KIT FOR IN VITRO OR EX VIVO MEASUREMENT OF SCD127 EXPRESSION IN A BIOLOGICAL SAMPLE

Abstract

A kit for in vitro or ex vivo measurement of sCD127 expression in a biological sample, the kit including: specific tools or reagents allowing measurement of sCD127 expression in said biological sample; and a positive standard sample which is a sample calibrated to contain an amount of sCD127 which corresponds to the mean amount measured in a pool of samples from patients who are known to have developed a nosocomial infection, and/or a negative standard sample which is a sample calibrated to contain the amount of sCD127 which corresponds to the mean amount measured in a pool of samples from patients who are known not to have developed a nosocomial infection.

Claims

1. A kit for in vitro or ex vivo measurement of sCD127 expression in a biological sample, the kit comprising: specific tools or reagents allowing measurement of sCD127 expression in said biological sample; and a positive standard sample which is a sample calibrated to contain an amount of sCD127 which corresponds to the mean amount measured in a pool of samples from patients who are known to have developed a nosocomial infection, and/or a negative standard sample which is a sample calibrated to contain the amount of sCD127 which corresponds to the mean amount measured in a pool of samples from patients who are known not to have developed a nosocomial infection.

2. The kit according to claim 1, wherein said specific tools or reagents allow a detection and/or a quantification of sCD127 expression.

3. The kit according to claim 2, wherein said specific tools or reagents allow the detection and/or the quantification of sCD127 expression at a protein level.

4. The kit according to claim 3, wherein said specific tools or reagents are anti-sCD127 antibodies.

5. The kit according to claim 3, wherein said specific tools or reagents are monoclonal anti-sCD127 antibodies.

6. The kit according to claim 1, wherein said patients are patients hospitalized in intensive care and/or having sustained an insult generating a systemic inflammatory response.

7. The kit according to claim 6, wherein said patients are patients with sepsis or severe sepsis.

8. The kit according to claim 6, wherein said patients are patients in septic shock.

Description

[0062] Various other characteristics will become apparent from the description given below with reference to the appended Figures which, as non-limiting examples, illustrate embodiments of the subject of the invention and in which:

[0063] FIG. 1A shows the IL-7 plasma concentration in 35 patients in septic shock at days 1-2 (1-2) and 3-4 (3-4) and in 30 healthy volunteers HV. ** p<0.005 vs. HV Mann Whitney U-test.

[0064] FIG. 1B shows a comparison of the results presented in FIG. 1A between NS and S patient groups.

[0065] FIG. 1C shows a comparison of the results presented in FIG. 1A between NI and No NI patient groups.

[0066] FIG. 2A shows the expression of CD127 in T CD4+ cells in 35 patients with septic shock at days 1-2 and 3-5 and in 30 healthy volunteers HV. p<0.05trend in time within one same group of patientsWilcoxon paired t-test.

[0067] FIG. 2B shows the expression of CD127 in T CD8+ cells in 35 patients with septic shock at days 1-2 and 3-5 and in 30 healthy volunteers HV. p<0.05 vs. HVMann Whitney U-test.

[0068] FIG. 3A shows the sCD127 plasma concentration in 35 patients with septic shock at days 1-2 and 3-4, and in 30 healthy volunteers. p<0.005 trend in time within one same group of patientsWilcoxon paired t-test.

[0069] FIG. 3B shows a comparison of the results presented in FIG. 1A between NS and S patient groups. p<0.05 p<0.005trend in time within one same group of patientsWilcoxon paired t-test.

[0070] FIG. 3C shows a comparison of the results presented in FIG. 1A between NI and No NI patient groups. # p<0.05 ## p<0.005 vs. No NIMann Whitney U-test; p<0.005trend in time within one same group of patientsWilcoxon paired t-test.

[0071] FIG. 4 shows the expression of the HLA-DR marker in 35 patients with septic shock at days 1-2 and 3-4, and in 30 healthy volunteers as per patient groups NI or No NI.

METHODS

Measurement of IL-7 Plasma Concentration

[0072] The IL-7 plasma concentration was measured with a kit using the LUMINEX technique marketed by Bio-Rad (Bio-Plex Pro Cytokine, Chemokine and Growth Factor Assays: BioPlex Pro Reagent kit, Bio-Rad #171-304070 and SinglePlex IL-7) following the supplier's directions.

Measurement of Cell Expression of the IL-7 Receptor (CD127)

[0073] In brief, 50 l of whole blood were incubated in the presence of 5 l of anti-CD4 antibody conjugated with PhycoerythinTexasRed (ECD) (Beckman Coulter #6604727) or 5 l of ECD-conjugated anti-CD8 antibody (Beckman Coulter #737659) and 10 l of anti-CD127 antibody conjugated with phycoerythrin (PE) (Beckman Coulter #IM1980U) for 15 minutes at ambient temperature and in the dark. The red blood cells were then lysed by hypotonic lysis and the cells fixed by automatic lysis on TQ-Prep automated system (Beckman Coulter). The membrane expression of CD127 on the surface of the T CD4+ or CD8+ lymphocytes was measured by flow cytometry.

Assay of the Soluble Form of the IL-7 Receptor (sCD127) by ELISA

Coating

[0074] A coating buffer was prepared to contain 0.8 g of Na.sub.2CO.sub.3, 1.4 g NaHCO.sub.3, 0.1 g NaN.sub.3 in 500 ml of water (pH 9.6).

[0075] 100 L of capture antibody (Ab) (Mouse Monoclonal Antibody Anti-CD127, human, R34.34, Beckman Coulter) diluted in coating buffer were deposited per well of an assay plate ([Ab]=8 g/mL). The plate was then covered for incubation at 4 C. overnight.

