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RESEARCH ARTICLEOpen Access
Interleukin-8 is the single most up-regulated
gene in whole genome profiling of H. pylori
exposed gastric epithelial cells
Lars L Eftang1,2*, Ying Esbensen1, Tone M Tannæs3, Ida RK Bukholm2,4and Geir Bukholm5
Background: The association between Helicobacter pylori infection and upper gastrointestinal disease is well
established. However, only a small fraction of H. pylori carriers develop disease, and there are great geographical
differences in disease penetrance. The explanation to this enigma lies in the interaction between the bacterium
and the host. H. pylori Outer Membrane Phospholipase A (OMPLA) has been suggested to play a role in the
virulence of this bacterium. The aim of this study was to profile the most significant cellular pathways and
biological processes affected in gastric epithelial cells during 24 h of H. pylori exposure, and to study the
inflammatory response to OMPLA+and OMPLA-H. pylori variants.
Results: Interleukin-8 was the most significantly up-regulated gene and appears to play a paramount role in the
epithelial cell response to H. pylori infection and in the pathological processes leading to gastric disease. MAPK and
NF-kappaB cellular pathways were powerfully activated, but did not seem to explain the impressive IL-8 response.
There was marked up-regulation of TP53BP2, whose corresponding protein ASPP2 may interact with H. pylori CagA
and cause marked p53 suppression of apoptosis. Other regulators of apoptosis also showed abberant regulation.
We also identified up-regulation of several oncogenes and down-regulation of tumor suppressor genes as early as
during the first 24 h of infection. H. pylori OMPLA phase variation did not seem to influence the inflammatory
epithelial cell gene response in this experiment.
Conclusion: In whole genome analysis of the epithelial response to H. pylori exposure, IL-8 demonstrated the most
marked up-regulation, and was involved in many of the most important cellular response processes to the
infection. There was dysregulation of apoptosis, tumor suppressor genes and oncogenes as early as in the first 24
h of H. pylori infection, which may represent early signs of gastric tumorigenesis. OMPLA+/-did not affect the acute
inflammatory response to H. pylori.
H. pylori is well established as the primary cause of pep-
tic ulcer disease and the initiator of the multistep cas-
cade leading to gastric adenocarcinoma. Although
gastric cancer is the fourth most common cancer world-
wide and second only to lung cancer in causing cancer
related deaths [1], there are remarkable differences in
the distribution of this disease between western and
eastern countries. The decrease in gastric cancer paral-
lels H. pylori prevalence in the western world, but this
phenomenon does not completely explain the great geo-
graphical differences in gastric cancer distribution. The
reason why only 1-2% of H. pylori-infected individuals
develop gastric malignancies remains unexplained, and
includes both differences in bacterial strains, most
importantly cagA status, host genetics and environmen-
tal aspects.
H. pylori carcinogenesis involves indirect action of the
bacteria through chronic inflammation of the gastric
corpus mucosa, and also direct action of H. pylori on
epithelial cells. Persistent inflammation is associated
with enhanced production of several pro-inflammatory
cytokines, such as IL-1b, TNF-a, IL-6, IL-7 and IL-8 [2]
which increase apoptosis, hyperproliferation and
* Correspondence: email@example.com
1Department of Clinical Molecular Biology (Epigen), Institute of Clinical
Medicine, University of Oslo, Akershus University Hospital, Lørenskog, Norway
Full list of author information is available at the end of the article
Eftang et al. BMC Microbiology 2012, 12:9
© 2012 Eftang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Page 2
production of reactive oxygen and nitrogen species
causing DNA damage and mutations. In addition, direct
action of H. pylori on epithelial cells may also promote
carcinogenesis. cagA+H. pylori strains inject bacterial
products into epithelial cells through a sophisticated
type IV injection process, which activates intracellular
signaling pathways, in particular the mitogen-activated
protein kinase family (MAPK) pathway [3] and nuclear
factor kappa B (NF-?B), and may facilitate epithelial-
mesenchymal transition [4], all of which may contribute
to neoplastic transformation. Furthermore, tumor devel-
opment is associated with proliferation and apoptosis
inhibition [5,6], whereas excessive apoptosis is thought
to promote gastric ulcer formation. The effect of H.
pylori on gastric epithelial apoptosis has showed con-
flicting evidence. Several in vitro studies have showed
that H. pylori stimulate apoptosis [7,8], whereas some in
vivo studies demonstrate inhibition of apoptosis [9,10].
CagA injection into gastric epithelial cells up-regulates
the anti-apoptotic MCL protein [11] and interferes with
apoptosis-stimulating protein 2 of p53 (ASPP2) [12].
ASPP2 inhibition causes enhanced degradation of p53,
in a way similar to DNA tumor viruses, thereby decreas-
ing apoptotic activity, which may explain the increased
risk of GC associated with cagA+H. pylori infection.
Tannæs et al. have previously reported that the H.
pylori pldA gene, coding for bacterial outer membrane
phospholipase A (OMPLA), displays phase variation
resulting in 'ON' (OMPLA+) and 'OFF' (OMPLA-)
switching of OMPLA activity due to a spontaneous slip-
page in a homopolymer (C) tract of the gene [13]. The
OMPLA+variant was associated with increased bacterial
survival in an acidic environment, adherence, hemolysis
and release of urease and VacA compared to the
OMPLA-variant [14]. OMPLA has also been implicated
in the virulence of other gastrointestinal pathogens [15],
and a link between OMPLA activity and gastric inflam-
mation has been suggested in a previous study [16].
Although the gastric epithelial cell response to H.
pylori exposure has been subjected to many experiments
since the discovery of the bacterium in 1984 [17], only a
few studies have utilized cDNA microarray technology
[18-29]. Almost all of these experiments have been per-
formed on Asian H. pylori strains, and no authors have
compared the epithelial cell response to OMPLA+
against OMPLA-bacteria. The aim of the current study
was to investigate the temporal gene expression
response of gastric epithelial cells exposed to a clinically
obtained H. pylori strain, and to examine the contribu-
tion of OMPLA on the inflammatory response. Empha-
sis has been put on the most important biological
responses using Gene Ontology (GO) terms and asso-
ciated cellular signaling pathways.
To study the cellular morphology following H. pylori
infection at 3 and 6 h, non-exposed and H. pylori
exposed cells were stained and examined with immuno-
fluorescence microscopy (Figure 1). At both 3 and 6 h
there was no significant difference in the ability between
the OMPLA+and OMPLA-H. pylori to adhere to AGS
cells, and there were no significant differences in the
morphological changes in the AGS cells in response to
exposure to the two variants. We were not able to iden-
tify any statistically significant differences in the gene
expression between the cells exposed to OMPLA+and
OMPLA-variants at any time point over the 24 h of co-
culture (p < 0.05). We therefore concluded that analysis
of the results could be performed without further con-
sideration of differences in phase variation.
The cDNA profile of H. pylori exposed AGS cells were
compared against non-infected control cells at six sepa-
rate time points within 24 h. 7498 chip probes corre-
sponding to 6237 human genes showed differential
expression in the infected cells compared to control
cells at no less than 1 time point (p < 0. 05) (Additional
file 1: Table S1). The number of significantly differen-
tially expressed genes at each time point compared to
non-infected AGS-cells, and how they overlap at differ-
ent time points are illustrated in Table 1 and Figure 2.
There were no significantly expressed genes at 0.5 h, a
moderate increase in the number of genes from 1 to 6
h, and a 20-fold increase from 6 to 24 h. From one sam-
pling point to the next, most genes overlap, however a
considerable number of unique genes were also differen-
tially regulated at each time point (Figure 2). Approxi-
mately 47% of the total number of significantly
expressed genes were up-regulated, and 53% showed
down-regulation compared to control. Among the more
than 6000 significantly expressed genes, IL-8 was the
single most differentially expressed gene (Figure 3).
The list of all significant genes was analyzed for asso-
ciated Kyoto Encyclopedia of Genes and Genomes
(KEGG) signal pathways by Pathway Express at each
time point. Significantly impacted pathways and corre-
sponding Impact Factor (IF) are presented in Table 2.
Early response signal pathways that were significantly
affected included the epithelial cell signaling in H. pylori
infection pathway, cytokine-cytokine receptor interac-
tion, Toll-like receptor (TLR) signaling pathways as well
as many cancer-related pathways and immunological
pathways. At 1 h, IL-8 was involved in most of the
affected signal pathways. At 3 and 6 h, most of the high-
est ranked pathways had several genes in common, such
as NFKB1, NFKB2, NFKBIA, NFKBIE, BIRC2, BIRC3,
JUND, CCND1 and AKT3. The phosphatidylinositol sig-
naling system is assigned a high IF at 6 h due to the
Eftang et al. BMC Microbiology 2012, 12:9
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significance of one single gene, PIK3C2B, which is
down-regulated by a log2FC of -0.58 and plays a key
role in this pathway. At 12 h, the most affected cellular
pathways were the leukocyte transendothelial migration,
cell adhesion molecules, DNA replication pathway, p53
signaling pathway as well as several cancer-related path-
ways. Relatively similar results are seen at 24 h, however
some of the cancer-related pathways are represented
Figure 1 Immunofluorescence images of AGS cells exposed to H. pylori. AGS cells were non-exposed, or exposed to OMPLA+and OMPLA-
H. pylori at a MOI of 300:1 and co-cultured for 3 and 6 h. The bacteria were stained with rabbit anti-Helicobacter antibody. Images were
captured by fluorescent microscopy.
