The research was on identifying genes coding
for the various human intestinal mucins (the MUC 1 to 7 family) using molecular-biology
techniques, regulating mucin synthesis and secretion under various pathological
conditions such as chronic inflammatory diseases of the intestine (ulcerative
colitis and Crohn's disease) or cancers (pancreas, stomach, colon, etc.), as
well as mucin's involvement in gallstone formation, and its role in the prevention
of gastric ulcers or Helicobacter pylori infection. To sum up, cytoprotection
of the mucosa seems to be one of the main physiological functions of the digestive
mucus.
OESOPHAGOPROTECTION
The protecting of the oesophageal mucosa from acid attack on gastric
reflux. They underlined the importance of the saliva's buffer value and of oesophageal mucus
pH in preventing the possible harmful effects of hydrochloric acid.
- The impact of chewing-stimuli (a "parafilm" ball) on the salivary secretion of mucins
in patients with gastro-oesophageal reflux (with or without complications: strictures,
Barrett's syndrome) and healthy volunteers. They showed that the chewing-stimulus
multiplied not only the volume of saliva by two, but also the quantity of mucins
produced, to a comparable degree both in healthy subjects and patients presenting
a gastro-oesophageal pathology, complicated or otherwise. On the other hand,
the oesophago-salivary reflex which varies
the volume of saliva during mechanical and/or chemical stimulation of the oesophagus
was seriously disturbed in patients with gastro-oesophageal reflux. Given the
importance ascribed to the salivary components in oesophagoprotection and the
deficit in salivary response to oesophageal stimuli in patients with reflux,
the authors suggest the use of non-nutritional chewing gum as a complement to
treating reflux oesophagitis.
- The importance
of the mucous layer's hydrophobic character in maintaining the integrity of
the oesophageal epithelium in cases of gastro-oesophageal reflux. It show
that, in healthy subjects, the hydrophobicity of the oesophageal secretion obtained
in response to HCl (pH 2.1) was significantly lower than that obtained by intraluminal
instillation of physiological solution. The drop was even greater when HCl was
instilled with pepsin (0.5 mg/ml; pH 2.1). Changes to the oesophageal secretions'
hydrophobic properties could therefore play a pathogenic role in the development
of mucosal lesions.
- It show a reflex increase in salivary mucin levels, largely
from the parotid glands, after both mechanical (intubation or intraluminal distension)
and chemical (HCl or HCl/pepsin) intraoesophageal stimulation, the latter representing
the main nociceptive stimulus in gastro-oesophageal reflux. Similar trial among patients with severe
reflux oesophagitis where they
demonstrated serious functional deterioration of the oesophageal mechanoreceptors
which regulate the rate of salivary mucin production, resulting in a lower production
of mucins in response to a mechanical or chemical nociceptive stimulation.
- The importance
of salivary pH which determines the pH at the surface of the oesophageal mucus.
Because of its buffer capacity, salivary secretion plays a very active role
against intraluminal hyperacidity resulting from gastro-oesophageal reflux.
The saliva's pH following oesophageal stimulation by HCl
or HCl/pepsin in asymptomatic subjects and in patients with reflux to greater
or lesser degrees of seriousness. Among the latter, those who had the most serious
attacks also exhibited a significant drop in their salivary pH. The authors
concluded that salivary pH could be a key factor in certain complications associated
with gastro-oesophageal reflux.
- Yang et al. (Iowa City, USA) (1636) studied the mediators and inhibitors
of oesophageal mucus secretion induced experimentally by capsaicin - used as
a model of nociceptive stimulus in the anaesthetised dog in vivo. They demonstrated
that capsaicin (5 ml, 0.5% solution, intraoesophageal) increased oesophageal
mucus secretion as much as substance P (1 µg/kg, IV) or bethanechol (30
µg/kg, IV). The effect of capsaicin was blocked by lidocaine (50 ml, 4%,
intraoesophageal), suggesting that the mucosal secretory response to the nociceptive
stimulus involved the afferent nerves. Confirmation of the hypothesis would
come from the effects of capsaicin being blocked by CP 96 345, an antagonist
of NK1 receptors of substance P, and by atropine, a muscarine antagonist. It
therefore seems that oesophageal mucus production would, in the dog at least,
be increased by antidromic stimulation of the afferent nerves, releasing substance
P, an axon reflex of one or more cholinergic synapses.
THE STOMACH AND MUCUS Mucin expression and synthesis
- Ichikawa et al. (Sagamihara, Japan) studied the regulatory mechanisms
of mucin biosynthesis, involving growth factors and/or stimulation of acid secretion,
on an in vitro model of the gastric mucosa (corpus and antrum) in the Wistar
rat. Mucin synthesis was measured by cultured explants in the presence of growth
factors such as EGF, TGF-beta, HGF, or bFGF, or acid secretagogues such as histamine,
carbachol or gastrin. They found that the two families of substances had different
effects on mucin synthesis in the gastric corpus and antrum tissues. Not only
EGF and HGF, but also histamine increased mucin synthesis in the corpus explant,
while bFGF and TGF-beta had no effect. Carbachol stimulated mucin production
in both regions, whereas the stimulant effect of gastrin was limited to the
oxyntic region. They concluded that mucin synthesis in the two regions was governed
by different regulatory mechanisms.
