This could be more crap but its another potenially interesting research article for some on leaky gut
Mechanisms Behind the Leaky Gut
Medical literature on the leaky gut provides useful information that may
help unify the picture of how diets used in autism are addressing some
issues that have been studied more carefully in celiac disease. This
mini-paper will review these mechanisms that may not yet be familiar to
many in the autism community.
Most people have heard of the term "leaky gut" but may not realize that
this term refers to how larger molecules may get into the blood when
structures called tight junctions that are there to seal the gaps between
intestinal cells, have instead, opened up. This opening creates a passage
that lacks the same type of regulation that happens when substances move
through intestinal cells. Scientists like to call this movement of fluid
and its solutes through this opening "paracellular transport". Other
tissues can be leaky like this, too, like the bladder, kidney cells and
even the blood brain barrier.
How does paracellular transport work?
Below you'll see a drawing of the epithelial cells that are the absorbing
part of the gut. It is important to notice that these cells are very
different on the food side versus the blood side where you are trying to
shuttle the nutrients that come from digestion.
Think of your gut as a big hose. That hose has an outside and an
inside. The picture below is a cross section, as if I cut through the hose
with a knife, and we are looking at the open edge of the hose as it would
be seen on the top side.
----------------------------------------------------------------------------
OUTSIDE of the "hose"
This is called the blood side or serosal side
or basolateral side
------------- -------------- -------------
| | | | | |
| | | | | |
| | | | | |
| |======| |======| |
]]]]]]]]]]]]] ]]]]]]]]]]]]]] ]]]]]]]]]]]]]
This is the brush border or apical side or
"lumen" where the food is.
INSIDE of the "hose"
-------------------------------------------------------------------------------
The big rectangles are intestinal cells, and the goods from food travel
from the bottom (or food side) to the top (or blood side).
The ]]]] represents the highly absorptive side that is touching the food
that is going through the intestine, and it has transporters on it that
help to absorb particular things across this membrane. The membrane also
excludes some things from crossing into cells. You'll hear this side
called the brush border, too, because of the villi that make this side look
very different. Here is a photograph of the brush border:
http://microvet.arizona.edu/Courses/MIC420/lecture_notes/ecoli/brush_border.html
The dashed line on top is the membrane that helps deliver things that were
absorbed into these cells to the circulation. Nutrients move across the
cell to get to this membrane that is on the blood side. Transporters within
this membrane will move only certain selected things across that membrane
in order to deliver them to blood.
In my drawing above, the line of ====== represents tight junctions which
keep things from crossing between cells to get to the space that has access
to the circulation. When this junction is closed, the nutrients HAVE to go
through the cells to get absorbed, and that is a process that is regulated
by the transporters on the top and bottom side working together.
Immediately below, I've put a link to someone else's cartoon of this scene,
but this time, we are viewing things from the bottom side of the
"hose". The point of the drawing is to show how glucose gets transported
into the cell, moves across the intestinal cell, and then crosses that last
membrane en route to the blood:
http://www.rpi.edu/dept/chem-eng/Biotech-Environ/Membranes/bauerp/co.gifAnother system of regulation exists that has to do with nutrients taking a
different route. Instead of going through cells, this time, the nutrients
are going around, cells using the gaps that exist between these cells that
are usually closed off by structures called tight junctions.
The substances that travel this route (called the paracellular route as
opposed to the transcellular route) can be peptides from foods (like
gluten or casein peptides or peptides from other foods), or non-protein
molecules like oxalate. Oxalate is a compound found mainly in plants which
is highly reactive and binds to calcium and other molecules.
So, when your tight junctions open up, those cells will change like this:
---------------------------------------------------------------
Blood side: serosal side: basolateral side
------------- -------------- -------------
| | | | | |
| | | | | |
| |* *| |* *| |
| | = =| |= =| |
]]]]]]]]]]]]] ]]]]]]]]]]]]]] ]]]]]]]]]]]]]
Brush border or apical side or "lumen" where
the food is.
---------------------------------------------------------------
When the gate is open, many molecules pass through those open junctions
just as your dog might go through an open gate in your fence. Remember
there is a flow of solute-loaded fluid moving through these open gates.
Perhaps we've had the impression that having a leaky gut means something is
broken, but that is not necessarily the case. The opening of the gates is
regulated, and that regulation can be called upon by systems like the
immune system. Certain of the immune system's cytokines open up the
"gates" in order to let cells of the immune system in the blood have access
to antigens that have been in the gut. Some new work has shown that this
opening and closing of the gate also has a lot to do with calcium which
makes this system of gatekeeping have an unusual relationship to dietary
oxalate.
What have scientists learned about this system of gatekeeping?
I've put a study at the end of this article whose authors discovered that
some of this process of opening and closing the tight junctions appeared to
be mediated through an interaction with calcium. This did not involve the
concentration of calcium that was inside the intestinal cells, but it only
involved the calcium that was outside the cell. Removing the calcium from
either side of that tight junction could really change things, but changing
the level of calcium inside the rectangle (representing the inside of the
cell) made no difference at all.
