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.

Impact of vector-priming on the immunogenicity of a live recombinant Salmonella enterica serovar typhi Ty21a vaccine expressing urease A and B from Helicobacter pylori in human volunteers.

Orally administered recombinant Salmonella vaccines represent an attractive option for mass vaccination programmes against various infectious diseases. Therefore, it is crucial to gather knowledge about the possible impact of preexisiting immunity to carrier antigens on the immunogenicity of recombinant vaccines. Thirteen volunteers were preimmunized with Salmonella typhi Ty21a in order to evaluate the effects of prior immunization with the carrier strain. Then, they received three doses of 1-2 x 10(10) viable organisms of either the vaccine strain S. typhi Ty21a (pDB1) expressing subunits A and B of recombinant Helicobacter pylori urease (n = 9), or placebo strain S. typhi Ty21a (n = 4). Four volunteers were preimmunized and boosted with the vaccine strain S. typhi Ty21a (pDB1). No serious adverse effects were observed in any of the volunteers. Whereas none of the volunteers primed and boosted with the vaccine strain responded to the recombinant antigen, five of the nine volunteers preimmunized with the carrier strain showed cellular immune responses to H. pylori urease (56%). This supports the results of a previous study in non-preimmunized volunteers where 56% (five of nine) of the volunteers showed a cellular immune response to urease after immunisation with S. typhi Ty21a (pDB1).

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.

Safety and immunogenicity of live recombinant Salmonella enterica serovar Typhi Ty21a expressing urease A and B from Helicobacter pylori in human volunteers.

Helicobacter pylori urease was expressed in the common live typhoid vaccine Ty21a yielding Ty21a(pDB1). Nine volunteers received Ty21a(pDB1) and three control volunteers received Ty21a. No serious adverse effects were observed in any of the volunteers. Ten out of 12 volunteers developed humoral immune responses to the Salmonella carrier as detected by antigen-specific antibody-secreting cells but only two volunteers seroconverted. A total of five volunteers showed responses in one or two out of three assays for cellular responses to the carrier (proliferation, IFN-gamma-secretion, IFN-gamma-ELISPOT). Three of the volunteers that had received Ty21a(pDB1) showed a weak but significant T-cell response to Helicobacter urease, while no volunteer had detectable humoral responses to urease. Ty21a(pDB1) is a suitable prototype to optimize Salmonella-based vaccination for efficient cellular responses that could mediate protective immunity against Helicobacter.

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.

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.

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.

18FDG-PET following treatment as valid predictor for disease-free survival in Hodgkin's lymphoma.

PURPOSE: The value of 18FDG-PET to predict the outcome after therapy in Hodgkin's lymphoma was compared to morphologic staging and ESR. PATIENTS AND METHODS: A total of 50 concurrent 18FDG-PET and CT studies were performed in 37 patients with Hodgkin's lymphoma. ESR was evaluated 32 times after treatment was completed. RESULTS: Out of 39 residual masses found by CT 8 relapses could be proven. Out of 11 CT exams with CR 3 relapses occurred. CT turned out to show a sensitivity, specificity, PPV, NPV, and accuracy of 72%, 21%, 21%, 73%, and 32%, with respect to predict disease-free survival (DFS). 18FDG-PET was positive in 22 examinations with 10 recurrences in this group. Out of 28 negative 18FDG-PET 1 relapse developed 3 years later. 18FDG-PET turned out to show promising sensitivity, specificity, PPV, NPV, and accuracy of 91%, 69%, 46%, 96%, 74%, with respect to predict DFS. ESR was elevated in 12 studies of which 5 relapses could be proven, while out of 20 normal ESR-studies 3 relapses occurred. Thus, ESR turned out to show sensitivity, specificity, PPV, NPV, and accuracy of 63%, 71%, 42%, 85%, and 75%, with respect to predict DFS. In summary, only 18FDG-PET was able to predict DFS statistically significant. CONCLUSION: 18FDG-PET can be very useful in patients with residual masses after treatment.

Adoptive transfer of CD4+ T cells specific for subunit A of Helicobacter pylori urease reduces H. pylori stomach colonization in mice in the absence of interleukin-4 (IL-4)/IL-13 receptor signaling.

Protection in the murine model of Helicobacter pylori infection may be mediated by CD4+ T cells, but the mechanism remains unclear. To better understand how protection occurs in this model, we generated and characterized H. pylori urease-specific CD4+ T cells from BALB/c mice immunized with Salmonella enterica serovar Typhimurium expressing H. pylori urease (subunits A and B). The CD4+ T cells were found to be specific for subunit A (UreA). Upon antigen-specific stimulation, expression of interleukin 4 (IL-4), IL-10, gamma interferon (IFN-gamma), and tumor necrosis factor alpha was induced. Immunocytochemical analysis showed that the majority of cells produced IFN-gamma and IL-10. Adoptive transfer of the UreA-specific CD4+ T cells into naive syngeneic recipients led to a threefold reduction in the number of bacteria in the recipient group when compared to that in the nonrecipient group. Stomach colonization was also reduced significantly after transfer of these cells into patently infected mice. Adoptive transfer of UreA-specific CD4+ T cells into IL-4 receptor alpha chain-deficient BALB/c mice indicated that IL-4 and IL-13 were not critical in the control of bacterial load. In addition, synthetic peptides were used to identify three functional T-cell epitopes present in subunit A which were recognized by the UreA-specific T cells. Analysis of H. pylori-specific cellular immune responses in recipient challenged and nonrecipient infected mice indicated a strong local restriction of the response in infected animals. The implications of these findings for the mechanism of protection and the development of peptide-based vaccination are discussed.

