Dig Dis Sci DOI 10.1007/s10620-015-4020-2

REVIEW

Gut Microbiota and Celiac Disease Giovanni Marasco1 • Anna Rita Di Biase2 • Ramona Schiumerini1 • Leonardo Henry Eusebi1 • Lorenzo Iughetti2 • Federico Ravaioli1 • Eleonora Scaioli1 • Antonio Colecchia1 • Davide Festi1

Received: 30 June 2015 / Accepted: 20 December 2015 Ó Springer Science+Business Media New York 2015

Abstract Recent evidence regarding celiac disease has increasingly shown the role of innate immunity in triggering the immune response by stimulating the adaptive immune response and by mucosal damage. The interaction between the gut microbiota and the mucosal wall is mediated by the same receptors which can activate innate immunity. Thus, changes in gut microbiota may lead to activation of this inflammatory pathway. This paper is a review of the current knowledge regarding the relationship between celiac disease and gut microbiota. In fact, patients

& Giovanni Marasco [email protected] Anna Rita Di Biase [email protected]

with celiac disease have a reduction in beneficial species and an increase in those potentially pathogenic as compared to healthy subjects. This dysbiosis is reduced, but might still remain, after a gluten-free diet. Thus, gut microbiota could play a significant role in the pathogenesis of celiac disease, as described by studies which link dysbiosis with the inflammatory milieu in celiac patients. The use of probiotics seems to reduce the inflammatory response and restore a normal proportion of beneficial bacteria in the gastrointestinal tract. Additional evidence is needed in order to better understand the role of gut microbiota in the pathogenesis of celiac disease, and the clinical impact and therapeutic use of probiotics in this setting. Keywords Celiac disease
Gut microbiota
Dysbiosis
Probiotic
Gluten-free diet

Ramona Schiumerini [email protected] Leonardo Henry Eusebi [email protected]

Introduction

Lorenzo Iughetti [email protected]

Increasing evidence strongly indicates that intestinal microbiota plays a pivotal role in maintaining the homeostasis of the human body. Its integrity and its dynamic interaction with the body are essential for maintaining a healthy state while its alterations may contribute to the development of diseases, not only those related to the gastrointestinal (GI) tract, but also on a metabolic [1] and systemic level. In particular, alterations of the gut microbiota have been hypothesized to play a pathogenic role in celiac disease (CD) [2]. The prevalence of CD is much higher than what it was believed to be in the recent past, and its pathogenesis has not yet been well defined. Recent data have suggested that gut microbiota leads to the activation of innate immunity in CD [3].

Federico Ravaioli [email protected] Eleonora Scaioli [email protected] Antonio Colecchia [email protected] Davide Festi [email protected] 1

Department of Medical and Surgical Science, University of Bologna, 40138 Bologna, Italy

2

Department of Pediatrics, University of Modena, 41124 Modena, Italy

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The aim of this review was to evaluate the pathogenetic relationship between gut microbiota and CD as well as the effects of a gluten-free diet (GFD) on this interaction. Furthermore, the in vitro and in vivo experimental data regarding the use of probiotics for CD patients during a gluten-free diet were also revised. A search of the literature was conducted using MEDLINE, EMBASE, Web of Science and Scopus with combinations of the terms: ‘‘CD,’’ ‘‘immunity CD,’’ ‘‘innate immunity CD,’’ ‘‘gut microbiota CD,’’ ‘‘dysbiosis CD,’’ ‘‘gut microbiota gluten disorder,’’ ‘‘gluten disorder microbiota,’’ and ‘‘probiotic CD.’’ The relevance of the studies was assessed using a hierarchical approach based on title, abstract, and full article. It was not an aim of this review to discuss the clinical presentation and the diagnostic hallmarks of the disease in depth; for this purpose, specific reviews concerning these topics can be used [4–7]. The Pathogenesis of Celiac Disease and Gut Microbiota: What’s Old and What’s New Genetic predisposition (HLA-DQ2 and HLA-DQ8 haplotypes) and exposure to a specific group of proteins called prolamines contribute to the complex pathogenesis of CD [8]. Gluten is able to activate the mechanisms of both innate immunity and adaptive immunity [9, 10]. Gluten peptides reach the intestinal lamina propria where tissue transglutaminase (tTG) mediates the formation of potent immunostimulatory epitopes [9], with a strong binding affinity for the HLA class II molecules of serotypes DQ2 and DQ8 present on the antigen presenting cells (APC) [8]. This leads to an increased presentation of the deamidated gluten peptide (DGP) to the T cells, stimulating the immune system [8]. The activated T cells secrete cytokines such as IFN-c, which induce the release and activation of matrix metalloproteinases (MMP) by the myofibroblasts, leading to a modeling of the mucosa [11–13]. However, the effect of these cells is not sufficient to induce atrophy of the villi. It is possible that the epithelial damage is mediated by cytotoxic intraepithelial lymphocytes which recognize ligands produced by inflammatory stimuli on the surface of the intestinal epithelium [14]. Indeed, gliadin peptide a-2 (p31–43) is responsible for the activation of innate immunity of the intestinal epithelial cells [15, 16]. Thus, the gliadin peptides can also directly stimulate the immune response of macrophages and dendritic cells through pattern recognition receptors (PRR), such as toll-like receptors (TLRs) 4 [17]. This activation leads to the maturation of these cells and to the secretion of inflammatory cytokines (e.g., IL-1b, IL-8, TNF-a and MCP-1). Consequently, the adaptive immune response directed against gluten [9, 18] is enhanced, and

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intestinal permeability is increased [17]. In CD, it seems that the innate immune response favors the development of the adaptive immunity response to gluten in patients with HLADQ2 or HLA-DQ8 haplotypes [15]. Bacterial products on the intestinal surface are detected by specific receptors for the pathogen-associated molecular pattern (PAMP) called TLRs. Each of these is able to recognize a specific bacterial product which can lead to the activation of various intracellular cascades [19]. The innate immune reaction is immediate and is usually directed against microbial antigens which bind to the toll-like receptors; in the case of CD, this reaction can also be directed against specific components of cereals [20, 21]. Interestingly, Szebeni et al. [3] observed that an increased expression of TLR4 and TLR2 was present in both inflammatory bowel diseases and in celiac disease, suggesting a role of dysbiosis in the initiation of intestinal barrier damage. The intestinal microbiota has an immunomodulatory effect directly on immunity, inflammation, and allergic responses [22]. The complex relationship between the gut and its microbial content allows the identification of dangerous and harmless bacteria as well as food antigens [23]. In addition, the microbiota contributes to maintaining both the intestinal barrier function, by increasing the proliferation of epithelial cells, and the integrity of the intestinal epithelium through the translocation of proteins forming tight junctions and stimulating the genes involved in the maintenance of desmosomes [24]. As a result, it regulates the development of the vascular architecture of the villi [25]. Currently, there are two main methods of carrying out gut microbiota analysis without the use of bacterial culture: small-subunit ribosomal RNA (rRNA) analysis, in which 16S/18S rRNA gene sequences are used as phylogenetic markers for defining which lineages are present in a sample [26], and metagenomic analysis, in which community DNA is subject to shotgun sequencing [27]. Generally, an initial analysis is carried out using fingerprinting methods which involve the use of a polymerase chain reaction (PCR) for the amplification of the 16S rRNA genes (16S DNA) in microbial DNA extracted directly from the samples: (1) DDGE (denaturing gradient gel electrophoresis) and TGGE (temperature gradient gel electrophoresis) which are denaturant/temperature gels used for the separation of 16S rRNA amplicons, (2) T-RFLP (terminal restriction length polymorphism) in which primers are fluorescent-labeled and amplified, followed by restriction enzymes which are used to digest the 16S rRNA amplicon, and (3) ARDA (amplified rDNA restriction analysis) which is the extension of the RFLP technique to the gene encoding the small (16s) ribosomal subunit of bacteria. These methods provide less information regarding the microbial community but are faster to carry out and are also less costly. The main

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advantage of the sequencing methods (metagenomic profiles) as compared to the fingerprinting methods is that sequences can be classified according to the taxonomy and functions encoded in the genomes while sequences of 16S rRNA allow the characterization of the bacteria only at a taxonomic level [28].