Right next to where that gate is located on the basolateral (or blood side)
are some molecules and a "sensor" that picks up calcium that is travelling
in the fluid on this basolateral or blood side. I've represented that
sensor as an asterisk. If there is adequate calcium at that sensor, then
the leaky gut closes, just as if it had been zipped up. In fact, calcium
is actually a key ingredient used to close the zipper. When there is not
enough calcium present to close the gate, the gate stays open so that
calcium from the food side can come in through the gap until there is
enough calcium to close the gate again. In fact, at times, there are
oscillations that occur as this gate opens and closes in response to calcium.
What happens if the calcium level gets low?
If after the gate opens to let calcium in, there is not enough calcium on
the food side to travel through and bind the sensor, then that means there
won't be enough calcium to zip up the zipper and close the gate again. The
gate will stay open, and until enough calcium comes around to close the
gate again, you've got a "leaky gut" that will stay leaky . This means it
is important to think about all the circumstances that might cause the
supply of calcium to diminish that is travelling from the lumen of the gut
or what might cause the calcium on the basolateral side to get low.
What is the connection between calcium and fat maldigestion?
Scientists have figured out that calcium in the lumen can be tied up in fat
when one has fat maldigestion and malabsorption. The fat left undigested
in the gut binds calcium and makes something of a soap, but this doesn't
provide that either the fat or the calcium will get absorbed. This also
means that the calcium would fail to make it to the basolateral side when
the gate was open, and that would mean the calcium wouldn't be there to
interact with the molecules that govern the tight junctions by sensing that
there is adequate calcium there. Scientists have done experiments to
quantify how this fat effects oxalate absorption, and they also have noted
that very often people with celiac disease have this very same maldigestion
of fat. This offers one reason people with celiac sprue have a predictable
problem with this leaky gut and a condition of excess oxalate absorption
that is reflected by high levels of oxalate excretion in the urine called
hyperoxaluria.
What is the connection between calcium and oxalic acid that is in the gut?
Undigested fat is not the only way to tie up that needed calcium. Calcium
could also be tied up by soluble oxalate that comes from food high in
oxalates that were eaten and are present on the inside of the gut.
Food is not the only source of oxalate in the gut. Nature has provided a
system to help the body get rid of excess oxalate. Intestinal cells become
loaded with oxalic acid when this acid is transferred into them from
circulation from the basolateral or blood side, and from there, this acid
is actually secreted into the gut. Why does this happen? It is nature's
way of ridding the body of a compound that is highly reactive and can be
damaging to organs in the body, especially after those organs have already
suffered some sort of injury. Oxalates seek injured tissue because they
bind to molecules that ordinarily may be hidden from them in healthy tissue.
The secretion of oxalate into the gut happens regardless of the source of
those oxalates. That oxalate may have come from the diet, or from chemical
or environmental exposure to precursors to oxalate (such as to glycolic
acids), or from excess production of oxalates by our own cells due to
vitamin deficiency, genetic defect, or some other reason. Scientists have
found our bodies make excess oxalate when deficient in vitamin B6, which is
a vitamin that has been under a lot of study in autism. Some people may
make excess oxalate from an excess of glycine. There are also genetic
defects that produce excess oxalate. If there are times when our bodies
produce extra oxalates for a good purpose, it has not been discovered yet,
but we will be looking for this good purpose in our oxalate project.
One factor that may determine the level of secretion of oxalate into the
intestine is its concentration gradient. Oxalate will try and move from
places where it is in a higher concentration to places where it is in lower
concentration. An excess level of oxalate on the food side of enteric
cells may hamper the secretion of oxalates from the blood side. The body
may use signals like angiotensin II to step up oxalate secretion from
intestinal cells, but sometimes, even though the level in blood might be
higher, the secretion may be disrupted by a biochemical signal. This
happened experimentally when the signal from angiotensin II was
disrupted....something that might happen with an ACE inhibitor or possibly
with a chelating agent. More work needs to be done here.
It makes good sense that the body sends excess oxalate to the gut, because
the gut is where calcium from the diet could bind the oxalate and that
would keep it from being reabsorbed. The oxalate can just stay in the stool
in the form of calcium oxalate because it is only the unbound form that is
readily absorbed. There are many studies about this.
How do the microbes in the gut affect oxalate absorption?
A different method of reducing oxalate absorption is provided by microbes
inside the gut whose role is to eat oxalates and turn them into something
else. Unfortunately, these same microbes are easily killed off by
antibiotics. Quite a number of studies have found a lack of these
specialized bacteria in people who develop oxalate-related health problems.
Trying to address this problem, a biotech company is currently working on a
probiotic/enzyme formula to "recolonize" the most capable oxalate-eating
bacteria, which is oxalobacter formigenes.
