Biomedical subjects

E Remaut

Publications and source records attributed to E Remaut.

At least 19 recordsLinked to original sources

PubMed2006

Altered gut transcriptome in spondyloarthropathy.

BACKGROUND: Intestinal inflammation is a common feature of spondyloarthropathy (SpA) and Crohn's disease. Inflammation is manifested clinically in Crohn's disease and subclinically in SpA. However, a fraction of patients with SpA develops overt Crohn's disease. AIMS: To investigate whether subclinical gut lesions in patients with SpA are associated with transcriptome changes comparable to those seen in Crohn's disease and to examine global gene expression in non-inflamed colon biopsy specimens and screen patients for differentially expressed genes. METHODS: Macroarray analysis was used as an initial genomewide screen for selecting a comprehensive set of genes relevant to Crohn's disease and SpA. This led to the identification of 2625 expressed sequence tags that are differentially expressed in the colon of patients with Crohn's disease or SpA. These clones, with appropriate controls (6779 in total), were used to construct a glass-based microarray, which was then used to analyse colon biopsy specimens from 15 patients with SpA, 11 patients with Crohn's disease and 10 controls. RESULTS: 95 genes were identified as differentially expressed in patients with SpA having a history of subclinical chronic gut inflammation and also in patients with Crohn's disease. Principal component analysis of this filtered set of genes successfully distinguished colon biopsy specimens from the three groups studied. Patients with SpA having subclinical chronic gut inflammation cluster together and are more related to those with Crohn's disease. CONCLUSION: The transcriptome in the intestine of patients with SpA differs from that of controls. Moreover, these gene changes are comparable to those seen in patients with Crohn's disease, confirming initial clinical observations. On the basis of these findings, new (genetic) markers for detection of early Crohn's disease in patients with SpA can be considered.

Adult↗

PubMed2005

CARD15 polymorphisms are associated with anti-Saccharomyces cerevisiae antibodies in caucasian Crohn's disease patients.

Carriage of CARD15 gene polymorphisms and the serological marker anti-Saccharomyces cerevisiae antibodies (ASCA) are two markers for Crohn's disease (CD). Similar phenotypes have been associated with both markers. In the present study we analysed whether both markers were associated with each other and, if so, whether this association could be explained by a direct link or by an indirect association with those phenotypes. Therefore, we included 156 consecutive Caucasian CD patients and assessed the prevalence of the three common single nucleotide polymorphisms in the CARD15 gene. Serum samples were analysed for IgA and IgG ASCA by ELISA. CD patients with CARD15 polymorphisms were more frequently ASCA positive (OR 2.7 (1.4-5.2); P = 0.002) and had higher titres for ASCA IgA (P = 0.005) and ASCA IgG (P < 0.001) compared to patients carrying the wild type polymorphisms. Multivariate analysis demonstrated that this association was independent from ileal disease, penetrating disease and stricturing disease, the need for resective bowel surgery, familial cases, smoking habits and early age at onset. Homozygotes or compound heterozygotes for CARD15 polymorphisms had significantly more frequent ASCA positivity compared to single heterozygotes (OR 9.1 (1.1-74.2), P(c) (corrected P-value) = 0.030). These data indicate that there is a significant association between the carriage of CARD15 polymorphisms and ASCA, independent of the described phenotypes. Moreover, ASCA positivity is more frequent in CD patients carrying 2 CARD15 polymorphisms compared to single heterozygotes.

Adolescent↗

PubMed2004

CARD15 gene polymorphisms in patients with spondyloarthropathies identify a specific phenotype previously related to Crohn's disease.