Gut Microbiota and Celiac Disease Gluten ingestion in genetically susceptible subjects is not always sufficient to develop CD since environmental factors can also play a pathogenetic role in the disease. Studies conducted on twins showed that, in 25 % of the cases, one of the two twins did not develop CD, supporting this environmental hypothesis [29]. Moreover, an increased prevalence of the disease was associated with the amount and the period of introduction of gluten into the diet of an infant [30, 31] even if, on the other hand, two recent large randomized controlled trials [32, 33] have shown that, in high-risk children, the delayed as well as the early introduction of gluten into the diet and breast feeding did not modify the risk of CD, although a delayed introduction of gluten was associated with a delayed onset of CD [32]. Furthermore, an increased prevalence of CD was associated with neonatal infections [34], with modifications of the intestinal microbiota during pediatric age [35, 36] and with recurrent infections involving rotavirus [37, 38]. In the last decade, two large retrospective studies [39, 40] found a positive association between elective cesarean delivery and CD, and infants born small for gestational age were at increased risk of CD [40]. Finally, there are conflicting data regarding Helicobacter pylori infection and celiac disease; two large studies found an inverse association [41, 42] while others found no association [43, 44]. All these factors are directly or indirectly potential modifiers of the gut microbiota, leading to the hypothesis that dysbiosis could be an important cofactor in the pathogenesis of CD [2, 35]. Studies regarding the role of gut microbiota in CD have been carried out first on fecal samples and, subsequently, on duodenal biopsy specimens. To date, there have only been a few studies carried out on animal models [45–48], and they have mainly regarded the effects of probiotic strains on CD mucosal models, which will be discussed below. Fecal Microbiota in CD Patients Human Studies The first study on fecal samples of CD patients was conducted using both culture-dependent and culture-independent methods [2]. In CD patients, a greater number of

Bacteroides–Prevotella, Clostridium Histolitycum, Eubacterium rectal–C. coccoides, and Atopobium was documented. A greater diversity of fecal microbiota and a reduction in Bifidobacterium population diversity in CD patients was also found using PCR and DGGE [49]. Furthermore, the same Authors found species which were specific for CD patients, such as Lactobacillus curvatus, Leuconostoc mesenteroides, and carnosum. This result pointed out the need to further study the gut microbiota of CD patients and suggested a potential role for probiotic and prebiotic administration. Indeed, similar results were found using real-time PCR [35], showing a reduction in Bifidobacterium levels in the feces of CD patients as compared to controls. They also found that the Bacteroides and Clostridium leptum groups were more abundant in both the feces and the biopsies of CD patients than in controls, while E. coli and Staphylococcus were higher in celiac patients on a gluten-containing diet (GCD); however, their levels were normalized after going on a gluten-free diet (GFD). To link intestinal dysbiosis with immune activation in CD patients, De Palma et al. [36] investigated the relationship between microbiota and mucosal surface immunoglobulin secretion (IgA, IgG, IgM antibodies), which represents the first-line of mucosal defense. The levels of Bacteroides– Prevotella were more abundant in CD patients than in controls, and IgA-coated bacteria were significantly reduced in both untreated and treated CD patients as compared to the controls, suggesting the presence of a mucosal barrier defect in CD patients; the Authors speculated that the mucosal layer of CD patients fails to stabilize the gut microbiota and fails to prevent the host from the invasion of harmful antigens and pathogens [36]. Nistal et al. [50] confirmed an incomplete restoration of the gut microbiota after a GFD (i.e., a reduction in the diversity of the Lactobacillus and Bifidobacterium species) and also found a significantly lower content of short-chain fatty acids (SCFAs) in healthy adults as compared to CD patients (on a GCD or a GFD), reflecting changes in microbial composition. Furthermore, the SCFA metabolism was only partially restored after a GFD. Similar results were obtained in previous studies on metabolomics in pediatric CD patients [51]. Together with lower defenses due to a loss of ‘‘healthy’’ bacteria, dysbiosis leads to an increase in opportunistic pathogens carrying virulent genes, such as S. epidermidis carrying the mecA gene present both in GCD and GFD patients as compared to controls [52]. Other studies [2, 35] have reported increased numbers of the Staphylococcus species in the gut microbiota of GCD patients, similar to inflammatory bowel diseases [53, 54] or allergies [55, 56]. The main findings related to the fecal microbial composition of CD patients are summarized in Table 1.

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In conclusion, fecal microbiota could be a good indicator of gut dysbiosis in CD patients and could be used to monitor the restoration of the microbiota during a GFD. Promising data from metabolomics together with the evaluation of the gut microbiota could help clinicians in assessing the disease stage for the subject or in referring the subject for additional investigation. Duodenal Mucosa-Associated Microbiota in Celiac Disease Animal Studies In an animal model of CD, Cinova et al. [45] attempted to link several intestinal bacteria strains with barrier integrity, gliadin translocation, and cytokine production. The Authors stimulated the intestinal mucosa of germ-free rats with gliadin, IFN-c, and different bacterial strains isolated from CD patients and healthy children. E. coli or Shigella were found to significantly decrease the number of globlet cells, lead to greater mucin secretion and greater impairment in the tight junction, and, consequently, lead to the translocation of gliadin fragments into the lamina propria. On the other hand, Bifidobacterium bifidum increased the number of globlet cells and enhanced both the production of chemotactic factors and the inhibition of metalloproteinases, increasing gut protection. Thus, dysbiosis seems to modify gut permeability, enhancing gliadin fragment translocation, thereby contributing to CD pathogenesis. Human Studies Several studies have suggested that subjects at risk of developing CD have altered microbiota. A recent study by Olivares et al. [57] which included infants at high risk of developing CD concluded that the HLA-DQ2 and HLADQ8 genotypes could select intestinal microbiota composition at an early stage. Indeed, in healthy infants with at least one first-degree relative with CD and with HLA-DQ2 and HLA-DQ8, higher proportions of Firmicutes and Proteobacteria and a lower proportion of Actinobacteria were observed as compared to low-risk infants. High-risk infants also had significantly less Bifidobacterium species compared to low-risk infants, and an inverse correlation was found with Proteobacteria (Escherichia/Shigella) and Firmicutes (Clostridium), both potential pathogen genera which are greater in high-risk infants [57]. Therefore, HLA genotypes in high-risk patients could select potentially harmful microbiota. Microbiota characterization from duodenal biopsy specimens was initially carried out on CD children several years ago, showing dysbiosis in CD patients as compared to controls. In fact, several Spanish studies [35, 58–61]

