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Keywords:

  • adhesion;
  • Caco-2;
  • HT29-MTX;
  • intestinal mucin;
  • probiotics

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aims:  To evaluate the adhesion ability of intestinal bacteria to different in vitro models of intestinal epithelia, and to estimate the suitability of these models and the type of interactions involved.

Methods and results:  The adhesion of probiotic (Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis Bb12), commensal (B. animalis IATA-A2 and B. bifidum IATA-ES2) and potentially pathogenic bacteria (E. coli and L. monocytogenes) was determined. The adhesion models used were polycarbonate-well plates, with or without mucin, and different configurations of Caco-2 and/or HT29-MTX cell cultures. All bacteria adhered to wells without mucin (2·6–27·3%), the values being highly variable depending on the bacterial strain. Adhesion percentages of potentially probiotic bacteria to Caco-2 cultures were remarkably lower (< 0·05) than those to mucin, and more similar to those of pathogenic strains. The lowest adhesion of different bacterial strains was detected on HT29-MTX (0·5–2·3%) cultures and Caco-2/HT29-MTX (0·6–3·2%) cocultures, while these values were increased in Caco-2 cultures plus mucin.

Conclusions:  The results suggested that bacterial strains exhibit different capacities to adhere to cellular components and several types of mucin present in different models, showing preferences for intestinal MUC2.

Significance and impact of the study:  The use of Caco-2 cells monolayer plus mucin (type II) better approaches the physiological characteristics of in vivo situation, providing a reliable and suitable in vitro model to evaluate bacterial adhesion.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

There is growing interest in the design of functional foods beneficial to human health. Within the context of functional foods, it is known that probiotic bacteria have desirable traits such as immunomodulatory properties and ability to inhibit pathogenic micro-organisms by different mechanisms (Sanz et al. 2007). Bacterial adhesion to the intestinal epithelium is considered a requisite for probiotic selection, because it may influence the residence time of the bacteria in the intestinal tract (Servin and Coconnier 2003). The ability of some probiotic strains to inhibit pathogen colonization and invasion and to modulate the immune response(s) has also been related to their ability to adhere to intestinal mucus and/or epithelial cells (Servin and Coconnier 2003). In a similar way as beneficial bacteria, the harmful effect of enteropathogen bacteria may be determined by their ability to adhere and colonize the intestinal epithelium.

Although human clinical trials are the definitive tool to establish probiotic functionality, the use of in vitro models is necessary to select the most promising strains prior to such trials. Several in vitro studies have been made to evaluate the adhesion ability of potential probiotic bacteria and their interactions with pathogens at the intestinal epithelial interface (Sanz et al. 2007; Izquierdo et al. 2008; Sánchez et al. 2008). The simplest model to evaluate the adherence of bacterial strains to intestinal mucus is based on the immobilization of commercially available mucin on a micro-well plate surface (Tuomola et al. 1999; Izquierdo et al. 2008). However, cultures of human intestinal epithelial cell lines, notably Caco-2 and HT29-MTX cells derived from colon adenocarcinoma, seem to better represent the in vivo situation. More concisely, Caco-2 cell cultures has allowed important advances in cellular interaction studies because they grow in culture forming a homogeneous and polarized cell monolayer, which resembles mature human enterocytes in the small intestine (Pinto et al. 1983; Lenaerts et al. 2007). A previous study proved that fluorescent labelling of bacteria in conjunction with Caco-2 cell monolayers is a suitable model for adhesion studies and provides a reliable and safer alternative to radioactive labelling of bacteria (Bianchi et al., 2004). However, the lack of an adequate mucus layer constitutes a major drawback for the use of this cell line. The HT29-MTX cell line resulted from the isolation of HT29 cells adapted to methotrexate (MTX) (Lesuffleur et al. 1990), which differentiate into goblet cells characterized by the secretion of mucin (Lesuffleur et al. 1993; Leteurtre et al. 2004). In the intestinal epithelium, enterocytes and goblet cells represent the two major cell phenotypes. To better approach, the cell population representing the intestinal epithelium, cocultures of Caco-2 cells and mucin-secreting HT29-MTX cells have also been developed (Walter et al. 1996; Pontier et al. 2001; Novellaux et al. 2006; Mahler et al. 2008). Nevertheless, the use of combined Caco-2/HT29-MTX culture systems to evaluate bacterial adhesion of potentially probiotic strains has not been reported.

