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Biomedical subjects

U B Göbel

Publications and source records attributed to U B Göbel.

At least 19 recordsLinked to original sources
PubMed2005

Chronic periodontal disease is associated with single-nucleotide polymorphisms of the human TLR-4 gene.

Periodontitis is an inflammatory disease affecting the connective tissue surrounding the teeth leading to tooth loss. Pathogens associated with periodontitis interact with Toll-like receptors (TLRs) to induce cytokines causing and aggravating disease. We screened 197 individuals suffering from generalized periodontitis for the presence of Asp299Gly and Thr399Ile of TLR-4 as well as Arg753Gln of TLR-2 in comparison to matched controls. Single-nucleotide polymorphisms (SNPs) of TLR-4 were elevated among patients (odd's ratio 3.650, 95% CI 1.573-8.467, P < or = 0.0001), while no difference was observed for TLR-2. TLR-4 SNPs were correlated with chronic periodontitis (odd's ratio 5.562, 95% CI 2.199-14.04, P < or = 0.0001), but not with aggressive periodontitis. This observation was confirmed employing a group of periodontally healthy probands over 60 years of age. These data demonstrate that genetic variants of TLR-4 may act as risk factors for the development of generalized chronic periodontitis in humans.

Adult↗
PubMed2005

Misidentification of Mycobacterium leprae as Mycobacterium intracellulare by the COBAS AMPLICOR M. intracellulare test.

Commercially available nucleic acid probe- and amplification-based systems for detection and differentiation of mycobacteria are widely used in clinical microbiology laboratories. Here we report two cases of human leprosy in which the COBAS AMPLICOR Mycobacterium intracellulare test led to false- positive results. Correct identification of Mycobacterium leprae was possible only by amplification and comparative sequence analysis of the 16S rRNA gene.

Adult↗
PubMed2004

Treponema putidum sp. nov., a medium-sized proteolytic spirochaete isolated from lesions of human periodontitis and acute necrotizing ulcerative gingivitis.

So far, little phenotypic heterogeneity has been detected in cultured oral treponemes with trypsin-like proteolytic activity, and all have been assigned to the species Treponema denticola. However, comparisons of protein patterns and antigen expression in our collection of proteolytic oral treponemes occasionally identified isolates with a unique phenotype; e.g. strain OMZ 830 (=ATCC 700768), which qualified as a 'pathogen-related oral spirochaete' due to the presence of a approximately 37 kDa protein reactive with the Treponema pallidum FlaA-specific mAb H9-2. In addition to such single isolates, a homogeneous group of seven independent strains is described that were highly motile, medium-sized, proteolytic but asaccharolytic spirochaetes and were cultured from human gingivitis, periodontitis and acute necrotizing ulcerative gingivitis in medium OMIZ-Pat supplemented with 1% human serum and antibiotics. Growth of these spirochaetes in OMIZ-Pat was not dependent on, but was stimulated by, human or bovine serum. Carbohydrates were neither required nor stimulatory for growth. The protein and antigen patterns of total cell extracts of these organisms separated by SDS-PAGE were distinct from those of all previously cultured spirochaetes, with highest similarity to T. denticola. The novel spirochaete has a 2 : 4 : 2 arrangement of the periplasmic flagella, similar to T. denticola. However, the flagellin pattern as detected by immunostaining or glycan staining of Western blots readily distinguished the novel group from T. denticola. Also, distinct from reference strains of T. denticola, none of the novel isolates displayed sialidase or dentilisin activities, both of which are expressed by most strains of T. denticola. Trypsin-like activity and other enzymes as detected by API ZYM test were similar to those of T. denticola. The status of a novel species is supported by the 16S rRNA gene sequence, with 98.5% similarity to its closest cultured relative, T. denticola. The name Treponema putidum sp. nov. is proposed (type strain OMZ 758T=ATCC 700334T=CIP 108088T).

Antigens, Bacterial↗
PubMed2001

Proteolytic activity among various oral Treponema species and cloning of a prtP-like gene of Treponema socranskii subsp. socranskii.