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compared CD patients on a gluten-containing diet and on a gluten-free diet to healthy controls, finding a higher incidence of Gram-negative bacteria in the GCD and the GFD groups as compared to the healthy controls. In particular, the Bacteroides, Escherichia coli, Bifidobacterium, Enterobacteriaceae, and Staphylococcus groups were significantly more abundant in GCD patients than in the controls with a greater diversity of these species [35, 58, 59, 61]. Furthermore, the Streptococcus and Prevotella genera were more frequent in healthy subjects than in celiac patients [60, 61]. The ratio of Lactobacillus-Bifidobacterium (beneficial) to Bacteroides-E. coli (potentially harmful) was significantly reduced in both GCD and GFD patients as compared to controls, suggesting that microbiota composition was not restored after a GFD and symptom normalization [58]. On the basis of these results, it was possible to speculate that dysbiosis could be linked to the disease and to symptom development. A Swedish study carried out by Ou et al. [62] on children born during the Swedish CD epidemic, also speculated about the predominant fraction of rod-shaped bacteria found, such as Clostridium, Prevotella, and Actinomycesas, which are a risk factors for CD. Rod-shaped bacteria have previously been observed in the mucosa of both GCD and GFD pediatric patients along with a distinctive lectin pattern [63]. In a recent study by Wacklin et al. [64], Proteobacterial genera, such as Acinetobacter and Neisseria, were found to be more abundant and associated with gastrointestinal symptoms in CD patients as compared to controls. Furthermore, Firmicutes and Bacteroides, such as Streptococcus and Prevotella, were most abundant in patients with dermatitis herpetiformis (DH) and the controls. The same study showed that the microbiota of DH patients was more similar to the controls (greater abundance of Firmicutes) than to the CD patients (greater abundance of Proteobacteria). Moreover, patients with GI symptoms or anemia had lower microbial diversity than those with DH, suggesting that gut microbiota may also have a role in the manifestation of CD. Recently, Kalliomaki et al. [65] demonstrated the decreased expression of TLRs and of the negative regulator of Toll-receptor signaling (Tollip), as well as an increased expression of TLR9 and of IL-8, which are markers of intestinal inflammation in CD patients [65]. The aim of the study was to attempt to link immunity in CD to gut microbiota; as matter of fact, intestinal epithelial homeostasis is directly dependent on TLR activation which serves to balance the response against pathogens and the excessive inflammatory response [66, 67]. The main findings related to the duodenal microbial composition of CD patients are summarized in Table 2. In conclusion, current data suggest that gut microbiota has already been altered before the clinical onset of CD,

Dig Dis Sci Table 1 The main findings related to fecal microbiota in celiac disease Authors

Year

Population

Number of subjects

Mean age CD patients (years)

Methods

Findings in CD patients

Collado et al. [2]

2007

CD ? HC

26 ? 23

2.2

Culture ? FISH

: Bacteroides–Prevotella, Clostriudium hystoliticum, Eubacterium rectale– C. coccoides, Atopobium and Staphylococcus

Sanz et al. [49]

2007

CD ? HC

10 ? 10

3

Culture ? qPCR ? DGGE

Collado et al. [35]

2009

CD ? tCD ? HC

8 ? 8?8

5

qPCR

Di Cagno et al. [76]

2009

CD ? tCD ? HC

7?7?7

6–12

PCR ? DGGE

De Palma et al. [36]

2010

Nistal et al. [50]

2012

Sanchez et al. [52]

2012

; Bifidobacterial strains (no significative) In CD patients higher diversity of fecal microbiota. L. curvatus, Leuconostoc mesenteroides and carnosum characteristic of CD patients : Bacteroides, C. leptum, E. coli, Staphylococcus ; Bifidobacteria ; Ratio of cultivable lactic acid bacteria and Bifidobacterium to Bacteroides and enterobacteria in tCD and in CD L. brevis, L. rossiae, and L. pentosus identified only in samples from tCD and HC. L. fermentum, L. delbrueckii subsp. bulgaricus, L. gasseri identified only in several samples from HC

CD ? HC

24 ? 18 ? 20

5.5

FISH ? flow cytometry

: Bacteroides–Prevotella group ; Bifidobacterium, C. histolyticum, C. lituseburense and Faecalibacterium praustnitzii

CD ? tCD ? HC

10 ? 11 ? 11

39.5

DGGE

: Bacterial diversity in CD : L. sakei in CD and HC compared to tCD : B. bifidum and catenulatum in CD compared to HC andtCD

CD ? tCD ? HC

20 ? 20 ? 20

5.2

PCR ? DNA sequencing

: Staphylococcus spp. diversity : Abundance of S. epidermidis in CD and tCD, of S. heamolyticus in CD versus HC ; S. aureus in CD

CD celiac disease, tCD treated celiac disease, HC healthy controls, FISH fluorescent in situ hybridization, PCR polymerase chain reaction, qPCR quantitative polymerase chain reaction, DGGE denaturing gradient gel electrophoresis

due to HLA-DQ2-8 selection for potentially pathogenic bacteria [57]. In patients on a GCD, almost all studies have found an unbalanced microbial composition with a reduction in healthy genera, such as Bifidobacteria, and an increase in potentially harmful Gram-negative bacteria, such as Proteobacterial genera (Staphylococcus spp. and Bacteroides). Moreover, a greater presence of Proteobacterial genera has been linked to gastrointestinal symptoms. The results obtained on fecal and duodenal samples suggest that dysbiosis in CD patients could have a pathogenetic role in inducing modification of the mucosal barrier which maintains persistent immune activation, thus leading to the development of symptoms. Additional studies are needed to confirm this pathogenetic hypothesis.

A Gluten-Free Diet and Gut Microbiota To date, a gluten-free diet is the only therapy for celiac patients [7]. A GFD reduces symptoms and restores the well-being of the individual [7]. Several studies have compared the gut microbiota of CD patients following a GFD with CD patients on a GCD and controls, while only very few studies have longitudinally evaluated the effect of a GFD in the same population. Preliminary studies have been conducted on Spanish children [35, 58]; in celiac patients, even after following a GFD (for at least 2 years), the duodenal mucosal microbiota was not completely restored and showed a less abundant bacterial richness as compared to healthy and

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Dig Dis Sci Table 2 The main findings related to duodenal-associated microbiota in celiac disease Authors

Year

Population

Number of subjects

Mean age of CD patients (years)

Methods

Findings in CD patients

Nadal et al. [58]

2007

CD ? tCD ? HC

20 ? 10 ? 8

5.4

FISH ? flow cytometry

: Bacteroides–Prevotella and E. coli, normalized after GFD

Collado et al. [35]

2009

CD ? tCD ? HC

8?8?8

5

qPCR

: Bacteroides, C. leptum, E. coli, Staphylococcus, Lactobacillus, Akkermansia m

Ou et al. [62]

2009

CD ? HC

45 ? 18

8

Culture ? Scanning electron microscopy

: Streptococcus and Neisseria

Schippa et al. [77]

2010

CD ? tCD ? HC

20 (before and after a GFD) ? 10

8.3

TTGE

:Bacteroides vulgatus and E. coli. Differences in biodiversity between CD and tCD

Sanchez et al. [59]

2010

CD ? tCD ? HC

20 ? 12 ? 8

5

PCR- DGGE

: Bacteroides dorei;

Di Cagno et al. [69]

2011

tCD ? HC

19 ? 15

9.6

PCR-DGGE

In treated CD: lower levels of Lactobacillus, Enterococcus and Bifidobacteria and increased levels of Bacteroides, Staphylococcus, Salmonella, Shigella and Klebsiella

Nistal et al. [60]

2012

CD ? tCD ? HC

13 ? 5?10

3.7 in children, 25 in adults

16SrRNA gene sequencing

; Streptococcus and Prevotella

Sanchez et al. [61]

2013

CD ? tCD ? HC

32 ? 17 ? 8

5.5

Culture ? PCR

: Proteobacteria, Enterobacteriaceae and Staphylococcaceae, in particular the species Klebsiellaoxytoca, Staphylococcus epidermidis and pasteuri

; Bifidobacterium and Lactobacilli

; Bifidobacterium, C. coccoides

; Bacteroides distasonis, Bacteroides fragilis/ Bacteroides thetaiotaomicron, Bacteroides uniformis and Bacteroides ovatus

; Streptococcaceae family, i.e., Streptococcus anginosus and mutans De Meij et al. [71]

