MD Alan W Steel

PhD Christopher M Mela

MD James Oliver Lindsay

MD Brian G Gazzard

PhD Martin R Goodier
email: m.goodier@imperial.ac.uk

Increased proportion of CD16 + NK cells in the colonic lamina propria of IBD patients but not after Azathioprine treatment

Keywords: Immunology < Topics, Inflammatory bowel disease < Diseasebased, Thiopurines < Topics, Ulcerative colitis < Disease-based, Crohn'

come

Increased proportion of CD16+ NK cells in the colonic

lamina propria of IBD patients but not after Azathioprine treatment.

INTRODUCTION

Peripheral blood human natural killer (NK) cells have been classically defined into two major functional subpopulations [START_REF] Cooper | The biology of human natural killer-cell subsets[END_REF]. CD56 + CD16 + NK cells constitute the most abundant blood NK cell population, expressing high levels of intracellular perforin, being enriched for cells expressing Killer Cell Immunoglobulin-like receptors (KIR) and having high levels of cytotoxic activity against target cell lines. CD56 + CD16 -NK cells represent a minor population of NK cells in the blood, have little or no expression of KIR and perforin and exhibit poor natural cytotoxicity whilst being effective in the production of cytokines, including IFN-γ, TNF-α, IL-10 and IL-13 [START_REF] Cooper | The biology of human natural killer-cell subsets[END_REF]. Previous studies from our group and others have, in contrast, demonstrated that tissue NK cells, including those detected in the human gastrointestinal tract, are predominantly of the latter CD56 + CD16 -phenotype, and have helper rather than cytotoxic function [START_REF] Chinen | Lamina propria c-kit+ immune precursors reside in human adult intestine and differentiate into natural killer cells[END_REF][START_REF] Leon | Human small-intestinal epithelium contains functional natural killer lymphocytes[END_REF][START_REF] Mela | Depletion of natural killer cells in the colonic lamina propria of viraemic HIV-1infected individuals[END_REF][START_REF] Goodier | Intestinal NK cells[END_REF].

Our previous studies demonstrated that chronic infection with HIV-1 resulted in an increase in the representation of CD56 + CD16 + NK cells in colonic lamina propria, which are virtually absent in HIV-1 negative healthy control individuals, possibly reflecting increased NK cell differentiation under conditions of chronic infection [START_REF] Mela | Depletion of natural killer cells in the colonic lamina propria of viraemic HIV-1infected individuals[END_REF].

Natural Killer cells have been implicated both in the pathogenesis of and recovery from inflammatory bowel diseases. Early studies indicate that only weak natural cytotoxicity can be detected lamina propria in patients with either Ulcerative Colitis (UC) or Crohn's disease (CD), implying a lack of conventional cytotoxic NK cell involvement [START_REF] Hogan | Lymphokine-activated and natural killer cell activity in human intestinal mucosa[END_REF][START_REF] Melgar | Cytolytic capabilities of lamina propria and intraepithelial lymphocytes in normal and chronically inflamed human intestine[END_REF]. Furthermore, in the [START_REF] Auer | Immune status in Crohn's disease. V. Decreased in vitro natural killer cell activity in peripheral blood[END_REF][START_REF] Ginsburg | Impaired natural killer cell activity in patients with inflammatory bowel disease: evidence for a qualitative defect[END_REF]. An increased frequency of colonic dendritic cells producing IL-6 and IL-12, has been observed in human Crohn's disease, suggesting an increased potential for dendritic cell (DC) mediated NK cell stimulation [START_REF] Hart | Characteristics of intestinal dendritic cells in inflammatory bowel diseases[END_REF].

An increase in NK cell markers has been demonstrated in the colonic mucosa of patients with active UC [START_REF] Del Mar Cabrera | Expression of the Kp43 (CD 94) receptor by natural killer (NK) cells in ulcerative colitis[END_REF]. However, TCR-αβ + and TCR-γδ + subsets also express NK cell markers in the colonic mucosa, and have subsequently been demonstrated to play an important role in driving the Th-2, IL-13 dominated pattern of cytokine production in UC [START_REF] Targan | Defects in mucosal immunity leading to ulcerative colitis[END_REF]. Conversely, a murine model of colitis, in which IL-10-/-mice are susceptible to a Th-1 mediated pathology, demonstrates a role for NK cells in controlling disease [START_REF] Fort | A role for NK cells as regulators of CD4+ T cells in a transfer model of colitis[END_REF].

There is some indication that IBD therapies can impact on NK cell function.

Corticosteroids have clearly been demonstrated to impede NK cytotoxicity [START_REF] Van Ierssel | Suppression of intestinal mucosal natural killer cells by corticosteroids[END_REF][START_REF] Van Ierssel | Contribution of plasma cortisol to corticosteroidsuppressed peripheral blood natural killer cell activity in Crohn's disease[END_REF]. Azathioprine (and its orally active metabolite mercaptopurine) are thought to impair the cytotoxity of peripheral NK cells in treated CD and rheumatoid arthritis patients [START_REF] Brogan | The effect of 6-mercaptopurine on natural killer-cell activities in Crohn's disease[END_REF][START_REF] Cseuz | The inhibition of NK cell function by azathioprine during the treatment of patients with rheumatoid arthritis[END_REF][START_REF] Czeuz | Natural killer cells in the blood of patients with rheumatoid arthritis treated with azathioprine[END_REF]. The other main classes of drugs used to treat IBD are aminosalicylates that have not been shown to affect NK cell numbers or function, and the recently adopted monoclonal anti TNF biologic therapies which have yet to be specifically linked to changes in NK cell frequency or function.

We therefore investigated whether chronic inflammatory conditions of the gastrointestinal tract (UC and CD) would, like chronic infectious inflammation influence the composition of functional NK cell subsets in the blood and

Study subjects.

For ex vivo studies 28 healthy control patients were selected from patients attending for endoscopic examination because of: personal history of colonic polyps, familial history of polyposis or for investigation of rectal bleeding where endoscopic examination was unremarkable. All healthy controls had no overt pathology on endoscopic examination. Studies were performed on colonic biopsy and blood samples from a total of 17 Individuals CD and 30 patients with UC with disease previously defined as extending at least to the splenic flexure. Patients on current or recent (four weeks) corticosteroid therapy and those ever treated with biologic therapies were excluded. All subjects receiving azathioprine therapy had done so at a stable dose for more than six months. Levels of thiopurine methyltransferase (TPMT) prior to commencement of azathioprine therapy were not routinely recorded at the time of this study.

To determine the effect of disease activity UC patients were scored using the Ulcerative Colitis Severity Score (UCSS), with a range 0-12 and a score of ≤6 corresponding to inactive/mildly active disease [START_REF] Schroeder | Coated oral 5-aminosalicylic acid therapy for mildly to moderately active ulcerative colitis. A randomized study[END_REF]. For CD patients the Harvey Bradshaw Index (HBI) was used with a score of ≤6 corresponding to inactive/mildly active disease [START_REF] Harvey | A simple index of Crohn's-disease activity[END_REF]. Since both of these measures include components that are subjective we also independently evaluated C reactive protein levels (CRP). Further details of patient characteristics are shown in table 1.

Biopsy sampling and processing

Biopsies for evaluation of LP NK populations were taken from the sigmoid colon (x6) circumferentially at 30cm from the anus, avoiding areas of ulceration where necrotic cells and fibroblastic proliferation predominate.

Biopsy forceps used were Olympus FB 242 radial jaw with external diameter of 3.7mm (Keymed, Southend-on-Sea, UK). Parallel blood samples were taken into Lithium Heparin Vacutainers (Becton Dickinson, Oxford, UK) and peripheral blood mononuclear cells were separated by density gradient centrifugation on Histopaque-1077 (Sigma, Poole, UK).

Biopsies obtained during colonoscopy were collected in ice-cold Dutch modification of RPMI 1640 supplemented with antibiotics. Epithelium and associated lymphocytes were removed by repeated treatment with EDTA and washing and lamina propria lymphocytes (LPL) were prepared either by enzymatic digestion or by 'walk out' methods as previously described [START_REF] Hart | Characteristics of intestinal dendritic cells in inflammatory bowel diseases[END_REF].

