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Background Farm-derived dust samples have been screened for bacteria with potential allergo-protective properties. Among those was Staphylococcus sciuri W620 ( S. sciuri W620), which we tested with regard to its protective capacities in murine models of allergic airway inflammation. Methods We employed two protocols of acute airway inflammation in mice administering either ovalbumin ( OVA) or house dust mite extract ( HDM) for sensitization. Mechanistic studies on the activation of innate immune responses to S. sciuri W620 were carried out using human primary monocytic dendritic cells (mo DC) and co-culture with autologous T cells. Results The allergo-protective properties of S. sciuri W620 were proven in a TH2-driven OVA model as well as in a mixed TH1/TH2 phenotype HDM model as demonstrated by abrogation of eosinophils and neutrophils in the airways after intranasal treatment. In the HDM model, lymph node cell TH1/ TH2 signature cytokines were decreased in parallel. Studies on human moDC revealed an activation of TLR2 and NOD2 receptors and initiation of DC maturation following incubation with S. sciuri W620. Cytokine expression analyses after exposure to S. sciuri W620 showed a lack of IL-12 production in mo DC due to missing transcription of the IL-12p35 mRNA. However, such DC selectively supported TH1 cytokine release by co-cultured T cells. Conclusion and clinical relevance Our proof-of-concept experiments verify the screening system of farm-derived dust samples as suitable to elucidate new candidates for allergo-protection. S. sciuri W620 was shown to possess preventive properties on airway inflammation providing the basis for further mechanistic studies and potential clinical implication. [ABSTRACT FROM AUTHOR]

To cite this article: Debarry J, Hanuszkiewicz A, Stein K, Holst O, Heine H. The allergy-protective properties of Acinetobacter lwoffii F78 are imparted by its lipopolysaccharide. Allergy 2010; 65: 690–697. Background: An increasing number of epidemiological studies show that exposure to farming environment during early childhood strongly influences the development of allergic reactions later in life (‘hygiene hypothesis’). Also, it had been shown that certain bacteria from this environment may have allergy-protective properties. In the present study, we further characterized one of these bacteria, namely Acinetobacter lwoffii F78, with regard to the bacteria-induced signaling and possible mechanisms of allergy protection. Methods: The impact of A. lwoffii F78 on human monocyte-derived dendritic cells especially with respect to their THelper cell polarization capacity was investigated by ELISA and real-time PCR experiments as well as confocal microscopy. The responsible molecule for these effects was further characterized and identified using blocking experiments. Results: It was shown that A. lwoffii F78 induced a TH1-polarizing program in human dendritic cells which led to TH1 differentiation. In addition, a positive influence on the TBet/GATA3 level could be detected. Blocking experiments revealed that the lipopolysaccharide (LPS) of A. lwoffii F78 was the responsible molecule promoting these effects. Conclusion: We found evidence that the allergy-protecting effects of A. lwoffii F78 are because of the activation of a TH1-polarizing program in human dendritic cells, and that the LPS of A. lwoffii F78 is responsible for these beneficial effects. [ABSTRACT FROM AUTHOR]

Background: Caspase recruitment domain protein (CARD) 4 has been recently identified as an intracellular pattern recognition receptor that interacts with muropeptides found in common Gram-negative bacteria. We therefore aimed to explore whether the previously observed inverse association between exposure to microbial products and asthma and allergies in childhood is modified by genetic variation in CARD4. Methods: We genotyped 668 children [mean age 9.3 (SD 1.5) years] enrolled in the cross-sectional ALEX study for seven haplotype tagging single nucleotide polymorphisms in CARD4. We studied the association of asthma, hay fever and allergen-specific serum immunoglobulin E with exposure to a farming environment and with levels of endotoxin and muramic acid measured in house dust samples. We tested whether these associations differed between the genotypes of the polymorphisms under study. Results: A strong protective effect of a farming environment on allergies was only found in children homozygous for the T allele in CARD4/-21596, but not in children carrying the minor allele (C). Among the former, farmers' children had a significantly lower frequency of sensitization against pollen (5.8%), hay fever (1.7%) and atopic asthma symptoms (1.7%) compared with children not living on a farm (19.4%, 13.0% and 7.6%, P < 0.01, < 0.01 and < 0.05, respectively). Conversely, no significant difference in prevalence of these phenotypes by farming status was found among children with a C allele in CARD4/-21596 (14.3%, 7.1% and 8.0% vs 16.5%, 9.0% and 5.7%, respectively). Conclusion: Polymorphisms in CARD4 significantly modify the protective effect of exposure to a farming environment. [ABSTRACT FROM AUTHOR]