- Okazaki et al. (Koshi, Japan) examined the in vitro inhibitory
effects of dexamethasone (corticoid) on mucin biosynthesis and expression of
the MUC1 gene by the human stomach. Assaying the mucins on samples of gastric
mucosa taken from 6 subjects undergoing gastrectomy, and cultured with different
concentrations of dexamethasone, showed that the corticoid had a dose-dependent
inhibitory effect on not only mucin secretion, but also MUC1 gene expression.
The result confirmed that one of the steroid's harmful effects on the gastric
mucosa could be its inhibition of gastric mucus production.
- In humans, expression of a gene known as MDR1, coding for glycoprotein P (P-Gly)
found in the membrane of various cells including liver tumour cells, is elicited
by anticarcinogenic agents. Hall et al. (Houston, USA) studied
the expression and role of P-Gly in phospholipid secretion by the rodent (mouse
and rat) liver and gastric mucosa. They showed that P-Gly was expressed in the
superficial cells of the gastric mucosa, i.e. those which secrete or are directly
exposed to the acid, and not in the lamina propria or myocytes. The location
points to a possible involvement of P-Gly in the secretion of gastroprotective
phospholipids.
- Konda et al. (Tokyo, Japan) examined the role of the protease
furine in the differentiation process of gastric mucosal cells from undifferentiated
stem cells. They found that furine, an endopeptidase involved in the cleavage
of protein precursors into active proteins, disappeared when a line of gastric
surface mucous cells (GSM06) differentiated in culture. By transfecting either
cDNA or the corresponding anti-sense, they found that cells expressing furine
secreted less mucin and, vice versa, those not expressing it increased their
mucus production. Furine could thus play a role in keeping cells in an undifferentiated
state.
- Yamada et al. (Kyoto and Tokyo, Japan) showed that EGF (Epithelial
Growth Factor) stimulated the proliferation and differentiation of gastric mucosa
cells in culture into epithelial cells. On the other hand, on the same cell
cultures, TGF-beta1 completely inhibited the EGF-elicited proliferation of mucus-secreting
cells and synthesis of mucins. Inhibition by TGF-beta1 could act by eliminating
the induction of early gene expression by EGF.
Gastroprotection
Work on gastroprotection concentrated more on the pharmacological and therapeutic
aspects of ulcer treatment, but also on trying to explain the factors aggravating
tissue attack.
- To compensate for the shortage of human in vitro models in long-term
studies of gastric mucus secretion, Wagner et al. (Hanover, Germany)
developed a primary culture system of epithelial cells isolated by collagenase/pronase
from samples of human gastric mucosa. The culture, considerably enriched in
mucus-producing cells (90 to 95%), stayed functional with no loss of secretory
efficacy for 10 to 14 days. This, the authors demonstrated by stimulating mucus
secretion by gastrin, prostaglandin E2 or carbachol. According to the authors,
the study was the first to show that gastric mucus secretion in human cell cultures
is regulated by gastrin.
- Synnerstad and Holm (Uppsala, Sweden) started from the premise that
the internal pressure of gastric mucosa glands increases on stimulation of acid
secretion. In the anaesthetised rat, high doses of omeprazole in long-term (400
µmoles/kg/day per os for 15-30 days) or acute treatment (40 µmoles/kg,
IV), blocked acid secretion and raised the intraglandular pressure with no change
to the thickness of the mucous gel adhering to the epithelial surface. Omeprazole
would thus act on the mechanisms regulating the glands' hydrostatic pressure
by limiting outward flux, and not by increasing the thickness of the adherent
mucus.
- Systemic alkalosis or topical administration of prostaglandin E2 (PGE2) protects
the gastric mucosa against luminal hyperacidity. Ranta-Knuutila et al.
(Helsinki, Finland) tried to find out whether the protection was related
to an increased secretion of HCO3- by the mucosal epithelium, the effect of
which would be to render the pre-epithelial mucus alkaline and increase its
buffer capacity. Rats treated with PGE2 and rats exhibiting HCO3 hyperalkalinaemia
tolerated a luminal acidification resulting from an perfusion of HCl (0, 10,
30, 50 and 100 mM) significantly better than their controls. On the other hand,
tissue resistance to the acid was low in the hyperalkalinaemic rats.
- Tanoue et al. (Long Beach and Irvine, USA and Fukuoka, Japan),
after immunohistochemically labelling gastric mucosal samples from rats with
portal hypertension, used an image analysis system to visualise the gastroprotective
effects of teprenone against aggression of the mucosa by ethanol (8 ml/kg, intragastric).
Teprenone is widely used in Japan for treating gastric ulcer and inflammation.