The proteolytic activity of 11 treponemal strains representing different phylogenetic groups was investigated by SDS-polyacrylamide gel electrophoresis with copolymerised casein, gelatin and fibrinogen as substrates. The activity was specified to be trypsin- and chymotrypsin-like by the cleavage of synthetic substrates BAPNA and SAAPFNA, respectively. Nine strains degrade casein and the synthetic substrate BAPNA. Chymotrypsin-like activity specifically inhibited by phenylmethylsulfonyl fluoride was found in four treponemes. Southern blot analysis using a Treponema socranskii subsp. socranskii-specific prtP probe confirmed the presence of prtP homologous genes in these four strains. The internal fragments of the chymotrypsin-like protease genes were cloned and sequenced after PCR amplification. Here we report the cloning of the complete prtP-like gene of T. socranskii subsp. socranskii, an organism shown to possess epidemiologic relevance in periodontitis.

Blotting, Southern↗
PubMed2001

Outer sheath associated proteins of the oral spirochete Treponema maltophilum.

We recently cloned the major outer membrane protein of Treponema maltophilum [Heuner, K., Choi, B.K., Schade, R., Moter, A., Otto, A., Göbel, U.B., J. Bacteriol. 181, 1025-1029]. Here we report the localization of the major sheath protein (Msp)A protein in T. maltophilum by immunogold electron microscopy and its expression. Northern blot analysis revealed that mspA is expressed constitutively as a monocistronic unit. The transcription initiation site of the mspA gene was identified by primer extension analysis. A further screening of a genomic library of T. maltophilum with an anti-outer membrane fraction antibody was done. We were able to clone DNA regions of T. maltophilum encoding putative sugar transport operons and putative outer membrane proteins of this oral treponeme which has a high prevalence in periodontal lesions.

Bacterial Outer Membrane Proteins↗
PubMed2001

Toll-like receptor-2 mediates Treponema glycolipid and lipoteichoic acid-induced NF-kappaB translocation.

Recently Toll-like receptors (TLRs) have been found to be involved in cellular activation by microbial products, including lipopolysaccharide, lipoproteins, and peptidoglycan. Although for these ligands the specific transmembrane signal transducers TLR-4, TLR-2, or TLR-2 and -6 have now been identified, the molecular basis of recognition of lipoteichoic acids (LTAs) and related glycolipids has not been completely understood. In order to determine the role of TLRs in immune cell activation by these stimuli, experiments involving TLR-2-negative cell lines, TLR-expression plasmids, macrophages from TLR-4-deficient C3H/HeJ-mice, and inhibitory TLR-4/MD-2 antibodies were performed. Glycolipids from Treponema maltophilum and Treponema brennaborense, as well as highly purified LTAs from Staphylococcus aureus and Bacillus subtilis exhibited TLR-2 dependence in nuclear factor kappaB activation and cytokine induction; however, T. brennaborense additionally appeared to signal via TLR-4. Fractionation of the T. brennaborense glycolipids by hydrophobic interaction chromatography and subsequent cell stimulation experiments revealed two peaks of activity, one exhibiting TLR-2-, and a second TLR-4-dependence. Furthermore, we show involvement of the signaling molecules MyD88 and NIK in cell stimulation by LTAs and glycolipids by dominant negative overexpression experiments. In summary, the results presented here indicate that TLR-2 is the main receptor for Treponema glycolipid and LTA-mediated inflammatory response.

Animals↗
PubMed2001

Validation of the sterilization procedure of allogeneic avital bone transplants using peracetic acid-ethanol.

Different procedures are available to inactivate bacteria and fungi, including their spores, as well as viruses in human bone transplants. The most efficient methods are considered to be gamma irradiation and thermal inactivation as well as chemical sterilization methods like the peracetic acid-ethanol treatment (PES). Following national and international standards or draft standards, the antimicrobial effectiveness of this procedure was evaluated. Due to the standardizable size as well as the clinical relevance, defatted human spongiosa cuboids (15x15x15 mm) served as model system. After treatment with PES for 2 and 4 hours, respectively, the titre of living micro-organisms was determined in the supernatant and the cuboid. A reduction in the titre of viable micro-organisms below the detection level (reduction factor >5 log10) was already achieved after an incubation time of 2 hours (Staphylococcus aureus, Enterococcus faecium, Pseudomonas aeruginosa, Bacillus subtilis, Clostridium sporogenes, Mycobacterium terrae, Candida albicans as well as spores of Bacillus subtilis). No viable micro-organisms could be detected in any of the PES-treated test cuboids. Spores of Aspergillus niger were also completely inactivated. The PES procedure proved to be a reliable method for the sterilization of human bone transplants derived from spongiosa.

Bacteria↗
PubMed2001

Plaque formation on surface modified dental implants. An in vitro study.

