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Exploratory, Randomized, Double-blind, Placebo-controlled Study on the Effects of Bifidobacterium infantis Natren Life Start Strain Super Strain in Active Celiac Disease


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: The aim of this exploratory trial was to establish if the probiotic Bifidobacterium natren life start (NLS) strain strain may affect the clinical course and pathophysiological features of patients with untreated celiac disease (CD). Positive findings would be helpful in directing future studies. : Twenty-two adult patients having 2 positives CD-specific tests were enrolled. Patients were randomized to receive 2 capsules before meals for 3 weeks of either Bifidobacterium infantis natren life start strain super strain (Lifestart 2) (2×10 colony-forming units per capsule) (n=12) or placebo (n=10), whereas they also consumed at least 12 g of gluten/day. A biopsy at the end of the trial confirmed CD in all cases. The primary outcome was intestinal permeability changes. Secondary endpoints were changes in symptoms and the Gastrointestinal Symptom Rating Scale, and in immunologic indicators of inflammation. : The abnormal baseline intestinal permeability was not significantly affected by either treatment. In contrast to patients on placebo, those randomized to B. infantis experienced a significant improvement in Gastrointestinal Symptom Rating Scale (P=0.0035 for indigestion; P=0.0483 for constipation; P=0.0586 for reflux). Final/baseline IgA tTG and IgA DGP antibody concentration ratios were lower in the B. infantis arm (P=0.055 for IgA tTG and P=0.181 for IgA DGP). Final serum macrophage inflammatory protein-1β increased significantly (P<0.04) only in patients receiving B. infantis. The administration of B. infantis was safe. : The study suggests that B. infantis may alleviate symptoms in untreated CD. The probiotic produced some immunologic changes but did not modify abnormal intestinal permeability. Further studies are necessary to confirm and/or expand these observations.
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Exploratory, Randomized, Double-blind, Placebo-controlled
Study on the Effects of Bifidobacterium infantis Natren Life
Start Strain Super Strain in Active Celiac Disease
Edgardo Smecuol, MD,*
Hui J. Hwang, MD,*Emilia Sugai, MD,*Laura Corso, MD,
Alejandra C. Chern
˜avsky, MD,
Franco P. Bellavite, MD,*Andrea Gonza
´lez, MD,
Florencia Voda
´novich, MD,
Marı´a L. Moreno, MD,*Horacio Va
´zquez, MD,*
Graciela Lozano, MD,*Sonia Niveloni, MD,*
Roberto Mazure, MD,*
Jon Meddings, MD,8Eduardo Maurin
˜o, MD,*and Julio C. Bai, MD*
Background/Aims: The aim of this exploratory trial was to establish
if the probiotic Bifidobacterium natren life start (NLS) strain strain
may affect the clinical course and pathophysiological features of
patients with untreated celiac disease (CD). Positive findings would
be helpful in directing future studies.
Methods: Twenty-two adult patients having 2 positives CD-specific
tests were enrolled. Patients were randomized to receive 2 capsules
before meals for 3 weeks of either Bifidobacterium infantis natren
life start strain super strain (Lifestart 2) (2 10
units per capsule) (n = 12) or placebo (n = 10), whereas they also
consumed at least 12 g of gluten/day. A biopsy at the end of the
trial confirmed CD in all cases. The primary outcome was intestinal
permeability changes. Secondary endpoints were changes in
symptoms and the Gastrointestinal Symptom Rating Scale, and in
immunologic indicators of inflammation.
Results: The abnormal baseline intestinal permeability was not sig-
nificantly affected by either treatment. In contrast to patients on pla-
cebo, those randomized to B. infantis experienced a significant
improvement in Gastrointestinal Symptom Rating Scale (P=
0.0035 for indigestion; P=0.0483 for constipation; P=0.0586 for
reflux). Final/baseline IgA tTG and IgA DGP antibody concentration
ratios were lower in the B. infantis arm (P= 0.055 for IgA tTG and
P= 0.181 for IgA DGP). Final serum macrophage inflammatory
protein-1bincreased significantly (P< 0.04) only in patients receiving
B. infantis. The administration of B. infantis was safe.
Conclusions: The study suggests that B. infantis may alleviate
symptoms in untreated CD. The probiotic produced some immu-
nologic changes but did not modify abnormal intestinal perme-
ability. Further studies are necessary to confirm and/or expand
these observations.
Key Words: celiac disease, probiotics, Bifidobacterium infantis NLS
super strain, gluten-free diet
(J Clin Gastroenterol 2013;47:139–147)
Celiac disease (CD) is an autoimmune intestinal disorder
affecting approximately 1% of the general population.
The disease is produced by an immune-mediated enter-
opathy triggered by ingested prolamins present in wheat,
barley, and rye (generically called gluten) occurring in
predisposal individuals carrying the characteristic HLA
haplotype DQ2 and/or DQ8. The disorder is characterized
by almost invariable mucosal damage as a consequence of
both the innate and adaptive immunologic response to the
offensive proteins in the small intestine mucosa.1
Until now, the only effective treatment for CD is
lifelong compliance with a gluten-free diet (GFD). Adher-
ence to treatment leads to clinical and histologic remission,
normalization of biochemical parameters, significant
reduction of long-term complications (eg, bone disease),
and improvement in quality of life.2,3 Consensus suggests
that the GFD must be absolutely strict to avoid CD-related
complications.2,4 However, most long-term assessments
suggest that absolute restriction of offensive proteins is
voluntarily achieved in only a variable proportion of cases,
usually <50%.5,6 Furthermore, assessing strict adherence
to dietary measures is complicated because hidden gluten is
found in many so-called “gluten-free foods.”7Considering
the risk induced by the continuing consumption of gluten,
and the fact that strict dietary restriction is not easy to
perform, the identification of new treatment alternatives is
Received for publication August 12, 2012; accepted October 3, 2012.
zDepartment of Alimentation, Hospital de Gastroenterologı´a“Dr.C.
Bonorino Udaondo”; yDepartment of Immunogenetics, Hospital de
Clı´nicas “Jose
´de San Martı´n”, Universidad de Buenos Aires; wConsejo
de Investigacio
´n en Salud, Ministerio de Salud, Ciudad de Buenos
Aires; #Department of Gastroenterology, Universidad del Salvador,
Buenos Aires, Argentina; and 8Gastrointestinal Research Group,
University of Calgary, Calgary, AB, Canada.
Funded, in part, by Consejo de Investigacio
´n en Salud; Ministerio de
Salud; Gobierno de la Ciudad de Buenos Aires; The National Institute
of Probiotics, Westlake Village, CA; and Research Grant from the
Research Committee of the Sociedad Argentina de Gastroenterologı´a.
This is an investigator-performed study and Natren Inc. had no direct
or indirect involvement in the design of the study, data collection,
nor preparation or submission of the manuscript. Inova Diagnostic
Inc. generously provided assays. Opinions and conclusions of the
study were exclusively produced by the authors. None of the
authors have a personal conflict of interest with the manufacturer.
Authors contribution: E.S.: Study design and study execution, analysis
of data, revision of manuscript. H.J.H.: Study execution, analysis of
data. E.S.: Laboratory procedures. L.C.: Dietary supervisio
´n for
patients. A.C.: Immunological tests and revisio
´n of manuscript.
F.P.B., M.L.M., S.N., R.M.: Study execution. A.G.: Study design
and dietary supervisio
´n for patients. F.V.: Immunological tests.
H.V.: Study design and analysis of data. G.L.: Pathology analysis.
J.M.: Permeability tests and revision of manuscript. E.M.: Study
design and critical revision of manuscript. J.C.B.: Study design,
supervision, analysis of data, writing of manuscript.
Reprints: Julio C. Bai, MD, Department of Medicine, Dr. C. Bonorino
Udaondo Gastroenterology Hospital, Av. Caseros 2061, Buenos
Aires 1264, Argentina (e-mail:
Supplemental Digital Content is available for this article. Direct URL
citations appear in the printed text and are provided in the HTML
and PDF versions of this article on the journal’s Website,
Copyright r2013 by Lippincott Williams & Wilkins
J Clin Gastroenterol Volume 47, Number 2, February 2013 |139
justified. Several new approaches targeting different options
are being explored as potential complementary or sub-
stitutive treatments of CD.8–10
The enteric microbiota plays a pivotal role in maintaining
health status in normal individuals, modulating the function of
the gut-associated lymphoid tissue (GALT).11 Dysbiosis, the
abnormal composition of the gut flora, has been associated
with autoimmune inflammatory disorders of the intestines
including CD.12 In this context, reduced concentrations of
Bifidobacterium species have been demonstrated in duodenal
biopsies and feces of active and nonactive CD patients.13
Furthermore, a very recent molecular study of the microbiota
of celiac patients confirms that bifidobacteria is not present in
the duodenum and exist in significantly lower levels in feces
compared with healthy controls.14
Probiotics, live or attenuated bacteria conferring a
significant health benefit to the host, are potential candi-
dates to influence pathophysiological mechanisms involved
in inflammatory disorders such as postinfective irritable
bowel syndrome (IBS)15 and CD,16–18 among others. In the
context of the CD pathogenesis, several studies have ex-
plored the role of Bifidobacterium species using in vitro and
ex vivo models. Some studies have reported that the use of
probiotic bacteria in sourdough fermentation increases the
degradation of gluten during the process. Furthermore,
bifidobacteria may change the gliadin-derived pattern by
in vitro intestinal digestion, attenuating the proinflam-
matory effect on intestinal epithelial cells.19 Secreted bio-
active factors from Bifidobacterium infantis have been
shown to normalize permeability defects of the intestinal
mucosa.20 Recently, Lindfors et al21 have demonstrated
that Bifidobacterium lactis reduces the toxic effects of
wheat-derived gliadin on epithelial cell cultures by inhibit-
ing the gliadin-induced increases in epithelial permeability.