BACKGROUND: The association between spondyloarthropathy and Crohn's disease is well known. A risk for evolution to Crohn's disease has already been shown in the subgroup of patients with spondyloarthropathy associated with chronic gut inflammation. OBJECTIVE: To investigate whether the reported polymorphisms in the CARD15 gene, a susceptibility gene for Crohn's disease, are associated with the presence of preclinical intestinal inflammation observed in spondyloarthropathies. METHODS: 104 patients with spondyloarthropathies were studied. All underwent ileocolonoscopy with biopsies between 1983 and 2004. The prevalence of three single nucleotide polymorphisms in the CARD15 gene (R702W, G908R, and 1007fs) was assessed using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR); the patients were compared with an ethnically matched Crohn's disease population and a control population. RESULTS: The carrier frequency of R702W, G908R, or 1007fs variants in the spondyloarthropathy populations (20%) was similar to the control population (17%), but increased to 38% in the spondyloarthropathy subgroup with chronic gut inflammation. This frequency was significantly higher than in the other spondyloarthropathy subgroups (p = 0.001) or the control group (p = 0.006), but not different from the Crohn's disease group (49%) (NS). This indicates that CARD15 polymorphisms are associated with a higher risk for development of chronic gut inflammation. CONCLUSIONS: CARD15 gene polymorphisms clearly identify a subgroup of patients with spondyloarthropathies associated with chronic intestinal inflammation.

Adolescent↗

PubMed2004

Radiological sacroiliitis, a hallmark of spondylitis, is linked with CARD15 gene polymorphisms in patients with Crohn's disease.

BACKGROUND: Sacroiliitis is a common extraintestinal manifestation of Crohn's disease but its association with the HLA-B27 phenotype is less evident. Polymorphisms in the CARD15 gene have been linked to higher susceptibility for Crohn's disease. In particular, associations have been found with ileal and fibrostenosing disease, young age at onset of disease, and familial cases. OBJECTIVES: To investigate whether the presence of sacroiliitis in patients with Crohn's disease is linked to the carriage of CARD15 polymorphisms. METHODS: 102 consecutive patients with Crohn's disease were clinically evaluated by a rheumatologist. Radiographs of the sacroiliac joints were taken and assessed blindly by two investigators. The RFLP-PCR technique was used to genotype all patients for three single nucleotide polymorphisms (SNP) in the CARD15 gene. Every SNP was verified by direct sequencing. The HLA-B27 phenotype was determined. RESULTS: Radiological evidence of sacroiliitis with or without ankylosing spondylitis was found in 23 patients (23%), of whom only three were HLA-B27 positive. In contrast, 78% of patients with sacroiliitis carried a CARD15 variant v 48% of those without sacroiliitis (p = 0.01; odds ratio 3.8 (95% confidence interval, 1.3 to 11.5)). Multivariate analysis (logistic regression) showed that the association between sacroiliitis and CARD15 polymorphisms was independent of other CARD15 related phenotypes (ileal and fibrostenosing disease, young age at onset of disease, familial Crohn's disease) (p = 0.039). CONCLUSIONS: CARD15 variants were identified as genetic predictors of Crohn's disease related sacroiliitis. An association was demonstrated between these polymorphisms and an extraintestinal manifestation of Crohn's disease.

Adolescent↗

PubMed2001

A novel replication element from an Antarctic plasmid as a tool for the expression of proteins at low temperature.

Genetic manipulation of Antarctic bacteria has been very limited so far. This article reports the isolation and molecular characterization of a novel plasmid, pMtBL, from the Antarctic gram-negative bacterium Pseudoalteromonas haloplanktis TAC 125. This genetic element, 4,081 bp long, appeared to be a multicopy cryptic replicon with no detectable transcriptional activity. By an in vivo assay, the pMtBL autonomous replication sequence was functionally limited to an AluI plasmid fragment of about 850 bp. This novel cold-adapted replication element showed quite a broad host range profile: it was cloned into a mesophilic genetic construction, obtaining a cold-adapted expression vector that was able to promote the production of P. haloplanktis A23 alpha-amylase in a psychrophilic bacterium. This study represents the first report of successful recombinant production of a cold-adapted protein in an Antarctic host.

Antarctic Regions↗

PubMed2000

Treatment of murine colitis by Lactococcus lactis secreting interleukin-10.

