinant staphylococcal cells were shown to have gained Ni(2+)- and Cd(2+)-binding capacity, suggesting that such bacteria could find use in bioremediation of heavy metals. This is, to our knowledge, the first time that recombinant, surface-exposed metal-binding peptides have been expressed on gram-positive bacteria. Potential environmental or biosensor applications for such recombinant staphylococci as biosorbents are discussed.

Cadmium↗
PubMed1999

Engineering of a Staphylococcus carnosus surface display system by substitution or deletion of a Staphylococcus hyicus lipase propeptide.

Surface display of recombinant proteins on bacteria and phages has become an important topic in bioscience. A system for the display of heterologous proteins on the surface of Staphylococcus carnosus employs the secretion signal and propeptide from a Staphylococcus hyicus lipase for translocation and since the propeptide is of considerable size (207 amino acids) and not processed in S. carnosus, we have investigated the possibility to delete or substitute the propeptide for smaller protein domains, to thereby improve the surface display system. A set of new vectors was constructed and the surface expression of model proteins was investigated by various methods, including fluorescence-activated cell sorting. The results suggest that the propeptide region indeed can be deleted when proteins which are easily secretable are displayed. In contrast, the propeptide seems to be advantageous for translocation of inefficiently secreted proteins. Moreover, our study also presents a rational strategy for how to monitor the engineering efforts for the optimization of a surface display system.

Base Sequence↗
PubMed1999

A surface-displayed cholera toxin B peptide improves antibody responses using food-grade staphylococci for mucosal subunit vaccine delivery.

The possibility of improving the antibody responses to a model streptococcal antigen, administered by intranasal immunization as surface-displayed on the food-grade bacterium Staphylococcus carnosus, by co-exposure of a peptide (CTBp) comprising amino acids 50-75 of the cholera toxin B subunit, was investigated. It was found that the introduction of the CTBp into the chimeric surface proteins, containing a serum albumin binding protein (ABP) from streptococcal protein G as model antigen, significantly increased serum IgG responses upon intranasal immunization. Similarly, elicited local IgA responses were also found to be improved. Furthermore, it was demonstrated that live delivery of the staphylococci was required to obtain this effect, since UV-irradiated or heat-killed bacteria exposing the same chimeric surface proteins did not show increased anti-ABP IgG responses.

Adjuvants, Immunologic↗
PubMed1999

Staphylococcal surface display of immunoglobulin A (IgA)- and IgE-specific in vitro-selected binding proteins (affibodies) based on Staphylococcus aureus protein A.

An expression system designed for cell surface display of hybrid proteins on Staphylococcus carnosus has been evaluated for the display of Staphylococcus aureus protein A (SpA) domains, normally binding to immunoglobulin G (IgG) Fc but here engineered by combinatorial protein chemistry to yield SpA domains, denoted affibodies, with new binding specificities. Such affibodies, with human IgA or IgE binding activity, have previously been selected from a phage library, based on an SpA domain. In this study, these affibodies have been genetically introduced in monomeric or dimeric forms into chimeric proteins expressed on the surface of S. carnosus by using translocation signals from a Staphylococcus hyicus lipase construct together with surface-anchoring regions of SpA. The recombinant surface proteins, containing the IgA- or IgE-specific affibodies, were demonstrated to be expressed as full-length proteins, localized and properly exposed at the cell surface of S. carnosus. Furthermore, these chimeric receptors were found to be functional, since recombinant S. carnosus cells were shown to have gained IgA and IgE binding capacity, respectively. In addition, a positive effect in terms of IgA and IgE reactivity was observed when dimeric versions of the affibodies were present. Potential applications for recombinant bacteria with redirected binding specificity in their surface proteins are discussed.

Antibodies, Bacterial↗
PubMed1998

Chromosomal sequencing using a PCR-based biotin-capture method allowed isolation of the complete gene for the outer membrane protein A of Klebsiella pneumoniae.

By employing a novel biotin- and PCR-assisted capture method, which allows determination of unknown sequences on chromosomal DNA, the gene for the outer membrane protein A (OmpA) of Klebsiella pneumoniae has been isolated and sequenced to completion. The method involves linear amplification of DNA from a biotinylated primer annealing to a region with known sequence. After capture of the amplified single-stranded DNA on to paramagnetic beads, unspecifically annealing primers, i.e. arbitrary primers, were used to generate sequences with only partly determined nt sequences. The homology of the sequenced gene to ompA of related bacteria is discussed, and the gene fragment was assembled for intracellular expression in Escherichia coli, and two different fusion proteins were produced and recovered with good yields. The importance of the novel chromosomal sequencing method for gene isolation in general and the potential use of the OmpA fusion proteins are discussed.

Amino Acid Sequence↗
PubMed1997

Surface display of the cholera toxin B subunit on Staphylococcus xylosus and Staphylococcus carnosus.

