JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
Psoriasis is not an autoimmune disease?
Exp. Dermatol.
PUBLISHED: 10-13-2014
Show Abstract
Hide Abstract
The concept that psoriasis is an autoimmune disease needs to be questioned. The autoimmune label has been based on molecular mimicry between streptococcal and keratin proteins and the existence of homologous peptides between these proteins. However, it is only peripheral blood CD8, and not CD4, T lymphocytes that respond to the homologous peptides. This ignores the fact that it is CD4 T cells which are necessary to initiate psoriasis. Recent studies on skin bacterial microbiota have found a variety of bacteria in both normal skin and psoriatic lesions. In biopsy specimens, the most common phylum was Firmicutes and the most common genus streptococcus in both psoriasis and normal skin. The innate immune system is activated in psoriasis, and recent genetic findings have shown the majority of susceptibility loci are associated with innate immunity. There is a known clinical relationship between both Crohn's disease (CD) and periodontitis, and psoriasis, and patients with psoriasis share mutations in some innate immunity genes with individuals with CD. It is now accepted that CD is due to a breakdown of immune tolerance (dysbiosis) to bacteria in the intestine. These findings suggest that psoriasis is initiated by an abnormal response to bacteria in the skin due to genetic factors.
Related JoVE Video
[In Process Citation].
Lakartidningen
PUBLISHED: 09-26-2014
Show Abstract
Hide Abstract
There is international consensus that patients younger than 50 years of age presenting with uninvestigated dyspepsia without any alarm symptoms or signs shall be managed according to the »test and treat« strategy and not with an initial upper endoscopy. This has however not been applied in Sweden. One reason might be the lack of health economic analysis performed according to the Swedish health care system. In this report we have applied costs according to Swedish conditions to a model with a decision tree analysis. Our health economic analysis indicate that the »test and treat« model is an alternative to including gastroscopy in the primary management of dyspepsia in younger patients, saves money and reduces the number of gastroscopies by 80%.  
Related JoVE Video
Susceptibility to Campylobacter infection is associated with the species composition of the human fecal microbiota.
MBio
PUBLISHED: 09-18-2014
Show Abstract
Hide Abstract
The gut microbiota is essential for human health, but very little is known about how the composition of this ecosystem can influence and respond to bacterial infections. Here we address this by prospectively studying the gut microbiota composition before, during, and after natural Campylobacter infection in exposed poultry abattoir workers. The gut microbiota composition was analyzed with 16S amplicon sequencing of fecal samples from poultry abattoir workers during the peak season of Campylobacter infection in Sweden. The gut microbiota compositions were compared between individuals who became culture positive for Campylobacter and those who remained negative. Individuals who became Campylobacter positive had a significantly higher abundance of Bacteroides (P = 0.007) and Escherichia (P = 0.002) species than those who remained culture negative. Furthermore, this group had a significantly higher abundance of Phascolarctobacterium (P = 0.017) and Streptococcus (P = 0.034) sequences than the Campylobacter-negative group, which had an overrepresentation of Clostridiales (P = 0.017), unclassified Lachnospiraceae (P = 0.008), and Anaerovorax (P = 0.015) sequences. Intraindividual comparisons of the fecal microbiota compositions yielded small differences over time in Campylobacter-negative participants, but significant long-term changes were found in the Campylobacter-positive group (P < 0.005). The results suggest that the abundance of specific genera in the microbiota reduces resistance to Campylobacter colonization in humans and that Campylobacter infection can have long-term effects on the composition of the human fecal microbiota.
Related JoVE Video
A repetitive DNA element regulates expression of the Helicobacter pylori sialic acid binding adhesin by a rheostat-like mechanism.
PLoS Pathog.
PUBLISHED: 07-01-2014
Show Abstract
Hide Abstract
During persistent infection, optimal expression of bacterial factors is required to match the ever-changing host environment. The gastric pathogen Helicobacter pylori has a large set of simple sequence repeats (SSR), which constitute contingency loci. Through a slipped strand mispairing mechanism, the SSRs generate heterogeneous populations that facilitate adaptation. Here, we present a model that explains, in molecular terms, how an intergenically located T-tract, via slipped strand mispairing, operates with a rheostat-like function, to fine-tune activity of the promoter that drives expression of the sialic acid binding adhesin, SabA. Using T-tract variants, in an isogenic strain background, we show that the length of the T-tract generates multiphasic output from the sabA promoter. Consequently, this alters the H. pylori binding to sialyl-Lewis x receptors on gastric mucosa. Fragment length analysis of post-infection isolated clones shows that the T-tract length is a highly variable feature in H. pylori. This mirrors the host-pathogen interplay, where the bacterium generates a set of clones from which the best-fit phenotypes are selected in the host. In silico and functional in vitro analyzes revealed that the length of the T-tract affects the local DNA structure and thereby binding of the RNA polymerase, through shifting of the axial alignment between the core promoter and UP-like elements. We identified additional genes in H. pylori, with T- or A-tracts positioned similar to that of sabA, and show that variations in the tract length likewise acted as rheostats to modulate cognate promoter output. Thus, we propose that this generally applicable mechanism, mediated by promoter-proximal SSRs, provides an alternative mechanism for transcriptional regulation in bacteria, such as H. pylori, which possesses a limited repertoire of classical trans-acting regulatory factors.