The cytokine interleukin-10 (IL-10) has shown promise in clinical trials for treatment of inflammatory bowel disease (IBD). Using two mouse models, we show that the therapeutic dose of IL-10 can be reduced by localized delivery of a bacterium genetically engineered to secrete the cytokine. Intragastric administration of IL-10-secreting Lactococcus lactis caused a 50% reduction in colitis in mice treated with dextran sulfate sodium and prevented the onset of colitis in IL-10(-/-) mice. This approach may lead to better methods for cost-effective and long-term management of IBD in humans.

Animals↗

PubMed2000

Secretion of biologically active murine interleukin-10 by Lactococcus lactis.

We investigated the ability of Lactococcus lactis to secrete biologically active, murine interleukin-10 (mIL-10). mIL-10 was synthesized as a fusion protein, consisting of the mature part of the eukaryotic protein fused to the secretion signal of the lactococcal Usp45 protein. The secreted protein was analyzed by PAGE, ELISA and bioassay.We show that L. lactis can efficiently secrete biologically active, murine IL-10. Determination of the N-terminal amino acid sequence confirmed correct processing of the fusion polypeptide by the lactococcal signal peptidase. The amount of mIL-10, accumulating in the medium, could be increased by a factor of ten by growing the cells in an optimized medium, buffered at near-neutral pH. Under these conditions, up to 30 mg of mIL-10 was obtained from a 10-litre fermentation.

Journal Article↗

PubMed1998

Functional display of a heterologous protein on the surface of Lactococcus lactis by means of the cell wall anchor of Staphylococcus aureus protein A.

In this study, we showed that the cell wall anchor of protein A from Staphylococcus aureus is functional in the food-grade organism Lactococcus lactis. A fusion protein composed of the lactococcal Usp45 secretion signal peptide, streptavidin monomer, and the S. aureus protein A anchor became covalently attached to the peptidoglycan when expressed in L. lactis. The streptavidin moiety of the fusion protein was functionally exposed at the cellular surface. L. lactis cells expressing the anchored fusion polypeptide could be specifically immobilized on a biotinylated alkaline phosphatase-coated polystyrene support.

Alkaline Phosphatase↗

PubMed1998

Mucosal delivery of murine interleukin-2 (IL-2) and IL-6 by recombinant strains of Lactococcus lactis coexpressing antigen and cytokine.

Lactococcus lactis is a nonpathogenic and noncolonizing bacterium which is being developed as a vaccine delivery vehicle for immunization by mucosal routes. To determine whether lactococci can also deliver cytokines to the immune system, we have constructed novel constitutive expression strains of L. lactis which accumulate a test antigen, tetanus toxin fragment C (TTFC), within the cytoplasmic compartment and also secrete either murine interleukin-2 (IL-2) or IL-6. When mice were immunized intranasally with various different expression strains of L. lactis, the anti-TTFC antibody titers increased more rapidly and were substantially higher in mice immunized with the bacterial strains which secreted IL-2 or IL-6 in addition to their production of TTFC. This adjuvant effect was lost when the recombinant strains of L. lactis were killed by pretreatment with mitomycin C and could therefore be attributed to the secretion of IL-2 or IL-6 by the recombinant lactococci. These results provide the first example of the use of a cytokine-secreting, noninvasive experimental bacterial vaccine vector to enhance immune responses to a coexpressed heterologous antigen and point the way to experiments which will test the possible therapeutic efficacy of this mode of cytokine delivery.

Administration, Intranasal↗

PubMed1996

The expression of the Photinus pyralis luciferase gene in Staphylococcus aureus Cowan I allows the development of a live amplifiable tool for immunodetection.

We expressed the luc gene, encoding luciferase from Photinus pyralis, in Staphylococcus aureus Cowan I downstream of the plasmid-borne promoter for protein A. Constitutive luciferase synthesis did not impair the growth rate of the host nor did it affect the stability of the plasmid. Light production started immediately after addition of luciferin. The kinetic profile is of the glowing rather than the peak type. Because S. aureus Cowan I produces large quantities of protein A, of which a substantial part becomes covalently attached to rigid cell walls, the bacterial cells could be specifically immobilized on a substrate to which immunoglobulin G molecules were adsorbed either directly or as secondary antibodies. Light production from these cells can be used as a reporter tool for the detection of antigen-antibody complexes. Fourfold amplifications of the emitted signals were obtained by in situ incubation of the bound cells in bacterial growth medium.