Recent publications have shown down regulation of the
immune response caused by Bifidobacterium species in the
proinflammatory milieu of CD.22–24
Despite a large volume of preclinical information with
regard to the effect of probiotics on underlying mechanisms
of CD damage, to our knowledge there are no clinical
studies assessing these hypotheses. Thus, we designed an
exploratory trial to determine the potential effect of B.
infantis natren life start strain (NLS) super strain on
intestinal permeability, the perception of symptoms, and
inflammatory immunologic markers present in patients
with active CD before starting treatment and while con-
suming a regular gluten-containing diet. Results of such
exploratory study would help guide future trials with regard
to effects, power calculations, and considerations about the
potential use of probiotics as a reasonable replacement and/
or adjuvant treatment to the well-established GFD.
Study Population
Patients were recruited at a single center, the Small
Intestinal Clinic of the Dr C. Bonorino Udaondo Gastro-
enterology Hospital. Patients aged between 18 and 75 years
initially fulfilling serological criteria suggestive of CD were
invited to participate in the screening for the trial. The CD
serological protocol included tissue transglutaminase (tTG)
and the deamidated gliadin-derived peptide (DGP) anti-
bodies, both of the IgA and IgG subclass, and total IgA
serum concentration. If 2 of these tests were positive a
potential diagnosis of CD was considered. Additional in-
clusion criteria were: body mass index between 18.5 and
35.0; patients not taking medications such as non-steroidal
anti-inflammatory drugs, aspirin, lactulose, probiotics, and
prebiotics in any form of administration (eg, yogurts or
other dairy products) or alcohol from the week prior the
enrollment to the end of the trial. Patients were excluded if
they were diagnosed with refractory CD or severe compli-
cations or had other active chronic gastrointestinal (GI)
pathologies or comorbidities whose participation, in the
investigator’s judgment, would be inadvisable. We also
excluded patients with symptomatic neurological or psy-
chiatric conditions that could potentially interfere with the
study, patients with a clinical severity requiring immediate
treatment, subjects not willing to participate, pregnant
women, and major alimentary allergies.
Trial Design
We designed a placebo-control, double-blind, random-
ized study. The trial consisted of a 2-week run-in period,
3 weeks of treatment, and a follow-up visit at day 50 after
having initiated treatment with the GFD. A graphical
explanation of the general trial design is observed in
Figure 1. After written consent, a full clinical history and
physical examination was obtained. Clinical laboratory
studies during this time included routine blood tests, CD-
specific antibodies, and a serum bhuman chorionic gona-
dotropin pregnancy test. At the end of the run-in period,
patients fulfilling inclusion criteria were blindly randomized
to receive one of the following treatments: (a) B. infantis
NLS super strain, 2 capsules 3 times per day 15 minutes
before meals (breakfast, lunch, and dinner) (Lifestart 2;
Natren Inc., Westlake Village, CA) (210
units per capsule) or (b) placebo 2 capsules containing rice
flour, dehydrated potato powder, cellulose powder, and
hydroxypropyl-methylcellulose with the same treatment
scheme. Randomization was produced by an external and
independent person using the blocked method. Two nutri-
tion experts in CD provided standardized nutritional coun-
seling and assessed patients at baseline and at each follow-up
visit to assure intake of a gluten-containing diet (con-
sumption of at least 12 g/d of gluten was required).
Trial visits performed on day 0 (baseline), day 10 and
day 21 (end of the drug administration) included the fol-
lowing procedures: report of symptoms, vital signs, clinical
safety reports, urine collection for intestinal permeability and
nitrite/nitrate measurements, blood draws for tTG and DGP
0 7 14 21
Períod Pre
Gluten containing diet (17g /day)
res Consent.
Inf. Cytokines
Dietary Control
Dietary Control
Dietary Control
14 2
FIGURE 1. A general study design. GSRS indicates Gastro-
intestinal Syndrome Rate Scale; IP, inflammatory protein.
Smecuol et al J Clin Gastroenterol Volume 47, Number 2, February 2013
140 | r2013 Lippincott Williams & Wilkins
antibody serum concentrations. At baseline and follow-up
visits, patients completed the Gastrointestinal Symptom
Rating Scale (GSRS) questionnaire. Blood samples obtained
at each time point were tested for cytokine concentrations
both in serum and cell-free supernatant from isolated
peripheral blood mononuclear cell (PBMC) after 24 hours of
culture. At each time point, the count of ingested capsules
was recorded. Capsules were preserved refrigerated (4 to 81C)
during transportation and throughout the study period.
Endoscopically procured duodenal biopsies were
obtained at the end of the treatment period in all patients.
After biopsy, patients started consuming a GFD that was
instructed and assessed by the expert dietitians. Final fol-
low-up visit (day 50) included a detailed assessment of
adverse events related with the study.
The primary endpoint of the study was to determine
the effect of administration of B. infantis NLS super strain
at a single-dose on intestinal permeability measured by the
lactulose/mannitol fractional excretion ratio. Secondary
endpoints were: (1) the assessment of the outcome of clin-
ical symptoms measured by both the GSRS questionnaire
and the perception of the outcome of the most prevalent
symptoms; (2) to evaluate modifications of immunologic
indicators of the gluten-driven inflammatory process, by the
production of CD-related antibodies and in the secretion to
plasma of a series of 17 cytokines, chemokines, and growth
factors, and the production of the same immunologic
mediators by nonstimulated 24-hour culture of PBMC.
Intestinal Permeability: Lactulose/Mannitol
After an overnight fast, patients ingested 5 g of lactulose
(Technilab, Montreal, Quebec, Canada) and 2g mannitol
(Sigma, St Louis, MO) in 450 mL of water (osmolality
approximately 1800 mOsmol/L). Patients collected all urine
passed over the ensuing 5 hours into a preweighed container
with 5 mL of 10% thymol in isopropanol. The fractional
excretion of lactulose and mannitol were calculated from
urinary concentrations determined by high pressure liquid
Clinical Assessment and GSRS Questionnaire
The presence of GI symptoms was assessed using the
GSRS questionnaire, which evaluates common symptoms
of GI disorders including CD.25 It is comprised of 15 items
grouped into 5 major GI syndromes: diarrhea, indigestion,
constipation, abdominal pain, and GI reflux syndromes.
Rating is based on a 7-point Likert scale, from 1 (no dis-
comfort) to 7 (very severe discomfort) where higher scores
indicate more severe GI symptoms. The scores for each
syndrome were calculated by taking the mean of the items
completed within an individual scale.25 In addition to the
analysis of the GSRS questionnaire, we explored patients’
perceptions of their most prevalent symptoms and changes
produced by the treatment. At each follow-up visit, patients
were asked about their perception of diarrhea, distension,
gas, and abdominal pain (patients reported symptoms as
improved, similar, or impaired).
CD Serology
Determination of CD-related antibodies was per-
formed after 2 different protocols. For the diagnosis of CD,
we used anti-tTG IgA and IgA and IgG antibodies against
synthetic DGPs (IgA DGP; IgG DGP) detected by enzyme-
linked immunosorbent assays (Inova Diagnostic Inc., San
Diego, CA) as reported in previous studies.26 Cut-off values
at 20 units (U)/mL were used as recommended by the
manufacturer. Because of the limitations of enzyme-linked
immunosorbent assay tests for the precision dealing with
higher antibody concentrations, analyses for the serologic
follow-up at each time point were performed at the Inova
Research laboratory using the chemiluminescent immuno-
assays (Inova Diagnostic Inc.). For this purpose, serum
samples were kept frozen at 201C until the assay was
performed. Assays were measured on the BIO-FLASH
instrument (Biokit SA, Barcelona, Spain), a fully auto-
mated chemiluminescent analyzer.
Blood Samples and Cytokine Detection
by Multiplex Microbead Immunoassay
Blood samples were collected from a peripheral vein
and kept on ice. Plasma was collected by centrifugation at
800gfor 15 minutes at 41C, aliquoted, and stored at 701C
until the analysis. A multiplex biometric immunoassay
was used for cytokine measurement according to the
manufacturer’s instructions (Bio-Plex Human Cytokine
Assay; Bio-Rad Inc., Hercules, CA). Cytokines and che-
mokines measured were: IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7,
CXCL8 (IL-8), IL-10, IL-12 (p70), IL-13, IL-17, gran-
ulocyte colony-stimulating factor (G-CSF), granulocyte
monocyte colony-stimulating factor (GM-CSF), monocyte
chemoattractive protein (MCP-1/CCL2), macrophage
inflammatory protein (MIP-1b/CCL4), and tumor necrosis
factor (TNF)-a. Cytokine levels were determined both in
cell-free supernatants and plasma using a multiplex array
reader from Luminex Instrumentation System (Bio-Plex
Workstation from Bio-Rad Laboratories).
