Biological dosage of peptidoglycans

Abstract

The present invention relates to a biological method for the dosage of peptidoglycans in a sample, especially a sample of glucose polymers.

Claims

1. A method of assaying peptidoglyeans (PGNs) in a sample of glucose polymer, comprising: a) enzymatic treatment of the sample of glucose polymer by a lysozyme, wherein the treatment of the sample comprises incubation of lysozyme at a concentration of approximately 250 U/ml in the sample at a glucose polymer concentration of 37.5% (weight/volume) for 2 h at a temperature of 37 C.; b) bringing the treated sample or a dilution thereof into contact with a recombinant cell expressing an exogenous TLR2 receptor (Toll-like Receptor 2) and a reporter gene under the direct dependence of the signaling pathway associated with the TLR2 receptor, the reporter gene coding for a fluorescent; c) measuring the reporter gene signal; and d) determining the amount of PGN in the sample using a calibration curve of the correspondence between the amount of PGN and the intensity of the reporter gene signal.

2. The method as claimed in claim 1, wherein the enzymatic treatment of the sample fragments and disaggregates the PGNs contained in the sample so as to make them capable of activating the TLR2 receptor.

3. The method as claimed in claim 1, wherein the enzymatic treatment of the sample generates PGNs having a size of approximately 120 kDa.

4. The method as claimed in claim 1, wherein the treatment of the sample comprises incubation of lysozyme at a concentration of approximately 250 to 2500 U/ml in the sample at a glucose polymer concentration of 37.5% (weight/volume) for 30 minutes to 16 h at a temperature of 37 C.

5. The method as claimed in claim 1, wherein the reporter gene is a secreted alkaline phosphatase.

6. The method as claimed m claim 1, wherein the cell IS a cell of the HEK-Blue hTLR2 line.

7. The method as claimed in claim 1, wherein the calibration curve of the correspondence between the amount of PGN and the intensity of the reporter gene signal is set up with an internal standard that is an agonist of TLR2.

8. The method as claimed in claim 7, wherein said agonist of TLR2 is PAM3 Cys-Ser-(Lys)4 trihydrochloride.

9. The method as claimed in claim 1, wherein the sample is diluted, if necessary, so as to generate a signal of the reporter gene corresponding to the linear portion of the calibration curve.

10. The method as claimed in claim 1, wherein the sample is a sample of a solution of icodextrin.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Theoretical curve of the cellular response as a function of increasing concentrations of PGN.

(2) FIG. 2: Calibration curve of the cellular response as a function of the S. aureus PGN level obtained with the HEK-Blue-hTLR2 cells.

(3) FIG. 3: Response of the HEK-Blue-hTLR2 cells as a function of increasing concentrations of PGN from different bacterial species.

(4) FIG. 4: Structure of PAM.sub.3Cys-Ser-(Lys)4 trihydrochloride (PAM3(cys)).

(5) FIG. 5: Comparison of the responses induced by different batches of PGNs of S. aureus and by PAM3(cys) in the HEK-Blue-hTLR2 cells.

(6) FIG. 6: Response of the HEK-Blue-hTLR2 cells as a function of the corrected PGN concentrations.

(7) FIG. 7: Calibration curve of the response of the HEK-Blue-hTLR2 cells as a function of the corrected active PGN concentrations.

(8) FIG. 8: Effect of the duration of treatment by the lysozyme on the cellular responses induced by the samples of glucose polymers.

(9) FIG. 9: Comparison of the cellular responses induced by the samples of glucose polymers after treatment by 10 and 100 g/ml of lysozyme.

EXAMPLES

(10) The assay is based on the specific recognition of PGNs by a line expressing the TLR2 receptor and on the production of an enzyme activity measurable via activation of the signaling pathway associated with TLR2.

(11) Cellular Material

(12) For the experiments relating to this assay, two lines are used: HEK-Blue hTLR2 (HEK-TLR2) line: specific response for the TLR2 ligands, with strong reactivity for the soluble PGNs. HEK-Blue Null2 (HEK-Null) line: nonspecific response connected with a cytotoxic effect of the sample.

(13) The cells are cultured according to the recommendations of the supplier (InvivoGen). At 75% confluence, the cells are resuspended at a density of 0.2810.sup.6 cells/ml. Before stimulation, 180 l of the cell suspension are distributed in the culture wells (96-well plate), that is to say 50 000 cells/well. The cells are then stimulated for 24 h by adding 20 l of the samples of glucose polymer at 37.5% (weight/volume) (i.e. a final dilution of the samples at 3.75%). After 24 h of stimulation, the cellular response is measured by quantification of the enzyme activity produced.