Animals↗

PubMed1996

Increased stability of phage T7g10 mRNA is mediated by either a 5'- or a 3'-terminal stem-loop structure.

The mRNA encoding the major capsid protein of phage T7 (T7g10) is highly expressed in Escherichia coli. In common with other highly expressed T7 genes, the 5' end of this mRNA contains a stem-loop structure, while transcription termination at the phage T7 T phi terminator generates a stable 3'-end stem-loop structure. We assessed the influence of these structures on the expression level of T7g10 and on the functional stability of the mRNA. Each one of the 5'- or 3'-hairpin structures was sufficient to increase the functional stability of the T7g10 mRNA more than twofold. A duplication of the 3' T phi-terminator slightly increased the mRNA stability further. Also, differences in the observed functional half-life could be correlated with the expression level of the T7g10 derivatives when these were partially induced. Our data suggest that mRNA stabilization by a 5' stem-loop structure can occur even in the absence of a stem-loop structure that protects RNA against 3' exonucleases.

Bacteriophage T7↗

PubMed1995

Versatile, multi-featured plasmids for high-level expression of heterologous genes in Escherichia coli: overproduction of human and murine cytokines.

We describe the construction, expression characteristics and some applications of a versatile dual-promoter expression plasmid for heterologous gene expression in Escherichia coli which contains both lambda pL and PT7 promoters. Furthermore, the plasmid is optimized to allow the expression of mature coding sequences without compromising the strength of the highly efficient PT7 or of the T7g10 ribosome-binding site. The effect of the the naturally occurring RNA loops at both the 5' and 3' ends of the T7g10 mRNA on expression was also examined. A double T7 RNA polymerase transcription terminator was inserted to ensure more reliable transcription termination and a higher expression level of the preceding gene. Further improvements involve a clockwise orientation of the promoters to minimize read-through transcription from plasmid promoters, a largely extended multiple cloning site, an antisense phage T3 promoter and a phage f1-derived, single-stranded replication origin. Variants of this plasmid allow for the production of fusion proteins with part of T7g10, a hexahistidine peptide and an enterokinase recognition site. The potential of these expression vectors is demonstrated by comparing the expression levels of a number of mammalian cytokines (human tumor necrosis factor, human immune interferon, human and murine interleukins 2, murine interleukin 4 and murine fibroblast interferon), using these expression plasmids.

Amino Acid Sequence↗

PubMed1995

Production in Escherichia coli of a functional murine and murine::human chimeric F(ab')2 fragment and mature antibody directed against human placental alkaline phosphatase.

We report the production in Escherichia coli of a murine antibody IgG2b, a murine::human chimeric antibody IgG3 and the corresponding F(ab')2 fragments, all directed against human placental alkaline phosphatase, a tumor marker. The cDNA of the heavy chain of the mature antibodies and their fragments were linked up to the bacterial alkaline phosphatase signal sequence and were placed under control of the inducible tac promoter. Coexpression with the murine kappa light chain resulted in production of functional dimeric, trimeric and tetrameric, mature antibodies and F(ab')2 fragments in the periplasm of E. coli in a yield of 200-300 micrograms l-1. High amounts of light and heavy chains were present also in the insoluble fraction.

Alkaline Phosphatase↗

PubMed1995

Production of soluble and active recombinant murine interleukin-2 in Escherichia coli: high level expression, Kil-induced release, and purification.