Isolation and Culture of PBMC: Cytokine
Detection in Cell-free Supernatants by Multiplex
Microbead Immunoassay
Blood samples were collected by venous puncture
in heparin. PBMC were prepared by Ficoll-Hypaque den-
sity gradient centrifugation at 2000 rpm at room temper-
ature for 20 minutes, washed twice in phosphate-buffered
saline, counted, and resuspended in RPMI 1640 with 10%
fetal bovine serum, 2 mmol/L L-glutamine, and 50 mg/mL
gentamicin to yield a final concentration of 1 10
PBMC were incubated, nonstimulated, for 24 hours at 371C
in a 5% CO
humidified atmosphere. Cell-free supernatants
were stored frozen at 701C and analyzed for cytokine
levels as above, but without previous dilution.
Duodenal Histology
Seven biopsy samples were obtained from the de-
scending duodenum at different levels distal to the papilla
(n = 6) and the duodenal bulb (n = 1) using conventional
endoscopic forceps (open cup: 8 mm) during a sedated
upper endoscopy. Morphologic and quantitative assess-
ments (intraepithelial lymphocyte density) were performed
by an experienced pathologist. Morphology was catego-
rized according to the modified Marsh classification.27
Statistical Analyses and Ethical Issues
The protocol was approved by the Research and
Ethical Committees of the “Dr. C. Bonorino Udaondo”
Gastroenterology Hospital. A written consent was given
after serologic screening. Patients rejecting participation in
the trial received standard care. The trial was registered at under the number NCT01257620.
J Clin Gastroenterol Volume 47, Number 2, February 2013 B. infantis NLS Super Strain in Untreated Celiac Disease
r2013 Lippincott Williams & Wilkins |141
The analysis of results was performed on the intention-
to-treat basis. On the basis of data distribution, descriptive
data are reported either as mean and 95% of confidence
interval (CI) or median and range. Some data (serology and
cytokine concentrations) are also reported and analyzed as
final/baseline ratios. Data were analyzed using MedCalc
version (MedCalc Software bvba, Mariakerke,
Belgium). Comparisons within groups were performed
using paired ttest or Wilcoxon test for paired samples
according to distribution of data. For comparisons between
groups we used unpaired ttest and Mann-Whitney test.
Demography and Baseline Features
From December 2010 to August 2011, a total of 54
patients had positive serology tests according to the
requirements of the protocol, thereby making them candi-
dates for enrollment in the trial. Only 22 of these patients
(18 female) fulfilled the strict inclusion and exclusion criteria.
Most patients were excluded because they were following
some form of gluten restriction at the time of screening
(n = 18) or they did not accept the proposed trial (n = 10).
After randomization, 12 patients received B. infantis NLS
super strain and 10 ingested a placebo. Table 1 depicts the
demographic and clinical data of patients. Randomization
was not stratified according to other variables such as sex or
baseline clinical characterization. Thus, there was a sex
imbalance between groups with the probiotic arm including
the 4 men enrolled. No differences were found in terms of
clinical characterization (symptomatic vs. subclinical CD) at
diagnosis in both treatment groups. Table 2 reports baseline
mean GSRS syndrome scores for patients in the probiotic
and placebo arms, respectively. No statistical differences
were observed between treatment groups. The most common
symptoms for both groups were bloating and abdominal
distension (20/22), abdominal pain (19/22), and diarrhea
(11/22). Prevalence of symptoms at diagnosis was similar
between groups (data not shown).
Lactulose/mannitol ratios determined at study entry
were abnormal (ratio >0.025) in 19 patients. The sugar test
was within the normal range in 3 other cases, 2 in the
B. infantis NLS super strain arm and 1 in the placebo
group. Mean values and dispersions determined at baseline
are reported in Table 1. No statistical differences were
observed between groups at baseline (P= 0.816).
At diagnosis, all patients were positive for serologic
assays. Table 1 shows mean serum concentrations (95% CI)
of antibodies at baseline. Using the reported normal phys-
iological levels of human cytokines as reference (Bio-Rad
Laboratories Inc., Tech note 6029, 2010), serum immunologic
mediators concentrations were increased at baseline, charac-
terized by high mean values of proinflammatory cytokines
(TNF-aand IL-6), Th1 cytokine (INF-g), Th-17 cytokine
(IL-17), and chemokines (G-CSF, GM-CSF, MCP-1, MIP-
1b), but normal levels for Th2 cytokines (IL-4, IL-5, and IL-
10) (data not shown).
Effects of Treatment
Primary Endpoint: Intestinal Permeability
The mean lactulose/mannitol fractional urinary ratio
at the end of the trial in the placebo group (0.253; 95% CI,
0.098-0.407) was higher than at baseline but the difference
was not significant (P= 0.342). Similarly, the mean final
lactulose/mannitol ratio in the B. infantis NLS super strain
treated group (0.179; 95% CI, 0.0526-0.306) was non-
significantly higher than values determined at baseline
(P= 0.064). The comparison of final lactulose/mannitol
mean ratios for both treatment groups shows comparable
results (P= 0.417). Table 2 reports that baseline/final ratios
were similar for both treatment groups (P= 0.693).
Secondary Endpoints
Clinical aspects: Table 2 and Figure 2 summarize the
outcome of clinical features as they were assessed using the
GSRS questionnaire. Comparing baseline with final scores
within groups, patients treated with B. infantis NLS super
strain experienced a significant reduction in indigestion
(P=0.0035) and constipation (P=0.0483) symptoms, but
were borderline for reflux symptoms (P=0.0586). Both,
abdominal pain and diarrhea symptom scores decreased
but did not change significantly over the course of treat-
ment. Figure 2 depicts the time course dynamics of the 5
syndromes expressed as a percentage decline with respect to
baseline. In the probiotic group, a continual decline of
scores was observed for all syndromes, except diarrhea.
Interestingly, the mean diarrhea syndrome score decreased
by visit 1 (10 d after randomization) averaging 57% of
the baseline estimation; however, this score increased at the
final visit to an average of 27% from baseline (Supple-
mentary Table 1, Analysis
of the individual cases showed that one of the patients
without diarrhea at entry reported symptoms suggestive of
an acute gastroenteritis.
Patients in the placebo group had a different outcome
with respect to the GSRS syndrome scores. As shown
in Table 2 and Figure 2, placebo did not produce significant
changes in any syndrome except the significant improvement
TABLE 1. Demography, and Clinical, Serological, and Histologic
Characteristics of Patients at Baseline and According to
Characteristics Probiotic Arm Placebo Arm
No. patients (females) 12 (8) 10 (10)
Age, median (range) (y) 46 (29-62) 40 (20-71)
BMI, mean (95% CI)
23.2 (19.9-26.4) 22.1 (19.2-25.0)
Clinical characterization (No. patients)
Symptomatic CD 8 8
Subclinical CD 4 2
Intestinal permeability
ratio, median
0.054 (0.015-0.488) 0.110 (0.012-0.503)
Serology, mean (SEM) (U/mL)
IgA tTG 2121.5 (445.9) 1407.1 (441.2)
IgA DGP 356,8 (189.6) 296.8 (74.4)
IgG DGP 330.8 (83.7) 496.8 (123.4)
Marsh categorization (No. patients)
Marsh 3a 0 0
Marsh 3b 4 2
Marsh 3c 8 8
IELs density,
mean (SEM)
42.3 (1.3) 44.9 (2.4)
CD indicates celiac disease; CI, confidence interval; DGP, deamidated
gliadin-derived peptide; IEL, intraepithelial lymphocyte.
Smecuol et al J Clin Gastroenterol Volume 47, Number 2, February 2013
142 | r2013 Lippincott Williams & Wilkins
of diarrhea (P=0.0035). Differences observed between
treatment groups for all syndromes were not statistically
At the final visit, patients were asked whether they
perceived any change in diarrhea, distension, gas, or
abdominal pain. At this time point, diarrhea was reported
as improved in 4 of 5 and 5 of 6 patients treated with
B. infantis NLS super strain or placebo, respectively.
Interestingly, subjective improvement of abdominal dis-
tension and bloating at the final assessment was higher in
TABLE 2. Mean Scores and 95% Confidence Interval for the 5 Syndromes of the Gastrointestinal Syndrome Rate Scale Questionnaire
and the Composite Index (Final/Baseline Ratio) for the Probiotic and Placebo Arms
Probiotic Arm Placebo Arm
Syndromes Baseline Final Baseline Final
Indigestion 4.3 (3.4-5.3) 2.9 (2.1-3.7)* 4.0 (2.7-5.3) 3.6 (2.6-4.6)
Diarrhea 3.3 (2.0-4.7) 2.7 (1.6-3.8) 2.9 (1.7-4.0) 1.6 (0.9-2.3)*
Constipation 3.6 (2.2-5.0) 2.5 (1.4-3.5)^ 2.7 (1.5-3.9) 2.4 (1.0-3.7)
Gastroesophageal reflux 2.6 (1.9-3.4) 1.7 (1.1-2.3)12.2 (1.0-3.4) 2.0 (0.7-3.4)
Abdominal pain 3.1 (2.1-4.2) 2.4 (1.5-3.4) 3.3 (2.1-4.6) 2.8 (2.1-3.5)
Composite score
Final/baseline ratio 0.77 (0.56-0.97) 0.90 (0.62-1.18)
*P=0.0035; ^P=0.0483; 1P=0.0586.
baseline visit 2
baseline abdominal pain
-10% visit 2
-20% GE reflux
-30% Indigestion
abdominal pain
GE reflux
visiit 1
visiit 1
FIGURE 2. Mean percentage (%) of change for scores of the 5 Gastrointestinal Syndrome Rate Scale syndromes in the placebo (A) and
probiotic (B) arms.
