Culture of a Gastric Non‐Helicobacter pylori Helicobacter from the Stomach of a 14‐Year‐Old Girl

Helicobacter felis belongs to the fastidious gastric non‐Helicobacter pylori helicobacter species that are typically found in the stomach of cats and dogs. These bacteria have the potential to colonize the human stomach and are then associated with gastritis, gastroduodenal ulcers, and MALT lymphoma. Strains cultured from the human stomach are rare. Here, we present the first isolation of H. felis from a gastric biopsy specimen of a 14‐year‐old girl who presented with persistent epigastric pain. The strain was cultured using our routine protocol for H. pylori and identified by phylogenetic analyses of partial urease AB and gyrB gene sequences.     

Investigations on Aberrant Glycosylation of Glycosphingolipids in Colorectal Cancer Tissues Using Liquid Chromatography and Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF-MS)

Cancer is a leading cause of death and alterations of glycosylation are characteristic features of malignant cells. Colorectal cancer is one of the most common cancers and its exact causes and biology are not yet well understood. Here, we compared glycosylation profiles of colorectal tumor tissues and corresponding control tissues of 13 colorectal cancer patients to contribute to the understanding of this cancer. Using MALDI-TOF(/TOF)-MS and 2-dimensional LC-MS/MS we characterized enzymatically released and 2-aminobenzoic acid labeled glycans from glycosphingolipids. Multivariate data analysis revealed significant differences between tumor and corresponding control tissues. Main discriminators were obtained, which represent the overall alteration in glycosylation of glycosphingolipids during colorectal cancer progression, and these were found to be characterized by (1) increased fucosylation, (2) decreased acetylation, (3) decreased sulfation, (4) reduced expression of globo-type glycans, as well as (5) disialyl gangliosides. The findings of our current research confirm former reports, and in addition expand the knowledge of glycosphingolipid glycosylation in colorectal cancer by revealing new glycans with discriminative power and characteristic, cancer-associated glycosylation alterations. The obtained discriminating glycans can contribute to progress the discovery of biomarkers to improve diagnostics and patient treatment.Worldwide, cancer is a leading cause of death. With estimated 1.2 million diagnoses in 2008, colorectal cancer is the third most common cancer in the world and the fourth most common cause of death with an annual mortality of ∼600 000 (1). The exact causes of colorectal cancer are unknown, but different risk factors such as age, polyps, personal and family history, ulcerative colitis, or Crohn''s colitis have been proposed (2). Standard screening procedures include flexible sigmoidoscopy, colonoscopy, and immunological fecal occult blood testing. Each of them has its advantages and drawbacks such as invasiveness or low sensitivity and specificity (3). The method of choice for the treatment of colorectal cancer is surgery and therapeutic decisions are based on the tumor, lymph node, and metastasis staging-system as a prognostic factor (4). Current research has led to improved treatment strategies of colorectal cancer, however, the clinical outcome, the progression of the disease, and the response to the treatment remain variable among individuals. The heterogeneity of colorectal cancer at the molecular level—caused by accumulation of multiple genetic changes—may be one of the main reasons for this variability (5). Genetic factors such as instabilities, but also expression levels (6) can explain part of the cancer biology, but glycomics is gaining importance to complement the overall picture as aberrant glycosylation of proteins and lipids has been shown to be correlated with disease and malignancy (7, 8).Glycosylation is involved in many biological processes and especially its functional role in cellular interaction with respect to adhesion, cell growth, and signaling is prone to be affected in cancer progression, invasion, and metastasis (9). Several cancer-associated alterations in protein glycosylation have been reported: (1) increased branching of N-glycans, (2) higher density of O-glycans, and (3) incomplete synthesis of glycans. More particularly, an increased or induced expression of GlcNAc transferase V resulting in N-glycan structures with β1–6GlcNAc antennae (5, 10), and the expression of (sialyl) Tn-antigens (11) as aberrant O-glycosylation have been reported (10).Altered glycosphingolipid (GSL)¹ glycosylation of the cell surface membrane during malignancy can affect cell recognition, adhesion, and signal transduction (12) and is found to reflect: (1) incomplete synthesis with or without precursor accumulation, (2) neosynthesis (9), (3) increased sialylation, and (4) increased fucosylation (13). In many cancers, including colorectal cancer, an overexpression of the (sialyl) Lewis X antigen (10, 14) and the expression of (sialyl) Lewis A (15) are considered to be related to malignant transformation—reflecting incomplete synthesis of sialyl 6-sulfo Lewis X and disialyl Lewis A (16) as well as neosynthesis (17). Studies on gangliosides showed an overexpression of these sialylated GSLs in human malignant melanoma (18). Furthermore, the involvement of gangliosides in cell adhesion and motility was reported, which contributes to tumor metastasis (19). Specifically, the gangliosides GD3 (Hex2NeuAc2ceramide) and GM2 (Hex2HexNAc1NeuAc1ceramide) have been found to be associated with tumor-angiogenesis (19). The up-regulation of fucosyltransferases in cancer was shown to cause a higher degree of fucosylation in malignant tissues (20) and Moriwaki et al. proposed that the increase in the fucosylation for GSLs was an early event in cancer (21). Misonou et al. investigated glycans derived from GSLs in colorectal cancer tissues showing aberrant glycan structures based on linkage differences as well as increased sialylation and fucosylation compared with control tissue (22), which is in line with observed changes in GSL glycosylation with regard to cancer progression (9, 13).Recently, we investigated the N-glycosylation profiles of colorectal tumors and correlating control tissues for biomarker discovery. Statistical analyses revealed an increase of sulfated glycan structures as well as paucimannosidic glycans and glycans containing sialylated Lewis type epitopes in the tumor tissue, whereas structures with bisecting GlcNAc were found to be decreased in malignancy (23). To further progress the understanding of colorectal cancer biology and the improvement of diagnostic tools and patient treatment, we complemented this recent study on N-glycosylation by an investigation of the glycosphingolipid-derived glycans (named GSL-glycans in the following) from frozen tumor tissues and corresponding control tissues from the same 13 colorectal cancer patients. GSL-glycans were enzymatically released, labeled with 2-aminobenzoic acid (AA) and analyzed by hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Employing multivariate statistical analysis, this approach revealed an intricate GSL-glycosylation pattern of tumor tissues and specific glycosylation differences in comparison to the corresponding control tissue.     