2013

CD ? HC

21 ? 21

5

IS-pro, profiling method

No differences among the groups. High overall abundance of Streptococcus, Lactobacillus and Clostridium

Cheng et al. [102]

2013

CD ? HC

10 ? 9

9.5

qRTPCR ? HITchip microarray

No differences in the total microbiota profile and in the abundance of individual genus-like bacterial groups

Wacklin et al. [64]

2013

CD ? HC

33 ? 18

39

PCR-DGGE

: Microbial diversity and richness. : Proteobacteria, such as Acinetobacter and Neisseria, in subjects with gastrointestinal symptoms. : Streptococcus and Prevotella in subjects with dermatitis herpetiformis

Wacklin et al. [78]

2014

tCD with symptoms versus tCD asymptomatic

18 ? 18

58.5

16SrRNA gene pyrosequencing

In patients with persistent symptoms: : Proteobacteria ; microbial richness, Bacteroides and Firmicutes

CD celiac disease, tCD treated celiac disease, HC healthy controls, GFD gluten-free diet, FISH fluorescent in situ hybridization, PCR polymerase chain reaction, qPCR quantitative polymerase chain reaction, TGGE temperature gradient gel electrophoresis, qRT-PCR real-time quantification polymerase chain reaction, HITchip human intestinal tract chip, DGGE denaturing gradient gel electrophoresis

untreated subjects, with a persistent imbalance of the ratio of potentially harmful/beneficial bacteria [35]. Although E. coli and Staphylococcus counts were restored after a GFD, Bifidobacteria counts remained lower in the feces of

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patients as treated with a GFD compared to controls. The same Authors [68] carried out a study targeted on gut Bifidobacteria composition from CD patients on both a GCD and a GFD, and from healthy controls. In all subjects, a

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correlation between the levels of the Bifidobacterium and B. longum species in fecal and bioptic samples was found. Moreover, an overall reduction in these bacterial populations was found in CD patients as compared to healthy children. Bacterial differences due to treatment with a GFD were also found by Nistal et al., as previously reported [60]. Di Cagno et al. [69] carried out a study on duodenal and fecal microbiota, and on the metabolome of healthy subjects as compared to CD patients on a GFD using PCRDGGE analysis. The metabolome is the complete set of small-molecule chemicals found in a biological sample, both endogenous and exogenous, and gives us information regarding the phenotype and the environment in which an organism lives. Following a GFD, the Authors observed lower levels of Lactobacillus, Enterococcus, and Bifidobacteria, and increased levels of Bacteroides, Staphylococcus, Salmonella, Shigella, and Klebsiella as compared to the controls. Moreover, using 1H-Nuclear Magnetic Resonance for metabolomics, they found that several molecules (ethyl-acetate and otyl-acetate, some short-chain fatty acids and free amino acids, and glutamine) appeared to be metabolic markers of CD. In particular, SCFAs were significantly higher in the fecal samples from CD patients on a GFD and healthy patients than in the fecal samples from celiac patients on a GCD. Major differences were found regarding butyric, isovaleric, and pentanoic acids. A more recent study [70] showed lower levels of Bifidobacterium genus strains from the fecal samples of celiac patients on a GFD as compared to the controls, regardless of the pH. On the contrary, a study conducted by de Meij et al. [71] showed no differences in duodenal-associated microbiota between CD patients on a GFD and controls, showing high concentrations of Streptococcus, Lactobacillus, and Clostridium in both groups; the study concluded that microbiota did not seem to play an important role in the pathogenesis of CD. However, the majority of the studies cited suggested the presence of an incomplete restoration of gut microbiota in GFD patients. Additional confirmation comes from a study which evaluated the effect of a GFD on healthy subjects [72] using fluorescence in situ hybridization (FISH) and quantitative PCR. De Palma et al. [72] unexpectedly observed a decrease in Bifidobacterium, Bifidobacterium longum, Clostridium lituseburense, Lactobacillus, and Faecalibacterium prausnitzii, and an increase in Enterobacteriaceae and E. coli strains. On the contrary, TNF-a, IFN-c, IL-10, and IL-8 production was reduced. The Authors justified their results as a consequence of the reduced production of both proinflammatory and regulatory cytokines due to a generalized reduction in the total luminal bacterial load of the large intestine caused by the GFD. The main finding was that a GFD influenced gut microbial composition and immune

activation regardless of the presence of a disease and that these effects were directly related to the reductions in polysaccharide intake. A comment by Jackson [73] on this paper pointed out the prebiotic stimulus brought on by a gluten-containing diet. The contribution of wheat and barley to fructans ranges from 70 [74] to 81 % of oligofructose and inuline [75]. Thus, dysbiosis could derive from this marked reduction in fructans which exerts a prebiotic action. An additional confirmation of the incomplete restoration of gut microbiota after a GFD comes from the few studies that carried out a dynamic follow-up of the same patients. An Italian study using PCR-DGGE showed that the Lactobacillus community was lower before than after a GFD and lower in CD patients than in healthy controls. There was also a lower ratio of lactic acid bacteria, Bifidobacterium to Bacteroides and Enterobacteria as compared to healthy controls [76]. Some Lactobacillus strains, such as L. brevis, L. rossiae and L. pentosus, were not identified in CD patients before a GFD. Other strains were identified only in healthy subjects. They also found that SCFA concentrations were significantly lower in CD patients before a GFD than after the introduction of a GFD and than in healthy controls; in particular, major differences were found for butyric, isovaleric, and pentanoic acids. The Authors concluded that a GFD only partially restores fecal microbiota balances and metabolism. Similar results were also obtained by Schippa et al. [77] in children, studying the mucosa-associated microbiota from 20 children before and after a GFD and from ten controls. Recently, a study from Sweden [78] included CD patients with an absence of serum antibodies and histological alterations, but having persistent symptoms after at least 3 years of a GFD. Despite small sample size and the inclusion of patients not screened for irritable bowel syndrome [79], the Authors found reduced bacterial richness, a higher abundance of Proteobacteria, and a reduced amount of Bacteroidetes and Firmicutes as compared to patients without persistent symptoms. In their opinion, dysbiosis could be responsible for the persistent symptoms in CD patients after a GFD, and they speculated that the manipulation of gut microbiota through the use of probiotics, antibiotics, or fecal transplantation could improve the symptoms. In conclusion, a GFD allows only a partial recovery of the gut microbiota in CD patients, in both microbiological and metabolomical studies [69, 77]. The reason is still unclear; however, several speculations could be made: a genetic influence on gut microbiota in patients with CD which persists after a GFD can be evoked [57]; furthermore, since gluten has a prebiotic action, its absence in a GFD induces a different gut microbiota composition as compared to healthy subjects [73]. However, following a

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GFD, a reduction in potentially harmful bacteria takes place and the Bifidobacteria concentrations are not completely restored; data regarding the Lactobacilli are still conflicting. Probiotics and Celiac Disease Adherence to a diet represents a difficult problem for many patients since traces of gluten are found in the majority of processed foods, and a strict gluten-free diet inevitably limits the social activities of patients [80]. Moreover, a GFD may be rich in high glycemic index foods which can increase insulin resistance and, thus, the risk of obesity and cardiovascular diseases. In children with type 1 diabetes, a GFD makes metabolic control more complex [81–84]. In the last decade, new therapies have been suggested to improve compliance to a GFD or to replace a GFD [85]. Dysbiosis in CD patients has shifted the focus of new therapies for CD regarding the use of probiotics. The majority of studies on this topic have concluded that probiotic and prebiotic administration could be useful in celiac disease patients. In fact, dysbiosis could facilitate a loss of gluten tolerance in genetically predisposed subjects by means of an increase in gut mucosal permeability and an increased recruitment of T cells [36, 52, 62]. Probiotic administration could modulate the composition and functions of the intestinal microbiota, both to defer or to avoid the onset of CD, and could also be useful following a GFD when the normal microbial composition has not been completely restored. Benefits could come from the regulation of the immune response, the degradation of toxin receptors, the competition for nutrients, the blockage of adhesion sites, and the production of inhibitory substances against pathogens [86]. Although data regarding the use of probiotics for celiac disease are encouraging, to date most of these data come from in vitro experimental models of celiac disease; only a few studies have been conducted on humans. In Vitro and In Vivo Studies Previous studies carried out both in vitro and in vivo showed that specific probiotic strains are able to prevent tight junction leakage detected during inflammation [87, 88] and to reduce the gliadin-induced increase in epithelial permeability [89]. Lindfors et al. [89] investigated whether probiotics, such as Lactobacillus fermentum or Bifidobacterium (B.) lactis, could inhibit the toxic effects of gliadin in intestinal cell cultures (Human colon Caco-2 cells). The main result was that B. lactis inhibited increased epithelial gliadin-induced permeability in a dose-dependent manner. Moreover, specific Bifidobacterium strains carry out regulatory and anti-inflammatory activity by means of the stimulation of IL-10 production in regulatory T cells [90, 91].