Related JoVE Video
Impact of lifestyle on the gut microbiota of healthy infants and their mothers - the ALADDIN birth cohort.
FEMS Microbiol. Ecol.
PUBLISHED: 06-18-2014
Show Abstract
Hide Abstract
An anthroposophic lifestyle, which has been associated with reduced allergy risk in children, has several characteristics that could influence gut microbiota. This study aimed to investigate the impact of anthroposophic lifestyle as well as specific early life exposures on the gut microbiota. In total, 665 stool samples from 128 mother-infant pairs from the ALADDIN birth cohort study were included. Samples collected from infants at ages 6 days, 3 weeks, 2 months and 6 months, and from their mothers before and after delivery, respectively, were analyzed using 454-pyrosequencing. Information regarding lifestyle exposures was collected prospectively through interviews and questionnaires. Six-month-old infants in anthroposophic families had a significantly higher abundance of Bifidobacterium and lower abundances of Bacteroides and Veillonella. Caesarean section and breastfeeding had a significant impact on the microbiota: caesarean section was primarily associated with delayed colonization of Bifidobacterium and Bacteroides, whereas breastfed children had a higher relative abundance of Bifidobacterium and a lower abundance of Clostridiales. However, despite large differences in lifestyle exposures, we determined no significant differences in the gut microbiota between the anthroposophic and non-anthroposophic mothers or their infants' before 6 months of age.
Related JoVE Video
DegePrime, a program for degenerate primer design for broad-taxonomic-range PCR in microbial ecology studies.
Appl. Environ. Microbiol.
PUBLISHED: 06-13-2014
Show Abstract
Hide Abstract
The taxonomic composition of a microbial community can be deduced by analyzing its rRNA gene content by, e.g., high-throughput DNA sequencing or DNA chips. Such methods typically are based on PCR amplification of rRNA gene sequences using broad-taxonomic-range PCR primers. In these analyses, the use of optimal primers is crucial for achieving an unbiased representation of community composition. Here, we present the computer program DegePrime that, for each position of a multiple sequence alignment, finds a degenerate oligomer of as high coverage as possible and outputs its coverage among taxonomic divisions. We show that our novel heuristic, which we call weighted randomized combination, performs better than previously described algorithms for solving the maximum coverage degenerate primer design problem. We previously used DegePrime to design a broad-taxonomic-range primer pair that targets the bacterial V3-V4 region (341F-805R) (D. P. Herlemann, M. Labrenz, K. Jurgens, S. Bertilsson, J. J. Waniek, and A. F. Andersson, ISME J. 5:1571-1579, 2011, http://dx.doi.org/10.1038/ismej.2011.41), and here we use the program to significantly increase the coverage of a primer pair (515F-806R) widely used for Illumina-based surveys of bacterial and archaeal diversity. By comparison with shotgun metagenomics, we show that the primers give an accurate representation of microbial diversity in natural samples.
Related JoVE Video
Decreased gut microbiota diversity, delayed Bacteroidetes colonisation and reduced Th1 responses in infants delivered by Caesarean section.
Gut
PUBLISHED: 08-07-2013
Show Abstract
Hide Abstract
The early intestinal microbiota exerts important stimuli for immune development, and a reduced microbial exposure as well as caesarean section (CS) has been associated with the development of allergic disease. Here we address how microbiota development in infants is affected by mode of delivery, and relate differences in colonisation patterns to the maturation of a balanced Th1/Th2 immune response.
Related JoVE Video
Helicobacter pylori and the gastric microbiota.
Best Pract Res Clin Gastroenterol
PUBLISHED: 01-14-2013
Show Abstract
Hide Abstract
The human microbiota along the gastrointestinal tract is currently extensively studied and a number of studies focuses on elucidating the association between a more or less diverse intestinal microbial community and health and disease. The human stomach is considered to be exclusively inhabited by Helicobacter pylori and further lacks a colonizing non-H. pylori bacterial flora due to the acidic environment. However, recently a limited number of studies using molecular-based methods have provided a broader picture of the stomach microbiota. The question is whether changes in gastric pH or antibiotic treatment can lead to significant shifts in the stomach microbiota that may be involved in disease development such as gastric cancer.
Related JoVE Video
Adaptive mutations and replacements of virulence traits in the Escherichia coli O104:H4 outbreak population.
