Biomedical subjects
Publications and source records attributed to Claus Koch.
Vaccination is a simple and effective way to protect against certain infectious diseases and is nearly always to be recommended when one is travelling to countries with lesser hygienic standards. This report provides guidance on immunization concerns and describes the individual vaccines most commonly used in travel medicine.
BACKGROUND: Individuals genetically deficient of properdin are more susceptible to meningococcal disease. Likewise low concentration or decreased biological activity of mannose-binding lectin (MBL) is associated with higher incidence of bacterial infections during childhood. In this study we report our findings in a Danish family with a remarkably high incidence of meningococcal meningitis-in total four cases, one of them fatal. METHODS: Properdin and MBL were quantified by ELISA and the properdin gene was screened for sequence variations using denaturing high-performance liquid chromatography (DHPLC) and subsequent sequencing of abnormal patterns. The MBL gene was genotyped for the three known variant alleles (B, C and D) as well as three promoter polymorphisms (-221Y/X, -550H/L and +4P/Q). RESULTS: Two out of six males with undetectable properdin activity had meningitis. They had also low MBL serum levels or carried an MBL variant allele, whereas high MBL concentrations were measured in three out of four properdin deficient males--without meningitis. A splice site mutation in exon 10 (c.1487-2A>G) was found in the properdin gene and co segregated with biochemically measured properdin deficiency. CONCLUSION: Our results indicate that a combined deficiency of both properdin and MBL increases the risk of infection with Neisseria meningitidis and stress the importance of epistatic genetic interactions in disease susceptibility.
Activation of small intestinal gluten-reactive CD4+ T cells is a critical event in celiac disease. Such cells predominantly recognise gluten peptides in which specific glutamines are deamidated. Deamidation may be catalysed by intestinal tissue transglutaminase (TG2), a protein which is also the main autoantigen in celiac disease. Our aim was to study how the two main catalytic activities of transglutaminase--deamidation and transamidation (cross-linking) of an immunodominant gliadin epitope--are influenced by the presence of acceptor amines in the intestinal mucosa, and thereby contribute to further elucidation of the pathogenetic mechanisms in celiac disease. We prepared monoclonal antibodies, reacting specifically with the non-deamidated epitope QPFPQPQLPYPQPQ-amide and/or the deamidated epitope QPFPQPELPYPQPQ-amide. A solid phase immunoassay combined with gel filtration chromatography was used to analyse deamidation and cross-linking of these peptides to proteins. Our results show that QPFPQPQLPYPQPQ-amide was deamidated when incubated with purified TG2, with fresh mucosal sheets and with mucosal homogenates. Of other transglutaminases tested, only Streptoverticillium transglutaminase was able to generate the deamidated epitope. A fraction of the non-deamidated epitope was cross-linked to proteins, including TG2. The results suggest that intestinal TG2 is responsible for generation of the active deamidated epitope. As the epitope often occurs in a repeat structure, the result may be cross-linking of a deamidated, i.e., activated cell epitope. Alternatively, the deamidation may occur by reversal of the cross-linking reaction. The results provide a basis for the suggestion that binding of a peptide to a protein, in connection to its modification to a T cell epitope, might be a general explanation for the role of TG2 in celiac disease and a possible mechanism for the generation of autoantigens.
BACKGROUND: The aim of this study was to develop site-specific antibodies as a tool to capture Plasmodium falciparum-dihydrofolate reductase (Pf-DHFR) from blood samples from P. falciparum infected individuals in order to detect, in a sandwich ELISA, structural alterations due to point mutations in the gene coding for Pf-DHFR. Furthermore, we wanted to study the potential use of homology models in general and of Pf-DHFR in particular in predicting antigenic malarial surface epitopes. METHODS: A homology model of Pf-DHFR domain was employed to define an epitope for the development of site-specific antibodies against Pf-DHFR. The homology model suggested an exposed loop encompassing amino acid residues 64-100. A synthetic peptide of 37-mers whose sequence corresponded to the sequence of amino acid residues 64-100 of Pf-DHFR was synthesized and used to immunize mice for antibodies. Additionally, polyclonal antibodies recognizing a recombinant DHFR enzyme were produced in rabbits. RESULTS AND CONCLUSIONS: Serum from mice immunized with the 37-mer showed strong reactivity against both the immunizing peptide, recombinant DHFR and a preparation of crude antigen from P. falciparum infected red blood cells. Five monoclonal antibodies were obtained, one of which showed reactivity towards crude antigen prepared from P. falciparum infected red cells. Western blot analysis revealed that both the polyclonal and monoclonal antibodies recognized Pf-DHFR. Our study provides insight into the potential use of homology models in general and of Pf-DHFR in particular in predicting antigenic malarial surface epitopes.
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.