Background: Recent epidemiological studies have shown that growing up on a traditional farm provides protection from the development of allergic disorders such as hay fever and allergic asthma. We present experimental evidence that substances providing protection from the development of allergic diseases can be extracted from dust collected in stables of animal forms. Methods: Stable dust was collected from 30 randomly selected farms located in rural regions of the Alps (Austria, Germany and Switzerland). The dust was homogenised with glass beads and extracted with physiological sodium chloride solution. This extract was used to modulate immune response in a well established mouse model of allergic asthma. Results: Treatment of mice by inhalation of stable dust extract during sensitisation to ovalbumin inhibited the development of airway hyperresponsiveness and airway eosinophilia upon challenge, as well as the production of interleukin 5 by splenocytes and of antigen specific IgG1 and IgE. Dust extract also suppressed the generation of human dendritic cells in vitro. The biological activity of the dust extract was not exclusively mediated by lipopolysaccharide. Conclusions: Stable dust from animal farms contains strong immune modulating substances. These substances can interfere with the development of both cellular and humoral immunity against allergens, thus suppressing allergen sensitisation, airway inflammation, and airway hyperresponsiveness in a murine model of allergic asthma. [ABSTRACT FROM AUTHOR]

A cultivation strategy combining the advantages of temperature-limited fed-batch and probing feeding control is presented. The technique was evaluated in fed-batch cultivations with E. coli BL21(DE3) producing xylanase in a 3 liter bioreactor. A 20% increase in cell mass was achieved and the usual decrease in specific enzyme activity normally observed during the late production phase was diminished with the new technique. The method was further tested by growing E. coli W3110 in a larger bioreactor (50 l). It is a suitable cultivation technique when the O2 transfer capacity of the reactor is reached and it is desired to continue to produce the recombinant protein. [ABSTRACT FROM AUTHOR]

Metabolic stress is a phenomenon often discussed in conjunction with recombinant protein production in Escherichia coli. This investigation shows how heterologous protein production and the presence of host cell proteases is related to: (1) Isopropyl-β-D-thiogalactopyranoside (IPTG) induction, (2) cell-mass concentration at the time of induction, and (3) the presence of metabolites (glutamic acid or those from tryptone soy broth) during the post-induction phase of high cell density fed-batch cultivations. Two thermostable xylanase variants and one thermostable cellulase, all originating from Rhodothermus marinus, were expressed in E. coli strain BL21 (DE3). A three-fold difference in the specific activity of both xylanase variants [between 7,000 and 21,000 U/(g cell dry weight)], was observed under the different conditions tested. Upon induction at high cell-mass concentrations employing a nutrient feed devoid of the metabolites above, the specific activity of the xylanase variants, was initially higher but decreased 2–3 h into the post-induction phase and simultaneously protease activity was detected. Furthermore, protease activity was detected in all induced cultivations employing this nutrient feed, but was undetected in uninduced control cultivations (final cell-mass concentration of 40 g/l–1), as well as in induced cultivations employing metabolite-supplemented nutrient feeds. By contrast, maximum specific cellulase activity [between 700 and 900 U/(g cell dry weight)] remained relatively unaffected in all cases. The results demonstrate that detectable host cell proteases was not the primary reason for the decrease in post-induction activity observed under certain conditions, and possible causes for the differing production levels of heterologous proteins are discussed. [ABSTRACT FROM AUTHOR]

The thermostable cellulase Cel12A from Rhodothermus marinus was produced at extremely low levels when expressed in Escherichia coli and was cytotoxic to the cells. In addition, severe aggregation occurred when moderately high concentrations of the enzyme were heat-treated at 65 °C, the growth optimum of R. marinus. Sequence analysis revealed that the catalytic module of this enzyme is preceded by a typical linker sequence and a highly hydrophobic putative signal peptide. Two deletion mutants lacking this hydrophobic region were cloned and successfully expressed in E. coli. These results indicated that the N-terminal putative signal peptide was responsible for the toxicity of the full-length enzyme in the host organism. This was further corroborated by cloning and expressing the hydrophobic N-terminal domain in E. coli, which resulted in extensive cell lysis. The deletion mutants, made up of either the catalytic module of Cel12A or the catalytic module and the putative linker sequence, were characterised and their properties compared to those of the full-length enzyme. The specific activity of the mutants was approximately three-fold higher than that of the full-length enzyme. Both mutant proteins were highly thermostable, with half-lives exceeding 2 h at 90 °C and unfolding temperatures up to 103 °C. [ABSTRACT FROM AUTHOR]