PLoS ONE
PUBLISHED: 01-01-2013
Show Abstract
Hide Abstract
The sequencing of highly virulent Escherichia coli O104:H4 strains isolated during the outbreak of bloody diarrhea and hemolytic uremic syndrome in Europe in 2011 revealed a genome that contained a Shiga toxin encoding prophage and a plasmid encoding enteroaggregative fimbriae. Here, we present the draft genome sequence of a strain isolated in Sweden from a patient who had travelled to Tunisia in 2010 (E112/10) and was found to differ from the outbreak strains by only 38 SNPs in non-repetitive regions, 16 of which were mapped to the branch to the outbreak strain. We identified putatively adaptive mutations in genes for transporters, outer surface proteins and enzymes involved in the metabolism of carbohydrates. A comparative analysis with other historical strains showed that E112/10 contained Shiga toxin prophage genes of the same genotype as the outbreak strain, while these genes have been replaced by a different genotype in two otherwise very closely related strains isolated in the Republic of Georgia in 2009. We also present the genome sequences of two enteroaggregative E. coli strains affiliated with phylogroup A (C43/90 and C48/93) that contain the agg genes for the AAF/I-type fimbriae characteristic of the outbreak population. Interestingly, C43/90 also contained a tet/mer antibiotic resistance island that was nearly identical in sequence to that of the outbreak strain, while the corresponding island in the Georgian strains was most similar to E. coli strains of other serotypes. We conclude that the pan-genome of the outbreak population is shared with strains of the A phylogroup and that its evolutionary history is littered with gene replacement events, including most recently independent acquisitions of antibiotic resistance genes in the outbreak strains and its nearest neighbors. The results are summarized in a refined evolutionary model for the emergence of the O104:H4 outbreak population.
Related JoVE Video
Composition of the vaginal microbiota in women of reproductive age--sensitive and specific molecular diagnosis of bacterial vaginosis is possible?
PLoS ONE
PUBLISHED: 01-01-2013
Show Abstract
Hide Abstract
Bacterial vaginosis (BV) is the most common vaginal disorder, characterized by depletion of the normal lactobacillus-dominant microbiota and overgrowth of commensal anaerobic bacteria. This study aimed to investigate the composition of the vaginal microbiota in women of reproductive age (healthy women and women with BV), with the view of developing molecular criteria for BV diagnosis.
Related JoVE Video
Performance of routine Helicobacter pylori tests in patients with atrophic gastritis.
J Gastrointestin Liver Dis
PUBLISHED: 12-22-2011
Show Abstract
Hide Abstract
Decreased density of H. pylori in atrophic gastritis may lead to low sensitivity of the routine tests.
Related JoVE Video
Titration-free 454 sequencing using Y adapters.
Nat Protoc
PUBLISHED: 08-18-2011
Show Abstract
Hide Abstract
We describe a protocol for construction and quantification of libraries for emulsion PCR (emPCR)-based sequencing platforms such as Roche 454 or Ion Torrent PGM. The protocol involves library construction using customized Y adapters, quantification using TaqMan-MGB (minor groove binder) probe-based quantitative PCR (qPCR) and calculation of an optimal template-to-bead ratio based on Poisson statistics, thereby avoiding the need for a laborious titration assay. Unlike other qPCR methods, the TaqMan-MGB probe specifically quantifies effective libraries in molar concentration and does not require specialized equipment. A single quality control step prior to emulsion PCR ensures that libraries contain no adapter dimers and have an optimal length distribution. The presented protocol takes ?7 h to prepare eight barcoded libraries from genomic DNA into libraries that are ready to use for full-scale emPCR. It will be useful, for example, to allow analyses of precious clinical samples and amplification-free metatranscriptomics.
Related JoVE Video
Comparison of bacterial microbiota in skin biopsies from normal and psoriatic skin.
Arch. Dermatol. Res.
PUBLISHED: 07-12-2011
Show Abstract
Hide Abstract
Microorganisms have been implicated in the pathogenesis of psoriasis. Previous studies of psoriasis and normal skin have used swabs from the surface rather than skin biopsies. In this study, biopsies were taken from 10 patients with psoriasis and 12 control subjects from unmatched sites. Samples were analysed with massive parallel pyrosequencing on the 454 platform targeting the 16S rRNA gene and the variable regions V3-V4. The samples grouped into 19 phyla, 265 taxon and 652 operational units (OTUs) at 97% identity. A cut-off abundance level was set at 1%. The three most common phyla in both normal and psoriasis skin were Firmicutes (39% psoriasis, 43% normal skin), Proteobacteria (38% psoriasis, 27% normal skin) and Actinobacteria (5% psoriasis, 16% normal skin, p = 0.034). In trunk skin, Proteobacteria were present at significantly higher levels in psoriasis compared to controls (52 vs. 32%, p = 0.0113). The commonest genera were Streptococci in both psoriasis (32%) and normal skin (26%). Staphylococci were less common in psoriasis (5%) than in controls (16%), as were Propionibacteria (psoriasis 0.0001669%, controls 0.0254%). Both Staphylococci and Propionibacteria were significantly lower in psoriasis versus control limb skin (p = 0.051, 0.046, respectively). This study has shown some differences in microbiota between psoriasis and normal skin. Whether these are of primary aetiological significance, or secondary to the altered skin of psoriasis remains to be determined.
Related JoVE Video
Low diversity of the gut microbiota in infants with atopic eczema.
J. Allergy Clin. Immunol.
PUBLISHED: 07-08-2011
Show Abstract
Hide Abstract
It is debated whether a low total diversity of the gut microbiota in early childhood is more important than an altered prevalence of particular bacterial species for the increasing incidence of allergic disease. The advent of powerful, cultivation-free molecular methods makes it possible to characterize the total microbiome down to the genus level in large cohorts.
Related JoVE Video
Accuracy and cut-off values of pepsinogens I, II and gastrin 17 for diagnosis of gastric fundic atrophy: influence of gastritis.
