Biomedical subjects

S Thiel

Publications and source records attributed to S Thiel.

At least 37 records · Page 2Linked to original sources

PubMed2003

Recombinant mannan-binding lectin (MBL) for therapy.

Mannan-binding lectin (MBL) is a plasma protein involved in the innate immune response. It binds to a number of micro-organisms and promotes killing of these through complement activation either directly or through opsonization. Clinical evidence indicates that in a variety of situations genetically determined low MBL levels are associated with increased susceptibility to infections. Infusions of plasma-derived MBL into MBL-deficient individuals was found to be safe in preliminary trials, but we considered that sufficient production and product safety could only be achieved through synthesis of recombinant MBL. A transfected human cell line produces MBL showing the same biological activity as plasma-derived MBL, and an essentially identical profile on MS. The production has been scaled up and clinical trials will start this year.

Animals↗

PubMed2003

Umbilical cord mannan-binding lectin and infections in early childhood.

To investigate whether mannan-binding lectin (MBL) in umbilical cord blood (UCB) is associated with the incidence rate of hospitalizations for infections during early childhood. A follow-up study from birth to 31 months of age, with endpoint data from the Danish National Hospital Discharge Registry. The concentration of MBL and immunoglobulin A (IgA) in UCB was measured in a time-resolved immunofluorometric assay. Information on possible confounding factors was obtained from questionnaires completed by mothers during their pregnancy, and characteristics of the child at birth were registered by a midwife on a structured coding sheet. A total of 2104 infants born in the period between 1 February 1990 and 25 May 1991 to mothers living in the municipality of Aarhus. Concentration of MBL and IgA in UCB, gestational age, birth weight, number of siblings less than 3 years old, mother's smoking habits, alcohol and coffee consumption, educational and marital status and previous spontaneous abortions and stillbirths. Of 2104 children, 626 were hospitalized at least once, and 346 of these were hospitalized with infection. The hazard ratio (HR) of hospitalization with infections in children with low levels of MBL (<120 ng/ml) was 1.4 (95% confidence interval (CI), 1.0-1.8), which was similar to our findings on hospitalization because of other diseases. The HR of hospitalization for viral infections was 2.8 (CI, 1.3-5.9). Low levels of MBL in UCB were associated with hospitalization in general and particularly with hospitalization for viral infections.

Adult↗

PubMed2003

On the site of C4 deposition upon complement activation via the mannan-binding lectin pathway or the classical pathway.

The mannan-binding lectin (MBL) pathway and the classical pathway of complement activation are initiated by the binding of the recognition structure of the initiator complexes, MBL and C1q, respectively, to their ligands, i.e. carbohydrate structures or immune complexes. Proenzymes associated with MBL or C1q are then activated and generate C3 convertase through the activation of C4 and C2. The cleavage product of C4, C4b, attaches covalently to nearby hydroxyl or amino groups. The current picture is that C2 must then attach to C4b before being cleaved by the same associated proteases into the enzymatically active fragment, C2b. This suggests a stringent requirement for the deposition of C4b very close to the initiator complex, or indeed onto the initiator complex. We examined the possibility of C4b being bound to the initiator complex by a solid-phase assay, allowing for the selective elution of the initiator complexes, followed by quantification of the C4b being eluted and the C4b remaining on the solid phase. Also, we estimated the generation of complexes between the released initiator complex and C4b. More than 99% of deposited C4b was bound directly to the solid phase rather than to the initiator complex. Our approach cannot answer the question of the whereabouts of the C2 when it is cleaved.

Binding Sites↗

PubMed2001

An assay for the mannan-binding lectin pathway of complement activation.

