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1.  Body Site Is a More Determinant Factor than Human Population Diversity in the Healthy Skin Microbiome 
PLoS ONE  2016;11(4):e0151990.
We studied skin microbiota present in three skin sites (forearm, axilla, scalp) in men from six ethnic groups living in New York City. Methods. Samples were obtained at baseline and after four days following use of neutral soap and stopping regular hygiene products, including shampoos and deodorants. DNA was extracted using the MoBio Power Lyzer kit and 16S rRNA gene sequences determined on the IIlumina MiSeq platform, using QIIME for analysis. Results. Our analysis confirmed skin swabbing as a useful method for sampling different areas of the skin because DNA concentrations and number of sequences obtained across subject libraries were similar. We confirmed that skin location was the main factor determining the composition of bacterial communities. Alpha diversity, expressed as number of species observed, was greater in arm than on scalp or axilla in all studied groups. We observed an unexpected increase in α-diversity on arm, with similar tendency on scalp, in the South Asian group after subjects stopped using their regular shampoos and deodorants. Significant differences at phylum and genus levels were observed between subjects of the different ethnic origins at all skin sites. Conclusions. We conclude that ethnicity and particular soap and shampoo practices are secondary factors compared to the ecological zone of the human body in determining cutaneous microbiota composition.
PMCID: PMC4835103  PMID: 27088867
2.  Effect of antibiotic pre-treatment and pathogen challenge on the intestinal microbiota in mice 
Gut Pathogens  2016;8:60.
More than 50 years after the discovery of antibiotics, bacterial infections have decreased substantially; however, antibiotics also may have negative effects such as increasing susceptibility to pathogens. An intact microbiome is an important line of defense against pathogens. We sought to determine the effect of orally administered antibiotics both on susceptibility to pathogens and on impact to the microbiome. We studied Campylobacter jejuni, one of the most common causes of human diarrhea, and Acinetobacter baumannii, which causes wound infections. We examined the effects of antibiotic treatment on the susceptibility of mice to those pathogens as well as their influence on the mouse gut microbiome.
In C57/BL6 mice models, we explored the effects of pathogen challenge, and antibiotic treatment on the intestinal microbiota. Mice were treated with either ciprofloxacin, penicillin, or water (control) for a 5-day period followed by a 5-day washout period prior to oral challenge with C. jejuni or A. baumannii to assess antibiotic effects on colonization susceptibility. Mice were successfully colonized with C. jejuni more than 118 days, but only transiently with A. baumannii. These challenges did not lead to any major effects on the composition of the gut microbiota. Although antibiotic pre-treatment did not modify pathogen colonization, it affected richness and community structure of the gut microbiome. However, the antibiotic dysbiosis was significantly reduced by pathogen challenge.
We conclude that despite gut microbiota disturbance, susceptibility to gut colonization by these pathogens was unchanged. The major gut microbiome disturbance produced by antibiotic treatment may be reduced by colonization with specific microbial taxa.
Electronic supplementary material
The online version of this article (doi:10.1186/s13099-016-0143-z) contains supplementary material, which is available to authorized users.
PMCID: PMC5116224  PMID: 27891184
Microbiome; Antibiotic treatment; Pathogen challenge; Penicillin; Ciprofloxacin; Campylobacter jejuni; Acinetobacter baumannii; High-throughput sequencing; Next-generation sequencing
3.  Systematic analysis of phosphotyrosine antibodies recognizing single phosphorylated EPIYA-motifs in CagA of East Asian-type Helicobacter pylori strains 
BMC Microbiology  2016;16(1):201.
Highly virulent strains of the gastric pathogen Helicobacter pylori encode a type IV secretion system (T4SS) that delivers the effector protein CagA into gastric epithelial cells. Translocated CagA undergoes tyrosine phosphorylation by members of the oncogenic c-Src and c-Abl host kinases at EPIYA-sequence motifs A, B and D in East Asian-type strains. These phosphorylated EPIYA-motifs serve as recognition sites for various SH2-domains containing human proteins, mediating interactions of CagA with host signaling factors to manipulate signal transduction pathways. Recognition of phospho-CagA is mainly based on the use of commercial pan-phosphotyrosine antibodies that were originally designed to detect phosphotyrosines in mammalian proteins. Specific anti-phospho-EPIYA antibodies for each of the three sites in CagA are not forthcoming.
This study was designed to systematically analyze the detection preferences of each phosphorylated East Asian CagA EPIYA-motif by pan-phosphotyrosine antibodies and to determine a minimal recognition sequence. We synthesized phospho- and non-phosphopeptides derived from each predominant EPIYA-site, and determined the recognition patterns by seven different pan-phosphotyrosine antibodies using Western blotting, and also investigated representative East Asian H. pylori isolates during infection. The results indicate that a total of only 9–11 amino acids containing the phosphorylated East Asian EPIYA-types are required and sufficient to detect the phosphopeptides with high specificity. However, the sequence recognition by the different antibodies was found to bear high variability. From the seven antibodies used, only four recognized all three phosphorylated EPIYA-motifs A, B and D similarly well. Two of the phosphotyrosine antibodies preferentially bound primarily to the phosphorylated motif A and D, while the seventh antibody failed to react with any of the phosphorylated EPIYA-motifs. Control experiments confirmed that none of the antibodies reacted with non-phospho-CagA peptides and in accordance were able to recognize phosphotyrosine proteins in human cells.