PLoS ONE
PUBLISHED: 04-15-2011
Show Abstract
Hide Abstract
To establish optimal cutoff values for serologic diagnosis of fundic atrophy in a high-risk area for oesophageal squamous cell carcinoma and gastric cancer with high prevalence of Helicobacter pylori (H. pylori) in Northern Iran, we performed an endoscopy-room-based validation study.
Related JoVE Video
A method for metagenomics of Helicobacter pylori from archived formalin-fixed gastric biopsies permitting longitudinal studies of carcinogenic risk.
PLoS ONE
PUBLISHED: 04-07-2011
Show Abstract
Hide Abstract
The human microbiota has come into focus in the search for component causes of chronic diseases, such as gastrointestinal cancers. Presumably long induction periods and altered local environments after disease onset call for the development of methods for characterization of microorganisms colonizing the host decades before disease onset. Sequencing of microbial genomes in old formalin-fixed and paraffin-embedded (FFPE) gastrointestinal biopsies provides a means for such studies but is still challenging. Here we report a method based on laser capture micro-dissection and modified Roche 454 high-throughput pyrosequencing to obtain metagenomic profiles of Helicobacter pylori. We applied this method to two 15 year old FFPE biopsies from two patients. Frozen homogenized biopsies from the same gastroscopy sessions were also available for comparison after re-culture of H. pylori. For both patients, H. pylori DNA dissected from FFPE sections had ~96.4% identity with culture DNA from the same patients, while only ~92.5% identity with GenBank reference genomes, and with culture DNA from the other patient. About 82% and 60% of the predicted genes in the two genomes were captured by at least a single sequencing read. Along with sequences displaying high similarity to known H. pylori genes, novel and highly variant H. pylori sequences were identified in the FFPE sections by our physical enrichment approach, which would likely not have been detected by a sequence capture approach. The study demonstrates the feasibility of longitudinal metagenomic studies of H. pylori using decade-preserved FFPE biopsies.
Related JoVE Video
Rapid screening of complex DNA samples by single-molecule amplification and sequencing.
PLoS ONE
PUBLISHED: 03-18-2011
Show Abstract
Hide Abstract
Microbial cloning makes Sanger sequencing of complex DNA samples possible but is labor intensive. We present a simple, rapid and robust method that enables laboratories without special equipment to perform single-molecule amplicon sequencing, although in a low-throughput manner, from sub-picogram quantities of DNA. The method can also be used for quick quality control of next-generation sequencing libraries, as was demonstrated for a metagenomic sample.
Related JoVE Video
Mutations in the ELANE gene are associated with development of periodontitis in patients with severe congenital neutropenia.
J. Clin. Immunol.
PUBLISHED: 01-26-2011
Show Abstract
Hide Abstract
Patients with severe congenital neutropenia (SCN) often develop periodontitis despite standard medical and dental care. In light of previous findings that mutations in the neutrophil elastase gene, ELANE, are associated with more severe neutropenic phenotypes, we hypothesized an association between the genotype of SCN and development of periodontitis.
Related JoVE Video
The complement regulator CD46 is bactericidal to Helicobacter pylori and blocks urease activity.
Gastroenterology
PUBLISHED: 01-20-2011
Show Abstract
Hide Abstract
CD46 is a C3b/C4b binding complement regulator and a receptor for several human pathogens. We examined the interaction between CD46 and Helicobacter pylori (a bacterium that colonizes the human gastric mucosa and causes gastritis), peptic ulcers, and cancer.
Related JoVE Video
Genome sequencing reveals a phage in Helicobacter pylori.
MBio
PUBLISHED: 01-01-2011
Show Abstract
Hide Abstract
Helicobacter pylori chronically infects the gastric mucosa in more than half of the human population; in a subset of this population, its presence is associated with development of severe disease, such as gastric cancer. Genomic analysis of several strains has revealed an extensive H. pylori pan-genome, likely to grow as more genomes are sampled. Here we describe the draft genome sequence (63 contigs; 26× mean coverage) of H. pylori strain B45, isolated from a patient with gastric mucosa-associated lymphoid tissue (MALT) lymphoma. The major finding was a 24.6-kb prophage integrated in the bacterial genome. The prophage shares most of its genes (22/27) with prophage region II of Helicobacter acinonychis strain Sheeba. After UV treatment of liquid cultures, circular DNA carrying the prophage integrase gene could be detected, and intracellular tailed phage-like particles were observed in H. pylori cells by transmission electron microscopy, indicating that phage production can be induced from the prophage. PCR amplification and sequencing of the integrase gene from 341 H. pylori strains from different geographic regions revealed a high prevalence of the prophage (21.4%). Phylogenetic reconstruction showed four distinct clusters in the integrase gene, three of which tended to be specific for geographic regions. Our study implies that phages may play important roles in the ecology and evolution of H. pylori.
Related JoVE Video
Helicobacter pylori defines local immune response through interaction with dendritic cells.
FEMS Immunol. Med. Microbiol.