Table 1 Number of differentially regulated genes
Time0.513612 24
02 91123 16792997
0126652034 2492
03117 188 37135489
Number of significantly differentially regulated genes (p < 0.05) at each of the
sampling time points after a period of co-incubation of H. pylori in AGS cells
Eftang et al. BMC Microbiology 2012, 12:9
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further down the list (data not shown, only top 10
shown in Table 2).
Because GO analysis simply associates differentially
expressed genes with the ontologies, there is no attempt
at ranking the true biological significance of individual
genes or ontologies. Therefore, we included only genes
with a log2FC > 1.5 in the GO analysis, excluding lesser
significantly expressed genes that were likely to result in
erroneous GO ranking. Only terms categorized under
Biological Processes are included (Table 2), as these
were the focus of the study. No GO terms were
enriched at 0.5 or 1 h time points. Among the up-regu-
lated genes at 3-6 h, the most frequently associated GOs
were anti-apoptosis, and several inflammatory and anti-
microbial processes such as regulation of retroviral gen-
ome replication, T-helper 1 cell differentiation, chemo-
taxis, neutrophil activation and immune activation. At
12-24 h, the up-regulated genes enriched ontologies like
cell cycle arrest, apoptosis, stress response, amino acid
transport, angiogenesis and keratinization, while certain
biosynthetic processes are among the down-regulated
Hierarchical clustering of the 245 genes with a log2FC
> 1.5 formed 5 distinct clusters (A-E), at a distance
threshold of 0.54, (Figure 3). Each cluster was examined
for GO and cellular signal pathway associations (Table
3). GO analysis provided significant terms for all clusters
(p < 0.05). Table 3 shows the top 10 significantly
impacted cellular signaling pathways within each cluster,
ranked according to IF. Cluster A contained 9 genes,
and demonstrated steady levels at 6-12 h before showing
a decline. Three genes were involved in anti-apoptotic
processes and two genes were involved in MAPK signal-
ing. Only 3 genes were assigned to cluster B, where
Figure 2 Venn diagrams of significantly regulated genes. Venn
diagrams of differentially expressed genes of H. pylori-infected AGS
cells compared to control cells (p < 0.05). The intersecting circles
indicate overlapping genes at the indicated time points. AGS =
non-infected control AGS cells.
Figure 3 Hiarchical clustering of the most significantly
differentially regulated genes. Hiarchical clustering of significantly
differentially regulated genes (log2FC > 1.5, p < 0.05). Arrow points
at IL-8.
Eftang et al. BMC Microbiology 2012, 12:9
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Table 2 Time course: KEGG cellular pathways and gene ontology
Time KEGG cellular pathway nameIF GO up-regulated genes GO down-regulated genes
No significant genesNo significant genesNo significant genes
Epithelial cell signaling in Helicobacter pylori
Cytokine-cytokine receptor interaction
Bladder cancer
Toll-like receptor signaling pathway
Base excision repair
Primary immunodeficiency
Pathways in cancer
16.6 No significant GONo significant genes
Epithelial cell signaling in Helicobacter pylori
Pathways in cancer
Small cell lung cancer
MAPK signaling pathway
17.8 anti-apoptosis No significant GO
16.9 regulation of retroviral genome
14.2 replication
14.2 T-helper 1 cell differentiation
12.5 negative regulation of LPS-mediated signaling
12.3 negative regulation of smooth muscle cell
11.4 regulation of MAP kinase activity chemotaxis
11.1 protein amino acid dephosphorylation
10.5 neutrophil activation
9.9 entrainment of circadian clock
Adipocytokine signaling pathway
Prostate cancer
Toll-like receptor signaling pathway
T cell receptor signaling pathway
B cell receptor signaling pathway
Phosphatidylinositol signaling system
Epithelial cell signaling in Helicobacter pylori
Small cell lung cancer
Pathways in cancer
Adipocytokine signaling pathway
Toll-like receptor signaling pathway
MAPK signaling pathway
Bladder cancer
B cell receptor signaling pathway
32.2 anti-apoptosis
15.5 regulation of retroviral genome
No significant GO
14.2 replication
12.4 T-helper 1 cell differentiation
11.6 neutrophil activation
10.1 negative regulation of I-kappaB
8.9 kinase/NF-kB cascade
8.7 induction of positive chemotaxis
8.5 myeloid dendritic cell differentiation
Leukocyte transendothelial migration
Cell adhesion molecules (CAMs)
309.7 cell cycle arrest
75.4 amino acid transport
response to unfolded protein
S-adenosylmethionine biosynthetic
DNA replication
Cell cycle
Pathways in cancer
p53 signaling pathway
Antigen processing and presentation
MAPK signaling pathway
Small cell lung cancer
Circadian rhythm
25.0 positive regulation of transcription
20.0 response to stress
19.4 regulation of MAP kinase activity
Leukocyte transendothelial migration
Cell cycle
p53 signaling pathway
Circadian rhythm
DNA replication
Adherens junction
Pathways in cancer
Nucleotide excision repair
80.3 keratinocyte differentiation
24.4 amino acid transport
20.9 keratinization
18.6 angiogenesis
18.0 apoptosis
16.1 response to stress
14.9 cell cycle arrest
14.3 pyrimidine nucleotide metabolic
cholesterol biosynthetic process
response to unfolded protein
isoprenoid biosynthetic process
creatine biosynthetic process
response to oxidative stress
Eftang et al. BMC Microbiology 2012, 12:9
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there was a rapid and potent increase in expression dur-
ing the first 3 h, followed by a decline. Of the 3 genes
in the cluster, IL-8 and CXCL2 seemed to dictate many
of the acute inflammatory processes like chemotaxis,
immune response and neutrophil activation.
Cluster C comprised the largest cluster, and contained
150 genes that did not show any change until after 6-12
h. The GO terms apoptosis, cell cycle arrest and stress
response genes were markedly enriched, and many of
these genes such as JUN, GADD45A, DDIT3, MKNK2,
DUSP1, RPS6KA5, FLNC, and RASGRP were also
involved in MAPK signaling. Furthermore, CSF2RA,
IL24, IL20R and the oncogene PIM1 were involved in
Jak-STAT signaling and cytokine-cytokine signaling.
Cluster D showed a moderate increase peaking at 12
h, followed by a decrease towards 24 h. 13 genes were
assigned to this cluster, including EDN1, one of the iso-
forms of the potent vasoconstrictor endothelin, which
enriched virtually all of the listed GOs. NFKB2, one of
two NF-?B subunits, HBEGF and ETS1 were also
included in this cluster.
Cluster E demonstrated 71 genes that showed down-
regulation after 6-12 h and included FGFR3 and several
heat shock protein genes that were involved in the
MAPK signaling pathway and apoptosis inhibition. Also,
several GO biosynthetic processes were enriched.
To confirm the microarray results, we chose to verify
IL-8, as this was the single most differentially regulated
gene in the study. mRNA and protein were sampled at
the same time points and studied by rt-PCR and ELISA
(Figures 4 and 5). There was an increase in IL-8 mRNA
noticeable after 1 h and peaking at around 3 h. The IL-8
mRNA response then dropped towards 6 and 12 h. At
24 h there was a second increase, however with note-
worthy variance between the two experiments. At 0.5
and 1 h of co-culture, IL-8 protein levels were low and
did not show any change. Between 3 and 6 h of co-cul-
ture, there was a significant IL-8 increase which showed
no further increase after 6 h.
Lastly, we wanted to ascertain that the chosen MOI
was stable with regard to AGS gene expression. We
used IL-8 response as an indicator of gene expression,
and AGS cells were co-incubated with H. pylori for 3 h
at various MOI in two separate experiments (Figure 6).
There was a modest IL-8 response at MOI 15:1 and
150:1, with a remarkable increase at MOI of 300:1.
There were then negligible changes in IL-8 expression
above 300:1, which suggested that the original inoculum
of 300:1 was adequate to elicit a biological response
without overloading the cell culture system.
In this study we demonstrate a significant, immediate
response from AGS cells to the exposure to a H. pylori
strain obtained from a clinical setting. More than 6000
human genes showed statistically significant differential
regulation during the first 24 h of co-incubation.