The heterologous surface expression of the cholera toxin B subunit (CTB) from Vibro cholerae in two staphylococcal species, Staphylococcus xylosus and Staphylococcus carnosus, has been investigated. The gene encoding native CTB (103 amino acids) was introduced into gene constructs encoding chimeric receptors designed to be translocated and anchored on the outer cell surface of the staphylococci. Since functionality of CTB is correlated with its ability to form pentamers and the capacity of the pentameric CTB to bind the GM1 ganglioside, both the surface accessibility and the functionality of the surface-displayed CTB receptors were evaluated. It could be concluded that the chimeric receptors were targeted to the cell wall of the staphylococci, since they could be released by lysostaphin treatment and, after subsequent affinity purification, identified as full-length products by immunoblotting. Surface accessibility of the chimeric receptors was demonstrated by a colorimetric assay and by immunofluorescence staining with a CTB-reactive rabbit antiserum. Pentamerization was investigated by using a monoclonal antibody described to be specific for pentameric CTB, and the functionality of the receptors was tested in a binding assay with digoxigenin-labelled GM1. It was concluded that functional CTB was present on both types of staphylococci, and for S. carnosus, the reactivity to the pentamer-specific monoclonal antibody and in the GM1 binding assay was indeed significant. The implications of the results for the design of live bacterial vaccine delivery systems intended for administration by the mucosal route are discussed.

Antibodies, Bacterial↗
PubMed1997

Flow cytometric quantification of surface-displayed recombinant receptors on staphylococci.

Surface display of recombinant proteins on bacteria and phages has become an important tool in bioscience. To evaluate the various host systems, a great need exists for quantitative methods to determine the densities of displayed proteins and peptides on the bacteria and phage surfaces. Here we describe how a method previously applied for quantification of surface proteins on mammalian cells has been adapted for quantification of chimeric receptors surface-displayed on bacteria; in this study, the bacteria being recombinant staphylococci. The presented method takes advantage of fluorescence-activated cell sorting (FACS) technology and a new type of nonfluorescent plastic beads, similar in size (2 microns in diameter) to bacterial cells, and thus suitable for generation of calibration curves from which the number of chimeric receptors can be obtained. The method was used to estimate the number of antigenic sites on two types of recombinant staphylococci, both carrying heterologous chimeric receptors, and it was found that the recombinant Staphylococcus carnosus cells carried approximately 10(4) surface-displayed antigenic sites, while recombinant Staphylococcus xylosus exposed approximately 3 x 10(3) sites per cell. The use of the deviced method for different applications is discussed.

Animals↗
PubMed1996

Surface display on staphylococci: a comparative study.

Two different host-vector expression systems, designed for cell surface display of heterologous receptors on Staphylococcus xylosus and Staphylococcus carnosus, respectively, were compared for the surface display of four variants of a 101 amino acid region derived from the G glycoprotein of human respiratory syncytial virus (RSV). Surface localization of the different chimeric receptors was evaluated by a colorimetric assay and by fluorescence-activated cell sorting. It was concluded that the S. carnosus system was better both in the ability to translocate inefficiently secreted peptides and in the number of exposed hybrid receptors. The potential use of the described staphylococci as live bacterial vaccine vehicles or alternatives to filamentous phages for surface display of protein libraries is discussed.

Flow Cytometry↗
PubMed1996

Surface display of a functional single-chain Fv antibody on staphylococci.

Two different host-vector expression systems designed for cell surface display of chimeric receptors on Staphylococcus xylosus and Staphylococcus carnosus have been evaluated for surface display of a mouse immunoglobulin G1(kappa) [IgG1(kappa)] anti-human IgE single-chain Fv (scFv) antibody fragment. To achieve surface anchoring of the chimeric receptors containing the scFv, the cell surface attachment regions from Staphylococcus aureus protein A were used in both expression systems. The different chimeric receptors could be recovered from cell wall extracts of both S. xylosus and S. carnosus, and surface localization was demonstrated by taking advantage of a serum albumin-binding reporter region present within the two types of receptors. In addition, the two different recombinant staphylococci carrying hybrid receptors containing the scFv were demonstrated to react with the antigen, which was human IgE, in whole-cell enzyme-linked immunosorbent assays. This is the first report of an antibody fragment expressed in a functional form anchored to the surface of gram-positive bacteria. The potential use of recombinant gram-positive bacteria as whole-cell diagnostic devices or alternatives to filamentous phages for surface display of scFv libraries is discussed.

Animals↗
PubMed1996

Cryotherapy after cruciate knee surgery. Skin, subcutaneous and articular temperatures in 8 patients.

We recorded temperature changes on the skin surface, subcutaneously and intra-articularly during cryotherapy after knee surgery by using Cryo-cuff compression dressings. Subcutaneous recordings on the contralateral knee were used as reference. 8 patients were examined. There was a reproducible decrease in skin temperature and subcutaneous temperature. Skin temperature had to be lowered to about 20 degrees C to obtain demonstrable intraarticular temperature changes.

Adolescent↗
PubMed1995

Hydrophobicity engineering to facilitate surface display of heterologous gene products on Staphylococcus xylosus.

Protein engineering has been employed to investigate the effect of specific amino acid changes on the targeting of heterologous proteins to the outer cell surface of the Gram-positive bacterium Staphylococcus xylosus. Three different variants, corresponding to a 101 amino acid region of the major glycoprotein (G protein) of human respiratory syncytial virus (RSV), were generated in which multiple hydrophobic phenylalanine residues were either substituted or deleted. The different G protein fragments were expressed as one part of recombinant receptors designed for surface display on S. xylosus cells. The engineered variants of the RSV G protein hybrid receptors were, in contrast to a non-engineered fragment, efficiently targeted to the outer cell surface of recombinant S. xylosus cells as determined by different methods, including fluorescence-activated cell sorting. In addition, immunization of mice with live recombinant S. xylosus demonstrated that surface exposure was required to generate receptor-specific antibodies. The present strategy of hydrophobic engineering should be of general interest in surface-display applications and for secretion of proteins otherwise difficult to translocate through host cell membranes.

Amino Acid Sequence↗