PUBLISHED: 12-22-2010
Show Abstract
Hide Abstract
The bacterial pathogen Helicobacter pylori colonizes the human gastric and duodenal mucosa, evades clearance by the host response and is associated with peptic ulcer disease and an increased risk of gastric adenocarcinoma. Dendritic cells (DCs) are initiators of the immune response to H. pylori. The aim of the current study was to investigate the interaction between H. pylori with DCs. To determine the impact of H. pylori on the maturation and the activation of monocyte-derived DCs, the effect of 20 clinical H. pylori strains with different inflammatory backgrounds on adenocarcinoma gastric epithelial cells was investigated. The inflammatory background was defined according to the degree of lymphocyte and granulocyte infiltration and the bacterial density at the site of infection. DC maturation and activation varied after exposure to the different strains. While maturation appeared to be independent of any virulence factor tested, a significant increase in the average level of cytokine production was observed for the proinflammatory cytokines interleukin-12 (IL-12), tumour necrosis factor-?, IL-6 and IL-1? when comparing strains with low inflammatory backgrounds with those of the medium or high backgrounds. In conclusion, the DC response towards different strains in vitro was associated with the clinical outcome of the individual host, suggesting a major role of this cell type in modulating strain-specific H. pylori infection.
Related JoVE Video
Expression of Helicobacter pylori TonB protein in transgenic Arabidopsis thaliana: toward production of vaccine antigens in plants.
Helicobacter
PUBLISHED: 11-19-2010
Show Abstract
Hide Abstract
The aim of this study was to produce a recombinant version of the highly antigenic Helicobacter pylori TonB (iron-dependent siderophore transporter protein HP1341) in transgenic plants as a candidate oral vaccine antigen.
Related JoVE Video
A pyrosequencing study in twins shows that gastrointestinal microbial profiles vary with inflammatory bowel disease phenotypes.
Gastroenterology
PUBLISHED: 08-21-2010
Show Abstract
Hide Abstract
The composition of the gastrointestinal microbiota is thought to have an important role in the etiology of inflammatory bowel diseases (IBDs) such as Crohns disease (CD) and ulcerative colitis (UC). Interindividual variation and an inability to detect less abundant bacteria have made it difficult to correlate specific bacteria with disease.
Related JoVE Video
Use of tobacco products and gastrointestinal morbidity: an endoscopic population-based study (the Kalixanda study).
Eur. J. Epidemiol.
PUBLISHED: 07-16-2010
Show Abstract
Hide Abstract
The impact of snus (smokeless tobacco or snuff) on gastrointestinal symptoms and pathological findings is largely unknown. The authors aimed to investigate whether the exposure to different forms of tobacco influences upper gastrointestinal symptoms, histology and frequency of Helicobacter pylori infection. A random sample (n = 2,860) of the adult population of two northern Swedish municipalities Kalix and Haparanda (n = 21,610) was surveyed between December 1998 and June 2001 using a validated postal questionnaire assessing gastrointestinal symptoms (response rate 74.2%, n = 2,122) (The Kalixanda Study). A random sub-sample (n = 1,001) of the responders was invited to undergo an esophagogastroduodenoscopy (participation rate 73.3%) including biopsies, Helicobacter pylori culture and serology and symptom assessment and exploration of present and past use of tobacco products. No symptom groups were associated with snus use. Snus users had a significantly higher prevalence of macroscopic esophagitis univariately but snus use was not associated with esophagitis in multivariate analysis. Snus use was associated with basal cell hyperplasia (OR = 1.74, 95% CI: 1.02, 3.00) and with elongation of papillae (OR = 1.79, 95% CI: 1.05-3.05) of the squamous epithelium at the esophago-gastric junction. Current smoking cigarettes was associated with overall peptic ulcer disease (OR = 2.32, 95% CI: 1.04, 5.19) whereas snus use was not. There were no significant association between current Helicobacter pylori infection and different tobacco product user groups. Snus significantly alters the histology of the distal esophagus but does not impact on gastrointestinal symptoms or peptic ulcer disease.
Related JoVE Video
Twelve-month endoscopic and histological analysis following proton-pump inhibitor-based triple therapy in Helicobacter pylori-positive patients with gastric ulcers.
Scand. J. Gastroenterol.
PUBLISHED: 06-01-2010
Show Abstract
Hide Abstract
To evaluate endoscopic and histological findings after Helicobacter pylori eradication therapy in gastric ulcer (GU) patients after 12 months follow-up.
Related JoVE Video
Titration-free massively parallel pyrosequencing using trace amounts of starting material.
Nucleic Acids Res.
PUBLISHED: 04-30-2010
Show Abstract
Hide Abstract
Continuous efforts have been made to improve next-generation sequencing methods for increased robustness and for applications on low amounts of starting material. We applied double-stranded library protocols for the Roche 454 platform to avoid the yield-reducing steps associated with single-stranded library preparation, and applied a highly sensitive Taqman MGB-probe-based quantitative polymerase chain reaction (qPCR) method. The MGB-probe qPCR, which can detect as low as 100 copies, was used to quantify the amount of effective library, i.e. molecules that form functional clones in emulsion PCR. We also demonstrate that the distribution of library molecules on capture beads follows a Poisson distribution. Combining the qPCR and Poisson statistics, the labour-intensive and costly titration can be eliminated and trace amounts of starting material such as precious clinical samples, transcriptomes of small tissue samples and metagenomics on low biomass environments is applicable.