H. pylori infection has been associated with both sti-
mulation and inhibition of apoptosis. Some cell culture
experiments demonstrate up-regulation of genes asso-
ciated with apoptosis [7,8], whereas some in vivo studies
demonstrate proliferation and apoptosis inhibition
[9,10]. VacA toxin has been shown to cause apoptosis in
several studies [30-33], whereas the role of CagA is con-
flicting. CagA has been associated with both stimulation
and inhibition of apoptosis [11,12,34]. Biliary cells
exposed to cagA+H. pylori at a very low inoculum
(MOI 1:1) demonstrated increased cell growth, whereas
at MOI of 200:1, apoptosis was stimulated [35]. CagA
may even directly antagonize the pro-apoptotic effect of
VacA, as seen in AGS cells [31]. Apoptosis occurs after
a number of cellular events, leading to activation of cas-
pase-3, which is thought to constitute the basic effector
of apoptosis. In the present study, both inhibitory and
stimulatory genes showed significant differential expres-
sion, demonstrating the complexity of the influence of
H. pylori on apoptosis: caspase inhibitors HSPA5 and
DHCR24 showed similar late down-regulation as heat
shock genes HSPA1B, HSPB1, which are also associated
with apoptosis stimulation (cluster E, Table 3). On the
other hand, TNFAIP3, BIRC2, BIRC3 and SERPINB2,
also associated with apoptosis inhibition, demonstrated
early and persistent up-regulation grouped together in
cluster A. However, positive regulators of apoptosis
PHLDA4, PP1R15A and SQSTM1 were all up-regulated
in similar pattern after 6-12 h (cluster C). MCL1, an
anti-apoptotic gene expressed in response to CagA
injection [11], demonstrated increasing up-regulation
over the course of the study. There were no significant
changes in BCL-2 and very little increase in BAX expres-
sion in our study, two important genes that determine
the sensitivity of cells to other apoptotic stimuli [36-39].
Noteworthy, there was marked up-regulation of
Table 2 Time course: KEGG cellular pathways and gene ontology (Continued)
Ubiquitin mediated proteolysis
Phosphatidylinositol signaling system
14.2 process
13.7 induction of positive chemotaxis
Significantly impacted KEGG cellular pathways and enriched Gene Ontology terms (biological processes only) (p < 0.05) at different time points following co-
culture of H. pylori and AGS cells. Top 10 pathways/ontologies included where number exceeds 10. IF = impact factor
Eftang et al. BMC Microbiology 2012, 12:9
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Table 3 Cluster profiling: KEGG cellular pathways and Gene Ontology
Temporal profile over 24 hCellular PathwayImpact
GO name
MAPK signaling pathway7.3 GO:0006916 anti-apoptosis
Apoptosis 7.1 GO:0045063 T-helper 1 cell differentiation
GO:0031665 negative regulation of LPS-mediated signaling
GO:0014912 negative regulation of smooth muscle cell migration
GO:0043405 regulation of MAP kinase activity
Epithelial cell signaling in
H. pylori infection
12.4 GO:0006935 chemotaxis
Cytokine-cytokine receptor
Bladder cancer
Toll-like receptor signaling
Pathways in cancer
10.2 GO:0006954 inflammatory response
6.8 GO:0006955 immune response
5.9 GO:0045091 regulation of retroviral genome replication
4.8 GO:0042119 neutrophil activation
GO:0050930 induction of positive chemotaxis
GO:0030593 neutrophil chemotaxis
GO:0030155 regulation of cell adhesion
GO:0019722 calcium-mediated signaling
Circadian rhythm20.0 GO:0006915 apoptosis
MAPK signaling pathway
mTOR signaling pathway
Tight junction
Jak-STAT signaling
Cytokine-cytokine receptor
Regulation of autophagy
p53 signaling pathway
Regulation of actin
TGF-beta signaling
Natural killer cell mediated
10.7 GO:0006950 response to stress
7.5 GO:0007050 cell cycle arrest
7.0 GO:0030216 keratinocyte differentiation
6.7 GO:0006865 amino acid transport
6.5 GO:0031424 keratinization
6.4 GO:0008652 amino acid biosynthetic process
5.6 GO:0006220 pyrimidine nucleotide metabolic process
Eftang et al. BMC Microbiology 2012, 12:9
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TP53BP2, an important tumor suppressor gene (TSG) in
human cancer, primarily stimulating p53 promotion of
apoptosis genes. On the other hand, TP53BP2 is coding
ASPP2 protein, which has also been shown to stimulate
apoptosis independently of p53 [40-42]. However, Buti
et al. recently demonstrated that CagA injected into gas-
tric epithelial cells targeted ASPP2 protein to inhibit
p53-mediated apoptosis [12]. The increased TP53BP2
expression seen in our study, might therefore potentiate
this effect by increasing the CagA-ASPP2 interaction to
cause increased inhibition of p53-mediated apoptosis. In
fact, the current study showed that p53 target genes
involved in apoptosis [43] such as FAS, DR4,
TNFRSF10B (also referred to as DR5/KILLER), DCR1,
DCR2, P53AIP1, CASP6, APAF1 and BNIP3L did not
show any significant increase, and BNIP3L, CASP6 and
APAF1, BID and BAX showed only little increase. p53
target genes regulating non-apoptotic cellular processes
including MDM2, GADD45A, CDKN1A (also known as
P21 WAF1/CIP1), EGFR, CCND1, CCNG2 and TGFA
demonstrated moderate to marked up-regulation. This
differential gene expression identified among the p53
target genes in this study, may indicate selective inhibi-
tion of p53-mediated apoptosis due to increased CagA-
ASPP2 interaction, consistent with Buti's findings.
Nevertheless, this study was not designed to assess
whether the overall sum of inhibitory and stimulatory
signals facilitated apoptosis or proliferation of epithelial
cells. The current results illustrate the complexity of
apoptosis regulation in epithelial cells in response to H.
pylori exposure, and the cluster analysis suggests that
there is some biological coordination of gene expression
Table 3 Cluster profiling: KEGG cellular pathways and Gene Ontology (Continued)
Melanogenesis 8.3 GO:0030146 diuresis
GnRH signaling pathway
ErbB signaling pathway
7.6 GO:0030147 natriuresis
6.7 GO:0048661 positive regulation of smooth muscle cell
6.4 GO:0002268 follicular dendritic cell differentiation
5.7 GO:0031583 activation of phospholipase D activity by G-protein
coupled receptor protein signaling
GO:0014826 vein smooth muscle contraction
GO:0002467 germinal center formation
GO:0030578 PML body organization
GO:0030195 negative regulation of blood coagulation
GO:0043507 positive regulation of JUN kinase activity
Pathways in cancer
Epithelial cell signaling in
H. pylori infection
Antigen processing and
13.7 GO:0006695 cholesterol biosynthetic process
MAPK signaling pathway
Bladder cancer
Pathways in cancer
9.7 GO:0006986 response to unfolded protein
6.2 GO:0006916 anti-apoptosis
6.1 GO:0006139 nucleobase, -side, -tide and nucleic acid metabolic
6.1 GO:0008299 isoprenoid biosynthetic processRegulation of actin
GO:0006601 creatine biosynthetic process
GO:0009416 response to light stimulus
GO:0043154 negative regulation of caspase activity
GO:0007566 embryo implantation
Temporal profiles of 5 main clusters identified by hiarchical clustering of the 245 most differentially expressed genes (p < 0.05) and associated gene ontologies
(biological processes only) and KEGG cellular signaling pathways in each cluster in H. pylori exposed AGS cells. Data points are at 0.5, 1, 3, 6, 12 and 24 h of co-
incubation. Error bars represent ± standard deviation of expression within the cluster. Top 10 ontologies listed where number is exceeding 10
Eftang et al. BMC Microbiology 2012, 12:9
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regulating apoptosis. This may explain some of the com-
plex carcinogenic mechanism of H. pylori in gastric ade-
nocarcinoma. There is strong association between H.
pylori infecton, in particular the cagA+genotype [44],
and gastric adenocarcinoma [45,46], and also other can-
cers have been suggested to harbour a role for H. pylori
[47,48]. Furthermore, the present study shows that sev-
eral cancer-related KEGG pathways are impacted in
AGS cells during 24 h of cagA+H. pylori infection, in
particular pathways in cancer, bladder cancer, prostate
cancer, small cell lung cancer and the MAPK pathway.
Several individual oncogenes and cancer related genes
were also increased during, and at the end of the study,
including ANGPT2, CEBPB, ECGF1, MMP7, MMP10,
KLK6, KRT7, LCN2, MYC, PIM1, PIM2, PIM3 and
IL-8 appears paramount in the acute inflammatory
response to H. pylori infection, as this gene is involved
in all significant response pathways in the initial cellular
response to infection. Several authors have demon-
strated increase in IL-8 in response to H. pylori in both
in vivo [49] and in vitro [50,51] studies. IL-8 is a key
chemokine in accumulating neutrophils. Gastric mucosal
IL-8 levels have shown a positive correlation with the
degree of stomach corpus inflammation [52], and IL-8 is
also highly increased in gastric cancer [53,54]. Our find-
ings are supported by other authors who have demon-
strated that IL-8 mRNA in vitro peaks between 2 and 4
h before decreasing over the next hours under similar
conditions [55,56]. Protein studies have shown steady
state IL-8 levels after 3 h [50,57,58], which is also in
harmony with our ELISA results, where marked IL-8
levels were detectable at 3 h and continuing to increase
at 6 h before reaching a steady level. H. pylori-induced
IL-8 secretion may be explained by both stimulation of
Figure 4 Time-course of IL-8 mRNA expression in AGS cells co-
cultured with H. pylori. Quantitative PCR analysis of IL-8 expression
in H. pylori-infected AGS cells at six different sampling points over
24 h. Data points are the values of three cell culture replicates from
two independent experiments, A and B. Lines represent the
calculated mean within each of the experiments.
Figure 5 Time-course of IL-8 protein expression in AGS cells
co-cultured with H. pylori. ELISA analysis of IL-8 protein expression
in H. pylori-infected AGS cells at six different sampling points over
24 h. Data points are the values of three cell culture replicates from
two independent experiments, A and B. Lines represent the
calculated mean within each of the experiments.