It would seem that teprenone effectively protects the gastric mucosa from the
effects of ethanol by acting on various mechanisms such as increasing the gastric
blood flow, but also on the secretion of gastric mucus, growth factors (basic
fibroblast growth factor) and prostaglandins.
- Following the stimulation of acid secretion, caffeine can cause gastric lesions.
On the other hand, nothing is known as to the potential effects of caffeine
on mucus secretion by the gastric epithelial cells. Hamada et al. (Tokyo
and Osaka, Japan) examined the effects of caffeine on JR1, a mucus-producing
epithelial cell line derived from human gastric carcinoma. They observed that
caffeine selectively counters the increase of intracellular calcium induced
by agents like acetylcholine, histamine or ATP, probably by blocking the hydrolysis
of PIP2 into IP3, resulting in a drop of mucus production. It would thus seem
that caffeine induces mucosal lesions, not only by increasing acid secretion,
but also by decreasing gastric mucus production.
- Working on the intracellular level (Golgi preparations with fetuin, a positive
substrate, and creatine kinase, a negative substrate), Slomiany et al. (Newark,
USA) investigated the mechanism of gastric mucosal lesion by ethanol.
They showed that, under ethanol, modified mucins with different substituents
in their carbohydrate chains were synthesised. They also observed substantial
changes to the glycosylation of proteins other than mucins which could, in cases
of chronic alcoholism, have serious consequences on the immunological and inflammatory
responses.
- Iaquinto et al. (Pisa, Italy) demonstrated that the gastroprotective
effects of 4-methylpyrazole (4-MP), known to be an alcohol dehydrogenase inhibitor,
were due neither to cessation of acid secretion in the stomach, nor to biosynthesis
of prostaglandins by the gastric mucosa. The authors therefore put forward the
hypothesis, and confirmed experimentally in the rat, that the compound's mode
of action could involve stimulating secretion of the mucus adhering to the gastric
mucosa. The effectiveness of 4-MP in gastroprotection would thus be related
to an increased secretion of mucus, yet another illustration of its cytoprotective
function.
- Takahashi et al. (Kyoto, Japan) underlined that not all gastroprotective
effects seemed to involve the gastric mucus itself. For example, the anti-ulcer
protective effects of leminoprazole (NC-1300-O-3), a proton-pump inhibitor,
on rabbit gastric mucosa cell culture attacked by indometacine did not seem
to be due to action on the mucus and/or prostaglandins E2, but to involve the
synthesis of specific proteins making the cells resistant to the harmful effects
of indometacine.
- On the other hand, under basal conditions on the same cell model (Takahashi
et al., Kyoto, Japan), leminoprazole was able to stimulate mucus
synthesis and secretion directly, independent of its effects on the proton-pump,
the stimulant effect being mediated by the nitric oxide produced by a constitutive
nitric oxide synthase.
- Tanaka et al. (Los Angeles, USA) studied the possible relations
between how gastrin and prostaglandins work in gastroprotection. Pentagastrin
strengthens the mucosal defence barrier in vivo, mainly by increasing the thickness
of the adherent mucous gel. These effects are reproduced by exogenous prostaglandins.
Consequently, the authors blocked the synthesis of endogenous prostaglandins
by indometacine and measured the intracellular pH and thickness of the gastric
mucus after pentagastrin. They showed that indometacine did not affect the increase
in acid secretion due to pentagastrin, but did partially prevent the increase
in mucus thickness. They thus demonstrated that strengthening the mucous barrier
by pentagastrin only partially resulted in the synthesis of endogenous prostaglandins.
- The same team studied the effects of the ranitidine-bismuth citrate
complex (GR 122,311X, Glaxo) on the permeability of the adherent mucous gel.
They measured the initial level of acidification in the rat gastric mucosa,
indicating the mucus's permeability to acid. They showed that GR 122,311X made
the mucus more impermeable to acid, almost certainly due to the bismuth binding
with the mucins.
- Bismuth binding with mucus in the gastric wall was also examined by O'Brien
et al. (Cincinnati, USA). The bonding was very fast (< 1 mn)
and stable (> 2 h) on a tissue model treated by bismuth subsalicylate in
an Ussing chamber, essentially involving the metabolite bismuth oxychlorhydrate.
The observations could be used in the therapeutic applications of bismuth in
treating dyspepsia and/or eradicating Helicobacter pylori.
- Lichtenberger et al. (Houston, USA) studied the effects of naproxen,
a non-salicylated NSAID, on the hydrophobicity of the gastric mucosa, and more
particularly its molecular interactions with surface phospholipids such as DPPC
(dipalmitoyl phosphatidylcholine). It would seem that the ability of NSAIDs
to cause lesions of the gastro-intestinal tract could be related to their properties
of rapid association with phospholipids on the mucosal surface.
