Biomedical subjects

Hans O Madsen

Publications and source records attributed to Hans O Madsen.

At least 19 recordsLinked to original sources

PubMed2006

Influence of candidate susceptibility genes on tuberculosis in a high endemic region.

Genetic susceptibility towards clinical tuberculosis has been proposed in several population studies. We investigated whether polymorphisms in the candidate genes encoding solute carrier 11a1 protein (SLC11A1 formerly NRAMP1), mannose-binding lectin (MBL2) and Vitamin D receptor (VDR) were associated with tuberculosis in an East-African setting. Four hundred and forty-three culture positive pulmonary tuberculosis patients and 426 controls from Mwanza district in the northern part of Tanzania were prospectively included. Polymorphisms in the candidate genes were detected by different PCR-based techniques. A significant association between pulmonary tuberculosis and a microsatellite marker in the 5'(CA)n locus in the SLC11A1 gene compared with controls (38% versus 30% odds ratio 1.45, 95% CI: 1.06-1.9, P=0.014) was observed. The association was apparent only in HIV negative tuberculosis patients. No association with tuberculosis was seen with 3 other SLC11A1 loci investigated, which previously have been associated with tuberculosis in other populations or with MBL2 and VDR polymorphisms. The tuberculosis associated microsatellite marker was situated on different SLC11A1 haplotypes. In this cohort a microsatellite marker in the 5'(CA)n locus situated in the SLC11A1 gene was associated with tuberculosis. The observed association was seen only in HIV negative patients suggesting that this genetic susceptibility for tuberculosis may be surpassed by co-infections.

Adolescent↗

PubMed2006

Multiple inflammatory markers in patients with significant coronary artery disease.

BACKGROUND: Several inflammatory biomarkers are linked to cardiovascular risk. In order to investigate their coexistence and relative responses, several established and two novel markers (lactoferrin and the terminal complement complex), representing infection and central components of inflammation, were measured simultaneously in patients undergoing first-time coronary angiography. METHODS AND RESULTS: Blood samples from patients with (n=131) or without (n=103) significant coronary artery stenosis were analyzed for plasma markers representing endothelium, platelets, neutrophils, monocytes, and complement, C-reactive protein, and antibodies against the infectious agents Chlamydia pneumoniae, Helicobacter pylori, and cytomegalovirus. In multivariate logistic regression analysis, hypercholesterolemia (p<0.001), increased concentrations of the neutrophil activation marker lactoferrin (p<0.001) and the monocyte activation marker neopterin (p=0.012), lower concentrations of the terminal complement complex (p<0.001), and antibodies against C. pneumoniae (p=0.023) were variables linked to coronary artery stenosis. In univariate analysis additional relationships were found to current smoking (p<0.001), increased plasma concentrations of vascular cell adhesion molecule-1 (p=0.015), E-selectin (p<0.01), myeloperoxidase (p=0.051) and endothelin-1 (p=0.053), as well as diabetes (p=0.039). CONCLUSIONS: Activation of multiple inflammatory pathways and C. pneumoniae infection may influence the inflammatory response in atherosclerosis. These pilot data provide an indication of the relative usefulness of various inflammatory biomarkers, indicating that the novel markers lactoferrin and the terminal complement complex warrant further investigation.

Analysis of Variance↗

PubMed2006

[Chromosome changes associated with childhood leukaemia occur prenatally].

Leukaemia is the most common cancer in childhood, yet only a few risk factors have been identified. Studies of monozygotic twins with concordant leukaemia and retrospective analyses of neonatal blood spots from children with leukaemia indicate that chromosomal translocations characteristic of childhood leukaemia often occur prenatally. The chromosomal translocations may be initiators of the leukaemia development but per se are insufficient to cause the disease. The findings provide a basic understanding of the natural history of childhood leukaemia and may make the development of preventive measures feasible.

Child, Preschool↗

PubMed2006

Molecular organization of human Ficolin-2.

