Slide 1
Helicobacter pyloriCommensal or Pathogen?
Vietnam 2004
Barry MarshallClinical Professor of Microbiology UWA
Slide 2
Marshall & Warren, Perth 1983Marshall & Warren, Perth 1983
It was very difficult for gastroenterologists and physicians to accept the
proposal of these two investigators, Marshall and Warren, who stated in 1983
that spiral bacteria in the human stomach were likely to be the cause of ulcers
and stomach cancer.
Established gastroenterologists and scientists found it hard to accept the
teachings of the two Australians! Instead, most gastroenterologists of the
time assumed that Helicobacter were commensals which merely colonized
people with ulcers or gastritis
Slide 3
Oct-04 © BJM
First Attempt at Publication: 1983
Score = bottom 20%
Dear Dr. Marshall,
I regret that your research paper was not accepted for presentation...
The number of abstracts we receive continues to increase and for this meeting 67 were submitted and we could only accept 56.
As shown here, our discovery was not “appreciated” by Australian
gastroenterologists (read text from slide). Our discovery was rated in the
lowest 20% and was not accepted even for a poster!
I only show this slide to remind young investigators that they should always
keep their rejection letters. Although it is too painful to read them now, you
may be proved correct in the future and then you can proudly display them.
Slide 4
Oct-04 © BJM
An attempt to Fulfill Koch’s Postulates for Campylobacter pyloridis
Med J. Aust 1984
Because of this rejection, in 1984 it was necessary for me to drink a culture of
Helicobacter pylori to prove to the skeptics that Hp could cause gastritis and
was not just a commensal present in people with ulcers or stomach cancer.
This was published in the Medical Journal of Australia and in an editorial for
The Lancet. Many gastroenterologists took notice but were determined to
prove me wrong! However, it stimulated much of the successful research
which we have seen presented since that time.
Slide 5
Gastric Mucosa, Silver Stain, Day 8
The self infection in 1984 fulfilled Koch’s postulates for H.pylori, proving that it
caused gastritis. This shows a silver stain of the gastric mucosa with the
acute infection. The black helical organisms are seen on damaged (brown)
epithelial cells. Note that the cells do not have the normal transparent mucus
content usually seen in the gastric epithelial layer.
Slide 6
Oct-04 © BJM
Repels waterdue to phospholipid
DamagedBy Hp
This high power view reminds us that the gastric wall has a mucus layer to
keep the acid from touching the mucosa and digesting it.
This diagram shows how the mucus barrier works. Here you see the mucus
above in green and the cells below. In the lumen of the stomach is the acid
represented by the H+. The surface of the mucus layer can actually repel
water and acid. This enables the cells below to remain healthy because even
though the stomach acid is strong, with pH of 2.0, the pH down on the cells
(below the mucus barrier) is about 6.0.
In addition, a greasy component of the mucus gel (hydrophobic phospholipid)
tends to repel water and acid from it.
H.Pylori damages this barrier in two ways. Directly by its phospholipase and
proteases which make the mucus layer less repellant to water and acid.
Indirectly by causing mucus cells to separate, become loosened from each
other, stimulating the inflammatory response causing cells from below the
mucosa to migrate through the epithelium.
Slide 7
Oct-04 © BJM
Epidemiology and Disease Associations of H.pylori
O ct-04 c BJM
H. pylori+H. pylori+
duodenal ulcerduodenal ulcer
gastric ulcergastric ulcer
gastric cancergastric cancer
gastric lymphomagastric lymphoma
Western (USA)30% Hp +
O ct-04 c BJM
H. pylori+H. pylori+
duodenal ulcerduodenal ulcer
gastric ulcergastric ulcer
gastric cancergastric cancer
gastric lymphomagastric lymphoma
Developing (Vietnam)60% Hp +
Epidemiology and Disease Associations of H.pylori Many studies have shown variants on the association with peptic ulcer, gastric
cancer, and gastric lymphoma. In any developed country, about 20-30% of
the population are infected with Helicobacter pylori (H. pylori). It is within this
group that peptic ulcer disease develops. The great majority of duodenal
ulcers and gastric ulcers are in the infected patients shown as the red inner
circle.