- Teprenone is an anti-ulcer agent which has been shown to protect the gastric
mucosa against lesions produced by irritants. Hirakawa et al. (Tokushima,
Japan) have developed a cell culture system in which cell growth can
be blocked and induction of mucin synthesis suppressed. They thus demonstrated
that the cytoprotective effects of teprenone act via the heat-shock proteins
secreted by the gastric epithelial cells, independently of mucin synthesis.
- The protective efficacy of antacids on the gastric mucosa is due to their
ability to neutralise HCl, although other mechanisms of action have also been
suggested recently. By comparing hydrotalcit with Talcid (a polymer suspension
of the former), Gruebel et al. (Boston, USA) demonstrated that
the polymers used in antacid formulations could play a major part in gastroprotection
and the remission of ulcers, due either to their own properties (comparable
visco-elasticity to that of mucins), or by interaction with the gastric mucins
(increasing their viscosity).
Mucus and Helicobacter pylori (Hp)
- Taha et al. (Glasgow, United Kingdom), showed significant variations
in the concentration of copper, zinc and iron ions in the gastric epithelium
and in the mucus according to the presence or not of Hp in 52 patients. For
example, the copper/nitrogen concentration ratio in the gastric epithelium rose
from 0.01 during Hp infection to 0.16 after its eradication. The same ratio
dropped from 0.12 to 0 in the mucus. Under the same conditions, the relative
concentrations of iron and zinc did not change significantly. Hp gastritis is
thus associated with a relative deficiency of copper (known, like zinc, to have
anti-inflammatory properties) in the gastric epithelium, probably by the metal's
diffusion into the mucus.
- Flemström et al. (Newcastle, United Kingdom) wanted to ascertain
whether the mucous layer adhering to the gastroduodenal mucosa was a barrier
to high-molecular-weight toxins migrating from the lumen to the surface of the
duodenal epithelium. On an in situ preparation of anaesthetised rat duodenum,
they observed that perfusing a suspension of Hp or cholera toxin (CT) only elicited
an increase of bicarbonated secretion if the adherent mucous gel had been eliminated
by papain beforehand. If the mucous gel was intact, Hp or CT were virtually
incapable of reaching the epithelial surface.
- Degradation of soluble mucins by Hp could be a major factor in the development
of gastritis and gastroduodenal ulcers. Byrd et al. (Detroit, USA) compared the purified soluble mucins from the gastric juices of patients infected
(Hp+) or not (Hp-) with Hp. They showed that there was no reduction in the concentration,
carbohydrate content or size of the soluble mucins in the Hp+ patients. However,
the same mucins bound more than those of the Hp- patients to the monoclonal
antibodies PS10 (pancreatic cancer) and GS2 (gastric cancer), which recognise
non-sialile carbohydrate epitopes on the mucins.
- It has been shown that one result of ingesting ethanol is a reduction in thickness
of the adherent mucous gel. Since Hp mainly attaches itself beneath the mucous
layer, Sarma et al. (Newington and Farmington, USA) investigated
the effects of regular ingestion of alcohol on frequency of Hp infection. The
frequency of Hp infection was determined (CLO test and histology) on biopsies
of 55 gastritic patients, classed into groups according to degree of alcohol
consumption: a significant reduction in Hp frequency was observed in patients
who drank the most. Ethanol thus seemed to alter the gastric surface's milieu
making it unsuitable for Hp colonisation.
- Tanaka et al. (Los Angeles, USA) showed that indometacine increased
the bismuth concentration in the mucus of rats treated with ranitidine-bismuth
citrate (GR 122,311X). Their objective was to maintain sufficient antibacterial
concentrations to eradicate Hp, without having to perform multiple administrations.
Under indometacine, the concentration of bismuth fixed onto the gastric mucus
was ten times higher than in the control untreated by the anti-inflammatory.
The effect could be the result of inhibition of pepsin activity by the bismuth
and stimulation of mucus synthesis by indometacine. Combining GR122,311X with
an NSAID would therefore prove very useful in eradicating Hp, without antibiotics
being added.
- Logan et al. (Nottingham, USA) sought to understand why Hp only
infected gastric type epithelia. They therefore studied the chemotactic response
of Hp to gastric mucus and acid. To do so, they used isolates of H. pylori
and H. mustelae in culture, and had them rise inside capillary tubes
containing chemotaxins. All isolates tested exhibited optimal chemotactism towards
slightly acidified mucus (pH 5.7-6.0) which, given the conditions in vivo,
could contribute to Hp's gastric epithelial tropism.
- Lastly, Gibson et al. (Washington, USA and Lausanne, Switzerland) established
a cell line, HAE, which they describe as an excellent cell model for studying
Hp's pathobiology. The HAE cells, obtained by collagenase digestion of human
gastric biopsies, were immortalised by introducing the gene coding for the antigen
T SV40. They secrete a mucus consisting essentially of neutral and some acid
mucins. When Hp was added to the culture medium, it very quickly adhered to
the HAE cells and specifically inhibited their growth.