The intestines might feel better when the secretion into the gut of oxalic
acid is reduced, because research has shown that oxalic acid is by nature
corrosive and burning to tissues. Even so, whenever the intestines lose
the ability to get rid of "waste" oxalate (using this secretion coupled
with binding calcium or being metabolized by oxalate-eating bacteria), then
the oxalate remaining in circulation can cause someone to suffer the
consequences of having higher levels of oxalic acid reaching other tissues.
What is the role of zonulin in opening up tight junctions?
A study a number of years ago found that the proteins from wheat and corn
could induce a leaky gut in rats which had first been made niacin
deficient. Since then, other work has found that there is a relationship
between exposure to the wheat protein gliadin and the excess production of
a talented disrupter of the tight junction called zonulin.
Zonulin is a physiological molecule which was discovered in 2000. Before
that discovery scientists had been studying a mimic of this molecule: a
toxin produced by a phage that infected a bacteria that could be infecting
a human. This toxin was called Zot, and all its talents at disruption of
the tight junction came from its being a mimic of zonulin. By watching
what Zot did, scientists learned a whole new set of interactions that were
governing paracellular transport in the gut.
Zonulin's presence (similar to lack of calcium) opens up the tight
junctions between cells. Scientists found that zonulin was elevated both
in serum and in the lumen of the gut in celiac disease. They also learned
by monitoring people with skin reactions to gluten called dermatitis
herpetiformis, that this leaky gut/zonulin phenomenon was a part of the
disease process that occurred before the flattening of the villi. They
learned that the disruption caused by zonulin could be set in motion by a
simple exposure to gliadin.
Apparently, zonulin keeps the gate open. I don't think they've figured out
exactly why or how it does that, but this may have to do with the fact that
the piece of gluten called gliadin mimics part of a molecule called
calreticulin that carries a huge load of calcium. Calreticulin is known to
be involved in the regulation of oxalate in plants, but any role for
calreticulin at this location at the tight junction has not been
characterized in animals. There also seems to be one other molecular mimic
of gluten at this site in intestinal cells.
Unfortunately, we are stuck with the order in which scientific discovery is
taking place because this work on zonulin and the leaky gut is brand new.
Antibodies to calreticulin have been found in celiac disease, and also in a
lot of other autoimmune diseases associated with a leaky gut that tend to
develop alongside celiac disease. This raises several questions:
-- Are the oxalates obtaining access to tissues in the body raising the
level of oxidative stress and tissue damage whenever they latch to damaged
tissues?
-- Is this what is exposing new antigens from organs like the pancreas, or
thyroid or connective tissues to the immune system, and helping to induce
autoimmune disease?
-- What happens when oxalates get into the brain if the blood brain
barrier is also leaky? Could this explain the origin of calcifications in
the brain associated with celiac disease which may be related to seizure?
Why is celiac disease the best model system for understanding these
mechanisms at the tight junction?
These new-found mechanisms confirm why hyperoxaluria is a well known
component of celiac disease. In fact, celiac disease is the main disease
emphasis circling around the study of zonulin. I've put abstracts from
three studies below that talk about this connection between zonulin and
celiac disease, but there are only thirteen articles on zonulin in the
whole of Medline so far.
How does this information relate to the diets that are now currently used
in autism?
Certainly, we need to consider that one of the benefits of gf/cf diet might
have been an improvement in the barrier function in the gut which would not
only reduce the opioid peptide absorption, but would also limit the
absorption of oxalates and allergy-provoking food peptides of all
sorts. We also need to consider that the introduction of very high
oxalate foods as a substitute for gluten and dairy may compromise some of
the benefits of removing gluten.
Disaccharidase deficiency apparently appears before villous atrophy shows
up in celiac disease. The order to this decline may suggest that something
related to the changes at the tight junction that come from exposure to
gliadin may furnish the reason that disaccharidase activity falls off so
early in celiac disease. Is there a parallel order of things in autism
that explains why disaccharidase activity would get low when it does? The
lack of disaccharidase activity is the reason for restricting disaccharides
in the SCD diet and that restriction does seem to quell the fire in the gut
for a significant set of people.
I find myself wishing that the SCD diet as it became used in autism hadn't
evolved with such an emphasis on high oxalate foods. There is nothing
about restricting disaccharides that means you HAVE to eat high oxalate
foods. What might have happened is that parents were seeking more calories
for their children, and wanted to make foods that were more like familiar
bread products because they couldn't use those complex carbohydrates in
cooking on SCD. Perhaps they found that their children really went after
these high oxalate foods once the high foods were introduced. The body
does accommodate somewhat to high oxalate foods, but the eagerness of these
children for these foods may have also been tied to a somewhat "addictive"
quality of oxalates that some parents have reported on our
website....addictions which seemed to diminish as the children's exposure
to oxalate was lowered.