Figure 6 Dose-response of IL-8 mRNA expression in AGS cells
co-cultured with H. pylori. Quantitative PCR analysis of IL-8
expression in H. pylori-infected AGS cells, co-incubated for 3 h. Data
points are the values of three cell culture replicates from two
independent experiments, A and B. Lines represent the calculated
mean within each of the experiments.
Eftang et al. BMC Microbiology 2012, 12:9
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the MAPK signaling system [59,60], and NF-?B activa-
tion through several pathways [61,62]. In the present
study, MAPK signaling was ranked relatively high from
3 h onwards, based on IF calculations, and the cluster
analysis showed that increasingly more genes in the
MAPK pathway were affected after 6 h of H. pylori
exposure. Regulators of NF-?B; TNFAIP3, RELB and
BIRC3, which could also have explained the IL-8 expres-
sion, show increasing expression after 3 h (Additional
file 1: Table S1), identical to the findings of Guillemin et
al. [29]. Therefore, it is interesting that the great
increase in IL-8 mRNA, which peaked as early as 3 h as
shown in both the microarray and rt-PCR data, occurs
before the onset of both MAPK and NF-?B signaling.
The rt-PCR data, but not the microarray analysis, also
demonstrated a second increase in IL-8 mRNA at 24 h,
although with noteworthy variance between experi-
ments. While it is possible that this second surge may
be explained by MAPK and/or NF-?B activation, it is
unlikely that MAPK or NF-?B signaling explain the
initial, powerful IL-8 mRNA peak seen at 3 h. The pre-
sent study is the first to demonstrate that among more
than 38 000 human genes, IL-8 was the single most up-
regulated gene by gastric epithelial cells in response to
H. pylori exposure in vitro, and it appears feasible that
mechanisms other than MAPK or NF-?B activation may
be responsible for this up-regulation.
Although histopathological studies indicate that MOI
around 10:1 appear in H. pylori-colonized gastric
mucosa, laboratory conditions can never replicate the
complex physiology of the human stomach. Much
higher MOI have normally been used to study in vitro
gastric epithelial cell response to H. pylori colonization,
and MOI of 300:1 was our incoulum of choice, as we
wanted a sufficient inoculum to induce a biological
response from AGS cells, both at the mRNA and pro-
tein levels, as indicated by other experiments [35,63-71].
However, it is worth noting that in a recent report by
Ritter et al., a marked IL-8 response from AGS cells
exposed to cagA+H. pylori was seen at MOI ranging
from 10:1 to 100:1 [61]. The IL-8 response was higher
at MOI 100:1 compared to 10:1 in all the bacterial
strains tested. The response to MOI 300:1 was not
assessed. Neither cagA nor vacA status seemed to affect
the IL-8 response at the higher inoculum. Ritter's study
also showed that different cellular pathways were acti-
vated in response to high or low MOI. In some other
studies, where non-gastric cells were exposed to cagA+
H. pylori, low MOI was associated with apoptosis inhibi-
tion and cell growth, whereas high MOI stimulated
apoptosis and inhibited survival [35,72,73]. Hence, the
choice of MOI may be crucial for the study outcome.
Nevertheless, based on our immunofluorescence studies,
where we found sufficient bacterial adhesion to AGS
cells, typical morphological changes, and most impor-
tantly, a marked IL-8 mRNA and protein response to
MOI 300:1, we concluded that under our experimental
conditions, 300:1 was adequate to elicit a biological
response without overloading the system.
You et al. performed a similar microarray study pub-
lished in 2010 [74], where AGS cells were exposed to H.
pylori for 6 h. A relatively stable number of 300-400
genes were reported to be differentially expressed at
each of the sample points, whereas our data showed a
progressive increase in the number of genes from 0.5 to
24 h. In addition, key biological processes like chemo-
taxis, TLR signaling and epithelial cell signaling were
reported as down-regulated. This is in contrast to our
results, and also the findings of most other similar
microarray studies [19-23,25,26,28,29,68,75], where these
particular processes are regularly increased. However,
many of these studies do indeed show somewhat con-
flicting results, possibly explained by differences in incu-
bation conditions, bacterial strains and obsolete or
proprietary cDNA arrays and technology.
We have previously suggested a potential role for
OMPLA in inflammation [14,16]. OMPLA+variants
were found to yield increased hemolysis, adherence and
release of urease and VacA compared to the OMPLA-
variant. One of the aims of the present study was there-
fore to investigate the role of OMPLA on the gastric
epithelial cell inflammatory response. We compared the
gene expression profile of H. pylori OMPLA+exposed
cells against OMPLA-exposed cells at the 6 different
time points. No significant difference was detected at
any of the time points.
No other studies have directly investigated the role of
OMPLA on the gastric epithelial cell inflammatory
response, as the pldA/OMPLA status is unknown in
most strains. Among the few full genome sequenced H.
pylori strains, G27 carries a C7 repeat in the pldA gene
[76] and B38 carries a C9 repeat, both giving rise to a
truncated and inactive OMPLA [77]. Several experi-
ments have demonstrated the ability of G27 to induce a
significant IL-8 response [29,78], supporting our current
observation that OMPLA-H. pylori is indeed capable of
inducing significant inflammation. One surprising result
has been reported in a study of pH-regulated gene
expression in the G27-strain [79], where Merrell et al.
reported that cagA was consistently suppressed by low
pH in H. pylori G27. Previous studies of other H. pylori
strains, however, had suggested that cagA expression
was induced at low pH.
Although the pldA phase variation did not appear to
affect the inflammatory response in this study, phase
variation of the pldA gene probably serves a purpose in
other aspects of H. pylori. OMPLA activity is associated
with increased survival at low pH [13,80]. The
Eftang et al. BMC Microbiology 2012, 12:9
Page 10 of 15
Page 11
mechanism behind this property is not yet known. One
possibility might be that OMPLA has adapted an as yet
unknown function needed for this specific environment,
in addition to phospholipase activity. Dorrell et al. have
showed that a pldA knockout mutant was unable to
colonize mice [81]. Salaün et al. have assessed changes
in a spectrum of H. pylori phase-variable genes in a
mouse model of gastric colonization [82]. pldA was
among the most rapidly changing genes, with changes
occurring within the first 3 days of colonization. The
change in pldA showed a phenotypic selection from an
initial inoculum which consisted of a mixture of ON
and OFF phenotypes, to an exclusively ON population.
Wernegreen et al. have postulated that evolutionary
selection will interrupt a slippery tract, such as the C-
tract in the pldA gene, thus removing the possibility of
phase variation [83]. When selection does not happen,
the sequence feature must be to some benefit for the
bacterium. It seems clear that the normal gastric envir-
onment is optimal for the OMPLA+phenotype, but for
what niche the OMPLA-phenotype is adaptive is cur-
rently unknown. One could speculate that the properties
of the OMPLA-variant could be useful when transfer-
ring from one human stomach to another.
In summary, we have confirmed important biological
processes and pathways affected by H. pylori infection of
gastric epithelial cells described by many other authors.
IL-8 was the single most differentially regulated gene
among more than 38 000 genes tested, and seems fun-
damental in the epithelial cell reaction to H. pylori
demonstrated by its involvement in the majority of the
response processes that we have identified. Several intra-
cellular signaling pathways are significantly impacted,
such as the epithelial cell signaling in H. pylori infection
pathway including the MAPK and NF-?B pathways,
however none of these pathways seem to explain the
very rapid up-regulation of IL-8 seen at 3 h. Further-
more, we have observed differential expression of both
stimulatory and inhibitory apoptosis genes, suggesting
dysregulation of apoptosis following H. pylori infection.
Apoptotic p53 target genes showed little changes in reg-
ulation, whereas many non-apoptotic p53 target genes
demonstrated a marked increase in expression. This
phenomenon may be explained by selective inhibition of
p53 caused by the ASPP2-CagA interaction.
Lastly, although gastric carcinogenesis is a very
delayed consequence of H. pylori infection, we have
seen up-regulation of cancer-related signaling, as well as
aberrant regulation of oncogenes and TSGs as early as
the first 24 h of infection.
The work presented in this study does not support the
previous suggestion that OMPLA enzyme activity
enhances inflammatory response induced by H. pylori in
epithelial cells. However, the phase shift seen in the
pldA gene probably plays a role in other aspects in the
life of the bacterium.
Human gastric epithelial cells were infected by the
OMPLA+and OMPLA-H. pylori, and mRNA and pro-
tein were sampled at 6 different time points within the
first 24 h. The co-cultures were studied by immuno-
fluorescent microscopy at 3 and 6 h to study bacterial
adhesion and cell morphological changes. First, human
whole genome cDNA microarray analysis was conducted
to study gene expression changes in the H. pylori-
exposed cells. Second, the epithelial cell response to the
OMPLA+variant was compared against the OMPLA-
variant. Third, IL-8 levels were analyzed by real-time
PCR and ELISA to verify the microarray results. Last, a
dose-response experiment was performed to ensure ade-
quate bacterial inocula.