A sandwich ELISA for quantification of the endometrial protein PP14 revealed false positive reactions in 81% of male sera (n = 54). The PP14 ELISA was based on two monoclonal antibodies (Mabs) with different epitope specificities--a catcher and a biotinylated indicator. The monoclonal antibodies were purified by protein G affinity chromatography from culture supernatant containing 10% (v/v) fetal calf serum (FCS). Human anti-animal IgG (bovine, mouse, horse, and swine) antibodies and human anti-bovine serum albumin antibodies were measured using an ELISA design, with direct bridging of the solid phase and biotinylated antigens. The false positive reactions were abolished by addition of 1% (v/v) bovine serum to the dilution buffer (DB). Human anti-bovine IgG antibodies (HABIA) were detected in 99 out of 104 sera from blood donors (50 females; 54 males). HABIA levels in male sera (n = 54) were positively correlated to the false positive signals in the PP14 ELISA (r = 0.923; p < 0.0001). Antibodies to IgG from other mammalian species (mouse, horse, and swine) were also detected in the donor sera, but levels and frequencies were lower compared to that of HABIA. Furthermore, HABIA were positively correlated to human anti-bovine serum albumin antibodies in the donor sera (r = 0.639; p < 0.0001; n = 103). HABIA (prevalence 95%) cause false positive reactions due to crossbinding of contaminating bovine IgG and/or crossreaction with mouse IgG in two-site immunoassays. The apparent presence of human anti-mouse IgG antibodies (HAMA), described to create false positive results, may be due to a crossreacting fraction of the polyclonal circulating antibodies against bovine IgG.
This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious advantages using this assay, are that it can be performed directly on culture supernatants in the early phase of monoclonal antibody production, and also works for antigens with repetitive epitopes. Moreover, the bonus effect, i.e., a signal in excess of the reference signal when sets of monoclonal antibodies with different epitope specificity are compared, gives a relative measure of affinity.
Lung surfactant protein A (SP-A) is a collectin produced by alveolar type II cells and Clara cells. It binds to carbohydrate structures on microorganisms, initiating effector mechanisms of innate immunity and modulating the inflammatory response in the lung. Reverse transcriptase-polymerase chain reaction was performed on a panel of RNAs from human tissues for SP-A mRNA expression. The lung was the main site of synthesis, but transcripts were readily amplified from the trachea, prostate, pancreas, and thymus. Weak expression was observed in the colon and salivary gland. SP-A sequences derived from lung and thymus mRNA revealed the presence of both SP-A1 and SP-A2, whereas only SP-A2 expression was found in the trachea and prostate. Monoclonal antibodies were raised against SP-A and characterized. One of these (HYB 238-4) reacted in Western blotting with both reduced and unreduced SP-A, with N-deglycosylated and collagenase-treated SP-A, and with both recombinant SP-A1 and SP-A2. This antibody was used to demonstrate SP-A in immunohistochemistry of human tissues. Strong SP-A immunoreactivity was seen in alveolar type-II cells, Clara cells, and on and within alveolar macrophages, but no extrapulmonary SP-A immunoreactivity was observed. In contrast to lung surfactant protein D (SP-D), which is generally expressed on mucosal surfaces, SP-A seems to be restricted to the respiratory system.
The serum opsonin mannose-binding lectin (MBL) has been shown to be involved in the handling of apoptotic cells. However, at what stage in the process this happens and whether this mediates activation of complement is unknown. Cells rendered apoptotic or necrotic were incubated with purified MBL/MBL-associated serine protease (MASP) complexes and assessed by flow cytometry and fluorescence microscopy. MBL bound specifically to late apoptotic cells, as well as to apoptotic blebs and to necrotic cells, but not to early apoptotic cells. Binding of MBL could be inhibited by EDTA as well as with an antibody against the CRD region. Addition of C1q, another serum opsonin involved in the handling of apoptotic cells, prior to MBL partly inhibited MBL binding to apoptotic cells and vice versa. MBL/MASP could initiate deposition of purified complement C4 on the target cells. However, addition of MBL/MASP to whole serum deficient for both C1q and MBL did not enhance deposition of C4, but MBL enhanced phagocytosis of apoptotic cells by macrophages. These results demonstrate that MBL interacts with structures exposed on cells rendered late apoptotic or necrotic and facilitates uptake by macrophages. Thus, MBL may promote non-inflammatory sequestration of dying host cells.
