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Finding Gluten Peptides Inside Bacteria - Part 1

Whether gut bacteria play a role in the pathogenesis of celiac disease is
an important question that needs to answered by celiac disease research.
If such bacteria exist, a cure for celiac disease might exist by the
elimination of these bacteria from the gut.  I present here a discussion of
a research method that might provide the answer to that question.  I
strongly urge that NIH funding be set aside for a study utilizing this
method.

My article, "Are Commensal Bacteria with a Taste for Gluten the Missing
Link in the Pathogenesis of Celiac Disease?", which appears in the current
Spring 2004 edition of Scott Adams' "Celiac.com's Guide to A Scott-Free
Life Without Gluten" is available free at:

http://www.celiac.com/st_prod.html?p_prodid=967

That article proposes that the immune system may become intolerant to
gluten via an unknown species of pathogenic bacteria which is able to
internalize gluten peptides.  The result is that when dendritic cells
(antigen presenting cells) sample the pathogenic bacteria and break it down
into peptides for presentation to naive T cells, the internalized gluten
peptides are mixed with the bacteria's own native peptides.  The immune
system would not be able to distinguish internalized gluten peptides from
the pathogenic bacteria peptides.  When the dendritic cell presents these
internalized gluten peptides to a T cell, the T cell would receive signals
which tell the T cell that the internalized gluten peptides are from a
pathogenic bacteria.  Hence the immune system would learn to respond to
gluten peptides as though a pathogenic bacteria were present, resulting in
gluten intolerance.

My article suggests that celiac research should try to find and identify
such bacteria by searching for gluten peptides within the cell membranes of
gut bacteria.  In the time since writing that article, I have been looking
for methods to detect gluten peptides within bacteria.  I have found that
an established method exists that can do this.  That method is called
immunogold electron microscopy.

Immunogold electron microscopy uses monoclonal antibodies, labelled with
fine gold particles, to probe for and bind to specific epitopes of a
specific antigens in biological specimens.  In this case, the antigen of
interest is a gluten peptide. The gold particles (colloidal gold) are
typically between 1 and 20 nanometers in size.  Monoclonal antibodies are
produced by a process that involves first immunizing animals with the
desired antigen and ending up by harvesting monoclonal antibodies from
hybridomas which have been created by fusing B cells from the animals with
tumor cells.  See:

http://www.accessexcellence.org/AB/GG/monoclonal.html

A number of biotechnical companies offer off-the-shelf antibodies and
custom services to produce monoclonal antibodies made-to-order for specific
peptides.  A great source of information on antibodies can be found at:

http://www.antibodyresource.com/educational.html

Prepared biological specimens are exposed to a solution containing the gold-
labelled monoclonal antibodies.  The antibodies will bind to target
antigens present on the specimen surface.  Specimens are sliced into
ultrathin sections of tissues or cell suspensions (which includes
suspensions of bacteria cells.)  By slicing the bacteria into ultrathin
sections, access is gained to any antigens (gluten peptides) that have been
internalized by the bacteria.  When that specimen is examined under a
transmission electron microscope (TEM), the colloidal gold particles will
show up as distinct opaque spots indicating the presence and the location
of the target antigen (gluten peptide) within the specimen.

The process of searching for gluten peptides within bacteria would begin
with fecal samples collected from a variety of subjects: with and without
celiac disease; on gluten free and non-gluten free diets; adults and
children; twins discordant with celiac disease; etc.  Fresh feces from
gluten free subjects can be incubated with gluten peptides to see if any
bacteria in the feces take up gluten peptides.

Using a centrifuge, bacteria is separated from fecal matter, concentrated,
and collected in a suspension.  The bacteria suspension is further
processed and embedded within a cured small block of epoxy resin, or,
alternately, the suspension is fixed in a special low temperature resin and
frozen into a solid small block.  That block is then cut into ultrathin
sections with a glass knife using an ultramicrotome.  If frozen, the
sections are thawed.  The ultrathin sections are supported on fine metal
grids and then treated with solutions of stains and gold-labelled
monoclonal antibodies to be made ready for viewing under the electron
microscope.  Ultrathin sections prepared by freezing usually allow better
access to the specimen's antigen binding sites for the monoclonal
antibodies than do ultrathin sections which have been embedded in epoxy
resin.  The epoxy resin process is called ultramicrotomy, and the low
temperature process is called cryoultramicrotomy.

Immunogold electron microscopy has already been used in celiac disease
research to study the distribution of gliadin within enterocytes of the
intestinal mucosa.  Hence monoclonal antibodies suitable for immunogold
studies to find gluten peptides within bacteria have already been
produced.  See:

K.-P. Zimmer, T. Mothes, E. Méndez, P. Ciclitira. Immunoelectron
Microscopical Analysis of Gliadin Transport Pathways within Enterocytes.
10th International Symposium on Coeliac Disease, June 2002, Paris, France
http://www.maladiecoeliaque.com/colloque/Lectures/c7-8.htm

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Eur J Gastroenterol Hepatol. 2001 Oct;13(10):1189-93

A monoclonal antibody that recognizes a potential coeliac-toxic repetitive
pentapeptide epitope in gliadins.

Osman AA, Uhlig HH, Valdes I, Amin M, Mendez E, Mothes T.

Department of Laboratory Medicine, Clinical Chemistry and Molecular
Diagnostics, University Hospital, Leipzig, Germany.

OBJECTIVES: Antibodies that detect coeliac-toxic prolamins from wheat,
barley and rye are important tools for controlling the diet of coeliac
disease patients. Recently, a monoclonal antibody R5 that recognizes wheat
gliadin, barley hordein and rye secalin equally was described. In this
study, the epitope recognized by R5 was investigated. METHODS: Both a phage-
displayed heptapeptide library and overlapping peptides spanning the
sequence of alpha- and gamma-type gliadins (pepscan) were screened for
binding of R5. RESULTS: Both techniques yielded comparable pentapeptide
consensus sequences (phage display QXPW/FP; pepscan QQPFP). According to
recent observations, this peptide stretch may be of key importance in the
pathogenicity of coeliac disease. This sequence occurs repetitively in
prolamins (in gamma- and omega-type prolamins more frequently than in alpha-
type prolamins) together with several homologous peptide stretches, which
are recognized less strongly. CONCLUSIONS: R5 seems to be a good candidate
for the specific detection of putative coeliac disease-active sequences in
prolamins and thus represents a valuable tool for the quality control of
gluten-free food.

PMID: 11711775 [PubMed - indexed for MEDLINE]

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Continued in Part 2

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