Related JoVE Video
Type I restriction-modification loci reveal high allelic diversity in clinical Helicobacter pylori isolates.
Helicobacter
PUBLISHED: 04-21-2010
Show Abstract
Hide Abstract
A remarkable variety of restriction-modification (R-M) systems is found in Helicobacter pylori. Since they encompass a large portion of the strain-specific H. pylori genes and therefore contribute to genetic variability, they are suggested to have an impact on disease outcome. Type I R-M systems comprise three different subunits and are the most complex of the three types of R-M systems.
Related JoVE Video
Short-term antibiotic treatment has differing long-term impacts on the human throat and gut microbiome.
PLoS ONE
PUBLISHED: 03-03-2010
Show Abstract
Hide Abstract
Antibiotic administration is the standard treatment for the bacterium Helicobacter pylori, the main causative agent of peptic ulcer disease and gastric cancer. However, the long-term consequences of this treatment on the human indigenous microbiota are relatively unexplored. Here we studied short- and long-term effects of clarithromycin and metronidazole treatment, a commonly used therapy regimen against H. pylori, on the indigenous microbiota in the throat and in the lower intestine. The bacterial compositions in samples collected over a four-year period were monitored by analyzing the 16S rRNA gene using 454-based pyrosequencing and terminal-restriction fragment length polymorphism (T-RFLP). While the microbial communities of untreated control subjects were relatively stable over time, dramatic shifts were observed one week after antibiotic treatment with reduced bacterial diversity in all treated subjects in both locations. While the microbiota of the different subjects responded uniquely to the antibiotic treatment some general trends could be observed; such as a dramatic decline in Actinobacteria in both throat and feces immediately after treatment. Although the diversity of the microbiota subsequently recovered to resemble the pre treatment states, the microbiota remained perturbed in some cases for up to four years post treatment. In addition, four years after treatment high levels of the macrolide resistance gene erm(B) were found, indicating that antibiotic resistance, once selected for, can persist for longer periods of time than previously recognized. This highlights the importance of a restrictive antibiotic usage in order to prevent subsequent treatment failure and potential spread of antibiotic resistance.
Related JoVE Video
Response of gastric epithelial progenitors to Helicobacter pylori Isolates obtained from Swedish patients with chronic atrophic gastritis.
J. Biol. Chem.
PUBLISHED: 09-01-2009
Show Abstract
Hide Abstract
Helicobacter pylori infection is associated with gastric adenocarcinoma in some humans, especially those that develop an antecedent condition, chronic atrophic gastritis (ChAG). Gastric epithelial progenitors (GEPs) in transgenic gnotobiotic mice with a ChAG-like phenotype harbor intracellular collections of H. pylori. To characterize H. pylori adaptations to ChAG, we sequenced the genomes of 24 isolates obtained from 6 individuals, each sampled over a 4-year interval, as they did or did not progress from normal gastric histology to ChAG and/or adenocarcinoma. H. pylori populations within study participants were largely clonal and remarkably stable regardless of disease state. GeneChip studies of the responses of a cultured mouse gastric stem cell-like line (mGEPs) to infection with sequenced strains yielded a 695-member dataset of transcripts that are (i) differentially expressed after infection with ChAG-associated isolates, but not with a "normal" or a heat-killed ChAG isolate, and (ii) enriched in genes and gene functions associated with tumorigenesis in general and gastric carcinogenesis in specific cases. Transcriptional profiling of a ChAG strain during mGEP infection disclosed a set of responses, including up-regulation of hopZ, an adhesin belonging to a family of outer membrane proteins. Expression profiles of wild-type and DeltahopZ strains revealed a number of pH-regulated genes modulated by HopZ, including hopP, which binds sialylated glycans produced by GEPs in vivo. Genetic inactivation of hopZ produced a fitness defect in the stomachs of gnotobiotic transgenic mice but not in wild-type littermates. This study illustrates an approach for identifying GEP responses specific to ChAG-associated H. Pylori strains and bacterial genes important for survival in a model of the ChAG gastric ecosystem.
Related JoVE Video
Is there a link between the lipopolysaccharide of Helicobacter pylori gastric MALT lymphoma associated strains and lymphoma pathogenesis?
PLoS ONE
PUBLISHED: 08-31-2009
Show Abstract
Hide Abstract
The aim of this study was to investigate the Lewis antigen expression in Helicobacter pylori gastric MALT lymphoma associated strains in comparison to chronic gastritis only strains. Forty MALT strains (19 cagPAI (-) and 21 cagPAI (+)) and 39 cagPAI frequency-matched gastritis strains (17 cagPAI (-) and 22 cagPAI (+)) were included in this study. The lipopolyssacharide for each strain was extracted using a hot phenol method and the expression of Le(x) and Le(y) were investigated using Western Blot. The data were analyzed according to the strains cagPAI status and vacA genotype. Le(x) was identified in 21 (52.5%) MALT strains and 29 (74.3%) gastritis strains. Le(y) was identified in 30 (75%) MALT strains and 31 (79.5%) gastritis strains. There was an association between cagPAI positivity and Le(x) expression among MALT strains (p<0.0001), but not in gastritis strains (p = 0.64). Among cagPAI (-) strains, isolates expressing solely Le(y) were associated with MALT with an odds ratio of 64.2 (95% CI 4.9-841.0) when compared to strains expressing both Le(x) and Le(y). vacA genotypes did not modify the association between Lewis antigen expression and disease status. In conclusion, cagPAI (-) MALT strains have a particular Lewis antigen profile which could represent an adaptive mechanism to the host response or participate in MALT lymphomagenesis.
