The study will be a prospective, randomized, placebo-controlled, single center clinical
trials. Patients diagnosed with SS, fulfilling the American-European Consensus Group
criteria for SS, will be recruited at the outpatient clinic of the Rheumatology Department,
University Hospital 'P. Giaccone' of Palermo, Italy.
Inclusion criteria of SS patients.
- - age >18 and <65 years;
- negativity of anti-deamidated gliadin protein (anti-DGP) immunoglobulins (Ig) class A
(IgA) and immunoglobulins (Ig)G, anti-tissue transglutaminase (anti-tTG) class IgA and
IgG, and Endomysium antibodies (EmA);
- absence of intestinal villous atrophy, documented in all the patients carrying the DQ2
and/or the DQ8 Human Leukocyte Antigen (HLA) haplotypes (thus irrespective of
CD-specific serum antibody negativity.
- - absence of WA (negative prick-test and/or specific serum immunoglobulin (Ig) E assay
for wheat, gluten, and gliadin).
Exclusion criteria of SS patients.
- - age <18 and >65 years;
- self-exclusion of gluten/wheat from the diet and refusal to reintroduce it, for
diagnostic purposes, before entering the study;
- pregnancy;
- alcohol and/or drug abuse;
- Helicobacter pylori and other bacterial and/or parasitic infections;
- diagnosis of chronic inflammatory bowel disease and other organic pathology affecting
the digestive system (e.g., severe liver disease), nervous system diseases, major
psychiatric disorders, immunological deficits, and impairments that limit physical
activity;
- diagnosis of cancer.
- - patients undergoing chemotherapy and/or radiotherapy.
Study design In a preliminary phase of the study, all patients who access the SS outpatient
clinic of the Rheumatology Department of the University Hospital 'P. Giaccone' of Palermo,
Italy, will be asked to answer, consecutively, to a questionnaire for the self-assessment
of wheat and other foods' intolerance.
After this evaluation, individual's enrollment will start. A database will be predisposed
to register demographic, clinical, laboratory, cytofluorimetric and immunohistochemistry
data. A repository bank will be used to collect samples from patients at different
timepoints.
Patients enrolled based on the inclusion and exclusion criteria and who have agreed to
enter the study, after having signed the informed consent, will be randomized, matched for
age/gender/racial origin, through a computerized system into an intervention group and a
control group. The two groups, each of 15 subjects, will be asked to follow a diet for 2
months. Intervention group will be asked to eliminate wheat and all cow's milk (both fresh
and aged) products from the diet (i.e., WFD plus CMPFD). The control group will be asked to
eliminate rice and turkey's meat products from the diet. The elimination diet in the
control group must be considered as a 'placebo diet' because both rice and turkey's meat
are foods known to be used in standard 'oligoantigenic' elimination diet.
Before starting the elimination diet (at time 0, T0), patients will be evaluated by
experienced rheumatologists to assess their clinical features, as well as by physicians
with expertise in the field of food intolerance about GI and extraintestinal symptoms which
could be related to foods intake. Moreover, at this time point, all subjects will be
subjected to:
- - salivary flux study, by standard sialometry, and salivary samples collection (treated
to block enzymatic digestion of proteins), to dose immunologic and inflammatory
markers;
- a blood sample, for the analysis of inflammatory markers, cytokine profile, and
intestinal permeability markers;
- a urine collection, after the administration of the lactulose/mannitol (LA/MA) test,
to define intestinal permeability;
- a collection of stools, for calprotectin assay and definition of the gut microbiota;
- a dietary consult to better explain the dietary approach and provide any information
useful to allow adherence to the elimination diet;
- finally, an alimentary (for the self-assessment of adherence to the diet) and
symptom's diary will be provided to all patients, which must be filled-in daily.
After 2 months of elimination diet (at time 1, T1), intervention and control patients will
be evaluated again both clinically and by laboratory techniques, repeating exactly what
have been done at T0. Moreover, they will have to deliver alimentary and symptom's diary,
and will discuss the results with physicians.
At this time-point, the subjects enrolled in the intervention group will go to an open
challenge, with reintroduction of wheat. After 2 weeks of open diet or whenever
rheumatologic, intestinal and/or extraintestinal symptoms should return or intensify
(T2int), patients will be valued again both clinically and by laboratory techniques,
repeating exactly what have been done at T0 and T1. Patients in this group will end the
study at this time-point.
At the same timepoint, patients enrolled in the control group will be asked to repeat the
elimination diet, this time removing from the diet wheat and all cow's milk (both fresh and
aged) products (i.e., i.e., WFD plus CMPFD, in the context of a cross-over design), for
further 2 months (T2con). At the end of these 2 months patients will be valued again both
clinically and by laboratory techniques repeating exactly what have been done at T0 and T1.
As described before for the intervention group, at this time-point patients of the control
group will go to an open challenge with reintroduction of wheat. After 2 weeks of open diet
or whenever rheumatologic, intestinal and/or extraintestinal symptoms should return or
intensify (T3con), patients will be valued again both clinically and by laboratory
techniques repeating exactly what have been done at T0, T1 and T2con. Patients in this
group will end the study at this time-point.
Sample size The sample size is difficult to determine as no precise data are known about
the effectiveness of a WFD and the response to wheat re-exposure after challenge in SS
patients, as well as NCWS frequency in these subjects; previously reported data suggest
that about 25% of NCWS patients suffer from an autoimmune disease, and in a study
evaluating the effect of a GFD on quality of life, GI symptoms, and immune system in
patients with fibromyalgia and NCWS, statistical significance was reached with 10 subjects.
Other studies assessing NCWS immunological pattern obtained statistical significance with
26, 30, 22, and 12 patients. Therefore, considering the nature of this prospective and
pilot study the investigators planned to enroll a total of 30 patients suffering from SS,
15 with GI symptoms and 15 without, fulfilling the American-European Consensus Group
criteria for SS.
Definition of 'positivity' to the open challenge During the open challenge period, patients
will be asked to note the possible onset of intestinal and/or extraintestinal symptoms
using the food and symptom's diary, using a 10-point Visual Analogic Scale (VAS). The
challenges will be stopped when a clinical reaction will occur for at least two consecutive
days with a >3-point increase in the patient's recorded symptom VAS scale. The challenges
will be considered positive if the same symptoms, which were initially present, will
reappear after their disappearance on elimination diet and if the GSRS score and/or the
extraintestinal symptoms rating scale will be 25% higher than the same score recorded on
elimination diet. Finally, from a strictly rheumatologic point of view, the challenge will
be considered positive if a 5% increase in the ESSPRI and ESSDAI score will be registered
compared to the same score recorded on the elimination diet.
Statistical Analysis Statistical analysis will be performed using commercial software.
Parametric and non-parametric statistical analysis will be performed calculating the mean ±
standard deviation (SD) and median, respectively. For comparison of parametric and
non-parametric data, the t-test and Mann-Whitney rank-sum test will be used where
appropriate. Spearman's correlation analysis will be used to quantify the association
between analysed variables. Data will be expressed as mean ± SD. P values less than 0.05
will be considered significant.
All subjects will agree to participate in the study. The study protocol will conform to the
ethical guidelines of the Declaration of Helsinki, will be preliminary approved by the
Human Research committee of the University Hospital of Palermo, Italy, and registered on
the CliniaclTrials.gov. All authors will have access to the study data and will review and
approve the final manuscript.