Search PubMed⌕ Search

Biomedical subjects

D H Krüger

Publications and source records attributed to D H Krüger.

At least 19 recordsLinked to original sources
PubMed2005

Detection of human hantavirus infections in Lithuania.

BACKGROUND: In Europe certain hantaviruses are known to cause hemorrhagic fever with renal syndrome of different severity. The objective of the present investigation was to study the presence of hantavirus infections in Lithuania. MATERIAL AND METHODS: Two different serum panels from cancer patients (n = 438) and blood donors (n = 299) from Lithuania were tested by monoclonal antibody capture IgG ELISA using yeast-expressed recombinant nucleocapsid (rN) proteins of Puumala virus (PUUV), Hantaan virus (HTNV) and Dobrava virus (DOBV). The reactivity of ELISA-positive sera was proven in Western blot tests using various hantavirus rN proteins. Selected serum samples were further analyzed by focus reduction neutralization assays. RESULTS: In the IgG ELISA 39 sera from the cancer patients and four sera from blood donors were found to be reactive with at least one of the rN proteins. By immunoblot using the three yeast-expressed rN proteins, the ELISA reactivity of 36 of 39 and two of four serum samples from cancer patients and blood donors, respectively, was confirmed; this corresponds to a seroprevalence of 8.2% and 0.7%, respectively. In ELISA, the majority of the samples reacted exclusively with rN proteins of HTNV and DOBV (31 of 36 and one of two in the two groups). In the group of sera selected for serotyping by focus reduction neutralization assay, this dominance was confirmed by the identification of eight DOBV but only four PUUV infections. No infection by HTNV or another hantavirus besides DOBV and PUUV was verified. Anti-hantavirus-positive human sera were detected in all seven investigated counties of Lithuania. CONCLUSION: In Lithuania at least two hantaviruses, DOBV and PUUV, circulate and cause human infections. Additional investigations are needed to study the seroprevalence more precisely and to search for clinical cases of hantavirus infections.

Antibodies, Viral↗
PubMed2004

[Prevalence of hantavirus infections in Germany].

Hantaviruses belong to the group of "emerging" viruses. Pathogenic European hantaviruses can cause a human disease designated "hemorrhagic fever with renal syndrome" of varying severity. In general, diagnostics of hantavirus infections are based on immunofluorescence assays using virus-infected cells or enzyme immunoassays and Western blot tests using recombinant nucleocapsid proteins. For highly sensitive detection of hantavirus-specific antibodies in the enzyme immunoassay, a homologous hantavirus nucleocapsid protein is needed as a diagnostic antigen. Serological typing of hantavirus infections can be obtained by neutralization assays, which in certain cases require the use of late convalescent sera. The seroprevalence in the normal German population is about 1%. In professionally exposed risk groups, e. g., forest workers, a seroprevalence higher than that in the normal population was observed. Endemic regions for hantavirus infections are located mainly in Baden-Württemberg. In the years 2001-2003 an annual number of about 200 clinically apparent hantavirus infections were registered in Germany. Neutralization assays detected almost exclusively human infections caused by Puumala and Dobrava viruses, only very rarely by Tula virus. Until this day in Germany mainly mild to moderate courses of human hantavirus infections have been documented. Besides infections caused by "German" hantaviruses, up to 10% of the clinically apparent hantavirus infections registered annually in Germany are caused by infections imported from other countries, mainly from Europe. So far only very limited molecular genetic data about the circulating hantaviruses in Germany are available. Additional investigations are needed to get a more precise picture about the distribution of hantaviruses in Germany and to calculate the resulting risk for the human population.

Communicable Disease Control↗
PubMed2004

Yeast-expressed hantavirus Dobrava nucleocapsid protein induces a strong, long-lasting, and highly cross-reactive immune response in mice.