The objective of this study was to compare the adhesion ability of probiotic, commensal and pathogenic bacterial strains to different in vitro models of intestinal epithelium. Polycarbonate-well plates treated or not with mucin and different configurations of Caco-2 and/or HT29-MTX cell cultures to determine their suitability and the possible type of interactions involved in adhesion were used.

Material and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial cultures

Bifidobacterium bifidum (IATA-ES1) and Bifidobacterium animalis (IATA-A2) strains were isolated from faeces of healthy human subjects and identified as described elsewhere (Medina et al. 2008). Bifidobacterium animalis subsp. lactis Bb12 (Chr. Hansen, Horsholm, Denmark) and Lactobacillus rhamnosus GG (ATCC 53103) were included as controls, because they were reported to show high adhesion ability (Izquierdo et al. 2008). Bifidobacteria and lactobacilli were grown in Man-Rogosa-Sharpe broth and agar (Scharlau, Barcelona, Spain) supplemented with 0·05% (w/v) cysteine (Sigma, St Louis, MO) and incubated at 37°C under anaerobic conditions (AnaeroGen; Oxoid, Basingstoke, UK). Escherichia coli strains, IATA-CBL2 and IATA-CBD10, were also isolated from faeces (Sánchez et al. 2008). They were grown in Brain-Heart broth and agar (Scharlau) and incubated at 37°C under aerobic conditions. Listeria monocytogenes CECT 935 was grown in Brain-Heart broth and agar and incubated at 37°C under aerobic conditions. All cultures were grown for 20 h to be used in adhesion experiments.

Mucin treatment

Crude mucin (Type II, Sigma-Aldrich) was diluted in a phosphate-buffered solution (pH 7·2) (PBS) (0·5 mg ml−1). An aliquot (0·5 ml) of this solution was loaded into polycarbonate 24-well plates (Costar, Cambridge, MA, USA), with and without Caco-2 cell cultures, and incubated at 37°C for 1 h. To remove unbound mucin, the wells were washed twice with 0·5 ml PBS.

Cell lines and culture configurations

The Caco-2 cell line was obtained from the American Type Culture Collection (Rockville, MD, USA) at passage 17 and used in experiments at passage 25–33. The HT29-MTX cell line was kindly provided by Dr T. Lesuffleur (INSERM U560, Lille, France) at passage 7 and used in experiments at passage 16–22. Caco-2, HT29-MTX cells and Caco-2 cocultured with HT29-MTX cells were grown in Dulbecco’s Modified Eagle Medium (DMEM Glutamax; Gibco, Rockville, MD) containing 4·5 g l−1 glucose and supplemented with 25 mmol l−1 HEPES buffer and 10% (v/v) fetal bovine serum (Gibco). The cells were maintained at 37°C in 5% CO2, 95% air, and the culture medium was changed every 2 days.

For adhesion experiments, Caco-2 and HT29-MTX cells were cultured separately, or together at 90 : 10, Caco-2:HT29-MTX, ratios to mimic the major cell types in the intestine. Cells were seeded at a density of 50 000 cells cm−2 onto 24-well plates (Costar). All cultures were grown in DMEM, and culture medium was changed every 2 days. Experiments were performed 15 days post seeding, after complete morphological and functional differentiation of Caco-2 cells (Jovaníet al. 2001; Mahler et al. 2008).