Bacterial adhesion on titanium implant surfaces has a strong influence on healing and long-term outcome of dental implants. Parameters like surface roughness and chemical composition of the implant surface were found to have a significant impact on plaque formation. The purpose of this study was to evaluate the influence of two physical hard coatings on bacterial adhesion in comparison with control surfaces of equivalent roughness. Two members of the oral microflora, Streptococcus mutans and Streptococcus sanguis were used. Commercially pure titanium discs were modified using four different surface treatments: physical vapour deposition (PVD) with either titanium nitride (TiN) or zirconium nitride (ZrN), thermal oxidation and structuring with laser radiation. Polished titanium surfaces were used as controls. Surface topography was examined by SEM and estimation of surface roughness was done using a contact stylus profilometer. Contact angle measurements were carried out to calculate surface energy. Titanium discs were incubated in the respective bacterial cell suspension for one hour and single colonies formed by adhering bacteria were counted by fluorescence microscopy. Contact angle measurements showed no significant differences between the surface modifications. The surface roughness (Ra) of all surfaces examined was between 0.14 and 1.00 microm. A significant reduction of the number of adherent bacteria was observed on inherently stable titanium hard materials such as TiN and ZrN and thermically oxidated titanium surfaces compared to polished titanium. In conclusion, physical modification of titanium implant surfaces such as coating with TiN or ZrN may reduce bacterial adherence and hence improve clinical results.

Bacterial Adhesion↗
PubMed2001

Bordetella petrii sp. nov., isolated from an anaerobic bioreactor, and emended description of the genus Bordetella.

A novel Bordetella species was isolated from an anaerobic, dechlorinating bioreactor culture enriched from river sediment. The only strain, Se-1111R(T) (= DSM 12804T = CCUG 43448T), for which the name Bordetella petrii is proposed, is designated the type strain of the novel species. Strain Se-1111R(T) was isolated from the dechlorinating mixed culture due to its ability to anaerobically reduce selenate to elemental selenium. Comparative 16S rDNA sequence analysis showed a close relationship between Se-1111R(T) and members of the genus Bordetella within the beta-Proteobacteria. This close phylogenetic relatedness was also reflected in several metabolic properties of Se-1111R(T), including its incapacity to utilize carbohydrates, by the high G+C content (63.8 mol%) of its DNA and by the presence of Q-8 as the major isoprenoid quinone. DNA-DNA hybridization experiments with type strains of all species of the genus Bordetella and closely related species Achromobacter xylosoxidans subsp. denitrificans provided further evidence for the assignment of strain Se-1111R(T) as a novel species of the genus Bordetella. This genus currently consists of seven aerobic species, all of which are known to occur in close pathogenic, opportunistic or possibly commensal relationships with various host organisms. B. petrii is the first member of this genus isolated from the environment and capable of anaerobic growth. The proposal of the novel species and an emended description of the genus Bordetella is presented.

Anaerobiosis↗
PubMed2000

Activation of mitogen-activated protein kinases p42/44, p38, and stress-activated protein kinases in myelo-monocytic cells by Treponema lipoteichoic acid.

We have shown previously that phenol/water extracts derived from two novel Treponema species, Treponema maltophilum, and Treponema brennaborense, resembling lipoteichoic acid (LTA), induce cytokines in mononuclear cells. This response was lipopolysaccharide binding-protein (LBP)-dependent and involved Toll-like receptors (TLRs). Here we show that secretion of tumor necrosis factor-alpha induced by Treponema culture supernatants and extracted LTA was paralleled by an LBP-dependent phosphorylation of mitogen-activated protein kinases (MAPKs) p42 and p44, and p38, as well as the stress-activated protein kinases c-Jun N-terminal kinases 1 and 2. Phosphorylation of p42/44 correlated with an increase of activity, and tumor necrosis factor-alpha levels were significantly reduced by addition of inhibitors of p42/44 and p38, PD 98059 and SB 203580, respectively. Treponeme LTA differed from bacterial lipopolysaccharide regarding time course of p42/44 phosphorylation, exhibiting a prolonged activation of MAPKs. Furthermore, MAPK activation and cytokine induction failed to be strictly correlated. Involvement of TLR-4 for phosphorylation of p42/44 was shown employing the neutralizing anti-murine TLR-4 antibody MTS 510. In TLR-2-negative U373 cells, the compounds studied differed regarding MAPK activation with T. maltophilum leading to a stronger activation. In summary, the data presented here show that treponeme LTA are able to activate the MAPK and stress-activated protein kinase pathway involving LBP and TLR-4.