In Europe, Dobrava virus (DOBV) carried by the yellow-necked field mouse Apodemus flavicollis is one of the hantaviruses that can cause severe hemorrhagic fever with renal syndrome in humans. For several hantaviruses, the nucleocapsid (N) protein has proven to be very immunogenic in humans and rodents and even can protect rodents against a virus challenge. To investigate the immunogenicity of DOBV N protein, BALB/c and C57BL/6 mice were immunized three times with a DOBV recombinant N (rN) protein expressed in yeast Saccharomyces cerevisiae together with complete Freund's, with incomplete Freund's, and without adjuvant, respectively. Mice of both strains elicited N-specific antibodies with end-point titers being as high as 1:1,000,000 in C57BL/6 mice. The antibodies induced by DOBV rN protein were highly cross-reactive to the rN proteins of hantaviruses Puumala and Hantaan. In both mice strains, DOBV rN protein induced N-specific antibodies of all IgG subclasses (IgG1, IgG2a, IgG2b, and IgG3), suggesting a mixed Th1/Th2 immune response. Taken together, yeast-expressed DOBV rN protein represents a promising vaccine candidate.

Animals↗
PubMed2004

Life-threatening Dobrava hantavirus infection with unusually extended pulmonary involvement.

In Europe, hantavirus infections usually present as hemorrhagic fever with renal syndrome and its mild form nephropathia epidemica, while clinical cases with severe pulmonary affections are extremely rare and appear to be confined to infections by New World hanta viruses in the Americas. We report on a female patient from Northern Germany, who suffered primarily from severe acute respiratory distress syndrome-like pulmonary failure due to Dobrava hantavirus infection that was complicated by acute renal insufficiency.

Female↗
PubMed2002

Mapping of B-cell epitopes in the nucleocapsid protein of Puumala hantavirus.

Hantavirus nucleocapsid protein (N) has been proven to induce highly protective immune responses in animal models. The knowledge on the mechanisms behind N-induced protection is still limited, although recent data suggest that both cellular and humoral immune responses are of importance. For a detailed B-cell epitope mapping of Puumala hantavirus (PUUV) N, we used recombinant N derivatives of the Russian strain CG18-20 and the Swedish strain Vranica/Hällnäs, as well as overlapping synthetic peptides corresponding to the Finnish prototype strain Sotkamo. The majority of a panel of monoclonal antibodies (mAbs) reacted with proteins derived from all included PUUV strains demonstrating the antigenic similarity of these proteins. In line with previous results, the epitopes of most mAbs were mapped within the 80 N-terminal amino acids of N. The present study further revealed that the epitopes of four mAbs raised against native viral N were located within amino acids 14-45, whereas one mAb raised against recombinant N was mapped to amino acids 14-39. Differences between the reactivity of the PUUV strains Vranica/Hällnäs and CG18-20 N suggested the importance of amino acid position 35 for the integrity of the epitopes. In line with the patterns obtained by the truncated recombinant proteins, mapping by overlapping peptides (PEPSCAN) confirmed a complex recognition pattern for most analyzed mAbs. Together, the results revealed the existence of several, partially overlapping, and discontinuous B-cell epitopes. In addition, based on differences within the same competition group, novel epitopes were defined.

Amino Acid Sequence↗
PubMed2001

DNA vaccination of mice with a plasmid encoding Puumala hantavirus nucleocapsid protein mimics the B-cell response induced by virus infection.

Inoculation of naked DNA has been applied for the development of prophylactic and therapeutic vaccines against different viral infections. To study the humoral immune response induced by DNA vaccination we cloned the entire nucleocapsid protein-encoding sequence of the Puumala hantavirus strain Vranica/Hällnäs into the CMV promoter-driven expression unit of the plasmid pcDNA3, generating pcDNA3-VR1. A single dose injection of 50 microg of plasmid DNA into each M. tibialis anterior of BALB/c mice induced a high-titered antibody response against the nucleocapsid protein as documented 6 and 11 weeks after immunisation. PEPSCAN analysis of a serum pool of the pcDNA3-VR1-vaccinated animals revealed antibodies reacting with epitopes covering the whole nucleocapsid protein. The epitope-specificity of the immune response induced by DNA vaccination seems to reflect the antibody response in experimentally virus-infected bank voles (the natural host of the Puumala virus) and humans. The data suggest that DNA vaccination could be used for the identification of highly immunogenic epitopes in viral proteins.

Animals↗
PubMed2001

DNA cleavage by type III restriction-modification enzyme EcoP15I is independent of spacer distance between two head to head oriented recognition sites.

