Lecture 31 Helicobacter pylori
Reading Assignments: (1) Text Chapters 22, 57 (pp. 523-524) (2) Handout: The Digestive System: Plate 85, In Kapit, W. and L.M. Elson. 1977. The Anatomy Coloring Book, Harper and Row Publishers, New York.
Summary:
1. A Primer on the Anatomy of the Stomach and Gastric Disease
a. Gross Anatomy of the Stomach
1. Fundus
2. Body – parietal cells (HCL and intrinsic factor) and chief cell
(secrete pepsinogen)
3. Pylorus
4. Pyloric Sphincter
b. Layers of the Wall of the Stomach (there is a similar arrangement of
tissues throughout the GI tract)
1. Mucosa (gut epithelium with subepithelium connective tissue; the lining
of the stomach directly involved in digestive activity)
a. Epithelial layer
b. Lamina propria
c. Muscularis mucosae (inner muscle layer)
2. Submucosa
4. Oblique muscle layer outer muscle layers with fibers oriented
5. Circular muscle layer in 3 directions; with the muscularis 6. Longitudinal muscle layer mucosa produce an agitator action
7. Serosa
c. Close Up of The Stomach Mucosa- organized into rugae (wrinkled folds),
with pockets called gastric pits.
1. Overlaid with a thick blanket of mucus (which has a pH gradient)
2. Epithelial Layer (cells held together by tight junctions)
1. Surface epithelial cells
2. Mucous cells – secrete mucus and bicarbonate 3. Parietal cells – secrete HCl and intrinsic factor - located in the
(necessary for absorption of vitamin B12) gastric pits
4. Chief cells – secrete pepsinogens – digests proteins
The cells of the gastric mucosa secrete about 2500 ml of gastric
juice each day. The HCl kills many ingested bacteria, aids protein digestion, provides the necessary pH for pepsin to start protein digestion, and stimulates the flow of bile and pancreatic juice. It is concentrated enough to cuase tissue damage, but in normal individuals the gastric mucosa does not become irritated or digested because of the mucus layer which overlays it. As the mucous cells secrete the mucus and bicarbonate, the bicarbonate is trapped in the mucus gel, so that a pH gradient is established that ranges from pH 1-2 at the luminal side to pH 6-7 at the surface of the epithelial cells (1).
d. Role of the Stomach in Digestion (see Handout: Anatomy Coloring Book)
e. Nonspecific Defenses of the Stomach
1. Mucus – production disrupted by nonsteroidal anti-inflammatory drugs
(NSAIDS) which inhibit prostaglandins synthesis and therefore inhibit
mucus secretion.
2. Stomach acid – destroys most foodborne bacteria; compromised by
hypo or achlorhydria from a variety of causes including use of antacids,
drug therapy (proton pump blockers), partial or total gastrectomy,
3. Tight Junctions
4. Peristalsis
5. Normal Flora – small numbers of Lactobacilli, Fusobacterium
f. Gastric Disease
1. Dyspepsia – gastric indigestion (upset stomach) due to
alterations of gastric function that are caused by various
disorders of the stomach. Symptoms include belching, nausea
hearburn, gnawing, burning abdominal pain. (not associated with H. pylori infection)
2. Gastritis – inflammation of the gastric mucosa
3. Ulcer – a lesion on the surface of the skin or mucous membranes
caused by superficial loss of tissue; often accompanied by inflammation.
4. Peptic Ulcers – discrete regions of erosion through the mucosa
which are surrounded by apparently normal tissue. Acid induced
damage can occur in:
a. the stomach – gastric ulcer
b. the duodenum – duodenal ulcer
The actual ulcer crater is often surrounded by an area of intact but
inflamed mucosa, suggesting the gastritis is a predispoisng lesion or
development of gastric ulcer. Both gastric and duodenal ulcers are
thought to be the result of a consequence of excessive acid and pepsin
secretion plus diminished mucus (2).
2. Helicobacter pylori and Gastric and Duodenal Ulcers
A. Slide 5 (Discovery of H. pylori)
1. Discovered by Barry Marshall and Robin Warren in 1983. Observed spiral organisms
in biopsy specimens and isolated the organisms.
2. These organisms had been seen previously in biopsies of animals and in humans.
3. Marshall fulfilled Koch's postulates by drinking a culture of H. pylori. He developed
gastritis and the organisms were reisolated from gastric biopsies.
B. Slide 7 (H. pylori the organism)
1. Named for its spiral shape and its anatomic location
2. Gram-negative, spiral-shaped, microaerophiles (grow in reduced
oxygen 5-10% rather than 20%. They will not grow anaerobically.
3. The colonies tend to become coccoid with age, and these forms
are degenerative and noninfectious. (This morphological change is
also seen with aging C. jejuni cultures.)
