Rut Ferrer
email: rutferrer@ub.edu

Juan J Moreno
email: jjmoreno@ub.edu

Role of eicosanoids on intestinal epithelial homeostasis

Keywords: arachidonic acid, COX, cyclooxygenase, EETs, epoxyeicosatrienoic acids, EGF, epidermal growth factor, HETEs, hydroxyeicosatetraenoic acids, HODE, hydroxyoctadecadienoic acids, IBD, inflammatory bowel disease, LOX, lipoxygenase, LTs, leukotrienes, PSC, prostaglandin-expressing stromal cells, PGs, prostaglandins, cPLA 2 , cytosolic, TJ, tight junction, ZO, zonula occludens

A c c e p t e d M a n u s c r i p t

The intestinal epithelium is a highly dynamic system that is continuously renewed by a process involving cell proliferation and differentiation. Moreover, it is the main interface with the external environment, and maintenance and regulation of the epithelial structure and epithelial barrier function are key determinants of digestive health and host well-being. The tight junction, a multiprotein complex composed of transmembrane proteins associated with the cytoskeletal peri-junctional ring of actin and myosin, is an essential component of this barrier that is strictly regulated in a spatio-temporal manner by a complex signaling network. Defects in the intestinal epithelial barrier function have been observed in inflammatory bowel disease, and a classic example of the connection between inflammation and cancer is the increased risk of colorectal cancer in patients with inflammatory bowel disease. In recent years, several molecules have emerged as critical players contributing to inflammationassociated colorectal cancer. For example, eicosanoids derived from arachidonic acid are proposed as mediators involved in the regulation of epithelial structure/function.

Interestingly, the tissue concentration of eicosanoids increases during mucosal inflammation and colorectal cancer development. This overview focuses on the physiological and physiopathological roles of eicosanoids in cell growth/cell differentiation/apoptosis and in the paracellular permeability of the intestinal epithelium. A better understanding of these processes will foster new ideas for the development of therapies for these chronic disorders. Keywords: prostaglandin, leukotriene, hydroxyeicosatetraenoic acid, cell proliferation, cell differentiation, barrier function A c c e p t e d M a n u s c r i p t

Introduction. The intestinal epithelium

The whole digestive tract is fenced by an epithelial monolayer composed of different cell types. The surface area of the small intestine is increased by luminal protrusions, termed villi, and invaginations into the mucosa, the crypts of Lieberkühn. In the mucosa of the large intestine there are no villi, only crypts. Three differentiated cell types (enterocytes, enteroendocrine, and goblet cells) populate the villi, whereas Paneth cells reside at the bottom of the crypts together with stem cells that can be considered the crypt/villus progenitors. Stem cells divide every 12-16 h, generating 200 cells per crypt every day. This cell production is compensated by cell shedding at the tip of the villi (small intestine) or at the surface of the epithelium (large intestine). This proliferative process pushes cells to the top of the villus. Thus, the top of the crypts and villi contains terminally differentiated cells, whereas Paneth cells and stem cells escape this movement and consequently remain permanently at the bottom of the crypts [START_REF] Sancho | Signalling pathways in intestinal development and cancer[END_REF]. The proliferative state of stem cells must therefore be regulated by extrinsic cues that report the tissue status and thus adjust the rate of tissue renewal. The nature of these signals, how they are regulated, and how they activate stem cells are mostly unknown. However, given their importance for understanding epithelium homeostasis, epithelium repair processes and colorectal cancer, it would be interesting to shed light on these issues.

The intestinal epithelium is thus a highly dynamic system that is continuously renewed by a process involving cell proliferation and differentiation. Moreover, it is the main interface with the external environment, and maintenance and regulation of the epithelial structure and epithelial barrier function are key determinants of digestive health and host well-being. For the maintenance of gut homeostasis it is imperative that the permeability characteristics of the epithelium are tightly regulated so that the seemingly paradoxical functions of allowing nutrient uptake, while excluding A c c e p t e d M a n u s c r i p t 4 potentially harmful antigens from entering the mucosa, can coexist. In situations in which enhanced or prolonged leakiness of the epithelial barrier occurs, the result can be the passage of excessive amounts of pathogenic bacteria and toxic luminal antigens including endotoxins [START_REF] Anderson | Tight junctions and the molecular basis of regulation of paracellular permeability[END_REF] into the mucosa, eliciting an immune response that, if not properly controlled, can lead to chronic inflammation [START_REF] Bruewer | Inflammatory bowel disease and the apical junctional complex[END_REF].

The mechanisms that regulate the intestinal paracellular permeability to luminal substances and preserve its functional integrity are not completely understood.

However, crucial for this integrity are cell polarity and the multifaceted dynamic interactions between the cell adhesion complexes and tight junctions (TJ) with the actin cytoskeleton [START_REF] Hartsock | Adherens and tight junctions: structure, function and connections to the actin cytoskeleton[END_REF]. Thus, paracellular permeability is regulated primarily by the most apical epithelial intercellular junction, the TJ or zonula occludens (ZO). An intact intestinal epithelial TJ barrier is crucial for providing a barrier function. TJ is a complex multiprotein that is composed of both intracellular and membrane-spanning proteins.

Four distinct types of membrane protein have been localized to TJ: occludin, claudins, junctional adhesion molecules and the coxsackie virus and adenovirus receptor, whereas the intracellular complex of TJ-associated proteins includes ZO-1, ZO-2, ZO-3, cingulin, 7H6, symplekin, and ZA-1 [START_REF] Furuse | Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarly to occluding[END_REF].

The present commentary will focus on the role of arachidonic acid (AA) cascade enzymes as well as mediators derived from this cascade on intestinal epithelial cell proliferation/differentiation, intestinal barrier function, and immune cells that are present in the epithelium, and consequently on intestinal epithelial homeostasis.

A c c e p t e d M a n u s c r i p t 5

Eicosanoids and the intestine

AA is an important polyunsaturated fatty acid of cell membrane phospholipids and also a cellular mediator that acts by itself or following its transformation to eicosanoids, its oxidized biologically active products. Under physiological conditions, the amount of free intracellular available AA is quite low. However, AA release from phospholipids occurs through the activation of phospholipases, primarily phospholipase A 2 (PLA 2 ). PLA 2 comprises a large superfamily of distinct enzymes that exhibit different substrate specificities, cofactor requirements, and subcellular localizations, and includes secreted PLA 2 s (sPLA 2 s), cytosolic PLA 2 s (cPLA 2 s) and calcium-independent PLA 2 s (iPLA 2 s). Upon release from biomembranes, AA can be metabolized via three enzymatic pathways: the cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P-450 pathways. The COX pathway leads to the formation of thromboxanes and prostaglandins (PGs). There are two COX isozymes: COX-1, which is expressed constitutively and COX-2, an inducible enzyme that is expressed in the majority of mammalian cells. Both isozymes catalyze the synthesis of PGH 2 from AA which is metabolized by specific prostaglandin synthases to release PGs. LOXs insert a hydroxyperoxyl group into the AA, which is subsequently reduced to produce hydroxyeicosatetraenoic acids (HETEs). Thus, 5-LOX leads to the synthesis of 5-HETE, whereas 12-and 15-LOX can form 12-and 15-HETE. 5-HETE can be metabolized to synthesize leukotrienes (LTs) such as LTB 4 and the cysteinyl LTs (LTC 4 , LTD 4 and LTE 4 ). Finally, the cytochrome P-450 monooxygenases also metabolize AA by allylic oxidation, ω/ω-1 hydroxylation or epoxydation to synthesize a wide range of eicosanoids such as HETEs (20-HETE) and epoxyeicosatrienoic acids (EETs) [START_REF] Goetzl | Specificity of expression and effects of eicosanoid mediators in normal physiology and human diseases[END_REF][START_REF] Capdevila | Cytochrome P-450 and arachidonic acid bioactivation. Molecular and functional properties of the arachidonate monooxygenase[END_REF] (Fig. 1). cysteinyl LTs activate at least two receptors referred to as the CysLT 1 and CysLT 2 receptors (Fig. 1). Although HETEs and EETs were first described several decades ago, relatively little is known about their mechanism of action. The fact that HETE and EET receptors have not been identified may have contributed to this. Finally, we must consider that peroxisome proliferator-activated receptors that are expressed in the intestinal mucosa and involved in the regulation of cell growth/differentiation and intestinal inflammation can be activated by eicosanoids [START_REF] Na | Peroxisome proliferator-activated receptor gamma (PPAR gamma) ligands as bifunctional regulators of cell proliferation[END_REF][START_REF] Rizzo | PPARs and other nuclear receptors in inflammation[END_REF].

Regarding the characterization of eicosanoid production within the intestinal tract, considerable variability exists among different species as well as different anatomic locations. Thus, important regional variations in eicosanoid profiles exist throughout the gut [START_REF] Smith | Synthesis of degradation of prostaglandin E 2 in the epithelial and sub-epithelial layers of the rat intestine[END_REF]. The cells most likely to be responsible for eicosanoid production include immune cells in the lamina propria and subepithelial mesenchymal cells [START_REF] Smith | Synthesis of degradation of prostaglandin E 2 in the epithelial and sub-epithelial layers of the rat intestine[END_REF]; however, intestinal epithelial cells have also been shown to be capable of eicosanoid production [START_REF] Smith | Synthesis of degradation of prostaglandin E 2 in the epithelial and sub-epithelial layers of the rat intestine[END_REF]. In cultured intestinal epithelial cells, prostanoid production has been shown to increase after treatment with growth factors and phorbol esters [START_REF] Dubois | Regulation of eicosanoid production and mitogenesis in rat intestinal epithelial cells by transforming growth factor-alpha and phorbol ester[END_REF]. In contrast to their production, PG degradation seems to occur in the epithelial layer rather than in the subepithelium [START_REF] Smith | Synthesis of degradation of prostaglandin E 2 in the epithelial and sub-epithelial layers of the rat intestine[END_REF]. This could contribute to establish a PGs gradient along the cryp/villus axis, although this has yet to be demonstrated (Fig. 2).