Human Ficolin-2 (L-Ficolin) is an oligomeric serum protein consisting of a collagen-like stalk and fibrinogen-like recognition domains. The protein binds to arrays of sugars present on different microorganisms, enhances phagocytosis and promotes activation of the lectin complement pathway. So far the detailed oligomeric structure and composition of human Ficolin-2 has not been determined. Recombinant human Ficolin-2 was expressed in Chinese hamster ovary cells and its structure and biological functions were investigated by gel filtration, sucrose density gradient ultracentrifugation, mass spectrometry and surface plasmon resonance spectroscopy. It was revealed that Ficolin-2 has a high molecular weight due to extensive disulfide bridge formation. It was able to bind to different ligands, interact with mannose-binding lectin associated serine proteases and activate the complement system. Mass values of 807 and 403 kDa were determined corresponding to a 24-mer and a 12-mer of 34.4 kDa polypeptides. However, the 24-mer was unstable and the 12-mer is likely the major functional form of the protein. Our results are consistent with the view that Ficolin-2 is built up by a mixture of stable homodimers and homotrimers. Based on our findings we propose a model in which disulfide bridges located in the N-terminal region of the polypeptides explain the oligomerization pattern of human Ficolin-2.

Animals↗

PubMed2006

Infections during induction therapy of childhood acute lymphoblastic leukemia--no association to mannose-binding lectin deficiency.

OBJECTIVES: Infection during the induction phase of childhood acute lymphoblastic leukemia (ALL) is a major cause of morbidity and mortality. Several studies have indicated that genetically determined low serum levels of mannose-binding lectin (MBL), a component of innate immunity, are associated with increased risk for infections in patients receiving chemotherapy. Thus, these patients have been proposed to be candidates for MBL replacement therapy. METHODS: In a population-based cohort of 137 children with ALL treated at a single pediatric hematology-oncology center with an almost identical chemotherapy regimen, we studied the relationship between polymorphisms in the MBL gene (MBL2) and the MBL2 promoter and the risk of infections during the first 50 d of induction therapy. RESULTS: No increased frequency of infection was seen for the children with genotypes encoding serum low levels of MBL. A higher incidence of fever (P < 0.004), infectious events (P = 0.025), days with neutropenia (P < 0.001) and a higher frequency of antimicrobial therapy (P = 0.0007) were seen in the young age group (<2.5 yr) compared with the older age group (> or =2.5 yr), independent of the MBL genotype. CONCLUSIONS: MBL deficiency did not influence the frequency of infections in children receiving induction chemotherapy for ALL, not even in the youngest children (<2.5 yr) whom we found to have the highest risk for infections.

Adolescent↗

PubMed2006

Cytokine gene expression in peripheral blood mononuclear cells and alloreactivity in hematopoietic cell transplantation with nonmyeloablative conditioning.

Cytokines are thought to play an important role in the pathophysiology of graft-versus-host disease (GVHD). To study the relationship between cytokines and GVHD, we obtained peripheral blood mononuclear cells (MNCs) from 21 patients with hematologic malignancies and their HLA-identical sibling donors before and sequentially after hematopoietic cell transplantation (HCT) with nonmyeloablative conditioning. The MNCs were cultured for 72 hours either alone or in mixed lymphocyte cultures with irradiated MNCs of recipient, donor, or HLA-mismatched third-party origin. The gene expression of interleukin (IL)-2, IL-4, IL-10, IL-18, tumor necrosis factor alpha, and transforming growth factor beta in each culture was then measured by real-time quantitative reverse transcriptase-polymerase chain reaction. The composition of the responder MNCs differed between patients and donors and changed after HCT, with a possible influence on the results. Early after transplantation (day +14), the IL-10 messenger RNA (mRNA) level in response to recipient or donor antigens was higher in patients who did not develop clinically significant acute GVHD when compared with the level in patients who subsequently developed acute GVHD grades II to IV (P = .005 and P = .004, respectively). The IL-10 mRNA level on day +14 was highly correlated with the pretransplantation mRNA level of the recipient MNCs but not with the level of the donor MNCs; this suggests that the IL-10 mRNA detected on day +14 originated from responder cells of recipient origin. A higher IL-10 mRNA level was found in MNCs obtained before transplantation from recipients whose disease progressed or relapsed after the transplantation when compared with the level in patients whose disease did not progress or relapse (P = .03). In conclusion, a high IL-10 gene expression in the recipient MNCs may be related to a reduced incidence of acute GVHD grades II to IV and a reduced graft-versus-tumor effect after HCT with nonmyeloablative conditioning.

Adult↗

PubMed2006

Preleukemic TEL-AML1-positive clones at cell level of 10(-3) to 10(-4) do not persist into adulthood.