Because these high oxalate foods were likely included in the SCD in an
attempt to enhance nutrition and the calorie count, including them in the
diet seemed the right thing to do. Most of the research showing the damage
that oxalates can do outside the kidney was and still is not widely
known. For this reason the originators of the SCD could not have
anticipated what oxalates were capable of doing when a leaky gut allowed
these oxalates to be absorbed in a higher than usual quantity.
When we began the oxalate project, we certainly didn't anticipate this
issue either, but we've learned this negative side of oxalates together as
we saw children improve in very surprising ways after getting off high
oxalate foods. Gastrointestinal issues were improving in these children
going lower oxalate. Yeast issues were getting better. Dysbiosis seemed
to be improving. Some of the children were getting rid of problems with
chronic diarrhea or constipation, in addition to having some relief from
urinary problems, if those had already developed. These changes were
amazing enough, but what really surprised us is when we started hearing
about changes in cognition and speech, fine motor and gross motor
improvement, and even catch-up growth in children who had not been
growing. Is there a chance these absorbed oxalates were also getting past
the blood brain barrier, a type of tissue whose barrier regulation is
similar to the gut?
Some moms and dads figured out the problem with high oxalate foods on their
own because they watched their children's reactions and took them off the
nuts and certain vegetables long before anyone started talking about
oxalates. These parents are the true scientists who were so very
observant! Our oxalate project gave them a vocabulary to describe what
they observed, but it also linked them to other parents who were finding
that their children were also affected by oxalates.
Do children on the low oxalate diet find improvements in previous intolerances?
We have found that some of the children who previously had horrible
reactions to rice and corn and even wheat and dairy are tolerating some of
these foods and other starches when sticking to a lower oxalate diet. The
increased tolerance to these foods may have come from getting the tight
junctions closed which would eliminate the overexposure of the immune
system to these food antigens. If this is truly the case, the change might
diminish allergic reactions to food and might possibly help restore the
disaccharidase activity. All this will have to be studied formally,
because right now these improvements are only the reports of
parents. Through this project we are getting closer to understanding what
it is that should be studied scientifically and what should be measured.
In summary, I hope all this will mean that we are getting closer to
understanding something important in the mechanisms that can set into
motion, or that will keep in motion, the gut disruption and
gastrointestinal pain in children with autism. Maybe a lot of the problem
circles around calcium regulation and tight junctions losing their grip,
but there may be other players and other places of disruption for these
molecules that scientists have not yet identified.
I find it fascinating how much we have been able to learn from celiac
disease research. Maybe we should think hard about how and why
gastroenterologists tell us that osteoporosis is often the first presenting
symptom of celiac disease, for that, of course, is another issue that has a
lot to do with calcium.
Our future goals of this project will go beyond the initial management of
diet issues or addressing related issues of tissue permeability. We are
currently recruiting scientists to specifically study oxalate issues in
autism. A further goal will be characterizing the functions of oxalates in
animals in areas that go beyond crystal formation, calcifications, or
kidney disease. Obviously, we are still at the very beginning of this project.
Please read the abstracts below. I hope they will help fill in the gaps in
this information, especially if you are comfortable with their technical
nature.
Susan Costen Owens
Research Associate, Husson Science Research Institute
A member of the DAN! Thinktank of the Autism Research Institute
Quote from: FASANO, A. Intestinal zonulin: open sesame! Gut,
2001;49;159-162
It has recently been reported that untreated CD predisposes
to autoimmune disorders such as insulin dependent
diabetes mellitus, Hashimoto's thyroiditis, autoimmune
hepatitis, and connective tissue diseases.28 One could
hypothesise that zonulin opens small intestinal TJs during
the early stages of CD and permits entry of putative allergens
into the intestinal submucosa where an autoimmune
response is elicited.
Alterations in intestinal TJ permeability
J Gen Physiol. 1997 Dec;110(6):727-40. Related Articles, Links
Click here to read
Calcium site specificity. Early Ca2+-related tight junction events.
Lacaz-Vieira F.
Department of Physiology and Biophysics, Institute of Biomedical
Sciences, University of Sao Paulo, 05508-900 Sao Paulo, Brazil.