Bacterial strain and variants
The bacterial strain, H. pylori 17B/RH, a representative
isolate displaying pldA phase variation, was isolated
from a non-ulcer dyspeptic patient referred to outpati-
ent endoscopy and maintained at -70°C [13]. The two
pldA phase variants gave rise to a functional phospholi-
pase A (OMPLA+) and a truncated, non-functional
phospholipase A (OMPLA-) respectively. The isogenicity
of the variants was previously confirmed by amplified
fragment length polymorphism [13]. The variants had
the s1a/m2 vacA genotype and were cagA positive dis-
playing an ABC EPIYA genotype [16,80]. The presense
of the caga, cagb, cagE, cagL, cagM, cagX and cagY
genes indicated that the variants harboured an intact
cag pathogenicity island (cagPAI) and were capable of
CagA translocation (unpublished data). Both variants
displayed a truncated LPS.
The bacteria were cultured on blood agar plates under
microaerobic conditions at 37°C for 48 h. After cultiva-
tion, the bacteria were harvested and suspended in
phosphate buffered saline (PBS). Bacterial concentra-
tions were estimated by measuring OD600. Aliquots of
the OMPLA+and OMPLA-bacterial suspensions were
transferred to separate cell culture flasks at appropriate
concentrations. Dilutions of the suspensions were also
plated onto blood agar plates. After 5 days of microaero-
bic incubation, the colonies were counted and inspected
for any OMPLA phase shifts.
AGS cell line and inoculation of cell cultures
The gastric epithelial cell line AGS (American Type
Culture Collection no: CRL 1739) was grown on RPMI
supplemented with 2 mM L-glutamine and 10% foetal
Eftang et al. BMC Microbiology 2012, 12:9
Page 11 of 15
Page 12
calf serum at 37°C in a CO2incubator at a gas composi-
tion of 5% CO2and 20% O2. When cells grew to a con-
fluent monolayer of approximately 5,1 × 106cells/flask
(60%) the medium was changed to RPMI supplemented
with 2 mM L-glutamine only. After an equilibration per-
iod of about 30 min, bacteria in PBS were added. To
study AGS cell gene expression during the first 24 h,
the cells were co-cultured with the H. pylori at a multi-
plicity of infection (MOI) of 300:1. The two phase var-
iants (OMPLA+and OMPLA-) were assigned to
separate co-cultures, to allow the investigation of the
whole genome response to H. pylori infection per se,
and also to study possible differences in the response to
the OMPLA+and OMPLA-variants. Co-cultured cells
were incubated for 30 min, 1, 3, 6, 12 and 24 h, before
RNA was stabilized by RNAlater (Applied Biosystems,
United States), and the cells were harvested.
To ensure that the obtained gene response was ade-
quate, a dose-response experiment was performed, add-
ing bacteria to AGS cells at a MOI of 15:1, 150:1, 300:1,
600:1, 900:1 and 1200:1. Cells were co-incubated for 3
h, before being immersed in RNAlater followed by har-
vesting of the cells. Non-infected AGS cells served as a
negative control. Both the time-course and the dose-
response experiments were carried out in three cell cul-
ture replicates and independently performed twice on
separate days.
Microscopy and immunofluorescent staining
Briefly, the bacteria were added to AGS cells grown on
glass coverslips at a MOI of 300:1. The cells were co-
incubated for 3 and 6 h and then fixed by 4% formalin.
Following blocking with 5% bovine serum albumin in
PBS for 30 min, the bacteria were stained with rabbit
anti-Helicobacter antibody (FITC, 1:200, ab30954,
ABCAM PLC, USA) for 1 h at room temperature. Sub-
sequently, the AGS cells were morphologically examined
using a fluorescent microscope (Olympus IX81, Olym-
pus, Japan) under a 40x objective.
RNA isolation, quality control and cDNA synthesis
Total RNA was isolated using RNeasy Mini (Qiagen
GmBH, Germany) according to the manufacturer's pro-
tocol. RNA concentration and quality were determined
using a NanoDrop ND-1000 spectrophotometer (Nano-
Drop Technologies, USA) and Agilent 2100 Bioanalyzer
(Agilent Technologies, USA). For real-time PCR, cDNA
was prepared using a First-Strand cDNA Synthesis Kit
(GE Healthcare, USA), according to standard protocol.
The Illumina TotalPrep RNA amplification Kit
(Ambion Inc., USA) was used to amplify RNA for hybri-
dization on Illumina BeadChips. To synthesize first
strand cDNA by reverse transcription, we used total
RNA from each sample collected above. Following the
second strand cDNA synthesis and cDNA purification
steps, the in vitro transcription to synthesize cRNA was
prepared overnight for 12 h.
Real-time PCR analysis
Each sample was tested in triplicate by real-time quanti-
tative PCR (rt-PCR) on the 7900HT Fast Real-Time
PCR system (Applied Biosystems). Expression of IL-8
was analyzed using custom IL-8 primer and probe (part
no: 4331348, assay ID: Hs00174103_m1, Applied Biosys-
tems). Mean cycle time (Ct) was calculated, and the
comparative Ct-method [84] was utilized to control for
background gene expression using reference gene
GADPH (part no: 4333764F, Applied Biosystems).
IL-8 protein was measured in the cell culture superna-
tant by the Quantikine Human CXCL8/IL-8 enzyme
linked immunosorbent assay (ELISA) kit, according to
manufacturer's instructions (R&D Systems, USA). The
test samples were not diluted. Serial dilutions of recom-
binant human IL-8 were used for standard curves. The
optical density of the wells was determined using a
microtitre plate reader (Varioskan, Thermo Scientific,
USA) set to a wavelength of 450 nm, with wavelength
correction set to 540 nm
cDNA oligonucleotide microarray analysis
The gene expression profiles were measured using Illu-
mina Human HT-12 v3 Expression BeadChip (Illumina,
USA), which enables genome-wide expression analysis
(48 800 transcripts, corresponding to approximately 37
800 genes) of 12 samples in parallel on a single microar-
ray. 35967 of the probes were designed using the RefSeq
(build 36.2, release 22) library and 12.837 probes were
derived from the UniGene (build 199) database [85,86].
Bioinformatics and statistics
R/BioConductor [87,88], with the package Beadarray
[82], were used for preprocessing of the microarray
text data from BeadStudio. Spatial artifacts were
removed using BASH [89] before the expression data
were log2-transformed and quantile normalized. Mod-
erated t-tests [90] were then performed for each probe
on the array to test whether the differential expression
between the starting point and the later time points
was significant. To account for multiple testing,
adjusted p-values were calculated by controlling the
false discovery rate (FDR), using the Benjamini-Hoch-
berg procedure [91]. The differential expression was
declared significant if the adjusted p-value (FDR q-
value) < 0.05. The analysis was performed using the R
statistical package [87] and the limma software package
from Bioconductor [88].
Eftang et al. BMC Microbiology 2012, 12:9
Page 12 of 15
Page 13
To produce a reasonable sized list of the most differ-
entially expressed genes, lesser expressed genes were fil-
tered out. A cutoff level at log2fold change (log2FC) >
1.5 was applied to the total genelist of 6237 significant
genes (Additional file 1: Table S1), producing a list of
the 245 most differentially expressed genes (Additional
file 2: Table S2). For the selected genes, all 6 corre-
sponding fold change values, including non-significant
values, were assigned to the genelist for hierarchical
clustering. Assuming that similarly expressed genes may
share some of the same biological functions, the goal of
hierarchical clustering is to group together genes with
similar expression. In a time course study, it is most
biologically relevant to cluster together genes that have
a similar expression pattern, rather than expression
magnitude. Consequently, the Pearson correlation coeffi-
cient was the appropriate distance measure in the clus-
tering of our results.
Data were imported into Multi Experiment Viewer v
4.6.0 (MeV) software [92] for hierarchical clustering,
and both non-clustered data and the clustered subsets
were entered into Onto-Express and Pathway Express
[93,94], part of the Onto-Tools software suite, for GO
and KEGG signal pathway analysis. Pathway Express cal-
culates an Impact Factor (IF) which is used to rank the
affected pathways, based on the FC and the number of
the involved genes, and the amount of perturbation of
downstream genes [95].
The microarray data are available under the accession
number E-MTAB-846 in the ArrayExpress database
Additional material
Additional file 1: Table S1. The list of genes that showed significant
differential expression at no less than 1 time point in H. pylori exposed
AGS cells (p < 0.05).
Additional file 2: Table S2. The list of genes that showed significant
log2fold change > 1.5 in H. pylori exposed AGS cells at no less than 1
time point (p < 0.05).
The Illumina service was provided by the Norwegian Microarray Consortium
(NMC) at the national technology platform, and supported by the functional
genomics program (FUGE) in the Research Council of Norway. We further
thank Torben Lüders and Bettina Kulle Andreassen at the Department of
Clinical Molecular Biology and Clara-Cecilie Gunther at the Norwegian
Computing Center for preprocessing of microarray data and statistical
assistance. Many thanks to Per Eftang and Soran Draghici for software
support and Armand Borovik at the Prince of Wales Hospital, Sydney, for
valuable comments. The University of Oslo financed the project.
Author details
1Department of Clinical Molecular Biology (Epigen), Institute of Clinical
Medicine, University of Oslo, Akershus University Hospital, Lørenskog,
Norway.2Department of Gastroenterological Surgery, Akershus University
Hospital, Lørenskog, Norway.3Department of Clinical Molecular Biology
(Epigen), Akershus University Hospital, Lørenskog, Norway.4Institute of
Clinical Medicine, Akershus University Hospital, University of Oslo, Lørenskog,
Norway.5Institute of Health and Society, University of Oslo, Oslo, Norway.