The mannan-binding lectin (MBL) pathway of complement activation has been established as the third pathway of complement activation. MBL is a carbohydrate-binding serum protein, which circulates in complex with serine proteases known as mannan-binding lectin associated serine proteases (MASPs). When bound to microorganisms, the MBL complex activates the complement components C4 and C2, thereby generating the C3 convertase and leading to opsonisation by the deposition of C4b and C3b fragments. This C4/C2 cleaving activity is shared with the C1 complex of the classical pathway of complement activation. Therefore, in a generally applicable complement activation assay specific for the MBL pathway, the activity of the classical pathway must be inhibited. This can be accomplished by exploiting the finding that high ionic strength buffers inhibit the binding of C1q to immune complexes and disrupt the C1 complex, whereas the carbohydrate-binding activity of MBL and the integrity of the MBL complex is maintained under hypertonic conditions. In the assay described here, the specific C4b-depositing capacity of the MBL pathway was determined by incubating serum diluted in buffer containing 1 M NaCl in mannan-coated microtiter wells before the addition of purified C4. The interassay coefficient of variation in the ELISA version was 7.3%. As expected no activity was found in MBL-deficient serum. When 100 normal serum samples were analysed we found that the MBL level correlated with the amount of C4b deposited on the mannan-coated surface. However, we also found a threefold variation in C4b-depositing capacity between individuals with similar MBL concentrations. The assay permits for the determination of MBL complex activity in serum and plasma samples and may thus be used to evaluate the clinical implications of complement activation via this pathway.

Carrier Proteins↗

PubMed2001

Association between deficiency of mannose-binding lectin and severe infections after chemotherapy.

The plasma protein mannose-binding lectin (MBL) activates the complement system by binding to carbohydrate structures presented by microorganisms and thus could be an important component of the innate immune defence system. We measured MBL in patients with leukaemia who were scheduled to undergo chemotherapy (ie, a population especially susceptible to infection) and related the results to severity of infection after chemotherapy. We showed a significant association between low concentrations of MBL and serious infections related to chemotherapy (p<0.0001). These results suggest that increasing concentrations of MBL in patients having chemotherapy could reduce susceptibility to infection.

Adult↗

PubMed2001

Three-dimensional ab initio quantum dynamics of the photodesorption of CO from Cr(2)O(3)(0001): stereodynamic effects.

Having performed the first three-dimensional ab initio quantum dynamical study of photodesorption from solid surfaces, we gained mechanistic understanding of the rotational alignment observed in the CO/Cr(2)O(3)(0001) system. Our study is based on potential energy surfaces obtained by embedded cluster calculations for both the electronic ground and excited state of the adsorbate substrate complex. Stochastic wave packet calculations demonstrate the importance of the angular degrees of freedom for the microscopic picture of the desorption process in addition to the desorption coordinate.

Journal Article↗

PubMed2001

Interaction properties of human mannan-binding lectin (MBL)-associated serine proteases-1 and -2, MBL-associated protein 19, and MBL.

The mannan-binding lectin (MBL) activation pathway of complement plays an important role in the innate immune defense against pathogenic microorganisms. In human serum, two MBL-associated serine proteases (MASP-1, MASP-2) and MBL-associated protein 19 (MAp19) were found to be associated with MBL. With a view to investigate the interaction properties of these proteins, human MASP-1, MASP-2, MAp19, as well as the N-terminal complement subcomponents C1r/C1s, Uegf, and bone morphogenetic protein-1-epidermal growth factor (CUB-EGF) segments of MASP-1 and MASP-2, were expressed in insect or human kidney cells, and MBL was isolated from human serum. Sedimentation velocity analysis indicated that the MASP-1 and MASP-2 CUB-EGF segments and the homologous protein MAp19 all behaved as homodimers (2.8-3.2 S) in the presence of Ca(2+). Although the latter two dimers were not dissociated by EDTA, their physical properties were affected. In contrast, the MASP-1 CUB-EGF homodimer was not sensitive to EDTA. The three proteins and full-length MASP-1 and MASP-2 showed no interaction with each other as judged by gel filtration and surface plasmon resonance spectroscopy. Using the latter technique, MASP-1, MASP-2, their CUB-EGF segments, and MAp19 were each shown to bind to immobilized MBL, with K:(D) values of 0.8 nM (MASP-2), 1.4 nM (MASP-1), 13.0 nM (MAp19 and MASP-2 CUB-EGF), and 25.7 nM (MASP-1 CUB-EGF). The binding was Ca(2+)-dependent and fully sensitive to EDTA in all cases. These data indicate that MASP-1, MASP-2, and MAp19 each associate as homodimers, and individually form Ca(2+)-dependent complexes with MBL through the CUB-EGF pair of each protein. This suggests that distinct MBL/MASP complexes may be involved in the activation or regulation of the MBL pathway.