MUCUS AND HEPATIC DUCTS
Mucus and lithogenesis
- The bile of patients with calcified cholesterol stones is not very acid. Although
the reason for this lack of acidity is not known, it is known that calcification
by CaCO3 makes the stones insensitive to all acid therapy. Tomida et al.
(Tsukuba and Ibaraki, Japan) looked at the hypothesis whereby high bile
pH could be due to hypersecretion of mucus glycoproteins which, by an increased
buffer effect, would prevent normal acidification of the bile. Be this as it
may, their research showed that the mucin levels of patients with calcified
or non-calcified cholesterol stones, or even with bile-pigment stones did not
differ. Similarly, no correlation was found between bile pH and mucin concentration.
Consequently, it would seem that insufficient acidification of the bile is due
to the gastric mucosa not secreting enough acid.
- Shiffman et al. (Richmond, USA) considered the hypothesis of
ursodesoxycholic acid (UDCA) having an inhibitory action on the concentration
of mucins in the bile. UDCA is acknowledged to dissolve cholesterol stones and,
moreover, to prevent their formation during the rapid weight loss process. The
authors treated 21 patients from a group of subjects with symptoms of chronic
cholelithiasis, confirmed by echography, with UDCA (300 mg/day, for 15-30 days).
A significant drop in bile mucin concentration was observed in the patients,
very probably due to a decrease in mucin secretion by the gallbladder epithelium
caused by the UDCA.
- Working on canine gallbladder epithelial cell cultures (DG8E), Klinkspoor
et al. (Amsterdam, Netherlands and Seattle, USA) studied the mechanisms
by which bile salts, cholesterol crystals and phospholipids could regulate the
secretion of mucins. They concluded that it was indeed the bile salts and not
the cholesterol, cholesterol crystals or the phospholipids, which stimulated
the secretion of mucins by the gallbladder. Here, the hydrophobic salts were
more powerful stimulants than the hydrophilic. Unlike prostaglandin E2, bile
salts did not stimulate the synthesis of mucins by an increase in intracellular
cAMP. Their mechanism of action remains to be determined.
- Eder et al. (Munich, Germany) considering the lithogenic role
of mucins secreted by the bile, examined the physiopathological mechanism behind
the induction of mucin hypersecretion observed in patients with lithiasis. Adding
lithogenic bile from these patients to cultured human gallbladder epithelium
cells, they found a significant increase (2 to 3 times more than the control)
in mucins produced. Adding bile-pigment stones to the same preparations only
elicited a slight increase in mucin secretion. Nevertheless, the authors put
forward no hypothesis as to what specific ingredient of lithogenic bile is responsible
for the mucosal reaction.
- The American prairie dog (Cynomys sp.) has the particularity of developing
lithiases very soon after being fed on a cholesterol-supplemented diet. It is
therefore a useful model for studying cholelithiases. Fisk et al. (Nashville,
USA) devised a culture technique for Cynomys gallbladder epithelial
cells allowing them to examine the role of mucins in the development of gallstones
on the cellular and molecular levels by immunohistochemical methods. Working
on the same species, Johnston et al. (Baltimore, USA) (169) researched
the possible role of the non-mucus glycoproteins which very soon appear in Cynomys
bile when its diet is cholesterol rich. They suggest that these particular glycoproteins
could be major early pathogenic factors in the lithogenic process.
- Lechene de la Porte et al. (Marseille, France and Amsterdam, Netherlands)
examined the location of protein molecules inside mixed gallstones (made
up of pigments, calcium salts and cholesterol crystals). They located the mucins
inside the stones using specific monoclonal antibodies. The specific arrangement
of mucins demonstrated their structural role in stone formation. Thus, the calcium
salts and pigments deposit onto a protein matrix (APF/CBP and aminopeptidase-N)
linked to the mucins, and cholesterol crystallises onto the mucins directly.
- Rege and Prystowsky (Chicago, USA) examined the inflammatory properties
of bile in dogs with pigment lithiases induced experimentally by a methionine-deficient
diet. The diet caused inflammation of the gallbladder and hypersecretion of
bile mucus. One millilitre of this bile caused inflammation of the gallbladder
of healthy guinea pigs, and the effect persisted after ultracentrifuging the
bile to eliminate crystals and stones. The bile supernatant thus contained soluble
factors able to elicit inflammation of the gallbladder wall.
MUCUS AND INFLAMMATION OF THE GALLBLADDER
- Epitope CA19-9 is a tumoral marker whose serum concentrations increase in
patients affected with gastro-intestinal tumours. Although CA19-9 is naturally
present in the healthy human bile, it is not in the colon or pancreas. Von Ritter
et al. (Munich, Germany) investigated why this epitope was present
in the bile. Having revealed mucins with the epitope on epithelial cell cultures
of normal human gallbladder, they concluded that the rise in serum epitope concentration
observed in inflammation of the bile tract could be due to the mucins passing
from the bile to the blood.
- Inflammation of the gallbladder mucosa results in hypersecretion of mucins,
which in turn promotes the lithogenic properties of bile. Eder et al.