The results of this study disclose the various binding preferences of commercial anti-phosphotyrosine antibodies for phospho-EPIYA-motifs, and are valuable in the application for further characterization of CagA phosphorylation events during infection with H. pylori and risk prediction for gastric disease development.
PMCID: PMC5009636  PMID: 27590005
c-Abl; c-Src; CagA; cagPAI; Dotblot; EPIYA motifs; Gastric cancer; Helicobacter pylori; Signaling; Type IV secretion; T4SS; Tyrosine kinases
4.  Role of childhood infection in the sequelae of H. pylori disease 
Gut Microbes  2013;4(6):426-438.
The persistence of Helicobacter pylori infection plays a fundamental role in the development of H. pylori-associated complications. Since the majority of infected persons acquire the bacteria during early childhood, an examination of the immunobiology of H. pylori infection in children compared with that of adults may help identify host factors that contribute to persistent infection. Therefore, we begin our review of the role of persistence in H. pylori disease with an assessment of the clinical features of H. pylori infection in children. We next review the bacterial factors that promote colonization and evasion of host defense mechanisms. We then focus our attention on the early host immunological factors that promote persistence of the infection and its complications in humans and mouse models. We also highlight topics in which further research is needed. An examination of how immunological factors cause divergent manifestations of H. pylori infection in children compared with adults may provide new insight for therapeutic modification or prevention of persistent H. pylori infection and its complications.
PMCID: PMC3928156  PMID: 24275060
Helicobacter pylori; cancer; infection; children
5.  The Cutaneous Microbiome in Outpatients Presenting With Acute Skin Abscesses 
The Journal of Infectious Diseases  2015;211(12):1895-1904.
Background. Previous studies have demonstrated an association between antibiotic use and the development of skin abscesses. We tested the hypothesis that alterations in the composition of the cutaneous microbiota may predispose individuals to skin abscesses.
Methods. We studied 25 patients with skin abscesses and 25 age-matched controls, who each completed a questionnaire. Skin swab samples were obtained for DNA analysis from 4 sites around the abscess site (hereafter, “peri-abscess specimens”) and from similar sites on the patient's contralateral side and on healthy control subjects. DNA was extracted and analyzed by quantitative polymerase chain reaction (qPCR) and high-throughput sequencing. The purulent abscess drainage was sent for culture.
Results. Fifteen patients with abscess were infected with Staphylococcus aureus. Use of nuc qPCR to quantitate S. aureus revealed a significantly greater frequency of positive results for peri-abscess and contralateral skin samples, compared with control skin specimens. Analysis of community structure showed greater heterogeneity in the control samples than in the peri-abscess and contralateral samples. Metagenomic analysis detected significantly more predicted genes related to metabolic activity in the peri-abscess specimens than in the control samples.
Conclusions. The peri-abscess microbiome was similar to the contralateral microbiome, but both microbiomes differed from that for control patients. Host characteristics affecting microbial populations might be important determinants of abscess risk.
PMCID: PMC4539909  PMID: 25583170
skin infection; MRSA; skin microbiome; abscess
6.  Helicobacter pylori Eradication Causes Perturbation of the Human Gut Microbiome in Young Adults 
PLoS ONE  2016;11(3):e0151893.
Accumulating evidence shows that Helicobacter pylori protects against some metabolic and immunological diseases in which the development of these diseases coincide with temporal or permanent dysbiosis. The aim of this study was to assess the effect of H. pylori eradication on the human gut microbiome.
As part of the currently on-going ESSAY (Eradication Study in Stable Adults/Youths) study, we collected stool samples from 17 H. pylori-positive young adult (18–30 years-old) volunteers. The same cohort was followed up 6, 12 and 18 months-post H. pylori eradication. The impact of H. pylori on the human gut microbiome pre- and post-eradication was investigated using high throughput 16S rRNA gene (V3-V4 region) sequencing using the Illumina Miseq followed by data analysis using Qiime pipeline.
We compared the composition and diversity of bacterial communities in the fecal microbiome of the H. pylori-positive volunteers, before and after H. pylori eradication therapy. The 16S rRNA gene was sequenced at an average of 150,000–170,000 reads/sample. The microbial diversity were similar pre- and post-H. pylori eradication with no significant differences in richness and evenness of bacterial species. Despite that the general profile of the gut microbiome was similar pre- and post-eradication, some changes in the bacterial communities at the phylum and genus levels were notable, particularly the decrease in relative abundance of Bacterioidetes and corresponding increase in Firmicutes after H. pylori eradication. The significant increase of short-chain fatty acids (SCFA)-producing bacteria genera could also be associated with increased risk of metabolic disorders.
Our preliminary stool metagenomics study shows that eradication of H. pylori caused perturbation of the gut microbiome and may indirectly affect the health of human. Clinicians should be aware of the effect of broad spectrum antibiotics used in H. pylori eradication regimen and be cautious in the clinical management of H. pylori infection, particularly in immunocompromised patients.
PMCID: PMC4798770  PMID: 26991500
7.  Gastric Helicobacter pylori infection affects local and distant microbial populations and host responses 
Cell reports  2016;14(6):1395-1407.