Authors' contributions
LLE, YE and TMT performed inoculation and co-incubation of cells and
bacteria, as well as performed ELISA and rt-PCR analysis. YE and TMT carried
out immunofluorescence and microscopy. IRKB participated in the design of
the study, and GB coordinated the study and helped to draft the
manuscript. LLE carried out the microarray data analysis and wrote the main
manuscript. All authors read and approved the final manuscript.
Received: 5 July 2011 Accepted: 17 January 2012
Published: 17 January 2012
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Expression of interleukin-8 correlates with vascularity in human gastric
carcinomas. Am J Pathol 1998, 152:93-100.
Bartels M, Schweda AT, Dreikhausen U, Frank R, Resch K, Beil W, et al:
Peptide-mediated disruption of NFkappaB/NRF interaction inhibits IL-8
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activates NF-kappa B in gastric epithelial cells. Gastroenterology 1997,
Shih YT, Wu DC, Liu CM, Yang YC, Chen IJ, Lo YC: San-Huang-Xie-Xin-Tang
inhibits Helicobacter pylori-induced inflammation in human gastric
epithelial AGS cells. J Ethnopharmacol 2007, 112:537-544.
Sharma SA, Tummuru MK, Blaser MJ, Kerr LD: Activation of IL-8 gene
expression by Helicobacter pylori is regulated by transcription factor
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Hisatsune J, Nakayama M, Isomoto H, Kurazono H, Mukaida N,
Mukhopadhyay AK, et al: Molecular characterization of Helicobacter pylori
VacA induction of IL-8 in U937 cells reveals a prominent role for
p38MAPK in activating transcription factor-2, cAMP response element
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Isomoto H, Moss J, Hirayama T: Pleiotropic actions of Helicobacter pylori
vacuolating cytotoxin, VacA. Tohoku J Exp Med 2010, 220:3-14.
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Zaidi SF, Ahmed K, Yamamoto T, Kondo T, Usmanghani K, Kadowaki M,
et al: Effect of resveratrol on Helicobacter pylori-induced interleukin-8
secretion, reactive oxygen species generation and morphological
changes in human gastric epithelial cells. Biol Pharm Bull 2009,
Chattopadhyay R, Bhattacharyya A, Crowe SE: Dual regulation by apurinic/
apyrimidinic endonuclease-1 inhibits gastric epithelial cell apoptosis
during Helicobacter pylori infection. Cancer Res 2010, 70:2799-2808.
Ding SZ, Fischer W, Kaparakis-Liaskos M, Liechti G, Merrell DS, Grant PA,
et al: Helicobacter pylori-induced histone modification, associated gene
expression in gastric epithelial cells, and its implication in pathogenesis.
PLoS One 2010, 5:e9875.
O'Hara AM, Bhattacharyya A, Mifflin RC, Smith MF, Ryan KA, Scott KG, et al:
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Ding SZ, Olekhnovich IN, Cover TL, Peek RM Jr, Smith MF Jr, Goldberg JB:
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Helicobacter pylori CagA activates NF-kappaB by targeting TAK1 for
TRAF6-mediated Lys 63 ubiquitination. EMBO Rep 2009, 10:1242-1249.
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Characterization of Helicobacter pylori PldA, a phospholipase with a role
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Schmittgen TD, Livak KJ: Analyzing real-time PCR data by the
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differential expression in microarray experiments. Stat Appl Genet Mol Biol
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Draghici S, Khatri P, Bhavsar P, Shah A, Krawetz SA, Tainsky MA: Onto-Tools,
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Cite this article as: Eftang et al.: Interleukin-8 is the single most up-
regulated gene in whole genome profiling of H. pylori exposed gastric
epithelial cells. BMC Microbiology 2012 12:9.
Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
Eftang et al. BMC Microbiology 2012, 12:9
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text:
Posts tagged "michelle williams"
Destiny's Child Reunites For Michelle Williams' Gospel Song "Say Yes"
Michelle Williams from Destiny's Child has a new single out and it's all about Jesus! Apparently she is super Christian and I had no idea. Anyway, both Beyoncé and Kelly Rowland appear on the gospel song and in the video above, although this isn't being billed as a Destiny's Child endeavor. Someone called in a…
Jessica Wakeman
June 18, 2014
New Louis Vuitton Ads Showcase Michelle Williams' Flawless Brows And Ability To Wear Any Lip Color
I've had last fall's Louis Vuitton ads starring Michelle Williams pinned on my eyebrow vision board for months now (What? You don't have a vision board dedicated to achieving your ideal brows?). I'm beyond thrilled that she's been tapped as the face of the brand's spring accessories campaign, if only because it give…
Winona Dimeo-Ediger
April 13, 2014
Queen Latifah, Michelle Williams & More Reflect On What Being A Queen Really Means
In a world where Black women struggle for honorable mentions and the accolades they deserve in Hollywood, there's one event that aims to make us all Queens for the evening. BET's annual BLACK GIRLS ROCK! event, dedicated to honoring exceptional women of color around the world who serve as a…
Hello Beautiful
November 9, 2013
Michelle Williams And Her Eyebrows Are The New Face Of Louis Vuitton
As a huge proponent of dark, bold eyebrows on blondes ("DARKER! DAARRRRKER!" -- me, to the woman who tints my naturally invisible brows), I am obsessed with these Louis Vuitton fall/winter ads starring Michelle Williams. I mean, the purse and the dress and the hair and the lips and the nail polish and the bone…
Winona Dimeo-Ediger
July 25, 2013
11 Child Stars Who Sought Emancipation
urns out Jaden Smith doesn't really want to be emancipated from his parents—but these 10 child stars, rounded up by the Huffington Post, did: Drew Barrymore petitioned for emancipation in 1991, when she was 15, in order to skirt child labor laws that were restricting the hours she could work. Alicia Silverstone did the same thing, at the same age, so…
June 2, 2013
Who Looked Best In Blue At Tiffany's Blue Book Ball?
Tiffany blue is one of the most famous colors in the world. So it's no surprise that Thursday night, as stars gathered at Tiffany & Co.'s Blue Book Ball at Rockefeller Center, there'd be a lot of blue on the red carpet. Gwyneth Paltrow wore a light blue Ralph Lauren number, while…
Julie Gerstein
April 19, 2013
The Pixie Cut Grow-Out Process Is Looking Cringeworthy On Michelle Williams
Michelle Williams or Kate Gosselin? Kate Gosselin or Michelle Williams? Either way, she's making a great case against getting a pixie cut if you ever intend on growing it out. This looks painful. [Photo: WENN]…
Rachel Krause
April 10, 2013
Taylor Swift Obviously Hates Her Fans & Michelle Williams Offends With Magazine Photo
Ooh dang, does Taylor Swift dump her fan mail without opening it? [Newser] Brad Goreski really, really wants to style Jennifer Aniston for her wedding -- here's what he has in mind for her big day look. [Your Tango] Chris Brown was photographed canoodling with someone and it ain't Rihanna. […
Amelia McDonell-Parry
March 14, 2013
Style Stealer: Michelle Williams Runs Errands In Style
The most stylish women in the world to me are those who look effortless no matter what they're wearing. "Oz The Great and Powerful" star Michelle Williams is one such woman -- she always manages to look cool and well put together, never sloppy. I'm seriously digging this outfit she wore to grab coffee in Brookly…
Amelia McDonell-Parry
March 5, 2013
Her 10 Best: Michelle Williams
Michelle Williams (who plays Glinda in the upcoming "Oz the Great and Powerful") is not only one of our favorite actresses -- hello, her performances in "Brokeback Mountain," "Blue Valentine," "My Week With Marilyn" are some of the best ever -- but she's also one we always watch for on the red carpet. What will…
Amelia McDonell-Parry
March 2, 2013
Rachel Weisz & Michelle Williams Hold Hands At "Oz" European Premiere
Rachel Weisz and Michelle Williams were the absolute cutest at the European premiere of "Oz: The Great And Powerful," held in London. The pair clasped hands for their requisite portraits -- I'd like to think that Weisz is counseling Williams through her breakup with Jason Segal. Oh also, Mila Kunis was there and…
Julie Gerstein
March 1, 2013
Style Stealer: Michelle Williams Is One Cool Cat
Fresh off her breakup with Jason Segel (so sad!), Michelle Williams stepped out in London looking like her usual lovely self in a classic khaki trenchcoat, cropped black pants, and wayfarer sunglasses. What makes her outfit extra special? The addition of those adorable Charlotte Olympia cat face loafers. Her feline footwear…
Winona Dimeo-Ediger
March 1, 2013
The Cast Of That New "Oz" Movie Is Pretty Good-Looking — Plus, Meet Their Characters!