Animals↗
PubMed2000

A novel technique for monitoring the development of bacterial biofilms in human periodontal pockets.

A new technique is presented for analyzing subgingival bacterial plaque. Different materials (polytetrafluoroethylene, gold, dentin) kept for several days in periodontal pockets of patients suffering from periodontitis were analyzed by electron microscopy and fluorescence in situ hybridization (FISH). Those parts of the carriers extending into the deepest zone of the pockets were predominantly colonized by spirochetes and Gram-negative bacteria whereas those segments in contact with a shallower region were colonized by streptococci. Independent of the material used, the bacterial colonization of the carriers appears to be similar. FISH using eubacteria- and species-specific oligonucleotides on semi-thin cross-sections of the carriers in combination with confocal laser scanning microscopy allowed detailed analysis of the architecture of biofilms and identification of putative periodontal pathogens with single cell resolution.

Bacteria↗
PubMed2000

Involvement of lipopolysaccharide binding protein, CD14, and Toll-like receptors in the initiation of innate immune responses by Treponema glycolipids.

Culture supernatants from Treponema maltophilum associated with periodontitis in humans and Treponema brennaborense found in a bovine cattle disease accompanied with cachexia caused a dose-dependent TNF-alpha synthesis in human monocytes increasing with culture time. This activity could be reduced significantly by blocking the CD14-part of the LPS receptor using the My 4 mAb and by polymyxin B. In the murine macrophage cell line RAW 264.7, Treponema culture supernatants induced TNF-alpha secretion in a LPS binding protein (LBP)-dependent fashion. To enrich for active compounds, supernatants were extracted with butanol, while whole cells were extracted using a phenol/water method resulting in recovery of material exhibiting a similar activity profile. An LPS-LBP binding competition assay revealed an interaction of the treponeme phenol/water extracts with LBP, while precipitation studies implied an affinity to polymyxin B and endotoxin neutralizing protein. Macrophages obtained from C3H/HeJ mice carrying a Toll-like receptor (TLR)-4 mutation were stimulated with treponeme extracts for NO release to assess the role of TLRs in cell activation. Furthermore, NF-kappaB translocation in TLR-2-negative Chinese hamster ovary (CHO) cells was studied. We found that phenol/water-extracts of the two strains use TLRs differently with T. brennaborense-stimulating cells in a TLR-4-dependent fashion, while T. maltophilum-mediated activation apparently involved TLR-2. These results indicate the presence of a novel class of glycolipids in Treponema initiating inflammatory responses involving LBP, CD14, and TLRs.

Acute-Phase Proteins↗
PubMed2000

Cloning and characterization of a Legionella pneumophila-specific gene encoding a member of the LysR family of transcriptional regulators.

Flagellin gene regulation in Legionella pneumophila is modulated by various environmental factors. The expression of the virulent phenotype seems to be linked genetically to flagellum expression. To better understand the mechanisms of flagellin gene expression in L. pneumophila (Lp), we screened a pool of plasmids from a L. pneumophila Corby genomic library for the ability to prevent or reduce luciferase activity in the Escherichia coli strain YK410, which harbours a Lp-pflaA-luxAB fusion. We cloned a DNA fragment encoding the N-terminal part of a protein with significant similarity to members of the LysR family of transcriptional regulators (LTTRs). The entire gene, cloned by inverse PCR, was named flaR. It encodes a protein of 302 amino acids, and computer-assisted analysis of the amino acid sequence revealed a helix-turn-helix motif located near the N-terminus of the protein. The FlaR protein exhibits 21-31% identity to various LTTRs. Furthermore, gel retardation experiments indicate that the FlaR protein is able to bind to its own promoter region and, to a lesser extent, to the flaA promoter of L. pneumophila. The flaR promoter region contains putative LysR binding motifs and two putative Fur boxes. Taken together, these results indicate that FlaR is a DNA-binding protein which belongs to the LTTR family. Southern analysis with a L. pneumophila Corby-specific flaR probe revealed homologous genes in various L. pneumophila strains, but not in the 12 nonpneumophila strains tested so far.

Amino Acid Sequence↗
PubMed2000

Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms.