(Munich, Germany and Seattle, USA) demonstrated that the polynuclear neutrophils
activated during the inflammation process stimulated mucin secretion by gallbladder
epithelial cells in the dog. The inhibitory effects of catalase and superoxide
dismutase in the model indicated that the hydroxyl radicals (active metabolites
secreted by the polynuclear neutrophils) could mediate the hypersecretion.
INTESTINAL OR COLON MUCINS
Synthesis and regulation - Trefoil peptides
- ITF (intestinal trefoil peptide : intestinal) and SP (spasmolytic peptide :
antral) "trefoil peptides" are a recently recognised family of epithelial
regulatory peptides associated with mucins. They are found widespread in gastro-intestinal
tissues and are implicated in protecting and repairing the gastric mucosa.
- The ITF and SP "trefoil peptides" are conserved and potently expressed
in the gastro-intestinal region during development. Using radio-immunological
and immunohistochemical techniques, Taupin et al. (Melbourne, Australia)
quantified expression of the two peptides in the rat between the 17th
day of foetal life (gestation: 22 days) and weaning (16 to 27 days post-partum).
They demonstrated the presence of appreciable quantities of "trefoil peptides"
long before the morphological or cytochemical appearance of traces of mucins
or gastrin. The trefoil peptides' concentration peak would thus be just before
weaning.
- IGFBP-4 is the binding protein for cytokine IGF which stimulates the proliferation
of intestinal mucosa in vivo and numerous cell lines in vitro.
Human colon carcinoma line HT29, for example, normally expresses IGFBP-4. By
transfecting in these cells, Corkins et al. (Omaha, USA) (1401) caused
protein IGFBP-4 to superexpress (multiplication by 2-3). They found that a)
the rate of proliferation of transfected HT29 cells was significantly higher
than that of non-transfected cells, and that b) their MUC3 mRNA production increased
(360%) compared with controls, whereas the MUC2 mRNA rate did not change.
- Ogata et al. (Boston, USA) examined the promoter region of the
gene coding for the ITF trefoil peptide specific to colon and small intestine
goblet cells in the rat. They identified a 37-nucleotide sequence (-153 to -116)
which seemed determinant for ITF gene expression in these cells.
- Genes MUC1 to MUC7 have been described in man. Among them, MUC2 and MUC3 have
been cloned from intestinal cDNA libraries. MUC2 is the commonest mucin in the
colon. Van Klinken et al. (Amsterdam, Netherlands) studied the
expression of MUC2 and MUC3 mRNA and protein synthesis in the human small intestine.
They showed that both genes were expressed there and resulted in the synthesis
and secretion of well-characterised mucins. Nevertheless, a third form of mucin,
as yet unidentified, of approx. 500 kDa was also revealed.
- Hunninghake et al. (Minneapolis and Athens, USA) tried to ascertain
whether there was a range of mucin genes in the mouse. By hybridising and cloning,
they identified a specific colon mucin different to that which expresses itself
in the mouse stomach.
- Cohen et al. (Cincinnati and St Louis, USA) studied the role
of guanyline on rat and human intestinal cell lines (Caco-2). They concluded
that, in vivo, the function of the mechanisms implicated by guanyline
on the intestinal cell level was to ensure hydration of the mucins secreted
by the goblet cells.
- Previous papers of Castagliuolo et al. (Boston, USA) have demonstrated
that stress due to immobilisation elicited mucin synthesis in the rat colon,
along with an increase of colon motricity and prostaglandin synthesis. They
now examined the roles of the nervous system, neurotensin and substance P on
the effects of stress on the colon. They blocked the effects by a ganglioplegic,
hexamethonium. More specifically, they inhibited the production of mucins and
PGE2 elicited by stress by means of either neurotensin receptor antagonists
(SR 48692), or antineurotensin antibody. Substance P did not seem to be implicated.
They raised questions as to the existence of neural pathways involving neurotensin
in the colon mucosa's response to stress.
- Until now, it was thought that the cholera toxin (CT) elicited mucin secretion
indirectly via the enteric nervous system. Epple et al. (Berlin, Germany)
wished to check the assertion by testing the effects of CT on highly
differentiated cell cultures of human colon carcinoma (HT29/B6). Mucin secretion
was increased in these cells by a factor of 2.6 by CT and by a factor of 8 by
forskoline compared to non-stimulated controls. The authors also demonstrated
that it was indeed mucins synthesised de novo, unlike what is observed
with carbachol on the same preparations, which only elicits the release of small
quantities of probably new-formed mucins. There is thus a direct mechanism whereby
the cholera toxin stimulates mucin synthesis, independent of the autonomic nervous
system.
- Keratocyte growth factor (KGF) is a mitogen specific to epithelial cells.
It has been well described for the skin, but its role in the intestine remains
obscure. Zech et al. (Torrance, USA) investigated the possible
involvement of KGF in restoring the integrity of the colon mucosa after lesion,
cell proliferation and mucus production in the rat colon. KGF (5 mg/kg) distinctly
improved the condition of the mucosal tissue aggressed by trinitrobenzene sulphonic
acid (0.1 to 5 mg/kg), promoting both the proliferation of epithelial cells
and the production of colon mucus.