Briefly, for phenotypic analysis, epithelium and intra epithelial lymphocyte

Flow cytometric analysis of NK cell subsets.

Total NK cells were identified within PBMC or LPL as CD3 -CD56 + cells and T cells as CD3 + cells. CD16 + CD56 + and CD16 + CD56 -NK cell subsets were identified within PBMC or LPMC by flow cytometry after labelling with Fluorescein (FITC) conjugated anti-CD45 (Sigma, Poole, UK), phycoerythrin (PE) conjugated anti-CD16, allophycocyanin (APC) conjugated anti-CD56 (both from Beckman Coulter, Marseille, France) and PerCP conjugated anti-CD3 (Becton-Dickinson, UK). For analysis, lymphocytes were identified as CD45 + side scatter lo cells and NK cells as CD3 -CD56 + CD16 + or CD3 - CD56 + CD16 -cells. Further flow cytometric analysis of activating and inhibitory NK cell receptor expression was performed using the following phycoerythrin conjugated antibodies: anti-CD161, anti-KIR3DL-1, anti-NKG2D (Becton Dickinson); anti-KIR2DL-1/2 (AbD Serotec, Oxford, UK) ant-NKG2C (R&D systems, Abingdon, UK); anti-NKp30, anti-NKp44 and anti-NKp46 (Beckman Coulter).

Degranulation assay.

NK cell specific degranulation was measured using CD107a as previously described [START_REF] Alter | CD107a as a functional marker for the identification of natural killer cell activity[END_REF]. Biopsy material was cultured after removal of epithelial cells in complete medium with 20ng/ml IL-15 (R&D systems). After 18 hours cells were removed into polypropylene cell culture tubes (BD Falcon, Oxford, UK) and cultured with anti-CD107a antibody and with K562 cells at a ratio of 2

Cytotoxicity assay

For natural cytotoxicity, PBMC were tested immediately after isolation. For cytokine activated cytotoxicity cells were cultured overnight in IL-2 (100U/ml) and IL-15 (20ng/ml) before testing. Natural and cytokine activated cytotoxicity were measured using a non-radioactive cytotoxicity assay using PBMC effectors and K562 target cells at ratios titrated from 50:1 to 5:1 (Biorad, Southampton, UK). Specific lysis of target cells was calculated using the following formula: % Cytotoxicity = Experimental -Effector spontaneous -Target spontaneous × 100 Target maximum -Target spontaneous

Studies on the effect of Azathioprine in-vitro

Resting PBMC from healthy control individuals were incubated with azathioprine at a concentration range between 1 and 50 µM and the absolute number and proportion of NK cell and T cell subsets remaining after 6 days invitro were assessed by viable cell counting and flow cytometry. Previous studies have defined azathioprine concentrations of 5µM to be pharmacologically relevant [START_REF] Ben-Horin | Early preservation of effector functions followed by eventual T cell memory depletion: a model for the delayed onset of the effect of thiopurines[END_REF][START_REF] Tiede | CD28dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes[END_REF].

To test the impact of azathioprine on NK cell activation and expansion PBMC were stimulated with 100U/ml recombinant human IL-2 (R&D systems, Interferon gamma production in gated CD56 + CD16 -and CD56 + CD16 + NK cell subsets was measured by intracellular staining and flow cytometry using a FITC conjugated anti-human IFN-γ monoclonal antibody (BD Biosciences).

Apoptosis was measured in azathioprine treated unstimulated or cytokine activated NK cell subsets using FITC conjugated Annexin V (Invitrogen, Molecular Probes, Paisley, UK) after gating out dead cells with a fixable livedead cell discriminatory dye (RVid, Invitogen). Apoptotic cells were defined as Annexin V + /RVid negative.

Cell proliferation was measured after labelling cells with carboxyfluorescein diacetate succinimidyl ester (CFSE, Invitrogen) [START_REF] Quah | Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester[END_REF] and stimulating with cytokines in the presence or absence of Azathioprine as above. The proportion of proliferating cells was calculated by assessing the proportion of CFSE negative/low cells within gated NK cell and T cell subsets.

Statistical analysis.

Statistical analyses were performed after consultation with an on-site statistician. Comparison of continuous variables was performed using nonparametric tests. Mann-Whitney U test was used for comparison between groups and Wilcoxon signed-rank test was used for intra-group comparisons.

RESULTS

Increased proportion of CD16 + NK cells in the colonic lamina propria of CD patients and reversal by azathioprine.

The proportions of total CD3 -CD56 + NK cells within gated CD45 + lymphocytes were first compared within blood and colonic biopsies from healthy control individuals and CD and UC patients (Figure 1a,b). No significant differences were observed in the proportion of total CD3 -CD56 + NK cells between HC (blood: median 7.5%, range 2.2-17.0%; colon: median 4.8%, range 1.7-17.4%) and IBD patients with either CD (blood: median, 4.4%, range 1.3-10.9%; colon: median 4.2%, range 2.3-8.8%) or UC (blood: median 9.4%, range 1.3-23.2%; colon: median 3.6%, 1.4-13.6%) (Figure1b). Furthermore, no significant difference was observed in the subset distribution of NK cells, as determined by the proportion of CD16 + NK cells, within total blood NK cells from untreated IBD patients compared to healthy control individuals (HC, median 91.1%, range 81.3-96.9%, CD, median 86.9%, range 64.3-97.7%, UC median, 89.7% range 70.6-98.2%) (Figure 1c). A significant increase was, however, observed in the representation of CD56 + CD16 + NK cells within colonic LPNK cells in CD (median, 14.7%, range 3.6-48.9%, p=0.04) and UC patients (median 18.3%, range 0.1-49.5%, p=0.037) compared to control individuals (median 5.7%, range 0.1-30.2%) (Figure 1c).

A reduction in the overall proportion of blood NK cells was observed In IBD patients receiving azathioprine (blood: CD median 1.7%, range 0.3-6.2%; UC median 1.4%, range 0.5-10.9%), compared to healthy control individuals (CD, p=0.001; UC, p=0.001) and to untreated individuals (CD, p=0.013; UC, p= A similar tendency towards a reduction in total NK cells was observed within colonic lamina propria NK cell populations (colon: CD median 3.2%, range 0.6-4.6%, p=0.04 vs untreated; UC median 3.5%, range 0.9-9.4%, p=0.123 vs untreated) (Figure 1b). Analysis of NK cell subsets revealed that azathioprine treatment resulted in preferential reduction in the proportion of CD56 + CD16 + NK cells in the blood of CD (blood: median 63.4%, range 25-84%, p= 0.025 vs untreated;) and UC patients (blood median 81.1%, range 25.5-95.2% p=0.033 vs untreated;) (Figure 1c). A similar trend towards a reduction in the proportion of CD56 + CD16 + NK cells in colonic lamina propria comparing patients with or without azathioprine treatment (CD patients median 1.6%, range 0.8-21.9%, p=0.044 vs untreated; UC patients colon: median, 10.1%, range 0.1-21.7%, p=0.089 vs untreated, Figure 1c). No significant differences were observed comparing confounding variables, including sex, age, time since diagnosis, disease activity (HBI or UCSS), Creactive protein levels and 5-aminosalicate therapy between azathioprine treated or untreated groups of patients indicating that these variables were unlikely to contribute to differences in NK cell populations observed between Azathioprine treated or untreated patient groups (Table S1). Together these data demonstrate that inflammatory bowel diseases result in a redistribution of NK cell subsets in the colonic lamina propria towards a CD16 + phenotype.

Azathioprine therapy results in an overall depletion of NK cells with a preferential impact on the CD56 + CD16 + NK cell subset both in the blood and colonic lamina propria.

The expression of activating and inhibitory NK cell receptor families were also examined within gated NK cell populations from blood and colon (Table S2). The distribution of NK cell receptors on colonic lamina NK cells was significantly different compared to peripheral blood for control and for IBD patients. In addition to a reduced proportion of LPNK cells expressing CD16 compared to PBNK (p< 0.0001 for HC, CD and UC), significantly fewer LPNK expressed KIR compared to PBNK (HC, p=0.040, CD, p=0.043, UC, p=0.4).

