Protection against Gluten-mediated Tight Junction Injury with a Novel Lignite Extract Supplement

Abstract

Background: Tight junctions are found in epithelial cells and function as selective gatekeepers to regulate absorption. PT-gliadin is the gluten protein segment that is known to impair the functioning of tight junctions. This study aimed to examine the effects of a lignite extract dietary supplement (RESTORE) on tight junction function in small intestine (IEC-6) and colon (Caco-2) epithelial cells. The study also evaluated the biologic safety of the same supplement as established by the rates of apoptosis in the intestinal and proximal renal tubule cell cultures treated with the supplement.
Methods: IEC-6 and Caco-2 cells were incubated until a stable trans-epithelial electrical resistance (TEER) was measured. The dietary supplement at 20% concentration or a control were placed on the cells and left overnight. These cells were then treated with and without PT-gliadin. Tight junction expression was determined by immunofluorescent microscopy. The rate of apoptosis was established in cell culture with the lignite extract at 20% concentration in order to assess a toxic concentration in normal cell lines: IEC-6, Caco-2, and human renal proximal tubule cell (RPTC) lines.
Results: The lignite extract supplement increased the TEER in IEC-6 (58%) and Caco-2 (15%) compared to control. PT-gliadin dramatically decreased the TEER in both control IEC-6 (49%) and control Caco-2 (27%) membranes. The lignite supplement prevented PT-gliadin-mediated decrease in TEER. The supplement reduced apoptosis in RPTC (44%), IEC-6 (13%), and Caco-2 (24%) cell cultures.
Conclusion: The lignite supplement blocked a PT-gliadin dependent decrease in TEER in small intestine and colon cell line membranes. The lignite extract was not toxic on intestinal or renal cells at high concentration, and demonstrated a statistically significant reduction in apoptosis in RPTCs. Human clinical trials are needed to evaluate the use of RESTORE to support health in gluten-sensitive individuals.

Full text

Protection against Gluten-mediated Tight Junction Injury with a Novel
Lignite Extract Supplement
John J Gildea1, David A Roberts2 and Zachary Bush3
1Department of Pathology, University of Virginia, 450 Ray C Hunt Drive, VA 22903, USA
2Chief Public Health Officer, Biomic Sciences, LLC, Charlottesville, VA 22905, USA
3Director of Clinical Affairs, Revolution Health Center, Charlottesville, VA 22902, USA
*Corresponding author: John J Gildea, Department of Pathology, University of Virginia, 450 Ray C Hunt Drive, VA 22903, USA, Tel: 4349249463; E-mail:
jjg5b@virginia.edu
Received date: July 22, 2016; Accepted date: August 25, 2016; Published date: August 29, 2016
Copyright: © 2016 Gildea JJ, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
Background: Tight junctions are found in epithelial cells and function as selective gatekeepers to regulate
absorption. PT-gliadin is the gluten protein segment that is known to impair the functioning of tight junctions. This
study aimed to examine the effects of a lignite extract dietary supplement (RESTORE) on tight junction function in
small intestine (IEC-6) and colon (Caco-2) epithelial cells. The study also evaluated the biologic safety of the same
supplement as established by the rates of apoptosis in the intestinal and proximal renal tubule cell cultures treated
with the supplement.
Methods: IEC-6 and Caco-2 cells were incubated until a stable trans-epithelial electrical resistance (TEER) was
measured. The dietary supplement at 20% concentration or a control were placed on the cells and left overnight.
These cells were then treated with and without PT-gliadin. Tight junction expression was determined by
immunofluorescent microscopy. The rate of apoptosis was established in cell culture with the lignite extract at 20%
concentration in order to assess a toxic concentration in normal cell lines: IEC-6, Caco-2, and human renal proximal
tubule cell (RPTC) lines.
Results: The lignite extract supplement increased the TEER in IEC-6 (58%) and Caco-2 (15%) compared to
control. PT-gliadin dramatically decreased the TEER in both control IEC-6 (49%) and control Caco-2 (27%)
membranes. The lignite supplement prevented PT-gliadin-mediated decrease in TEER. The supplement reduced
apoptosis in RPTC (44%), IEC-6 (13%), and Caco-2 (24%) cell cultures.