J Clin Gastroenterol Volume 47, Number 2, February 2013 B. infantis NLS Super Strain in Untreated Celiac Disease
r2013 Lippincott Williams & Wilkins |143
patients of the probiotic arm (7/10) compared with those
with placebo (3/10). These differences were not significant.
No serious adverse effects or significant biochemical
changes were reported by patients in either treatment arm.
Immunologic markers, serum antibodies: Compared
with baseline values, serum antibody concentrations were
reduced at the final assessment for patients receiving pro-
biotics (averaging 10% for both IgA tTG and IgA DGP
antibodies) (data not shown). In contrast, patients in the
placebo arm had 7% and 10% increased antibody serum
concentrations at the end of the trial (for IgA tTG and IgA
DGP, respectively). Thus, the result expressed as final/
baseline ratio for IgA tTG serum concentrations had a
borderline significant reduction (P= 0.0558) in patients
receiving B. infantis NLS super strain compared with those
on placebo. A similar trend was shown for IgA DGP but, in
this case, such reduction was not statistically significant
(P= 0.1809; Table 3).
Outcome of inflammatory mediators: Globally, the
baseline Th1 biased profile of serum cytokines shown in
plasma did not change significantly in the analysis within
groups after both treatments (Supplementary Table 2, Similarly, no significant
changes were detected in the serum concentration of the
chemokines tested. However, we observed that the high
baseline serum concentration of MIP-1bincreased sig-
nificantly in patients treated with B. infantis NLS super
strain (P< 0.04), but not in the placebo group (Table 3).
No significant differences were observed comparing treat-
ment groups.
No significant changes were detected in the analysis
within groups in the production of cytokines and chemo-
kines detected in the supernatant of nonstimulated PBMC
culture. The only exception was an increased production of
IL-12p70 in the placebo group (P< 0.02) as seen by the
final/baseline ratio reported in Table 3. No changes were
demonstrated in patients treated with the probiotic. We
also did not detect significant changes in the final/baseline
ratios of proinflammatory cytokines (eg, IL-6) and in the
ratio of anti-inflammatory/proinflammatory mediators
(IL-10/IL-12p70) within groups (Table 3). No significant
differences were observed between groups.
This study is the first clinical trial assessing the effect of
a probiotic as the sole therapeutic intervention in patients
with untreated CD consuming gluten during the study
period. We specifically intended to explore the impact of
administering the probiotic bacterium B. infantis, which
was previously found in low concentrations in the intestine
of patients with untreated and treated CD.12–14 Our study
aimed to establish the effect of the probiotic independently
of the beneficial effect produced by a GFD. Thus, we
administered the study drug in the period between serologic
testing for CD and confirmation by the diagnostic duodenal
Our primary endpoint was to determine changes in
intestinal permeability. At baseline, 86% of patients
enrolled in the trial had abnormal lactulose/mannitol ratios
as evidence of mucosal damage, a proportion similar to that
found in our former publication.28 The present study shows
that the impaired barrier function was not significantly
modified by the probiotic. Thus, we were not able to
confirm former preclinical studies with probiotics of the
same family, which showed improvement in permeability
defects induced by gliadin administration.21 Potential
explanations for the evident lack of effect of B. infantis NLS
super strain on intestinal permeability could be related
to several factors such as the relatively short course of
treatment, the doses of probiotic used (we tested only a
single-dose regimen), or that the demonstrated preclinical
effects could be strictly dependent on the probiotic strain
In this study, we demonstrated that consumption of B.
infantis NLS super strain for 3 weeks improves perception
of some clinical syndromes (indigestion, constipation, and
gastroesophageal reflux) evaluated by the GSRS. However,
this effect was not observed with other syndromes (diarrhea
and abdominal pain). In contrast, patients in the placebo
group did not experience changes in any symptoms except
for the diarrhea syndrome, where a significantly improved
perception was demonstrated at the end of the trial. How-
ever, a more detailed analysis of GSRS for the diarrhea
syndrome at the middle of the trial shows that diarrhea
improved similarly in both study arms. Although such
TABLE 3. Final/Baseline Ratios for Intestinal Permeability (Lactulose/Mannitol Ratio), Serology (IgA tTG and IgA DGP), and Immunologic
Parameters (in Serum and in PBMC 24 h Culture Supernatant) in the Probiotic and Placebo Arms
Parameter Probiotic Arm Placebo Arm P
Lactulose/mannitol ratio
Final/baseline ratio, median (range) 1.11 (0.65-2.13) 0.99 (0.48-6.79)
Immunologic markers
Celiac disease serology (final/baseline antibody concentration ratio), median (range)
IgA tTG 0.90 (0.26-1.19) 1.07 (0.78-2.40) 0.0558
IgA DGP 0.90 (0.57-1.71) 1.10 (0.68-2.07) 0.1809
Inflammatory mediators
In serum (serum concentrations)
MIP-1b(pg/mL), median (range)
Baseline 99.3 (75.5-219.5) 104.8 (81.9-139.5)
Final 129.9 (78.3-379.2)* 98.8 (52.4-136.5)
In PBMC 24 h culture supernatant (final/baseline ratio), median (range)
IL-12p70 0.9 (0.1-4.2) 3.5 (1.2-4.4) <0.02
IL-6 0.8 (0.1-1.4) 1.0 (0.2-7.2)
IL-10/IL-12p70 ratio 1.0 (0.1-14.9) 0.5 (0.3-5.3)
*P< 0.04.
PBMC indicates peripheral blood mononuclear cell.
Smecuol et al J Clin Gastroenterol Volume 47, Number 2, February 2013
144 | r2013 Lippincott Williams & Wilkins
improvement persisted during the second half in patients
receiving placebo, increased diarrhea was perceived
between days 10 and the end of the trial in patients treated
with probiotics. Whether this was a consequence of a
reduced number of patients enrolled in both arms (berror)
or is the result of a lack of persistence of the response to the
probiotic is not clear. Supporting these perceptions based
on the quantitative analysis of the GSRS questionnaire, the
qualitative estimation by patients of the outcome of major
symptoms also found that probiotic therapy improved
distension, bloating, and gas. More than 70% of patients
reported that these symptoms had improved with pro-
biotics, whereas improvement occurred in 30% of patients
with placebo. Once again, diarrhea was perceived as im-
proved at the end of the trial by 80% of patients in both
treatment arms. Interestingly, these observations on dis-
tension, bloating, and gas are very similar to a previous
study of administration of B. infantis 35624 in women with
IBS (constipation predominant and diarrhea predom-
inant).29 Furthermore, studies using the referred Bifido-
bacterium strain (which seems equivalent to that used in the
present trial) in IBS also suggested a superior effect on
symptoms compared with those obtained with single bac-
teria or even a blend of probiotics.15,30,31 If this beneficial
effect of B. infantis NLS super strain in the management of
the most prevalent symptoms in patients with CD is con-
firmed in further studies, the mechanism(s) by which they
induce such improvement need(s) to be identified. A strong
body of evidence has explored potentially common patho-
biology of these overlapping symptoms affecting IBS and
gluten-sensitive patients. It has been suggested that neuro-
endocrinologic and immunologic factors might activate
efferent pathways, increasing acetylcholine release and pro-
ducing activation of peristaltic and secretory reflexes, which
may affect gut function generating symptom.32 Studies in
animal models of IBS and gluten sensitivity suggest that
gluten may contain a critical antigen triggering all these
abnormalities and symptoms, and that these disturbances
could, at least in part, involve innate immunity. It has been
hypothesized that the benefits produced by probiotics could
be induced by the anti-inflammatory effect of bacteria in the
inflammatory milieu.32
In this study we observed some changes in regard
to antibody production, which might have resulted from
the treatment. As expected, all patients had abnormally
increased serum concentrations of the 3 antibodies at base-
line. After the 3-week trial, patients in the probiotic arm
experienced a decrease of serum values of anti-tTG IgA
(averaging 10% reduction); contrarily, a 7% increment was
observed in the placebo arm. Compared with baseline val-
ues, the IgA DGP serum concentration decreased after the
probiotic (mean reduction 10%) but not with placebo (10%
increase). These differences did not reach statistical sig-
nificance in the comparison between within groups and
between groups. However, the consistent behavior of both
antibodies suggests that the effect could be associated with
the administration of B. infantis NLS super strain. Could
probiotic treatment of CD produce changes in antibody
concentration only 3 weeks after initiation? In this context,
although there is no data on shorter time frames, a previous
study by our group has shown that effective treatment with
a GFD induces a rapid serum antibody decline over a
3-month time course.26 Further studies should explore
whether these observations represent a genuine therapeutic
effect of B. infantis NLS super strain or not.