The type III restriction-modification enzyme EcoP15I requires the interaction of two unmethylated, inversely oriented recognition sites 5'-CAGCAG in head to head configuration to allow an efficient DNA cleavage. It has been hypothesized that two convergent DNA-translocating enzyme-substrate complexes interact to form the active cleavage complex and that translocation is driven by ATP hydrolysis. Using a half-automated, fluorescence-based detection method, we investigated how the distance between two inversely oriented recognition sites affects DNA cleavage efficiency. We determined that EcoP15I cleaves DNA efficiently even for two adjacent head to head or tail to tail oriented target sites. Hence, DNA translocation appears not to be required for initiating DNA cleavage in these cases. Furthermore, we report here that EcoP15I is able to cleave single-site substrates. When we analyzed the interaction of EcoP15I with DNA substrates containing adjacent target sites in the presence of non-hydrolyzable ATP analogues, we found that cleavage depended on the hydrolysis of ATP. Moreover, we show that cleavage occurs at only one of the two possible cleavage positions of an interacting pair of target sequences. When EcoP15I bound to a DNA substrate containing one recognition site in the absence of ATP, we observed a 36 nucleotide DNaseI-footprint that is asymmetric on both strands. All of our footprinting experiments showed that the enzyme did not cover the region around the cleavage site. Analyzing a DNA fragment with two head to head oriented recognition sites, EcoP15I protected 27-33 nucleotides around the recognition sequence, including an additional region of 26 bp between both cleavage sites. For all DNA substrates examined, the presence of ATP caused altered footprinting patterns. We assume that the altered patterns are most likely due to a conformational change of the enzyme. Overall, our data further refine the tracking-collision model for type III restriction enzymes.

Adenosine Triphosphate↗
PubMed2001

CCAAT/enhancer-binding proteins alpha and beta negatively influence the capacity of tumor necrosis factor alpha to up-regulate the human cytomegalovirus IE1/2 enhancer/promoter by nuclear factor kappaB during monocyte differentiation.

Recently we demonstrated that the ability of tumor necrosis factor alpha (TNFalpha) to stimulate the human cytomegalovirus (HCMV) IE1/2 enhancer/promoter activity in myeloid progenitor-like cells decreases when these cells differentiate into promonocytic cells. In addition, TNFalpha stimulation in the progenitor-like cell line HL-60 was shown to be mediated by nuclear factor kappaB (NF-kappaB) activation and its binding to the 18-base pair sequence motifs of the IE1/2 enhancer. We demonstrate here that the cell differentiation-dependent reduction of TNFalpha stimulation is not due to insufficient NF-kappaB activation but correlates with increased synthesis of the monocyte differentiation-associated factors CCAAT/enhancer-binding protein (C/EBP) alpha and beta. Overexpression of C/EBPalpha/beta in HL-60 cells, which normally produce only very small amounts of C/EBP, stimulated the basal activity of the promoter in the absence of NF-kappaB but suppressed the stimulatory effect of TNFalpha. A novel C/EBP-binding site was identified in the IE1/2 enhancer directly downstream of a NF-kappaB site. In order to understand the mechanisms of interaction, we used an IE1/2 promoter mutant that failed to bind C/EBP at this position and several constructs that contained exclusively NF-kappaB- and/or C/EBP-binding sites upstream of the minimal IE1/2 promoter. We could demonstrate that C/EBPalpha/beta interacts with NF-kappaB p65 and displays inhibitory activity even in the absence of direct DNA binding by forming p65-C/EBP-containing protein complexes bound to the NF-kappaB site. Moreover, C/EBP binding to the DNA adjacent to NF-kappaB supports the down-regulatory effect of C/EBPs possibly due to stabilization of a multimeric NF-kappaB-C/EBP complex. Our results show that cell differentiation factors may interfere with TNFalpha-induced human cytomegalovirus gene (re)activation.

Base Sequence↗
PubMed2001

A chemiluminescence detection method of hantaviral antigens in neutralisation assays and inhibitor studies.

A protocol for chemiluminescence detection of hantaviral antigens in infected cell foci is described. This focus detection is based on the conversion of a substrate into a luminescent product by peroxidase-antibody conjugates; the emitted light of infected cell foci can easily be recorded by autoradiography or video imaging providing a hard copy for documentation. The main advantage of this method as compared to conventional immunochemical staining is a higher detection sensitivity due to the inherent magnification effect of luminescence causing an obvious boost in focal image and intensity. This enables reduction of (i) incubation time of virus-infected cells and (ii) amount of needed antibody for antigen detection in foci. This method is applied to a chemiluminescence focus reduction assay for the serotyping of hantavirus-specific neutralising antibodies in infected persons and for the determination of activity of antiviral agents against hantavirus.