Although the majority of persons with H. pylori are asymptomatic at any point
in time, many of them eventually develop disease. In prospective studies, the
conversion rate to active peptic ulcer is about 1% per annum. Thus, during a
lifetime, about one third of infected persons develop symptomatic disease. In
addition, persons with H.pylori have a 1%-5% chance of developing gastric
cancer in their lifetime.
In Vietnam, with a population fo 80 million people, there are probably 40
million with Hp.
The fact that so many people with Hp never actually develop disease has
been reason for continuing controversy about Helicobacter.
Perhaps Hp is not always so bad. Actually there are several factors which
affect the ultimate outcome of Hp infection, some in the organism, some in the
host, and some in the environment.
Slide 8
Oct-04 © BJM
In many cases of H.pylori infection, the mucosa appears normal or only slightly irregular.
Antral “gooseflesh”also called “chicken skin”
The association between H.pylori and peptic ulcer is well known, but in many
persons the endoscopy is normal. Peptic ulcer disease is a condition
characterised by remissions and relapses so that the ulcer crater is not
always present at endoscopy. One visible lesion found to be the best
predictor of gastritis is the cobblestone, “gooseflesh” or “chicken skin”
appearance of the antral mucosa. Other appearances such as redness
remain for many years after treatment of the gastritis so they cannot be used
as indicators of active Helicobacter infection.
Slide 9
Oct-04 © BJM
All persons with hp have some degree of chronic gastritis and this becomes more severe and extensive in the stomach with time.
Chronic Gastritis.
Gastritis is always present in persons with H.pylori. Before discussing variations in the pathogenicity of Hp, I wish to emphasize
that Gastritis is always present in persons with Hp. Here is the histology characteristic of Hp showing infiltration of the gastric
mucosa with chronic inflammation – lymphocytes macrophages and plasma
cells. These cells are making IgG antibody which allows serological detection
to work quite well. Also there is acute inflammation present with scattered
neutrophils.
Even though some Hp are worse than others, ALL PERSONS with Hp HAVE
SOME DEGREE OF CHRONIC GASTRITIS AND THIS BECOMES MORE
SEVERE AND EXTENSIVE IN THE STOMACH WITH TIME.
Slide 10
Oct-04 © BJM
Mortality from Gastric Cancer by Country
Ferlay J, Bray F, Pisani P, Markin D. 2001. GLOBOCAN 2000: Cancer Incidence, Mortality and Prevalence Worldwide. V. 1.0. Lyon: IARCPress.
Cancer Incidence and Mortality The countries are listed sorted both by cancer mortality and by cancer
incidence. Note that in Japan, incidence is far greater than mortality. This is
because effective screening programs detect gastric cancer at an early stage
and cure is possible.
Although high gastric cancer usually correlates with H. pylori prevalence,
there are some notable exceptions. UAR and Kuwait are known to have a 50-
70% prevalence of H.pylori, but have rather low cancer mortality. Kenya is
the representative African country with a rather low gastric cancer rate but a
figh H. pylori prevalence. This paradox, referred to as the “African enigma” by
some, demonstrates that cancer causation from H. pylori must be modulated
by many factors. These could be dietary, ethnic and bacterial strain related.
Slide 11
Oct-04 © BJM
Variation in Pathogenicity of Hp
All Hp cause gastritisGastritis is worse if CagA pathogenicity island is presentCagA has various pathogenic potentials
• East Asian (Japan - severe)• Asian (Vietnam - mixed)• Western (mild)
But the pathogenicity of Hp varies according to the presence and type of
CagA toxin.
Slide 12
Oct-04 © BJM
Distribution of the Diversity of the CagA Protein in Japan
015354Gastric cancer
6117087Chronic gastritis
cagA (-)WesternEast Asian
n
Azuma T, Ohtani M, Yamazaki Y, Higashi H, Hatakeyama M. Meta-analysis of the relationship between CagA seropositivity and gastric cancer. Gastroenterology 2004;126(7):1926-7.
Distribution of the Diversity of the CagA Protein in Japan Note that in patients with gastric cancer in Japan, almost all will have the East
Asian type of CagA. (Azuma T, Ohtani M, Yamazaki Y, Higashi H,
Hatakeyama M. Meta-analysis of the relationship between CagA seropositivity
and gastric cancer. Gastroenterology 2004;126(7):1926-7.)