Conclusion: The lignite supplement blocked a PT-gliadin dependent decrease in TEER in small intestine and
colon cell line membranes. The lignite extract was not toxic on intestinal or renal cells at high concentration, and
demonstrated a statistically significant reduction in apoptosis in RPTCs. Human clinical trials are needed to evaluate
the use of RESTORE to support health in gluten-sensitive individuals.
Keywords: Lignite; Transepithelial electrical resistance (TEER);
Caco-2; IEC-6; Gluten sensitivity; Zonulin; RESTORE; PT-gliadin;
Tight junctions; Renal proximal tubule cell (RPTC)
Introduction
Tight junctions are expressed by epithelial and endothelial cells to
form the macro membranes of the digestive tract, vascular system, and
the blood-brain barrier. ese tight junctions function as selective
gatekeepers that regulate the absorption of macronutrients, and
compose a frontline of defense. e increased gut permeability that
results from tight junction dysfunction is increasingly recognized as an
early step in the pathogenesis of many acute and chronic inammatory
diseases, including celiac disease and inammatory bowel disease
(Crohn’s Disease and ulcerative colitis) [1-6]. e chronic
inammatory underpinnings of these conditions point to the chronic
immune system activation of the gastrointestinal-associated lymphoid
tissue that becomes exposed with tight junction dysfunction.
Gliadin is a component of gluten created during digestion that is
known to impair the functioning of tight junctions via the zonulin
occludin pathway. e common syndrome of gluten sensitivity now
aects more than 18 million individuals in the US alone. Celiac
disease, an autoimmune reactivity to gliadin, also aects a rapidly
growing number of individuals worldwide. e rapid rise of these
epidemics over the last 30 years raises the possibility of a progressive,
widespread biologic shi in the human intestinal microenvironment.
Healthy soil, similar to a healthy human intestinal ecosystem,
contains a vast library of nutrients, minerals, amino acids, and other
complex metabolites that are released through the digestive processes
of bacteria and fungi. As the nutrient density has waned in the soils of
our modern agricultural system, health practitioners around the world
have increasingly turned to fossil soil (lignite) extracts to supplement
human nutrition. Naturally-oxidized lignite extracts including shilajit,
humic acids, and fulvic acids have been used as dietary supplements to
deliver soil-based minerals and amino acids in China and India for
hundreds of years. eir clinical use has been limited by their oxidative
nature, frequent contamination by pathogenic bacteria, or inorganic
Journal of Nutrition & Food Sciences Gildea et al., J Nutr Food Sci 2016, 6:5
DOI: 10.4172/2155-9600.1000547
Research Article Open Access
J Nutr Food Sci, an open access journal
ISSN:2155-9600
Volume 6 • Issue 5 • 1000547

chemicals in these acidic compounds. Signicant contaminants that
occur during mining and manufacturing of these products in India
and China have been discovered to be present in numerous
commercial sources [7,8].
RESTORE is the rst lignite-derived dietary supplement that
delivers a stabilized family of carbon-based redox molecules as the
active ingredient resulting in an alkaline liquid form that carries only
trace minerals and amino acids. While various clinical trials have been
performed with some lignite-containing compounds, rigorous testing
of standardized extracts is needed to better establish the dierent
biologic eects and safety of these distinct classes of lignite extracts [9].
In this study, we examine the biologic eects of RESTORE lignite
extract on the tight junction barrier system of the gut via trans-
epithelial electrical resistance (TEER) of polarized epithelial
membranes of normal small bowel epithelium cells (IEC-6) and a
colon adenocarcinoma cell line that retains many characteristics of
normal colon epithelium cells (Caco-2) [10-12]. To our knowledge,
there are no previously published studies looking at lignite extract
eects on polarized epithelial cell lines as performed in this study.
Additionally, no lignite extract has been shown to be protective against
a known and prevalent intestinal toxin, PT-gliadin, an element of
gluten. Because gluten has been implicated as a causative agent in the
pathophysiology of celiac disease as well as in non-celiac gluten
sensitivity, we examined whether the lignite extract had protective
eects toward this pervasive and common food-borne toxic compound
on intestinal epithelial cell model systems [13,14].
e use of primary cultured renal proximal tubule cells (RPTCs) for
toxicity to xenobiotics is a well-established method for
in vitro
testing,
and therefore was used in the safety testing of the lignite extract
supplement [15]. Cells in culture show a constant, but low level of
apoptosis, and if a xenobiotic has even low levels of toxicity, the rate of
apoptosis is increased. erefore, a sensitive-ow cytometric assay for
apoptosis was chosen for testing RESTORE human RPTC lines.
