Kinetics of carbon sharing in a bacterial consortium revealed by combining stable isotope probing with fluorescence-activated cell sorting.

AIMS: To determine the kinetics of substrate fluxes in a microbial community in order to elucidate the roles of the community members. METHODS AND RESULTS: The kinetics of substrate sharing in a bacterial consortium were measured by a new analytical approach combining immunostaining, stable isotope probing and fluorescence-activated cell sorting (FACS). The bacterial consortium, consisting of four strains and growing on 4-chlorosalicylate (4-CS), was pulse-dosed with the degradation intermediate [U-(13) C]-4-chlorocatechol (4-CC). Cells were stained with strain-specific antibodies sorted by FACS and the (13) C-incorporation into fatty acids of the two most abundant members of the community was determined by isotope ratio mass spectrometry. From the two most abundant strains, the primary degrader Pseudomonas reinekei MT1 incorporated the labelled substrate faster than strain Achromobacter spanius MT3 but the maximal incorporation in strain MT3 was almost three times higher than in MT1. CONCLUSIONS: It has been reported that strain MT1 produces 4-CC as an intermediate but has a lower LD50 for it than strain MT3; therefore, MT3 still degrades 4-CC when the concentrations of 4-CC are already too toxic, even lethal, for MT1. By degrading 4-CC, produced by MT1, MT3 protects the entire community against this toxin. The higher affinity but lower tolerance of strain MT1 for 4-chlorocatechol compared to strain MT3 explains the complementary function these two strains have in the consortium adding exceptional stability to the entire community. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel approach can reveal carbon fluxes in microbial communities generating quantitative data for systems biology of the microbial community.

Correlation of T cell response and bacterial clearance in human volunteers challenged with Helicobacter pylori revealed by randomised controlled vaccination with Ty21a-based Salmonella vaccines.

BACKGROUND: Helicobacter pylori remains a global health hazard, and vaccination would be ideal for its control. Natural infection appears not to induce protective immunity. Thus, the feasibility of a vaccine for humans is doubtful. METHODS: In two prospective, randomised, double-blind, controlled studies (Paul Ehrlich Institute application nos 0802/02 and 1097/01), live vaccines against H pylori were tested in human volunteers seronegative for, and without evidence of, active H pylori infection. Volunteers (n = 58) were immunised orally with Salmonella enterica serovar Typhi Ty21a expressing H pylori urease or HP0231, or solely with Ty21a, and then challenged with 2x10(5) cagPAI(-) H pylori. Adverse events, infection, humoral, cellular and mucosal immune response were monitored. Gastric biopsies were taken before and after vaccination, and postchallenge. Infection was terminated with antibiotics. RESULTS: Vaccines were well tolerated. Challenge infection induced transient, mild to moderate dyspeptic symptoms, and histological and transcriptional changes in the mucosa known from chronic infection. Vaccines did not show satisfactory protection. However, 13 of 58 volunteers, 8 vaccinees and 5 controls, became breath test negative and either cleared H pylori (5/13) completely or reduced the H pylori burden (8/13). H pylori-specific T helper cells were detected in 9 of these 13 (69%), but only in 6 of 45 (13%) breath test-positive volunteers (p = 0.0002; Fisher exact test). T cells were either vaccine induced or pre-existing, depending on the volunteer. CONCLUSION: Challenge infection offers a controlled model for vaccine testing. Importantly, it revealed evidence for T cell-mediated immunity against H pylori infection in humans.

Dietary iron enhances the tumor rate in dimethylhydrazine-induced colon carcinogenesis in mice.

Treatment of male mice with 20 mg/kg dimethylhydrazine (DMH) s.c. for 10 weeks caused a mean tumour rate of 3.5 after 20 weeks. Dietary iron (3.5% Fefumarate for 10 weeks) enhanced the mean tumour rate to 13.9. All tumours detected were localized exclusively in the distal colon and rectum. The iron load caused a 6.5-fold increase in the mucosal Fe-concentration in the proximal as well as distal colon. DMH-demethylase activity was not influenced by iron and did not differ between proximal and distal segments. Cytosolic alcohol dehydrogenase (ADH) activity was also not altered by iron, but was 3.3-fold higher in the distal colon and rectum as compared to proximal segments; this might explain the DMH-induced tumorigenesis in the distal colon only. It is suggested that iron ions might evoke cocarcinogenic activity by a stimulation of cell proliferation.

Influence of dietary iron overload on cell proliferation and intestinal tumorigenesis in mice.