C. Slide 8 (TEM of H. pylori)
4. They have multiple, sheathed bipolar flagella and have rapid, darting
motility.
D. Slide 9 (Campylobacter to Helicobacter)
Following its initial isolation in 1984, Marshall and Warren named the organism pyloric campylobacter because of its many similarities to the genus Campylobacter (morphology, microaerophilic, coccoid appearance of older cells). The name was formalized to Campylobacter pyloridis and then Campylobater pylor. Eventually, it was determined by molecular analysis (16S ribosomal RNA) and by biochemical analysis (unique cellular fatty acids) that these gastric organisms were not campylobacters and they were given the novel genus, Helicobacter.
E. Association of H. pylori with Disease
The Big Picture: H. pylori colonizes the stomach for years and decades
producing a low-grade gastric inflammation which may progress (in some
people) over time to cause more serious disease. Infection with H. pylori is not
associated with dyspepsia.
1. Chronic Superficial Gastritis – usually asymptomatic; now estimated
that greater than 10% of those with gastritis will develop peptic ulcer
disease.
2. Peptic Ulcer Disease
a. 70-80% Gastric Ulcers
b. 90% Duodenal Ulcers
(While in the past peptic ulcer disease was known to be associated with the long term use of aspirin and NSAID's and a few other inflammatory conditions, 60-90% of all peptic ulcer disease were idiopathic – of unknown causes. We now known that H. pylori is the cause of nearly all of these idiopathic cases in adults and that treatment that eradicates H. pylori leads to cure of the ulcers.)
3. Atrophic Gastritis- chronic gastritis with atrophy of the mucous
membranes and glands; a precursor lesion to .....
4. Gastric Cancer (The association between H. pylori and this disease is
important since gastric cancer is the second leading cause of cancer death in the world. Can you imagine, treatment with antibiotics can prevent the second leading cause of cancer deaths in the world?)
5. Low grade MALT Lymphomas
F. Pathogenesis of H. pylori
1. Additional Evidence that Colonization with H. pylori Produces
Gastric Inflammation:
a. A second human volunteer study.
b. Eradication of H. pylori with antibiotics clears gastritis.
c. Untreated individuals show evidence of persistent immunological
responses; titers decrease after eradication.
d. Animal models have been developed.
e. H. pylori is associated with specific pathology.
f. There have been outbreaks of epidemic hypochlorhydria with
gastritis (iatrogenic from contaminated gastroenterological
equipment).
How does this organism cause gastritis, and peptic ulcer disease? What are the virulence factors that allow the organism to survive and grow in the low acid environment of the stomach?
2. Steps in H. pylori pathogenesis
a. Entry into the gastrointestinal tract.
b. Reaching the epithelial mucosa:
1. Secretion of urease creates an ammonia cloud that
allows the bacteria to survive the acid pH of the stomach
long enough to reach the mucus layer (where there is a
gradient of pH).
Slide 11 – From Urea to Ammonia ....to Neutralization
Slide 12 – Protected by the Ammonia Cloud
The protective cloud of ammonia effectively neutralizes
acid in the immediate vicinity, protecting H. pylori from gastric acid and facilitating its passage through the acidic environment to the site of colonization.
2. Slide 13 - H. pylori Infects the Gastric Mucosa
Migration through the thick viscous mucus facilitated by H.
pylori's spiral shape and by flagella – allowing the bacteria to
bore through the mucus.
The production of urease and motility imparted by flagella are essential for colonization of the stomach. Mutants defective in urease or mutants which are nonmotile cannot infect experimental animals.
c. Once the bacteria bore through the mucus,
two patterns of association with the epithelial mucosa have
been seen:
1. H. pylori may colonize the mucus in close proximity to the
epithelial cell surface (but without adherence), and/or
2. it may directly adhere to the epithelial cells primarily at
intercellular junctions. A number of putative adhesins have been identified.
(Slide 14 – H. pylori on the Mucosal Surface)
3. Another factor that may help H. pylori maintain itself in its
biological niche:
a. Microaerophilic growth pattern – the oxygen
tension at the gastric surface and within mucus is
reduced compared with that of the aerated gastric
tissue.
d. Long term colonization on or near the epithelial cell surface
is thought to lead to....
e. Direct Damage to the Epithelial Cell Surface by the bacteria
and/or.....
Virulence Factors???
1. Vacuolating cytotoxin- produces vacuoles in the
cytoplasm of eukaryotic cells in vitro; associated with
inflammation.
2. CagA protein – its presence is strongly associated with
the vacuolating cytotoxin activity (produced by 60% of
isolates Cag A +), and higher degrees of inflammation
and damage to the gastric mucosa.