(14) 1Establishment of the Calibration Curve for Biological Assay of PGNs with an Internal Standard

(15) The dose-response curves were constructed by diluting the PGNs of different bacterial species in a solution of uncontaminated maltodextrin (referenced P-11.11) prepared at 37.5% (weight/volume). The PGNs assayed are extracted from Staphylococcus aureus (Sigma, Cat No 77140), Micrococcus luteus (Sigma, Cat No 53243), Bacillus subtilis (InvivoGen, # tlrl-pgnb2), Escherichia coli K12 (InvivoGen, # tlrl-pgnek) and Alicyclobacillus acidocaldarius (strain CNCM I-4689).

(16) The curves obtained are conventional for the responses observed in the assays performed with a cellular material (bioassay) (FIGS. 1 and 2). The absorbance values below 0.2 are evidence of PGN concentrations that are too low to induce a cellular response, whereas values above 2 show a plateau effect connected with saturation of the TLR2 receptors. Consequently, only the zone between these two limit values of absorbance allows correlation of the production of SEAP with the amount of PGN present in the samples.

(17) The responses observed show a large variability in the cellular reactivity associated with each type of PGN. Indeed, the concentrations giving a response equal to 50% of the maximum response (EC50) are 20 ng/ml for the PGNs of S. aureus and B. subtilis, 1500 ng/ml for M. luteus, and more than 2000 ng/ml for those extracted from A. acidocaldarius and E. coli K12 (FIG. 3).

(18) However, these differences were expected, since the PGNs have different structures depending on their bacterial origin, which is responsible for large variations in inflammatory reactivity. These observations emphasize the importance of defining an internal standard so as to be able to express the results in equivalent units of PGN.

(19) Another factor likely to alter the response of the HEK-TLR2 cells is the size of the PGNs, which will influence their solubility and reactivity with respect to TLR2. Thus, the procedure for purification of these macromolecules may have a considerable influence on the response of the cells, since the conditions of extraction could alter the size of the PGNs, or even cause partial degradation.

(20) It therefore seems necessary to introduce an internal standard for the calibration curve, so as to avoid errors relating to the variability of the PGNs and to express the results as amount of active PGN.

(21) PAM.sub.3Cys-Ser-(Lys)4 trihydrochloride (PAM.sub.3(cys)) is a triacylated synthetic lipopeptide that mimics the structure of the bacterial lipopeptides and acts as a strong agonist of TLR2. Being of homogeneous structure, it is often used as positive control for calibrating the responses of cells expressing the TLR2 receptor (FIG. 4).

(22) To test this hypothesis, the responses of the HEK-TLR2 cells, induced by three separate batches of PGNs extracted from S. aureus: 2 Sigma batches (Cat No 77140: batch 1, 0001442777; batch 2, BCBH7886V) and 1 InvivoGen batch (# tlrl-pgnsa), were compared to those induced by PAM.sub.3(cys) (FIG. 5).

(23) The results show variability of reactivity between the three batches of PGNs. Indeed, the EC50 values are 4, 20 and 400 ng/ml respectively for the three batches. These data indicate that there is a risk that PGNs extracted from the same bacterial species might show differences in reactivity, even if the batches were obtained from the same supplier and were in theory extracted by the same procedure. As expected, the cells show strong reactivity with respect to PAM.sub.3(cys), with an EC50 of 8 ng/ml.

(24) Each batch of PGN may be calibrated relative to PAM.sub.3(cys) by comparing the slopes of the linear portions of each dose-response curve, and by calculating a correction factor for superimposing the curves of the PGNs on that of PAM.sub.3(cys). In the example presented in FIG. 6, the correction factors were estimated at 0.4, 2 and 40 for batches 1, 2 and 3, respectively. This means that 2.5 times less PGN from batch 1 is required for obtaining responses identical to those induced by PAM.sub.3(cys), but 2 times more PGN from batch 2 and 40 times more PGN from batch 3. After correcting the raw quantities of PGN, it can be seen that all the points are aligned on one and the same curve, which is superimposed on that obtained with PAM3(cys) (FIG. 6). The use of this internal standard therefore makes it possible to obtain corrected concentrations for all the batches of PGN and to establish a dose-response curve calibrated for active PGN.