Slide 13
Oct-04 © BJM
36.7105Korea24.56020China12.83010VietnamAsia3.31913Thailand3.8330India
13.0730IrelandThe West14.1210Austria
27.8410Italy17.6610England6.1560USA8.6430Australia
CagA type Mortality rate of gastric cancer (/100,00males in 2000)WesternEast AsianCountry
58.3912123JapanEast Asia
The Distribution of CagA Protein Diversity Worldwide
Type of CagA and the Risk of Cancer Azuma and co-workers suggest that cancer rates are related to the CagA type
predominating in each country.
This table is preliminary data and hasnot been confirmed yet. It was just a
letter to a journal. The number of strains tested from many countries is quite
low.
However, the bad news is that strains from Vietnam appear to be very similar
to Japanese strains – East Asian Type in the majority. This means that they
have quite a strong cancer potential.
Contrast this with Thailand where Westen style CagA strains are more
common and cancer incidence is lower.
(Azuma T, Ohtani M, Yamazaki Y, Higashi H, Hatakeyama M. Meta-analysis
of the relationship between CagA seropositivity and gastric cancer.
Gastroenterology 2004;126(7):1926-7)
Slide 14
Oct-04 © BJM
Location of Location of H. pyloriH. pyloripH=2
pH=6
aerobic
micro-aerophilic
anaerobic
Oxygen +++Oxygen +++
No OxygenNo Oxygen
bacteria are stuck herecannot move away}
Microaerophilic metabolism directs H. H. pyloripylori away from the lumen, towards the
mucosa
Hp is unique – almost a commensal This cartoon emphasizes the location of Helicobacter beneath the gastric
mucus layer, attached to the stomach mucus cells. The bacteria actually
avoid the very acidic stomach contents (at top) where the pH is 2.0, instead,
they stay at the bottom of the mucus layer.
But Helicobacter do not survive where oxygen levels are high such as in the
tissues. Therefore they remain half way between the oxygenated tissue and
the low oxygen environment of the stomach contents. This means that
Helicobacter can never become invasive like other pathogens.
This location creates problems for the immune system which cannot eradicate
gastric bacteria. If the immune response is too aggressive, the mucosal
barrier will break and ulceration will occur.
There is some evidence that Hp can down-regulate the immune system, thus
allowing it to persist.
Slide 15
Oct-04 © BJM
Virulence FactorsFactor Function DistributionUrease Buffers stomach acid All strainsFlagella Motility All strainsNAP Neutrophil activation All strainsBabA Adhesion for Leb Prevalent on
type I strainsLPS Low toxicity All strainsLewisx,y antigens Molecular mimicry Some strainsIceA Homolog of Nla III restriction Some strains
endonucleaseVacA Cytotoxicity (two alleles) All strainscag PAI 31 genes coding for type IV Type I strains
secretion systemCagA Immunodominant antigen Type I strains (part of cag PAI)PicB Equivalent to CagE Type I
Covacci et al, Science 1999 284: 1328-1333
Virulence Factors of H.pylori In-vitro studies using cultured epithelial cells have identified many factors
secreted by H.pylori which cause pathologic changes. Most of these factors
are present in the majority of Hp strains, but they may vary in their
pathogenicity.
Urease is essential for colonization because it protects from acid allowing the
bacterium to survive long enough to reach the mucosa.
Nap increases inflammation by attracting neutrophils.
BabA, is an adhesin responsible for attaching Hp to the epithelial cells. If the
adhesion is strong, then the other toxins have far more effect.
Lewis antigens affect the ability of Hp to down-regulate the immune response
perhaps by mimicing carbohydrates in the mucosa.
VacA and CagA, I will discuss in detail below. CagA toxin is part of the “Cag
pathogenicity island” also refered to as the “CagPAI”.
PicB is a component of the CagPAI which is an ATPase responsible for
driving the CagPAI machinery. It causes release of IL-8 and attracts
neutrophils.
Slide 16
Oct-04 © BJM
Secretion Systems
Covacci et al, Science 1999 284: 1328-1333
CagPAIis Type IV
Secretion Systems CagA is a toxin associated with the Cag pathogenicity Island (CagPAI).