INTESTINAL CYTOPROTECTION
- Although mainly dealing with bicarbonate secretion in the duodenum, Safsten
et al. (Uppsala, Sweden) demonstrated that endogenous benzodiazepins
(endozepins) play a major part in regulating mucus secretion intended to protect
the duodenal wall against gastric hydrochloric acid.
- Sababi et al. (Uppsala, Sweden) studied the effects of lumen
acidity, endogenous prostanoids and nitrogen monoxide (NO) on mucus and bicarbonate
production in the anaesthetised rat duodenum. They demonstrated that secretion
of duodenal mucus was strongly stimulated by HCl (10 mM, isotonic), by prostanoids
and, to a lesser extent, by NO. On the other hand, bicarbonate secretion was
stimulated by acid and endogenous prostanoids, but inhibited by NO.
- Following a reminder that prostaglandins' cytoprotective properties in the
intestine are partly due to the release of active phospholipids resulting in
increased hydrophobicity of the intestinal mucosa, Lugea et al. (Barcelona,
Spain) showed that, in the rat, the non-steroid anti-inflammatory drugs
(NSAIDs), indometacine (2 and 5 mg/kg, SC) and diclofenac (5 and 10 mg/kg, SC)
inhibited the formation of phospholipid surfactant at the surface of the duodenal
mucosa and thus reduced the cytoprotective hydrophobic properties. These observations
contribute towards a better understanding of NSAIDs' ulcerogenic mechanisms.
- The same authors (Lugea et al., Barcelona, Spain) also researched
cytoprotective mechanisms independent of prostaglandins which implicated the
stimulation of phospholipid surfactant synthesis. Working on the rat, they used
an agent which stimulates pneumocytes to synthesise and release pulmonary surfactant:
ambroxol (40 mg/kg, intraduodenal). Ambroxol elicited phosphatidylcholine secretion
by a mechanism independent of prostaglandins (resistant to indometacine). This
increased the resistance of the duodenal mucosa to experimental aggression by
hydrochloric acid (1 ml, 0,4 M, intraduodenal).
MUCUS ET MICI
In ulcerative colitis (UC), mucins may be deteriorated, thus affecting the
mucous barrier's cytoprotective function. Are mucins the cause or the consequence
of UC?
- Young et al. (New York, USA) reviewed the prevalence of the antigen
sialosyl-Tn (STn) in human UC. Since STn expression occurs earlier than dysplasia
may be identified, they stressed the value of studying the antigen alongside
monitoring the colitis by endoscopy.
- The above conclusion was added to by Karlen et al. (Stockholm, Sweden
and New York, USA) who studied STn expression in patients suffering from
chronic UC. They observed that the antigen only expressed itself in these patients
when the UC was compounded by dysplasia or cancer of the colon.
- Pladdet et al. (Amsterdam, Netherlands) considered mucin genes
as risk markers of predisposition to UC. Gene MUC2, for example, would be a
risk marker of genetic predisposition to ulcerative colitis. The presence of
these mucins in the colon of patients suffering from UC would thus not be the
result of the disorder but a predisposing factor.
- IL-11 is an interleukin whose anti-inflammatory properties now seem well established.
Pfeiffer et al. (Blacksburg, USA) tested 312 rats for the effects
of various doses of rhIL-11 (recombinant human) on experimental TNBS (trinitrobenzene
sulphonic acid) colitis. They observed that IL-11 increased mucus production
by the colon, significantly attenuated the increase in myeloperoxydase (MPO)
enzyme activity elicited by the TNBS and decreased the ulceration index of the
colon mucosa. The anti-inflammatory effects of IL-11 would thus be partly related
to the cytokine's stimulation of mucus production.
- Weiss et al. (New York, USA), by in situ hybridisation, studied
the expression of MUC2's and MUC3's messenger RNAs in the intestinal tissues
in the constitutive, tumoral or inflammatory state. MUC2 was expressed in the
goblet cells across the entire thickness of the mucosa in both the small intestine
and the colon. MUC3 was especially expressed by differentiated cells in the
villi (goblet and columnar cells) and surface epithelium. Expression of genes
MUC2 and MUC3 was kept during intestinal inflammation (Crohn and ulcerative
colitis) but decreased significantly in cancers of the colon.
- MUC2 is the main colon mucin secreted in UC. Tytgat et al. (Amsterdam,
Netherlands) isolated mucins from UC patients and compared them with
those identified from normals. It appeared that MUC2 was still the main mucin
biosynthesised in the colon, in both the healthy subject and the patient suffering
from UC. In addition, there seemed to be no quantitative differences in the
mucin concentrations between controls and UCs, although this remains to be confirmed.