lacaz@... The molecular mechanisms by which Ca2+ and metal ions interact with
the binding sites that modulate the tight junctions (TJs) have not been
fully described. Metal ions were used as probes of these sites in the frog
urinary bladder. Basolateral Ca2+ withdrawal induces the opening of the
TJs, a process that is abruptly terminated when Ca2+ is readmitted, and is
followed by a complete recovery of the TJ seal. Mg2+ and Ba2+ were
incapable of keeping the TJ sealed or of inducing TJ recovery. In addition,
Mg2+ causes a reversible concentration-dependent inhibition of the
Ca2+-induced TJ recovery. The effects of extracellular Ca2+ manipulation on
the TJs apparently is not mediated by changes of cytosolic Ca2+
concentration. The transition elements, Mn2+ and Cd2+, act as Ca2+
agonists. In the absence of Ca2+, they prevent TJ opening and almost
immediately halt the process of TJ opening caused by Ca2+ withdrawal. In
addition, Mn2+ promotes an almost complete recovery of the TJ seal. Cd2+,
in spite of stabilizing the TJs in the closed state and halting TJ opening,
does not promote TJ recovery, an effect that apparently results from a
superimposed toxic effect that is markedly attenuated by the presence of
Ca2+. The interruption of TJ opening caused by Ca2+, Cd2+, or Mn2+, and the
stability they confer to the closed TJs, might result from the interaction
of these ions with E-cadherin. Addition of La3+
microM) to the
basolateral Ca2+-containing solution causes an increase of TJ permeability
that fully reverses when La3+ is removed. This effect of La3+, observed in
the presence of Ca2+ (1 mM), indicates a high La3+ affinity for the
Ca2+-binding sites. This ability of La3+ to open TJs in the presence of
Ca2+ is a relevant aspect that must be considered when using La3+ in the
evaluation of TJ permeability of epithelial and endothelial membranes,
particularly when used during in vivo perfusion or in the absence of fixatives.
PMID: 9382899 [PubMed - indexed for MEDLINE]
FEBS Lett. 2005 Aug 29;579(21):4851-5. Related Articles, Links
Click here to read
Rapid disruption of intestinal barrier function by gliadin involves
altered expression of apical junctional proteins.
Sander GR, Cummins AG, Henshall T, Powell BC.
Tissue Development and Repair, Epithelial Biology Laboratory, Child
Health Research Institute, 72 King William Road, North Adelaide, SA 5006,
Australia.
guy.sander@... Coeliac disease is a chronic enteropathy caused by the ingestion of
wheat gliadin and other cereal prolamines derived from rye and barley. In
the present work, we investigated the mechanisms underlying altered barrier
function properties exerted by gliadin-derived peptides in human Caco-2
intestinal epithelial cells. We demonstrate that gliadin alters barrier
function almost immediately by decreasing transepithelial resistance and
increasing permeability to small molecules (4 kDa). Gliadin caused a
reorganisation of actin filaments and altered expression of the tight
junction proteins occludin, claudin-3 and claudin-4, the TJ-associated
protein ZO-1 and the adherens junction protein E-cadherin.
PMID: 16099460 [PubMed - indexed for MEDLINE]
Scand J Gastroenterol. 2001 Feb;36(2):163-8. Related Articles, Links
Intestinal disaccharidase deficiency without villous atrophy may
represent early celiac disease.
Murray IA, Smith JA, Coupland K, Ansell ID, Long RG.
Dept. of Gastroenterology, City Hospital, Nottingham, UK.
iainmurray10@... BACKGROUND: Intestinal disaccharidase activities are decreased in
untreated celiac disease and also in other conditions without villous
atrophy. Of 908 patients examined for suspected malabsorption, 37 (4.1%)
had generalized disaccharidase deficiency without villous atrophy. The aim
was to determine if generalized disaccharidase deficiency without villous
atrophy represented latent celiac disease. METHODS: Case notes and
histology of the 37 patients were reviewed. History and blood
investigations including antigliadin and endomysial antibodies were
checked. Where celiac disease was suspected, endoscopic duodenal biopsies
for histology and disaccharidase estimation were repeated. RESULTS: Of the
initial 37 patients, 6 patients had had repeat endoscopic biopsies; one
having celiac disease. A further 18 patients were reviewed. The remainder
declined further investigation. Eight had repeat endoscopic duodenal
biopsies; one had celiac disease. Two with positive celiac serology also
had enteroscopy with jejunal biopsies; both had celiac disease.
CONCLUSIONS: At least 11% of patients with generalized disaccharidase
deficiency without villous atrophy develop celiac disease. Enteroscopic
biopsies from distal duodenum and proximal jejunum should be considered as
the next investigation if endomysial or antigliadin antibodies are positive.
PMID: 11252408 [PubMed - indexed for MEDLINE]
J Cell Sci. 2000 Dec;113 Pt 24:4435-40. Related Articles, Links
Click here to read
Human zonulin, a potential modulator of intestinal tight junctions.
Wang W, Uzzau S, Goldblum SE, Fasano A.
Division of Pediatric Gastroenterology and Nutrition, Gastrointestinal
Pathophysiology Section, Center for Vaccine Development, Baltimore, MD
21201, USA.