Slide 16 Mucosa Fights Back- Immune/Inflammatory Response
f. Damage to the Epithelial Cell Surface by the Host's
Inflammatory Response to H. pylori colonization.
1. H. pylori infection results in an acute inflammatory
response with the influx of neutrophils, and the resulting
inflammatory response (including the release of
cytokines, enzymes and free oxygen radicals) damages
the gastric epithelial cells.
Two bacterial virulence factors that may allow H.
pylori to resist killing by phagocytes??
1. Catalase
2. Superoxide Dismutase
Subsequently, the acute inflammatory response diminishes,
monocytes and lymphocytes arrive, and a chronic inflammatory response is set up. There is local production of antibody, but it is ineffective in clearing the bacteria.
Is it direct damage by the microbe and/or the ongoing
local inflammatory response that is responsible for the
destruction of the mucosa and formation of ulcers? What
causes the progression to atrophic gastritis in some
patients??? Does the damage produced by the chronic
infection increase a person's susceptibility to
mutagens ultimately resulting in metaplasia and gastric
cancer???
How does H. pylori cause ulcers in the duodenum when it
does not colonize the duodenum? (see Text p. 227)
g. Immune Response to H. pylori
There is a vigorous local and systemic immune response after gastric colonization by H. pylori. Neutrophils, monocytes and macrophages are seen in in the gastric mucosa of infected children along and plasma cells are part of the inflammatory inflitrate. T cells do not seem to play a major role in the inflammation, but there are elevated elevels of IL-1, Il-2, Il-6, Il-8 and TNF alpha are seen in the gastric mucosa. Circulating IgG antibodies are easily detectable and form the basis of serological diagnosis. Why don't these responses clear the organisms???
G. Slide 28 – Epidemiology of H. pylori Infections
1. Responsible for one of the world's most common infections. It is
usually acqured without your knowledge from and unknown source. The infection persists slilently in most infected people and casues superficial gastitis that may persist for years, leading to chronic inflammation.
2. Slide 33- Prevalence is linked to Geography
a. Developing Countries- more prevalent, acquired early in
childhood with infection rates of 50-60% by age 10 and 90%
in adults.
b. Developed Countries – few infections occur in childhood;
gradual increase in prevalence (0.5% - !% per year), leading to
infection rates of 20-30% by age 20 and 50% at 50-60 years.
3. Slide 31- Socioeconomic Status Influences H. pylori infection
Prevalence is linked to socioeconomic status
4. Prevalence is linked to Ethnicity in the United States
a. Higher in Hispanic and black populations (unknown – related to
lower socioeconomic conditions?)
5. Slide 35 – Possible H. pylori Genetic Links
Identical twins reared apart have higher incidence of disease compared
to fraternal twins reared apart. This suggests that there may be genetic
factors influencing infection.
6. Slide 36 – Within a Family
Method of Transmission – Fecal-oral or oral-oral. Transmission within
families has been demonstrated.
7. Slide 29 – A variety of factors influence the rate of acquisition of
H. pylori
H. Diagnosis
1. Invasive Tests – based on biopsies of gastric tissue
a. Histological exam
diagnosis is made by a pathologist who examines
specimens.
b. Slide 21 - Culture (pros and cons)
1. Grown on BAP, 37C, microaerophilic conditions 2. Sensitivity 75-90% because technical expertise is
required to culture these fastidious, slow growing,
organisms.
3. Can detect for antibiotic resistance.
c. Slide 22 – Urease Test
Urease Test is performed on biopsy specimens. The specimen is placed into a sample with urea and a color indicator. If ammonia is produced, the pH increases, and the indicator changes color.
2. Noninvasive Tests
a. Slide 23 – Urea Breath Test
Patient ingest urea laeled with 13C or 14C. If H. pylori is
present in the stomach, the labeled urea will be converted to labeled CO2 which will then be absorbed into the bloodstream, and will appear in the expired breath. The labeled CO2 is detected and measured. (Slide 24). This test is used to monitor patients after they have had antibiotic therapy to determine if the organism has been eradicated.
b. Serology
Measures circulating IgG antibody against H. pylori antigens.
Can't tell whether the presence of antibody indicates present
or past infection.
I. Treatment
1. Treatment is difficult because of the niche the bacteria occupy.
Many antibiotics are inactivated by the stomach acid.
2. Initial therapy was with bismuth (pepto-bismol) and a single antibiotic-
not successful.
3. Currently, multiple antibiotic therapy is used because of resistance to
one of the drugs – metronidazole.
a. Triple Therapy – bismuth, metronidazole and amoxycillin or
tetracycline – 90% eradication (drops to 60% with resistance to
metronidazole)
b. New strategy – proton pump inhibitor and two antibiotics
(amoxycillin and clarithromycin)
4. Patient compliance is a problem.
J. Prevention – A Vaccine???