Genomic analysis reveals key aspects of prokaryotic symbiosis in the phototrophic consortium “Chlorochromatium aggregatum”

Background

‘Chlorochromatium aggregatum’ is a phototrophic consortium, a symbiosis that may represent the highest degree of mutual interdependence between two unrelated bacteria not associated with a eukaryotic host. ‘Chlorochromatium aggregatum’ is a motile, barrel-shaped aggregate formed from a single cell of ‘Candidatus Symbiobacter mobilis”, a polarly flagellated, non-pigmented, heterotrophic bacterium, which is surrounded by approximately 15 epibiont cells of Chlorobium chlorochromatii, a non-motile photolithoautotrophic green sulfur bacterium.

Results

We analyzed the complete genome sequences of both organisms to understand the basis for this symbiosis. Chl. chlorochromatii has acquired relatively few symbiosis-specific genes; most acquired genes are predicted to modify the cell wall or function in cell-cell adhesion. In striking contrast, ‘Ca. S. mobilis’ appears to have undergone massive gene loss, is probably no longer capable of independent growth, and thus may only reproduce when consortia divide. A detailed model for the energetic and metabolic bases of the dependency of ‘Ca. S. mobilis’ on Chl. chlorochromatii is described.

Conclusions

Genomic analyses suggest that three types of interactions lead to a highly sophisticated relationship between these two organisms. Firstly, extensive metabolic exchange, involving carbon, nitrogen, and sulfur sources as well as vitamins, occurs from the epibiont to the central bacterium. Secondly, ‘Ca. S. mobilis’ can sense and move towards light and sulfide, resources that only directly benefit the epibiont. Thirdly, electron cycling mechanisms, particularly those mediated by quinones and potentially involving shared protonmotive force, could provide an important basis for energy exchange in this and other symbiotic relationships.     