The effect of Bifidobacterium strains on the immune
response in the proinflammatory milieu of CD has been
explored by ex vivo studies and in vivo in animal models of
gluten sensitivity. On the basis these data, one of our sec-
ondary outcome measurements was to determine the impact
of B. infantis NLS super strain on the immunologic status of
patients by assessing both the serum production of cytokines
and chemokines and the release of the immune factors in
the supernatant of nonstimulated PMBC cultures from
samples obtained at baseline, and after the administration
of the probiotic and placebo and the production
of CD-related antibodies. At baseline, the assessment of
serum samples confirmed former observations on the
behavior of immunologic mediators in CD. Thus, our study
showed an increased concentration of proinflammatory
cytokines (TNF-aand IL-6), Th1 cytokines (INF-g), Th-17
cytokines (IL-17), and chemokines (G-CSF, GM-CSF,
MCP-1, MIP-1b, and IL-8), but normal levels for Th2
cytokines (IL-4, IL-5) and IL-10 (Va
´zquez H, Nachman FD,
Sugai E, unpublished personal observations).33 At the end of
the trial, the baseline proinflammatory status persisted in
both the group of patients, with similar concentrations for
cytokines and chemokines (data not shown). Furthermore,
no significant change was observed for Th2-type mediators
mean concentrations after treatment, findings that are similar
to those reported after 1 year on a GFD (Va
´zquez H,
Nachman FD, Sugai E, unpublished observations). The only
significant change we detected was that the high baseline
concentration of the MIP-1bwas further significantly
arm, but not in those on placebo. The potential importance
of the increased chemokine serum concentration after pro-
biotic treatment deserves further consideration. We suggest
that this finding in the probiotic treatment arm might be
relevant to the clinical response and comparable with the
reported down-modulation of CC chemokine receptors
recently described in human blood monocytes.34 MIP-1
proteins are potent chemoattractants of monocytes, but also
eosinophils, basophils, and lymphocytes, which are important
components of the inflammatory process, enhancing innate
immunity and T-cell responses in the GALT.35 However, the
effect of MIP-1 members in the release of cytokines by
immunologic cells is different depending on the inflammatory
activation. Animal studies have demonstrated a role for
CCR5; the receptor for CCL3 (MIP-1a), CCL4 (MIP-1b),
and CCL5, as essential to the induction of oral tolerance, and
that the lack of oral tolerance seen in CCR5
mice is
related to CCL5 regulation of CCL2 expression.36–38 Two
series of observations seem to be of value for interpretation of
our findings. Firstly, an experimental animal study showed
that CCR2 and CCR5 receptors have an important role
inducing symptoms (neuropathic pain associated to inflam-
mation) in rodents and that the agonist chemokine MIP-1b
39 This observation could be
relevant to our observation of improvement of symptoms in
patients of the probiotic arm coincident with the significant
increasedconcentrationofMIP-1b. Secondly, it has been
suggested that the use of a CCR5 agonist (eg, MIP-1b)in
conjunction with high-dose oral antigen might be able to
establish the GALT cytokine balance during ongoing auto-
immune disease toward anti-inflammatory state, and result in
a diminution of autoreactivity.35 In the context of these
observations, we consider that the potential relevance of the
probiotic treatment on MIP-1b/CCR5 axis deserves further
J Clin Gastroenterol Volume 47, Number 2, February 2013 B. infantis NLS Super Strain in Untreated Celiac Disease
r2013 Lippincott Williams & Wilkins |145
Our study has limitations. First, although the longi-
tudinal design allowed patients to serve as their own con-
trol, increasing the statistical power, this does not reduce
the importance of the low number of patients enrolled. The
length of the study period, the type or species of probiotics
to be explored (a single bacterium or a blend), and the doses
of probiotics to be used (the present trial was based on
single dose) are variables to be considered in further studies.
All these observations mean that our results should be
carefully considered. Finally, we recommend that, given the
results of this trial, further studies should explore surro-
gates of innate immunity as a potential pathogenic factor
involved in symptom generation and as targets for the
suggested role of probiotics. If abnormal, this observation
could give insight to the symptomatic treatment of both CD
and other gluten-related disorders such as non-CD gluten
In summary, data from the present exploratory study
suggest that administration of B. infantis NLS super strain to
untreated CD patients for 3 weeks does not modify inflam-
matory protein abnormalities, but might improve symptoms
and produce some immunologic changes. We conclude that
our data suggest that future trials are necessary to confirm
whether the use of B. infantis NLS super strain in patients
with untreated CD is warranted. The design used in this
study also provides insights to future clinical research trials
designed to generate information independent of the effect of
gluten restriction. Whether the chosen probiotic and the
doses used are optimal needs additional evaluation. Fur-
thermore, future studies should also consider using a greater
number of patients. If further studies confirm the benefit of
B. infantis NLS super strain, the addition of a probiotic to a
GFD might help symptomatic recovery or, alternatively,
could provide protection to the small intestinal mucosa
against the involuntary consumption of traces of gluten or in
voluntary transgressions. Furthermore, the results of this
study suggests that use of probiotics should be explored to
treat symptoms in other gluten-related disorders.
The authors thank Natasha Trenev from the National
Institute of Probiotics (Westlake Village, CA) who stimu-
lated our project and provided us with the probiotic and
placebo capsules; Victoria Thon, PhD from Natren Inc., for
critically reading the project; Gary Norman from Inova
Diagnostic Inc. (San Diego, CA) for testing sera for anti-
body concentrations.
1. Schuppan D, Dennis MD, Kelly CP. Celiac disease: epidemiol-
ogy, pathogenesis, diagnosis, and nutritional management.
Nutr Clin Care. 2005;8:54–69.
2. National Institutes of Health Consensus. Development Confer-
ence Statement on Celiac Disease. Gastroenterology. 2005;128:
3. Nachman F, Maurin
´zquez H, et al. Quality of life in
celiac disease patients. Prospective analysis on the importance
of clinical severity at diagnosis and the impact of treatment.
Dig Liver Dis. 2009;41:15–25.
4. Fasano A, Catassi C. Current approaches to diagnosis and
treatment of celiac disease: an evolving spectrum. Gastro-
enterology. 2001;120:636–651.
5. Hall NJ, Rubin G, Charnock A. Systematic review; adherence
to a gluten-free diet in adult patients with coeliac disease. Alim
Pharmacol Ther. 2009;30:315–320.
6. Ludvigsson JF, Green PH. Clinical management of coeliac
disease. J Intern Med. 2011;269:560–571.
7. Catassi C, Fabiani F, Iacono G, et al. A prospective, double-
blind, placebo-controlled trial to establish a safe gluten
threshold for patients with celiac disease. Am J Clin Nutr.
8. Paterson BM, Lammers KM, Arrieta MC, et al. The safety,
tolerance, pharmacokinetics and pharmacodynamics effects of
single doses of AT-1001 in celiac disease subjects: a proof of
concept. Alim Pharmacol Ther. 2007;26:757–766.
9. Gass I, Bethune MT, Siegel M, et al. Combination enzyme
therapy for gastric digestion of dietary gluten in patients with
celiac sprue. Gastroenterology. 2007;133:472–480.
10. Brown GJ, Daveson J, Marjaso JK, et al. A phase I study to
determine safety, tolerability and bioactivity of Nexvax2sin
HLA-Dq2 + volunteers with celiac disease following a long-
term, strict gluten-free diet. Gastroenterology. 2011;140(suppl
11. Preidis GA, Versalovic J. Targeting the human microbioma with
antibiotics, probiotics, and prebiotics: gastroenterology enters
the metagenoma era. Gastroenterology. 2009;136:2015–2031.
12. Collado MC, Donat E, Ribes-Koninckx C, et al. Imbalances in
faecal and duodenal Bifidobacterium species composition in
active and non-active coeliac disease. BMC Microbiol. 2008;8:232.
13. Nistal E, Caminero A, Herra
´n AR, et al. Differences of small
intestinal bacterial populations in adults and children with/
without celiac disease: effect of age, gluten diet and disease.
Inflamm Bowel Dis. 2011;8:649–656.
14. Di Cagno R, De Angelis M, De Pasquale I, et al. Duodenal and
faecal microbiota of celiac children: molecular, phenotype and
metabolome characterization. BMC Microbiol. 2011;11:219.
15. O
´Mahony L, McCarthy J, Kelly P, et al. Lactobacillus and
Bifidobacterium in irritable bowel syndrome: symptom
responses and relationship to cytokine profiles. Gastroentero-
logy. 2005;128:541–551.
16. Rollan G, De Angelis M, Gobbetti M, et al. Proteolytic activity
and reduction of gliadin-like fractions by sourdough lactoba-
cilli. J Appl Microbiol. 2005;99:1495–1502.
17. Di Cagno R, De Angelis M, Lavermicco P, et al. Proteolysis by
sourdough lactic acid bacteria: effects on wheat flour protein
fractions and gliadin peptides involved in human cereal
intolerance. Appl Environ Microbiol. 2002;68:623–633.
18. Di Cagno R, De Angelis M, Auricchio S, et al. Sourdough
bread made from wheat nontoxic flours and started with
selected lactobacilli is tolerated in celiac sprue patients. Appl
Environ Microbiol. 2004;70:1088–1096.
19. Laparra JM, Sanz Y. Bifidobacteria inhibit the inflammatory
response induced by gliadins in intestinal epithelial cells via
modifications of toxic peptide generation during digestion.
J Cell Biochem. 2010;109:801–807.
20. Ewaschuk JB, Diaz H, Meddings L, et al. Secreted bioactive
factors from Bifidobacterium infantis enhance epithelial cell
barrier function. Am J Physiol Gastrointest Liver Physiol.
21. Lindfors K, Blomqvist T, Juuti-Uusitalo K, et al. Live
probiotic Bifidobacterium lactis bacteria inhibit the toxic effects
induced by wheat gliadin in epithelial cell culture. Clin Exp
Immunol. 2008;152:552–558.
22. Medina M, De Palma G, Ribes-Koninckx C, et al. Bifido-
bacterium strains suppress in vitro the pro-inflammatory milieu
triggered by the large intestinal microbiota of coeliac disease
patients. J Inflamm. 2008;5:19.
23. D
´Arienzo R, Maurano F, Lavermicocca P, et al. Modulation
of the immune response by probiotic strains in a mouse model
of gluten sensitivity. Cytokine. 2009;48:254–259.