Furthermore, the expression of NCR NKp44 was elevated in LPNK compared to PBNK (HC, p=0.028, CD, p=0.028, UC, p=0.031) (Table S2). However, other than the expression of CD16, no significant differences were observed in the expression of these receptors comparing blood or colonic lamina propria between controls and IBD patients or comparing groups of IBD patients untreated or treated with azathioprine (Table S2).

IL-15 activated NK cells from HC and IBD patients degranulate in response to target cells.

The functional capacity NK cells from untreated UC patients and UC patients receiving azathioprine was then tested by measuring their ability to degranulate in response to target cells. Degranulation, as measured by CD107a expression was observed both in peripheral blood and lamina propria NK cells from HC individuals and from UC patients in response to K562 target cells (Figure 2a,2b). Interestingly, the remaining PBNK and LPNK cells from UC treated individuals receiving azathioprine were also capable of degranulation, indicating that these cells remained functional (Figure 2a,2b).

These data indicate that CD16 -NK cells remaining after azathioprine therapy maintain in-vitro responsiveness to target cell lines. Although NK cells from azathioprine treated individuals were capable of degranulating in response to effective in both natural and cytokine activated cytotoxicity (Figure 2c). Poor cytotoxicity is consistent with a reduced proportion of perforin expressing CD16 + 'cytotoxic' NK cells in Azathioprine treated individuals compared to healthy control or untreated individuals (Figure S1)

Resting and activated NK cells are sensitive to azathioprine in-vitro

Azathioprine and its metabolites 6-mercaptopurine and 6-thioguanine triphosphate have been shown in previous studies to inhibit T cell proliferation in-vitro at pharmacologically relevant concentrations [START_REF] Ben-Horin | Early preservation of effector functions followed by eventual T cell memory depletion: a model for the delayed onset of the effect of thiopurines[END_REF][START_REF] Tiede | CD28dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes[END_REF]. In view of the selective depletion of CD56 + CD16 + NK cells measured ex-vivo in PBMC or LPMC from azathioprine treated CD or UC patients, we tested whether similar pharmacologically relevant effects could be observed within resting or PBNK cells in-vitro activated NK cells in the presence or absence of azathioprine .

Surprisingly, incubation of resting PBMC with azathioprine for 6 days in culture resulted in a significant preferential loss in both the absolute number and proportion of CD3 -CD56 + NK cells in all donors tested at a pharmacologically relevant concentration of azathioprine concentration of 5µM whereas no reduction was observed in the proportion of resting CD3 + T-cells even at suprapharmacological concentrations of up to 50µM (Figure 3a). In contrast to ex-vivo observations on NK cells from treated patients, in-vitro treatment of resting NK cells with azathioprine had no impact on the relative proportions of CD56 + CD16 -, CD56 + CD16 + or CD56 + KIR + NK cell subsets (data not shown). We therefore tested the impact of azathioprine on cytokine indicating that cytokine activated NK cells were less sensitive to azathioprine than resting NK cells (figure 3b). No difference was observed in the overall proportions of CD56 + CD16 -and CD56 + CD16 + NK cells subsets within the remaining NK cells. However, in contrast to resting T-cells, the proportions of cytokine activated T cells were also reduced in the presence of high concentrations of azathioprine (figure 3b).

Azathioprine preferentially inhibits early activation, IFN-γ γ γ γ production and proliferation of CD16+ NK cells.

To test whether azathioprine impacted on cytokine driven early NK cell activation and proliferation we studied CD69 expression and the distribution of CFSE staining in CD56 + CD16 -and CD56 + CD16 + NK cell subsets. Only a small fraction of NK cells within freshly isolated PBMC expressed CD69 prior to stimulation with cytokines (median 4.9%, range 1.9-11.2%). High levels of CD69 were induced after 16 hours of cytokine stimulation and azathioprine reduced the proportion of NK cells expressing CD69 within both NK cell subsets (Figure 4a,b). The impact of azathioprine was greater on the activation of CD56 + CD16 + NK cells (inhibition at concentrations ≥5µM) compared to CD56 + CD16 -NK cells (inhibition at concentrations ≥20µM)(Figure 4d). The impact on the level of CD69 expression (mean fluorescence activity) gave similar results (data not shown). Azathioprine significantly inhibited cytokine driven T cell activation only at concentrations of 10µM or greater (Figure 4d).

The impact of azathioprine on IFN-γ production by resting and activated NK cells was also assessed using intracellular cytokine staining. Similarly to the effects on CD69 expression, azathioprine inhibited IFN-γ production by NK cell subsets in a concentration dependent manner, when added simultaneously with IL-12 and IL-15 (Figure 4e) whereas no effect was observed on IFN-γ production by NK cells which had been previously activated and then restimulated with IL-12+ IL-15 (Figure 4f).

The proliferative response of NK cells to cytokines was also dramatically reduced by azathioprine (Figure 5). CD56 + CD16 + NK cells were particularly sensitive to inhibition by azathiopine with a significant reduction in proliferating (CFSE low/negative) cells being observed at concentrations of 5µM or greater (Figure 5b). Significant Inhibition of cytokine driven CD56 + CD16 -NK cell proliferation and T cell proliferation was observed only at higher concentrations of azathioprine (≥10µM) (Figure 5b). Taken together these data indicate that whilst resting NK cell subsets are equally sensitive to azathioprine, activation and proliferation of CD56 + CD16 + NK cells is preferentially inhibited, providing a potential mechanism for our ex-vivo observations in azathioprine treated subjects.

Azathioprine induces apoptosis in resting NK cells.

In order to assess whether the impact of azathioprine on resting NK cells was associated with the induction of apoptosis, as shown previously by an increase in the proportion of apoptotic activated T-cells ( 22), we measured apoptosis in unstimulated or cytokine activated NK cell subsets after 5 days of culture (Figure 6). Resting NK cells from both CD56 + CD16 -and CD56 + CD16 + subsets were susceptible to apoptosis in the presence of azathioprine, with a progressive increase in Annexin V + ,RVid -cells being observed at concentration of 5mM or greater. However, a higher overall proportion of the resting CD56 + CD16 + subset was undergoing apoptosis after 5 days of culture compared to CD56 + CD16 -cells. In contrast to resting NK cells, cytokine activated NK cells were more resistant to apoptosis, in keeping with a maintenance in culture at lower concentrations of azathioprine (5mM or less). the GI tract [START_REF] Chinen | Lamina propria c-kit+ immune precursors reside in human adult intestine and differentiate into natural killer cells[END_REF][START_REF] Mela | Depletion of natural killer cells in the colonic lamina propria of viraemic HIV-1infected individuals[END_REF][START_REF] Fehniger | CD56bright natural killer cells are present in human lymph nodes and are activated by T cell-derived IL-2: a potential new link between adaptive and innate immunity[END_REF][START_REF] Ferlazzo | The abundant NK cells in human secondary lymphoid tissues require activation to express killer cell Ig-like receptors and become cytolytic[END_REF][START_REF] Hanna | Decidual NK cells regulate key developmental processes at the human fetal-maternal interface[END_REF]. Recent studies have demonstrated a predominant role for mucosal CD56 + CD16 -NKp44 + NK cells from human tonsils in promoting repair of epithelial tissue via production of growth and antiapoptotic factors [START_REF] Cella | A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity[END_REF]. Indeed, in animal models such cells play a role in protection from inflammatory bowel disease [START_REF] Fort | A role for NK cells as regulators of CD4+ T cells in a transfer model of colitis[END_REF].