Methods
Cell culture
Human colorectal carcinoma (Caco-2) and rat ileum epithelial
(IEC-6) cell lines were obtained from the American Type Culture
Collection (Manassas, VA, USA; ATCC catalog HTB-37 [Caco-2
cells]); ATCC catalogue CRL-1592 [IEC-6 cells]). Both cell lines were
propagated in their respective specic media according to
manufacturer protocols.
Primary cultured human renal proximal tubule cells (RPTC,
Lifeline), were the third polarized epithelial cell type used and were
propagated in its media according to manufacturer protocols. During
experiments each of the cells were cultured in the same media,
Dulbecco’s Modied Eagle Medium (DMEM-F12, Invitrogen,
Waltham, MA, USA) with 10% fetal bovine serum (FBS, Sigma-
Aldrich, St. Louis, MO, USA) at 37°C in 5% CO2 with and without
treatments.
Cell viability
Apoptotic cells were measured by incubation with Alexa 647
Annexin V (Invitrogen Waltham, MA, USA) in suspension and
measured by ow cytometry. Cells were trypsinized and incubated
with Annexin V according to the manufacturer’s protocol and
measured using a BD Accuri Flow Cytometer in 96 well plates using
the autosampler. Unstained and unlabeled cells were used for
background gaiting and apoptotic cell positive control cells were
identied by incubation with 10 µM staurosporine (Sigma-Aldrich, St.
Louis, MO, USA). ree dierent polarized epithelial cell types were
exposed to the lignite extract supplement at a 20% vol/vol
concentration in media for 18 hours and tested for Annexin V binding
as a measure of toxicity via induction of apoptosis. A 20%
concentration was used because it was the concentration which is
considered a maximal dose to determine if apoptosis is increased due
to toxicity.
RESTORE itself is comprised of the 150 mg of Terrahydrite™ lignite
extract at pH 8.7 and is therefore not considered fulvic or humic acid.
e supplement also has puried water, as well as a proprietary blend
the following ingredients, which comprise less than 1500 ppm:
(inorganic compound) chloride, sodium, lithium, calcium,
phosphorus, sulfur, bromide, potassium, iron, antimony, zinc, copper,
gold, magnesium, (organic compounds) alanine, glycine, histidine,
isoleucine, methionine, threonine, and valine.
Exposure of cells to gluten
One gram Gliadin (G3375, Sigma-Aldrich, St. Louis, MO, USA) was
digested with 20 mg Pepsin (7012, Sigma-Aldrich, St. Louis, MO, USA)
in 10 ml 100 mM HCl for two hours, then adjusted to pH 8.0 with 5 M
NaOH, then 20 mg Trypsin (8642, Sigma-Aldrich, St. Louis, MO, USA)
digested over four hours at room temperature. e trypsin was heat
inactivated at 90°C for three minutes. e PT-Gliadin peptides
responsible for tight junction disassembly-
QVLQQSTYQLLQELCCQHLW (151-170) and
QQQQQQQQQQQQILQQILQQ (111-130)-that bind to CXCR3 and
release zonulin have been previously identied [2].
Trans-epithelial electrical resistance
Trans-epithelial electrical resistance (TEER) was measured in
Caco-2 and IEC-6 cells seeded in 24-well transwell plates (1 micron
pore size, Becton Dickinson), incubated until a stable TEER was
measured three days in a row. TEER was measured using the epithelial
Volt-ohmmeter tted with a planar electrode (World Precision
Instruments). Water alone or water with RESTORE were used to make
media from powder (DMEM-F12, Invitrogen), and were placed on the
cells and incubated at 5% CO2 in a humidied 37°C incubator for the
appropriate amount of time. Peptic tryptic digest of gliadin (G3375,
Sigma), was made according to standard protocol, and added to apical
media at 1 mg/ml. Measurements were made at the two-hour time
point [16,17].