Hey, check out the cast of "Oz the Great and Powerful" at the Russian premiere in Moscow! Mila Kunis looks gorgeous, and let me tell you, I am so relieved. For a second there I was concerned that Ashton had siphoned all the pretty out of her and was using it for hi…
Rachel Krause
February 27, 2013
Michelle Williams And Jason Segel Are Dunzo
Nooooo! Michelle Williams and Jason Segel have broken up. Boo! We liked them as a couple. [Us Weekly] Whoopsies! Miss Delaware Teen USA Melissa King has resigned after her porn tape surfaced. [Gawker] Kathy Bates is joining "American Horror Story" season three! Yay! [Entertainment Weekly] A former NYC restaurant…
Jessica Wakeman
February 26, 2013
Suck It, Haters! Destiny's Child's Michelle Williams Scores Reality Show
Ha! Michelle Williams is usually kind of mocked for being the, ahem, least important member of Destiny's Child (and her hilarious pop up on stage at the Super Bowl didn't help), but she's not letting it keep her down! She's scored her own reality show. [Uptown Magazine] Kristen Stewart was named the "least…
Amelia McDonell-Parry
February 20, 2013
Michelle Williams Grows Out Short Hair Just As Awkwardly As Everyone Else
See, even Michelle Williams has to deal with that awkward stage between having a pixie cut and… not having a pixie cut. It's pretty cool that she's choosing to wear the length as is (which is admittedly kind of Kate Gosselin-esque, no?) without going the extensions route, which she could so easily do. Don't…
Rachel Krause
February 20, 2013
Michelle Williams In Crazy Blue Eyeshadow & Red Lipstick
I couldn't place my finger on who Michelle Williams looks like on the cover of AnOther magazine until — damn! I got it! Mimi from "The Drew Carey Show." Only Michelle and her sweet face make electric blue eyeshadow look demonstrably less evil. [AnOther]…
Jessica Wakeman
February 18, 2013
Every Time Destiny's Child Is Photographed Together, An Angel Gets Its Wings
I can't help but feel like things are just a little more right with the world when Beyonce finds time to hang with her Destiny's Child sisters Kelly Rowland and Michelle Williams. If you listen closely, I'm pretty sure you can hear the sound of babies laughing, birds chirping, and, oh yeah, the cha-ching! of more money entering…
Amelia McDonell-Parry
February 7, 2013
Destiny's Child Releasing New Song, "Nuclear," On January 29
Destiny's Child is releasing new music! Beyoncé, Kelly Rowland and Michelle Williams are dropping a new track, "Nuclear," in a compilation of their music called Love Songs which comes out January 29. Anyone else pumped for the "Say My Name" remix? [Evil Beet Gossip, Telegraph UK] A new Tina Fey "Seven Minute…
Jessica Wakeman
January 11, 2013
Check Out The New Poster For "Oz: The Great And Powerful"!
There's no story like "The Wizard of Oz," but the upcoming film "Oz: The Great and Powerful" adds a new twist on the classic tale, telling the story of the Wizard himself, before Dorothy came skipping up the Yellow Brick Road. James Franco plays the future Wizard, while Michelle Williams, Mila Kunis and Rachel Weisz…
Amelia McDonell-Parry
December 21, 2012
Simply Irresistible
All »
6 Movies Based On A True Story That Left Out Important…
Over the years, we've poked fun at a lot of "based on a true story" movies that turned out to be bullshit. But then we thought that maybe we should stop giving Hollywood shit for embellishing history a bit if it lets them tell a slightly better story. So instead, we decided to give Hollywood…
March 31, 2015
Michael Jackson Pranked Russell Crowe For Years
As you slog your way through the myriad not-yet-April Fools' Day pranks that plague this nation, revel in this: no one has ever pranked someone better than Michael Jackson pranked Russell Crowe, and he did it for years. In an interview with The Guardian, Crowe revealed that despite having never met Jackson,…
Beejoli Shah
March 31, 2015
Trevor Noah Reminds Us Why You Should Delete Your Old,…
Oh No(ah)!
Amelia McDonell-Parry
March 31, 2015
Joe Manganiello Holds Baby, World Swoons
You know you needed this.
Claire Hannum
March 30, 2015
You'll Love
If These Style Blogger Poses Could Talk...
8 More Realistic Types Of Female Orgasms
6 Terrible Date Ideas Brought To You By Celebrity Guys

subset: commoncrawl


text:
Details about Nokia Lumia 2520 32GB WiFi + 4G Unlocked Black 10.1in Windows Tablet PC ComputerSee original listing
Item condition:
New other (see details)
"This is a brand new item. However the box has previously been opened to check the contents."
27 Aug, 2014 16:24:33 BST
Item location:
Colchester, Essex, United Kingdom
|New other (see details)
A new, unused item with absolutely no signs of wear. The item may be missing its original packaging, or the original packaging has been opened or is no longer sealed. The item may be a factory second, or a new and unused item with minor defects. See the seller's listing for full details and description of any imperfections. See all condition definitions- opens in a new window or tab
|Seller notes:
|"This is a brand new item. However the box has previously been opened to check the contents."
|Product Line:
|Storage Capacity:
|Screen Size:
|10.1in (25.65 cm)
|Internet Connectivity:
|Wi-Fi + 4G
|Operating System:
|Windows RT
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After receiving the item, cancel the purchase within
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14 days
Buyer pays return postage
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If you need to return an item to us, please talk to our Customer Service team available Monday - Friday 9am to 4:30pm on 01206 851722 or at firstname.lastname@example.com, please ensure you have the eBay item number from the listing so we can assist you more readily. We warrant to you that the goods will materially conform with the descriptions given in the entire listing and that we agree and are able to sell the goods to you. Goods purchased via auction may only be returned for mis-description if you notify us by email within 14 days of receiving the goods setting out why the goods are mis-described. Returns must be pre-agreed in writing. Unless listed as such, our items do not have a manufacturer warranty or warranty offered by XS Items. Fixed price/buy it now returns: Goods purchased via fixed price listings may be returned if you notify us by email within 14 days of receiving the goods and return them within 14 days together with any packaging provided. We require that you inform us of the return in email/writing. Returns must be pre-agreed. Unwanted items: If the item is as described, the purchaser may be responsible for the cost of returning the goods to us for a unit cost refund. If an item is delivered in its original packaging, it will need to be returned in the same packaging and condition - please do not dispose of it as we cannot accept returns that are not returned in this way. Collected items: Items that are collected cannot be returned once removed from the premises - please inspect them thoroughly before taking away.Please ask if you wish to inspect the items before taking them away. We do have facilities to power items up if required. If you have purchased a new item and it develops a fault more than 28 days after purchase, please contact the manufacturer for repair/replacement via their warranty. Unless otherwise stated, items are in full working order Business Address: XS Items Ltd, Centenary House, 7 Crown Gate, Wyncolls Road, Colchester, Essex, CO4 9HZ.
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By Synergenics, LLC
Open iTunes to buy and download apps.
Please note: This app is for licensed Amateur Radio operators only. See www.echolink.org for more information.
EchoLink for iOS provides access to the EchoLink network for validated EchoLink users. You can use this app to connect to the EchoLink system from almost anywhere, using an iPhone, iPad, or iPod touch.
If you are not already a validated EchoLink user, you will be required to register (using your Amateur Radio callsign and a password of your choice) the first time you run the app. Please see the iPhone FAQ at www.echolink.org.
If you are interested in becoming a licensed Amateur Radio operator, see www.arrl.org for more information.
EchoLink is a registered U.S. trademark of Synergenics, LLC.
What's New in Version 2.1
This version includes feature suggestions and bug fixes from Version 2.0.
Customer Reviews
Can't Reliably Connect - A Step Backwards
This version is unusable. The previous version wasn't as fancy in the functionality and interface department, but it worked quite well. This version provides only constantly intermittent, broken audio. It's nice that they added all those other features, but the fundamental reason for using the program, Echolink access, doesn't work at all. At first I thought that there might be a problem with the new version and my satellite Internet connection, but I had the same problem on a cable broad band connection. I would recommend that the developer revert to the previous version until the bugs are worked out in this version. I don't think it's possible for this version to have been properly beta tested.
Texting problem ver. 2.1 on Ipad Air...still a problem!
No carriage return and line feed in 2.0 and 2.1 ..also in landscape mode keyboard covers up new text.
Please fix for 5 stars
Still a problem with linefeeds in new version 2.1..have to manually scroll up after page gets filled to bottom to continue getting new text..please fix!
Won't stay connected to proxies
I don't know what happen with this last update but EchoLink just refuses to stay connected to proxies on the Apple IPhones.
It seems like you take two steps forward and then one step back. I am hoping this issue that's effecting so many Hams right now is fixed here very,very soon.
- Free
- Category: Lifestyle
- Updated: Dec 08, 2014
- Version: 2.1
- Size: 10.2 MB
- Language: English
- Seller: Synergenics, LLC
- © 2002-2014 Synergenics, LLC
Compatibility: Requires iOS 7.0 or later. Compatible with iPhone, iPad, and iPod touch. This app is optimized for iPhone 5, iPhone 6, and iPhone 6 Plus.


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- Shylo Shy's real hot big, big dick X-Tube solo show for y'all. Video should be about 8 minutes and it is correct speed as uploaded. But this one seems to lack click to advance so you can't skip parts of it.
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Beulah Beacon News
Yields up, protein down as harvest continues in decreasing autumn
The wet spring and summer is paying dividends for farmers as small grain yields around the area are excellent. But that same cool wet weather could be costing some money as protein levels are low.