This study demonstrates no significant difference in the overall proportion of NK cells present in the peripheral blood or colonic lamina propria or individuals with either CD or UC compared to unaffected healthy control individuals however, significant enrichment of LPNK cells for the cytotoxic CD16 + CD56 + NK cell subset is observed in IBD patients not on immunomodulatory therapy. The biological impact of this shift in NK cell phenotype remains to be demonstrated although the expression of perforin and enhanced cytotoxic capacity in the CD56 + CD16 + NK cell subset may potentially contribute to tissue damage and inflammatory processes in the GI tract. A relative reduction of CD56 + CD16 -helper NK cells may conversely impact on repair processes in the gastrointestinal tract. In this respect we observe no direct correlation between disease severity and the proportion of CD16 + CD56 + NK cells present in colonic lamina propria from both CD and UC patients. However, UC patients with active disease have a significantly higher proportion of lamina propria CD16 + NK cells compared to those with inactive disease (p=0.035), suggesting that there may be some causal relationship. Several reports have established blood CD56 -CD16 + NK cells differentiate into CD56 + CD16 + cells on activation with various stimuli [START_REF] Fehniger | CD56bright natural killer cells are present in human lymph nodes and are activated by T cell-derived IL-2: a potential new link between adaptive and innate immunity[END_REF][START_REF] Ferlazzo | The abundant NK cells in human secondary lymphoid tissues require activation to express killer cell Ig-like receptors and become cytolytic[END_REF][START_REF] Chan | CD56bright human NK cells differentiate into CD56dim cells: role of contact with peripheral fibroblasts[END_REF][START_REF] Romagnani | CD56brightCD16-killer Ig-like receptor-NK cells display longer telomeres and acquire features of CD56dim NK cells upon activation[END_REF]. IL-2 and IL-15 promotes the differentiation of blood CD56+CD16-NK cells into CD56 + CD16 + NK cells that acquire the expression of cytolytic machinery including perforin and granzymes [START_REF] Ferlazzo | The abundant NK cells in human secondary lymphoid tissues require activation to express killer cell Ig-like receptors and become cytolytic[END_REF][START_REF] Romagnani | CD56brightCD16-killer Ig-like receptor-NK cells display longer telomeres and acquire features of CD56dim NK cells upon activation[END_REF][START_REF] Liu | IL-15 is highly expressed in inflammatory bowel disease and regulates local T cell-dependent cytokine production[END_REF]. Increased telomere length in CD56 -CD16 + cells compared to CD56 + CD16 + NK cells indicates that these cells are less mature, having undergone fewer cell divisions [START_REF] Romagnani | CD56brightCD16-killer Ig-like receptor-NK cells display longer telomeres and acquire features of CD56dim NK cells upon activation[END_REF]. A fraction of human lamina propria CD56 + CD16 -NK cells express c-kit, a marker of immature progenitor cells and a receptor for stem cell factor (SCF-1) and can be derived from lineage marker negative, c-kit+ precursors. (2). IL-15, which is elevated in expression in IBD, could potentially act to promote the differentiation of CD56 + CD16 -into CD56 + CD16 + lamina popria NK cells, although the relationship between these substes in the lamina propria CD56 + CD16 -and CD56 + CD16 + NK cells. Consistent with such a cytokine induced differentiation of NK cells in the gastrointestinal tract, IL-21, which is also elevated in expression in IBD, was recently reported to induce cytotoxic capacity in colonic LPNK cells and to promote the differentiation of regulatory T cell subsets [START_REF] Liu | Il-21 enhances NK cell activation and cytolytic activity and induces Th17 cell differentiation in inflammatory bowel disease[END_REF]. An increased proportion of cytotoxic type CD56 + CD16 + NK cells in the lamina propria during IBD could also, however, result from emigration of peripheral blood NK cells in response to inflammatory signals.

Here we report a differential effect of azathioprine on NK cell subsets. It is therefore likely that azathioprine acts preferentially on mature lymphocytes while permitting the generation or persistence of immature cell populations. Such an effect is consistent with a precursor role for CD56 -CD16 - cells in the development of mature cytotoxic CD56 + CD16 + NK cells.

Interestingly, we observed that IL-15 stimulated CD56 + CD16 -NK cells from the blood and colonic lamina propria of azathioprine treated UC patients were capable of degranulating in the presence of MHC class I negative target cells, indicating that these cells retained the potential to mature and potentially acquire cytotoxic capability. However, few perforin expressing NK cells are detected within PBMC from azathioprine treated individuals, in contrast to healthy control individuals and UC patients who are not receiving azathioprine, consistent with a preferential depletion CD16 + cytotoxic NK cells (figure 3). IL-15 treated NK Cells from azathioprine treated patients also responded similarly to the enterocyte lines HT29 and Caco2, indicating a potential for these cells to interact with intestinal epithelium (data not shown).

The Azathioprine metabolite 6-thioguanine triphosphate (6-thio-GTP) mediates its proapoptotic effects on CD28 costimulated CD4 + T cells via interaction with the small rho dependent GTPase Rac1 [START_REF] Tiede | CD28dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes[END_REF]. Rac 1 plays an important role in NK cell integrin signalling leading to P38 mitogen activated kinase induced chemokine secretion and in PI3-kinase and CD16 dependent NK cell cytotoxicity [START_REF] Jiang | Pivotal role of phosphoinositide-3 kinase in regulation of cytotoxicity in natural killer cells[END_REF][START_REF] Mainiero | RAC1/P38 MAPK signaling pathway controls beta1 integrin-induced interleukin-8 production in human natural killer cells[END_REF][START_REF] Galandrini | Role for the Rac1 exchange factor Vav in the signaling pathways leading to NK cell cytotoxicity[END_REF]. Binding of azathioprine-derived 6-Thio-GTP to Rac 1 could therefore also impact on NK cell function and survival.

Consistent with the depletion of the entire NK cell population by azathioprine in treated patients, we observed that total CD3 -CD56 + NK cells are 1.4 (0.5-10.9) 3.5 (0.9-9.4) 0.12 0.51

Statement of Interests

  is impaired NK cytotoxicity in CD, but no change in total numbers of NK cells found

  and whether treatment of these diseases with immunomodulatory therapy (azathioprine) affects these cell populations. This is a cross sectional ex-vivo and in-vitro study of blood and intestinal mucosa in UC and CD to determine NK subset phenotype and function and the effect of azathioprine therapy. The study was approved by the relevant Research Ethics Committee with written informed consent obtained from all subjects.

(

  IEL)-free tissue was digested with 1 mg/ml collagenase D (Roche Molecular Products, Basel, Switzerland) in Dutch modification of RPMI 1640 containing 20 mg/ml DNase I (Roche Molecular Products) and 2% FCS to release lamina For functional assays, epithelium and IEL free tissue explants were left overnight in complete medium to permit emigration of LPL.

  cells (LPMC) or peripheral blood mononuclear cells (PBMC) per target cell for one hour. Brefeldin A and monensin were added and the samples incubated for a further 4 hours. Samples were then washed once in complete medium and stained for flow cytometric analysis.

  ) in combination with 25ng/ml recombinant human IL-15 (Peprotec, London, UK) in the presence of varying concentration of azathioprine (Imuran, GlaxoSmithKline, UK). The impact of azathioprine on earlier cellular activation was measured by the expression of CD69 (anti-CD69 PE, Becton Dickinson) on NK cells after overnight incubation. The distribution of NK cell subsets was measured after 6 days of incubation.

11 Comparison

 11 of discrete variables was performed using a Chi-squared test.StatView 5.01 software was used (Abacus, Berkley, California, USA) and p values<0.05 were considered as significant.

  lines, blood NK cells from Azathioprine treated individuals were less

  within PBMC. A reduction in the absolute number and proportion of CD3-CD56+ NK cells was observed only at suprapharmacological concentrations of azathioprine (20µM or greater),

  are universally of a dominant CD56 + CD16 -phenotype as observed in human tonsils, secondary lymphoid tissue, uterine decidua and in

21 T

 21 Although azathioprine resulted in overall reduction in the proportion of NK cells in the blood and GI tract of treated individuals, our ex-vivo studies demonstrate a preferential depletion of CD56 + CD16 + NK cells. Azathioprine inhibits human T-cell proliferation in-vitro and ablates antigen-specific memory -cell responses on extended administration in an in-vivo murine model[START_REF] Ben-Horin | Early preservation of effector functions followed by eventual T cell memory depletion: a model for the delayed onset of the effect of thiopurines[END_REF].

  culture with increasing concentration of azathioprine invitro. However, no preferential depletion of CD16 + NK cells was observed invitro. Global NK cell depletion in azathioprine treated individuals may therefore be compensated by the generation of CD56 + CD16 -cells from bone marrow-derived NK cell precursors and from resident precursors in the gut (2).We observed no preferential impact of azathioprine on overall proportions of IL-2 + IL-15 stimulated CD56 + CD16 + and CD56 + CD16 -NK cell subsets, however, activation and proliferation in CD56 + CD16 + NK cells were more sensitive to azathioprine, again consistent with a preferential effect on mature NK cell subsets. Azathioprine could therefore potentially promote the maintenance of CD56 + CD16 -cells in-vivo by preventing the cytokine driven differentiation of CD56 + CD16 -into CD56 + CD16 + NK cells or by inhibiting the proliferation and expansion of CD56 + CD16 + NK cells. Further studies on differentiation and function of NK cells within the gastrointestinal tract will clarify the role of these cells in the immunopathogenesis of inflammatory bowel diseases and the potential of therapeutic interventions in preventing aberrant innate immune responses.