We describe the production of soluble murine interleukin-2 (mIL2) and its purification following regulated release in the growth medium of Escherichia coli. The system is based on the ability of the Kil protein of pMB9 to release periplasmic proteins into the growth medium. As the kil gene is under control of the strong, but well regulatable pL promoter, the kil bearing plasmid is stably maintained in the cell. mIL2, fused to the outer membrane protein A (OmpA) signal peptide, was secreted into the periplasm and subsequently released into the growth medium after induction of the kil gene. This strategy allows a quick and easy purification of the heterologous protein without using strong denaturants or detergents, yielding a native protein with a specific biological activity equal to the natural mIL2. The system permits the production of mIL2 at levels up to 16 mg/liter. From a 12-liter fermentation, a final yield of about 30 mg of pure mIL2 was obtained.

Animals↗

PubMed1995

Tight transcriptional control mechanism ensures stable high-level expression from T7 promoter-based expression plasmids.

One of the more efficient systems for high-level expression of cloned genes in Escherichia coli makes use of a phage T7 late promoter whose activity depends on a regulatable transcription unit supplying the specific T7 RNA polymerase. Using various T7 RNA polymerase/T7 promoter-based vector host systems with differential control on expression of the T7 RNA polymerase, we document that leaky expression of the latter is responsible for the frequently observed loss of the culture's ability to express genes of interest. We further show that the inability to achieve detectable expression levels can be overcome by using a tightly repressed expression system. We describe a novel and efficient control system in which basal level expression of T7 RNA polymerase is attenuated by a series of tandemly arranged transcription terminators. The plasmids also incorporate the phage lambda-derived nutL/N protein antitermination function, allowing conditional reversion of attenuation upon induction. The applicability of the system is illustrated by the strictly regulatable, high-level production of several cytokines of human and murine origin.

Animals↗

PubMed1995

Secretion of biologically active murine interleukin-2 by Lactococcus lactis subsp. lactis.

Secretion of functional recombinant murine interleukin-2 (mIL2) by Lactococcus lactis was achieved by fusion of the sequence encoding mature mIL2 to the secretion signal leader of the lactococcal usp45 gene placed under transcriptional control of the phage T7 promoter-T7 RNA polymerase expression system. The recombinant mature mIL2 was one of only a few proteins which accumulated in the growth medium. Sequence analysis revealed correct processing at the first amino acid of the mature protein. A T-cell proliferation assay showed that the recombinant protein has the same specific biological activity as mIL2 obtained from a natural source.

Amino Acid Sequence↗

PubMed1993

Folding of the MS2 coat protein in Escherichia coli is modulated by translational pauses resulting from mRNA secondary structure and codon usage: a hypothesis.

Possible translational pauses within the coat protein of the RNA bacteriophage MS2 were located on the basis of a distribution plot of rare codons and RNA secondary structure. It appeared that the position of certain codon pauses corresponds with the size of some nascent polypeptide intermediates, which have been isolated from MS2-infected cells. Other accumulated polypeptide intermediates seemed to be related to RNA regions, where double-stranded secondary structures occur, which probably impede the movement of ribosomes during chain elongation. We assume that a discontinuous translation rate is designed to allow optimal folding of this (and other) polypeptide(s).

Capsid↗

PubMed1993

High-level expression, purification, and renaturation of recombinant murine interleukin-2 from Escherichia coli.

A murine interleukin-2 (mIL-2)-encoding cDNA, isolated from a stimulated EL4 mRNA library, was used to construct several expression plasmids directing synthesis of the mature protein in Escherichia coli. The expression was under control of either the PTrp or the PL promoter. Using these systems, a high-level expression of between 10 and 35% of the total cellular protein was obtained. The mIL-2 protein, present as insoluble inclusion bodies, could be solubilized in a chaotropic mixture and was partially purified by preparative gel filtration under denaturing conditions. After renaturation, the protein was further purified to homogeneity by anion-exchange chromatography. Depending on the fermentation, induction, and renaturation conditions, the yield ranged between 0.35 and 1 mg of purified mIL-2/g wet cells. The specific biological activity was about 10(7) units/mg and the endotoxin content < 4 ng/mg pure recombinant protein.

Amino Acid Sequence↗