Iron-enriched diets caused an increase of tumor rate in two models of dimethylhydrazine(DMH)-induced colon tumorigenesis in mice. The effect was independent of the time the iron-diet was fed, i.e. during DMH-treatment or following the DMH-treatment period. The increase of tumor rate depended on the iron concentration in the diet (0.5-3.5%). The concentration-dependent iron accumulation in the colonic mucosa of mice was paralleled by increments of malonaldehyde contents indicating lipid peroxidation, another factor known to be involved in tumor development. It is suggested that iron exerts cocarcinogenic activity in the DMH-model by stimulating cell proliferation and inducing oxidative stress in the colonic mucosa. This effect of iron is independent of the time of tumor-initiation by DMH, as it is also observed in the period of tumor-promotion/progression after DMH-treatment.

Recombinant live Salmonella spp. for human vaccination against heterologous pathogens.

Live attenuated Salmonella spp. are promising candidates as oral vaccine delivery systems for heterologous antigens. Clinical trials have demonstrated that this approach is feasible for human vaccinations but further optimisation is necessary to obtain a better efficacy. Here, we discuss how existing clinical and pre-clinical data can be used to guide such optimisation efforts.

How CD4(+) T cells may eliminate extracellular gastric Helicobacter?

Helicobacter pylori is recognised as a causal agent in the pathogenesis of gastritis, gastric and duodenal ulcer disease as well as gastric cancers. Eradication of the bacteria with antibiotics is currently used to treat symptomatic, infected individuals. Theoretically the infection could also be controlled by vaccination. Several immunisation protocols were developed in small animal models and primates in order to validate this approach. Recently making use of mice with defined genetic defects, H. pylori-specific CD4(+) T cells were found to be crucial for protective vaccination. This was unexpected and poses the question of how activation of CD4(+) T cells leads to the elimination of bacteria that reside primarily in the mucin layer behind a barrier of epithelial cells. CD4(+) T cells fulfil their effector function by secreting lymphokines and by engaging specific surface ligands on interacting cells. Here we propose that phagocytes and epithelial cells stimulated either by direct interaction with CD4(+) T cells or by soluble mediators such as cytokines or neuropeptides are the ultimate effector populations in protective immunity induced by vaccination.

The effect of desferrioxamine on cell proliferation in human tumour cell lines.

Iron is known to have a stimulatory effect on cell proliferation whereas the iron-complexing agent desferrioxamine (DFO) has an inhibitory influence on the growth of cultured cells. The effect of iron salts and DFO on cell proliferation and DNA synthesis was studied on two established human tumour cell lines, a colon carcinoma (Caco-2) and a hepatoma-derived cell line (Hep.G2). Cell proliferation was estimated by the neutral red method, DNA synthesis by measuring the [(3)H]thymidine incorporation into the cells. For the analysis of the cell cycle the cells were marked with bromodeoxyuridine for S-phase cells and the monoclonal antibody Ki-67 for all proliferating cells. Ferric chloride stimulated cell proliferation in the Caco-2 line whereas there was no effect in the Hep.G2 line. DFO inhibited cell proliferation and DNA synthesis in both cell lines. Cell cycle analysis revealed a prolongation of the cell cycle by DFO, thereby reducing the number of cells entering the proliferating phases of the cell cycle in both cell lines. The data support the essential role of iron in cell proliferation and tumour growth.

[Computerized tomography and F-18-FDG positron emission tomography in staging of malignant lymphomas: a comparison]. / Computertomographie und F-18-FDG-Positronen-Emissions-Tomographie im Staging maligner Lymphome: ein Vergleich.

PURPOSE: To determine the value of F-18-FDG-positron emission tomography (FDG-PET) compared with computed tomography (CT) in the staging of malignant lymphomas. MATERIAL AND METHOD: 50 patients with biopsy-proven lymphoma were studied with FDG-PET and CT. The results in initial, posttherapeutic and staging of recurrence were compared. RESULTS: 37 of 65 FDG-PET were identical with CT. 28 studies showed differences. 14 post-therapeutically and one of the initial studies led to downstaging by FDG-PET were as upstaging resulted in one case of initial staging. In two cases false positive pulmonary FDG accumulations caused an upstaging. CONCLUSION: FDG-PET was at least comparable to CT in recording the extension of a newly diagnosed lymphoma, or its recurrence. Upstaging according to FDG-PET occurred only once in initial staging. FDG-PET plays its most important role in the evaluation of residual mass in CT after therapy by accumulating FDG in viable tumour rather than in fibrotic tissue. 14 cases of downstaging according to FDG-PET resulted.

[Importance of digital thoracic radiography in the diagnosis of pulmonary infiltrates in patients with bone marrow transplantation during aplasia]. / Wertigkeit der digitalen Thoraxaufnahme bei der Detektion von Lungeninfiltraten knochenmarktransplantierter Patienten in der Aplasie.