A c c e p t e d

M a n u s c r i p t 7 Shoji et al. [START_REF] Shoji | Downregulation of prostaglandin E receptor subtype EP 3 during colon cancer development[END_REF] reported that four PGE 2 receptor (EP 1 -EP 4 ) mRNAs were expressed in human colon tissues. Moreover, they observed EP 1 , EP 2 and EP 4 mRNA expression in intestinal epithelial cells. Interestingly, a different regional distribution of PG receptors was reported in intestinal mucosa.

Transformed and non-transformed intestinal epithelial cell lines [START_REF] Kamitani | Regulation of 12-lipoxygenase in rat intestinal epithelial cell during differentiation and apoptosis induced by sodium butyrate[END_REF][START_REF] Wächtershäuser | Expression of 5lipoxygenase by human colorectal carcinoma Caco-2 cells during butyrate-induced cell differentiation[END_REF][START_REF] Paruchuri | Endogenous production of leukotriene D 4 mediates autocrine survival and proliferation via CysLT1 receptor signalling in intestinal epithelial cells[END_REF] as well as the human intestine [START_REF] Dreyling | Leukotriene synthesis by human gastrointestinal tissues[END_REF][START_REF] Collins | Induction of arachidonate 12-lipoxygenase (Alox 15) in intestine of iron-deficient rats correlates with the production of biologically active lipid mediators[END_REF] also produce LOX metabolites such as LTs and HETEs, whereas there is no information about HETE/EET production by cytochrome P-450 in intestinal epithelial cells or the intestine. With respect to the presence of LT receptors in the intestine, the CysLT 1 receptor is expressed in intestinal epithelial cells and tumorderived intestinal cells [START_REF] Paruchuri | Endogenous production of leukotriene D 4 mediates autocrine survival and proliferation via CysLT1 receptor signalling in intestinal epithelial cells[END_REF] whereas the CysLT 2 receptor is expressed in differentiated intestinal epithelial cells but not in tumor-derived intestinal cell lines [START_REF] Magnusson | An increased expression of cysteinyl leukotriene 2 receptor in colorectal adenocarcinomas correlates with high differentiation[END_REF]. The BLT 1 receptor is also expressed in human colon cancer cell lines and colon cancer tissue but not in normal colon tissue [START_REF] Ihara | Blockade of leukotrienes B 4 signaling pathway induced apoptosis and suppresses cell proliferation in colon cancer[END_REF].

Role of eicosanoids in the control of intestinal epithelial cell growth

Our previous findings demonstrating that AA release by iPLA 2 participates in the signaling pathways involved in the control of intestinal epithelial proliferation [START_REF] Sanchez | Calcium-independent phospholipase A 2 through arachidonic acid mobilization is involved in Caco-2 cell growth[END_REF] showed that iPLA 2 and COX-2 are involved in PGE 2 release in proliferative epithelial cells, whereas differentiation appears to depress this pathway. These events could also be necessary for the completion of epithelial cell differentiation, or at least for the development of the epithelial barrier function that is characteristic of differentiated epithelium [START_REF] Martín-Venegas | Arachidonic acid cascade and epithelial barrier function Turing Caco-2 cell differentiation[END_REF]. Defining the molecular mechanisms of action of PGs on cell growth is an area of intense research. Epidermal growth factor (EGF) appears to be a key constituent in the maintenance, growth, repair and barrier integrity of the gastrointestinal mucosa. Recent studies have demonstrated the transactivation of the Furthermore, PGE 2 could be involved in the control of the intestinal epithelial cell cycle machinery through the up-regulation of cyclin B 1 and down-regulation of p21 expression independently of p53 [START_REF] Dvory-Sobol | Celecoxib leads to G 2 /M arrest by induction of p21 and down-regulation of cyclin B1 expression in a p53-independent manner[END_REF]. Thus, this proliferative effect of PGE 2 contributes to the development of the epithelium and could be involved in the intestinal epithelial response to injury (Fig. 2). These events could be related to the observation that shortterm administration of PGE 2 causes significant stimulation of DNA synthesis and that prolonged PGE 2 treatment markedly increases the weight and DNA content of the intestinal mucosa [START_REF] Dembinski | Effects of E, F, and I series prostaglandins and analogues on growth of gastroduodenal mucosa and pancreas[END_REF].

Interactions between intestinal epithelial cells and stromal cells, which include fibroblasts, myofibroblasts, endothelial cells and other cell types, may dramatically influence the growth and transformation of the intestinal epithelium [START_REF] Elenbaas | Heterotypic signaling between epithelial tumor cells and fibroblasts in carcinoma formation[END_REF]. PGs and other eicosanoids are synthesized by these cells [START_REF] Lloret | Proliferation-dependent changes in arachidonic acid mobilization from phospholipids of 3T6 fibroblasts. Pflugers Archv[END_REF][START_REF] Nieves | Hydroxyeicosatetraenoic acids released through the cytochrome P-450 pathway regulate 3T6 fibroblast growth[END_REF]. Eicosanoids derived from both stromal and epithelial cells may stimulate stromal cells to release growth factors, which, in turn, provide a pro-proliferative and pro-neoplastic environment for the intestinal epithelium. Shao et al. [START_REF] Shao | Roles of myofibroblasts in prostaglandin E 2 -stimulated intestinal epithelial proliferation and angiogenesis[END_REF] found that exogenous PGE 2 induced the expression and secretion of several pro-proliferative and pro-angiogenic growth factors such as amphiregulin, vascular endothelial growth factors, hepatocyte growth factor and neuregulins by intestinal subepithelial myofibroblasts, which may mediate intestinal epithelial growth and transformation. Finally, we must consider that activated macrophages also produce eicosanoids [START_REF] Vivancos | Role of Ca 2+ -independent phospholipase A 2 and cyclooxygenase pathways in the nitric oxide production by murine macrophages stimulated by lipopolysaccharides[END_REF] that are present near the epithelial progenitor niche [START_REF] Pull | Activated macrophages are an adaptive element of the colonic epithelial progenitor niche necessary for regenerative responses to injury[END_REF]. All these cells, in addition to eicosanoids and growth factors In agreement with the above mentioned papers, Stenson [START_REF] Stenson | Prostaglandins and epithelial response to injury[END_REF] addressed these effects of PGs, particularly PGE 2 , in the intestinal epithelial response to injury in a radiation injury model and in colitis models. Brown et al. [START_REF] Brown | Myd88-dependent positionary of Ptgs2-expresiong stromal cells maintains colonic epithelial proliferation during injury[END_REF] reported that epithelial tissue contains cells that express COX-2 at high levels, named prostaglandin-expressing stromal cells (PSCs) (Fig. 2). They observed that the majority of PSCs were located in the lamina propria lining the upper and middle third of the rectal crypt. However, after injury, the number of PSCs at the crypt bottom, adjacent to the intestinal stem cells compartment, increased, and consequently the PGE 2 levels. These authors proposed that local production of PGE 2 by PSCs may be involved in regulating epithelial proliferation after injury. However, other authors proposed that the PGE 2 involved in epithelial tissue repair is due to COX-1 rather than COX-2 [START_REF] Houchen | Disruption of cyclooxygenase-1 gene results in an impaired response to radiation injury[END_REF]. In this way, radiation injury results in increased COX-1 levels in crypt stem cells and their progeny, and that PGE 2 produced through COX-1 promotes crypt stem cell survival and proliferation [START_REF] Cohn | Crypt stem cell survival in the mouse intestinal epithelium is regulated by prostaglandins synthesized through cyclooxygenase-1[END_REF]. Although it is increasingly accepted that the COX pathway is involved in physiological and pathophysiological cell growth, the role of COX isoforms is still a matter of debate.

The human colon is also able to produce lipoxygenase metabolites [START_REF] Dreyling | Leukotriene synthesis by human gastrointestinal tissues[END_REF] and these AA-derived metabolites have also been implicated in the control of intestinal epithelial cell growth. Thus, endogenous production of LTD 4 mediates autocrine survival and proliferation via the nuclear-and membrane-located CysLT 1 receptor, triggering a proliferative ERK1/2 signal in non-transformed and transformed intestinal epithelial cells [START_REF] Paruchuri | Endogenous production of leukotriene D 4 mediates autocrine survival and proliferation via CysLT1 receptor signalling in intestinal epithelial cells[END_REF][START_REF] Nielsen | A novel localization of the G-protein-coupled cysLT1 receptor in the nucleus of colorectal adenocarcinoma cells[END_REF].

There is little information about HETE/EET production by intestinal epithelial cells or the effect of these eicosanoids on intestinal epithelial growth, although many recent A c c e p t e d M a n u s c r i p t 10 papers have focused on the emerging effects of HETEs on cell signaling and physiological/pathological cell growth [START_REF] Moreno | New aspects of the role of hydroxyeicosatetraenoic acids in cell growth and cancer development[END_REF]. Given that 5-LOX [START_REF] Wächtershäuser | Expression of 5lipoxygenase by human colorectal carcinoma Caco-2 cells during butyrate-induced cell differentiation[END_REF], 12-LOX [START_REF] Kamitani | Regulation of 12-lipoxygenase in rat intestinal epithelial cell during differentiation and apoptosis induced by sodium butyrate[END_REF] and 15-LOX [START_REF] Nixon | 15-lipoxygenase-1 has anti-tumorigenic effects in colorectal cancer[END_REF] are expressed by intestinal epithelial cells and that there is differentiated expression of these enzymes during epithelial cell differentiation, these eicosanoids may also have an important role in the physiology of the epithelium. Thus, Kamitani et al. [START_REF] Kamitani | Regulation of 12-lipoxygenase in rat intestinal epithelial cell during differentiation and apoptosis induced by sodium butyrate[END_REF] suggested that 12-LOX, and/or the lipid products synthesized by this enzyme are involved in intestinal epithelial cell differentiation and apoptosis. Recently, Collins et al. [START_REF] Collins | Induction of arachidonate 12-lipoxygenase (Alox 15) in intestine of iron-deficient rats correlates with the production of biologically active lipid mediators[END_REF] reported that 12-LOX was increased by up to > 100-fold throughout the entire length of the intestine of iron-deficient animals, inducing a strong increase in 12-HETE and 13-hydroxyoctadecadienoic acid (13-HODE) intestinal levels that was correlated with an increase in the number of mitotic cells. Interestingly, these changes in the 12-LOX pathway induce a series of structural changes in the intestine such as increased villus height, width and crypt depth. This elegant experimental model leads to related changes in the expression of AA cascade enzymes and subsequent eicosanoid production, with epithelial cell growth and significant morphological changes in the intestinal mucosa.