Helicobacter pylori is a late-in-life human pathogen with potential early-life benefits. Although H. pylori is disappearing from the human population, little is known about the influence of H. pylori on the host’s microbiota and immunity. Studying the interactions of H. pylori with murine hosts over six months, we found stable colonization accompanied by gastric histologic and antibody responses. Analysis of gastric and pulmonary tissues revealed increased expression of multiple immune response genes, conserved across mice and over time in the stomach, and more transiently in the lungs. Moreover, H. pylori infection led to significantly different population structures in both the gastric and intestinal microbiota. These studies indicate that H. pylori influences the microbiota and host immune responses not only locally in the stomach, but distantly as well, affecting important target organs.
Graphical abstract
PMCID: PMC4758874  PMID: 26854236
8.  Longitudinal analysis of serological responses of adults to Helicobacter pylori antigens 
The Journal of infectious diseases  2010;202(6):916-923.
Since Helicobacter pylori persist for decades in the human stomach, the aim of this study was to examine the long-term course in H. pylori-specific serum IgG responses with respect to subclass and antigenic target. We studied paired serum samples obtained in 1973 and in 1994 in Vammala, Finland from 64 healthy H. pylori-positive adults and from other healthy controls. H. pylori serum IgA, IgG, and IgG subclass responses were determined by antigen-specific ELISAs. H. pylori-specific IgG1 and IgG4 subtype responses from 47 subjects were similar in 1973 and 1994, but not when compared to unrelated persons. H. pylori-specific IgG1/IgG4 ratios amongst the participants varied > 1000-fold; however, 89.4% had an IgG1/IgG4 ratio >1.0, consistent with a predominant IgG1 (Th1) response. Furthermore, ratios in individual hosts were stable over the 21-year period (r=0.56, p< 0.001). The immune response to heat shock protein HspA was unchanged in 49 (77%) of the 64 subjects tested; of the 15 who changed serostatus, all seroconverted and were significantly younger than those who did not change status. These findings indicate that H. pylori-specific antibody responses are host-specific with IgG1/IgG4 ratios stable over 21 years, IgG1 responses predominating, and HspA seroconversion with aging.
PMCID: PMC2924458  PMID: 20698790
9.  Factors associated with H pylori epidemiology in symptomatic children in Buenos Aires, Argentina 
AIM: To determine prevalence of H pylori infection in symptomatic children in Buenos Aires, Argentina, and to investigate factors associated with H pylori positivity.
METHODS: A total of 395 children with upper gastrointestinal symptoms referred to the Gastroente-rology Unit of the Children Hospital “Sor Maria Ludovica” were evaluated for the presence of H pylori by the 13C-Urea Breath Test (13C-UBT). A questionnaire was applied to the recruited population.
RESULTS: Prevalence of H pylori infection was 40.0% in this population (mean age 9.97 ± 3.1 years). The factors associated with H pylori positivity were number of siblings (P < 0.001), presence of pet cats (P = 0.03) and birds (P = 0.04) in the household, and antecedents of gastritis among family members (P = 0.01). After multivariate analysis, number of siblings [Odds ratio (OR) = 1.39; 95% CI, 1.20-1.61] and contact with pet cats (OR = 1.76; 95% CI, 1.00-3.09) remained as variables associated with H pylori infection.
CONCLUSION: The prevalence of H pylori infection in children with upper gastrointestinal symptoms in Argentina was similar to that reported in developed countries. Children from families with a higher crowding index and presence of pet cats have a higher risk of being colonized with H pylori.
PMCID: PMC4088210  PMID: 16981273
H pylori; Children; Epidemiology; Urea breath test; Prevalence
10.  Changes in Metabolic Hormones in Malaysian Young Adults following Helicobacter pylori Eradication 
PLoS ONE  2015;10(8):e0135771.
More than half of the world’s adults carry Helicobacter pylori. The eradication of H. pylori may affect the regulation of human metabolic hormones. The aim of this study was to evaluate the effect of H. pylori eradication on meal-associated changes in appetite-controlled insulinotropic and digestive hormones, and to assess post-eradication changes in body mass index as part of a currently on-going multicentre ESSAY (Eradication Study in Stable Adults/Youths) study.
We enrolled 29 H. pylori-positive young adult (18–30 year-old) volunteer subjects to evaluate the effect of H. pylori eradication on meal-associated changes on eight gastrointestinal hormones, using a multiplex bead assay. Changes in body mass index and anthropometric measurements were recorded, pre- and post-eradication therapy.
Pre-prandial active amylin, total peptide YY (PYY) and pancreatic polypeptide (PP) levels were significantly elevated 12 months post-eradication compared with baseline (n = 18; Wilcoxon's signed rank test, p<0.05). Four of the post-prandial gut metabolic hormones levels (GLP-1, total PYY, active amylin, PP) were significantly higher 12 months post-eradication compared to baseline (n = 18; p<0.05). Following H. pylori eradication, the BMI and anthropometric values did not significantly change.
Our study indicates that H. pylori eradication was associated with long-term disturbance in three hormones (active amylin, PP and total PYY) both pre- and post-prandially and one hormone (GLP-1) post-prandially. Longer post-eradication monitoring is needed to investigate the long-term impact of the observed hormonal changes on metabolic homeostasis.