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In a similar study carried out by Medina et al. [92], Bifidobacterium strains, such as B. longum ES1 and B. bifidum ES2, increased IL-10 production and suppressed proinflammatory cytokine production when administered in vitro on peripheral blood mononuclear cells and co-incubated with fecal samples from CD patients. The cytokine effects induced by fecal microbiota seemed to be mediated by the NFkB pathway. The Authors concluded that the altered gut microbiota of CD patients could contribute to the inflammatory response and that Bifidobacterial strains could reverse this effect. Other studies conducted in cell cultures and experimental models showed that Bifidobacterial strains reduce the severity of the toxic effects of gluten in CD patients [89, 93]. In a study by Laparra et al. [93], a mixture of gliadin-digested fragments and Bifidobacteria inoculated on Caco-2 cell culture induced a down-regulation of the mRNA expression of pro-inflammatory cytokines, such as NFkB, TNF-alpha, and IL-1beta. More recently, the same group [46] hypothesized that the administration of Bifidobacterium longum CECT 7347, which reduces gliadin immunotoxic effects in vitro, could exert a protective effect in an animal model of gliadininduced enteropathy. The effects of this bacterium were evaluated in newborn rats fed with gliadin alone or sensitized with interferon (IFN)-c and fed with gliadin. The results showed that feeding with gliadin alone reduced enterocyte height and peripheral CD4? cells, but increased CD4?/Foxp3? T and CD8? cells; the simultaneous administration of B. longum CECT 7347 exerted the opposite effect. Moreover, B. longum CECT 7347 administration increased NFjB expression and reduced TNF-a production. In sensitized gliadin-fed animals, the administration of B. longum CECT 7347 reduced the CD4? and CD4?/Foxp3? cell populations and increased the CD8? T cell populations. On the basis of these results, the Authors speculated that B. longum could reduce the production of inflammatory cytokines and the CD4? T cell-mediated immune response. The Authors [94] also tried to evaluate the effects of the same probiotic strain on gliadin perturbations of the iron metabolism in rats fed with gliadin and/or Bifidobacterial strains. The results showed that liver transferrin receptor (TfR)-2 expression decreased after gliadin intake and was restored with B. longum CECT 7347. Liver expression levels of Hamp decreased with the administration of the probiotic. The Authors concluded that another effect of B. longum administration was the improvement of gliadinmediated perturbations, such as liver iron deposition and mobilization. Moreover, in another study [48], the administration of Lactobacillus casei in a mouse model of gliadin-sensitive enteropathy seemed to be useful in recovering the Gut-

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Associated Lymphoid Tissue (GALT) homeostasis and a normal mucosal structure since the probiotic use induced the recovery of villus blunting, a delay in weight decrease and a normalization of TNF-a levels. A pivotal study conducted by De Palma and colleagues [95] directly linked immune response in CD and dysbiosis. The Authors evaluated the effects of Bifidobacterium, such as B. bifidum and B. longum, compared to Gram-negative bacteria, such as Bacteroides fragilis and Escherichia coli, on peripheral blood mononuclear cells alone or with CD triggers. Gram-negative bacteria induced a higher secretion of TH-1 proinflammatory cytokines and activation mechanisms (HLA-DR, CD40, IL-12, and IFN-c) than the Bifidobacterium strains while Bifidobacterium strains upregulated CD83 expression, a marker of mature dendritic cells. The Authors assumed that gut microbiota could be a local factor which regulates the response of monocytes to gliadins and IFN-c in CD patients. The probiotic modulatory effect on dendritic cells was also shown in other previous studies [91, 96] by using probiotic strains of VSL#3. VSL#3 is a mixture of bacterial strains, such as S. thermophilus, L. plantarum, L. acidophilus, L. casei, L. delbrueckii spp. Bulgaricus, B. Breve, B. longum, and B. infantis specifically proposed for CD by De Angelis et al. [97]. The aim of the study was to document the capacity of this probiotic mixture for decreasing the toxicity of wheat flour during long-time fermentation through a dough fermentation and gliadin polypetide hydrolysis. There was almost complete hydrolysis of the gliadin polypeptides as compared to other products, such as Oxadrop, Florisia, and Yovis, and only alpha2-gliadin fragments 62–75 were found after hydrolysis at a very low concentration. Moreover, there was also a lesser reorganization of intracellular F-actin with a decreased release of zonulin, leading to decreased intestinal permeability. The Authors suggested that the proteolytic activity of probiotic VSL#3 has a role in the production of pre-digested and tolerated gliadins, increasing the palatability of gluten-free products and that probiotic strains must be used together to exert a beneficial effect in CD. Human Studies Among studies in humans, a study [98] on the effects of Bifidobacterium infantis natren life start strain has recently been conducted on CD patients on a gluten-containing diet. They were asked to take 2 capsules before each meal for 3 weeks. The first goal of the study was to assess changes in intestinal permeability. The results showed that probiotics did not modify intestinal permeability probably due to an insufficient dose or the short time of administration. However, probiotic administration improved gastrointestinal symptoms, improving digestion and reducing

constipation. Moreover, in the treatment arm, a reduction in IgA tTG and IgA DGP antibody concentrations was also noted, even if without statistical significance (p = 0.055 for IgA tTG and p = 0.181 for IgA DGP). A similar study evaluated the effects of Bifidobacterium longum CECT 7347 for 3 months in addition to a GFD in children newly diagnosed with CD [99]. Probiotic intake was associated with a greater height percentile, a decrease in CD3 T cells, and a reduction in the Bacteroides fragilis group and in the content of IgA in stools as well as with improving symptoms in CD patients. Recently, a study carried out in Argentina identified a significant lack of Lactobacilli in symptom-free CD children. Thus, the Authors isolated five different Lactobacilli in the stools of healthy children and proposed Lactobacillus rhamnosus and Lactobacillus paracasei as potential probiotic strains owing to their high resistance to gastrointestinal tract conditions [100]. On the other hand, there is also active research regarding the quality and the preparation of gluten-free foods. Some Authors have tried to add increasing levels of prebiotic inulintype fructans to assess the sensory and nutritional quality of gluten-free bread (GFB). They concluded that prebiotics are promising for improving a GFD and can provide benefits for patients with celiac disease since they are ingredients which can also increase calcium absorption [101]. In conclusion, data from in vitro and animal studies have shown that Bifidobacteria and Lactobacilli administration not only seems to restore gut microbiota composition but could also pre-digest gluten in the intestinal lumen and reduce the inflammation associated with gluten intake by means of a reduction in both intestinal permeability, and cytokine and antibody production. This could lead to an improvement in symptoms. To date, the evidence regarding the use of probiotics in patients with celiac disease is still insufficient to justify their use in clinical practice.