Colon cancer is the third most common cancer and the second leading cause of cancer-related deaths. The incidence of colon cancer doubles with each decade of life after the age of fifty. Although a great deal of effort has been made toward developing screening strategies, aggressive surgical therapy, and other therapies, there has been little improvement in the outcome for patients with advanced disease.

Today, the causal relationship between inflammation, innate immunity and cancer is more widely accepted; nevertheless, many of the molecular and cellular mechanisms mediating this relationship remain unresolved. However, there is now evidence that inflammatory mediators have a powerful effect on tumor development. Early in the A c c e p t e d M a n u s c r i p t 11 neoplastic process, eicosanoids could be powerful tumor promoters, producing an attractive environment for tumor growth and promoting angiogenesis. Thus, studies in the early 1980s indicated that non-steroidal anti-inflammatory drugs (NSAIDs) were chemopreventive in animal models of colon cancer [START_REF] Pollard | Effect of indomethacin on intestinal tumors induced in rats by the acetate derivative of dimethylnitrosamine[END_REF]. In 1991, Thun et al. [START_REF] Thun | Aspirin use and reduced risk of fatal colon cancer[END_REF] reported that aspirin use reduce the relative risk of colon cancer and colon cancer mortality among 600,000 individuals, whereas acetaminophen, which does not affect COX activity, did not provide as protective an effect. Even more relevant for the clinician were subsequent studies that demonstrated that NSAID therapy can cause the regression of adenoma in patients with familial adenomatous polyposis [START_REF] Koehne | COX-2 inhibition and colorectal cancer[END_REF]. Furthermore, COX-2 is elevated in colorectal cancers [START_REF] Eberhart | Upregulation of cyclooxygenase 2 gene expression in human colorectal adenomas and adenocarcinomas[END_REF], with 50% of adenomas and 80-90% of adenocarcinomas exhibiting increased COX-2 expression, and elevated PGE 2 and 6-keto PGF 1α levels [START_REF] Pugh | Patients with adenomatous polyps and carcinomas have increased colonic mucosal prostaglandin E 2[END_REF][START_REF] Moran | Apc deficiency is associated with increased Egfr activity in the intestinal enterocytes and adenomas of C57BL/6J-Min/+ mice[END_REF]. However, conflicting data exist regarding the localization of COX-2 in the epithelium or stromal components of colorectal tumors.

Using an elegant experimental model, Oshima et al. [START_REF] Oshima | Suppression of intestinal polyposis in Apc delta716 knockout mice by inhibition of cyclooxygenase 2 (COX-2)[END_REF] reported that COX-2 is located in the stromal component and may promote tumor growth by producing bioactive PGs that affect tumor growth in a paracrine fashion. This is consistent with the model postulated by Kinzler and Vogelstein [START_REF] Kinzler | Landscaping the cancer terrain[END_REF], which states that COX-2 affects tumor growth by acting as a landscaping tumor promoter in the stromal component of the adenoma.

Despite the very strong evidence of a causative role of prostaglandins in general, and PGE 2 in particular, in intestinal cancer, the underlying molecular mechanisms have remained obscure until recently. However, studies by several research groups are now clarifying this point. Dietary manipulation of AA content in APC min animals suggests that AA is involved in tumorigenesis [START_REF] Petrik | Highly unsaturated (n-3) fatty acids, but not alpha-linolenic, conjugated linoleic or gamma-linolenic acids, reduce tumorigenesis in Apc (Min/+) mice[END_REF]. Using APC min induced tumorigenesis and Research on PPARs has revealed that they play a fundamental role in cellular proliferation/differentiation processes [START_REF] Kliewer | The PPARs and PXPs: nuclear xenobiotic receptor that define moved hormone signaling pathways[END_REF]. In human colon cancer cell lines and tumor cells, PGE 2 transactivates PPARδ through the PI3 kinase/Akt pathway. PGE 2 treatment of APC min mice increased the intestinal adenoma burden; however, PGE 2 had no effect on the adenoma burden in APC min mice deficient in PPARδ. Other findings indicate that the proliferative effects of PGE 2 are also mediated through PPARδ [START_REF] Wang | Prostaglandin E 2 promotes colorectal adenoma growth via transactivation of the nuclear peroxisome proliferators-activated receptor δ[END_REF].

Cianchi et al. [START_REF] Cianchi | Inhibition of 5-lipoxygenase by MK886 augments the antitumor activity of celecoxib in human colon cancer cells[END_REF] reported that the two major metabolic pathways of the AA cascade, COX and 5-LOX, are simultaneously up-regulated in human colorectal cancer.

Interestingly, inhibition of either COX or 5-LOX alone resulted in activation of the other pathway, and consequently, combined treatment with COX and 5-LOX modulators produced greater inhibition of tumor cell proliferation. High expression of the BLT 1 receptor [START_REF] Ihara | Blockade of leukotrienes B 4 signaling pathway induced apoptosis and suppresses cell proliferation in colon cancer[END_REF] and the CystLT 1 receptor was detected in human colon cancer tissues, whereas CysLT 2 receptor expression was reduced in colon cancer and was associated with poor prognosis, due to its capacity to induce differentiation and growth inhibition [START_REF] Magnusson | An increased expression of cysteinyl leukotriene 2 receptor in colorectal adenocarcinomas correlates with high differentiation[END_REF]. Thus, the CysLT 2 receptor could have more anti-tumorigenic activity than the CysLT 1 receptor in intestinal epithelial cells, and the balance between the two receptors is important for the outcome of tumor progression. These findings indicate that AA cascade enzyme expression and eicosanoid production are important, but that the balance between eicosanoid receptors is also important for tumor progression and disease outcome.

Dietary fiber (non-starch polysaccharides) bypasses digestion in the stomach and small intestine, as do oligosaccharides and some resistant starches. In the large intestine, symbiotic bacteria ferment these carbohydrates releasing butyrate, a short chain fatty A c c e p t e d M a n u s c r i p t 14 acid, as a by-product. At physiological concentrations, butyrate has been shown to induce growth inhibition, differentiation and apoptosis in colorectal tumor cells in vitro [START_REF] Heerdt | Potentiation by specific short-chain fatty acids of differentiation and apoptosis in human colonic carcinoma cell lines[END_REF]. These observations may explain, in part, the correlation between a high fiber diet and a low incidence of colorectal cancer. Crew et al. [START_REF] Crew | A cyclooxygenase-2 (COX-2) selective nonsteroidal anti-inflammatory drugs enhances the growth inhibitory effects of butyrate in colorectal carcinoma cells expressing COX-2 protein: regulation of COX-2 by butyrate[END_REF] observed that COX-2 inhibitors sensitize the cell to growth inhibition induced by butyrate in colorectal carcinoma cells expressing COX-2 protein. These findings may mean that high fiber diet content would enable lower doses of COX-2 inhibitors to be used in chemoprevention.

Despite progress in knowledge of the effect of PGs and LTs on intestinal epithelial cell growth, we are a long way from fully understanding the role of the AA cascade in intestinal epithelial cell growth/differentiation/apoptosis, which will hopefully lead to a greater understanding of colorectal cancer in terms of the molecular pathways involved, and thus provide potential applications for the clinical setting.

Role of eicosanoids in the control of intestinal barrier function

Inflammatory bowel disease (IBD) including Crohn's disease and ulcerative colitis, is characterized by chronic, relapsing inflammation of the gastrointestinal tract, affecting at least 1 in 1000 people in Western countries [START_REF] Blumberg | Animal models of mucosal inflammation and their relation to human inflammatory bowel disease[END_REF]. Although the molecular mechanisms underlying the pathogenesis of IBD have not been identified, at least two events in which eicosanoids are involved have been described. The first is defects in the mucosal barrier, which may allow the continuous stimulation of the mucosal immune system due to impairment of the epithelial barrier function. Increased paracellular permeability of the epithelium has been well documented both in acutely and chronically damaged areas of the intestine [START_REF] Medding | Intestinal permeability in patients with Crohn´s disease[END_REF]. Moreover, it is well known that intestinal permeability is regulated directly through alteration of TJ proteins, or A c c e p t e d M a n u s c r i p t 15 indirectly through effects on the cytoskeleton. However, although altered intestinal permeability has been reported in patients with IBD, and the grade of barrier defect related to the onset of symptoms, the mechanism responsible and the mediators have not been completely identified. In fact, TNF-α and IFN-γ have been identified as being responsible, at least in part, for the increase in paracellular permeability [START_REF] Andus | Activation of moncytes during inflammatory bowel diease[END_REF][START_REF] Lu | Epithelial ion transport and barrier abnormalities evoked by superantigen-activated immune cells are inhibited by interleukin-10 bot not interleukin-4[END_REF], and the mechanisms underlying the regulation of TJ permeability by TNF-α and IFN-γ either directly through the alteration of TJ proteins, or indirectly through effects on the cytoskeleton have been well documented [START_REF] Turner | Molecular basis of epithelial barrier regulation: from basic mechanisms to clinical application[END_REF].

The second event that occurs in IBD is an increase in the inflammatory response with an associated increase in cytokine, eicosanoid and free radical production. Eicosanoids play a significant role in IBD as mediator of inflammation. Thus, the levels of prostanoids such as PGE 2 , PGF 2α and PGD 2 , as well as 12-HETE, 15-HETE and LTB 4 are higher in inflamed compared to normal mucosa [START_REF] Lauritsen | In vivo profiles of eicosanoids in ulcerative colitis, Crohn's colitis, and Clostridium difficile colitis[END_REF][START_REF] Wardle | Use of coculture of colonic mucosal biopsies to investigate the release of eicosanoids by inflamed and uninflamed mucosa from patients with inflammatory bowel disease[END_REF]. Moreover, the increase in PGE 2 levels is correlated with disease activity [START_REF] Carty | Measurement of in vivo rectal mucosal cytokine and eicosanoids production in ulcerative colitis using filter paper[END_REF]. Furthermore, during remission, PGE 2 and LTB 4 levels return to values comparable with normal colorectal mucosa [START_REF] Lauritsen | In vivo profiles of eicosanoids in ulcerative colitis, Crohn's colitis, and Clostridium difficile colitis[END_REF].