Zona occludens protein 1 immunouorescence microscopy
Directly following TEER measurements, IEC-6 cells were
simultaneously xed and permeablized in 4% paraformaldehyde
(Sigma-Aldrich, St. Louis, MO, USA) 1% Triton X100 (Sigma-Aldrich,
St. Louis, MO, USA) and incubated for 5 minutes. Cells were washed
and then blocked in 2% bovine serum albumin (BSA) and incubated
with 1 to 20 dilution of cell culture supernatant from Hybridoma clone
R26.4c, producing anti-ZO1 monoclonal antibody (Developmental
Studies Hybridoma Bank, Iowa City, IA, USA). Alexa 488 labeled
donkey anti-mouse IgG (Invitrogen, Waltham, MA, USA) was used at
4 µg/ml to make the antibody uorescent. Nuclei were stained with
Hoechst 5 µg/ml. Cells were imaged with a Zeiss (Oberkochen,
Citation: Gildea JJ, Roberts DA, Bush Z (2016) Protection against Gluten-mediated Tight Junction Injury with a Novel Lignite Extract
Supplement. J Nutr Food Sci 6: 547. doi:10.4172/2155-9600.1000547
Page 2 of 6
J Nutr Food Sci, an open access journal
ISSN:2155-9600
Volume 6 • Issue 5 • 1000547

Germany) Axiovert automated 6 D uorescent microscope and 100 ×
1.4 NA plan apochromatic objective.
Statistics
All experiments were run ve times and values shown are results of
each of the ve. Data are presented as mean values ± the standard error
from the mean. P-values were obtained by performing one-way
analysis of variance between groups.
Results
e two bowel-derived polarized epithelial cell lines, IEC-6 and
Caco-2, were able to consistently form a stable, electrically resistant
barrier on transwell inserts, and thus were tested for changes in TEER
when exposed to the lignite extract supplement. In both of these cell
types the TEER was signicantly increased in the presence of
RESTORE in comparison to the VEH (Figures 1 and 2) or vehicle
which is the carrier compound used to make the stock solution and is
used as a control in the experiment. In IEC-6 cells the TEER was
increased by 58%, n=8, p<0.05. In Caco-2 cells the TEER was increased
by 15%, n=4, p<0.01.
Figure 1: Eects of PT-gliadin (1 mg/ml) and RESTORE (Rstr)
lignite extract (20% vol/vol concentration in media) on the trans-
epithelial electrical resistance (TEER) of IEC-6 monolayers. Data
are presented for two independent experiments, with four replicates
each. Results are written as mean ± standard deviation [*represents
a TEER value that is statistically signicantly dierent from the
TEER of the vehicle (p<0.05)].
When testing the lignite extract’s eect in conjunction with a known
intestinal barrier toxin, PT-gliadin, PT-gliadin was found to decrease
TEER in IEC-6 by 49%, n=4, p<0.05. e lignite supplement
completely blocked PT-gliadin’s decrease on TEER, n=4, p<0.05.
Likewise, PT-gliadin decreased TEER in Caco-2 by 27%, n=4, p<0.05.
Again, the lignite extract completely blocked PT-gliadin’s decrease on
TEER, n=4, p<0.05.
A known mechanism by which PT-gliadin has been shown to
decrease TEER in IEC-6 and Caco-2 cells is by disruption of tight
junctions [18]. e eect of the lignite extract and PT-gliadin on tight
junction formation is seen by examining the tight junction localization
of ZO1 by immunouorescence microscopy in IEC-6 (Figure 3).
Figure 2: Eects of PT-gliadin (1 mg/ml) and RESTORE (Rstr)
lignite extract (20% vol/vol concentration in media) on the trans-
epithelial electrical resistance (TEER) of Caco-2 monolayers. Data
are presented for two independent experiments, with four replicates
each. Results are written as mean ± standard deviation [*represents
a TEER value that is statistically signicantly dierent from the
TEER of the vehicle (p<0.05)].
Figure 3: ZO1 immunouorescence of IEC-6 cells. e cells were
grown and then exposed to Hybridoma clone R26.4c to produce
anti-ZO1 monoclonal antibody. Alexa 488 labeled donkey anti-
mouse IgG was used at 4 µg/ml to make the antibody uorescent.
Nuclei were stained with Hoechst 5 µg/ml. Cells were imaged with a
Zeiss (Oberkochen, Germany) Axiovert automated 6 D uorescent
microscope and 100 × 1.4 NA plan apochromatic objective. Images
were taken of (a) the vehicle, (b) exposure to lignite extract (20%
vol/vol concentration in media) only, (c) exposure to PT-gliadin (1
mg/ml) only, and (d) exposure to both lignite extract (20% vol/vol
concentration in media) and PT-gliadin (1 mg/ml).