PURPOSE: Evaluation of digitized chest x-ray for the detection of pulmonary infiltrations in bone marrow transplant patients during aplasia. METHODS: Digitized chest x-rays of 40 patients (21 female, 19 male) with "Fever of unknown origin" (FUO) were evaluated concerning radiological signs of pulmonary infiltrations and correlated to clinical findings, blood chemistry, microbiology and bronchoscopy. Additionally, an individual risk profile was established. RESULTS: In 11/40 patients pulmonary infiltrations were detected in digitized chest x-rays (group 1). 10/11 developed an infectious pulmonary infiltration. 29/40 patients developed no pulmonary infiltration (group 2). When fever increased for the first time (initial chest x-ray) a sensitivity, specificity, positive and negative predictive value of 46%, 86%, 56%, 81% and for the chest x-rays in progress of 61%, 79% 68% and 73% was found. C-reactive protein and temperature increase occurred statistically significantly earlier (p < 0.05) in group 1 compared to group 2. The average latency of digital chest x-rays in comparison to c-reactive protein and temperature increase was 6 days. The incidence of risk factors was significantly higher in group 1 in comparison to group 2 (p < 0.05). CONCLUSION: Digitized chest x-rays are not a reliable method for primary detection of pulmonary infiltrations after bone marrow transplantation. Individual risk factors have to be taken into consideration to indicate further diagnostic methods such as computed tomography at an earlier time.

Regulated antigen expression in live recombinant Salmonella enterica serovar Typhimurium strongly affects colonization capabilities and specific CD4(+)-T-cell responses.

Regulated antigen expression can influence the immunogenicity of live recombinant Salmonella vaccines, but a rational optimization has remained difficult since important aspects of this effect are incompletely understood. Here, attenuated Salmonella enterica serovar Typhimurium SL3261 strains expressing the model antigen GFP_OVA were used to quantify in vivo antigen levels by flow cytometry and to simultaneously follow the crucial early steps of antigen-specific T-cell responses in mice that are transgenic for a T-cell receptor recognizing ovalbumin. Among seven tested promoters, P(pagC) has the highest activity in murine tissues combined with low in vitro expression, whereas P(tac) has a comparable in vivo and a very high in vitro activity. Both SL3261 (pP(pagC)GFP_OVA) and SL3261 (pP(tac)GFP_OVA) cells can induce potent ovalbumin-specific cellular immune responses following oral administration, but doses almost 1,000-fold lower are sufficient for the in vivo-inducible construct SL3261 (pP(pagC)GFP_OVA) compared to SL3261 (pP(tac)GFP_OVA). This efficacy difference is largely explained by impaired early colonization capabilities of SL3261 (pP(tac)GFP_OVA) cells. Based on the findings of this study, appropriate in vivo expression levels for any given antigen can be rationally selected from the increasing set of promoters with defined properties. This will allow the improvement of recombinant Salmonella vaccines against a wide range of pathogens.

In vivo visualization of bacterial colonization, antigen expression, and specific T-cell induction following oral administration of live recombinant Salmonella enterica serovar Typhimurium.

Live attenuated Salmonella strains that express a foreign antigen are promising oral vaccine candidates. Numerous genetic modifications have been empirically tested, but their effects on immunogenicity are difficult to interpret since important in vivo properties of recombinant Salmonella strains such as antigen expression and localization are incompletely characterized and the crucial early inductive events of an immune response to the foreign antigen are not fully understood. Here, methods were developed to directly localize and quantitate the in situ expression of an ovalbumin model antigen in recombinant Salmonella enterica serovar Typhimurium using two-color flow cytometry and confocal microscopy. In parallel, the in vivo activation, blast formation, and division of ovalbumin-specific CD4(+) T cells were followed using a well-characterized transgenic T-cell receptor mouse model. This combined approach revealed a biphasic induction of ovalbumin-specific T cells in the Peyer's patches that followed the local ovalbumin expression of orally administered recombinant Salmonella cells in a dose- and time-dependent manner. Interestingly, intact Salmonella cells and cognate T cells seemed to remain in separate tissue compartments throughout induction, suggesting a transport of killed Salmonella cells from the colonized subepithelial dome area to the interfollicular inductive sites. The findings of this study will help to rationally optimize recombinant Salmonella strains as efficacious live antigen carriers for oral vaccination.

Purification and characterization of oxygen-evolving photosystem II core complexes from the green alga Chlamydomonas reinhardtii.

Oxygen-evolving photosystem II complexes were isolated from the green alga Chlamydomonas reinhardtii by selective solubilization of thylakoid membranes with dodecyl maltoside followed by density gradient centrifugation and anion-exchange chromatography. In the presence of CaCl2 and K3[Fe(CN)6] the complexes evolved oxygen at rates exceeding 1000 mumol (mg of chl)-1 h-1. The particles contained 40 chlorophylls a and had properties very similar to those of PSII isolated from higher plants. Chlamydomonas reinhardtii is now the first organism which can be used for both site-directed mutagenesis and detailed biochemical and biophysical characterization of oxygen-evolving photosystem II. It seems therefore to be an ideal model organism for investigation of structure-function relationships in photosynthetic oxygen evolution.