Amino Acid Motifs↗

PubMed2001

Altered DNA repair capacity and bleomycin sensitivity as risk markers for non-small cell lung cancer.

DNA repair capacity in human peripheral blood lymphocytes was monitored by the repair rate of bleomycin-induced DNA damage using an alkaline single-cell gel electrophoresis assay (comet assay). DNA repair capacity, after 15 min repair time, in lymphocytes of non-small cell lung cancer patients (n = 160) and controls (n = 180) was 67% and 79.3%, respectively (p < 0.0004). Bleomycin sensitivity defined as the tail moment of bleomycin-treated peripheral blood lymphocytes, without allowing time for DNA repair, was significantly higher in lung cancer patients than in tumor-free hospital controls (p < 0.0001). There was no correlation, in either patient or control group, between the bleomycin sensitivity and DNA repair capacity with age or gender. The median values of DNA repair capacity and sensitivity in controls were used as the cut-off points for calculating odds ratios (OR). After adjustment for age, gender and smoking status, the cases vs. controls had reduced DNA repair capacity (OR = 2.1; 95% confidence limit [CL] 1.1-4.0) and increased bleomycin sensitivity (OR = 4; 95% CL 2.2-7.4). For current smokers, the adjusted risk associated with bleomycin sensitivity was 2.3 (95% CL 1.1-4.9). We conclude that our standard comet assay as a phenotypical repair test has sufficient sensitivity and rapidity allowing application to both native and cryopreserved lymphocytes. Bleomycin sensitivity and DNA repair capacity were found to be 2 independent susceptibility markers for non-small cell lung cancer, confirming similar investigations with different marker end points. The latter were much more time consuming than the method used in our study. Thus, the comet assay is more suitable for screening large numbers of individuals in epidemiological studies. Validation of this assay in large prospective studies for the identification of subjects at high risk for non-small cell lung cancer is now warranted.

Adult↗

PubMed2001

Tail-vein injection of mannan-binding lectin DNA leads to high expression levels of multimeric protein in liver.

The human plasma protein mannan-binding lectin (MBL) is an essential part of the innate immune defense system. Low levels of MBL are associated with recurrent infections and other clinically significant signs of a compromised immune defense. Previous studies have addressed the possibility of reconstitution therapy by the use of recombinant or plasma-derived protein. Natural MBL is a multimeric protein, which consists of up to 18 identical polypeptide chains. Synthesis by in vitro methods of MBL with the proper multimeric structure is difficult. We here report that mice obtain MBL levels comparable to those found in normal human plasma when injected with an MBL expression construct as naked plasmid DNA contained in a large volume of physiologic salt solution. The expression was confined to the liver and high MBL expression levels were obtained with less than 5% of the liver cells transfected. The multimeric structure of the MBL found in plasma of injected mice was similar to that of natural MBL. Thus, liver expression following injection of naked DNA is an alternative to reconstitution therapy with a protein having a complex quaternary structure.

Animals↗

PubMed2001

Swallowing function after endoscopic resection of supraglottic carcinoma with the carbon dioxide laser.

From April 1998 to May 2000, 14 patients with supraglottic cancer underwent transoral laser surgery (T-stage T1-2: 11 patients, T3: 3 patients). In three patients, an epiglottectomy or hemi-epiglottectomy was performed. In 11 patients, further structures (false cords, the valleculae and the base of the tongue and/or parts of the arytenoid cartilage) had to be resected. Thirteen patients had to undergo neck dissection and post-operative irradiation. Tracheostomy was carried out prophylactically in two cases. Every patient received a nasogastric tube perioperatively. One week after surgery, an evaluation of dysphagia was performed by video endoscopy (VEED). Aspiration was the main problem; in no case did dysphagia occur. The aspiration was graded according to videolaryngoscopical classification. Four patients had an occasional and ten patients a permanent aspiration after surgery. According to this assessment, an individual deglutition therapy management was established. Ten patients with permanent aspiration received a temporary percutaneous endoscopic gastrostomy (PEG) and were integrated in a rehabilitation programme (stimulation of the swallowing reflex, training of compensatory swallowing manoeuvres, dietary regime). Due to this training programme, the PEG could be removed in eight patients after 2-9 months. No patient needed a laryngectomy or a tracheostomy due to aspiration. There were no cases of aspiration-associated pneumonia. To obtain satisfying functional results after transoral laser surgery of supraglottic cancers with resection of the epiglottis, post-operative deglutition management, consisting of video endoscopy, a training programme and often a PEG, is necessary.