The enhancement of PGs is a consequence of the overexpression of COX and prostaglandin synthases in inflamed intestinal mucosa [START_REF] Subbaramaiah | Microsomal prostaglandin E synthase-1 is overexpressed in inflammatory bowel disease[END_REF]. This supports the role of eicosanoids in IBD. Further evidence that supports the importance of eicosanoids in IBD is that sulfasalazine, mesalamine and glucocorticoids, effective treatments, reduce eicosanoid synthesis [START_REF] Fretland | Eicosanoids and inflammatory bowel disease: regulation and prospects for therapy[END_REF][START_REF] Schreiber | The role of the mucosal immune system in inflammatory bowel disease[END_REF].

Enteroinvasive bacteria that are responsible for diarrheal diseases also activate the expression of genes such as COX-2 in the intestinal epithelium, provoking PGE 2 production and consequently impaired barrier function [START_REF] Resta-Lenert | Enteroinvasive Bacteria alter barrier and transport properties of human intestinal epithelium: role of iNOS and COX-2[END_REF]. On the other hand, we must consider that intestinal epithelial cells have the capacity to recognize and respond to commensal microbiota. Elements of the microbiota inhibit the NF-kB pathway by Activation of PPAR-γ by butyrate has also been shown to reduce colonic permeability, probably through the promotion of intestinal epithelial cell differentiation and the consequent reinforcement of the TJs [START_REF] Kinoshita | Butyrate reduces colonic paracellular permeability by enhancing PPARgamma activation[END_REF]. The microflora seems to be regulated to ensure protection from injury and induction of repair. An attractive hypothesis is that under normal physiological conditions the coordinated induction of various protective mechanisms helps to control pivotal elements involved in the AA cascade, thus maintaining normal epithelial cell homeostasis and the interplay with commensal bacteria.

In summary it seems that AA release and PGE 2 synthesis by the COX pathway could be involved in the regulation of intestinal paracellular permeability (Fig. 2). Thus, we observed that intestinal epithelial differentiation induces a decrease in both iPLA 2 activity and COX-2 expression and, consequently, a decrease in AA release and PGE 2 synthesis in parallel with a reduction in paracellular permeability and consequently the development of a barrier function that can be disrupted by the exogenous addition of PGE 2 [START_REF] Martín-Venegas | Arachidonic acid cascade and epithelial barrier function Turing Caco-2 cell differentiation[END_REF]. Recently, we observed that this modulation of the epithelial barrier function is mediated by the interaction with PGE 2 receptors EP 1 and EP 4 . Events that activate PLC-IP 3 -Ca 2+ and cAMP-PKA pathways that lead to an intracellular calcium concentration and the redistribution of TJ proteins such as occludin and the perijunctional actin ring that may be mediated these effects [START_REF] Rodríguez-Lagunas | PGE 2 receptors involved in the regulation of epithelial barrier function in Caco-2 cell monolayers[END_REF].

Given that NSAIDs, which are COX pathway inhibitors, might exacerbate the extent of IBD as well as epithelial barrier disruption [START_REF] Bjarnasson | Side effects of nonsteroidal anti-inflammatory drugs on the small and large intestine in humans[END_REF], we must consider whether other eicosanoids have any effect on the elements that regulate epithelial barrier function.

There is no information about the evolution of LOX expression in IBD, an important step in the AA cascade in this pathophysiological condition. Moreover, to the date, there (Fig. 3). These findings might explain the negative effects of NSAID treatments on IBD evolution.

Eicosanoids as immunoregulatory factors in intestinal mucosa

Another important aspect of the actions of eicosanoids in the intestine is their immunoregulatory effect. The close proximity of a wide array of antigens and lymphocytes in the gastrointestinal tract emphasizes the need to maintain immunological homeostasis and avoid an inappropriate immune response to nonpathogenic antigens, with subsequent epithelial alterations. Eicosanoids may also be involved in this process. PGE 2 is known to have immunomodulatory effects: it downregulates the major histocompatibility complex class II and cytokine receptor expression as well as cytokine production [START_REF] Van Der | Prostaglandin E 2 is a potente inhibitor of human interleukin 12 production[END_REF][START_REF] Wu | Prostaglandin E 2 and dexamethasone inhibit IL-12 receptor expression and IL-12 responsiveness[END_REF], events that might be involved in controlling an inflammatory immune response to dietary antigens. Consequently, PGs produced by epithelial cells, fibroblasts or lamina propria mononuclear cells could downregulate the intestinal immune system (Fig. 3). The importance of COX-dependent AA metabolites as immunoregulatory factors in the intestinal mucosa is supported by the observation that NSAIDs exacerbate clinical activity in human inflammatory bowel disease [START_REF] Bjarnasson | Side effects of nonsteroidal anti-inflammatory drugs on the small and large intestine in humans[END_REF]. In addition, some COX-2 -/-mice show peritonitis and intestinal abscess A c c e p t e d M a n u s c r i p t 18 formation, possibly secondary to inappropriate immune activation in the absence of high levels of PGE 2 [START_REF] Morham | Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse[END_REF]. Newberry et al. [START_REF] Newberry | Cyclooxygenase-2-dependent arachidonic acid metabolites are essential modulators of the intestinal immune response to dietary antigen[END_REF] proposed that 1 nM PGE 2 could have this immunomodulatory effect, such a concentration being reached in the intestinal mucosa.

It will be necessary to study the effects of other eicosanoids such as HETEs and EETs on intestinal immunoregulation to clarify their role in this important aspect of intestinal epithelial homeostasis.

Conclusions and perspectives

An attractive hypothesis is that under normal physiological conditions the coordinated induction of various protective mechanisms helps to control pivotal elements involved in the AA cascade, but the increase in eicosanoid mucosal levels may contribute to the development of inflammatory processes as well as colorectal cancer.

Answers to the question considered during this commentary may help us to determine the role of eicosanoids in the maintenance of intestinal homeostasis as well as to design protocols for using dietary modifications or pharmacological treatments in the prevention or treatment of IBD and colorectal cancer.

Given the different effects of eicosanoids we must consider that pharmacological manipulation of the AA cascade may have contradictory effects. For example, NSAID treatments reduce the risk of colorectal cancer whereas they exacerbate IBD. In the same way, treatment with an EP 4 receptor antagonist protected against the development of colon cancer [START_REF] Mutoh | Involvement of prostaglandin E receptor EP(4) in colon carcinogenesis[END_REF], but exacerbated experimental colitis [START_REF] Kabashima | The prostaglandin receptor EP4 suppresses colitis, mucosal damage and CD4 cell activation in the gut[END_REF]. The rational design of treatments that allow the impairment of cell growth or barrier dysfunction induced by eicosanoids without upregulation of the immune response may be a useful approach for IBD and colorectal cancer.

A c c e p t e d M a n u s c r i p t 6 Eicosanoids

 6 have pleitropic effects on cell physiology and signal transduction and exerts their actions through binding to specific members of the G protein-coupled seven transmembrane domain receptor family. The main prostanoid receptors are the DP (DP 1 -DP 2 ) receptors to PGD 2 , EP (EP 1 -EP 4 ) receptors to PGE 2 , FP receptor to PGF 2α , IP receptor to PGI 2 and TP receptor to thromboxanes. Two LTB 4 receptors have been characterized: BLT 1 is a high-affinity receptor whereas BLT 2 is a low-affinity receptor;

A c c e p t e d M a n u s c r i p t 8 EGF

 8 receptor by PGE 2 in intestinal epithelial cells[START_REF] Pai | Prostaglandin E 2 transactivates EGF receptor: a novel mechanism for promoting colon cancer growth and gastrointestinal hypertrophy[END_REF]. Moreover, the Wnt signaling cascade and the activation of PPARδ are involved in the effects of PGs on the proliferation of non-transformed and transformed intestinal epithelial cells.

  A c c e p t e d M a n u s c r i p t 9 produced are strategically located leading an instructive communication with the epithelial stem cells and their descendants.

M a n u s c r i p t 12 AA

 12 cPLA 2 -/-mice, Hong et al.[START_REF] Hong | Deletion of cytosolic phospholipase A 2 suppresses Apc Min -induced tumorigenesis[END_REF] demonstrated the pivotal role of enzymes involved in A c c e p t e d release in small intestine polyp formation whereas they observed a trend in the opposite direction in the small intestine, probably, by limiting proapoptotic signals[START_REF] Ilsley | Cytoplasmic phospholipase A 2 deletion enhances colon tumorigenesis[END_REF].Furthermore, a number of animal studies involving APC-mutant mice are converging on a central pathway involving the COX pathway. In addition, a recent study by Castellone et al.[START_REF] Castellone | Prostaglandin E 2 promotes colon cancer cell growth through a Gs-axin-beta-catenin signaling axis[END_REF] demonstrated that PGE 2 increases the activation of the Tcf/Lef transcription factor and activated components of the canonical Wnt signaling cascade. Thus, PGE 2 EP receptor interaction results in the displacement of APC and the loss of phosphorylation of β-catenin. In the absence of phosphorylation, β-catenin is not degraded, but instead translocates to the nucleus and activates Tcf/Lef. A possible alternative mechanism by which PGs may induce cell growth is the EGF pathway. EGF receptor (EGFR) signaling is more relevant to early APC-dependent tumorigenesis, and PGE 2 can transactivate EGFR in intestinal epithelial cells and consequently the EGFR-PI3K-Akt cell survival/proliferative pathway in vivo, as reported recently by Moran et al. [43]. These findings support the original hypothesis that the anti-tumorigenesis effects of NSAIDs work through decreased PG production. However, these explanations have lacked molecular detail, in large part because of a poor understanding of which PG receptors are involved. In the last decade, important findings were obtained with respect to this point. Targeted deletion of the EP 1 receptor reduced colonic lesions by 60% in a colon cancer experimental model and APC min -induced polyp formation was also reduced by 57% following treatment with a specific EP 1 antagonist [50]. Considering all together, we now have a complete picture of the main elements involved in the effects of PGs such as PGE 2 on epithelial cell growth in physiological and pathophysiological conditions: AA is released by PLA 2 , then metabolized by COX-1/COX-2 to produce bioactive eicosanoids such as PGE 2 , whichA c c e p t e d M a n u s c r i p t 13 interact with specific receptors (EP 1 and EP 4 ) and activate the cell signaling involved in the control of the cell cycle.