Conclusions In recent years, an increasing amount of attention has been paid to and, therefore, a growing number of publications have appeared regarding the role of gut microbiota in CD patients. Although the majority of the studies available have been conducted using different methods, small sample sizes, and patients of varying ages, the majority of the studies have confirmed low levels of Lactobacilli and Bifidobacteria in the gut microbial composition of CD patients. Moreover, an increase in Gram-negative bacteria has often been reported and, among the potential pathogens present in CD, the most reported is the Proteobacterial genera which has also been linked to gastrointestinal symptoms.

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Experimental data have suggested that dysbiosis in CD patients could result in a modification of the mucosal barrier which maintains persistent immune activation causing clinical symptoms. Additional studies with adequate sample sizes and methods for carrying out accurate gut microbiota analysis are needed in order to identify the exact microbial pattern associated with CD. In the future, researchers should focus not only on the study of intestinal microbiota but also on the study of the molecules and mechanisms associated with the pathogenesis of the disease in order to clarify the exact role of dysbiosis in celiac disease, that is, whether it is implicated in the pathogenesis of the disease or whether it is an epiphenomenon of the disease which could lead to the appearance of clinical symptoms. As far as a GFD is concerned, there is general consensus regarding the influence this diet has on gut microbiota, leading to a reduction in the concentration of both potential pathogenic bacteria and Bifidobacteria. Moreover, a GFD should be well balanced in order to provide an appropriate dose of prebiotics. In addition to a GFD, the administration of probiotics, such as Bifidobacterium and Lactobacilli, in CD patients could be useful in restoring altered gut microbiota, thus reducing both gliadin toxicity and immune activation. Their use could improve the daily ingestible gluten limit in celiac patients so that patients will better tolerate the diet. Finally, considering the alterations of gut microbiota as an environmental factor involved in CD pathogenesis and that the genetic background may influence gut microbial composition in subjects at high risk for CD, probiotic administration may have a role in primary prevention for subjects at high risk for CD. Even if there are promising data from in vivo and in vitro studies regarding the use of probiotics in CD, data from human studies are still lacking and uncertain; therefore, additional studies are needed in this area. Compliance with ethical standards Conflict of interest

The authors declare no conflict of interest.

5.

6. 7. 8.

9.

10.

11.

12.

13.

14. 15.

16.

17.

18.

19.

20. 21. 22.

References 23. 1. Festi D, Schiumerini R, Eusebi LH, Marasco G, Taddia M, Colecchia A. Gut microbiota and metabolic syndrome. World J Gastroenterol. 2014;20:16079–16094. 2. Collado MC, Calabuig M, Sanz Y. Differences between the fecal microbiota of coeliac infants and healthy controls. Curr Issues Intest Microbiol. 2007;8:9–14. 3. Szebeni B, Veres G, Dezsofi A, et al. Increased mucosal expression of Toll-like receptor (TLR)2 and TLR4 in coeliac disease. J Pediatr Gastroenterol Nutr. 2007;45:187–193. 4. Husby S, Koletzko S, Korponay-Szabo´ IR, et al. European Society for Pediatric Gastroenterology, Hepatology, and

123

24.

25.

26. 27.

Nutrition guidelines for the diagnosis of coeliac disease. J Pediatr Gastroenterol Nutr. 2012;54:136–160. Rubio-Tapia A, Hill ID, Kelly CP, Calderwood AH, Murray JA. ACG clinical guidelines: diagnosis and management of celiac disease. Am J Gastroenterol. 2013;108:656–676. (quiz 677). Fasano A, Catassi C. Clinical practice. Celiac disease. N Engl J Med. 2012;367:2419–2426. Guandalini S, Assiri A. Celiac disease: a review. JAMA Pediatr. 2014;168:272–278. Harris LA, Park JY, Voltaggio L, Lam-Himlin D. Celiac disease: clinical, endoscopic, and histopathologic review. Gastrointest Endosc. 2012;76:625–640. Schuppan D, Junker Y, Barisani D. Celiac disease: from pathogenesis to novel therapies. Gastroenterology. 2009;137: 1912–1933. Abadie V, Sollid LM, Barreiro LB, Jabri B. Integration of genetic and immunological insights into a model of celiac disease pathogenesis. Annu Rev Immunol. 2011;29:493–525. Pender SL, Tickle SP, Docherty AJ, Howie D, Wathen NC, MacDonald TT. A major role for matrix metalloproteinases in T cell injury in the gut. J Immunol. 1997;158:1582–1590. Daum S, Bauer U, Foss HD, et al. Increased expression of mRNA for matrix metalloproteinases-1 and -3 and tissue inhibitor of metalloproteinases-1 in intestinal biopsy specimens from patients with coeliac disease. Gut. 1999;44:17–25. Ciccocioppo R, Di Sabatino A, Bauer M, et al. Matrix metalloproteinase pattern in celiac duodenal mucosa. Lab Invest. 2005;85:397–407. Green PHR, Jabri B. Coeliac disease. Lancet. 2003;362: 383–391. Maiuri L, Ciacci C, Ricciardelli I, et al. Association between innate response to gliadin and activation of pathogenic T cells in coeliac disease. Lancet. 2003;362:30–37. Londei M, Ciacci C, Ricciardelli I, Vacca L, Quaratino S, Maiuri L. Gliadin as a stimulator of innate responses in celiac disease. Mol Immunol. 2005;42:913–918. Lammers KM, Lu R, Brownley J, et al. Gliadin induces an increase in intestinal permeability and zonulin release by binding to the chemokine receptor CXCR3. Gastroenterology. 2008;135:e3. Thomas KE, Sapone A, Fasano A, Vogel SN. Gliadin stimulation of murine macrophage inflammatory gene expression and intestinal permeability are MyD88-dependent: role of the innate immune response in Celiac disease. J Immunol. 2006;176:2512– 2521. Round JL, Mazmanian SK. The gut microbiota shapes intestinal immune responses during health and disease. Nat Rev Immunol. 2009;9:313–323. Meresse B, Cerf-Bensussan N. Innate T cell responses in human gut. Semin Immunol. 2009;21:121–129. Palmer E. The generation of T cell tolerance. Swiss Med Wkly. 2007;137:99S–100S. Sharma R, Young C, Neu J. Molecular modulation of intestinal epithelial barrier: contribution of microbiota. J Biomed Biotechnol. 2010;2010:305879. Nagler-Anderson C. Man the barrier! Strategic defences in the intestinal mucosa. Nat Rev Immunol. 2001;1:59–67. Ashida H, Ogawa M, Kim M, Mimuro H, Sasakawa C. Bacteria and host interactions in the gut epithelial barrier. Nat Chem Biol. 2012;8:36–45. Stappenbeck TS, Hooper LV, Gordon JI. Developmental regulation of intestinal angiogenesis by indigenous microbes via Paneth cells. Proc Natl Acad Sci USA. 2002;99:15451–15455. Pace NR. A molecular view of microbial diversity and the biosphere. Science. 1997;276:734–740. Rondon MR, August PR, Bettermann AD, et al. Cloning the soil metagenome: a strategy for accessing the genetic and functional

Dig Dis Sci

28.

29. 30.

31.

32.

33.

34.

35.

36.

37.