24. De Palma G, Cinova J, Stepankava R, et al. Pivotal advance:
Bifidobacteria and gram-negative bacteria differentially influ-
ence immune response in the pro-inflammatory milieu of celiac
disease. J Leukoc Biol. 2010;87:765–778.
25. Nachman F, del Campo MP, Gonza
´lez A, et al. Long-term
deterioration of quality of life in adult patients with celiac
Smecuol et al J Clin Gastroenterol Volume 47, Number 2, February 2013
146 | r2013 Lippincott Williams & Wilkins
disease is associated with treatment noncompliance. Dig Liver
Dis. 2010;42:685–691.
26. Sugai E, Nachman F, Vazquez H, et al. Dynamics of celiac
disease-specific serology after initiation of a gluten-free diet
and use in the assessment of compliance with treatment. Dig
Liver Dis. 2010;42:352–358.
27. Rostami K, Kerckhaert J, Tiemessen R, et al. Sensitivity of
antiendomysium and antigliadin antibodies in untreated celiac
disease: disappointing in clinical practice. Am J Gastroenterol.
28. Smecuol E, Bai JC, Vazquez H, et al. Intestinal permeability in
celiac disease. Gastroenterology. 1997;112:1129–1136.
29. Whorwell PJ, Altringer L, Morel J, et al. Efficacy of an
encapsulated probiotic Bifidobacterium infantis 35624 in
women with irritable bowel syndrome. Am J Gastroenterol.
30. Kim HJ, Va
´zquez Roque MI, Camilleri M, et al. A randomised
controlled trial of probiotic VSL 3 and placebo in IBS with
bloating. Neurogatroenterol Motil. 2005;17:687–696.
31. Bausserman M, Michail S. The use of Lactobacillus GG in
irritable bowel syndrome in children. Results of a double-blind
randomized control trial. J Pediatr. 2005;147:197–201.
32. Verdu E, Armstrong D, Murray J. Between celiac disease and
irritable bowel syndrome. The “no man’s land” of gluten
sensitivity. Am J Gastroenterol. 2009;104:1587–1594.
33. Manavalan JS, Hernandez L, Shah JG, et al. Serum cytokine
elevations in celiac disease: association with disease presenta-
tion. Human Immunol. 2010;71:50–57.
34. Fox JM, Letellier E, Oliphant CJ, et al. TLR2-dependent
pathway of heterologus down-modulation for the CC chemokine
receptors 1, 2 and 5 in human blood monocytes. Blood. 2011;117:
35. DePaolo RW, Lathan R, Karpus WJ. CCR5 Regulates high
dose oral tolerance by modulating CC chemokine ligand 2
levels in the GALT. J Immunol. 2004;173:314–320.
36. Schall TJ, Bacon K, Camp RDR, et al. Human macrophage
inflammatory Protein a(MIP-la) and MIP-Ibchemokines
attract distinct populations of lymphocytes. J Exp Med.
37. Lerner CG, Horton MR, Schwartz RH, et al. Distinct
requirements for C-C chemokine and IL-2 production by
naive, previously activated, and anergic T cells. J Immunol.
38. Nath A, Chattopadhya S. Macrophage inflammatory protein
(MIP) 1aand MIP 1bdifferentially regulate release of
inflammatory cytokines and generation of tumoricidal mono-
cytes in malignancy. Cancer Immunol Immunother. 2006;55:
39. Padi SSV, Shi XQ, Zhao YQ, et al. Attenuation of rodent
neuropathic pain by an orally active peptide, RAP-103, which
potently blocks CCR2- and CCR5-mediated monocyte chemo-
taxis and inflammation. Pain. 2012;153:95–106.
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... A placebo-control, double-blind, randomized study including adult patients with positive serology for CD showed a reduction in symptoms, particularly constipation, in patients treated with Bifidobacterium infantis [62]. Furthermore, in children Bifidobacterium infantis decreases Paneth cell counts and the expression of α-defensin-5 in CD, both of which are also related to the modulation of innate immunity [63]. ...
Full-text available
Celiac disease (CD) is a chronic enteropathy caused by the ingestion of gluten in a genetically susceptible individual. Currently, a gluten-free diet (GFD) is the only recommended treatment. However, unintentional gluten ingestion or a persistent villous atrophy with malabsorption (regardless of a strict GFD) as in the case of Refractory Celiac Disease (RCD) represents a major issue. In this review, we have analysed and discussed data from both randomized controlled trials and observational studies concerning adjunctive therapies as well as novel therapies for the treatment of CD and RCD. The literature search was carried out through Medline and Scopus. In total, 2268 articles have been identified and 49 were included in this review (36 studies resulting from the search strategy and 13 from other sources). Today, GFD remains the only effective treatment, although steroids, mesalamine, and more recently biological therapies have found space in the complex management of RCD. Currently, studies evaluating the effectiveness of novel therapies are still limited and preliminary results have been controversial.
... Recent studies demonstrated that Bifidobacterium-based probiotic interventions effectively delayed CD progression by reducing tumor necrosis factor-α production (Klemenak et al., 2015). Contrastingly, Smecuol et al. (2013) reported opposite observations. In our study, an increased relative abundance of Bifidobacterium was causally linked to the risk of CD, indicating its detrimental effect on CD. ...
Full-text available
Background A growing number of studies have implicated that gut microbial abundance and metabolite concentration alterations are associated with celiac disease (CD). However, the causal relationship underlying these associations is unclear. Here, we used Mendelian randomization (MR) to reveal the causal effect of gut microbiota and metabolites on CD. Methods Genome-wide association study (GWAS) summary-level data for gut microbiota, metabolites, and CD were extracted from published GWASs. Causal bacterial taxa and metabolites for CD were determined by two-sample MR analyses. The robustness of the results was assessed with sensitivity analyses. Finally, reverse causality was investigated with a reverse MR analysis. Results Genetically, increased genus Bifidobacterium was potentially associated with higher CD risk (odds ratio [OR] = 1.447, 95% confidence interval [CI]: 1.054–1.988, p = 0.022) while phylum Lentisphaerae (OR = 0.798, 95% CI: 0.648–0.983, p = 0.034) and genus Coprobacter (OR = 0.683, 95% CI: 0.531–0.880, p = 0.003) were related to lower CD risk. Moreover, there were suggestive associations between CD and the following seven metabolites: 1-oleoylglycerophosphoethanolamine, 1-palmitoylglycerophosphoethanolamine, 1,6-anhydroglucose, phenylacetylglutamine, tryptophan betaine, 10-undecenoate, and tyrosine. Sensitivity analyses deemed the results reliable without pleiotropy. Conclusion We investigated the causal relationships between gut microbiota, metabolites, and CD with two-sample MR. Our findings suggest several novel potential therapeutic targets for CD treatment. Further understanding of the underlying mechanism may provide insights into CD pathogenesis.
... Children receiving the probiotic supplement showed an increased height velocity that paralleled a reduction in the intestinal inflammation, as measured by peripheral CED3 + T and TNF levels [40] . In a another double-blind randomized placebo-controlled study on untreated CeD patients at the start of the GFD ( n = 12), Bifidobacterium infantis (Natren Life Start, NLS) treatment for three weeks determined a significant improvement in Gastrointestinal Symptom Rating Scale and reduction of IgA anti-tTG antibodies but had no effects on intestinal permeability [41] . The effect of Bifidobacterium breve strains B632 and BRO3 was evaluated in a double-blind placebo-controlled trial on 49 children showing a reduction of TNF and IL-10, and a restoration of the physiological Firmicutes / Bacteriodetes ratio in the probiotictreated group [42 , 43] . ...
Objective: To evaluate the efficacy of a multispecies probiotic on clinical and laboratory recovery of children with celiac disease (CeD) at diagnosis. Methods: Children with newly diagnosed CeD entered a randomized double-blind placebo-controlled trial. A gluten-free diet (GFD) plus a multispecies probiotic or placebo were administered for 12 weeks. Growth, laboratory, and clinical parameters were recorded at enrollment, after 3 and 6 months of follow-up. Results: Overall, 96 children completed the study: 49 in group A (placebo) and 47 in group B (probiotic). A significant increase of BMI-Z score was found in both groups after 3 and 6 months of treatment (p < 0.001), however the increase of BMI-Z score was significantly higher and faster in Group B than in Group A. Other clinical and laboratory parameters improved in both groups after 3 and 6 months (p<0.001), but no difference was found between the groups and a comparable time trend was observed in both groups. Conclusions: Treatment with a multispecies probiotic induced a higher and faster increase of BMI in children with newly diagnosed CeD. The mechanism of this positive effect remains to be elucidated.
... It has been shown that Bifidobacterium infantis can alleviate gastrointestinal symptoms if administered to CD patients before meals for three weeks. 68 This can explain the occurrence and persistence of gastrointestinal manifestations among the majority of participants in the current study. ...