Posted 9/23/09 (Wed) read more »
County public health flu immunizations localized to Beulah
Changes have been made to the usual routine for flu shots provided by Mercer County Public Health offices, a Custer Health District service. The Beulah Civic Center will be the only location of the public health Seasonal Immunization Clinic that in past years traveled to each community in the county.
Posted 9/23/09 (Wed) read more »
Property line problem moving toward an estimated $12,000 survey solution
Survey errors that prevented a home sale on Beulah's western edge should be cleared up soon as the affected landowners have agreed to all chip in to replat the Moens Parcel Addition subdivision, which borders the west side of State Highway 49.
Posted 9/16/09 (Wed) read more »
Work begins on water pipeline
Motorists taking State Highway 200 past Beulah may have noticed a large stockpile of pipes sitting near the intersection with State Highway 49. They might have also noticed more pipe sitting in the ditches as they headed west to Zap. All that pipe is finally going to be installed.
Posted 9/16/09 (Wed) read more »
Residents bring drainage issues to county
Throughout 10 years of relatively little moisture, water flowed mostly where it was supposed to flow. Huge amounts of snowmelt this past spring, however, exposed drainage problem areas that must be remedied before another flood event occurs – and two Beulah residents visited the Mercer County Commission last Wednesday to express a few of their concerns.
Posted 9/09/09 (Wed) read more »
Wanted – warm, dry sunny days
An extended stretch of warm, dry days would be a big help to area farmers, Craig Askim, Mercer County Extension Agent said. Cool damp evenings have pushed an already late crop even further behind.
Posted 9/09/09 (Wed) read more »
Hoffner tapped to head banker's group
Gordon H. Hoffner, president of the Union Bank of Beulah has been selected to be president of the Independent Community Banks of North Dakota.
Posted 9/02/09 (Wed) read more »
A different agenda for the Farmers Union
Every year in September, members of the North Dakota Farmers Union participate in an annual fly-in to Washington, D.C., to lobby members of Congress. Most years, the agenda is usually directly related to agricultural issues such as the farm bill or disaster assistance. This year, other issues are overshadowing the group's traditional agenda.
Posted 9/02/09 (Wed) read more »
Lightning shatters Golden Valley farm home and barn
Jim Erdman was just getting up. His wife Gayle already had a cup of coffee and was watching television. It seemed like a normal day at the farmhouse a mile east of Golden Valley.
Posted 8/26/09 (Wed) read more »
Setting a high bar – no dropouts
Beulah, like many schools in North Dakota, has an impressive record of graduating its seniors so they are equipped to pursue further education or enter the workforce. But the school district could still do even better, said Superintendent Rob Lech.
Posted 8/26/09 (Wed) read more »


text:
but the 5200 I have is a PPPoA (not pppoe) modem
Also, if I removed the user name and password, wouldn't Frontier ISP disallow my connection to the internet?
You should be able to put the routers ip address in the speedstreams dmz or use the speedstreams ip passthru for the router
Does the DMZ do the same thing as turning off the 5200's firewall?
When you say "ip passthru", do you mean "Static Route" (see pic)?
When the 5200 says "the address", does it mean that my Belkin router is not using the WAN address that the modem sees?
Does the "Lease time" mean the IP passthru will be abandoned after 10 minutes?
--from the ip passthru screenI elect to use the 192.168.254.1 device (my wifi router).Upon hitting the 'Apply' button the message in red print (see above) shows.At that point I set my wifi router to a 'Dynamic' WAN connection, then reboot the wifi router
The router's wan connection should be set to dynamic when you first connect it to the speedstream
I hope Frontiernet ISP is not doing anything to block FTP servers
I tried the DMZ option and it too made no difference. I hope Frontiernet ISP is not doing anything to block FTP servers. I can access the Frontiernet FTP server where I keep some backup files, but the FTP server I am trying to host here behind the 5200 will respond from a WAN connection.
quote:The World Wide Web and FTPThe web space and public FTP space included with a dialup or DSL account may not be resold or used for adult-oriented material. We reserve the right to require that sites using such Web or FTP space which receive high amounts of traffic be moved to other servers. Web pages and FTP files may not contain any material, text, or images, whether hosted on our servers or "transclusioned" (images from another site displayed on the page) which violate or infringe any copyright, trademark, patent, statutory, common law, or proprietary rights of others. Web pages and FTP files may not contain links that initiate downloads of copyright-infringing or other illegal material. Those who believe users of our services are infringing their copyrights must submit their complaints in writing to our Designated Agent to Receive Notifications of Claimed Infringement, Gregg Sayre, email@example.com, Frontier, 180 S. Clinton St., Rochester, NY 14646, 585-777-7270, 585-263-9986 fax.Servers, Proxies, and NetworksUsers may not run any program which makes a service or resource available to others, including but not limited to port redirectors, proxy servers, chat servers, MUDs, file servers, and IRC bots. Users may not run such programs on their own machines to make such services or resources available to others through one of our dialup or DSL accounts; a dedicated access account is required for such purposes. Customers are responsible for the security of their own networks and machines. We will assume neither responsibility nor accountability for failures or breach of customer-imposed protective measures, whether implied or actual. Abuse that occurs as a result of a compromised customer's system or account may result in suspension of services or account access.


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Welcome to a free stock market game from MarketWatch.
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News & Tools
- Live scores and win probabilities of every NCAA tournament game
- Japan kicks its 'loophole herb' drug habit
- Health care, financial stocks looking best going into tough earnings season
- When firing your financial adviser is best for your retirement
- 3 reasons to use a Roth IRA to save for college
- It's all love for Elton John in Massachusetts
- 6 things your tax preparer doesn't want you to know
- Indiana governor defends 'religious freedom' law
Editors' Picks
Stocks to WatchDuPont, Nike, KB Home are stocks to watch
Among the companies whose shares are expected to see active trade in Friday's session are DuPont, Nike, and KB Home.
The StockpickersIliad to forge ahead with T-Mobile bid
Collapse of Sprint offer may strengthen Paris telecoms operator's hand.
The Ratings GameKraft-Heinz deal seen as expensive
In Kraft, 3G Capital Partners is buying a business that has missed Wall Street's consensus on sales in eight of the last 10 quarters and is losing market share in key categories.


text:
The Stream
Can Someone Please Explain Why Californication is Still on the Air?
- Tags:
- Californication,
- Criticism,
- David Duchovny,
- Showtime
The Q
"Very Nice to Meet You. I've Heard the Wu-Tang Clan Ain't Nuthin' to F*&!# Wit'"
- Tags:
- ?uestlove,
- Amy Poehler,
- Parks and Recreation,
- Rza,
- Wu-Tang
News + Politics
- The Movie That Inspired This Season's Look: Jaws
- Stampd is What You Love About New York, Paris, and the Beach
- The Hill-Side Wants to Bring High-Quality Fabrics to the Gap
- NSF Destroys Your Clothes Before You Even Buy Them (And That's Good)
- Designer David Hart Took a Sewing Class to Meet Girls, the Rest is History
- Stampd is What You Love About New York, Paris, and the Beach
How To
Food + Travel
- David Chang's Kitchen: Don't Get All "Fresh" with Me
- David Chang's Kitchen: Your Idea of Bologna is Total Baloney
- Chart: The Family Tree of Bourbon Whiskey
- Recipe: Semi-Summer Salad - Chef Jeff Taibe of Oak + Almond
- The Great Franco-Italian Pasta Experiment
- How to Pick from an Overpriced Restaurant Wine List


text:
The Top 10 In Action Sports
01Welcome to Qasr Al-Farid: The Rock With A Door That Will Rock Your World
0218 Things That Will Give Anyone Who Did Duke Of Edinburgh A Serious Flashback
0316 Things That Will Give 90s Skater Kids A Serious Flashback
04This 10 Year Old Kid Just Nailed A Dub Cork 1080... On His Second Attempt
05The 10 Most Spectacular Road Trips on Earth
06Water Slides, Plane Wings and Danny MacAskill: This is Road Cycling Like Never Before...
0724 Reasons Why Snowboarding Was Better In The Early Noughties
08Could Scientists Be About to Build a Terminator? They're Closer Than You Think…
09Win a Holiday for 2 in Chamonix with Three #holidayspam
1017 Holiday Beer Photos That Every Skier Takes to Make Their Friends Jealous
Almost Live - Episode 5 - All Stomps Kicker
Behind the scenes from the filming of TGR's new film, in the making, Re: Session. Nestled deep in the heart of the San Juan Mountain Range lies the sleepy town of Silverton,
CO. The posse consisted of athletes Dash Longe, Dylan Hood, Wiley Miller, still photographer Adam Clark and TGR cinematographers Dustin Handley and Josh Nielson. The crew lucked out with six days of blue bird sky's, warm weather, great terrain, bomber snow pack, and very nice snow. The Boys built up a kicker and stomped everything they threw down... impressive! For more check out TetonGravity.com
What Do You Think?
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"Food52, a blog that shares tons of cooking tips, also runs a well-curated Instagram account full of simple food snapshots from the staff." - Chelsea Stark
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Quiz: Remember the definition of mal de mer?
late 14c., "praise, present as worthy," from Medieval Latin recommendare, from Latin re-, here probably an intensive prefix, or else from a sense now obscure (see re-), + commendare "commit to one's care, commend" (see commend). Meaning "advise as to action, urge (that something be done)" is from 1746. Related: Recommended; recommending.