Electron Microscopic,Genetic and Protein Expression Analyses of Helicobacter acinonychis Strains from a Bengal Tiger

Colonization by Helicobacter species is commonly noted in many mammals. These infections often remain unrecognized, but can cause severe health complications or more subtle host immune perturbations. The aim of this study was to isolate and characterize putative novel Helicobacter spp. from Bengal tigers in Thailand. Morphological investigation (Gram-staining and electron microscopy) and genetic studies (16SrRNA, 23SrRNA, flagellin, urease and prophage gene analyses, RAPD DNA fingerprinting and restriction fragment polymorphisms) as well as Western blotting were used to characterize the isolated Helicobacters. Electron microscopy revealed spiral-shaped bacteria, which varied in length (2.5–6 µm) and contained up to four monopolar sheathed flagella. The 16SrRNA, 23SrRNA, sequencing and protein expression analyses identified novel H. acinonychis isolates closely related to H. pylori. These Asian isolates are genetically very similar to H. acinonychis strains of other big cats (cheetahs, lions, lion-tiger hybrid and other tigers) from North America and Europe, which is remarkable in the context of the great genetic diversity among worldwide H. pylori strains. We also found by immunoblotting that the Bengal tiger isolates express UreaseA/B, flagellin, BabA adhesin, neutrophil-activating protein NapA, HtrA protease, γ-glutamyl-transpeptidase GGT, Slt lytic transglycosylase and two DNA transfer relaxase orthologs that were known from H. pylori, but not the cag pathogenicity island, nor CagA, VacA, SabA, DupA or OipA proteins. These results give fresh insights into H. acinonychis genetics and the expression of potential pathogenicity-associated factors and their possible pathophysiological relevance in related gastric infections.     

T Cell-Specific Overexpression of TGF?1 Fails to Influence Atherosclerosis in ApoE-Deficient Mice

Clinical data have indicated a negative correlation between plasma TGFß1 concentrations and the extent of atherosclerosis and have thus led to the hypothesis that the pleiotropic cytokine may have anti-atherogenic properties. T-cells are currently discussed to significantly participate in atherogenesis, but the precise role of adaptive immunity in atherogenesis remains to be elucidated. TGFß1 is known to strongly modulate the function of T-cells, however, inhibition of TGFß1 signalling in T-cells of atherosclerosis-prone knock-out mice failed to unequivocally clarify the role of the cytokine for the development of atherosclerosis. In the present study, we thus tried to specify the role of TGFß1 in atherogenesis by using the murine CD2-TGFß1 transgenic strain which represents a well characterized model of T-cell specific TGFß1 overexpression. The CD2-TGFß1 transgenic mice were crossed to ApoE knock-out mice and quantity and quality of atherosclerosis regarding number of macrophages, smooth muscle cells, CD3 positive T-cells and collagen was analyzed in CD2-TGFß1 ApoE double mutants as well as non-transgenic ApoE controls on both normal and atherogenic diet of a duration of 8, 16 or 24 weeks, respectively. In all experimental groups investigated, we failed to detect any influence of TGFß1 overexpression on disease. Total number of CD3-positive T-lymphocytes was not significantly different in atherosclerotic lesions of CD2-TGFß1 ApoE^−/− females and isogenic ApoE^−/− controls, even after 24 weeks on the atherogenic diet. The synopsis of these data and our previous study on TGFß1 overexpressing macrophages suggests that potential effects of TGFß1 on atherosclerosis are most probably mediated by macrophages rather than T-cells.     

Cognitive Factors Mediate Placebo Responses in Patients with House Dust Mite Allergy

Background

Placebo effects have been reported in type I allergic reactions. However the neuropsychological mechanisms steering placebo responses in allergies are largely unknown. The study analyzed whether and to what extend a conditioned placebo response is affecting type I allergic reactions and whether this response can be reproduced at multiple occasions.