The TEL-AML1 translocation, t(12;21)(p13;q22), is one of the most frequent genetic aberrations in childhood B-cell precursor acute lymphoblastic leukemia (ALL), where it occurs in 25% of all cases. In contrast, the translocation is seen in only 3% of adult ALL cases. Evidence suggests that the TEL-AML1 translocation occurs in utero in 1% of all newborn children at cell levels of 10 to 10. In this study, we explore the prevalence of TEL-AML1-positive cells in 2 cohorts of healthy blood donors by real-time and nested reverse transcription-polymerase chain reaction. Overall, TEL-AML1-positive cells were demonstrated in 10 of 2005 healthy donors, that is, a prevalence of 0.5% (95% confidence interval, 0.2-0.3%). The level of TEL-AML1-positive cells was estimated to 10 to 10. The observed prevalence of TEL-AML1-positive cells in healthy adults is of the same order of magnitude as the prevalence reported in healthy newborns, but the observed cell level of 10 to 10 is much lower. These data indicates that prenatal TEL-AML1 subclones does not persist throughout adult life at cell levels of 10 to 10. The findings are compatible with the risk of t(12;21)(p13;q22) ALL correlating with the total number of TEL-AML1-positive cells in peripheral blood in both childhood and adulthood.

Adult↗

PubMed2006

Elevated complement C3 is associated with early restenosis after eversion carotid endarterectomy.

Early restenosis following carotid endarterectomy (CEA) is an inflammatory process leading to myointimal hyperplasia of smooth muscle cells. The risk for restenosis is increased in homozygous carriers of the normal (A) allele of mannose-binding lectin (MBL2) gene. Our objective was to study the associations of C3 and as control three non-complement acute-phase reactants (APRs) (C-reactive protein, haptoglobin and alpha2HSglycoprotein) with early restenosis following CEA. We also considered, whether MBL2 genotype relates to C3 levels and to the risk of restenosis. Concentrations of the APRs were determined by radial immunodiffusion or immunoturbidimetric methods in 64 patients who underwent eversion CEA and were followed up with carotid duplex scan (CDS) examinations for at least one year. MBL2 genotypes were determined by a PCR-SSP method. C3 levels increased during the follow-up and correlated with the percentage of restenosis detected by CDS at 14 months postsurgery, in MBL2 A/A allele carriers. Patients with high C3 levels had nearly five-fold higher odds for the presence of significant restenosis (>50% reduction in diameter) even after adjusting for MBL2 genotype, age and gender. By contrast, no such associations were detected between the non-complement APRs and early restenosis. C3 is associated with and might have a direct role in the development of an early restenosis following CEA, which is partially related to an intact MBL lectin pathway, thus determining C3 levels might have clinical importance. On the other hand, our results indicate that the regulation of C3 differs from non-complement APRs.

Adult↗

PubMed2005

The 'involution' of mannose-binding lectin.

Mannose-binding lectin (MBL) acts as a serum opsonin in innate immune defense and induces complement activation by the lectin pathway. In humans, low levels of functional serum MBL are caused by the dominant action of three single nucleotide substitutions in exon 1 that disrupt the glycine-rich backbone structure of the protein. The presence of common MBL variant alleles is associated with both infectious and autoimmune diseases. Conversely, it has also been suggested that MBL variants are maintained because of selective advantages for the host. In man, the MBL genetic system comprises one functional gene (MBL2) and one expressed pseudogene (MBL1P1), whereas the lower primate, the rhesus monkey resembles rodents with two functional MBL genes. We have investigated the molecular mechanisms behind the evolutionary loss of MBL expression from lower primates to man, including silencing of the MBL1P1 gene and the generation of MBL2 variant structural alleles and promoter polymorphisms leading to the present human MBL2 haplotypes. We present data showing that the MBL1P1 gene has been repeatedly hit throughout evolution and silenced eventually by mutations in the glycine residues of the collagen-like region. Our results indicate that the MBL1P1 gene has been selectively turned off during evolution through the same molecular mechanisms causing the MBL2 variant alleles in man, suggesting an evolutionary selection for low-producing MBL genes.

Alleles↗

PubMed2005

Extra-hepatic transcription of the human mannose-binding lectin gene (mbl2) and the MBL-associated serine protease 1-3 genes.