PMCID: PMC4546342  PMID: 26291794
11.  Intergenerational change in Helicobacter pylori colonization in children living in a multi-ethnic Western population 
Gut  2014;64(8):1200-1208.
Helicobacter pylori (H. pylori) colonization rates in childhood have declined in Western populations, but it is unknown whether this trend is similar in children of non-Western ethnic backgrounds, born in a Western country. We aimed to identify H. pylori status in children, and determine both mother-to-child transmission and risk factors for colonization.
Antibodies against H. pylori and cytotoxin-associated gene A (CagA) were measured in children participating in a population-based prospective cohort study in Rotterdam, the Netherlands. Information on demographics and characteristics was collected using questionnaires.
We analysed the serum of 4,467 children (mean age 6.2 years ±0.5 SD) and compared the results with the H. pylori status of their mothers (available for 3,185 children). Overall, 438 (10%) children were H. pylori-positive, of whom 142 (32%) were CagA-positive. Independent risk factors for colonization were: maternal H. pylori positivity (OR 2.12; 95%CI 1.62–2.77), non-Dutch ethnicity (OR 2.05; 95%CI 1.54–2.73), female gender (OR 1.47; 95%CI 1.20–1.80), and lower maternal education level (OR 1.38; 95%CI 1.06–1.79). Comparing mothers and children, we found an intergenerational decrease of 76% and 77% for Hp+CagA− and Hp+CagA+-strains, respectively, consistent across all nine ethnic groups studied. Male gender, higher maternal educational level, and no older siblings, were independently associated with loss of H. pylori.
Although the highest H. pylori and CagA prevalence was found in children of non-Dutch ethnicities, the decreased colonization rates were uniform across all ethnic groups, implying the importance of environmental factors in H. pylori transmission in modern cities, independent of ethnicity.
PMCID: PMC4492887  PMID: 25192563
children; epidemiology; H. pylori; transmission
12.  Quantitation and Composition of Cutaneous Microbiota in Diabetic and Nondiabetic Men 
The Journal of Infectious Diseases  2013;207(7):1105-1114.
Background. Diabetic foot infections are a leading cause of lower extremity amputations. Our study examines the microbiota of diabetic skin prior to ulcer development or infection.
Methods. In a case-control study, outpatient males were recruited at a veterans hospital. Subjects were swabbed at 4 cutaneous sites, 1 on the forearm and 3 on the foot. Quantitative polymerase chain reaction (qPCR) with primers and probes specific for bacteria, Staphylococcus species, Staphylococcus aureus, and fungi were performed on all samples. High-throughput 16S ribosomal RNA (rRNA) sequencing was performed on samples from the forearm and the plantar aspect of the foot.
Results. qPCR analysis of swab specimens from 30 diabetic subjects and 30 control subjects showed no differences in total numbers of bacteria or fungi at any sampled site. Increased log10 concentrations of Staphylococcus aureus, quantified by the number of nuc gene copies, were present in diabetic men on the plantar aspect of the foot. High-throughput 16S rRNA sequencing found that, on the foot, the microbiota in controls (n = 24) was dominated by Staphylococcus species, whereas the microbiota in diabetics (n = 23) was more diverse at the genus level. The forearm microbiota had similar diversity in diabetic and control groups.
Conclusions. The feet of diabetic men had decreased populations of Staphylococcus species, increased populations of S. aureus, and increased bacterial diversity, compared with the feet of controls. These ecologic changes may affect the risk for wound infections.
PMCID: PMC3583274  PMID: 23300163
microbiota; microbiome; diabetic foot; cutaneous; Staphylococcus; Staphylococcus aureus
13.  Comparative Genome Analysis of Campylobacter fetus Subspecies Revealed Horizontally Acquired Genetic Elements Important for Virulence and Niche Specificity 
PLoS ONE  2014;9(1):e85491.
Campylobacter fetus are important animal and human pathogens and the two major subspecies differ strikingly in pathogenicity. C. fetus subsp. venerealis is highly niche-adapted, mainly infecting the genital tract of cattle. C. fetus subsp. fetus has a wider host-range, colonizing the genital- and intestinal-tract of animals and humans. We report the complete genomic sequence of C. fetus subsp. venerealis 84-112 and comparisons to the genome of C. fetus subsp. fetus 82-40. Functional analysis of genes predicted to be involved in C. fetus virulence was performed. The two subspecies are highly syntenic with 92% sequence identity but C. fetus subsp. venerealis has a larger genome and an extra-chromosomal element. Aside from apparent gene transfer agents and hypothetical proteins, the unique genes in both subspecies comprise two known functional groups: lipopolysaccharide production, and type IV secretion machineries. Analyses of lipopolysaccharide-biosynthesis genes in C. fetus isolates showed linkage to particular pathotypes, and mutational inactivation demonstrated their roles in regulating virulence and host range. The comparative analysis presented here broadens knowledge of the genomic basis of C. fetus pathogenesis and host specificity. It further highlights the importance of surface-exposed structures to C. fetus pathogenicity and demonstrates how evolutionary forces optimize the fitness and host-adaptation of these pathogens.