Figure 1 .

 1 Figure 1. Increased proportion of CD16 + NK cells in the colonic lamina

Figure 2 . 3 F 29 untreated

 2329 Figure 2. Functional activity of NK cells. The ability of PBNK and LPNK cells

Figure 3 .

 3 Figure 3. In vitro impact of Azathioprine. (A) resting (n=16) or (B) cytokine (IL-

Figure 4 .

 4 Figure 4. Azathioprine inhibits early NK cell activation and IFN-γ production.

Figure 5 .

 5 Figure 5. Azathioprine inhibits NK cell proliferation. The impact of

Figure 6 .

 6 Figure 6. Induction of apoptosis in CD56+CD16-and CD56-CD16+ NK cell

  This work was funded by grants from the St Stephen's AIDS Trust, the Campbell Foundation and the trustees of Chelsea and Westminster Hospital,

	F o r F o r F o r F o r F o r F o r
	P P P P P P
	e e e e e e
	e r e r e r e r e r e r
	R R R R R R
	e e e e e e
	v i e v i e v i e v i e v i e v i e
	w w w w w w
	London (to MRG).

Table 1 .

 1 Patient characteristics for the study.Disease stage for Crohn's disease was assessed using the Harvey Bradshaw Index (HBI) and for Ulcerative Colitis using the Ulcerative Colitis Severity Score. Inactive Crohn's disease was defined as HBI ≤6, whilst inactive/mildly active UC was defined as UCSS ≤6

				Healthy	Crohn's Disease	Ulcerative Colitis
				Control	(n= 17)	(n=30)
				(n=28)
	Age,years median	45 (27-70)	42 (23-74)	42.5 (16-78)
	(Range)
	Time since		n.a.	12 (0.5-26)	8.5 (0.5-38)
	diagnosis,years median
	(range)
	Inactive/mildly	active	n.a.
	disease n(%)			11 (65)	19 (65)
	CRP,	mg/l	median	n.a.	4 (2-37)	4 (2-112)
	(range)
	Azathioprine (%, n)	n.a	35.3 (6)	33.3 (10)

Table S2 :

 S2 Assessment of NK cell receptor expression in the blood and colon * Significant differences are observed in the proportions of NKp44 + cells between blood and gut within all groups and are maintained with Azathioprine treatment. (Wilcoxon signed rank test). Significant p values comparing Healthy control and Crohn's disease and ulcerative colitis groups without Azathioprine treatment are depicted in bold.

	% of subset,	Healthy Control	Crohn's Disease	Crohn's disease	Ulcerative Colitis	Ulcerative Colitis	Kruskal Wallis
	median(range)					Azathioprine			Azathioprine	p value
		PBMC	LP	PBMC	LP	PBMC	LP	PBMC	LP	PBMC	LP	PBMC	LP
	Total CD3-	7.5	4.8	4.4	4.2	1.7	3.2	9.4	3.6
	CD56+ NK	(2.2-17.0)	(1.7-17.4)	(1.3-10.9)	(2.3-8.8)	(0.3-6.2)	(0.6-4.6)	(1.3-22.0)	(1.4-13.6)
	cells

Alimentary Pharmacology & Therapeutic


MD Germán ; Soriano
email: gsoriano@santpau.cat

Óscar Esparzia

Michel Montemayor

Carlos Guarner-Argente

Roser Pericas

Xavier Torras

Núria Calvo

E Roman

Ferran Navarro

Carlos Guarner

Óscar Esparcia

Carlos Guarner- Argente

Eva Román

Pere Coll

CLINICAL STUDY: BACTERIAL DNA IN DIAGNOSIS OF SPONTANEOUS BACTERIAL PERITONITIS

Keywords: Ascites < ascites, cirrhosis, inflammation, microbiology

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

SBP is defined by the peritoneal inflammatory response. An ascitic fluid neutrophil count ≥250/mm 3 is currently considered the cut-off for SBP diagnosis. [START_REF] Runyon | Management of adult patients with ascites due to cirrhosis: an update[END_REF] Ascitic fluid culture, however, is not positive in all patients with ascitic fluid neutrophil count ≥250/mm 3 . When an alternative cause for the increased ascitic fluid neutrophil count (i.e. bloody ascites, pancreatitis, carcinomatosis or tuberculosis) is absent, these patients are considered to present "culture negative neutrocytic ascites" (CNNA) [START_REF] Runyon | Management of adult patients with ascites due to cirrhosis: an update[END_REF][START_REF] Runyon | Culture-negative neutrocytic ascites: a variant of spontaneous bacterial peritonitis[END_REF] or the more recently introduced designation of "culture negative SBP" . [START_REF] Rimola | Diagnosis, treatment and prophylaxis of spontaneous bacterial peritonitis: a consensus document[END_REF] Culture negative SBP is considered a variant of SBP in which bacteria are present in a low concentration and therefore not detected with microbiological culture methods. [START_REF] Runyon | Culture-negative neutrocytic ascites: a variant of spontaneous bacterial peritonitis[END_REF][START_REF] Runyon | Optimization of ascitic fluid culture technique[END_REF] In support of this hypothesis, it has been demonstrated that bedside inoculation of large amounts of ascitic fluid (10 and preferably 20 ml) into blood culture bottles increases culture positivity to approximately 70-90% as compared to 30-40% with conventional cultures.

11- [START_REF] Ortiz | Early microbiologic diagnosis of spontaneous bacterial peritonitis with BacT/ALERT[END_REF] However, in recent years, even using inoculation of ascitic fluid in blood culture bottles, the rates of bacterial detection in SBP have dropped to less than 50%. This decrease is probably related to the widespread use of antibiotic prophylaxis and to the greater awareness of SBP, leading to diagnosis at earlier stages. [START_REF] Fernández | Bacterial infections in cirrhosis: epidemiological changes with invasive procedures and norfloxacin prophylaxis[END_REF][START_REF] Soriano | Secondary bacterial peritonitis in cirrhosis: a retrospective study of clinical and analytical characteristics, diagnosis and management[END_REF] Moreover, a significant increase in the incidence of SBP caused by multiresistant bacteria has recently been reported to worsen the prognosis of this complication due to the lack of efficacy of current empirical antibiotics. [START_REF] Umgelter | Failure of current antibiotic first-line regimens and mortality in hospitalized patients with spontaneous bacterial peritonitis[END_REF][START_REF] Acevedo | Current efficacy of recommended empirical antibiotic therapy in patients with cirrhosis and bacterial infection[END_REF] This setting makes identification of the responsible bacteria in SBP even more necessary.

Bacterial DNA detection and sequencing is increasingly used in the diagnosis of many infectious diseases. Molecular techniques can detect minimal amounts of bacterial DNA and improve the rates and velocity of bacterial identification. [START_REF] Woo | Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories[END_REF][START_REF] Andrade | Advances in the microbiological diagnosis of sepsis[END_REF][START_REF] Sontakke | Use of broad range 16S rDNA PCR in clinical microbiology[END_REF][START_REF] Picard | Rapid molecular theranostics in infectious diseases[END_REF][START_REF] Baron | Medical Microbiology: Implications of new technology for infectious diseases practice[END_REF] Nevertheless, few studies to date have examined the usefulness of these molecular techniques in the microbiological diagnosis of SBP in cirrhotic patients. Furthermore, these reports enrolled a limited number of patients and results were controversial.