Adhesion assay

Bacteria from 20-hour-old cultures were collected by centrifugation (4000 g for 5 min at 4°C), washed twice and resuspended in PBS to reach an optical density of 0·5 (Aλ600). Colony forming unit (CFU ml−1) equivalence was determined by plate count, and the size of inoculum ranged between 6·0 ml−1× 107 ml−1 and 8·2 ml−1× 108 CFU ml−1. Suspensions of different bacteria were incubated with 75 μmol l−1 carboxyfluorescein diacetate (CFDA) (Sigma-Aldrich), at 37°C for 30 min. CFDA serves as substrate for intracellular esterases releasing the fluorescent compound 2,7-dichlorofluorescein, thereby, evidencing the presence of live bacteria (Bianchi et al., 2004). Then, the mixtures were washed twice and resuspended in PBS. Afterwards, a volume of 0·5 ml working labelled suspensions was loaded into 24-well plates and incubated at 37°C for 1 h. After the incubation period, the medium was removed, and wells were washed twice with 0·5 ml PBS. Then, wells were added with 1 ml 1% (w/v) sodium dodecyl sulphate in 0·1 mol l−1 NaOH and incubated at 37°C for 1 h. Afterwards, the mixtures were homogenized by pipetting, and 0·3 ml of the supernatants was transferred to black 96-well plates. The fluorescence was read in a multiscan fluorometer (Fluoroskan Ascent; Labsystem, Oy, Finland) at λex 485 and λem 538 nm. In parallel, the bacterial adhesion was also evaluated in 24-well plates without mucin. To evaluate the potential CFDA unspecific adsorption to the wells, negative controls without bacteria were used throughout the experiment.

Adhesion was expressed as the percentage of fluorescence recovered after binding to mucin and/or cell cultures relative to the fluorescence of the bacterial suspension added to the wells. Each assay was performed in triplicate and conducted in two independent experiments.

Statistical analysis

One-way analysis of variance (anova) and the Tukey post hoc test were applied. Statistical significance was established at P < 0·05. spss ver. 15 (SPSS Inc., Chicago, IL, USA) was used for the statistical analysis.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial adhesion to mucin

The adhesion percentages of different bacterial strains tested, estimated using the classical mucin adhesion assay, are shown in Table 1. All bacteria exhibited adhesion capacity to untreated wells, and B. lactis Bb12 showed the highest adhesion percentage. The adhesion percentages of probiotic (L. rhamnosus GG and B. lactis Bb12) and potentially probiotic (B. animalis IATA-A2 and B. bifidum IATA-ES2) strains (9·9–27·3%) were significantly higher (< 0·05) than those of potentially pathogenic strains such as E. coli (IATA-CBL2 and -CBD10) and L. monocytogenes CECT 935 (2·6–5·9%). The adhesion values of bacterial strains to mucin (type II)-treated wells were similar as those (2·7–31·1%) detected in untreated wells. Bifidobacterium lactis Bb12 was also the strain showing the highest adhesion capacity to mucin-treated wells. Bifidobacterium lactis Bb12 and B. animalis IATA-A2 showed higher (< 0·05) adhesion percentages to mucin than to untreated wells. In contrast, B. bifidum IATA-ES2, E. coli IATA-CBL2 and L. monocytogenes CECT 935 showed lower (P < 0·05) adhesion percentages to mucin than to untreated polycarbonate wells.

Table 1.   Adhesion of bacterial strains to mucin (type II), Caco-2 cell cultures, HT29-MTX cell cultures and Caco-2:HT29-MTX cell cocultures (seeded in 90:10 ratio). Results are expressed as mean ± standard deviation (n = 5)
Relative bacterial adhesion to in vitro model (%)
BacteriaPolycarbonate*Mucin†Caco-2‡HT29-MTX‡Caco-2/HT29-MTX§Caco-2/Mucin¶
  1. a-eDifferent superscript letter indicates statistically significant (P < 0·05) differences in a row. v-zDifferent superscript letter indicates statistically significant (P < 0·05) differences in a column.

  2. *Untreated 24-well plates.

  3. †Mucin (type II)-treated 24-well plates.

  4. ‡Differentiated (15 days post seeding).

  5. §Differentiated (15 days post seeding) cocultures in a 90 : 10 ratio to mimic the major cell phenotypes encountered in the intestine.

  6. ¶Differentiated (15 days post seeding) Caco-2 cultures plus mucin (type II).

  7. **IATA-CBL2.

  8. ††IATA-CBD10.