PMCID: PMC3887049  PMID: 24416416
14.  Community differentiation of the cutaneous microbiota in psoriasis 
Microbiome  2013;1:31.
Psoriasis is a common chronic inflammatory disease of the skin. We sought to characterize and compare the cutaneous microbiota of psoriatic lesions (lesion group), unaffected contralateral skin from psoriatic patients (unaffected group), and similar skin loci in matched healthy controls (control group) in order to discern patterns that govern skin colonization and their relationship to clinical diagnosis.
Using high-throughput 16S rRNA gene sequencing, we assayed the cutaneous bacterial communities of 51 matched triplets and characterized these samples using community data analysis techniques. Intragroup Unifrac β diversity revealed increasing diversity from control to unaffected to lesion specimens. Likewise, principal coordinates analysis (PCoA) revealed separation of the lesion samples from unaffected and control along the first axis, suggesting that psoriasis is a major contributor to the observed diversity. The taxonomic richness and evenness decreased in both lesion and unaffected communities compared to control. These differences are explained by the combined increased abundance of the four major skin-associated genera (Corynebacterium, Propionibacterium, Staphylococcus, and Streptococcus), which present a potentially useful predictor for clinical skin type. Psoriasis samples also showed significant univariate decreases in relative abundances and strong classification performance of Cupriavidus, Flavisolibacter, Methylobacterium, and Schlegelella genera versus controls. The cutaneous microbiota separated into two distinct clusters, which we call cutaneotypes: (1) Proteobacteria-associated microbiota, and (2) Firmicutes-associated and Actinobacteria-associated microbiota. Cutaneotype 2 is enriched in lesion specimens compared to control (odds ratio 3.52 (95% CI 1.44 to 8.98), P <0.01).
Our results indicate that psoriasis induces physiological changes both at the lesion site and at the systemic level, which select for specific differential microbiota among the assayed clinical skin types. These differences in microbial community structure in psoriasis patients are potentially of pathophysiologic and diagnostic significance.
PMCID: PMC4177411  PMID: 24451201
Cutaneous microbiota; Psoriasis markers; Microbiome analysis; Cutaneotypes
15.  Microbiomic Signatures of Psoriasis: Feasibility and Methodology Comparison 
Scientific Reports  2013;3:2620.
Psoriasis is a common chronic inflammatory disease of the skin. We sought to use bacterial community abundance data to assess the feasibility of developing multivariate molecular signatures for differentiation of cutaneous psoriatic lesions, clinically unaffected contralateral skin from psoriatic patients, and similar cutaneous loci in matched healthy control subjects. Using 16S rRNA high-throughput DNA sequencing, we assayed the cutaneous microbiome for 51 such matched specimen triplets including subjects of both genders, different age groups, ethnicities and multiple body sites. None of the subjects had recently received relevant treatments or antibiotics. We found that molecular signatures for the diagnosis of psoriasis result in significant accuracy ranging from 0.75 to 0.89 AUC, depending on the classification task. We also found a significant effect of DNA sequencing and downstream analysis protocols on the accuracy of molecular signatures. Our results demonstrate that it is feasible to develop accurate molecular signatures for the diagnosis of psoriasis from microbiomic data.
PMCID: PMC3965359  PMID: 24018484
16.  The Effect of Laparoscopic Gastric Banding Surgery on Plasma Levels of Appetite-Control, Insulinotropic, and Digestive Hormones 
Obesity surgery  2008;18(9):1089-1096.
We hypothesized that laparoscopic adjustable gastric banding (LAGB) reduces weight and modulates ghrelin production, but largely spares gastrointestinal endocrine function. To examine this hypothesis, we determined plasma concentrations of appetite-control, insulinotropic, and digestive hormones in relation to LAGB.
Twenty-four patients undergoing LAGB were prospectively enrolled. Body mass index (BMI) was measured and blood samples obtained at baseline and 6 and 12 months post-surgery. Plasma concentrations of leptin, acylated and total ghrelin, pancreatic polypeptide (PP), insulin, glucose-dependent insulinotropic peptide (GIP), active glucagon-like peptide-1 (GLP-1), gastrin, and pepsinogens I and II were measured using enzyme-linked immunoassays.
Median percent excess weight loss (%EWL) over 12 months was 45.7% with median BMI decreasing from 43.2 at baseline to 33.8 at 12 months post-surgery (p<0.001). Median leptin levels decreased from 19.7 ng/ml at baseline to 6.9 ng/ml at 12 months post-surgery (p<0.001). In contrast, plasma levels of acylated and total ghrelin, PP, insulin, GIP, GLP-1, gastrin, and pepsinogen I did not change in relation to surgery (p>0.05). Pepsinogen II levels were significantly lower 6 months after LAGB but returned to baseline levels by 12 months.
LAGB yielded substantial %EWL and a proportional decrease in plasma leptin. Our results support the hypothesis that LAGB works in part by suppressing the rise in ghrelin that normally accompanies weight loss. Unchanged concentrations of insulinotropic and digestive hormones suggest that gastrointestinal endocrine function is largely maintained in the long term.
PMCID: PMC3743550  PMID: 18408980
Obesity; Weight loss; Bariatric surgery; Adjustable gastric banding; Ghrelin; Leptin; GIP; GLP-1
Helicobacter  2012;17(3):232-237.