22-25

The aim of the present study was to determine, in a large series of patients, whether broadrange polymerase chain reaction (PCR) using primers targeting the 16S ribosomal DNA (16S

PATIENTS AND METHODS

Patients and study design

We prospectively included all cirrhotic patients with SBP diagnosed in our hospital during a 3year period (between November 2005 and January 2009). Cirrhosis was diagnosed by biopsy or based on clinical, analytical and ultrasonographic findings. SBP diagnosis was established when ascitic fluid neutrophil count was >250/mm 3 and there was no evidence of an intraabdominal source of infection. [START_REF] Runyon | Management of adult patients with ascites due to cirrhosis: an update[END_REF][START_REF] Soriano | Secondary bacterial peritonitis in cirrhosis: a retrospective study of clinical and analytical characteristics, diagnosis and management[END_REF] Paracentesis was performed in sterile conditions. We excluded patients with an alternative cause for increased ascitic fluid neutrophil count (i.e.

bloody ascites, pancreatitis, tuberculosis or carcinomatosis) and also those without a complete microbiological study (i.e. without ascitic fluid inoculation in blood culture bottles). An ascitic fluid aliquot was stored at -80º C until RT-PCR and sequencing of 16S rDNA gene were performed.

We considered SBP as community-acquired when infection was present at admission and nosocomial when diagnosis was made after 72 hours of admission or when a previous neutrophil count during admission was <250/mm 3 . [START_REF] Fernández | Bacterial infections in cirrhosis: epidemiological changes with invasive procedures and norfloxacin prophylaxis[END_REF][START_REF] Soriano | Secondary bacterial peritonitis in cirrhosis: a retrospective study of clinical and analytical characteristics, diagnosis and management[END_REF][START_REF] Guarner | Risk of a first community-acquired spontaneous bacterial peritonitis in cirrhotics with low ascitic fluid protein levels[END_REF] Renal failure at diagnosis of SBP or prior to SBP resolution was considered when serum creatinine values were above 133 µmol/l.

Systemic inflammatory response syndrome (SIRS) and septic shock were defined according to previously established criteria.

2,28

Patients with SBP were initially treated with intravenous ceftriaxone (2 g/day) , unless they were already receiving this antibiotic at SBP diagnosis. In this case, they were treated with imipenem. [START_REF] Umgelter | Failure of current antibiotic first-line regimens and mortality in hospitalized patients with spontaneous bacterial peritonitis[END_REF][START_REF] Acevedo | Current efficacy of recommended empirical antibiotic therapy in patients with cirrhosis and bacterial infection[END_REF] Treatment was changed when needed according to the patient's response and the in vitro antibiotic sensitivity. Albumin was administered in patients with urea > 11 mmol/l and/or bilirubin > 68 µmol/l. BacT/ALERT blood culture bottles. In the BacT/ALERT blood culture system (BioMérieux, Marcy l'Etoile, France), an aerobic and an anaerobic BacT/ALERT blood culture bottle were each inoculated with 10 ml of ascitic fluid at the bedside. The bottles were placed in the BacT/ALERT instrument and were processed according to the manufacturer's instructions.

Each bottle was treated independently, and only bottles flagged as positive by the instrument were further processed.

13 Isolated bacteria were identified by standard methods. RT-PCR. RT-PCR was performed with a Rotor-Gene 3000 (Corbett Robotics, Australia). Rotor-Gene has four light-emitting diodes that detect up to four targets in a single amplification reaction. This characteristic was used to develop specific Taqman® probes to detect all bacterial DNA with a universal probe (5'-CCACACTGGGACTGAGACACGG-3'-Cy5), E. coli probe (5'-CAGCGGGGAGGAAGGGAGTAAAGT-3'-JOE), and also an internal control probe

(5'-TTAGCCGCTTCCTTCAACTTCACCT-3'-Cy5) corresponding to a soja lecitin gene fragment (EMBL#K00821) that could be amplified with universal primer adaptors (Biotools, Madrid, Spain). Probes were selected based on alignments of the 16S rDNA region with sequences from the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov). Primers and probes were synthesized at Chipron (Chipron GmbH, Berlin, Germany) and at Roche Diagnostics (Mannheim, Germany), respectively.

RT-PCR was performed in two 25-µl reaction mixtures containing 10 µl of DNA, 12.5 µL of Quantitect® Multiplex PCR NoROX (Qiagen, Hilden, Germany), 4 µM of primer 27f and 20 µM of biotinylated primer 519r. [START_REF] Hall | Evaluation of the MicroSeq system for identification of mycobacteria by 16S ribosomal DNA sequencing and its integration into a routine clinical mycobacteriology laboratory[END_REF] DNA from the internal control and 20 µM of both E. coli and IC Taqman® probes were added to one mixture. In the other reaction mixture, 20 µM of universal probe and 0.5 µl of Sau3A1 (1U/µl) were added followed by a digestion at 37ºC for 30 minutes and 95ºC for 5 minutes to inactivate the restriction enzyme. The amplification reaction profile included a hot start at 95ºC for 15 minutes followed by 35 cycles of 94°C for 60 seconds and 60°C for 60 seconds. The acquisition of a fluorescent signal was performed at the end of 60ºC step in all cycles.

To assess the lower limit of detection in ascitic fluid, the approach was performed on 10-fold serial dilutions of Escherichia coli and Enterococcus faecalis ranging from 10 6 to 10 CFU/ml of pooled ascitic fluid previously proven to be culture negative. Bacteria used for spiking were quantified by agar dilution.

DNA

RESULTS

Patients characteristics

A total of 69 SBP episodes were diagnosed during the 3-year study period. After excluding cases with conventional ascitic fluid culture but without culture in blood culture bottles (n=14), 55 episodes in 48 cirrhotic patients were finally included. Of these, 25 had a positive ascitic fluid culture (45.4%) and 30 had a negative culture (54.5%).

A group of 20 cirrhotic outpatients with sterile ascites or culture-negative nonneutrocytic ascites and a group of 27 non-cirrhotic outpatients with neoplasic ascites were also included. The characteristics of cirrhotic patients with SBP and sterile ascites are shown in Table 1.

Patients with SBP showed a higher incidence of hepatocellular carcinoma, worse renal function and higher blood leukocyte and ascitic fluid neutrophil counts than patients with sterile ascites.

16S rDNA gene detection by RT-PCR and sequencing

Tables 2 to 4 show the results of 16S rDNA gene detection by RT-PCR and further DNA identification by sequencing. The lowest detection limits in ascitic fluid for E. coli and E. faecalis were 5900 CFU/ml and 3400 CFU/ml, respectively. Bacterial DNA was detected in 23/25 (92%) culture positive SBP episodes (Table 2), in 16/30 (53%) culture negative SBP (p=0.002 respect to culture positive SBP cases) (Table 3), in 12/20 (60%) sterile ascites (p=0.01 respect to culture positive SBP and p=0.64 respect to culture negative SBP) (Table 4) and in 0/27 patients with neoplasic ascites (p<0.001 respect to other groups).

RT-PCR or sequencing of amplified fragments identified one microorganism to genus or species level in 12 cases from the culture positive SBP group (Table 2). The molecular identification agreed with the culture in 8 of these 12 cases but not in 4. Six of the 30 culture negative SBP cases gave positive results by molecular techniques to genus or species level (4 Escherichia coli and 2 Streptococcus) (Table 3). And in 6 of the 20 patients with sterile ascites, positive results to genus or species level were obtained (all six were E. coli) (Table 4). In the remaining cases with positive RT-PCR, sequencing did not yield a definitive bacterial identification.

Characteristics and outcome of patients with SBP according to presence or absence of bacterial DNA in ascitic fluid

Analyzing all SBP episodes (n=55, Table 5) and comparing positive (n=39) and negative bacterial DNA patients (n=16), the former showed more frequent alcoholic etiology, worse liver function and less frequent treatment with beta-blockers. Moreover, SBP cases with positive bacterial DNA showed a trend to a higher inflammatory response (as observed by higher blood leukocyte count and incidence of SIRS and a tendency to higher ascitic fluid neutrophil count)

and more frequent positive ascitic fluid culture.