Bacteria have numerous secretion systems. Type IV secretion refers to the
ability of some bacteria to create a “molecular syringe”, which is a modified
flagellum or pilus structure, through which bacterial proteins and even genes
can be injected into a hoist cell.
The prototype of this mechanism is the Type IV system of Agrobacterium
which is extensively used to create new GM varieties of plants such as
Vitamin enriched rice etc.
Slide 17
Oct-04 © BJM
Type IV Secretion Homologues
S
B9B9
D4
NTPNTP
B4
NTPNTP
B6
B8
B11
NTPNTP
B10
B3
B 7
S
B 7
S
S
B5 B5
Covacci et al, Science 1999 284: 1328-1333
B2B2
Escherich
ia coli
Agrobacteriu tumefacie
ns
Brucella suis
Bordetella pertussi
s
Helicobacte
r pylo
ri
Rickettsi
a prowazekii
Legionella pneumophila
D4
G
F
O
N
D
Eex
KorA
M
L
C
B
A
D4 D4
DotBB11 292B11 525H
B10 291 IcME527B10 G
B9 B9 F 528 290
B8 B8E E 287
B7 IB7 CagT
B6 DB6
B5 B5
B4 B4 C CagE 103
B3 B3 B
B2 AB2
B1 B1
tra(pKM101)
vir vir ptl cag RP ORF
Dot/icm
B1
core
subu
nits
Transfer of secreted components across the bacterial envelope.
Genes present in Hp and various other Type IV systems
Slide 18
Oct-04 © BJM
urea
vacuoles
Action of vacA Toxin
VacA Toxin: VacA toxin is present in all Helicobacters. But when the CagA pathogenicity
island is present, then a more virulent form of VacA is present.
VacA is secreted from the Hp bacterium where, in the presence of low pH, it
polymerizes to form a membrane channel. It inserts into the epithelial cell
membrane and creates a pore through which anions can pass. The cell
becomes leaky, perhaps to the advantage of the attached Hp organism. The
channels also insert into intracellular organelles causing vacuoles to form.
Slide 19
Oct-04 © BJM
Redundancy in the Genetic Code
• CGTA in triplets• 64 combinations
for 20 AA’s• Mutations in third
codon may be “synonymous”
mRNA codes
The Molecular epidemiology of Hp The Molecular epidemiology of Hp can be studied as shown here. The
principle relies on the fact that there is redundency in the genetic code. This
slide shows the genetic codes which can form each amino acid. Notice that
changes in the third base of a codon often does not change the amino acid.
So, taking the case of Glycine, mutations affecting the third base, which can
be U, C, A or G, all code for Glycine. Thus mutations here do not affect the
Hp bacterium and go unnoticed in evolution. However, they can be counted
to measure the number of years since strains have diverged from one
another.
Slide 20
Oct-04 © BJM
Hp and Human Migrations
Covacci et al, Science 1999 284: 1328-1333
10 – 40 00012 000
40 000
60 – 70 00060 – 100 000
40 000
Human and Helicobacter Migrations If Hp was merely a pathogen, we might find that it was only recently acquired
by humans. However, if Hp was a commensal, then we might expect that Hp
has evolved with man for millions of years, as has the rest of the intestinal
flora.
By studying the sequence variations of CagA, VacA and various other
essential “housekeeping” genes (present in all Hp strains), several groups
have mapped the global migration of Hp in parallel to the known human
migrations.
This world map indicates the direction of human migrations (arrows) and time
range (years since migrations happened), as taken from Cavalli-Sforza (47)
and Diamond (48), The geographic centers of the major Hp genotypes known
today are indicated by concentric circles of different colors. It appears that Hp
followed man during the migrations indicated by the arrows, giving rise to the
present genotype distribution (indicated by circles).
Light green areas indicate the locations where the development of agriculture
and animal breeding was initially started, resulting in the expansion of the
initial human populations (46-48).
So far, it appears that Hp has followed humans for at least 50,000 years.
Thus, perhaps Hp resembles a commensal in some ways.
Slide 21
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Pathophysiology of Gastritis, Ulcers and Cancer
Saurbaum michetti
Natural History of Helicobacter pylori Infection.