The prevalence of Helicobacter pylori in Western populations has steadily decreased. This has been suggested as one of the factors involved in the recent increase of asthma and allergy. Some studies have reported a negative association between H.pylori and asthma and allergy, but data are inconsistent and there are a few studies in children.
We investigated whether the prevalence of H.pylori was associated with asthma symptoms, allergic rhinitis, and atopic dermatitis in childhood.
We determined IgG anti-H.pylori and CagA antibodies in serum of Dutch children, who took part in the PIAMA birth cohort study. Serum was collected from 545 children, aged 7–9 yrs (Dutch ethnicity 91.5%). Symptoms of asthma and atopy were assessed by yearly questionnaires. Chi-square tests and logistic regression were used.
We found 9% H.pylori and 0.9% CagA seropositivity. Twelve (5.9%) children with reported wheezing ever were H.pylori positive, compared to 37 (10.9%) of the non-wheezers (p=0.05). No significant differences in H.pylori prevalence were found between children with or without allergic rhinitis (8.5% vs. 9.5%), atopic dermatitis (8.7% vs. 9.2%), and physician-diagnosed asthma (7.1% vs. 9.4%). Multivariate analysis showed no significant associations between H.pylori seropositivity and wheezing (OR 0.52; 95% CI 0.25–1.06), allergic rhinitis (OR 0.96; 95% CI 0.51–1.81), atopic dermatitis (OR 1.05; 95% CI 0.56–1.98) or physician-diagnosed asthma (OR 0.87; 95% CI 0.37–2.08).
We found a borderline significantly lower H.pylori seropositivity in children with wheezing compared to non-wheezers, but no association between H.pylori serum-antibody status and allergic rhinitis, atopic dermatitis, or asthma.
PMCID: PMC3335763  PMID: 22515362
Helicobacter pylori; children; asthma; wheezing; atopy; disappearing microbiota hypothesis
18.  Genotypic and phenotypic variation of Lewis antigen expression in geographically diverse Helicobacter pylori isolates 
Helicobacter  2011;16(6):475-481.
Helicobacter pylori is a persistent colonizer of the human gastric mucosa, which can lead to the development peptic ulcer disease and gastric adenocarcinomas. However, H. pylori can asymptomatically colonize a host for years. One factor that has been hypothesized to contribute to such persistence is the production of Lewis (Le) antigens in the lipopolysaccharide layer of the bacterial outer membrane as a form of molecular mimicry, since humans also express these antigens on their gastric mucosa. Humans and H. pylori both are polymorphic for Le expression, which is driven in H. pylori by variation at the Le synthesis loci. In this report we sought to characterize Le genotypic and phenotypic variation in geographically diverse H. pylori isolates.
Materials and Methods
From patients undergoing endoscopy in 29 countries, we determined Le phenotypes of 78 H. pylori strains, and performed genotyping of the galT and β-(1,3)galT loci in 113 H. pylori strains.
Le antigen phenotyping revealed a significant (p <0.0001) association between type 1 (Lea and Leb) expression and strains of East-Asian origin. Genotyping revealed a significant correlation between strain origin and the size of the promoter region upstream of the Le synthesis gene, galT (p <0.0001).
These results indicate that the heterogeneity of human Le phenotypes are reflected in their H. pylori colonizing strains, and suggest new loci that can be studied to assess variation of Le expression.
PMCID: PMC3228314  PMID: 22059399
19.  Novel gastric helicobacters and oral campylobacters are present in captive and wild cetaceans 
Veterinary microbiology  2011;152(1-2):138-145.
The mammalian gastric and oral mucosa may be colonized by mixed Helicobacter and Campylobacter species, respectively, in individual animals. To better characterize the presence and distribution of Helicobacter and Campylobacter among marine mammals, we used PCR and 16S rDNA sequence analysis to examine gastric and oral samples from ten dolphins (Tursiops gephyreus), one killer whale (Orcinus orca), one false killer whale (Pseudorca crassidens), and three wild La Plata river dolphins (Pontoporia blainvillei). Helicobacter spp. DNA was widely distributed in gastric and oral samples from both captive and wild cetaceans. Phylogenetic analysis demonstrated two Helicobacter sequence clusters, one closely related to H. cetorum, a species isolated from dolphins and whales in North America. The second related cluster was to sequences obtained from dolphins in Australia and to gastric non-Helicobacter pylori helicobacters, and may represent a novel taxonomic group. Dental plaque sequences from four dolphins formed a third cluster within the Campylobacter genus that likely represents a novel species isolated from marine mammals. Identification of identical Helicobacter spp. DNA sequences from dental plaque, saliva and gastric fluids from the same hosts, suggests that the oral cavity may be involved in transmission. These results demonstrate that Helicobacter and Campylobacter species are commonly distributed in marine mammals, and identify taxonomic clusters that may represent novel species.
PMCID: PMC3142288  PMID: 21592686
Helicobacter; Campylobacter; marine mammals; cetaceans; gastritis
20.  Serologic host response to Helicobacter pylori and Campylobacter jejuni in socially housed Rhesus macaques (Macaca mulatta) 
Gut Pathogens  2012;4:9.