- In ulcerative colitis, the mucolytic activity of colon proteinases is high,
resulting in a thinning out and discontinuity of the adherent mucous gel. Rankin
et al. (Newcastle, USA) looked at the interaction of a polyacrylic
acid, Carbopol 934P, with the mucus colon. Patients suffering from UC, treated
with the product for 4 weeks (400 mg, 3 times a day), showed a distinct improvement
in the sigmoidoscopic symptoms, and a significant drop in colon protease activity.
The polyacrylic acids would thus seem to prevent mucolysis by preventing proteases
from breaking down the pre-epithelial protective barrier.
- Pitcher et al. (Cambridge, USA) studied the role of sulphur-reducing
bacteria (SRB) in the colon. SRB produce sulphites which generate inflammation
in subjects genetically predisposed to colon disorders. They demonstrated that
UC patients had a higher prevalence of SRB in the colon than healthy volunteers,
and a higher level of free sulphates, probably from enzyme desulphatation of
the mucus by faecal bacteria.
MUCUS AND CANCER
Gastric cancer
- Schmidt et al. (Hanover, Germany) used highly differentiated
gastric cancer cell lines (HM02 and HS746T) which produce phospholipid-enriched
mucus. Under normal conditions, the phospholipids are transported in the blood
by lipoproteins. The authors checked the hypothesis according to which the synthesis
and secretion of mucus phospholipids are regulated by apolipoproteins. The lines
used secreted mucus containing phospholipids and expressed apoE but not apoA-I,
apoB-48 or apoB-100 unlike the intestinal or hepatic cells. The intracellular
role of apoE remains to be determined.
- Working on stomach cancer, Yong et al. (Osaka and Nagoya, Japan)
studied the deterioration of human mucins occurring alongside malignant transformations
identified by the expression of antigens Tn, sialyl Tn and T in cancer of the
colon. The authors identified a new antigen (F1 alpha) associated with the mucins
during experimental research on 141 patients with gastric cancer. The F1 alpha
antigen expressed itself very early on, at the same time as carcinogenesis in
the gastric epithelial cells.
Cancer of the Pancreas
- Raoul et al. (Rennes and Kremlin-Bicêtre, France) emphasise
the prognostic value of determining the type of mucin (sialomucin, sulphomucin,
etc.) encountered in, for example, Vater's ampulla carcinoma. The identification
can be a useful complement and back up the well-established classical criteria
for prognosis of this type of carcinoma.
- CAM17.1 is a murine antimucin antibody which binds to the sialyl I group of
blood antigens. Yiannakou et al. (Liverpool, United Kingdom) demonstrated
that the antibody can be used as serum tracer for detecting mucins released
into the blood circulation by a pancreatic carcinoma.
Cancer of the Colon
- The mucins secreted by colon tumoral tissues have been characterised by Cho
et al. (San Francisco, USA) on cell lines derived from colon adenocarcinomas
(HM3) and colloid cancers (C1a) using cDNA probes coding for MUC1, 2, 3, 5 and
6. Colloid cancer cells secrete more MUC2-type mucins than adenocarcinomas,
and present a higher level of antigen expression associated with short-side-chain
mucins (CHO) such as sialyl-Tn, T and Tn.
- Yeh et al. (San Francisco, USA) examined the possible alterations
in expression of mucin structural peptides and corresponding carbohydrate antigens
on colorectal carcinomas (Duke's cancer) compared to metastases of lymph nodes.
It would seem that expression of the mucins, which is specific to each tissue,
is deregulated in cancerous tissues.
- In animal models, mucin production by cancerous colon cells is often related
to the presence of hepatic metastases. Bresalier et al. (New York, USA)
examined the relations between the structure of mucins' carbohydrate
moieties and their adhesive properties. It is thus the carbohydrate chains,
truncated at the mucin's body, which ensure cancerous colon cells adhere to
the basement membranes. On the other hand, it is the long glycoprotein chains
that are needed for colonising the liver, by means of interactions between the
selectins and the sinusoid endothelium.
- Patients with pancreatic carcinoma produce killer T cells and antibodies which
recognise certain peptide regions of the polypeptide core of MUC1-type mucins.
Ho et al. (San Francisco, USA) evaluated the accessibility of
MUC1-type peptide regions of intracellular, surface or secreted mucins on a
pancreatic cancer cell line (SW1990). They observed that the highly glycosylated
MUC1 peptide regions were accessible, eventhough they are normally protected
by molecules of sialic acid.
- The MUC2 human gene codes for a mucin specific to goblet cells in the colon
and small intestine. Gum et al. (San Francisco, USA) isolated
and sequenced a genomic DNA clone of some 8000 base pairs on the 5' end region
of gene MUC2, allowing them to characterise the MUC2 gene promoter in detail.
The site is understood to include the first 500 base pairs starting from the
transcription initiation site and consist in 63% G+C base pairs. The sequence
isolated contains a TATA box located 24 bases above the initiation site. The
research is geared towards producing transgenic mice expressing the MUC2 promoter
as a model for studying various cell types in vivo.