H. pylori is usually acquired in childhood. Acute H. pylori infection causes transient
hypochlorhydria and is rarely diagnosed.
Chronic gastritis will develop in virtually all persistently colonized persons, but 80 to
90 percent will never have symptoms.
The further clinical course is highly variable and depends on bacterial and host
factors. Patients with higher acid output are likely to have antral predominant gastritis,
which predisposes them to duodenal ulcers. Patients with lower acid output are more
likely to have gastritis in the body of the stomach, which predisposes them to gastric
ulcer and can initiate a sequence of events that, in rare cases, leads to gastric
carcinoma.
H. pylori infection induces the formation of mucosa -associated lymphoid tissue
(MALT) in the gastric mucosa.
Malignant lymphoma arising from such acquired MALT is another rare complication
of H. pylori infection.
Slide 22
Oct-04 © BJM
No cancer in patients with DU
Helicobacter pylori infection and the development of gastric cancer.Uemura N, Okamoto S, Yamamoto S, et al. N Engl J Med 2001;345:784-789.
By itself, H.pylori rarely causes cancer. Cancer only occurs in long term infection where acid secretion is suppressed or removed by atrophic gastritis
HP+, >3% ca. in 12 yrs
Here is an important paradox about H.pylori and Cancer Perhaps there is some reason for a little optimism about Hp. Helicobacter is
said to be a risk factor for stomach cancer. But this important observation has
been largely overlooked. Stomach cancer rates for persons with a history of
duodenal ulcer are normal, or even lower than normal.
In this prospective study reported in the New England Journal of Medicine,
Uemura performed surveillance endoscopy on patients with Hp for up to 12
years. He observed that about 4% developed stomach cancer during that
time.
But there was no stomach cancer in patients who had duodenal ulcer, even
though duodenal ulcer is associated with the more harmful, toxin producing,
strains of Helicobacter.
Thus, by itself, Helicobacter does not always cause cancer. Cancer probably
only occurs in long term infection where acid secretion is suppressed or
removed by atrophic gastritis.
When stomach acid is normal or high, as it is in duodenal ulcer patients and
most persons with Helicobacter, cancer risk is actually low. This observation
has also been made in seroepidemiologic studies in California (Parsonnet
NEJM) and Hawaii (Nomura NEJM).
Slide 23
Oct-04 © BJM
9/97
0102030405060708090
100
0 10 20 30 40 50 60
age
% in
fect
edDevelopingWestern
Epidemiology of H. pylori - Western vs. Developing
Whole of life
The upper graph shows that even in a Western country, older persons have a high prevalence of H. pylori because they acquired the infection in childhood. The lower graph illustrates a developing country where the infection rate is
20% per annum and the loss of the infection rate is 3% per annum.
Vietnam is in transition so we expect to see the pattern change over the next
generation.
Things which will decrease the new Hp cases are smaller families and
improved public health measures, particularly drinking water quality.
Slide 24
Oct-04 © BJM
Population Demographics: VietnamAge structure: – 0-14 years: 29.4%– 15-64 years: 65%– 65 years and over: 5.6%
Median age: – total: 24.9 years Life expectancy at birth: – male: 67.86 years – female: 73.02 years (2004 est.)
Demographics
As you know even more than I do, Vietnam has a young population so most
people do not yet experience a cancer risk from Hp. As shown here, in 2004,
only 5.6% of the population is above the age of 65 years. The average age is
25 years.
Slide 25
Oct-04 © BJM
Age related incidence and mortality from gastric cancer for Japanese males
Ferlay J, Bray F, Pisani P, Markin D. 2001. GLOBOCAN 2000: Cancer Incidence, Mortality and Prevalence Worldwide. V. 1.0. Lyon: IARCPress.
As lifespan increasesin Vietnam, cancer risk
is more important
Gastric cancer and Age – Data from Japan More than 2800 new cases of stomach cancer occur in persons under the age
of 44 year but most (10,000 or so) are in persons above the age of 60.
As the population ages, and lifespan increases, gastric cancer will become
more and more important. That is why Hp needs attention now.
Slide 26
Oct-04 © BJM
End
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