Related JoVE Video
Twin studies reveal specific imbalances in the mucosa-associated microbiota of patients with ileal Crohns disease.
Inflamm. Bowel Dis.
PUBLISHED: 08-12-2009
Show Abstract
Hide Abstract
Large interindividual variation in the composition of the intestinal microbiota between unrelated individuals has made it challenging to identify specific aspects of dysbiosis that lead to Crohns disease (CD).
Related JoVE Video
Interleukin 1-beta gene polymorphisms and risk of gastric cancer in Sweden.
Scand. J. Gastroenterol.
PUBLISHED: 05-06-2009
Show Abstract
Hide Abstract
Helicobacter pylori (H. pylori) infection stimulates the production of interleukin (IL)-1 beta, a pro-inflammatory cytokine and suppressor of gastric acid secretion. As both inflammation and hypochlorhydria, which might facilitate proximal colonization of H. pylori and other bacterial species alike, have been implicated in gastric carcinogenesis, much attention has been directed to functional genetic polymorphisms that affect the production of IL-1 beta. The purpose of this study was to clarify the role of these polymorphisms.
Related JoVE Video
A changing gastric environment leads to adaptation of lipopolysaccharide variants in Helicobacter pylori populations during colonization.
PLoS ONE
PUBLISHED: 04-24-2009
Show Abstract
Hide Abstract
The human gastric pathogen Helicobacter pylori colonizes the stomachs of half of the human population, and causes development of peptic ulcer disease and gastric adenocarcinoma. H. pylori-associated chronic atrophic gastritis (ChAG) with loss of the acid-producing parietal cells, is correlated with an increased risk for development of gastric adenocarcinoma. The majority of H. pylori isolates produce lipopolysaccharides (LPS) decorated with human-related Lewis epitopes, which have been shown to phase-vary in response to different environmental conditions. We have characterized the adaptations of H. pylori LPS and Lewis antigen expression to varying gastric conditions; in H. pylori isolates from mice with low or high gastric pH, respectively; in 482 clinical isolates from healthy individuals and from individuals with ChAG obtained at two time points with a four-year interval between endoscopies; and finally in isolates grown at different pH in vitro. Here we show that the gastric environment can contribute to a switch in Lewis phenotype in the two experimental mouse models. The clinical isolates from different human individuals showed that intra-individual isolates varied in Lewis antigen expression although the LPS diversity was relatively stable within each individual over time. Moreover, the isolates demonstrated considerable diversity in the levels of glycosylation and in the sizes of fucosylated O-antigen chains both within and between individuals. Thus our data suggest that different LPS variants exist in the colonizing H. pylori population, which can adapt to changes in the gastric environment and provide a means to regulate the inflammatory response of the host during disease progression.
Related JoVE Video
Molecular characterization of the stomach microbiota in patients with gastric cancer and in controls.
J. Med. Microbiol.
PUBLISHED: 03-11-2009
Show Abstract
Hide Abstract
Persistent infection of the gastric mucosa by Helicobacter pylori can initiate an inflammatory cascade that progresses into atrophic gastritis, a condition associated with reduced capacity for secretion of gastric acid and an increased risk of developing gastric cancer. The role of H. pylori as an initiator of inflammation is evident but the mechanism for development into gastric cancer has not yet been proven. A reduced capacity for gastric acid secretion allows survival and proliferation of other microbes that normally are killed by the acidic environment. It has been postulated that some of these species may be involved in the development of gastric cancer; however, their identities are poorly defined. In this study, the gastric microbiota from ten patients with gastric cancer was characterized and compared with that from five dyspeptic controls using the molecular profiling approach terminal restriction fragment length polymorphism (T-RFLP), in combination with 16S rRNA gene cloning and sequencing. T-RFLP analysis revealed a complex bacterial community in the cancer patients that was not significantly different from that in the controls. Sequencing of 140 clones revealed 102 phylotypes, with representatives from five bacterial phyla (Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria and Fusobacteria). The data revealed a relatively low abundance of H. pylori and showed that the gastric cancer microbiota was instead dominated by different species of the genera Streptococcus, Lactobacillus, Veillonella and Prevotella. The respective role of these species in development of gastric cancer remains to be determined.
Related JoVE Video
Composition of the ANTIGENome of Helicobacter pylori defined by human serum antibodies.