A c c e p t e d M a n u s c r i p t 16 hijacking

 16 the PPAR-γ pathway, and consequently modulate the COX pathway[START_REF] Artis | Epithelial-cell recognition of commensal bacteria and maintenance of immune homeostasis in the gut[END_REF].

A c c e p t e d M a n u

  s c r i p t is little information on the effects of eicosanoids produced by this pathway on TJ and consequently on barrier function. However, Rodríguez-Lagunas et al.[START_REF] Rodríguez-Lagunas | Role of N-6 PUFA derived lípido mediator son epithelial barrier function in intestinal Caco-2 cell monolayers[END_REF] have recently reported that LTD 4 and 5-, 12-and 15-HETE produced by the LOX pathway alter the epithelial barrier function to a greater extent than prostanoids. Thus, the administration of NSAIDs could allow LT and HETE synthesis by the LOX pathway to proceed unchecked while blocking PG formation, and hypothetically could shift the net effect of prevailing eicosanoids toward proinflammation and the impairment of barrier function

A c c e p t e d M a n u s c r i p t 19 In

 19 conclusion, more studies are needed to clarify the pivotal questions that remain unanswered in this exciting field. In particular, additional work is required to explore mechanistic issues involving the effects of LTs and HETEs/EETs on epithelial cell growth/differentiation, barrier function, and on the intestinal immune response.

u s c r i p t 31 Figure 1 .

 311 Figure 1. Scheme of the main enzymes and metabolites of the arachidonic acid cascade.

Figure 2 .

 2 Figure 2. Structure of the small and large intestine considering epithelial cell

Figure 3 .

 3 Figure 3. Effects of eicosanoid balance on immunoregulation,

A c c e p t e d M a n u s c r i p

Figure 1 A c c e p t e d M a n u s c r i p

 1 Figure 1

Figure 2 A c c e p t e d M a n u s c r i p

 2 Figure 2

Figure 3 A

 3 Figure 3

Acknowledgements

The authors gratefully acknowledge the support of the Generalitat de Catalonia (2005SGR00269 and 2009SGR00438) and the financial support of the Ministry of Sciences and Technology (BFI2004-0496/BFI and BFI2007-61727/BFI) for grant funding.


Dr Julián Benito-León

Diana Pisa

Ruth Alonso

Patricia Calleja

M Díaz-Sánchez

Luis Carrasco

Dr Julian Benito-Leon

Benito-Leon Julian

Maria Diaz-Sanchez

Association between multiple sclerosis and Candida species: evidence from a case-control study

Keywords: Candida, multiple sclerosis, serology, case-control study Candida species and multiple sclerosis Candida, multiple sclerosis, serology, case-control study

Objective. Candida infection among multiple sclerosis (MS) patients has not been studied in depth. We determined whether there is an association between serological evidence of Candida infection and MS. Methods. Blood specimens were obtained from 80 MS patients and 240 matched controls. Immunofluorescence analysis and ELISA were used to detect Candida species antibodies and slot-blot to detect antigens. Results. Using immunofluorescence analysis, moderate to high concentrations of serum antibodies to Candida famata were present in 30 (37.5%) MS patients vs. 30 (12.5%) controls (p<0.001). Results for Candida albicans were 47.5% (38/80 MS patients) vs. 21.3% (51/240 controls) (p<0.001), Candida parapsilosis 37% (28/80 MS patients) vs. 17.1% (41/240 controls) (p<0.001) and, Candida glabrata, 46.3% (37/80 MS patients) vs. 17.5% (42/240 controls) (p<0.001). After adjusting for age and gender, the odds ratios (95% confidence intervals) for MS, according to the presence of Candida antigens were: 2.8 (0.3-23.1, p=0.337) for Candida famata; 1.5 (0.7-3.4, p=0.290) for Candida albicans; 7.3 (3.2-16.6, p<0.001) for Candida parapsilosis; and 3.0 (1.5-6.1, p = 0.002) for Candida glabrata. The results were similar after excluding 10 patients on immunosuppressants. Conclusions. The results of this single study suggest that Candida species infection may be associated with increased odds of MS.

Response to Reviewers: Reviewer 1: 1. "The main objective of this study was to analyze the association between the serological evidence of Candida infection and MS. This is a well designed, written article with clear methodology and results. However, in my opinion it is very confusing to conclude that a prior Candida infection increases the odds for MS, when there is an increasing number of publications looking at the association between multiple viral infections, vaccines...It looks that the background of prior infections is very complex and we still do not know if it is due to a single or multiple infections with an abnormal inmunological system. Based on the data of a single study the conclusion of the abstract and article should be changed and just "suggesting it".

Following the suggestions of the reviewer, the conclusions of the abstract and article have therefore been toned down. We now write in the conclusions: "the results of this single study suggest that Candida species infection may be associated with increased odds of MS. The clinical importance of this association is uncertain. We therefore propose future studies that should clarify whether and, if so how, yeasts from the genus Candida act in the pathogenesis of this disease".

Reviewer 2: 1. This is an interesting and well-written paper on the association between candida species and multiple sclerosis )MS). After adjusting for age and gender, the odds for MS were elevated for several candida species. The rationale for studying this link is provide in the second page of the introduction. The finding appears to be novel. The selection of cases seems reasonable and was not biased. The diagnosis and clinical assessment was reasonable. There was 1:3 matching with controls. The analytic plan is well presented. Several additional analyses were performed in which they excluded cases who were on immuosuppressive therapies. The limitations are discussed appropriately. The results seemed to be dependent to some measure on the assay used and all results were not significant. Perhaps the authors can discuss why this was the case.

We acknowledge the value of this comment. We now write the following paragraph in the discussion section: "In this case-control study we found a significant association between elevated antibodies of four species of Candida and MS using indirect immunofluorescence. However, these differences were not observed using ELISA, but for Candida glabrata. We feel detection of antibodies by ELISA in this large sample of participants showed poor specificity. Although it is true that antibodies to Candida species may occur in normal individuals as a result of commensal colonization of several anatomically distinct sites including skin, oral, gastrointestinal tract, and vagina [START_REF] Southern | C. albicans colonization of human mucosal surfaces[END_REF][START_REF] Yang | Virulence factors of Candida species[END_REF], MS patients had a significant seroprevalence of Candida species using indirect immunofluorescence".

Introduction

Multiple sclerosis [MS) is one of the most common chronic neurological disorders among young adults, especially in high northern latitudes, and the most common cause of non-trauma related disability in this group of age [START_REF] Pugliatti | The worldwide prevalence of multiple sclerosis[END_REF].

The broad spectrum of symptoms of MS considerably impact upon the quality of life experienced by patients and their families to a greater extent than several other chronic diseases [START_REF] Benito-León | Health-related quality of life and its relationship to cognitive and emotional functioning in multiple sclerosis patients[END_REF][3][START_REF] Mitchell | Quality of life and its assessment in multiple sclerosis: integrating physical and psychological components of wellbeing[END_REF][START_REF] Morales-Gonzalez | GEDMA Study Group (2004) A systematic approach to analyse healthrelated quality of life in multiple sclerosis: the GEDMA study[END_REF][START_REF] Rivera-Navarro | Informal caregiving in multiple sclerosis patients: data from the Madrid Demyelinating Disease Group study[END_REF]. Although etiological factors are not clearly established, there are data which suggest that non-genetic (environmental] factors, especially infectious agents [e.g. virus or Chlamydia pneumoniae), may play a role in MS [START_REF] Ascherio | Epidemiology of multiple sclerosis: from risk factors to prevention[END_REF][START_REF] Gilden | Infectious causes of multiple sclerosis[END_REF][START_REF] Contini | Molecular detection of Parachlamydia-like organisms in cerebrospinal fluid of patients with multiple sclerosis[END_REF][START_REF] Ascherio | Epstein-Barr virus in the development of multiple sclerosis[END_REF]. Among these, Epstein-Barr virus (EBV) stands out as one of the most consistent risk factors [START_REF] Ascherio | Epstein-Barr virus in the development of multiple sclerosis[END_REF]. Nevertheless, some features of MS epidemiology are not explained by EBV and strongly suggest a key role of other infectious agents [START_REF] Contini | Molecular detection of Parachlamydia-like organisms in cerebrospinal fluid of patients with multiple sclerosis[END_REF]. For instance, Butcher [START_REF] Butcher | The distribution of multiple sclerosis in relation to the dairy industry and milk consumption[END_REF] had suggested a global correlation between MS incidence and milk consumption, as taken from data provided by the Food and Agriculture Organization of the United Nations (FAO).

In addition, in an analysis of 27 countries and 29 populations worldwide, Malosse et al [START_REF] Malosse | Correlation between milk and dairy product consumption and multiple sclerosis prevalence: a worldwide study[END_REF] found a good correlation (rho = 0.836) between MS prevalence and fresh cow milk consumption. Milk may contain potentially pathogenic yeast species such as Candida parapsilosis or Candida glabrata, both able to grow at 40ºC [START_REF] Lagneau | Isolation of yeasts from bovine milk in Belgium[END_REF].