Full-text available
Background : Celiac disease is an autoimmune disorder triggered by gluten, that occurs in susceptible individuals and is associated with dietary restriction and subsequent nutritional deficiencies. This study investigated the diet quality, nutrition imbalances and nutrition status among young children,adolescents and adults with CD who were referred to several hospitals in Lebanon. Methods: A cross-sectional study in 50 individuals (31.74 ± 15.64 years) with CD who follow a gluten free diet was conducted, using biochemical parameters, anthropometric measurements, dietary and physical activity assessments. Results : Of the 50 participants, 38% and 16% were presenting low serum levels of iron and vitamin B12, respectively. The majority of participants were physically inactive and around 40% of them had low muscle mass. A weight loss of 10% to 30% indicating mild to moderate malnutrition was shown in 14% of individuals. The assessment of food-related behaviors shows that 80% of participants were reading nutrition labels and 96% of them were following gluten-free diets (GFD). Some barriers including family ignorance (6%), language of the nutrition labels (20%) and expensive GF products (78%) were limiting the adherence to GFD. The inadequacy of the daily energy intake along with insufficient intakes of calcium and vitamin D were remarked among individuals with CD. However, protein and iron intake were exceeding the recommendations among all age groups, except in males aged 4-8 years and 19-30 years. Half the study participants were using dietary supplements where 38%, 10%, 46%, 18%, 16% and 4% used vitamin D, vitamin B12, iron, calcium, folate and probiotics, respectively. Conclusion: GFD is the key treatment for CD. However, it is not without inadequacies and may cause certain deficiencies such as calcium and vitamin D leading to reduced bone density. This underlines the critical role of dietitians in education and maintenance of healthy GFD among individuals with CD.
Coeliac disease (CD) is associated with alterations in gut microbiota composition. This study evaluated the effects of probiotics on gut microbiota composition and clinical symptoms of treated CD patients. In this double-blind, placebo-controlled trial study, 31 CD patients that were randomly classified as probiotics (n = 15) and placebo (n = 16) groups received 109 colony-forming units/capsule for 12 weeks. Fecal samples were collected before and after probiotics, or placebo administration and the changes in intestinal microbiota were assessed by quantitative real-time PCR. Probiotic administration improved the patients' clinical symptoms when compared to the placebo group. Fatigue score was significantly reduced by the intake of probiotic supplements (P = 0.02). Except for Staphylococcus spp., the relative abundances of Bacteroidetes, Lactobacillus spp., Bifidobacterium spp., Clostridium cluster I, Enterobacteriaceae, and Firmicutes were higher in probiotics group. Accordingly, a 12-week multi-strain probiotic treatment regimen may modify the composition of intestinal microbiota and improve GI symptoms in CD patients.
Celiac disease is an autoimmune disorder in which the immune system of genetically susceptible individuals elicits a reaction to gluten causing small intestine damage. If left undiagnosed and untreated, the resulting nutritional malabsorption can lead to anemia, bone disease, growth faltering or other consequences. The condition is lifelong and lacks a cure; the only treatment is lifelong adherence to a gluten-free diet (GFD). This diet is challenging to follow and adversely impacts quality of life; however, it is essential to ensure intestinal recovery and prevent future negative health consequences. The Academy of Nutrition and Dietetics convened an expert panel complemented by a celiac disease patient advocate to evaluate evidence for six topics including Medical Nutrition Therapy (MNT), the GFD, oat consumption, micronutrients, pro-/prebiotics and the low FODMAP (Fermentable Oligosaccharides, Disaccharides, Monosaccharides and Polyols) diet. This publication outlines the Academy's Evidence Analysis Library methods used to complete the systematic review and guideline development, and summarizes the recommendations and supporting evidence. The guidelines affirm that all individuals with celiac disease should follow a GFD (1C, Imperative) that may include gluten-free oats in adults (2D, Conditional). Children should follow a nutritionally adequate GFD that supports healthy growth and development (Consensus, Imperative) and does not unnecessarily restrict gluten-free oats (Consensus, Conditional). The guidelines indicate nutritional care should include routine nutritional assessment (Consensus, Imperative) and MNT (Consensus, Imperative). At this time, the guidelines do not support a recommendation for the addition of the low-FODMAP diet (2C, Conditional), prebiotic or probiotic supplementation (2D, Conditional) or micronutrient supplementation (in the absence of nutritional deficiency) (Consensus, Conditional). The 2021 Celiac Disease Evidence-Based Nutrition Guideline will assist Registered Dietitian Nutritionists in providing appropriate evidence-based MNT to support people with celiac disease in achieving and maintaining nutritional health and avoiding adverse celiac disease consequences throughout their lives.
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Background: Probiotics play a vital role in treating immune and inflammatory diseases by improving intestinal barrier function; however, a comprehensive evaluation is missing. The present study aimed to explore the impact of probiotics on the intestinal barrier and related immune function, inflammation, and microbiota composition. A systematic review and meta-analyses were conducted. Methods: Four major databases (PubMed, Science Citation Index Expanded, CENTRAL, and Embase) were thoroughly searched. Weighted mean differences were calculated for continuous outcomes with corresponding 95% confidence intervals (CIs), heterogeneity among studies was evaluated utilizing I2 statistic (Chi-Square test), and data were pooled using random effects meta-analyses. Results: Meta-analysis of data from a total of 26 RCTs (n = 1891) indicated that probiotics significantly improved gut barrier function measured by levels of TER (MD, 5.27, 95% CI, 3.82 to 6.72, P < 0.00001), serum zonulin (SMD, -1.58, 95% CI, -2.49 to -0.66, P = 0.0007), endotoxin (SMD, -3.20, 95% CI, -5.41 to -0.98, P = 0.005), and LPS (SMD, -0.47, 95% CI, -0.85 to -0.09, P = 0.02). Furthermore, probiotic groups demonstrated better efficacy over control groups in reducing inflammatory factors, including CRP, TNF-α, and IL-6. Probiotics can also modulate the gut microbiota structure by boosting the enrichment of Bifidobacterium and Lactobacillus. Conclusion: The present work revealed that probiotics could improve intestinal barrier function, and alleviate inflammation and microbial dysbiosis. Further high-quality RCTs are warranted to achieve a more definitive conclusion. Clinical trial registration:, identifier CRD42021281822.
As one of the most prevalent autoimmune diseases, celiac disease (CD) affects 1% of people globally and is frequently linked to the HLA-DQ2/DQ8 polymorphism and gut microbiota dysbiosis. The possibility of different symptom severities between pediatric and adult populations, as well as the heterogeneity amongst them, might make diagnosis difficult. However, there are currently accessible diagnostic techniques, such as duodenal mucosal biopsies, serological screening for IgA and IgG as transglutaminase 2 specific antibodies, HLA haplotypes DQ8 and DQ2 and endoscopic assessment that is assessed on both healthy and vulnerable individuals. The only effective treatment for CD at the moment is rigorous, lifetime adherence to a glu-ten-free diet (GFD), which is sometimes a difficult challenge. The symptoms of accidental gluten intake cannot be controlled or mucosal damage prevented by a GFD alone. Additionally, many people may experience long-term consequences. There is hence an unmet demand for further therapies that can contain a reasonable combination of dietary-and non-dietary-based therapies for the management of CD. In this review, while exploring various aspects of etiology, pathophysiology, and nutritional issues related to CD, the latest findings of preclinical and clinical studies related to dietary-and non-dietary-based therapies are discussed.
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Celiac disease is a complex polygenic systemic disorder caused by dietary gluten exposure that selectively occurs in genetically susceptible people. The potential celiac disease is defined by the presence of celiac disease-specific antibodies and compatible human leukocyte antigen but without histological abnormalities in duodenal biopsies. At present, the only treatment is lifelong adherence to a gluten-free diet. Despite its effectiveness, the diet is difficult to maintain due to its cost, availability of gluten-free foods, and hidden gluten. The need to develop non-dietary treatment methods is widely recognized, but this is prevented by the absence of a pathophysiologically relevant preclinical model. Nonetheless, in vitro and in vivo models have made it possible to investigate the mechanisms of the disease and develop new treatment approaches: The use of foods with neutralized gluten, microbiota correction, cocktails of specific endoproteinase, polymer gluten binders, specific inhibitors of transglutaminases and inflammatory cytokines, and a vaccine based on allergen-specific therapy.