Helicobacter pylori are successful colonizers of the human gastric mucosa. Colonization increases the risk of peptic ulcer disease and adenocarcinoma. However, potential benefits of H. pylori colonization include protection against early-onset asthma and against gastrointestinal infections. Campylobacter jejuni are a leading cause of bacterial diarrhea and complications include Guillain-Barré syndrome. Here, we describe the development of reliable serological assays to detect antibodies against those two bacteria in Rhesus macaques and investigated their distribution within a social group of monkeys.
Two cohorts of monkeys were analyzed. The first cohort consisted of 30 monkeys and was used to establish an enzyme-linked immunosorbent assay (ELISA) for H. pylori antibodies detection. To evaluate colonization of those macaques, stomach biopsies were collected and analyzed for the presence of H. pylori by histology and culture. C. jejuni ELISAs were established using human serum with known C. jejuni antibody status. Next, plasma samples of the 89 macaques (Cohort 2) were assayed for antibodies and then statistically analyzed.
An H. pylori IgG ELISA, which was 100% specific and 93% sensitive, was established. In contrast, the IgA ELISA was only 82% specific and 61% sensitive. The CagA IgG assay was 100% sensitive and 61% of the macaques were positive. In cohort 2, 62% macaques were H. pylori sero-positive and 52% were CagA positive. The prevalence of H. pylori IgG and CagA IgG increased with monkey age as described for humans. Of the 89 macaques 52% showed IgG against C. jejuni but in contrast to H. pylori, the sero-prevalence was not associated with increasing age. However, there was a drop in the IgG (but not in IgA) mean values between infant and juvenile macaques, similar to trends described in humans.
Rhesus macaques have widespread exposure to H. pylori and C. jejuni, reflecting their social conditions and implying that Rhesus macaques might provide a model to study effects of these two important human mucosal bacteria on a population.
PMCID: PMC3499398  PMID: 22920270
Helicobacter pylori; Campylobacter jejuni; Rhesus macaques; Antibodies; Sero-prevalence; CagA
21.  Serum pepsinogens and Helicobacter pylori in relation to the risk of esophageal squamous cell carcinoma in the ATBC Study 
Helicobacter pylori (H. pylori) can induce gastric atrophy in humans, which in turn increases gastric cancer risk. Whether H. pylori and gastric atrophy also affect the risk of esophageal squamous cell carcinoma (ESCC), however, remains unresolved.
We performed a nested case-control study within the prospective ATBC Study to assess these relationships. The ATBC Study is composed of 29,133 Finnish male smokers, aged 50–69, who were recruited during 1985–1988. Using baseline sera, we assessed H. pylori status (via IgG antibodies against whole-cell and CagA antigens) and gastric atrophy status (via the biomarkers pepsinogen I (PGI) and II (PGII)) in 79 ESCC cases and 94 controls. Logistic regression with adjustment for age, date of blood draw, education, cigarette smoking, alcohol, body mass index, and fruit and vegetable intake was used to estimate odds ratios (OR) and 95% confidence intervals (95%CI).
Gastric atrophy (PGI:PGII <4) was associated with ESCC (OR=4.58, 95%CI:2.00–10.48). There was no evidence for an association between H. pylori and ESCC (OR=0.94, 95%CI:0.40–2.24).
These results could be explained by misclassification of H. pylori status due to serologic amnesia, ESCC risk being dependent upon the functional consequences or interactions of H. pylori, rather than the infection per se, gastric atrophy having a different histogenesis in ESCC without being primarily dependent upon H. pylori acquisition, or a lack of statistical power to detect an effect.
Validation of these results may warrant mechanistic studies to determine the route of association between gastric atrophy and ESCC.
PMCID: PMC2919643  PMID: 20647397
Atrophy; Esophageal Neoplasms; Helicobacter pylori; Nested Case-Control Studies; Pepsinogens; Prospective Studies
22.  The effect of H. pylori eradication on meal-associated changes in plasma ghrelin and leptin 
BMC Gastroenterology  2011;11:37.
Appetite and energy expenditure are regulated in part by ghrelin and leptin produced in the gastric mucosa, which may be modified by H. pylori colonization. We prospectively evaluated the effect of H. pylori eradication on meal-associated changes in serum ghrelin and leptin levels, and body weight.
Veterans referred for upper GI endoscopy were evaluated at baseline and ≥8 weeks after endoscopy, and H. pylori status and body weight were ascertained. During the first visit in all subjects, and during subsequent visits in the initially H. pylori-positive subjects and controls, blood was collected after an overnight fast and 1 h after a standard high protein meal, and levels of eight hormones determined.
Of 92 enrolled subjects, 38 were H. pylori-negative, 44 H. pylori-positive, and 10 were indeterminate. Among 23 H. pylori-positive subjects who completed evaluation after treatment, 21 were eradicated, and 2 failed eradication. After a median of seven months following eradication, six hormones related to energy homeostasis showed no significant differences, but post-prandial acylated ghrelin levels were nearly six-fold higher than pre-eradication (p = 0.005), and median integrated leptin levels also increased (20%) significantly (p < 0.001). BMI significantly increased (5 ± 2%; p = 0.008) over 18 months in the initially H. pylori-positive individuals, but was not significantly changed in those who were H. pylori-negative or indeterminant at baseline.