Mannose-binding lectin (MBL) is a part of the innate immune defense and activates complement via MBL associated serine proteases (MASPs). Human MBL is expressed by hepatocytes, but recent evidence in mice indicates a substantial extra-hepatic transcription. Therefore, we investigated whether mRNA transcribed from the human mbl2 gene as well as the masp genes was present in different tissues. The transcription of human mbl2 is regulated by two alternative promoters (named 0 and 1) where promoter 0 derived transcripts include an additional 5' untranslated part encoded by an extra exon (exon 0). Low extra-hepatic levels of mbl2 mRNA were predominantly found in small intestine and testis tissue, and were quantitatively dominated by promoter 1 transcripts. Moreover, these transcripts varied due to the use of alternative acceptor splice sites positioned inside exon 1. The mRNA distribution of masp1 and masp2 were found very similar to that of mbl2, while masp3 mRNA seemed ubiquitous present at quite high levels when compared to liver. These results indicate that the regulation of mbl2 gene expression in man is more complex than previously anticipated and that local expression of MBL may be relevant in local immune defense, particularly in restricted areas of the gastrointestinal and reproductive tracts.

Alternative Splicing↗

PubMed2005

A functional polymorphism in the Eta-1 promoter is associated with allele specific binding to the transcription factor Sp1 and elevated gene expression.

Early T lymphocyte activator 1 (Eta-1), also known as Osteopontin, is a cytokine produced by macrophages and T lymphocytes. It is involved in the regulation of IL-12 and IL-10 expression in macrophages and stimulates the polarization of T cells to the Th1 subset. Three promoter polymorphisms of the human Eta-1 gene, -443T/C, -156delG/G, -66T/G, were investigated for possible influence on gene expression. Electrophoretic mobility shift assays (EMSA) with nuclear extract from the human myeloid leukaemia premonocyte cell line, THP-1, revealed sequence specific binding of the transcription factor Sp1 to the -66T allele but not the -66G allele, and haplotype -443C/-156G/-66T showed a marked increase in promoter activity of a luciferase reporter gene. Thus, a substitution of the T-base with G at position -66 in the Eta-1 promoter modulates the promoter activity of the Eta-1 gene, which might influence the Th1 versus Th2 balance. These observations are discussed in relation to a recently reported related observation on the same gene, and it is argued that discrepancies between reporter gene assays in the two studies may be due to the use of different cell lines and may reflect requirements for different transcription factors in cells involved in immune responses compared with other cells.

Alleles↗

PubMed2005

Polymorphisms in the FCN2 gene determine serum variation and function of Ficolin-2.

The ficolin 1, 2 and 3 (derived from the FCN1, 2 and 3 genes, respectively) are homologous soluble pattern recognition molecules of importance for innate immunity, comprising collagen-like and fibrinogen-like domains, binding to sugar groups on different types of microorganisms. Serum concentration of Ficolin-2 varies considerably in healthy individuals. Thus, we speculated whether this could be due to variations in the FCN2 gene. We sequenced the promoter region and the exons and intron-exon boundaries of FCN2 in Danish Caucasians. For comparison, FCN1 and FCN3 were also investigated. Ficolin-2 concentrations were measured in serum and the functional relevance of amino acid substituting polymorphisms in FCN2 was investigated by binding to and recovery from N-acetylglucosamine (GlcNAc). Both FCN1 and FCN2 contained polymorphisms in the promoters and structural parts of the genes, but only polymorphisms in FCN2 resulted in amino acid exchanges. FCN2 promoter polymorphisms were associated with marked changes in the Ficolin-2 serum concentration, whereas two polymorphisms clustered in the exon encoding the fibrinogen-like domain were associated with increased and decreased GlcNAc binding, respectively. In FCN3, only a single frame-shift deletion in exon 5 was detected. These results show that the FCN genes are polymorphic and that particularly FCN2 harbors functional polymorphic sites that regulate both the expression as well as the function of Ficolin-2, which may have pathophysiological implications for innate immunity.

Acetylglucosamine↗

PubMed2005

High rate of early restenosis after carotid eversion endarterectomy in homozygous carriers of the normal mannose-binding lectin genotype.