L. rhamnosus GG9·89 ± 0·17a,v10·21 ± 0·36a,v5·17 ± 1·15c,vw0·84 ± 0·09b,v0·85 ± 0·07b,v3·19 ± 0·59d,v
B. lactis Bb1227·25 ± 0·45a,w31·07 ± 1·02b,w8·89 ± 0·49d,x0·76 ± 0·06c,v0·92 ± 0·08c,v7·96 ± 0·29d,w
B. animalis10·92 ± 1·56a,v17·62 ± 0·99b,x6·21 ± 1·01d,w0·54 ± 0·05c,v0·72 ± 0·06c,v4·53 ± 0·23d,xy
B. bifidum11·11 ± 0·71a,v5·28 ± 0·73b,y5·91 ± 0·11b,vw2·33 ± 0·19c,w3·15 ± 0·30c,w9·82 ± 0·20d,z
E. coli**5·97 ± 0·25a,x5·06 ± 0·31b,y6·45 ± 0·51a,w1·40 ± 0·09c,x1·34 ± 0·10c,x5·07 ± 0·02b,y
E. coli††2·60 ± 0·16ab,y2·70 ± 0·3b,z5·43 ± 0·25d,vw1·88 ± 0·11ac,w1·89 ± 0·29c,y3·79 ± 0·47e,vw
L. monocytog6·56 ± 0·93a,x3·51 ± 0·14b,z4·09 ± 0·37b,v0·68 ± 0·01c,v0·59 ± 0·02cv4·07 ± 0·34b,vwx

Bacterial adhesion to cell cultures

The relative adhesion of different bacterial strains to several cultures of human intestinal epithelial cells is given in Table 1. The adhesion values of different bacterial strains to Caco-2 cell cultures showed less variability than those detected with well-plates treated or not with mucin. Adhesion percentages of L. rhamnosus GG, B. lactis Bb12 and B. animalis IATA-A2 to Caco-2 cultures were remarkably lower (< 0·05) than those to mucin and more similar to those of the E. coli strains and L. monocytogenes. The adhesion percentages of B. bifidum IATA-ES2 and L. monocytogenes CECT 935 to Caco-2 and mucin did not differ (> 0·05). In contrast, adhesion of both E. coli strains tested was higher (< 0·05) to Caco-2 cultures than to mucin, indicating that interaction with cellular components could be more relevant than to mucin to E. coli adhesion.

The use of HT29-MTX cell cultures, a mucin secreting cell line, markedly reduced the adhesion values of all bacterial strains in relation to those obtained with Caco-2 cell cultures (Table 1). Similarly, low adhesion values to Caco-2:HT29-MTX (90:10) cocultures were detected for all strains without showing statistically significant differences among them. As shown, the relatively high adhesion percentages of both commercial probiotics L. rhamnosus GG and B. lactis Bb12 to mucin were also markedly reduced and resulted similar (P > 0·05) in both HT29-MTX cultures or Caco-2:HT29-MTX (90:10) cocultures. The addition of mucin to Caco-2 cells significantly reduced (P > 0·05) the adhesion of L. rhamnosus GG and two E. coli strains, probably because of the ability of this glycoprotein to overlap adhesion sites in Caco-2 cells.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial adhesion to the intestinal epithelium influences the residence time and the ability of probiotic strains to modulate the immune response(s) and, thereby, to exert health effects in the gut (Servin and Coconnier 2003). The results indicate that interactions with glycoproteins partly explain the adhesion ability of probiotic, commensal and pathogen bacterial strains and that this feature is partially shared within the same species. In addition, adhesion percentages to untreated polycarbonate wells suggest that hydrophobic interactions are relevant to their adhesion ability. In fact, B. bifidum IATA-ES2 and L. monocytogenes CECT 935 showed adhesion capacities 2·1- and 1·9-fold higher to untreated wells than to mucin-treated wells. The adhesion percentages to mucin of the commercially available probiotic bacteria, L. rhamnosus GG (10·2%vs 19·0%) and B. lactis Bb12 (31·1%vs 38·5), resembled those reported by using human mucus extracted from faecal samples (He et al. 2001). This adherence ability was highly variable among strains in accordance with previous data (Izquierdo et al. 2008) and, in particular, the adhesion of B. animalis IATA-A2 and B. bifidum IATA-ES2 to mucin was significantly different (P < 0·05). It has also been reported that faecal bifidobacteria exhibited a widely variable (0·9–14·6%) adhesion ability to human faecal mucus (He et al. 2001), and the reported adhesion values are in good accordance with those detected in this study for the B. animalis and B. bifidum strains tested.