Intercellular tight junctions are dynamic structures involved in
vectorial transport of water and electrolytes across the intestinal
epithelium. Zonula occludens toxin derived from Vibrio cholerae interacts
with a specific intestinal epithelial surface receptor, with subsequent
activation of a complex intracellular cascade of events that regulate tight
junction permeability. We postulated that this toxin may mimic the effect
of a functionally and immunologically related endogenous modulator of
intestinal tight junctions. Affinity-purified anti-zonula occludens toxin
antibodies and the Ussing chamber assay were used to screen for one or more
mammalian zonula occludens toxin analogues in both fetal and adult human
intestine. A novel protein, zonulin, was identified that induces tight
junction disassembly in non-human primate intestinal epithelia mounted in
Ussing chambers. Comparison of amino acids in the active zonula occludens
toxin fragment and zonulin permitted the identification of the putative
receptor binding domain within the N-terminal region of the two proteins.
Zonulin likely plays a pivotal role in tight junction regulation during
developmental, physiological, and pathological processes, including tissue
morphogenesis, movement of fluid, macromolecules and leukocytes between the
intestinal lumen and the interstitium, and inflammatory/autoimmune disorders.
PMID: 11082037 [PubMed - indexed for MEDLINE]
Display Show
J Biol Chem. 2004 Oct 22;279(43):44785-94. Epub 2004 Jul 30. Related
Articles, Links
Click here to read
Establishment and characterization of cultured epithelial cells
lacking expression of ZO-1.
Umeda K, Matsui T, Nakayama M, Furuse K, Sasaki H, Furuse M, Tsukita S.
Department of Cell Biology, Kyoto University Faculty of Medicine,
Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan.
In well polarized epithelial cells, closely related ZO-1 and ZO-2 are
thought to function as scaffold proteins at tight junctions (TJs). In
epithelial cells at the initial phase of polarization, these proteins are
recruited to cadherin-based spotlike adherens junctions (AJs). As a first
step to clarify the function of ZO-1, we successfully generated mouse
epithelial cell clones lacking ZO-1 expression (ZO-1-/- cells) by
homologous recombination. Unexpectedly, in confluent cultures, ZO-1-/-
cells were highly polarized with well organized AJs/TJs, which were
indistinguishable from those in ZO-1+/+ cells by electron microscopy. In
good agreement, by immunofluorescence microscopy, most TJ proteins
including claudins and occludin appeared to be normally concentrated at TJs
of ZO-1-/- cells with the exception that a ZO-1 deficiency significantly
up- or down-regulated the recruitment of ZO-2 and cingulin, another TJ
scaffold protein, respectively, to TJs. When the polarization of ZO-1-/-
cells was initiated by a Ca2+ switch, the initial AJ formation did not
appear to be affected; however, the subsequent TJ formation (recruitment of
claudins/occludin to junctions and barrier establishment) was markedly
retarded. This retardation as well as the disappearance of cingulin were
rescued completely by exogenous ZO-1 but not by ZO-2 expression.
Quantitative evaluation of ZO-1/ZO-2 expression levels led to the
conclusion that ZO-1 and ZO-2 would function redundantly to some extent in
junction formation/epithelial polarization but that they are not
functionally identical. Finally, we discussed advantageous aspects of the
gene knock-out system with cultured epithelial cells in epithelial cell
biology.
PMID: 15292177 [PubMed - indexed for MEDLINE]
J Neurochem. 2000 Jan;74(1):320-6. Related Articles, Links
Click here to read
Affinity purification and partial characterization of the
zonulin/zonula occludens toxin (Zot) receptor from human brain.
Lu R, Wang W, Uzzau S, Vigorito R, Zielke HR, Fasano A.
Division of Pediatric Gastroenterology and Nutrition and Center for
Vaccine Development, University of Maryland School of Medicine, Baltimore
21201, USA.
The intercellular tight junctions (TJs) of endothelial cells represent
the limiting structure for the permeability of the blood-brain barrier
(BBB). Although the BBB has been recognized as being the interface between
the bloodstream and the brain, little is known about its regulation.
Zonulin and its prokaryotic analogue, zonula occludens toxin (Zot)
elaborated by Vibrio cholerae, both modulate intercellular TJs by binding
to a specific surface receptor with subsequent activation of an
intracellular signaling pathway involving phospholipase C and protein
kinase C activation and actin polymerization. Affinity column purification
revealed that human brain plasma membrane preparations contain two Zot
binding proteins of approximately 55 and approximately 45 kDa. Structural
and kinetic studies, including saturation and competitive assays,
identified the 55-kDa protein as tubulin, whereas the 45-kDa protein
represents the zonulin/Zot receptor. Biochemical characterization provided
evidence that this receptor is a glycoprotein containing multiple sialic
acid residues. Comparison of the N-terminal sequence of the zonulin/Zot
receptor with other protein sequences by BLAST analysis revealed a striking
similarity with MRP-8, a 14-kDa member of the S-100 family of calcium
binding proteins. The discovery and characterization of this receptor from
human brain may significantly contribute to our knowledge on the
pathophysiological regulation of the BBB.
PMID: 10617135 [PubMed - indexed for MEDLINE]
: Biochim Biophys Acta. 2005 Nov 30;1762(1):80-93. Epub 2005 Oct
21. Related Articles, Links
Click here to read
VSL#3 probiotic preparation has the capacity to hydrolyze gliadin
polypeptides responsible for Celiac Sprue probiotics and gluten intolerance.