Renal failure was present at SBP diagnosis in 13/39 (

DISCUSSION

In the present study, RT-PCR detected bacterial DNA in ascitic fluid from most patients with culture positive SBP (23/25, 92%). Our results are in agreement with the four previous studies including small series of patients with culture positive SBP, that showed positivity of bacterial DNA in ascitic fluid in 9/9, [START_REF] Francés | Bacterial DNA in patients with cirrhosis and noninfected ascites mimics the soluble immune response established in patients with spontaneous bacterial peritonitis[END_REF] 3/4, [START_REF] Sugihara | Rapid identification of bacterial species with bacterial DNA microarray in cirrhotic patients with spontaneous bacterial peritonitis[END_REF] 5/5, 24 and 4/4 [START_REF] Vieira | Amplification of bacterial DNA does not distinguish patients with ascitic fluid infection from those colonized by bacteria[END_REF] patients. Bacterial DNA was negative in two of our patients. A low percentage of false negative cases has consistently been described in studies using molecular methods in infections other than SBP, mainly in those caused by grampositive cocci. 18, 33, 34 This has been attributed to a number of factors. Technical difficulties in disrupting the cell wall of these organisms may result in failure in DNA extraction. 18,33,34 Contamination during sample extraction or processing may lead to a false positive culture in an otherwise DNA negative sample. [START_REF] Andrade | Advances in the microbiological diagnosis of sepsis[END_REF][START_REF] Vieira | Amplification of bacterial DNA does not distinguish patients with ascitic fluid infection from those colonized by bacteria[END_REF][START_REF] Harris | Development of broad-range 16S rDNA PCR for use in the routine diagnostic clinical microbiology service[END_REF][START_REF] Jordan | Evaluating the near-term infant for early onset sepsis: progress and challenges to consider with 16S rDNA polymerase chain reaction testing[END_REF] Indeed, in our study, cultures grew gram-positive cocci in the two cases with positive culture and negative bacterial DNA. Another factor that could also contribute is an inoculum effect, as 10 ml of ascitic fluid was inoculated for culture whereas only 400 µl were used for molecular techniques.

22,23

Considering culture negative SBP patients, bacterial DNA was not detected in almost half of them (14/30, 46.6%). This finding could perhaps be due to a low DNA concentration in ascitic fluid, although an alternative explanation could be that bacteria were not the cause of the increased ascitic fluid neutrophil count in these cases. In support of these hypotheses, when we analyzed all SBP patients according to the presence or absence of bacterial DNA, we observed a trend to a less severe inflammatory response and better outcome in terms of SBP resolution and survival in the latter. Data about culture negative SBP are controversial in the four previous studies that used molecular techniques in SBP. Bacterial DNA was detected in ascitic fluid in 3/4 [START_REF] Vieira | Amplification of bacterial DNA does not distinguish patients with ascitic fluid infection from those colonized by bacteria[END_REF] and 13/13 [START_REF] Francés | Bacterial DNA in patients with cirrhosis and noninfected ascites mimics the soluble immune response established in patients with spontaneous bacterial peritonitis[END_REF] patients with culture negative SBP using conventional PCR, in 1/6 patients using multiplex PCR [START_REF] Bruns | Identification of bacterial DNA in neutrocytic and nonneutrocytic cirrhotic ascites by means of a multiplex polymerase chain reaction[END_REF] and 1/5 with DNA microarray.

23

Molecular techniques can increase the rates and velocity of bacterial identification in comparison with microbiological cultures. [START_REF] Andrade | Advances in the microbiological diagnosis of sepsis[END_REF][START_REF] Sontakke | Use of broad range 16S rDNA PCR in clinical microbiology[END_REF] They could therefore allow a more suitable choice of antibiotic and consequently lead to a better patient outcome. [START_REF] Umgelter | Failure of current antibiotic first-line regimens and mortality in hospitalized patients with spontaneous bacterial peritonitis[END_REF][START_REF] Acevedo | Current efficacy of recommended empirical antibiotic therapy in patients with cirrhosis and bacterial infection[END_REF] In the present study, however, the results of bacterial DNA analysis would not have determined a change in the antibiotic treatment in any patient. Indeed, one limitation of the present methodology is its inability to assess bacterial resistance. Other molecular methods, however, such as DNA microarrays, may be able to assess such resistances in the future.

18,20

We detected bacterial DNA in ascitic fluid in 60% of patients with sterile ascites. This is consistent with previous studies by Such et al [START_REF] Such | Detection and identification of bacterial DNA in patients with cirrhosis and culture-negative, nonneutrocytic ascites[END_REF][START_REF] González-Navajas | Presence of bacterial-DNA in cirrhosis identifies a subgroup of patients with marked inflammatory response not related to endotoxin[END_REF][START_REF] Zapater | Serum and ascitic fluid bacterial DNA: a new independent prognostic factor in noninfected patients with cirrhosis[END_REF] and Viera et al [START_REF] Vieira | Amplification of bacterial DNA does not distinguish patients with ascitic fluid infection from those colonized by bacteria[END_REF] that showed that some cirrhotic patients with sterile ascites or culture-negative, nonneutrocitic ascites present bacterial DNA in blood and ascitic fluid, and that this presence is associated to an increased inflammatory response [START_REF] Francés | Bacterial DNA in patients with cirrhosis and noninfected ascites mimics the soluble immune response established in patients with spontaneous bacterial peritonitis[END_REF][START_REF] González-Navajas | Presence of bacterial-DNA in cirrhosis identifies a subgroup of patients with marked inflammatory response not related to endotoxin[END_REF] and worse prognosis. [START_REF] Zapater | Serum and ascitic fluid bacterial DNA: a new independent prognostic factor in noninfected patients with cirrhosis[END_REF] The presence of bacterial DNA in noninfected cirrhotic patients possibly reflects subclinical bacterial translocation, not leading to overt infection but impairing liver function, immune response and hemodynamics. [START_REF] Francés | Bacterial DNA in patients with cirrhosis and noninfected ascites mimics the soluble immune response established in patients with spontaneous bacterial peritonitis[END_REF][START_REF] Guarner | Bacterial translocation and its consequences in patients with cirrhosis[END_REF][START_REF] Albillos | Increased lipopolysaccharide binding protein in cirrhotic patients with marked immune and hemodynamic derangement[END_REF][START_REF] Albillos | Multifactorial gut barrier failure in cirrhosis and bacterial translocation: working out the role of probiotics and antioxidants[END_REF] We found a higher percentage of patients with sterile ascites and positive bacterial DNA (60%) than the figures previously reported by Such et al. and Vieira et al (approximately 30%). [START_REF] Vieira | Amplification of bacterial DNA does not distinguish patients with ascitic fluid infection from those colonized by bacteria[END_REF][START_REF] Such | Detection and identification of bacterial DNA in patients with cirrhosis and culture-negative, nonneutrocytic ascites[END_REF][START_REF] Zapater | Serum and ascitic fluid bacterial DNA: a new independent prognostic factor in noninfected patients with cirrhosis[END_REF] It should be noted, however, that our methodology differed from that used in these studies as we performed RT-PCR with specific and universal Taqman probes instead of conventional PCR. It Accordingly, we hypothesize that the low percentages of bacterial identification among samples with positive bacterial DNA could be the consequence of previous polybacterial translocations leading to the presence of polybacterial DNA (with or without viable bacteria) in ascitic fluid.

Indeed, polybacterial cultures have been described in up to 37% of rats with experimental cirrhosis and bacterial translocation. [START_REF] Runyon | Translocation of gut bacteria in rats with cirrhosis to mesenteric lymph nodes explains the pathogenesis of spontaneous bacterial peritonitis[END_REF][START_REF] Llovet | Translocated intestinal bacteria cause spontaneous bacterial peritonitis in cirrhotic rats: molecular epidemiologic evidence[END_REF][START_REF] Pérez-Páramo | Effect of propranolol on the factors promoting bacterial translocation in cirrhotic rats with ascites[END_REF][START_REF] Chiva | Intestinal mucosal oxidative damage and bacterial translocation in cirrhotic rats[END_REF] Further studies with other methodologies able to separate and identify polymicrobial DNA could be useful in these cases with multiple amplification products. The use of techniques such as denaturing gradient gel electrophoresis (DGGE), [START_REF] Schabereiter-Gurtner | 16S rDNA-based identification of bacteria from conjunctival swabs by PCR and DGGE fingerprinting[END_REF] denaturing high-performance liquid chromatography (DHPLC), [START_REF] Domann | Culture-independent identification of pathogenic bacteria and polymicrobial infections in the genitourinary tract of renal transplant recipients[END_REF]

INTRODUCTION

  Spontaneous bacterial peritonitis (SBP) is a frequent and severe complication of cirrhotic patients with ascites. Morbidity and mortality remain relevant even though advances in the management of these patients have improved outcome in recent years.1-8

4 rDNA)

 4 by real-time PCR (RT-PCR) and sequencing increases the efficacy of culture in the microbiological diagnosis of SBP.