The ctenophore Mnemiopsis leidyi has a flow-through system for digestion with three consecutive phases of extracellular digestion.

The ctenophore (comb jelly) Mnemiopsis leidyi is a periodically abundant and voracious predator in U.S. coastal waters. Mnemiopsis leidyi is especially competitive at high prey concentrations because of its very efficient extracellular digestion. We investigated the functional basis for these outstanding digestion capabilities. Extracellular digestion takes place in the pharynx and consists of three distinct and consecutive phases. The three phases take place in different regions of the pharynx so that various prey items can be treated simultaneously in each phase. The first phase is acidic, while the second and the third are alkaline. Extracellular digestion is completed by ciliary currents that mechanically disrupt the predigested food. Bulky indigestible food fragments are expelled through the mouth. Except for a small area, the paths for ingestion and egestion are separate. Hence, both ingestion and egestion can occur simultaneously. The flattened and elongated shape of the pharynx provides the morphological basis for this flow-through system with various regions for different digestive treatments of the food. This system is highly elaborated compared with those of other lower invertebrates and allows for an efficient, fast, and simultaneous digestion of many prey items, which accounts for the outstanding feeding capabilities of M. leidyi.

Destruction of a single chlorophyll is correlated with the photoinhibition of photosystem II with a transiently inactive donor side.

Pigments destroyed during photoinhibition of water-splitting photosystem II core complexes from the green alga Chlamydomonas reinhardtii were studied. Under conditions of a transiently inactivated donor side, illumination leads to an irreversible inhibition of the electron transfer at the donor side that is paralleled by the destruction of chlorophylls a absorbing maximally around 674 and 682 nm. The observed stochiometry of 1 +/- 0.1 destroyed chlorophyll per inhibited photosystem II suggests that chlorophyll destruction could be the primary photodamage causing the inhibition of photosystem II under these conditions.

[Tertiary yaws--skeletal manifestations at the distal tibia]. / Tertiäre Frambösie--Skelettmanifestation an der distalen Tibia.

We describe the case of a 16-year old male patient from African presenting tertiary yaws at the distal tibia. Clinical signs, biochemical results and the different diagnostic imaging methods are demonstrated. Differential diagnosis and therapy are described.

[Primary pleural metastasis of osteosarcoma]. / Primäre pleurale Metastasierung eines Osteosarkoms.

We describe the case of a 18-year-old male patient presenting primary pleural metastasis with osteosarcoma at the proximal right tibia. Clinical signs, therapy and imaging methods are demonstrated. Differential diagnosis is described.

The attenuated Salmonella vaccine approach for the control of Helicobacter pylori-related diseases.

The Gram-negative bacterium Helicobacter pylori is a widespread human pathogen that colonizes the gastric mucosa and is associated with gastro-intestinal illnesses such as gastritis, peptic ulcer, gastric lymphoma and gastric cancer. Current pharmacological therapies are becoming less reliable for the control of H. pylori due to the elevated costs and to the increasing number of antibiotic resistant strains. New vaccination strategies utilizing H. pylori antigens combined with adjuvants or delivery of antigens by attenuated Salmonella strains have been successful in protecting mice against H. pylori infections. Oral immunization with single doses of urease-expressing Salmonella vaccine strains elicits mucosal and systemic antibody responses and fully protects different mouse strains against challenge infections with H. pylori. The high efficacy in the mouse model, combined with remarkable immunogenicity, safety and low-cost production, makes attenuated live recombinant Salmonella promising vaccine candidates for the control of H. pylori-related diseases in humans.

Proteome analysis of the common human pathogen Helicobacter pylori.

The common human pathogen Helicobacter pylori is the major cause of gastric and duodenal ulcers. Based on the complete genome sequences of two independent isolates more than 1800 protein species have been resolved by two-dimensional gel electrophoresis and more than 200 of them have been identified (http://www.mpiib-berlin.mpg.de/2D-PAGE). Using these data, a large range of research areas including strain fingerprinting, protein composition and subcellular localization, gene regulation, and pathogen-host interactions have been investigated. The results that have been obtained led to a more detailed understanding of the Helicobacter biology and pathology and open further interesting fields for future work.

Multiparameter selection of Helicobacter pylori antigens identifies two novel antigens with high protective efficacy.

A multiparameter selection of Helicobacter pylori antigens for vaccine development identified 15 candidates, 6 of which are known protective antigens. Two novel antigens with low homology to other organisms (HP0231 and HP0410) were overexpressed and purified with high yields. Both confer protective immunity in the mouse Helicobacter infection model.