Angelis MD, Rizzello CG, Fasano A, Clemente MG, Simone CD, Silano M,
Vincenzi MD, Losito I, Gobbetti M.
Department of Plant Protection and Applied Microbiology, University of
Bari, 70126 Bari, Italy.
The native structure and distribution of gliadin epitopes responsible
for Celiac Sprue (CS) may be influenced by cereal food processing. This
work was aimed at showing the capacity of probiotic VSL#3 to decrease the
toxicity of wheat flour during long-time fermentation. VSL#3 (10(9) cfu/ml)
hydrolyzed completely the alpha2-gliadin-derived epitopes 62-75 and 33-mer
(750 ppm). Two-dimensional electrophoresis, immunological (R5 antibody) and
mass spectrometry analyses showed an almost complete degradation of
gliadins during long-time fermentation of wheat flour by VSL#3. Gliadins
non-hydrolyzed during fermentation by VSL#3 were subjected to
peptic-tryptic (PT) digestion and analyzed by CapLC-ESI-Q-ToF-MS (Capillary
Liquid Chromatography-Electrospray Ionization-Quadrupole-Time of
Flight-Mass Spectrometry). Search for several epitopes showed the only
presence of alpha2-gliadin-fragment 62-75 at a very low concentration
(sub-ppm range). Compared to IEC-6 cells exposed to intact gliadins
extracted from the chemically acidified dough (control), VSL#3 pre-digested
gliadins caused a less pronounced reorganization of the intracellular
F-actin which was mirrored by an attenuated effect on intestinal mucosa
permeability. The release of zonulin from intestinal epithelial cells
treated with gliadins was considerably lower when digested with VSL#3.
Agglutination test on K 562 (S) cells showed that the PT-digest of wheat
flour treated with VSL#3 increased the Minimal Agglutinating Activity of
ca. 100 times. Wheat proteins were extracted from doughs and subjected to
PT digestion. Compared to PT-digest from chemically acidified dough, celiac
jejunal biopsies exposed to the PT-digest from the dough fermented by VSL#3
did not show an increase of the infiltration of CD3(+) intraepithelial
lymphocytes. Proteolytic activity by probiotic VSL#3 may have an importance
during food processing to produce pre-digested and tolerated gliadins for
increasing the palatability of gluten-free products.
PMID: 16311022 [PubMed - in process]
2: Clin Gastroenterol Hepatol. 2005 Apr;3(4):335-41. Related Articles, Links
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Permeability, zonulin production, and enteropathy in dermatitis
herpetiformis.
Smecuol E, Sugai E, Niveloni S, Vazquez H, Pedreira S, Mazure R,
Moreno ML, Label M, Maurino E, Fasano A, Meddings J, Bai JC.
Dr. C. Bonorino Udaondo Gastroenterology Hospital, Av. Caseros 2061,
(1264) Buenos Aires, Argentina.
BACKGROUND & AIMS: Dermatitis herpetiformis (DH) is characterized by
variable degrees of enteropathy and increased intestinal permeability.
Zonulin, a regulator of tight junctions, seems to play a key role in the
altered intestinal permeability that characterizes the early phase of
celiac disease. Our aim was to assess both intestinal permeability and
serum zonulin levels in a group of patients with DH having variable grades
of enteropathy. METHODS: We studied 18 DH patients diagnosed on the basis
of characteristic immunoglobulin (Ig)A granular deposits in the dermal
papillae of noninvolved skin. Results were compared with those of classic
celiac patients, patients with linear IgA dermatosis, and healthy controls.
RESULTS: According to Marsh's classification, 5 patients had no evidence of
enteropathy (type 0), 4 patients had type II, 2 patients had type IIIb
damage, and 7 patients had a more severe lesion (type IIIc). Intestinal
permeability (lactulose/mannitol ratio [lac/man]) was abnormal in all
patients with DH. Patients with more severe enteropathy had significantly
greater permeability ( P < .05). The serum zonulin concentration
(enzyme-linked immunosorbent assay) for patients with DH was 2.1 +/- .3
ng/mg with 14 of 16 (87.5%) patients having abnormally increased values. In
contrast, patients with linear IgA dermatosis had normal histology, normal
intestinal permeability, and negative celiac serology. CONCLUSIONS:
Increased intestinal permeability and zonulin up-regulation are common and
concomitant findings among patients with DH, likely involved in
pathogenesis. Increased permeability can be observed even in patients with
no evidence of histologic damage in biopsy specimens. Patients with linear
IgA dermatosis appear to be a distinct population with no evidence of
gluten sensitivity.
PMID: 15822038 [PubMed - indexed for MEDLINE]
3: Lancet. 2000 Apr 29;355(9214):1518-9. Related Articles, Links
Click here to read
Comment in:
* Lancet. 2001 Nov 17;358(9294):1729-30.