The interest in the intestinal microbiota of humans has increased in the last 20 years and significant advances have been achieved with regard to its structure and functions. Since gut microbiota is involved in a range of complex interactions with the host, its manipulation for promoting human health has been the object of several studies. Probiotics, with their long history of safety and effectiveness against harmful microorganisms, are a strategy to not only maintain or restore the correct balance in the microbial population of the intestinal tract, but also to prevent or treat a range of disease conditions. Personalized administration of selected probiotic strains is necessary to ensure the success of their application and is critical to implement their clinical use as a strategy to support preventive, personalized, and predictive medicine (PPPM).The aim of this review is to explore preclinical and clinical studies focused on the use of probiotics for the prevention and treatment of gastrointestinal, metabolic and neurological diseases. In vitro and in vivo studies on probiotic administrations allow the screening of strains for clinical application and the development of personalized probiotic treatments, in accordance with the PPPM perspective. The results achieved in this field showed the positive effects of probiotic supplementation, especially for Lactobacillus and Bifidobacterium genera, but new strains belonging to different genera such as Akkermansia are gaining interest. The examined studies support the concept of “precision probiotics” that represent the future of probiotic-based intervention, according to which individuals will be recommended specific diets and probiotics administration tailored to their unique gut microbiome structure.KeywordsPPPMGut microbiotaProbioticsTargeted microorganismsHealthDiseases
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Epidemiology of celiac disease (CD) is increasing. CD mainly presents in early childhood with small intestinal villous atrophy and signs of malabsorption. Compared to healthy individuals, CD patients seemed to be characterized by higher numbers of Gram-negative bacteria and lower numbers Gram-positive bacteria. This study aimed at investigating the microbiota and metabolome of 19 celiac disease children under gluten-free diet (treated celiac disease, T-CD) and 15 non-celiac children (HC). PCR-denaturing gradient gel electrophoresis (DGGE) analyses by universal and group-specific primers were carried out in duodenal biopsies and faecal samples. Based on the number of PCR-DGGE bands, the diversity of Eubacteria was the higher in duodenal biopsies of T-CD than HC children. Bifidobacteria were only found in faecal samples. With a few exceptions, PCR-DGGE profiles of faecal samples for Lactobacillus and Bifidobacteria differed between T-CD and HC. As shown by culture-dependent methods, the levels of Lactobacillus, Enterococcus and Bifidobacteria were confirmed to be significantly higher (P = 0.028; P = 0.019; and P = 0.023, respectively) in fecal samples of HC than in T-CD children. On the contrary, cell counts (CFU/ml) of presumptive Bacteroides, Staphylococcus, Salmonella, Shighella and Klebsiella were significantly higher (P = 0.014) in T-CD compared to HC children. Enterococcus faecium and Lactobacillus plantarum were the species most diffusely identified. This latter species was also found in all duodenal biopsies of T-CD and HC children. Other bacterial species were identified only in T-CD or HC faecal samples. As shown by Randomly Amplified Polymorphic DNA-PCR analysis, the percentage of strains identified as lactobacilli significantly (P = 0.011) differed between T-CD (ca. 26.5%) and HC (ca. 34.6%) groups. The metabolome of T-CD and HC children was studied using faecal and urine samples which were analyzed by gas-chromatography mass spectrometry-solid-phase microextraction and 1H-Nuclear Magnetic Resonance. As shown by Canonical Discriminant Analysis of Principal Coordinates, the levels of volatile organic compounds and free amino acids in faecal and/or urine samples were markedly affected by CD. As shown by the parallel microbiology and metabolome approach, the gluten-free diet lasting at least two years did not completely restore the microbiota and, consequently, the metabolome of CD children. Some molecules (e.g., ethyl-acetate and octyl-acetate, some short chain fatty acids and free amino acids, and glutamine) seems to be metabolic signatures of CD.
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During innate immune responses, the inflammatory CC chemokine receptors CCR1, CCR2, and CCR5 mediate the recruitment of blood monocytes to infected tissues by promoting cell migration in response to chemokines CCL2-5. Toll-like receptors also play an essential role, allowing pathogen recognition by the recruited monocytes. Here, we demonstrate that Toll-like receptor 2 (TLR2) stimulation by lipoteichoic acid (LTA) from Staphylococcus aureus leads to gradual down-modulation of CCR1, CCR2, and CCR5 from the plasma membrane of human blood-isolated monocytes and inhibits chemotaxis. Interestingly, LTA does not promote rapid desensitization of chemokine-mediated calcium responses. We found that the TLR2 crosstalk with chemokine receptors is not dependent on the Toll/interleukin-1 receptor domain-containing adaptor protein, but instead involves phospholipase C, the small G protein Rac1, and is phorbol ester sensitive. Activation of this pathway by LTA lead to β-arrestin-mediated endocytosis of Ser349-phosphorylated CCR5 into recycling endosomes, as does CCL5 treatment. However, LTA-induced internalization of CCR5 is a slower process associated with phospholipase C-mediated and phorbol ester-sensitive phosphorylation. Overall, our data indicate that TLR2 negatively regulates CCR1, CCR2, and CCR5 on human blood monocytes by activating the machinery used to support chemokine-dependent down-modulation and provide a molecular mechanism for inhibiting monocyte migration after pathogen recognition.
Lymphocyte trafficking is an essential process in immune and inflammatory functions which can be thought to contain at least two main components: adhesion and migration. Whereas adhesion molecules such as the selections are known to mediate the homing of leukocytes from the blood to the endothelium, the chemoattractant substances responsible for the migration of specific subsets of lymphocytes to sites of infection or inflammation are largely unknown. Here we show that two molecules in the chemokine (for chemoattractant cytokine) superfamily, human macrophage inflammatory protein 1 alpha (MIP-1 alpha) and MIP-1 beta, do not share identical attractant activities for lymphocyte subpopulations. When analyzed in vitro in microchemotaxis experiments, HuMIP-1 beta tends to attract CD4+ T lymphocytes, with some preference for T cells of the naive (CD45RA) phenotype. HuMIP-1 alpha, when tested in parallel with HuMIP-1 beta, is a more potent lymphocyte chemoattractant with a broader range of concentration-dependent chemoattractant specificities. HuMIP-1 alpha at a concentration of 100 pg/ml attracts B cells and cytotoxic T cells, whereas at higher concentrations (10 ng/ml), the migration of these cells appears diminished, and the migration of CD4+ T cells is enhanced. Thus, in this assay system, HuMIP-1 alpha and -1 beta have differential attractant activities for subsets of immune effector cells, with HuMIP-1 alpha having greater effects than HuMIP-1 beta, particularly on B cells.
Chemokine signaling is important in neuropathic pain, with microglial cells expressing CCR2 playing a well-established key role. DAPTA, a HIV gp120-derived CCR5 entry inhibitor, has been shown to inhibit CCR5-mediated monocyte migration and to attenuate neuroinflammation. We report here that as a stabilized analog of DAPTA, the short peptide RAP-103 exhibits potent antagonism for both CCR2 (half maximal inhibitory concentration [IC50] 4.2 pM) and CCR5 (IC50 0.18 pM) in monocyte chemotaxis. Oral administration of RAP-103 (0.05-1 mg/kg) for 7 days fully prevents mechanical allodynia and inhibits the development of thermal hyperalgesia after partial ligation of the sciatic nerve in rats. Administered from days 8 to 12, RAP-103 (0.2-1 mg/kg) reverses already established hypersensitivity. RAP-103 relieves behavioral hypersensitivity, probably through either or both CCR2 and CCR5 blockade, because by using genetically deficient animals, we demonstrated that in addition to CCR2, CCR5 is also required for the development of neuropathic pain. Moreover, RAP-103 is able to reduce spinal microglial activation and monocyte infiltration, and to inhibit inflammatory responses evoked by peripheral nerve injury that cause chronic pain. Our findings suggest that targeting CCR2/CCR5 should provide greater efficacy than targeting CCR2 or CCR5 alone, and that dual CCR2/CCR5 antagonist RAP-103 has the potential for broad clinical use in neuropathic pain treatment.
Scientific evidence has revealed microecological changes in the intestinal tract of celiac infants. The objective of this work is the study of bacterial differences in the upper small intestine in both adults (healthy, untreated celiac disease [CD], and CD treated with a gluten-free diet) and children (healthy and untreated CD). Intestinal bacterial communities were identified by 16S rRNA gene sequencing of DNA extracted from duodenal biopsies. Analysis of the sequences from adults and children showed that this niche was colonized by bacteria affiliated mainly with three phyla: Firmicutes, Proteobacteria, and Bacteroidetes. In total, 89 different genera were identified in adults and 46 in children. Bacterial richness was significantly lower in the children than in the adults. A global principal component analysis of the bacterial communities of both healthy and untreated CD patient groups (including both children and adults) revealed a strong effect of age in principal component 1--clustering all adults and children separately--and a possible effect of the disease in adults with untreated patients clustering separately. There are bacterial differences in the upper small intestine between untreated children CD patients and untreated CD adults due to age. There are bacterial differences in the upper small bacteria microbiota between treated and untreated CD adults due to treatment with a gluten-free diet.
To describe the prevalence of Coeliac disease (CD) and its clinical management. Narrative review. Coeliac disease (CD) is an immune-mediated disorder that primarily affects the gastrointestinal (GI) tract. Recent data suggest a prevalence of about 1% in most Western countries, a figure that likely represents an increase in the prevalence of CD. Risk groups include those who are members of families with individuals who have CD as well as those with Type I diabetes and a variety of autoimmune diseases. Whereas biopsy is the gold standard in diagnosis, serological tests are crucial in determining who should undergo endoscopy and biopsy. HLA testing should be used only to rule out CD. Currently, a gluten-free diet is the only available therapy. In conclusion, CD is one of the most common immune-mediated disorders in the Western world. It should be considered in patients with a number of varying GI and non-GI symptoms, as well as in high-risk groups that include first-degree relatives.
Deterioration of quality of life in the long term has been suggested for celiac disease patients on a gluten-free diet. To determine long-term quality of life of celiac disease patients and to assess the benefits of gluten-free diet compliance. We prospectively evaluated 53 newly diagnosed adult celiac disease patients. The Short Form 36 Health Survey, the Gastrointestinal Symptoms Rating Scale and the Beck Depression Inventory were employed at the time of diagnosis, 1 year, and beyond 4 years (median: 53 months) on treatment. At 1 year, a significant improvement from baseline in quality of life indicators was observed (p<0.001 to p<0.0001) with comparable scores to healthy subjects. At 4 years, the Short Form 36 Health Survey scores (p<0.002 to p<0.0002) and Beck Depression Inventory score (p<0.002) show significant deterioration compare with 1 year. Most scores remained significantly better than those at diagnosis (p<0.03 to p<0.0005). No changes were detected in the Gastrointestinal Symptoms Rating Scale scores. The long-term impairment of quality of life was attributable to the deterioration of most dimensions in patients who were not strictly compliant with the gluten-free diet (p<0.05 to p<0.001). Long-term deterioration of quality of life outcomes after the first year of gluten-free diet was associated with the lack of strict compliance with the diet.