In 1993, Gass [START_REF] Gass | Acute zonal occult outer retinopathy[END_REF] described a new syndrome, the so-called acute zonal occult outer retinopathy (AZOOR) in 13 patients who were predominantly young women. This enigmatic syndrome is characterized by rapid loss of one or more large zones of outer retinal function, photopsia, minimal fundoscopic changes, and electroretinographic abnormalities affecting one or both eyes [START_REF] Gass | Acute zonal occult outer retinopathy[END_REF]. Recent evidences from case reports and case series suggest a link between AZOOR and MS [START_REF] Gass | Acute zonal occult outer retinopathy: a long-term follow-up study (2002)[END_REF][START_REF] Hintzen | Acute zonal occult outer retinopathy and multiple sclerosis[END_REF]. The only available long-term follow-up study of patients with AZOOR showed the presence of white matter abnormalities on cerebral magnetic resonance imaging in a substantial subgroup [START_REF] Gass | Acute zonal occult outer retinopathy: a long-term follow-up study (2002)[END_REF]. Both AZOOR and MS share common characteristics, such as a relapsing course, often followed by gradual worsening, a skew to female sex, inflammatory lesions localized predominantly in areas around venules, along with signs of secondary neuronal cell loss [START_REF] Gass | Acute zonal occult outer retinopathy[END_REF][START_REF] Gass | Acute zonal occult outer retinopathy: a long-term follow-up study (2002)[END_REF][START_REF] Hintzen | Acute zonal occult outer retinopathy and multiple sclerosis[END_REF]. Moreover, for both AZOOR and MS, there seems to be an association with generalized autoimmune disorders [START_REF] Gass | Acute zonal occult outer retinopathy[END_REF][START_REF] Gass | Acute zonal occult outer retinopathy: a long-term follow-up study (2002)[END_REF][START_REF] Hintzen | Acute zonal occult outer retinopathy and multiple sclerosis[END_REF]. In the last three years, an association between AZOOR and Candida species infection has been suggested [START_REF] Carrasco | Isolation of Candida famata from a patient with acute zonal occult outer retinopathy[END_REF][START_REF] Pisa | Evolution of antibody response and fungal antigens in the serum of a patient infected with Candida famata[END_REF][START_REF] Pisa | Fungal infection in patients with serpiginous choroiditis or acute zonal occult outer retinopathy[END_REF].

Although pathogenic yeasts from the genus Candida are well recognized infectious agents [START_REF] Trick | National Nosocomial Infections Surveillance System Hospitals (2002) Secular trend of hospital-acquired candidemia among intensive care unit patients in the United States during 1989-1999[END_REF][START_REF] Pfaller | Role of sentinel surveillance of candidemia: trends in species distribution and antifungal susceptibility[END_REF], they have never been systematically studied in MS.

Demyelinated lesions in the central nervous system have been described in patients with Candida infection [START_REF] Lipton | Candidal infection in the central nervous system[END_REF]. The previous associations encouraged us to test the hypothesis that some yeast species of medical importance (including Candida famata, Candida albicans, Candida parapsilosis, and Candida glabrata) are associated with MS. Traditional methods of Candida species detection, which include blood culture and biopsy, usually lack both sensitivity and specificity. Therefore, diverse non-culture-based methods were also applied in the current study [START_REF] Laín | Contribution of serum biomarkers to the diagnosis of invasive candidiasis[END_REF].

Methods

1. Study population

MS patients were included in the study if they had met the McDonald criteria [START_REF] Mcdonald | Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the diagnosis of multiple sclerosis[END_REF]. Exclusion criteria included antibiotic therapy within the previous six months, any corticosteroid therapy within the previous 12 months, and major systemic conditions (including hematologic or solid-organ malignancies, recent surgery, gastrointestinal tract surgery, endocrinopathies, and acute or chronic renal failure).

Our sample of patients with MS was derived from the MS association of Madrid (Spain). This association was chosen because a) there was a close relationship between our research group and the association; and b) the association maintained a computer-based registry of MS patients from the community of Madrid. In this computer-based registry, basic demographic data of the members (e.g., age and gender) were recorded. We obtained permission from the association to access this confidential information. The association included more than 1,000 MS patients from the community of Madrid (Spain).

We recruited MS patients without regard to age or gender or other demographic factors (i.e., every 5 th name was selected from patient lists so as to achieve the needed number of recruits) from the MS association. MS patients were recruited between June 2007 and May 2008.

A 20 minute interview was conducted on each MS patient in which information on age of MS onset and disease evolution was also obtained. Three neurologists with expertise in MS (JBL, PC, and MDS) participated in the clinical assessment and applied the EDSS scale to rate the severity of disease (range = 0 -10) [START_REF] Kurtzke | Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS)[END_REF]. Three subgroups were formed according to EDSS score: a) low physical disability (EDSS 0-2.5); moderate physical disability (EDSS 3.0-5.5); and high physical disability (EDSS ≥ 6.0). Healthy controls were selected from those subjects who were occasional blood donors at different health centers in Madrid (Spain) between June 2007 and May 2008. MS patients were 1:3 frequency-matched with control subjects.

Frequency matching was based on age (±5 years) and gender. Control subjects were excluded if they had symptoms or a history of a major systemic disease or MS, and none was being treated with any type of antibiotics.

A 10-ml venous blood sample was obtained from each individual. In the case of MS patients, blood samples were obtained just after clinical assessment. In all analyses, control samples were interspersed with samples from patients. Laboratory studies (see below) were performed by experienced scientists (DP, RA and LC) who were blinded to the diagnosis (patient vs. control). This study was approved by both the research ethics board at University Hospital "12 de Octubre" and at the Autónoma University of Madrid.

Signed consent was obtained for all participants.

2.2. Candida infection detection.

Candida species growth

All the blood samples were cultured for yeasts in a YEPD medium (1% yeast extract, 2% peptone, and 2% glucose) by incubation at 30°C. The same medium, containing agar, was used to test growth of individual yeast colonies.

Antibodies detection

Rabbit antisera against different yeast species were obtained by inoculation of 0.5 ml phosphate-buffered saline (PBS) containing 1 or 2 mg yeast after autoclaving and lyophilization. Each inoculum had been mixed previously with the same volume of Freund's adjuvant. Rabbits were inoculated up to four times and the antibody titre and specificity of the sera were tested by indirect immunofluorescence and Enzyme-Linked ImmunoSorbent Assay (ELISA).

Indirect immunofluorescence

For Candida famata, 1 ml of culture was placed in 1.5 ml microcentrifuge tubes.

Cells were washed with PBS, incubated with 50 mM ammonium chloride for 10 min, and washed three times with PBS-Tween 20. Cells were then treated with the different serum samples diluted 1:500 in PBS-Tween 20 at 37°C for 2 hours, washed again with PBS-Tween 20, and incubated with the secondary antibody.

Rabbit anti-human fluorescein-conjugated antibody (immunoglobulin A [IgA]

plus IgM plus IgG) was added at a dilution of 1:500. The samples were incubated at 37 °C for another hour. The cells were then washed, resuspended in PBS, and mounted on slides with a drop of Depex (Serva). For the remaining Candida species, an Euroimmun commercial kit (Medizinische Labordiagnostika AG) was used in accordance with the manufacturer's instructions and using the same serum dilutions as for Candida famata [START_REF] Carrasco | Isolation of Candida famata from a patient with acute zonal occult outer retinopathy[END_REF][START_REF] Pisa | Evolution of antibody response and fungal antigens in the serum of a patient infected with Candida famata[END_REF][START_REF] Pisa | Fungal infection in patients with serpiginous choroiditis or acute zonal occult outer retinopathy[END_REF]. Finally, the cells were observed under a fluorescence microscope [START_REF] Carrasco | Isolation of Candida famata from a patient with acute zonal occult outer retinopathy[END_REF][START_REF] Pisa | Evolution of antibody response and fungal antigens in the serum of a patient infected with Candida famata[END_REF][START_REF] Pisa | Fungal infection in patients with serpiginous choroiditis or acute zonal occult outer retinopathy[END_REF].

Positivity percentage of each one of the results was compared to the positive control provided by the manufacturer (Euroimmune, Germany). Results were scored as follows: 0% (clearly negative in contrast with the positive control provided by the manufacturer), 20% (low antibody concentrations), 40% and 60% (moderate antibody concentrations), 80% and 100% (clearly positive as the positive control provided by the manufacturer) [START_REF] Carrasco | Isolation of Candida famata from a patient with acute zonal occult outer retinopathy[END_REF][START_REF] Pisa | Evolution of antibody response and fungal antigens in the serum of a patient infected with Candida famata[END_REF][START_REF] Pisa | Fungal infection in patients with serpiginous choroiditis or acute zonal occult outer retinopathy[END_REF].

Enzyme-Linked ImmunoSorbent Assay (ELISA).

ELISA was carried out as described previously [START_REF] Carrasco | Isolation of Candida famata from a patient with acute zonal occult outer retinopathy[END_REF][START_REF] Pisa | Evolution of antibody response and fungal antigens in the serum of a patient infected with Candida famata[END_REF]. Briefly, yeast suspension diluted in PBS was seeded in ELISA microtitre plates (Maxisorp; Nunc). The plates were blocked with PBS containing 3% low-fat dried milk and 0.2 % Tween 20. Sera from the study participants were added at different dilutions and the value obtained at 1:500 dilution was selected for data analysis.

Plates were subsequently incubated with goat anti-human (heavy and light chain) Ig horseradish peroxidase-conjugated antibodies (Pierce) and washed five times. Colour development was accomplished by incubation with ophenylenediamine (Sigma) and measured at 490 nm in a microplate reader (EL340; Bio-Tek Instruments). Results were scored as absolute values [START_REF] Carrasco | Isolation of Candida famata from a patient with acute zonal occult outer retinopathy[END_REF][START_REF] Pisa | Evolution of antibody response and fungal antigens in the serum of a patient infected with Candida famata[END_REF].