Vaccine
PUBLISHED: 02-05-2009
Show Abstract
Hide Abstract
Helicobacter pylori is the most prevalent human pathogen and although, it remains silent in most individuals for lifetime, colonization may develop into severe gastric and duodenal conditions. Rapidly developing resistance to antibiotic treatment urgently calls for the development of effective vaccines. We determined the ANTIGENome of two clinical isolates of H. pylori, KTH-Ca1 and KTH-Du, derived from patients with gastric cancer and duodenal ulcer, respectively. Using disease-relevant human sera from well-characterized donors we identified 124 annotated ORFs and 54 non-annotated peptides as antigens. Through in vitro validation assays we selected the 20 most promising vaccine candidates. Importantly, two candidates represent proteins that were previously shown to provide protection in models of H. pylori infection. One of the most frequently selected and conserved protein, the siderophore-dependent transporter HP1341, was confirmed to show high reactivity with human serum IgGs. These analyses provide the means to identify novel antigens for the selection of vaccine candidates, as well as disease associated biomarkers.
Related JoVE Video
The prevalence of the duodenal ulcer promoting gene (dupA) in Helicobacter pylori isolates varies by ethnic group and is not universally associated with disease development: a case-control study.
Gut Pathog
PUBLISHED: 01-20-2009
Show Abstract
Hide Abstract
The putative H. pylori pathogenicity-associated factor dupA has been associated with IL-8 induction in vitro, and duodenal ulcer (DU) and gastric cancer (GC) development in certain populations, but this association is inconsistent between studies. We aimed to investigate dupA prevalence in clinical isolates from Sweden, Australia and from ethnic Chinese, Indians and Malays resident in Malaysia and Singapore and to examine the association with DU and GC. In addition we investigated the sequence diversity between isolates from these diverse groups and compared the level of IL-8 secretion in isolates possessing and lacking dupA.
Related JoVE Video
Acute effects of Helicobacter pylori extracts on gastric mucosal blood flow in the mouse.
World J. Gastroenterol.
PUBLISHED: 01-10-2009
Show Abstract
Hide Abstract
To investigate the mechanisms underlying the reduction in gastric blood flow induced by a luminal water extract of Helicobacter pylori (HPE).
Related JoVE Video
Inhibition of bacterial thioredoxin reductase: an antibiotic mechanism targeting bacteria lacking glutathione.
FASEB J.
Show Abstract
Hide Abstract
Increasing antibiotic resistance makes the identification of new antibacterial principles an urgent task. The thioredoxin system including thioredoxin reductase (TrxR), thioredoxin (Trx), and NADPH plays critical roles in cellular DNA synthesis and defense against oxidative stress. Notably, TrxR is very different in structure and mechanism in mammals and bacteria. Ebselen [2-phenyl-1,2 benzisoselenazol-3(2H)-one], a well-known antioxidant and a substrate for mammalian TrxR and Trx, is rapidly bacteriocidal for methicillin-resistant Staphylococcus aureus by an unknown mechanism. We have discovered that ebselen is a competitive inhibitor of Escherichia coli TrxR with a Ki of 0.52 ± 0.13 ?M, through reaction with the active site dithiol of the enzyme. Bacteria lacking glutathione (GSH) and glutaredoxin, in which TrxR and Trx are essential for DNA synthesis, were particularly sensitive to ebselen. In growth-inhibited E. coli strains, Trx1 and Trx2 were oxidized, demonstrating that electron transfer via thioredoxin was blocked. Ebselen and its sulfur analog ebsulfur were bactericidal for GSH-negative pathogens. Ebsulfur inhibited a clinically isolated Helicobacter pylori strain with a minimum inhibitory concentration value as low as 0.39 ?g/ml. These results demonstrate that bacterial Trx and TrxR are viable antibacterial drug targets using benzisoselenazol and benzisothiazol derivates.
Related JoVE Video
Intestinal microbial profiles in extremely preterm infants with and without necrotizing enterocolitis.
Acta Paediatr.
Show Abstract
Hide Abstract
Necrotizing enterocolitis (NEC) represents one of the gravest complications in premature infants. The suggested role of intestinal microbiota in the development of NEC needs to be elucidated.
Related JoVE Video
In vivo sequence variation in HopZ, a phase-variable outer membrane protein of Helicobacter pylori.
Infect. Immun.
Show Abstract
Hide Abstract
The Helicobacter pylori outer membrane protein HopZ is regulated by a phase-variable CT repeat and occurs in two distinct allelic variants. Whole-genome comparisons of isolates from one human volunteer recently provided evidence for in vivo selection for the hopZ ON status. We explored the frequency of sequence variation in hopZ during acute and chronic human infection and studied the association of hopZ with the phylogeographic population structure of H. pylori. hopZ ON variants were cultured from 24 out of 33 volunteers challenged with the hopZ OFF strain BCS 100. Transmission of H. pylori within families was also frequently associated with a status change of hopZ. In contrast, hopZ sequences obtained from 26 sets of sequential isolates from chronically infected individuals showed no changes of status, suggesting that the hopZ status selected during early infection is subsequently stable. Mutations leading to amino acid changes in HopZ occurred more frequently in ON than in OFF status isolates during chronic infection, indicating that sequence changes are more likely the result of positive selection in ON isolates than of a loss of negative selection pressure in OFF isolates. Analysis of 63 isolates from chronically infected individuals revealed no significant correlation of hopZ status with chronic atrophic gastritis. hopZ sequences were obtained from a globally representative collection of 54 H. pylori strains. All H. pylori populations contained hopZ-positive isolates. The data suggest that hopZ has been acquired and split into the two variants before the human migration out of Africa.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.