38. 39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

diversity of uncultured microorganisms. Appl Environ Microbiol. 2000;66:2541–2547. Hamady M, Knight R. Microbial community profiling for human microbiome projects: tools, techniques, and challenges. Genome Res. 2009;19:1141–1152. Greco L, Romino R, Coto I, et al. The first large population based twin study of coeliac disease. Gut. 2002;50:624–628. Ivarsson A, Myle´us A, Norstro¨m F, et al. Prevalence of childhood celiac disease and changes in infant feeding. Pediatrics. 2013;131:e687–e694. Norris JM, Barriga K, Hoffenberg EJ, et al. Risk of celiac disease autoimmunity and timing of gluten introduction in the diet of infants at increased risk of disease. JAMA. 2005;293:2343–2351. Lionetti E, Castellaneta S, Francavilla R, et al. Introduction of gluten, HLA status, and the risk of celiac disease in children. N Engl J Med. 2014;371:1295–1303. Vriezinga SL, Auricchio R, Bravi E, et al. Randomized feeding intervention in infants at high risk for celiac disease. N Engl J Med. 2014;371:1304–1315. Myle´us A, Hernell O, Gothefors L, et al. Early infections are associated with increased risk for celiac disease: an incident case-referent study. BMC Pediatr. 2012;12:194. Collado MC, Donat E, Ribes-Koninckx C, Calabuig M, Sanz Y. Specific duodenal and faecal bacterial groups associated with paediatric coeliac disease. J Clin Pathol. 2009;62:264–269. De Palma G, Nadal I, Medina M, et al. Intestinal dysbiosis and reduced immunoglobulin-coated bacteria associated with coeliac disease in children. BMC Microbiol. 2010;10:63. Stene LC, Honeyman MC, Hoffenberg EJ, et al. Rotavirus infection frequency and risk of celiac disease autoimmunity in early childhood: a longitudinal study. Am J Gastroenterol. 2006;101:2333–2340. Pavone P, Nicolini E, Taibi R, Ruggieri M. Rotavirus and celiac disease. Am J Gastroenterol. 1831;2007:102. Decker E, Engelmann G, Findeisen A, et al. Cesarean delivery is associated with celiac disease but not inflammatory bowel disease in children. Pediatrics. 2010;125:e1433–e1440. Ma˚rild K, Stephansson O, Montgomery S, Murray JA, Ludvigsson JF. Pregnancy outcome and risk of celiac disease in offspring: a nationwide case-control study. Gastroenterology. 2012;142:e3. Lasa J, Zubiaurre I, Dima G, Peralta D, Soifer L. Helicobacter pylori prevalence in patients with celiac disease: results from a cross-sectional study. Arq Gastroenterol. 2015;52:139–142. Lebwohl B, Blaser MJ, Ludvigsson JF, et al. Decreased risk of celiac disease in patients with Helicobacter pylori colonization. Am J Epidemiol. 2013;178:1721–1730. Simondi D, Ribaldone DG, Bonagura GA, et al. Helicobacter pylori in celiac disease and in duodenal intraepithelial lymphocytosis: active protagonist or innocent bystander? Clin Res Hepatol Gastroenterol. 2015;39:740–745. Jozefczuk J, Bancerz B, Walkowiak M, et al. Prevalence of Helicobacter pylori infection in pediatric celiac disease. Eur Rev Med Pharmacol Sci. 2015;19:2031–2035. Cinova J, De Palma G, Stepankova R, et al. Role of intestinal bacteria in gliadin-induced changes in intestinal mucosa: study in germ-free rats. PLoS One. 2011;6:e16169. Laparra JM, Olivares M, Gallina O, Sanz Y. Bifidobacterium longum CECT 7347 modulates immune responses in a gliadininduced enteropathy animal model. PLoS One. 2012;7:e30744. Papista C, Gerakopoulos V, Kourelis A, et al. Gluten induces coeliac-like disease in sensitised mice involving IgA, CD71 and transglutaminase 2 interactions that are prevented by probiotics. Lab Invest. 2012;92:625–635. D’Arienzo R, Stefanile R, Maurano F, et al. Immunomodulatory effects of Lactobacillus casei administration in a mouse model

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

of gliadin-sensitive enteropathy. Scand J Immunol. 2011;74: 335–341. Sanz Y, Sa´nchez E, Marzotto M, Calabuig M, Torriani S, Dellaglio F. Differences in faecal bacterial communities in coeliac and healthy children as detected by PCR and denaturing gradient gel electrophoresis. FEMS Immunol Med Microbiol. 2007;51: 562–568. Nistal E, Caminero A, Vivas S, et al. Differences in faecal bacteria populations and faecal bacteria metabolism in healthy adults and celiac disease patients. Biochimie. 2012;94:1724– 1729. Tjellstro¨m B, Stenhammar L, Ho¨gberg L, et al. Gut microflora associated characteristics in children with celiac disease. Am J Gastroenterol. 2005;100:2784–2788. Sa´nchez E, Ribes-Koninckx C, Calabuig M, Sanz Y. Intestinal Staphylococcus spp. and virulent features associated with coeliac disease. J Clin Pathol. 2012;65:830–834. Chiba M, Hoshina S, Kono M, et al. Staphylococcus aureus in inflammatory bowel disease. Scand J Gastroenterol. 2001;36: 615–620. Nguyen GC, Patel H, Chong RY. Increased prevalence of and associated mortality with methicillin-resistant Staphylococcus aureus among hospitalized IBD patients. Am J Gastroenterol. 2010;105:371–377. Ong PY, Patel M, Ferdman RM, Dunaway T, Church JA. Association of staphylococcal superantigen-specific immunoglobulin e with mild and moderate atopic dermatitis. J Pediatr. 2008; 153:803–806. Garza-Gonza´lez E, Morfı´n-Otero R, Llaca-Dı´az JM, RodriguezNoriega E. Staphylococcal cassette chromosome mec (SCC mec) in methicillin-resistant coagulase-negative staphylococci. A review and the experience in a tertiary-care setting. Epidemiol Infect. 2010;138:645–654. Olivares M, Neef A, Castillejo G, et al. The HLA-DQ2 genotype selects for early intestinal microbiota composition in infants at high risk of developing coeliac disease. Gut. 2015;64:406–417. Nadal I, Donat E, Donant E, Ribes-Koninckx C, Calabuig M, Sanz Y. Imbalance in the composition of the duodenal microbiota of children with coeliac disease. J Med Microbiol. 2007;56:1669–1674. Sa´nchez E, Donat E, Ribes-Koninckx C, Calabuig M, Sanz Y. Intestinal Bacteroides species associated with coeliac disease. J Clin Pathol. 2010;63:1105–1111. Nistal E, Caminero A, Herra´n AR, et al. Differences of small intestinal bacteria populations in adults and children with/ without celiac disease: effect of age, gluten diet, and disease. Inflamm Bowel Dis. 2012;18:649–656. Sa´nchez E, Donat E, Ribes-Koninckx C, Ferna´ndez-Murga ML, Sanz Y. Duodenal-mucosal bacteria associated with celiac disease in children. Appl Environ Microbiol. 2013;79:5472–5479. Ou G, Hedberg M, Ho¨rstedt P, et al. Proximal small intestinal microbiota and identification of rod-shaped bacteria associated with childhood celiac disease. Am J Gastroenterol. 2009;104: 3058–3067. Forsberg G, Fahlgren A, Ho¨rstedt P, Hammarstro¨m S, Hernell O, Hammarstro¨m M-L. Presence of bacteria and innate immunity of intestinal epithelium in childhood celiac disease. Am J Gastroenterol. 2004;99:894–904. Wacklin P, Kaukinen K, Tuovinen E, et al. The duodenal microbiota composition of adult celiac disease patients is associated with the clinical manifestation of the disease. Inflamm Bowel Dis. 2013;19:934–941. Kallioma¨ki M, Satokari R, La¨hteenoja H, et al. Expression of microbiota, Toll-like receptors, and their regulators in the small intestinal mucosa in celiac disease. J Pediatr Gastroenterol Nutr. 2012;54:727–732.