In the classical mucin adhesion assay, it is assumed that the used mucin concentration (0·5 mg ml−1) covers the surface area into the well to interact with bacteria (Tuomola et al. 1999; Izquierdo et al. 2008). However, the results shown that adhesion to the well represents a high proportion of the adhesion detected in mucin-treated wells. Therefore, the results obtained by using this model could not represent exclusively those resulting from mucin–bacteria interactions, which are supposed to be one of the main determinants of in vivo bacterial adhesion.

The use of human intestinal cell lines in culture for evaluating bacterial adhesion provides useful advantages in cellular interaction studies, because they resemble the small intestine forming homogeneous cell monolayers (Pinto et al. 1983; Lenaerts et al. 2007). In general, probiotic bacterial adhesion percentages to Caco-2 cell monolayers were remarkably lower (< 0·05) than those to mucin and more similar to commensals and pathogens such as E. coli and L. monocytogenes, respectively. These results seem to suggest the unspecific adhesion of probiotic bacteria to mucin, although we cannot rule out the implication of mucus-binding elements similar to those identified and characterized in Lactobacillus (Perea et al. 2007). This observation evidences that mucin could be critical to determine the residence of commensals instead of pathogenic bacteria. The adhesion values of B. lactis Bb12 to this Caco-2 model were relatively higher (8·9%) than those (2%) detected for the same strain and model in previous reports (Fernandez et al. 2008), which can be explained by differences in methodology regarding the surface area accessible for bacterial adhesion (24-well vs 96-well plates). Interestingly, it has been reported that the proportion of adherent relative to the added bacteria over Caco-2 cultures would likely remain unaltered when using concentrations of inocula between 106 and 107 CFU ml−1 (Bianchi et al., 2004). The use of HT29-MTX cell cultures provides some advantages to study the adhesion ability of bacterial strains to the intestinal epithelia. They constitute a mucin secreting cell culture and express similar protein patterns as Caco-2 cells and the human intestinal epithelium (Lenaerts et al. 2007). However, the adhesion ability of the different bacterial strains to independent cultures of the HT29-MTX cells was markedly lower (< 0·05) than that to Caco-2 cell cultures (Table 1). These results suggest that the mucin glycoproteins expressed by this cell type are not the only major determinants of adhesion but probably other proteins in the cell plasmatic membrane of mature enterocytes.

Fully differentiated Caco-2 cells exhibit many of the characteristics of mature enterocytes (Pinto et al. 1983), while HT29-MTX cells differentiate into goblet cells (Lesuffleur et al. 1990). To better simulate the major presence of columnar absorptive cells relative to the goblet cells in the intestinal epithelium, cocultures of the two human intestinal cell lines (Caco-2 and HT29-MTX) have been tested to produce a more physiological model, mimicking the major cell phenotypes present in the intestinal epithelium (Pontier et al. 2001; Novellaux et al. 2006). Mahler et al. (2008) have reported that a firmly adherent mucus layer is formed over both HT29-MTX cultures and Caco-2:HT29-MTX (90:10) cocultures. The fact that the bacterial adhesion values to Caco-2:HT29-MTX (90:10) cocultures were markedly lower than those to Caco-2 cultures indicate again that mucin layer produced by HT29-MTX cells could cover potential recognition components in the plasmatic membrane of Caco-2 cells making them inaccessible for bacterial adhesion.