Methods

62 patients with house dust mite allergy were randomly allocated to either a conditioned (n = 25), sham-conditioned (n = 25) or natural history (n = 12) group. During the learning phase (acquisition), patients in the conditioned group received the H₁-receptor antagonist desloratadine (5mg) (unconditioned stimulus/US) together with a novel tasting gustatory stimulus (conditioned stimulus/CS). Patients in the sham-conditioned control group received the CS together with a placebo pill. After a wash out time of 9 days patients in the conditioned and sham-conditioned group received placebo pills together with the CS during evocation. Allergic responses documented by wheal size after skin prick test and symptom scores after nasal provocation were analyzed at baseline, after last desloratadine treatment and after the 1^st and 5^th CS re-exposure.

Results

Both conditioned and sham-conditioned patients showed significantly decreased wheal sizes after the 1^st CS-evocation and significantly decreased symptom scores after the 1^st as well as after the 5^th evocation compared to the natural history control group.

Conclusions

These results indicate that placebo responses in type I allergy are not primarily mediated by learning processes, but seemed to be induced by cognitive factors such as patients’ expectation, with these effects not restricted to a single evocation.     

Copper residues in meat from wild artiodactyls hunted with two types of rifle bullets manufactured from copper

Copper content in muscle and the presence of bullet fragments were assessed in samples from red deer (Cervus elaphus), roe deer (Capreolus capreolus), fallow deer (Dama dama) and wild boar (Sus scrofa) (total of 46 animals) which had been hunted with two types of solid copper bullets. Also, the release of copper from bullets or fragments remaining in muscle was tested. For bullet type “B”, a fragment was detected in only 1 out of 34 carcasses whereas for type “A”, fragments were detected in all 12 carcasses, with up to 24 fragments (maximum size, 5?×?7?×?2 mm). Median copper concentrations around the shot wound (0–30 mm distance) were 1.25 and 1.77 mg/kg fresh weight for bullet types B and A, respectively, and thus in the expected range for venison. Around bullet fragments that had remained in muscles, the copper content increased significantly. In roe deer longissimus muscle that had been exposed for 7 days at +7 °C to bullets of type B, up to 1,000 mg/kg copper (fresh weight) was found in a distance of 0–2 mm. However, in a distance of 10–20 mm, maximum copper contents were <10 mg/kg fresh weight. Bullet fragments can constitute physical hazards and will release copper under acidic conditions as those prevailing in meat. Removal of bullet fragments prior to culinary preparation should ensure that a recommended dietary copper intake of 1.25 mg per adult consumer per day is not exceeded. From a food hygiene viewpoint, non-fragmenting bullets seem to be preferable.     

Flow infusion electrospray ionisation mass spectrometry for high throughput,non-targeted metabolite fingerprinting: a review

Producing a comprehensive overview of the chemical content of biologically-derived material is a major challenge. Apart from ensuring adequate metabolome coverage and issues of instrument dynamic range, mass resolution and sensitivity, there are major technical difficulties associated with data pre-processing and signal identification when attempting large scale, high-throughput experimentation. To address these factors direct infusion or flow infusion electrospray mass spectrometry has been finding utility as a high throughput metabolite fingerprinting tool. With little sample pre-treatment, no chromatography and instrument cycle times of less than 5 min it is feasible to analyse more than 1,000 samples per week. Data pre-processing is limited to aligning extracted mass spectra and mass-intensity matrices are generally ready in a working day for a month’s worth of data mining and hypothesis generation. ESI-MS fingerprinting has remained rather qualitative by nature and as such ion suppression does not generally compromise data information content as originally suggested when the methodology was first introduced. This review will describe how the quality of data has improved through use of nano-flow infusion and mass-windowing approaches, particularly when using high resolution instruments. The increasingly wider availability of robust high accurate mass instruments actually promotes ESI-MS from a merely fingerprinting tool to the ranks of metabolite profiling and combined with MS/MS capabilities of hybrid instruments improved structural information is available concurrently. We summarise current applications in a wide range of fields where ESI-MS fingerprinting has proved to be an excellent tool for “first pass” metabolome analysis of complex biological samples. The final part of the review describes a typical workflow with reference to recently published data to emphasise key aspects of overall experimental design.