In VEH control cells the localization of ZO1 shows intermittent
gaps in ZO1 between cells (a). When RESTORE was incubated with
cells for only two hours, an increase in abundance of ZO1 between
Citation: Gildea JJ, Roberts DA, Bush Z (2016) Protection against Gluten-mediated Tight Junction Injury with a Novel Lignite Extract
Supplement. J Nutr Food Sci 6: 547. doi:10.4172/2155-9600.1000547
Page 3 of 6
J Nutr Food Sci, an open access journal
ISSN:2155-9600
Volume 6 • Issue 5 • 1000547

cells can be visualized as represented by (b). PT-gliadin dramatically
decreased the continuity of ZO1 localization between cells (c), and
RESTORE prevented this loss of ZO1 localization between cells (d).
In all three cell types exposed to the lignite extract supplement and
tested for toxicity, there was no increase in apoptosis (Figure 4) when
measured for Annexin V binding by ow cytometry. In both IEC-6
(13%) and Caco-2 (24%) cells there was a trend toward lower apoptotic
rates, but these did not reach statistical signicance. In RPTCs there
was a statistically signicant decrease in the rate of apoptosis (-44 ±
5%; N=4; p<0.001).
Figure 4: Eects of lignite extract (20% vol/vol concentration in
media) on apoptosis of IEC-6, Caco-2 and RPT cells was measured
incubation with Alexa 647 Annexin V in suspension and measured
by ow cytometry. Annexin V binding as a measure of toxicity via
induction of apoptosis with (grey bars) and without (black bars)
lignite extract (Rstr) [*represents a value that is statistically
signicantly dierent from the vehicle (p<0.05)].
Discussion
Intercellular tight junctions are an integral part of forming a
functional polarized epithelial layer and allowing vectorial transport of
water and electrolytes across the intestinal epithelium. e anatomical
and functional arrangement of the gastrointestinal tract regulates
passage of micro-and macro-molecules between the environment and
the host through transcellular transport (micromolecules) and
paracellular diusion (macromolecules) via modulation of the
intercellular tight junctions. To prevent harm to the host and reduce
inammation, a fully functional paracellular pathway minimizes
antigen presentation and toxin exposure of the gut-associated
lymphoid tissue (GALT) adjacent to the bowel epithelium. ese tight
junctions, also called zonula occludens, form a regulatory barrier
throughout the digestive tract that acts as an active transport pathway
of macronutrients into the body, and a rewall against unwanted toxins
and host pathogens [19].
A growing number of manufactured and naturally-occurring
elements in processed foods and mono-crop farming are being
implicated in tight junction damage [20]. In the developed world, the
unintentional chronic stimulation of zonulin-mediated intestinal
permeability from food elements, such as the gluten-derived peptides
that include the PT-gliadin used in this study, compromises tight
junction integrity and leads to unregulated absorption of organic and
inorganic material. Gluten is a protein found in foods processed from
wheat and other related grains (e.g. kamut, barley, and rye). e
quantity of rened gluten products has markedly increased in the
American diet in recent decades, and the rates of clinically-recognized
gluten sensitivity and allergy are rapidly on the rise. e clinical
manifestations of gluten sensitivity illustrate the chronic inammatory
repercussions of gluten-mediated membrane permeability, with
symptoms including arthralgia, fatigue, cognitive decits, irritable
bowel, neurologic dysfunction, and chronic pain [21,22]. e ndings
in this study demonstrate a common mechanism by which gluten-
mediated tight junction damage can predispose the host to
unregulated antigen presentation at the GALT.
ere are only a handful of studies that have shown improvements
in tight junction formation and trans-epithelial electrical resistance of
polarized epithelial cells including small and large intestine cells. Some
of these substances include the bioavonoid quercetin and indole,
butyrate, nicotine, the amino acid L-glutamine, the mineral zinc, the
pharmaceutical compound and zonulin-inhibitor larazotide, and now
the novel lignite extract studied here [19-30]. e lignite extract
supplement has unique biologic eects among these reported
compounds in both the speed of response in the TEER functional
analysis of the tight junction, which occurred within 60 min from
introduction to the membranes, and the extent of the response in
regard to tight junction protein expression as seen by
immunouorescence.
is study also demonstrates that the addition of the lignite extract
supplement to the intestinal membranes can stabilize tight junction
integrity in the face of PT-gliadin exposure. ese results suggest a
previously unrecognized biologic factor in the widespread
development of tight junction dysfunction and the resulting disease
epidemics in the developed world. e cumulative usage of antibiotics
in humans and meat production coupled with the rapid use and
accumulation of herbicides and pesticides in our agricultural system
over the last 30 years has had a major impact on the bacterial
biodiversity in the human gut [31]. ese bacteria play a signicant
role in maintaining the tight junction integrity.