123

Dig Dis Sci 66. Maynard CL, Elson CO, Hatton RD, Weaver CT. Reciprocal interactions of the intestinal microbiota and immune system. Nature. 2012;489:231–241. 67. Abreu MT. Toll-like receptor signalling in the intestinal epithelium: how bacterial recognition shapes intestinal function. Nat Rev Immunol. 2010;10:131–144. 68. Collado MC, Donat E, Ribes-Koninckx C, Calabuig M, Sanz Y. Imbalances in faecal and duodenal Bifidobacterium species composition in active and non-active coeliac disease. BMC Microbiol. 2008;8:232. 69. 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. 70. Golfetto L, de Senna FD, Hermes J, Beserra BTS, Franc¸a FDS, Martinello F. Lower bifidobacteria counts in adult patients with celiac disease on a gluten-free diet. Arq Gastroenterol. 2014;51:139–143. 71. De Meij TGJ, Budding AE, Grasman ME, Kneepkens CMF, Savelkoul PHM, Mearin ML. Composition and diversity of the duodenal mucosa-associated microbiome in children with untreated coeliac disease. Scand J Gastroenterol. 2013;48: 530–536. 72. De Palma G, Nadal I, Collado MC, Sanz Y. Effects of a glutenfree diet on gut microbiota and immune function in healthy adult human subjects. Br J Nutr. 2009;102:1154–1160. 73. Jackson FW. Effects of a gluten-free diet on gut microbiota and immune function in healthy adult human subjects—comment by Jackson. Br J Nutr. 2010;104:773. 74. Moshfegh AJ, Friday JE, Goldman JP, Ahuja JK. Presence of inulin and oligofructose in the diets of Americans. J Nutr. 1999;129:1407S–1411S. 75. Van Loo J, Coussement P, de Leenheer L, Hoebregs H, Smits G. On the presence of inulin and oligofructose as natural ingredients in the western diet. Crit Rev Food Sci Nutr. 1995; 35:525–552. 76. Di Cagno R, Rizzello CG, Gagliardi F, et al. Different fecal microbiotas and volatile organic compounds in treated and untreated children with celiac disease. Appl Environ Microbiol. 2009;75:3963–3971. 77. Schippa S, Iebba V, Barbato M, et al. A distinctive ‘‘microbial signature’’ in celiac pediatric patients. BMC Microbiol. 2010;10:175. 78. Wacklin P, Laurikka P, Lindfors K, et al. Altered duodenal microbiota composition in celiac disease patients suffering from persistent symptoms on a long-term gluten-free diet. Am J Gastroenterol. 2014;109:1933–1941. 79. Marasco G, Colecchia A, Festi D. Dysbiosis in Celiac disease patients with persistent symptoms on gluten-free diet: a condition similar to that present in irritable bowel syndrome patients? Am J Gastroenterol. 2015;110:598. 80. Roma E, Roubani A, Kolia E, Panayiotou J, Zellos A, Syriopoulou VP. Dietary compliance and life style of children with coeliac disease. J Hum Nutr Diet. 2010;23:176–182. 81. Sonti R, Green PHR. Celiac disease: obesity in celiac disease. Nat Rev Gastroenterol Hepatol. 2012;9:247–248. 82. Kabbani TA, Goldberg A, Kelly CP, et al. Body mass index and the risk of obesity in coeliac disease treated with the gluten-free diet. Aliment Pharmacol Ther. 2012;35:723–729. 83. Mariani P, Viti MG, Montuori M, et al. The gluten-free diet: a nutritional risk factor for adolescents with celiac disease? J Pediatr Gastroenterol Nutr. 1998;27:519–523. 84. Scaramuzza AE, Mantegazza C, Bosetti A, Zuccotti GV. Type 1 diabetes and celiac disease: the effects of gluten free diet on metabolic control. World J Diabetes. 2013;4:130–134. 85. Kaukinen K, Lindfors K, Ma¨ki M. Advances in the treatment of coeliac disease: an immunopathogenic perspective. Nat Rev Gastroenterol Hepatol. 2014;11:36–44.

123

86. Vanderpool C, Yan F, Polk DB. Mechanisms of probiotic action: implications for therapeutic applications in inflammatory bowel diseases. Inflamm Bowel Dis. 2008;14:1585–1596. 87. Zeng J, Li Y-Q, Zuo X-L, Zhen Y-B, Yang J, Liu C-H. Clinical trial: effect of active lactic acid bacteria on mucosal barrier function in patients with diarrhoea-predominant irritable bowel syndrome. Aliment Pharmacol Ther. 2008;28:994–1002. 88. Seth A, Yan F, Polk DB, Rao RK. Probiotics ameliorate the hydrogen peroxide-induced epithelial barrier disruption by a PKC- and MAP kinase-dependent mechanism. Am J Physiol Gastrointest Liver Physiol. 2008;294:G1060–G1069. 89. 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. 90. Medina M, Izquierdo E, Ennahar S, Sanz Y. Differential immunomodulatory properties of Bifidobacterium logum strains: relevance to probiotic selection and clinical applications. Clin Exp Immunol. 2007;150:531–538. 91. Baba N, Samson S, Bourdet-Sicard R, Rubio M, Sarfati M. Commensal bacteria trigger a full dendritic cell maturation program that promotes the expansion of non-Tr1 suppressor T cells. J Leukoc Biol. 2008;84:468–476. 92. Medina M, De Palma G, Ribes-Koninckx C, Calabuig M, Sanz Y. Bifidobacterium strains suppress in vitro the pro-inflammatory milieu triggered by the large intestinal microbiota of coeliac patients. J Inflamm (Lond). 2008;5:19. 93. 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. 94. Laparra JM, Olivares M, Sanz Y. Oral administration of Bifidobacterium longum CECT 7347 ameliorates gliadin-induced alterations in liver iron mobilisation. Br J Nutr. 2013; 110:1828–1836. 95. De Palma G, Cinova J, Stepankova R, Tuckova L, Sanz Y. Pivotal advance: bifidobacteria and gram-negative bacteria differentially influence immune responses in the proinflammatory milieu of celiac disease. J Leukoc Biol. 2010;87:765–778. 96. D’Arienzo R, Maurano F, Lavermicocca P, Ricca E, Rossi M. Modulation of the immune response by probiotic strains in a mouse model of gluten sensitivity. Cytokine. 2009;48:254–259. 97. De Angelis M, Rizzello CG, Fasano A, et al. VSL#3 probiotic preparation has the capacity to hydrolyze gliadin polypeptides responsible for Celiac Sprue. Biochim Biophys Acta. 2006; 1762:80–93. 98. Smecuol E, Hwang HJ, Sugai E, Corso L, et al. Exploratory, randomized, double-blind, placebo-controlled study on the effects of Bifidobacterium infantis natren life start strain super strain in active celiac disease. J Clin Gastroenterol. 2013; 47:139–147. 99. Olivares M, Castillejo G, Varea V, Sanz Y. Double-blind, randomised, placebo-controlled intervention trial to evaluate the effects of Bifidobacterium longum CECT 7347 in children with newly diagnosed coeliac disease. Br J Nutr. 2014;112:30–40. 100. Lorenzo Pisarello MJ, Vintin˜i EO, Gonza´lez SN, Pagani F, Medina MS. Decrease in lactobacilli in the intestinal microbiota of celiac children with a gluten-free diet, and selection of potentially probiotic strains. Can J Microbiol. 2015;61:32–37. 101. Capriles VD, Areˆas JAG. Effects of prebiotic inulin-type fructans on structure, quality, sensory acceptance and glycemic response of gluten-free breads. Food Funct. 2013;4:104–110. 102. Cheng J, Kallioma¨ki M, Heilig HGHJ, et al. Duodenal microbiota composition and mucosal homeostasis in pediatric celiac disease. BMC Gastroenterol. 2013;13:113.