As a technique allowing simultaneous visualization, identification, enumeration and localization of individual microbial cells, fluorescence in situ hybridization (FISH) is useful for many applications in all fields of microbiology. FISH not only allows the detection of culturable microorganisms, but also of yet-to-be cultured (so-called unculturable) organisms, and can therefore help in understanding complex microbial communities. In this review, methodological aspects, as well as problems and pitfalls of FISH are discussed in an examination of past, present and future applications.

Animals↗
PubMed2000

Phylogenetic analysis of rhizosphere-associated beta-subclass proteobacterial ammonia oxidizers in a municipal wastewater treatment plant based on rhizoremediation technology.

In wastewater treatment plants based on the rhizosphere zone (rhizoremediation technology), ammonia-oxidizing bacteria (AOB) play an important role in the removal of fixed nitrogen. However, the diversity of these bacteria in rhizoremediation wastewater treatment plants is largely unknown. We employed direct PCR amplification and cloning of 16S rRNA genes to determine the phylogenetic affiliation of AOB occurring in root and soil samples of a wastewater treatment plant (Merzdorf plant, Brandenburg, Germany). 16S rDNA clone libraries were screened by hybridization using an oligonucleotide probe specific for AOB of the beta subclass of proteobacteria. Comparative sequence analysis of all hybridization-positive clones revealed that the majority of rDNA sequences was affiliated to members of the genus Nitrosospira and formed a novel subcluster (SM cluster), whereas only three sequences were most closely related to Nitrosomonas species. Affiliation of the novel Nitrosospira-like sequences with those of isolates from soil and rhizosphere suggests that phylogenetic clusters reflect physiological differences between members of this genus.

Ammonia↗
PubMed2000

Bacteria of an anaerobic 1,2-dichloropropane-dechlorinating mixed culture are phylogenetically related to those of other anaerobic dechlorinating consortia.

A 16S-rDNA-based molecular study was performed to determine the bacterial diversity of an anaerobic, 1,2-dichloropropane-dechlorinating bioreactor consortium derived from sediment of the River Saale, Germany. Total community DNA was extracted and bacterial 16S rRNA genes were subsequently amplified using conserved primers. A clone library was constructed and analysed by sequencing the 16S rDNA inserts of randomly chosen clones followed by dot blot hybridization with labelled polynucleotide probes. The phylogenetic analysis revealed significant sequence similarities of several as yet uncultured bacterial species in the bioreactor to those found in other reductively dechlorinating freshwater consortia. In contrast, no close relationship was obtained with as yet uncultured bacteria found in reductively dechlorinating consortia derived from marine habitats. One rDNA clone showed >97% sequence similarity to Dehalobacter species, known for reductive dechlorination of tri- and tetrachloroethene. These results suggest that reductive dechlorination in microbial freshwater habitats depends upon a specific bacterial community structure.

Anaerobiosis↗
PubMed2000

Peptide nucleic acid-mediated PCR clamping as a useful supplement in the determination of microbial diversity.

Peptide nucleic acid (PNA)-mediated PCR clamping (H. Orum, P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, and C. Stanley, Nucleic Acids Res. 21:5332-5336, 1993) was introduced as a novel procedure to selectively amplify ribosomal DNAs (rDNAs) which are not frequently found in clone libraries generated by standard PCR from complex microbial consortia. Three different PNA molecules were used; two of these molecules (PNA-ALF and PNA-EUB353) overlapped with one of the amplification primers, whereas PNA-1114F hybridized to the middle of the amplified region. Thus, PCR clamping was achieved either by competitive binding between the PNA molecules and the forward or reverse primers (competitive clamping) or by hindering polymerase readthrough (elongation arrest). Gene libraries generated from mixed rDNA templates by using PCR clamping are enriched for clones that do not contain sequences homologous to the appropriate PNA oligomer. This effect of PCR clamping was exploited in the following two ways: (i) analysis of gene libraries generated by PCR clamping with PNA-ALF together with standard libraries reduced the number of clones which had to be analyzed to detect all of the different sequences present in an artificial rDNA mixture; and (ii) PCR clamping with PNA-EUB353 and PNA-1114F was used to selectively recover rDNA sequences which represented recently described phylogenetic groups (NKB19, TM6, cluster related to green nonsulfur bacteria) from an anaerobic, dechlorinating consortium described previously. We concluded that PCR clamping might be a useful supplement to standard PCR amplification in rDNA-based studies of microbial diversity and could be used to selectively recover members of undescribed phylogenetic clusters from complex microbial communities.

Bacteria↗