The anaerobic sulfur-reducing archaeon Pyrococcus furiosus was investigated regarding its capacity to desulfurize rubber material. The microorganism's sensitivity towards common rubber elastomers and additives was tested and several were shown to be toxic to P. furiosus. The microorganism was shown to utilize sulfur in vulcanized natural rubber and an increase in cell density was obtained when cultivated in the presence of spent tire rubber. Ethanol-leached cryo-ground tire rubber treated with P. furiosus for 10 days was vulcanized together with virgin rubber material (15% w/w) and the mechanical properties of the resulting material were determined. The increase in the stress at break value and the decrease in swell ratio and stress relaxation rate obtained for material containing microbially treated rubber (compared to untreated material) show the positive effects of microbial desulfurization on rubber. [ABSTRACT FROM AUTHOR]

Amyloglucosidase (AMG) was produced by Aspergillus niger in solid-state fermentation (SSF), submerged fermentation (SmF) and an aqueous, two-phase system of polyethyleneglycol (PEG) and salt. In SSF, a fed-batch mode of operation gave a yield of 64 U/ml compared with 44 U/ml in batch mode. Similar trends were observed for SmF, where fed-batch cultivation gave a yield of 102 U/ml compared with 66 U/ml in batch. Shorter cultivation times (66 h) were required for SmF than for SSF (96 h). In the aqueous, two-phase cultivation, the productivity and yield of AMG were both twice those in the control fermentation. [ABSTRACT FROM AUTHOR]

Three strains of thermophilic bacteria producing extracellular protease have been isolated from hot springs in Chiang Mai, Thailand. The bacterium producing the highest amount of protease has been selected and identified as belonging to the genus Thermus, and is tentatively called Thermus 2S. The isolate is a Gram-negative, rod shaped bacterium. It exhibited maximum growth around 60°C at pH 7. Entrapment of the microbial cells in calcium alginate maintained the cell viability. Protease production from immobilized cells using 2 g wet cells per 10 ml 3% (w/v) sodium alginate was higher than that from a free-cell system using 2% inoculum. [ABSTRACT FROM AUTHOR]

Thiobacillus ferrooxidans, T. thioparus, Acidianus brierleyi and TH2 Lund, an archaeal isolate, were tested for sulphur oxidising capacity on ground rubber from spent car tyres. T. ferrooxidans converted 8 % (w/w) of the sulphur in cryoground spent rubber to sulphate in 20 days. Desulphurization of fresh made natural rubber was studied with scanning electron microscope with energy-dispersive x-ray spectrometer and electron spectroscopy for chemical analysis. The average depletion of sulphur on the treated surface was 24 % compared to the bulk region. Oxidised sulphur species were detected on the surface after microbial treatment. © Rapid Science Ltd. 1998 [ABSTRACT FROM AUTHOR]

The structure of the repeating unit of the O-antigenic polysaccharide from the lipopolysaccharide (LPS) of Serratia marcescens strain 111 was determined by compositional and methylation analyses and NMR spectroscopy as →6)-α-D-Glcp-(1→2)-α-L-Rhap-(1→2)-α-L-Rha p-(1→2)-α-L-Rhap-(1→ (Rha, rhamnose) which represents a new O-antigenic structure in LPS of S. marcescens but is similar to the structure of the repeating unit of the O-antigen of S. marcescens 018 →6)-α-D-GlcpNAc-(1→2)-α-L-Rhap-(1→2)-α-L-Rha p-(1→2)-α-L-Rhap-(1→ (Oxley D., Wilkinson S.G. Structure of a neutral polymer isolated from the lipopolysaccharide of the reference strain for S. marcescens serogroup 018. Carbohydr Res 1989; 195: 111-115). Serological investigations using S. marcescens O- and K-specific ELISA tests and immunoblotting showed that S111 reacted with anti-O18 and anti-K4 sera, and that the anti-S111 serum reacted with serotype strain 018. The K4 reaction was confirmed as capsular by the Quellung reaction. After absorption with the heterologous strain, S. marcescens 018 and strain 111 were shown to be immunologically distinct, probably because of the presence of the GlcNAc residue in 018. We propose to add S111 to the O-serotype strains of S. marcescens as 029. [ABSTRACT FROM PUBLISHER]