BACKGROUND AND PURPOSE: Mannose-binding lectin (MBL) is thought to influence the pathophysiology of cardiovascular disease by decreasing the risk of advanced atherosclerosis and by contributing to enhanced ischemia reperfusion injury. Thus, we investigated the role of MBL in restenosis after eversion endarterectomy in patients with severe carotid atherosclerosis. METHODS: In a prospective study, 123 patients who underwent carotid endarterectomy were followed-up by carotid duplex scan (CDS) sonography for 14 months. In a retrospective study, we examined 17 patients and 29 patients, respectively, who had or had not at least 50% restenosis 29 months after carotid eversion endarterectomy. MBL genotypes were analyzed by a polymerase chain reaction-based method, and MBL serum concentrations were measured. RESULTS: In the prospective study in the patients homozygous for the normal MBL genotype, CDS values were significantly higher after 14 months of follow-up compared with the values measured 6 weeks after surgery (P<0.001). In contrast, only a slight increase was registered in patients carrying MBL variant alleles. The differences were much more pronounced in female than in male patients. Similar differences were observed when patients with high and low MBL serum concentrations were compared. In the retrospective study, a significant increase in the frequency of MBL variant genotypes was observed in patients not experiencing restenosis compared with the patients with restenosis (P=0.007). CONCLUSIONS: These results indicate that reoccurrence of stenosis after carotid endarterectomy is partially genetically determined and imply that MBL contributes significantly to the pathophysiology of this condition.

Aged↗

PubMed2005

A common polymorphism in the SFTPD gene influences assembly, function, and concentration of surfactant protein D.

Surfactant protein D (SP-D) plays important roles in the host defense against infectious microorganisms and in regulating the innate immune response to a variety of pathogen-associated molecular pattern. SP-D is mainly expressed by type II cells of the lung, but SP-D is generally found on epithelial surfaces and in serum. Genotyping for three single-nucleotide variations altering amino acids in the mature protein in codon 11 (Met(11)Thr), 160 (Ala(160)Thr), and 270 (Ser(270)Thr) of the SP-D gene was performed and related to the SP-D levels in serum. Individuals with the Thr/Thr(11)-encoding genotype had significantly lower SP-D serum levels than individuals with the Met/Met(11) genotype. Gel filtration chromatography revealed two distinct m.w. peaks with SP-D immunoreactivity in serum from Met/Met(11)-encoding genotypes. In contrast, Thr/Thr(11) genotypes lacked the highest m.w. form. A similar SP-D size distribution was found for recombinant Met(11) and Thr(11) expressed in human embryonic kidney cells. Atomic force microscopy of purified SP-D showed that components eluting in the position of the high m.w. peak consist of multimers, dodecamers, and monomers of subunits, whereas the second peak exclusively contains monomers. SP-D from both peaks bound to mannan-coated ELISA plates. SP-D from the high m.w. peak bound preferentially to intact influenza A virus and Gram-positive and Gram-negative bacteria, whereas the monomeric species preferentially bound to isolated LPS. Our data strongly suggest that polymorphic variation in the N-terminal domain of the SP-D molecule influences oligomerization, function, and the concentration of the molecule in serum.

Alleles↗

PubMed2005

Evaluation and automation of hematopoietic chimerism analysis based on real-time quantitative polymerase chain reaction.

Abstract Chimerism analysis is an essential tool in the follow-up of patients after allogeneic stem cell transplantation. High-resolution methods for chimerism analysis based on real-time quantitative polymerase chain reaction (RQ-PCR) with a detection limit of 0.1% marker-specific cells are especially valuable in the detection of patient-derived subpopulations for the monitoring of minimal residual disease. Using artificial chimeric mixtures of genotypically different cells, we optimized and evaluated the intrasample variation, accuracy, and detection limit of chimerism analysis based on RQ-PCR of short insertion and deletion polymorphisms. Furthermore, automated setup by robot was evaluated. The results were accurate, with acceptable intrasample variation at and above 0.1% marker-specific cells. The sensitivity was mainly limited by background values. Chimerism results based on RQ-PCR were similar to results based on PCR of short tandem repeats when samples from recipients of transplants with nonmyeloablative conditioning were analyzed. Furthermore, automated setup was feasible in a time-, labor-, and reagent-conserving manner.

Evaluation Studies as Topic↗

PubMed2005

Locked nucleic acid inhibits amplification of contaminating DNA in real-time PCR.