(25) By applying this method, the standard curve of response of the HEK-TLR2 cells has a detection threshold of 0.07 ng/ml (i.e. 2 ng/g of glucose polymers) and a zone of linearity for concentrations of active PGN of between 0.3 and 200 ng/ml (i.e. between 8 and 5400 ng/g of glucose polymers). (FIG. 7)

(26) 2. Enzymatic Treatment of the Samples of Glucose Polymers

(27) The aim of the enzymatic treatment by the lysozyme is to fragment and/or disaggregate the PGNs contained in the sample, so as to make them capable of inducing an inflammatory response via the TLR2 receptor. In particular, the enzymatic treatment of the sample causes a partial depolymerization of the PGNs to generate soluble PGNs of sizes of between 30 and 5000 kDa, especially of a size of approximately 120 kDa. However, the enzymatic treatment must not affect the capacity of the PGNs to interact with the TLR2 receptors. It is preferably optimized for releasing a maximum amount of soluble PGNs capable of interacting with TLR2 and for storing a maximum amount of PGN already active on TLR2.

(28) Tests of enzymatic treatments were carried out on four standard samples:

(29) (1) a preparation of uncontaminated maltodextrin (reference P-11.11),

(30) (2) a preparation of P-11.11 maltodextrin, artificially contaminated with a sub-optimal dose of PGN from S. aureus (20 ng/ml final) (referred to as P-11.11+PGN),

(31) (3) a preparation of contaminated icodextrin (reference I-209J), and

(32) (4) a glucose polymer matrix (reference E1242).

(33) TABLE-US-00001 P-11.11 I-E209J E1242 LAL assay LPS (EU/g) <0.15 0.6 2.4 SLP-HS assay PGN (ng/g) <3 393 21

(34) The treatments were carried out on 37% (weight/volume) solutions of glucose polymers. The solutions were then diluted to 1/10.sup.th in the presence of the cells, by adding 20 l of the solution to be tested to 180 l of cell suspension.

(35) The lysozyme used in these experiments is sold by Euromedex (ref: 5934; origin: egg white; activity: 25 000 units/mg).

(36) The first tests were carried out by incubating preparations of P-11.11 maltodextrin contaminated with PGN (20 ng/ml), I-209J icodextrin or 1920-E1242 lab matrix, in the presence of lysozyme at a concentration of 250 U/ml (10 g/ml). The treatment was carried out at 37 C. for times varying from 30 min to 16 h (FIG. 8). After stimulating the cells, the results show that the enzymatic treatment increases the reactivity of the PGNs with respect to the HEK-TLR2 cells. Moreover, no response is observed in the HEK-Null cells, which indicates that the treatment by the lysozyme does not have a cytotoxic effect.

(37) The increase in the cellular response induced by the lysozyme is observed for short time periods, with optimal effect at 2 h of treatment. On the contrary, a longer hydrolysis time reduces the cellular response. This observation indicates that prolonged treatment in the presence of lysozyme causes too great a depolymerization of the PGNs, which reduces their capacity to interact with the TLR2 receptors.

(38) The experiments were then reproduced with the same samples, but using the lysozyme at concentrations of 10 and 100 g/ml (FIG. 9). After 2 h of treatment at 37 C., no significant difference in the cellular responses is observed between the two concentrations of 10 and 100 g/ml. Indeed, the enzymatic treatments similarly increase the reactivity of the PGNs for the HEK-TLR2 cells, and no cytotoxicity is observed in the HEK-Null cells, even at the higher lysozyme concentration.

(39) These results show that a treatment by the lysozyme at a concentration of 250 U/ml for 2 h at 37 C. is effective for bringing about partial depolymerization of the PGNs present in samples of glucose polymer and for increasing their reactivity with respect to the HEK-TLR2 cells.

(40) In conclusion, under the optimal conditions described above, a treatment by the lysozyme is effective for increasing the reactivity of the PGNs present in preparations of glucose polymers.

(41) Depending on the type of PGN and the nature of the sample of glucose polymer (end product versus matrix derived from a process step), the activating effect brought about by the enzymatic treatment increases the reactivity of the HEK-TLR2 cells by a factor of up to 1.5. Thus, this treatment is particularly suited for converting all the traces of PGN present in samples to active PGN, and for allowing their biological assay.