There is an emerging interest for mannose-binding lectin (MBL) due to its role in innate immunity. In this survey we present a mixture of old and new data describing the effect MBL polymorphisms may have on the level and function of the molecule. Three single nucleotide substitutions in exon 1 of the mbl2 gene cause a dominant decrease of functional MBL in the circulation. Additionally, promoter variants influence expression of MBL. It has been assumed that the structural variant alleles may disrupt the assembly of MBL trimers or accelerate the degradation of the protein, thereby causing the decrease in MBL serum concentrations. We have analysed 1183 different sera in a double sandwich antibody ELISA using the same antibody to capture and detect MBL and find the same results as have been presented previously showing that different MBL promoter alleles have profound effect of on the MBL serum concentration. The use of a new anti-MBL monoclonal antibody, however, has shown that the amount of MBL in the circulation is less dependent on the presence of structural variant alleles than previously anticipated. Molecular characterisation of MBL revealed that sera from donors homozygous for the normal MBL genotype predominantly contained high molecular weight MBL, while sera from individuals heterozygous for the variant alleles contained both high and low molecular weight MBL. The ratio between high and low molecular weight MBL was dependent on the MBL promoter type on the normal haplotype. Sera deriving from individuals homozygous for MBL variant alleles contained mainly low molecular weight MBL. Of the different oligomers of MBL only the high molecular weight forms bound mannan efficiently and activated complement. In contrast to a previous notion, we demonstrate that variant alleles give rise to relatively high levels of MBL in the circulation. However, the variant MBL has lower molecular weight and is dysfunctional compared to normal MBL. The physiological relevance of variant MBL remains to be established.
SP-D is a lectin involved in the first line of defense against microorganisms. It is primarily found in the lung but also at extrapulmonary sites and in the circulation. An immunoassay for the quantification of SP-D in serum was established and the SP-D concentration was measured in consecutive blood samples from 61 patients hospitalized for community-acquired pneumonia of suspected bacterial origin. On the day of admission to the hospital the serum SP-D concentration was significantly lower than that in healthy subjects. On day 5, the SP-D concentration had increased on average three times the concentration on admission and then slowly declined toward normal levels. CRP was measured simultaneously but no correlation was observed between concentrations of SP-D and CRP. The results show a wide range of serum SP-D concentration in healthy volunteers and indicate that significant changes occur during pulmonary infection.
PURPOSE: To estimate the clinical significance of low serum concentrations of mannose-binding lectin (MBL) in patients with acute myeloid leukaemia (AML) during initial cancer chemotherapy. PATIENTS AND METHODS: 80 consecutive, newly diagnosed, and unselected AML patients (age 18-77 yr) undergoing remission induction chemotherapy. The patients were examined for 28 d. MAIN FINDINGS: Low levels of serum MBL (<1,000 microg/L) were found in 16/80 patients at diagnosis. This frequency is similar to what is found in the general population. In the remaining 64 patients, MBL concentrations were significantly higher than in controls and showed only a slight rise during the period of antineoplastic chemotherapy with its associated infectious complications. Low levels of MBL did not affect overall survival or morbidity in terms of incidence or duration of fever, or occurrence of septicaemia or pneumonia. Long-term survival was likewise independent of MBL concentration. CONCLUSION: MBL levels have no discernible influence on the occurrence or course of infections in AML patients during the initial hospitalisation. The predominant immunodeficiency during this phase is the profound granulocytopenia, which also compromises important effector functions of MBL. The finding in most AML patients of elevated MBL concentrations on admission is most likely because of the role of MBL as an acute phase reactant.
Mannan-binding lectin (MBL) is a collectin synthesized by the liver and secreted into the bloodstream. It has a receptor for microbial structures in its C-type lectin domain and a separate receptor(s) located within its collagen-like region for autologous phagocytic cells. Here we demonstrate that human peripheral blood adherent cells (monocytes) and monocyte-derived dendritic cells are a source of MBL, and that a novel calcium-dependent and sugar-specific MBL receptor is up-regulated in immature (CD1a-positive) dendritic cells. These findings suggest a previously unsuspected autologous function for MBL, perhaps a regulatory role within the immune system.
Deficiency of mannose-binding lectin has been shown to be a risk factor for cystic fibrosis (CF) patients. We, therefore, decided to treat a patient with CF, mannose-binding lectin deficiency, severe bronchopulmonary Pseudomonas aeruginosa infection, and rapid deterioration of lung function with purified mannose-binding lectin in an attempt to ameliorate the course of the lung disease. The mannose-binding lectin used originated from pooled human donor plasma and was given as an intravenous infusion twice a week for a period of 3 months. The patients's clinical condition was stabilized during the treatment period, but was not improved. No adverse events were observed. However, the lung function assessed as percent forced expiratory volume in 1 sec (FEV1%) and percent forced vital capacirt (FVC%) correlated significantly with the mannose-binding serum lectin levels (rho=+0.68, P=0.008, and rho=+0.73, P=0.004). Additionally, an inverse correlation with the acute phase-reactant C-reactive protein and the proinflammatory cytokine IL-6 was observed (rho=-0.49, P=0.007 and rho=-0.41, P=0.04, respectively). The results emphasize the importance of mannose-binding lectin as a secondary disease modifier in CF. Moreover, purified mannose-binding lectin can safely be administered to chronically ill patients, and may be a potential treatment in CF and other diseases in which mannose-binding lectin deficiency plays a pathophysiological role.