Circulating meal-associated leptin and ghrelin levels and BMI changed significantly after H. pylori eradication, providing direct evidence that H. pylori colonization is involved in ghrelin and leptin regulation, with consequent effects on body morphometry.
PMCID: PMC3089783  PMID: 21489301
23.  Quantitation of Major Human Cutaneous Bacterial and Fungal Populations▿  
Journal of Clinical Microbiology  2010;48(10):3575-3581.
Because the human skin microbiota may play roles in the causation or modification of skin diseases, we sought to provide initial quantitative analysis from different cutaneous locations. We developed quantitative PCRs to enumerate the total bacterial and fungal populations, as well as the most common bacterial and fungal genera present in six locales, in eight healthy subjects. We used a set of primers and TaqMan MGB probes based on the bacterial 16S rRNA and fungal internally transcribed spacer region, as well as bacterial genus-specific probes for Propionibacterium, Corynebacterium, Streptococcus, and Staphylococcus and a fungal genus-specific probe for Malassezia. The extent of human DNA contamination of the specimen was determined by quantitating the human housekeeping GAPDH gene. The highest level of 16S rRNA copies of bacteria was present in the axilla (4.44 ± 0.18 log10 copies/μl [mean ± standard error of the mean]), with normalization based on GAPDH levels, but the other five locations were similar to one another (range, 2.48 to 2.89 log10 copies/μl). There was strong symmetry between the left and right sides. The four bacterial genera accounted for 31% to 59% of total bacteria, with the highest percent composition in the axilla and the lowest in the forearm. Streptococcus was the most common genus present on the forehead and behind the ear. Corynebacterium spp. were predominant in the axilla. Fungal levels were 1 to 2 log10 lower than for bacteria, with Malassezia spp. accounting for the majority of fungal gene copies. These results provide the first quantitation of the site and host specificities of major bacterial and fungal populations in human skin and present simple methods for their assessment in studies of disease.
PMCID: PMC2953113  PMID: 20702672
24.  Repeat-Associated Plasticity in the Helicobacter pylori RD Gene Family▿ †  
Journal of Bacteriology  2009;191(22):6900-6910.
The bacterium Helicobacter pylori is remarkable for its ability to persist in the human stomach for decades without provoking sterilizing immunity. Since repetitive DNA can facilitate adaptive genomic flexibility via increased recombination, insertion, and deletion, we searched the genomes of two H. pylori strains for nucleotide repeats. We discovered a family of genes with extensive repetitive DNA that we have termed the H. pylori RD gene family. Each gene of this family is composed of a conserved 3′ region, a variable mid-region encoding 7 and 11 amino acid repeats, and a 5′ region containing one of two possible alleles. Analysis of five complete genome sequences and PCR genotyping of 42 H. pylori strains revealed extensive variation between strains in the number, location, and arrangement of RD genes. Furthermore, examination of multiple strains isolated from a single subject's stomach revealed intrahost variation in repeat number and composition. Despite prior evidence that the protein products of this gene family are expressed at the bacterial cell surface, enzyme-linked immunosorbent assay and immunoblot studies revealed no consistent seroreactivity to a recombinant RD protein by H. pylori-positive hosts. The pattern of repeats uncovered in the RD gene family appears to reflect slipped-strand mispairing or domain duplication, allowing for redundancy and subsequent diversity in genotype and phenotype. This novel family of hypervariable genes with conserved, repetitive, and allelic domains may represent an important locus for understanding H. pylori persistence in its natural host.
PMCID: PMC2772487  PMID: 19749042
25.  Effect of Helicobacter pylori eradication on gastric carcinogenesis 
Chronic gastritis induced by Helicobacter pylori is the strongest known risk factor for gastric adenocarcinoma, yet the effects of bacterial eradication on carcinogenesis remain unclear. Animal models provide important insights into factors that are involved in gastric carcinogenesis, and we previously utilized such a model to demonstrate that an in vivo adapted H. pylori strain, 7.13, rapidly and reproducibly induces inflammation-mediated gastric carcinoma. In the current study, we used this bacterial strain as a prototype to define the role of targeted antimicrobial therapy in gastric carcinogenesis. Mongolian gerbils were infected with H. pylori for 4 or 8 weeks, treated with antimicrobial agents or vehicle, and then euthanized at 8 weeks after the completion of therapy. All infected gerbils developed gastritis; however, inflammation was significantly attenuated in animals receiving antimicrobial therapy. Gastric dysplasia or cancer developed in > 60% of the gerbils that remained persistently colonized with H. pylori, but in none of the animals treated with antibiotics following 4 weeks of infection. Infection with H. pylori for 8 weeks prior to therapy resulted in an attenuation, but not complete prevention, of pre-malignant and malignant lesions. Similarly, antibiotic therapy initiated at 4, but not 8, weeks after H. pylori challenge significantly reduced expression of the Th1 pro-inflammatory cytokine interferon-γ within colonized gastric mucosa. These results indicate that treatment of H. pylori in this model decreases the incidence and severity of lesions with carcinogenic potential. The effectiveness of eradication is dependent upon the timing of intervention, providing insights into mechanisms that may regulate the development of malignancies arising within the context of inflammatory states.
PMCID: PMC2833422  PMID: 18180700
inflammation; cytokine; dysplasia; gastric cancer; antibiotics

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