Animals↗
PubMed2001

Hantavirus infections and their prevention.

Hantaviruses are rodent-borne bunyaviruses which cause haemorrhagic fever with renal syndrome and Hantavirus pulmonary syndrome in humans. This review covers the host interactions of the viruses, including the rodent reservoirs, the clinical outcome of human infections as well as the pathogenesis and laboratory diagnosis of infections. The current stage in prophylaxis and therapy of hantaviral diseases is described and different approaches in vaccine development are discussed.

Animals↗
PubMed2001

First molecular evidence for Puumala hantavirus in Slovakia.

We report on the first Puumala hantavirus nucleotide sequence (strain Opina-916) amplified from a bank vole trapped in Slovakia, central Europe. Phylogenetic analysis of the S-segment sequence grouped the virus within the western/central European sublineage of Puumala virus. In the neighborhood of the rodent trapping site two cases of human infection by the Puumala virus were verified.

Animals↗
PubMed2001

Dobrava hantavirus causes hemorrhagic fever with renal syndrome in central Europe and is carried by two different Apodemus mice species.

In central Europe, hemorrhagic fevers with renal syndrome (HFRS) in humans are caused by the hantavirus species Puumala (transmitted by voles) and a second, Hantaan-related species (transmitted by mice). The second virus could be identified as Dobrava virus. To date, 19 clinical cases of Dobrava infection have been found in Germany and Slovakia. All patients exhibited a mild/moderate clinical course and no case fatality occurred. Screening for infected rodents revealed that the striped field mouse (Apodemus agrarius) represents the main reservoir for Dobrava virus in central Europe. Nucleotide sequence comparisons and phylogenetic analysis based on complete and partial genomic S segment nucleotide sequences placed the Slovakian A. agrarius-derived hantavirus strains within the Dobrava species, forming a cluster on the Dobrava phylogenetic tree. In east Slovakia, a single Dobrava virus-infected yellow-necked mouse (Apodemus flavicollis) was trapped in a locality that predominantly showed Dobrava-infected A. agrarius. Comparison of the S segment sequence (nucleotides 381-935) revealed that the Dobrava strain from A. flavicollis shows only 84.3% nucleotide homology to A. agrarius-derived strains from this location but 96.3% homology to A. flavicollis-derived Dobrava strains from the Balkans (southeast Europe). Phylogenetic analysis of the partial S segment placed the A. flavicollis-derived Dobrava strain from Slovakia on a distinct Dobrava lineage (DOB-Af) together with the south-east European A. flavicollis-derived strains. The results indicate that Dobrava strains from A. agrarius (DOB-Aa) vs. A. flavicollis (DOB-Af) could develop different degrees of virulence in humans.

Adolescent↗
PubMed2001

Clinical characterization of Dobrava hantavirus infections in Germany.

There is increasing evidence that Dobrava (DOBV) but not Hantaan (HTNV) hantavirus is a hemorrhagic fever with renal syndrome (HFRS) causing agent in Central Europe. However, only single clinical cases of HFRS due to acute DOBV infection have been described so far. We report on three male patients from a non-endemic hantavirus focus in Northern Germany who suffered from mild to moderate HFRS strongly resembling nephropathia epidemica. Serotyping by detection of hantavirus species-specific neutralizing antibodies revealed acute infections by the HTNV-related hantavirus DOBV in all three cases. Since DOBV infections in the Balkans frequently present as severe HFRS, our cases suggest that Central-European DOBV infections have a different, less severe clinical outcome. These differences in DOBV virulence towards humans might be due to the existence of different genetic lineages of DOBV.

Acute Disease↗
PubMed2000

Puumala (PUU) hantavirus strain differences and insertion positions in the hepatitis B virus core antigen influence B-cell immunogenicity and protective potential of core-derived particles.