Antigen detection

The presence of Candida species antigens in plasma was determined by slot-blot analyses [START_REF] Pisa | Evolution of antibody response and fungal antigens in the serum of a patient infected with Candida famata[END_REF]. Different serum dilutions (200 µl) in TBS were added to each well. Samples were blotted onto a 45 mm nitrocellulose membrane (Bio-Rad) previously hydrated in TBS for 10 min using a Bio-Dot SF apparatus (Bio-Rad). After blotting, the membrane was processed and developed. The primary rabbit polyclonal antibodies against Candida famata, Candida albicans, Candida parapsilopsis and Candida glabrata were used at a 1:1000 dilution. A donkey anti-rabbit IgG horseradish peroxidase-conjugated antibody (Amersham Biosciences) at a 1:5000 dilution was used as secondary antibody [START_REF] Pisa | Evolution of antibody response and fungal antigens in the serum of a patient infected with Candida famata[END_REF]. Titres ≥20 were counted positive and ≤19, counted negative or doubtful [START_REF] Pisa | Evolution of antibody response and fungal antigens in the serum of a patient infected with Candida famata[END_REF][START_REF] Pisa | Fungal infection in patients with serpiginous choroiditis or acute zonal occult outer retinopathy[END_REF].

Data analysis and Sample Size

Statistical analyses were performed in SPSS Version 15.0 (SPSS, Inc., Chicago, IL). All tests were two sided, and significance was accepted at the 5% level (alpha = 0.05). The continuous variables age and ELISA were not normally distributed, and thus Mann-Whitney test was used to calculate differences between the patients and controls. Chi square was used for categorical data (gender, immunofluorescence and slot-blot analyses). Spearman's correlation coefficient was used to determine the correlations between the clinical and serological parameters. Odds ratios (ORs) and 95% confidence intervals (CIs) assessing the risk of MS associated with infection by each type of Candida, using indirect immunofluorescence and slot-blot, were estimated by use of binary logistic regression, adjusted for age and gender.

The targeted sample size (80 participants in the patient group and 240 participants in the control group) had 100% power to detect a 30% difference in slot-blot analysess between patients and controls (assuming alpha = 0.05).

Results

Of 85 eligible patients with MS, 5 (5.9%) were excluded due to insufficient volume of blood drawn. None were excluded because they had major comorbidities or had received antibiotic or corticosteroid therapy. The final study sample consisted of 80 patients with MS and 240 controls who were frequency-matched by age and gender. Forty (50%) MS patients were taking immunomodulatory (beta-interferons and glatiramer acetate) or immunosuppressant (azathioprine, methotrexate and mitoxantrone) medications (table 1). The remaining 33 (41.25%) had never been on immunomodulatory or immunosuppressant medications or their drugs had been withdrawn due to inefficacy at least one year before clinical assessment (7/80, 8.75%). The patients with MS and controls were similar in age and gender (table 1). Disease duration ranged from 1 to 39 years (median = 10.5 years) (table 1). No yeasts were cultured from the blood samples of patients and controls.

Moderate to high concentrations of serum antibodies to Candida famata

were present in 30 (37.5%) MS patients vs. 30 (12.5%) controls (p < 0.001), using indirect immunofluorescence analysis (table 2). In a regression analysis that adjusted for age and gender, subjects with moderate to high antibody concentrations were more than four times more likely to have MS than were those with negative or low concentrations of antibodies (OR = 4.4, 95% CI = 2.4 -8.1, p < 0.001) (table 3). However, the median concentrations of antibodies using ELISA were similar in the group of patients with MS compared with the control group [0.5 ± 0.5 (mean ± SD), 0.3 (median) vs. 0.5 ± 0.5 (mean ±SD), 0.4 (median), p = 0.101] (table 2). Similarly, the proportion of MS patients with positivity for Candida famata antigens, detected by slot-blot analysis, was similar to that in the control group (79 MS patients, 98.8% vs. 232 controls, 96.7%, p = 0.459) (adjusted OR = 2.8, 95% CI = 0.3 -23.1, p = 0.337) (Tables 3 and4). Similar results were obtained for Candida albicans and Candida parapsilosis (Tables 2 to 4), except that the proportion of MS patients with positivity for Candida parapsilosis antigens, detected by slot-blot analysis, was significantly higher than that of the control group (73 MS patients, 91.3% vs. 142 controls, 59.2%) (adjusted OR = 7.3, 95% CI = 3.2 -16.6, p < 0.001) (Tables 3 and4).

For Candida glabrata, moderate to high concentrations of serum antibodies were detected by indirect immunofluorescence in 37 of 80 patients with MS compared with 42 of 240 controls (46.3% versus 17.5%, p < 0.001; Table 2) (adjusted OR = 4.2, 95% CI = 2.4 -7.4, p < 0.001) (Table 3). Similarly, the concentrations of antibodies, using ELISA, were significantly higher in the patient group than in the control group [0.4 ± 0.3 (mean ± SD), 0.3 (median) vs. 0.3 ± 0.1 (mean ± SD), 0.3 (median), p = 0.001]. In addition, the proportion of MS patients with positivity for Candida glabrata antigens, detected by slot-blot analysis, was significantly higher than in the control group (18 MS patients, 22.5% vs. 21 controls, 8.7%) (adjusted OR = 3.0, 95% CI = 1.5 -6.1, p = 0.002) (Tables 3 and4).

No correlations were found between the clinical and the serological parameters with any type of Candida species. The above results were also similar after excluding 10 MS patients who were on immunosuppressant therapies or after excluding 47 MS patients who were receiving (40 cases) or ever received (7 cases) immunosuppressant or immunomodulatory therapies (data not shown). In addition, after excluding 40 MS patients whose MS duration was > 10 years, the results remained similar (data not shown).

Discussion

Several Candida species are widely distributed throughout the human population as both commensal organisms and as intermittent pathogens [START_REF] Ellepola | Laboratory diagnosis of invasive candidiasis[END_REF][START_REF] Southern | C. albicans colonization of human mucosal surfaces[END_REF]. In the clinical arena, blood cultures are the main assay for routine detection of candidiasis [START_REF] Ellepola | Laboratory diagnosis of invasive candidiasis[END_REF]. However, microbiological confirmation is difficult because blood cultures can be negative in up to 50% of autopsy-proven cases of deepseated candidiasis or may only become positive late in the infection [START_REF] Ellepola | Laboratory diagnosis of invasive candidiasis[END_REF]. Thus, different approaches are needed to demonstrate disseminated fungal infection.

Given the lack of a single definitive test to detect Candida species, different assays were carried out in the current study. First, search for antibodies against Candida species in blood by means of immunofluorescence and ELISA tests, and second, by detection of Candida species proteins in blood by means of slotblot analyses [START_REF] Carrasco | Isolation of Candida famata from a patient with acute zonal occult outer retinopathy[END_REF][START_REF] Pisa | Evolution of antibody response and fungal antigens in the serum of a patient infected with Candida famata[END_REF][START_REF] Pisa | Fungal infection in patients with serpiginous choroiditis or acute zonal occult outer retinopathy[END_REF].

To our knowledge, this is the first large epidemiologic study to test for an association between MS and infection with Candida species. In this casecontrol study we found a significant association between elevated antibodies of four species of Candida and MS using indirect immunofluorescence. However, these differences were not observed using ELISA, but for Candida glabrata. We feel detection of antibodies by ELISA in this large sample of participants showed poor specificity. Although it is true that antibodies to Candida species may occur in normal individuals as a result of commensal colonization of several anatomically distinct sites including skin, oral, gastrointestinal tract, and vagina [START_REF] Southern | C. albicans colonization of human mucosal surfaces[END_REF][START_REF] Yang | Virulence factors of Candida species[END_REF], MS patients had a significant seroprevalence of Candida species using indirect immunofluorescence. However, the most striking result of our study is a near-complete (91.3%) presence of Candida parapsilosis antigens in patients with MS compared with only 59.2% of subjects in the control group. Subjects with positivity for Candida parapsilosis antigens were seven times more likely to have MS than were those with negative or doubtful concentrations of antigens. The basis for the case-control differences we observed is not clear. The prevalence hypothesis, proposed by Kurtzke [START_REF] Kurtzke | Epidemiologic evidence for multiple sclerosis as an infection[END_REF],

suggests that MS is caused by a pathogen, which may be more common in regions of high MS prevalence. This pathogen would be widespread and, in most individuals, would cause an asymptomatic persistent infection and, only rarely and years after the primary infection, does this agent would cause MS.

Milk consumption has been correlated with higher worldwide prevalence rates of MS [START_REF] Butcher | The distribution of multiple sclerosis in relation to the dairy industry and milk consumption[END_REF][START_REF] Malosse | Correlation between milk and dairy product consumption and multiple sclerosis prevalence: a worldwide study[END_REF] and pathogenic Candida species may be isolated from milk products [START_REF] Lagneau | Isolation of yeasts from bovine milk in Belgium[END_REF]. One could hypothesize that chronic or reactivated latent infection by pathogenic Candida species drives or initiates autoimmunity. However, longitudinal studies of incident MS patients are required to assess whether the observed associations are due to a pathogenic effect of Candida species on the risk of developing incident MS or a susceptibility on the part of patients with MS to be infected by yeasts.

We acknowledge several limitations of this study. First, we recruited a group of patients with MS (i.e., patients seen in a patient-based association) and, as a consequence, our results might not be generalized to populationdwelling MS patients. Therefore, we acknowledge the need to replicate these findings in a population-based survey of unselected patients. Second, while we frequency-matched patients and controls based on age and gender we were not able to adjust for other potential unmeasured confounders, such as socioeconomic status, dietary intakes or hygiene habits. Third, there is also evidence that immunosuppressant therapies could interfere with the generation of antibodies [START_REF] Belgi | Traditional therapies: glucocorticoids, azathioprine, methotrexate, hydroxyurea[END_REF]. However, after excluding 10 MS patients who were receiving immunosuppressant therapies, the concentrations of Candida species antibodies remained significantly higher in MS patients than controls. Fourth, therapeutic use of repeated injectable beta-interferons or glatiramer acetate (immunomodulatory therapies) may prone to skin colonization by Candida species that could be followed by candidemia. However, the results were similar after excluding 47 MS patients who were receiving (40 cases) or ever received (7 cases) immunosuppressant or immunomodulatory therapies. In addition, we enrolled 40 MS patients whose MS duration was > 10 years. Patients with longlasting disease duration (> 10 years) may be exposed to corticosteroids or they could have a higher frequency of urinary tract infections and hence antibiotic treatment. However, the results were similar after excluding these 40 MS patients whose MS duration was longer than 10 years. This supports that the reported association is present early in MS course.