Adult↗

PubMed2001

The mannan-binding lectin pathway of complement activation: biology and disease association.

Mannan-binding lectin (MBL) is a plasma protein found in association with several serine proteases (MASPs) forming the MBL complex. MBL recognises carbohydrate structures arranged in a particular geometry, such as those found on the surface of micro-organisms. When bound to e.g. bacteria the MBL complex will initiate the activation of the complement cascade. Mounting evidence supports the importance of the MBL pathway of complement activation in innate immunity. In this review, we focus on the structure and function of the proteins within the MBL pathway and address the properties of the pathway as an initiator of the host response against potential pathogenic micro-organisms.

Amino Acid Sequence↗

PubMed2001

The mannan-binding-lectin pathway of the innate immune response.

Dramatic progress has been achieved during the past year in our understanding of how the complement system is activated via the mannan-binding-lectin pathway. Surprising discoveries have changed our concepts of the complexes that are formed upon engagement of mannan-binding lectin with its serine proteases.

Animals↗

PubMed2001

MASP-3 and its association with distinct complexes of the mannan-binding lectin complement activation pathway.

The mannan-binding lectin (MBL) pathway of complement activation is part of the innate immune defense. The binding of MBL to microbial carbohydrates activates the MBL-associated serine proteases (MASPs), which recruit the complement factors, C4 and C2, to generate the C3 convertase or directly activate C3. We present a phylogenetically highly conserved member of the MBL complex, MASP-3, which is generated through alternative splicing of the MASP-1/3 gene. The designation of MASP-3 as a protease is based on homology to known MASPs. Different MBL oligomers were found to have distinct MASP composition and biological activities. MASP-1, MAp19, and direct C3-cleaving activity are associated with smaller oligomers whereas MASP-3 is found together with MASP-2 on larger oligomers. MASP-3 downregulate the C4 and C2 cleaving activity of MASP-2.

Alternative Splicing↗

PubMed2001

Recombinant expression of human mannan-binding lectin.

Mannan-binding lectin (MBL) constitutes an important part of the innate immune defence by effecting the deposition of complement on microbial surfaces. MBL deficiency is among the most common primary immunodeficiencies and is associated with recurrent infections and symptoms of poor immune complex clearance. Plasma-derived MBL has been used in reconstitution therapy but concerns over viral contamination and production capacity point to recombinant MBL (rMBL) as a future source of this protein for clinical use. Natural human MBL is an oligomer of up to 18 identical polypeptide chains. The synthesis of rMBL has been accomplished in several mammalian cell lines, however, the recombinant protein differed structurally from natural MBL. In this, study we compare rMBL produced in myeloma cells, Chinese hamster ovary (CHO) cells, human hepatocytes, and human embryonic kidney (HEK) cells. We report that rMBL structurally and functionally similar to natural MBL can be obtained through synthesis in the human embryonic kidney cells followed by selective carbohydrate affinity chromatography.

Carrier Proteins↗

PubMed2001

The human gene for mannan-binding lectin-associated serine protease-2 (MASP-2), the effector component of the lectin route of complement activation, is part of a tightly linked gene cluster on chromosome 1p36.2-3.

The proteases of the lectin pathway of complement activation, MASP-1 and MASP-2, are encoded by two separate genes. The MASP1 gene is located on chromosome 3q27, the MASP2 gene on chromosome 1p36.23-31. The genes for the classical complement activation pathway proteases, C1r and C1s, are linked on chromosome 12p13. We have shown that the MASP2 gene encodes two gene products, the 76 kDa MASP-2 serine protease and a plasma protein of 19 kDa, termed MAp19 or sMAP. Both gene products are components of the lectin pathway activation complex. We present the complete primary structure of the human MASP2 gene and the tight cluster that this locus forms with non-complement genes. A comparison of the MASP2 gene with the previously characterised C1s gene revealed identical positions of introns separating orthologous coding sequences, underlining the hypothesis that the C1s and MASP2 genes arose by exon shuffling from one ancestral gene.