Zonulin, a newly discovered modulator of intestinal permeability, and
its expression in coeliac disease.
Fasano A, Not T, Wang W, Uzzau S, Berti I, Tommasini A, Goldblum SE.
We identified zonulin, a novel human protein analogue to the Vibrio
cholerae derived Zonula occludens toxin, which induces tight junction
disassembly and a subsequent increase in intestinal permeability in
non-human primate intestinal epithelia. Zonulin expression was raised in
intestinal tissues during the acute phase of coeliac disease, a clinical
condition in which tight junctions are opened and permeability is increased.
Publication Types:
* Letter
PMID: 10801176 [PubMed - indexed for MEDLINE]
J Autoimmun. 2003 Dec;21(4):383-92. Related Articles, Links
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Epitopes of calreticulin recognised by IgA autoantibodies from
patients with hepatic and coeliac disease.
Sanchez D, Tuckova L, Mothes T, Kreisel W, Benes Z, Tlaskalova-Hogenova H.
Department of Immunology, Institute of Microbiology, Czech Academy of
Sciences, 142204 Prague, Czech Republic.
sanchez@... Calreticulin (CRT) was identified as a frequent target of serum
autoantibodies (Ab) in various diseases, but anti-CRT Ab of IgA isotype
were described only in coeliac (CLD) and some hepatic diseases. Employing
ELISA with recombinant CRT we found significantly higher (P<0.001) levels
of IgA anti-CRT Ab in sera of patients with primary biliary cirrhosis (PBC)
(77.6+/-8.9 AU/mean+/-SE), autoimmune hepatitis (AIH) (105.1+/-9.2 AU) and
alcoholic liver cirrhosis (ALC) (193.5+/-21.0 AU) relative to healthy
controls (38.6+/-2.0 AU). The levels of IgG anti-CRT Ab in sera of patients
with PBC (59.5+/-3.4 AU), AIH (89.7+/-7.9 AU) and ALC (86.4+/-6.2 AU) were
also significantly increased (P<0.001) when compared with controls
(38.5+/-2.1 AU). Pepscan technique with decapeptides of CRT (each
overlapping by eight amino acids) revealed antigenic epitopes of this
molecule recognised by IgA Ab of almost all tested patients-KGKNVLINKD and
QVKSGTIFDNFL. We also identified disease specific antigenic epitopes on CRT
molecule, predominantly recognised by IgA Ab of patients suffering from a
particular disease: GGYVKLFPNS and YVKLFPNSLD in AIH (83%, 92% of
patients), GLQTSQDARF and EQRLKEEEED in CLD (both 75%) and ASKPEDWDER in
ALC (67%). Identification of disease specific CRT epitopes contributes to
clarification of autoreactivity against this molecule.
PMID: 14624761 [PubMed - indexed for MEDLINE]
Eur J Immunol. 2001 Mar;31(3):918-28. Related Articles, Links
Click here to read
IgA cross-reactivity between a nuclear autoantigen and wheat proteins
suggests molecular mimicry as a possible pathomechanism in celiac disease.
Natter S, Granditsch G, Reichel GL, Baghestanian M, Valent P, Elfman
L, Gronlund H, Kraft D, Valenta R.
Department of Pathophysiology, AKH, University of Vienna, Vienna, Austria.
Celiac disease patients display IgA antibody reactivity to wheat as
well as to human proteins. We used serum IgA from celiac patients and, for
control purposes, from patients with Crohn's disease, ulcerative colitis
and from healthy individuals to identify celiac disease-specific IgA
autoantigens in nitrocellulose-blotted extracts from various human cell
types (epithelial, endothelial, intestinal cells, fibroblasts). The
pattern, recognition intensity and time course of IgA autoreactivity was
monitored using serial serum samples obtained from celiac children before
and under gluten-free diet. By immunoblot inhibition and subcellular
(cytosolic, nuclear) cell fractionation we identified a 55 kDa nuclear
autoantigen expressed in intestinal, endothelial cells and in fibroblasts
which was recognized by IgA antibodies of approximately half of the celiac
disease patients and cross-reacted with wheat proteins. IgA reactivity to
the 55 kDa autoantigen disappeared during gluten-free diet and was
inhibited after pre-absorption of sera with wheat proteins but not with
tissue transglutaminase, previously reported as the unique celiac
disease-specific autoantigen. In conclusion, we defined a novel 55 kDa
celiac disease-specific nuclear IgA autoantigen which shares epitopes with
wheat proteins and which is different from tissue transglutaminase and
calreticulin. Although the newly defined autoantigen was recognized much
less frequently than tissue transglutaminase, our data suggest molecular
mimicry between wheat and human proteins as a possible pathomechanism for
the induction and/or maintenance of mucosal tissue damage in celiac disease.