Because numerous products utilizing geologic sediments have tested
positive for toxic levels of heavy metals, the lignite supplement was
tested by ultra-sensitive mass spectrometry based heavy metal
detection and found to be free of potentially toxic heavy metals or soil
minerals [7,8]. It was further tested for toxicity on a cell type known to
be very sensitive to toxins, namely human primary cultured RPTCs
[15]. Results from this toxicity testing were surprising; in that even at
very high levels of exposure, there was never any measurable toxicity.
Even more unusual was nding improved vitality in the cultured cells
as measured by a decrease in apoptosis.
Lignite extracts have been used in traditional medicine practices all
around the world, including shilajit, humic acid, and fulvic acid. No
studies have been found that examine these traditional lignite extracts
on proximal tubule, small intestine, and large intestine cells in culture.
Fulvic acid has been recognized to penetrate cell membranes and aect
cellular biology more directly than the larger-particle humic and
shilajit compounds. In this study we demonstrate that the lignite
extract decreases apoptosis in the RPTC cultures, suggesting a unique
safety prole.
Citation: Gildea JJ, Roberts DA, Bush Z (2016) Protection against Gluten-mediated Tight Junction Injury with a Novel Lignite Extract
Supplement. J Nutr Food Sci 6: 547. doi:10.4172/2155-9600.1000547
Page 4 of 6
J Nutr Food Sci, an open access journal
ISSN:2155-9600
Volume 6 • Issue 5 • 1000547

e public health implications of these ndings may be profound as
gut membrane permeability via tight junction damage is increasingly
being recognized as a root cause source for systemic inammation and
immune dysregulation [32]. e clinical manifestations of the ‘leaky
gut’ phenomenon that are specic to the gliadin-mediated damage
demonstrated in this study include the current epidemics of gluten
sensitivity and celiac sprue. Tight junction injury is also implicated in a
broad spectrum of seemingly disparate diseases including asthma,
allergies, autism spectrum disorders, mood disorders, Parkinson’s
disease, and Alzheimer’s dementia. ere is on-going debate as to
whether the apparent increases in the incidence of these conditions are
simply reective of increasing public and practitioner awareness and
screening, or if in fact there has been a system wide environmental
change in the food system and gut microbiome that have allowed for
widespread vulnerability to tight junction damage. e data from this
study demonstrate a clear mechanism by which the public’s dietary
consumption of rened wheat products may result in gut membrane
permeability, unregulated antigen presentation to the GALT, and an
increase in chronic inammatory conditions in our population.
Clinical studies are needed to establish the population response and
potential clinical applications of lignite extract supplements in clinical
practice.
Acknowledgement
is study, including the experiments, analysis, and publication was
funded by Biomic Sciences, LLC (Charlottesville, VA, USA) which
produces RESTORE.
Competing Interests
DAR and ZB are shareholders and employees of Biomic Sciences,
LLC, the entity that produces the lignite extract supplement used in the
study. JJG is a consultant and shareholder of Biomic Sciences, LLC.
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Citation: Gildea JJ, Roberts DA, Bush Z (2016) Protection against Gluten-mediated Tight Junction Injury with a Novel Lignite Extract
Supplement. J Nutr Food Sci 6: 547. doi:10.4172/2155-9600.1000547
Page 5 of 6
J Nutr Food Sci, an open access journal
ISSN:2155-9600
Volume 6 • Issue 5 • 1000547

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Citation: Gildea JJ, Roberts DA, Bush Z (2016) Protection against Gluten-mediated Tight Junction Injury with a Novel Lignite Extract
Supplement. J Nutr Food Sci 6: 547. doi:10.4172/2155-9600.1000547
Page 6 of 6
J Nutr Food Sci, an open access journal
ISSN:2155-9600
Volume 6 • Issue 5 • 1000547