Base Sequence↗

PubMed2001

Characterization and quantification of mouse mannan-binding lectins (MBL-A and MBL-C) and study of acute phase responses.

Rat monoclonal antibodies (MoAbs) against mouse mannan-binding lectin (MBL)-A and MBL-C were generated and assays for MBL-A and MBL-C were constructed. This allowed for the quantitative analysis of both proteins for the first time. Previously only MBL-A has been quantified using less standardized methods. In a mouse serum pool the concentrations were now determined at 7.5 microg MBL-A and 45 microg MBL-C per ml. On gel permeation chromatography of mouse serum, MBL-A eluted corresponding to a M(r) of 850 kDa whereas the majority of MBL-C eluted corresponding to a Mr of 950 kDa. On sucrose density gradient centrifugation the sedimentation velocities of MBL-A and MBL-C were estimated at 7.3 S and 10.8 S, respectively. The MBL-A and MBL-C levels in 10 laboratory mice strains were compared and found to vary between 4 microg/ml to 12 microg/ml, and 16 microg/ml to 118 microg/ml, respectively. After the induction of acute phase responses by intraperitoneal injection of either casein or lipopolysaccharide (LPS), MBL-A was found to increase approximately two-fold, with a maximum after 32 h, while MBL-C did not increase significantly. In comparison, serum amyloid A component (SAA) peaked at 15 h with an approximate 100-fold increase.

Acute-Phase Proteins↗

PubMed2001

Rapid screening assay for mutagen sensitivity and DNA repair capacity in human peripheral blood lymphocytes.

Individual susceptibility to carcinogens, an important determinant of disease risk, is influenced by host factors such as the ability to repair DNA lesions. In order to identify subjects who are at high risk, we have developed a microgel electrophoresis assay for use in molecular epidemiological studies. The assay was validated in a pilot case-control study: Peripheral blood lymphocytes were collected from 100 patients with lung cancer and 110 control patients without cancer and from the same hospital, and stored at -80 degrees C. After thawing, phytohaemagglutinin-stimulated cells were treated with bleomycin at 20 microg/ml for 30 min and the extent of DNA damage and DNA repair capacity were determined by microgel electrophoresis. Peripheral blood lymphocytes from patients with lung cancer were significantly more sensitive to mutagens than those from controls and showed reduced DNA repair capacity (both P < 0.001). Both endpoints were independent risk factors for smoking-related lung cancer. Repeated analysis of peripheral blood lymphocytes from the same individual demonstrated good reproducibility of the assay. Cryopreservation of the lymphocytes for less than or = 12 months did not significantly affect their sensitivity. Our standardized microgel electrophoresis assay is suitable for determining individual sensitivity to mutagens and DNA repair capacity: it is sensitive and faster than cytogenetic assays, and can be applied to native and cryopreserved peripheral blood lymphocytes.

Adult↗

PubMed2001

Determination of the calibration factor of polysulphone film UV dosemeters for terrestrial solar radiation.

Polysulphone film is used as a personal UV dosemeter in dermatological or epidemiological studies. The relative efficiency of this detector does not exactly match the action spectrum as proposed by the International Commission on Non-Ionizing Radiation Protection (ICNIRP) and to which the UV dose and exposure limits refer. Therefore, the calibration of the dosemeter depends on the spetrum. In the present paper the variation of the calibration factor for terrestrial solar UV spectra is analysed on the basis of a two year observation period at a site near Munich. Germany. A detailed error estimation is included. It is shown that the variation of the calibration factor within this class of spectra is the main contribution to the total uncertainty of the dose determination, which can be up to 40%. The shape of the spectrum of terrestrial solar UV radiation is mainly determined by the total ozone column and the solar elevation angle. It is shown how the calibration depends on these two parameters and how this additional information can help to reduce the measurement error to a residual uncertainty of 17%. Exposure studies of terrestrial solar UV radiation using polysulphone film as a dosemeter would gain in accuracy if total ozone column values at the study's site could be measured or taken from satellite or weather service data. The interpretation of the magnitude of the dose uncertainty depends on the further use of these data.

Biocompatible Materials↗