The differences in bacterial ability to adhere to mucin, HT29-MTX cultures and Caco-2:HT29-MTX (90:10) cocultures could be attributed to the different mucin type present in these models. Differentiated goblet cells secrete several types of mucins including MUC5AC and MUC5B, which are the main expressed mucins, and MUC2, which is less abundant (Lesuffleur et al. 1993; Leteurtre et al. 2004). In contrast, the mucin used for adhesion assays was purely type II. While MUC2 is predominantly expressed in the large intestine and reduced in the upper intestinal tract, MUC5AC and MUC6 are highly expressed in the stomach and upper intestinal tract. The increased adhesion detected in the presence of mucin, rich in MUC2, in comparison with cell cultures expressing other mucin types is in agreement with other studies showing that lactobacilli adhesion to extracted ileal mucus was higher than to duodenal and jejunal mucus, less rich in MUC2 (Li et al. 2008). In fact, the addition of mucin (type II) to Caco-2 cell cultures increased the bacterial adhesion values (P < 0·05) with respect to those obtained with HT29-MTX and Caco-2:HT29-MTX cocultures. However, the addition of mucin to Caco-2 cell cultures could also mask adhesion sites present in Caco-2 cells. In general, the adherence of bacteria to mucin-treated Caco-2 cultures was slightly lower compared to that detected using the untreated Caco-2 cultures except for B. bifidum IATA-ES2 (< 0·05), which suggests that this strain might bind more preferentially to MUC2 than to other Caco-2 cellular components.

Overall, the differences detected in adhesion depending on the strain and the type of in vitro model system used for the evaluation could be because of both differences in the presence of molecules involved in the interaction as well as on the strain aggregation patterns. The nature of the bacterial cell-associated components involved in their adherence to intestinal epithelial cells has not been completely elucidated, and different components seemed to be involved. Most studies have indicated that in strains of the genera Lactobacillus (Sanchez et al. 2008; Perea et al., 2007) and Bifidobacterium components of protein nature are the major responsible for bacterial adhesion to the intestinal mucin types and/or epithelial cells (Izquierdo et al. 2008). Several exported proteins such as the mucus-binding protein Mub of Lactobacillus reuteri, the lectin-like mannose adhesin Msa of Lactobacillus plantarum and a sortase-dependent and other surface layer proteins have been identified as mediators of adhesion. In addition, the involvement of some other compounds such as carbohydrates (Greene and Klaenhammer 1994) and/or lipoteichoic acids (Granato et al. 1999) has also been suggested, whose expression and composition may be strain and species dependent. In addition, differences on aggregation patterns of bacterial strains might contribute to explain the differences on their adhesion abilities. For example, lactobacilli seem to adhere forming pairs and clusters, favouring this process (Coconnier et al. 1992; Gopal et al. 2001; Li et al. 2008), and a similar effect has been reported for several B. longum strains (Del Re et al. 2000).

Although the models used for in vitro adhesion assays present structures probably involved in vivo adhesion and, for instance, Caco-2 cell cultures express biologically significant proteins similarly to those expressed in small intestinal scrapings (Lenaerts et al. 2007), in vivo studies are required to confirm in vitro results. Some comparative evaluations of in vitro and in vivo adhesion ability of probiotic strains have been published (Crociani et al. 1995). While in some cases, the adhesion experiments of B. longum strains (BB536 and ATCC 15707) to Caco-2 cells were in agreement with the in vivo intestinal colonization, particularly for B. longum BB536 ingested in the form of a fermented milk (Crociani et al. 1995), the adhesion test of B. animalis to Caco-2 cultures underestimated the bacterial adhesion determined in vivo. These differences between the in vitro and in vivo situation could be because of the lack of suitability of a unique model system to predict adhesion ability of every strain.

In summary, interactions with mucin (MUC2) seemed to be more relevant to the adhesion of potentially probiotic strains than to pathogens. Although the use of cell cultures instead of extracted mucin better mimic the in vivo situation, the selection of the cell type and culture configuration also seems to be important because it determines the nature of adhesion sites in the system. Therefore, the use of different methods to study adhesion in vitro could provide more complete information on different bacterial adhesion ability and could help to elucidate the type of interactions and molecules that mediate the host-microbe interactions.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by grants PIF08-010-4 form CSIC and Consolider Fun-C-Food CSD2007-00063 from the Spanish Ministry of Science and Innovation (Spain) for which the authors are greatly indebted. Dr José Moisés Laparra was sponsored as a researcher within the programme ‘Juan de la Cierva’ (Spain). HI29-MTX cell cultures were kindly supplied by Dr Thécla Lesufflew (INSERM U843, Paris, France).

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  2. Abstract
  3. Introduction
  4. Material and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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