In summary, the results of this single study suggest that Candida species infection may be associated with increased odds of MS. The clinical importance of this association is uncertain. We therefore propose future studies that should clarify whether and, if so how, yeasts from the genus Candida act in the pathogenesis of this disease.

Table 1 .

 1 Characteristics of study population. SD (median) and frequency (%) are reported. a Mann-Whitney U test and b chi-square test. EDSS = expanded disability status scale.

	MS Patients	Controls	p value
	(N = 80)	(N = 240)

Table 2 .

 2 Seroprevalence of Candida species antibodies in patients and controls, using indirect immunofluorescence and ELISA tests. Chi-square test and b Mann-Whitney U test. * Mean ± SD (median) are reported.

	MS Patients	Controls	p value
	(N = 80)	(N = 240)

Table 4 .

 4 Candida species antigens in patients and controls, using slot-blot analyses.

	MS Patients	Controls	p value
	(N = 80)	(N = 240)

a Chi-square test.

Acknowledgments

We wish to express our sincere thanks to Dr. Nancy Grajales Hernández and Gerardo García Perales, as well as other members of the Multiple Sclerosis association of Madrid (Spain). We also thank Drs. Mari Luz Borbolla and Manuel Algora (Transfusion Center of the Community of Madrid) for providing the control samples. Fátima Palomares is also acknowledged for her involvement during the early stages of this study. Finally, we also acknowledge the contribution provided by Dr. Elan D. Louis who critiqued late stage drafts of the paper. The study was supported by grants from the Fundación de Investigación Médica Mútua Madrileña and Fundación Ramón Areces.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

JB-L and LC conceived, coordinated and designed the original study. JB-L conducted the analysis of data and drafted the manuscript. JBL, DP, RA, PC, and MDS collected data and samples. DP and RA conducted the serological testing. All authors offered critical input into the manuscript and all have read and approved the final version.


T Bruele

MD Petronella E C Mourad-Baars

PhD; Eric C J Claas

R N Plas

MD, PhD Ed J Kuijper

MD, PhD Robbert G M Bredius
email: r.g.m.bredius@lumc.nl

MD Tjitske Van Den Bruele

MD, PhD; Roos N Van Der Plas

Kuijper

CAMPYLOBACTER JEJUNI BACTEREMIA AND HELICOBACTER PYLORI IN A PATIENT WITH X-LINKED AGAMMAGLOBULINEMIA

Keywords: Bacteremia, Campylobacter jejuni, Helicobacter pylori, hypogammaglobulinemia, X-linked agammaglobulinemia

bacteremia and in a patient with X-linked agammaglobulinemia

Dear Dr. van Leeuwen, Thank you for reviewing our manuscript entitled "Campylobacter jejuni bacteremia and Helicobacter pylori in a patient with X-linked agammaglobulinemia" (EJCMID-D-10-00198).

Hereby you find the revised version of our manuscript. We have considered all the points raised by the reviewer, and gave all the details of the way in which we have handled each of the points below.

We kindly ask you to reconsider our manuscript for publication in your journal. Answers to comments and questions from reviewer #1 -The reviewer suggested adding a sentence to the abstract to prompt physicians to use adapted culture methods and adequate prolonged therapy in the case of antibody deficient patients. Added in Abstract, page 4, line 10.

Kind regards,

-Both in the Abstract and the text information is added to describe the microbiological work-up and a description of the antibiogram of the different strains. Added in Abstract, page 4, and the Case Report, page 6, line 17.

-We agree with the reviewer that the association of Helicobacter and Campylobacter infection in a child with XLA is rarely reported, and could be explained by the geographic origin of the patient. A comment including a new reference to address this is added. Added in the Discussion, page 8, line 17.

-We agree with the reviewer that immunoglobulin substitution of XLA patients should be aimed at higher through levels than we initially reached in this patient (see publication Eijkhout et al). A sentence stressing this is added in text, plus the reference to support this notion. Added in the Case Report, page 6 line 8 & Discussion, page 8, line 9.

-The possibility of neutropenia in XLA is indeed described. Our patient had normal neutrophil counts during the entire course of his disease; this is now added to the Case Report (page 6, line 21).

-Enterovirus infections are potential causes of gastrointestinal complaints and neurological abnormalities in XLA patients. Other pathogens were repeatedly searched for in the feces, blood and the cerebrospinal fluid but never found.

  d e p a r t m e n t Department of Pediatrics to Dr W. van Leeuwen p o s t a l z o n e J6-S Reviews Editor EJCMID R.G.M. Bredius, Pediatrician a d d r e s s phone +31 71 526 4131 fa x +31 71 524 8198 e -m a i l r.g.m.bredius@lumc.nl o u r r e f e r e n c e RB: H. pylori and C. jejuni in XLA y o u r r e f e r e n c e EJCMID-D-10-00198 d a t e May 18, 2010 s u b j e c t Revised manuscript, van den Bruele et al. EJCMID-D-10-00198

  Bredius Dr R.G.M. Bredius, pediatric-immunologist Bone Marrow Transplantation Unit Leiden, I.H.O.B.A. Dept. of Pediatrics, L.U.M.C., J6-S PO Box 9600 / 2300 RC Leiden / the Netherlands tel. +31-71-5269111 / secr. +31-71-5264131 / fax. +31-71-5248198 e-mail: r.g.m.bredius@lumc.nl d e p a r t m e n t Department of Pediatrics to Dr W. van Leeuwen, Reviews Editor o u r r e f e r e n c e RB: H. pylori and C. jejuni in XLA d a t e 18 May 2010 page 2 s u b j e c t Manuscript EJCMID-D-10-00198

Table 1 . Summary of published case reports and series of patients with hypogammaglobulinemia or XLA with Campylobacter or Helicobacter bacteremia

 1 year old Iranian boy with a history of recurrent infections and an absence of peripheral 3 blood B-lymphocytes was diagnosed with XLA. The boy was living in The Netherlands since his fifth.

	ABSTRACT

In the Case Report the results of PCR, culture, et cetera are given (page 6, line 25 & page 7, line 15). with bacteremia caused by Helicobacter and Campylobacter species, see Table 1.[2;3] 9 Recently, we treated a boy with XLA who developed fever, loss of appetite and failure to thrive 10 caused by a combination of H. pylori gastritis and recurrent C. jejuni bacteremia. 8 He had no detectable IgA and IgM. At the age of 10 years he developed epilepsy caused by left 9 occipital lesions, presumably acquired by infections in the past. 10 At the age of 15 years he was admitted to our hospital with progressive loss of appetite, initially 11 ascribed to his anti-epileptic medication. He was severely malnourished (weight / length-ratio -3 Dutch 12 Standard Deviation Score, comparable to Iranian growth charts). Stool antigen test for H. pylori was

13 positive. Endoscopy and biopsies of the gastric antrum, corpus and duodenum showed chronic active 14 inflammation with atrophy and intestinal metaplasia. Cultures of the antrum and corpus were positive 15 for H. pylori as well as for C. jejuni. Biopsies of anthrum and corpus were inoculated on non-selective 16 blood agar media and onto specific H. pylori media (Pylori agar, Biomérieux, France) in a micro-17 aerophilic environment for 7 days. Identification was performed by conventional biochemical reactions 18 according to standard operation procedures.[6] During hospitalization the patient had periods of fever.

negative for enteric parasites, viruses including enteroviruses and pathogenic bacteria. The patient was 25 treated with intravenous meropenem for two weeks, followed by oral erythromycin for another 4 weeks.

26

Repeated blood cultures after ten days were negative. For eradication of H. pylori he additionally received triple therapy with amoxicillin, clarithromycin and omeprazol for one week. H. meropenem (MIC 0.008 mg/L). Ten months later, he again developed a C. jejuni bacteremia. The strain 9 had a similar pattern as the stool isolate, with high level resistance to erythromycin by E-testing (MIC ≥ 10 256 mg/L), but the strain was sensitive to tetracycline. Sequence analysis of part of the 23S rRNA gene 11 revealed an A2058C mutation (position of nucleotide reported as the Escherichia coli equivalent), 12 known to be associated with erythromycin resistance.[7;8] The strain isolated 8 weeks earlier from the 13 feces did not have this mutation. No other pathogens were isolated. He again had complaints of loss of 14 appetite, no diarrhea or skin lesions and some weight loss. Co-trimoxazole prophylactic therapy was 15 switched to azithromycin (for 2 weeks) and later to doxycycline (for 4 weeks), which resulted in clinical 16 improvement and negative stool and blood cultures; now, with a follow-up of 6 months.

(children and adults).[12] C. jejuni is the most frequently isolated pathogen of the Campylobacter 12 species; C. coli, C. lari and C. upsaliensis are less frequently encountered pathogens. Transmission of 13 C. jejuni occurs by ingestion of contaminated food (i.e. chicken and pork), water and unpasteurized 14 milk. Helicobacter infections in children are acquired early in life, mainly via oral-fecal contact. In the 15 Netherlands the prevalence of H. pylori in children is very low (1.2%); most infected children are 16 offspring of non-Dutch parents like our patient.

[13] Our patient had a double infection, which may be 17 coincidental but could also be based on his Iranian background. In Iran, the prevalence of H. pylori was 18 reported to be 58% in healthy 15 year old children.

[14]

19 Bacteremia with Campylobacter and Helicobacter species is frequently found in XLA patients. sensitive for amoxicillin-clavulanic acid (resistance rate 1 -2%).[9] The strain of our patient had high-25 level resistance to erythromycin (MIC ≥ 256 mg/L). Because of reported difficulties in the phenotypic

Table

recognition of erythromycin resistant isolates, the presence of the erythromycin resistant C. jejuni was

Mourad-Baars PE, Verspaget HW, Mertens BJ, Mearin ML (2007) Low prevalence of