Locked nucleic acid (LNA) is a modified DNA with increased binding affinityfor complementary DNA sequences. Our strategy was to use this property of LNA to inhibit undesired PCR amplification (e.g.,from contaminating genomic DNA) in a cDNA-based assay. By placing a short complementary LNA sequence in intronic DNA, the aim was to inhibit the amplification of genomic DNA without affecting the amplification of reverse-transcribed spliced mRNA. LNA was designed to bind within intron 5 in the x-box binding protein 1 (XBP1) gene. An irrelevant LNA oligonucleotide served as a negative control. In both PCR and real-time PCR, the addition of LNA showed blocking of the amplification of genomic XBP1 but not cDNA XBP1. To test the effect of melting temperature (Tm) on the LNA, we investigated the number of LNA nucleotides that could be replaced with DNA nucleotides and still retain the blocking activity. More than three DNA nucleotides reduced the LNA inhibition ability. The sequence specificity of the LNA was tested by investigating the number of LNA nucleotide mismatches permitted. The introduction of one mismatch maintained the inhibition of genomic amplification whereas two mismatches reduced the amplification. Our results show that LNA may be used to enhance the specificity of PCR by eliminating unwanted PCR products.

Base Pair Mismatch↗

PubMed2004

Mannose-binding lectin variant alleles and the risk of arterial thrombosis in systemic lupus erythematosus.

BACKGROUND: Cardiovascular disease is an important complication in patients with systemic lupus erythematosus (SLE). Variant alleles of the mannose-binding lectin gene are associated with SLE as well as with severe atherosclerosis. We determined whether mannose-binding lectin variant alleles were associated with an increased risk of arterial thrombosis among patients with SLE. METHODS: Mannose-binding lectin alleles were genotyped by means of a polymerase-chain-reaction assay in 91 Danish patients with SLE. Arterial and venous thromboses occurring after the diagnosis of SLE were assessed in a prospective study. Arterial and venous thromboses were confirmed by appropriate diagnostic methods. RESULTS: Fifty-four patients had no mannose-binding lectin variant alleles (A/A genotype), 30 were heterozygous (A/O genotype), and 7 were homozygous (O/O genotype). During a median follow-up of 9.1 years, arterial thromboses (cerebral or myocardial infarction or leg embolus) developed in 6 of the 7 patients with the O/O genotype, as compared with 18 of the 84 patients with the other two genotypes (hazard ratio, 5.8; 95 percent confidence interval, 2.2 to 15.2; overall incidence, 26 percent). After correction for other known risk factors, the hazard ratio was 7.0 (95 percent confidence interval, 1.9 to 25.4). Venous thromboses, which occurred in 14 patients, were statistically unrelated to the mannose-binding lectin genotype. CONCLUSIONS: Among patients with SLE, homozygosity for mannose-binding lectin variant alleles is associated with an increased risk of arterial thrombosis. The risk of venous thrombosis is not increased, indicating that mannose-binding lectin has a specific role in providing protection against arterial thrombosis.

Adolescent↗

PubMed2004

Disease-associated mutations in human mannose-binding lectin compromise oligomerization and activity of the final protein.

Deficiency of human mannose-binding lectin (MBL) caused by mutations in the coding part of the MBL2 gene is associated with increased risk and severity of infections and autoimmunity. To study the biological consequences of MBL mutations, we expressed wild type MBL and mutated MBL in Chinese hamster ovary cells. The normal MBL cDNA (WT MBL-A) was cloned, and the three known natural and two artificial variants were expressed in Chinese hamster ovary cells. When analyzed, WT MBL-A formed covalently linked higher oligomers with a molecular mass of about 300-450 kDa, corresponding to 12-18 single chains or 4-6 structural units. By contrast, all MBL variants formed a dominant band of about 50 kDa, with increasingly weaker bands at 75, 100, and 125 kDa corresponding to two, three, four, and five chains, respectively. In contrast to WT MBL-A, variant MBL formed noncovalent oligomers containing up to six chains (two structural units). MBL variants bound ligands with a markedly reduced capacity compared with WT MBL-A. Mutations in the collagenous region of human MBL compromise assembly of higher order oligomers, resulting in reduced ligand binding capacity and thus reduced capability to activate complement.

Amino Acid Sequence↗