Hepatitis B virus (HBV) core-derived chimeric particles carrying a Puumala (PUU) hantavirus (strain Vranica/Hällnäs) nucleocapsid (N) protein sequence (aa 1-45), alternatively inserted at three distinct positions (N-, C-terminus, or the internal region), and mosaic particles consisting of HBV core as well as core/PUU (Vranica/Hällnäs) N (aa 1-45) readthrough protein were generated. Chimeric particles carrying the insert at the N-terminus or the internal region of core induced some protective immune response in bank voles (Clethrionomys glareolus) against a subsequent PUU virus (strain Kazan) challenge; 40-50% of the animals showed markers of protection. In contrast, internal insertion of PUU strain CG18-20 N (aa 1-45) into the HBV core caused a highly protective immune response in the bank vole model. Immunizations with particles carrying aa 75-119 of PUU (CG18-20) N at the C-terminus of core verified the presence of a second, minor protective region in the N protein. A strong PUU N-specific antibody response was detected not only in bank voles immunized with chimeric particles containing internal and N-terminal fusions of PUU N protein but also in animals immunized with the corresponding mosaic particles. Except for the exclusive occurrence of antibodies directed against aa 231-240 of N in non-protected animals post virus challenge, there was no additional obvious difference in the epitope-specificity of N-specific antibodies from immunized animals prior and post virus challenge.

Amino Acid Sequence↗
PubMed2000

Imaging DNA loops induced by restriction endonuclease EcoRII. A single amino acid substitution uncouples target recognition from cooperative DNA interaction and cleavage.

EcoRII is a type IIE restriction endonuclease characterized by a highly cooperative reaction mechanism that depends on simultaneous binding of the dimeric enzyme molecule to two copies of its DNA recognition site. Transmission electron microscopy provided direct evidence that EcoRII mediates loop formation of linear DNA containing two EcoRII recognition sites. Specific DNA binding of EcoRII revealed a symmetrical DNase I footprint occupying 16-18 bases. Single amino acid replacement of Val(258) by Asn yielded a mutant enzyme that was unaffected in substrate affinity and DNase I footprinting properties, but exhibited a profound decrease in cooperative DNA binding and cleavage activity. Because the electrophoretic mobility of the mutant enzyme-DNA complexes was significantly higher than that of the wild-type, we investigated if mutant V258N binds as a monomer to the substrate DNA. Analysis of the molecular mass of mutant V258N showed a high percentage of protein monomers in solution. The dissociation constant of mutant V258N confirmed a 350-fold decrease of the enzyme dimerization capability. We conclude that Val(258) is located in a region of EcoRII involved in homodimerization. This is the first report of a specific amino acid replacement in a restriction endonuclease leading to the loss of dimerization and DNA cleavage while retaining specific DNA binding.

Asparagine↗
PubMed2000

Interaction of wild-type and naturally occurring deleted variants of hepatitis B virus core polypeptides leads to formation of mosaic particles.

The simultaneous presence of hepatitis B virus (HBV) genomes carrying wild-type (wt) and in-frame deleted variants of the HBV core gene has been identified as a typical feature of HBV-infected renal transplant patients with severe liver disease. To investigate possible interactions of wt and deleted core polypeptides a two-vector Escherichia coli expression system ensuring their concomitant synthesis has been developed. Co-expression of wt and a mutant core lacking 17 amino acid residues (77-93) within the immunodominant region led to the formation of mosaic particles, whereas the mutant alone was incapable of self-assembly.

Blotting, Western↗
PubMed2000

A novel link between stress and human cytomegalovirus (HCMV) infection: sympathetic hyperactivity stimulates HCMV activation.

Recently, inflammatory mediators such as TNFalpha were identified as triggering active human cytomegalovirus (HCMV) infection. Here, we demonstrate that a highly stressful event in the absence of systemic inflammation, as observed in patients with acute myocardial infarction, leads to the development of an active HCMV infection in latently infected patients. Elucidating the molecular mechanism of virus activation, we could show that catecholamines directly stimulate the HCMV immediate-early (IE) enhancer/promoter in monocytic cells via beta-2 adrenergic receptors. Subsequent activation of the cAMP/PK-A-signaling pathway results in enhanced synthesis and binding of the transcription factor CREB-1/ATF-1 to the cAMP-responsive elements within the IE enhancer. Epinephrine also enhanced HCMV gene expression in infected THP-1 cells by about 50% in three of four experiments. These data suggest that HCMV, like HSV-1 and VZV, can be (re)activated under stress conditions.

Activating Transcription Factor 1↗