31 RT-PCR and sequencing of 16S rDNA gene (figure 1 ) 6 were

 3116 DNA extraction. DNA samples were isolated with QIAamp DNA Blood Mini kit (Qiagen, Hilden, Germany) according to manufacturer's instructions with few modifications: 400 µl of ascitic fluid centrifuged at 7500 rpm for 10 minutes, resuspended with 180 µl of a home-made lysis solution [Lisozime (20 mg/ml), 20 mM Tris-Hcl, pH 8.0, and 1.2% Triton X-100] and incubated at 37ºC for 30 minutes. Lastly, proteinase K was added to a final concentration of 20 mg/ml and incubated at 56ºC for 30 minutes and at 95ºC for 15 minutes before DNA extraction.

  Molecular techniques identified one bacteria in 12 culture positive SBP, eight of which correlated with the culture. The lack of agreement in the other 4 cases could be due to contamination during sample extraction or processing, or to the presence of DNA from nonviable bacteria in addition to the viable bacteria detected in the culture. The latter could be the consequence of previous bacterial translocations, as pointed out below. Again, cases of and molecular techniques have been reported previously.

  point out that bacterial DNA was not detected in any patient with neoplasic ascites, supporting the reliability of the positive results in cirrhotic patients.Another interesting finding was that molecular techniques, and specially sequentiation, did not yield a definitive bacterial identification in approximately half the cases in which bacterial DNA was detected. These unexpected results could be explained by a suboptimal sequencing reaction due to a low initial DNA concentration, or by a mix of amplification products corresponding to different bacterial species. The latter situation could be attributed to the presence of DNA from multiple bacteria in ascitic fluid or polybacterial contamination during paracentesis or later processing. Although contamination may have occurred in a few cases it would not explain the high percentage of non-conclusive identification, especially taking into account that bacterial DNA was not detected in 27 ascitic fluid samples from neoplasic noncirrhotic patients.

  Standard PCR was performed in a 50-µl reaction mixture containing 6 µl of DNA from the RT-PCR product (tube containing the universal probe), 5 µl of reaction buffer 10X with MgCl 2

	(Expand High Fidelity PCR System, Roche Diagnostics GmbH, Mannheim, Germany), 1µM of
	primer 27f and 519r, 10 µM of DNTPs and 1.75 U of Taq DNA Polimerase (Expand High Fidelity
	PCR System, Roche Diagnostics GmbH, Mannheim, Germany). An initial denaturation at 80ºC
	for 5 minutes was performed, followed by 28 cycles of 94ºC for 45 seconds, 53ºC for 1 minute,
	and 72ºC for 90 seconds, and a final extension at 72ºC for 10 minutes. PCR products were
	purified with the Exosap-IT PCR System (USB Corporation, Cleveland, Ohio, USA) and
	sequenced in both directions using the universal 27f and 519r primers by Macrogen Inc. (Seoul,
	Korea). Each sequence was compared with GenBank sequences by using the basic local F r o alignment scratch tool (BLAST) algorithm.
	P
	e
	e r
	R
	e
	v i e
	w
	7

Sequencing. Ascitic fluid samples with a universal positive signal and negative for the specific E. coli probe in the RT-PCR were re-analysed by standard PCR and DNA sequencing.

Statistical analysis

Statistical analysis was performed by

Student's t test and Mann-Whitney test to analyze quantitative variables, and Chi 2 test and Fisher test for qualitative variables. Results are expressed as mean±standard deviation or frequencies. To analyze survival of SBP patients according to the presence or absence of bacterial DNA in ascitic fluid, Kaplan-Meier method with log rank test was used. A p value <0.05 was considered statistically significant. Calculations were performed with the SPSS Statistical Package (version 17.0, SPSS Inc., Chicago, IL).

Table 1 .

 1 Molecular techniques to detect bacterial DNA still present several limitations and challenges that need to be addressed, such as false positive results due to contamination, the lack of standardized methods and reagents, and difficulties and variability in interpretation of results. Figure 1. Flow-chart showing procedures used in the present study. SBP: spontaneous bacterial peritonitis; RT-PCR: real-time polymerase chain reaction. E. coli: Escherichia coli. Clinical and analytical characteristics of cirrhotic patients with SBP and sterile ascites.

	bacterial tag

Table 3 .

 3 Ascitic fluid real-time PCR (RT-PCR) and sequencing results in cases with negative culture SBP.

	Nº of	Culture	RT-PCR	Sequentiation
	cases
	14	negative	negative	ND
	10	negative	positive	undetermined
	2	negative	positive E. coli probe	ND
	2	negative	positive	Streptococcus
	2	negative	positive	Escherichia coli
	SBP: spontaneous bacterial peritonitis; ND: not done.

Table 4 .

 4 Ascitic fluid real-time PCR (RT-PCR) and sequencing results in cases with sterile ascites.

	Nº of	Culture	RT-PCR	Sequentiation
	cases
	8	negative	negative	ND
	6	negative	positive	Escherichia coli
	6	negative	positive	undetermined
	ND: not done.

Table 5 .

 5 Clinical and analytical characteristics of patients with SBP and positive or negative ascitic fluid bacterial DNA. Data are mean±SD or frequency (%). Previous antibiotic treatment consisted of norfloxacin in the 3 patients with negative bacterial DNA. In patients with positive bacterial DNA, previous antibiotic treatment consisted of norfloxacin in 6 patients, amoxicillin/clavulanic acid in 4, ceftriaxone in 3 and cloxacillin in one.

		Negative bacterial DNA	Positive bacterial DNA	p
		n=16	n=39
	Age (yr)	69.1±13.7	67.1±9.9	0.54
	Sex (male/female)	7 (43.7%)/9	24 (61.5%)/15	0.22
	Etiology
	Alcohol	1 (6.2%)	17 (43.5%)	0.007
	HCV	11 (68.7%)	13 (33.3%)	0.01
	Hepatocellular carcinoma	6 (37.5%)	12 (30.7%)	0.62
	Child-Pugh AB/C	13 (81.2%)/3	19 (48.7%)/20	0.02
	Child-Pugh score	8.7±1.2	9.6±2.1	0.06
	MELD score	14.3±5.7	18.5±7.9	0.06
	Previous complications of	14 (87.5%)	30 (76.9%)	0.47
	cirrhosis
	Previous SBP	5 (31.2%)	5 (12.8%)	0.13
	Previous ascites	14 (87.5%)	28 (71.7%)	0.30
	Previous variceal bleeding	4 (25%)	7 (17.9%)	0.71
	Previous encephalopathy	5 (31.2%)	13 (33.3%)	0.88
	Previous antibiotic	3 (18.7%)	14 (35.8%)	0.33
	treatment 1
	Beta-blockers	11 (68.7%)	14 (35.8%)	0.02
	Diuretics	11 (68.7%)	27 (69.2%)	1
	Nosocomial	3 (18.7%)	14 (35.8%)	0.33
	Asymptomatic	5 (31.2%)	10 (25.6%)	0.73
	Abdominal pain	6 (37.5%)	13 (33.3%)	0.64
	Fever (>38ºC)	7 (43.7%)	13 (33.3%)	0.46
	Renal failure	3 (18.7%)	13 (33.3%)	0.34

HCV: hepatitis C virus; MELD: model for end stage liver disease score; SIRS: systemic inflammatory response syndrome; MAP: mean arterial pressure; INR: international normalized ratio. 1 F o r P e e r R e v i e w

ACKNOWLEDGEMENTS

We thank Carolyn Newey for English language revision.

STATEMENT OF INTERESTS

This study was supported by the Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III: FEDER, Spanish Network for the Research in Infectious Diseases (REIPI RD06/0008/0013), CIBERehd and grant PI060431. The authors do not have other personal or funding interests.