Lipopolysaccharide (LPS) of the polymyxin B (PmB)-sensitive variant of Serratia marcescens smooth-type strain 111 (senLPS) binds significantly more PmB than that of the resistant variant (resLPS), following mild acid hydrolysis this difference is abolished. The main compositional differences between these strains are: (i) the amount of 4-amino-4-deoxy-arabinose (Ara4N); and (ii) the substitution patterns of the phosphate groups. ResLPS contains three times more Ara4N than senLPS, mainly phosphodiesters, whereas senLPS contains mainly (di)-phosphomonoesters. Thus, the senLPS, and the bacterial surface, contain a higher negative charge density. Mild acid hydrolysis decreases the PmB-binding capacity of senLPS and increases that of resLPS. The resulting change in LPS composition was a reduction in the amount of phosphate (about 40%) and a loss of nearly all the Ara4N residues, from both LPS. Mild alkaline hydrolysis removes Ara4N from both LPS, and the hydrolysate contains Ara4N-phosphate, but no Ara4N-(1 →8)-Kdo was detected. The results suggest that the 4'-phosphate of lipid A mediates the binding of PmB, whereas its substitution by Ara4N prevents it. [ABSTRACT FROM PUBLISHER]

Five phosphorylated saccharides were isolated from the deacylated then N-acetylated lipopolysaccharide (LPS) of Proteus mirabilis R45 (Re-chemotype) by anion-exchange chromatography. Their structures were determined by 1- and 2-dimensional 1H- and 3C-nuclear magnetic resonance spectroscopy as α-Kdo-(2-4)-[β-L-ArapNAc-(1-8)-]-α-Kdo-(2-6)-β-D-GIc pNAc-(1-6)-α-D-GIcpNAc 1-phosphate (1), α-Kdo-(2-4)-[β-L-Ara pNAc-(1-8)-]-α-Kdo-(2-6)-β-D-GlcpNAc-(1-6)-α-D-Glc pNAc 1,4'-bisphosphate (2), α-Kdo-(2-4)-α-Kdo-(2-6)-β-D-Glc pNAc-(1-6)-α-D-GlcpNAc 1-phosphate (3), α-Kdo-(2-4)-α-Kdo-(2-6)-β-D-Glc pNAc-(1-6)-α-D-GlcpNAc 1,4'-bisphosphate (4), and β-L-Ara pNAc 1-phosphate (5) (ArapNAc, 4-acetamido-4-deoxy-L-arabinopyranose; Kdo, 3-deoxy-D-manno-octulopyranosonic acid). Compounds 1-5 were obtained from deacylated LPS in a molar ratio of ~1.1:0.8:1.1:1.0:0.6 as determined by high-performance anion-exchange chromatography. Although we cannot exclude that in the deacylated LPS (without N-acetylation) the molar ratio may be different, the result can be taken as indication that in LPS about 50% of the 4'-phosphates of lipid A are substituted by (β-L-AraρN. Furthermore, our data prove that the first Kdo in the carbohydrate chain is partially substituted by a second residue of ArapN.The de-O-acylated LPS was additionally investigated using fast atom bombardment and B/E linked scan mass spectrometry which confirmed the above findings and also identified minor amounts of another LPS species possessing a third pentosamine residue. [ABSTRACT FROM PUBLISHER]

A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxymethylcellulose. Three plasmids conferring cellulase activity were purified and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan, but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a nickel nitrilotriacetate column. The enzyme had a pH optimum of 6–7 and its highest measured initial activity at 100 °C. The heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after 8 h at 90 °C. [ABSTRACT FROM AUTHOR]

The use of isopropyl-β- d-thiogalactoside (IPTG) for induction of the lac-promoter in small-scale cultivations is well established. However, for large-scale microbiological processes the cost of this inducer is a severe limitation. Here is described a method by which lactose is used as inducer of the lac promoter with the same efficiency as that of IPTG. It was found that after growth on glucose the time of the addition of lactose is important for the quality of induction. the resulting yield of the recombinant protein increased when lactose was added to the culture if the glucose concentration was rather low. By careful monitoring of the glucose level in the fermentation, using a biosensor, it was possible to add the inducer when the carbon source was nearly depleted. Using Escherichia coli BL21 (pET3), in which was cloned the main antigen coat protein of the foot and mouth disease virus, induction of the gene led to expression of the target protein at a level exceeding 20% of the total cell protein. [ABSTRACT FROM AUTHOR]