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.

Comparative proteome analysis of Helicobacter pylori.

Helicobacter pylori, the causative agent of gastritis, ulcer and stomach carcinoma, infects approximately half of the worlds population. After sequencing the complete genome of two strains, 26695 and J99, we have approached the demanding task of investigating the functional part of the genetic information containing macromolecules, the proteome. The proteins of three strains of H. pylori, 26695 and J99, and a prominent strain used in animal models SS1, were separated by a high-resolution two-dimensional electrophoresis technique with a resolution power of 5000 protein spots. Up to 1800 protein species were separated from H. pylori which had been cultivated for 5 days on agar plates. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) peptide mass fingerprinting we have identified 152 proteins, including nine known virulence factors and 28 antigens. The three strains investigated had only a few protein spots in common. We observe that proteins with an amino acid exchange resulting in a net change of only one charge are shifted in the two-dimensional electrophoresis (2-DE) pattern. The expression of 27 predicted conserved hypothetical open reading frames (ORFs) and six unknown ORFs were confirmed. The growth conditions of the bacteria were shown to have an effect on the presence of certain proteins. A preliminary immunoblotting study using human sera revealed that this approach is ideal for identifying proteins of diagnostic or therapeutic value. H. pylori 2-DE patterns with their identified protein species were added to the dynamic 2D-PAGE database (http://www.mpiib-berlin.mpg.de/2D-PAGE/). This basic knowledge of the proteome in the public domain will be an effective instrument for the identification of new virulence or pathogenic factors, and antigens of potentially diagnostic or curative value against H. pylori.

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.

[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.

How important is the route of reconstruction after esophagectomy: a prospective randomized study.

OBJECTIVE: A prospective randomized trial was performed to compare retrosternal and posterior mediastinal gastric tube reconstruction with regard to postoperative function and quality of life. METHODS: Twenty-six patients were randomly allocated to either retrosternal (n = 14) or posterior mediastinal (n = 12) reconstruction after gastric tube formation. Radionuclide transit studies were applied to obtain objective functional data and a standardized quality-of-life assessment was performed. RESULTS: Retrosternal reconstruction showed an increased morbidity (15 vs 13 major complications) and mortality (14.2 vs 8.3%). Radionuclide clearance in the supine position was delayed in the gastric tube in general, compared with normal controls (retention index > 40% vs < 10%). There was a significantly higher retention (p < 0.005) in the retrosternal group in the middle third of the tube and the whole tube after intake of the liquid tracer. The retention of the first solid tracer was also higher in the retrosternal group in the middle third of the tube (p = n.s.) and was significantly higher in the whole tube after 30 (p < 0.05) and 60 (p < 0.01) s. This had no significant impact on the patients' quality of life. CONCLUSIONS: The posterior mediastinal route of reconstruction is recommended but curative resection (R0) is mandatory to avoid possible complications due to local tumor relapse. After incomplete resection (R1 or R2) we recommend retrosternal reconstruction for better palliation.

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.

[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.

Whole-body positron emission tomography (PET) for diagnosis of residual mass in patients with lymphoma.

BACKGROUND: PET using 18fluorodesoxyglucose (FDG) may offer the possibility of differentiating vital from necrotic residual masses. PATIENTS AND METHODS: Seventeen patients with HD and 17 patients with NHL underwent FDG-PET following therapy. According to staging by routine methods at diagnosis, 7 patients presented stage I, 13 stage II, 5 stage III, and 9 stage IV. A dose of 250-400 MBq FDG was injected and whole-body PET was performed 30-60 minutes later. RESULTS: Residual mass was found in 32 patients with routine methods. FDG-PET was negative in 17 patients, who were considered to be in CR. None of them relapsed (median follow-up 63 weeks ). FDG-PET was positive in 17 patients. Sixteen patients had residual mass with routine methods. Four patients received radiation after PET. Their median follow-up is 58 weeks without relapse. Two other patients with lasting CR had FDG uptake outside the residual mass--one with confirmed pneumonia. Five patients had histologically confirmed lymphoma, 2 patients relapsed according to routine methods. One patient is likely to be false positive because of fracture at lymphoma site. Seven of 10 patients with FDG uptake in the residual mass after completed therapy relapsed. According to routine restaging, 2 patients achieved CR. In 1 patient an additional focus was found in the humerus in spite of normal scintigraphy with histologically confirmed lymphoma. There were no false-negative results, but 3 false-positive results inside and 2 false-positive results outside the residual mass after completed therapy. CONCLUSIONS: PET performed for evaluation of residual mass after treatment of lymphoma has a high predictive value.

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.

[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.