[0076] The content of the wells was then aspirated and the wells washed 3 times with at least 300 L of 0.05% PBS-Tween.sub.20 wash buffer. All the liquid was carefully removed at each wash. After the last wash the plate was turned over onto absorbent paper to remove all traces of buffer.

Blocking

[0077] Non-specific fixing was blocked using 150 L of blocking buffer per well (10% foetal bovine serum (FBS)/0.05% PBS-Tween.sub.20), and the plate incubated for 1 h at 37 C.

[0078] The content of the wells was again aspirated and the wells washed 3 times with at least 300 L of 0.05% PBS-Tween20 wash buffer. All the liquid was carefully removed at each wash. After the last wash, the plate was turned over onto absorbent paper to remove all traces of buffer.

Samples and Standards

[0079] A standard range was obtained with recombinant human IL-7R/CD127 Fc chimera (R&D SystemsCatalog Number: 306-IR) diluted in PBS containing 5% FCS diluting buffer, as described in Table 1 below and in accordance with C. Janot-Sardet et al Journal of Immunological Methods, 2010, 28, 115-123.

TABLE-US-00001 TABLE 1 rh IL-7R/CD127 Fc chimera [c] (ng/mL) 500 250 125 62.5 31.25 15.7 7.85 0 Diluent (L) 0 100 100 100 100 100 100 100 500 ng/mL 100 100 Successive dilutions 0 solution (L)

[0080] 100 L of sample or standard (extemporaneously-reconstituted solution of CD127 Fc chimera aliquoted at concentrations of 60 ng/ml and 10 ng/ml) were added to each well and the plate incubated for 1 h at 37 C.

[0081] The content of the wells was again aspirated and the wells washed 3 times with at least 300 L of 0.05% PBS-Tween.sub.20 wash buffer. All the liquid was carefully removed at each wash. After the last wash the plate was turned over onto absorbent paper to remove all traces of buffer.

Detection Antibody

[0082] 100 L of detection antibody (biotinylated polyclonal anti-CD127 goat antibody reconstituted with 1 mL of 1% TBS-BSA, R&D Systems) diluted in PBS/5% FBS, were added to each well ([Ab]=200 ng/mL), and the plate then incubated for 1 h at 37 C.

[0083] The content of the wells was again aspirated and the wells washed 3 times with at least 300 L of 0.05% de PBS-Tween.sub.20 wash buffer. All the liquid was carefully removed at each wash. After the last wash the plate was turned over onto absorbent paper to remove all traces of buffer.

Detection

[0084] 100 L of Streptavidin-HRP were added to each well ([Streptavidin-HRP]=8 L/mL). The plate was covered to be incubated for 30 min at ambient temperature.

[0085] The content of the wells was again aspirated and the wells washed 3 times with at least 300 L of 0.05% PBS-Tween.sub.20 wash buffer. All the liquid was carefully removed at each wash. After the last wash the plate was turned over onto absorbent paper to remove all traces of buffer. At this washing step the wells were impregnated with the wash buffer 1 to 2 min before aspiration.

[0086] The two bottles of TMB colorimetric substrate solution (3,3,5,5-tetramethylbenzidine, bioMrieux #XX7LF1UC) were volume/volume mixed. 100 L of this substrate solution were deposited in each well. The plate was covered to be incubated 30 min at ambient temperature.

[0087] Finally, plate read-off was performed by absorbance measurement at 450 nm.

Measurement of the Monocyte Expression of HLA DR

[0088] Measurement was performed by the flow cytometry technique, using direct staining with EDTA whole blood.

[0089] The two following antibodies were incubated with 50 L of whole blood (30 minutes at ambient temperature and in the dark): [0090] 5 L of fluorescein-conjugated anti-CD14 antibody (Beckman Coulter ImmunotechRef.: IM0645), allowing identification of the monocytes among the white blood cells); [0091] 10 L of phycoerythrin-conjugated anti-HLA-DR antibody (Becton DickinsonRef.: 347401), allowing quantification of HLA-DR expression on the surface of the cells.

[0092] The red blood cells were then removed: lysis by adding 1 ml of lysis solution (marketed by Becton Dickinson under reference 349202), 1:10 dilution, 15 minutes at ambient temperature and in the dark.

[0093] The cells were then analyzed on a flow cytometer (FC500Beckman Coulter).

[0094] Results are expressed as a % of positive cells, the positivity threshold being defined by isotype control (Mouse IgG2a PE Becton DickinsonRef.: 349053).

RESULTS

[0095] Plasma samples were taken from 35 patients in septic shock at days 1-2 (D1-2) and 3-4 (D3-4) after septic shock and then stored (retrospective cohort). Different parameters or markers were measured such as IL-7 and sCD127 plasma concentrations, and CD127 expression on T CD4.sup.+ cells, and HL-DR expression on the monocytes. At 28 days after admission into intensive care for septic shock, 13 patients did not survive (NS) i.e. 37% whereas 22 patients did survive (S) out of 35 patients. 6 patients contracted a nosocomial infection (NI) i.e. 17%, whereas 29 patients remained free of any nosocomial occurrence (No NI).

[0096] The same measurements were performed in 30 healthy volunteers (HV).

[0097] The results obtained were compared using a Mann Whitney U-test in these different populations of volunteers or patients, and are grouped together in appended FIGS. 1 to 4.

[0098] These results show that the IL-7 plasma concentration is significantly reduced (at D1-2), but not significantly at D3-4 in patients who develop secondary nosocomial infection compared with patients who did not suffer any nosocomial infection occurrence (FIG. 1C), whereas it remained unchanged between the surviving patients S and non-surviving patients NS.

[0099] In addition, the cell expression of the IL-7 receptor (CD127) was maintained after septic shock (FIGS. 2A and 2B), and without any difference between the surviving patients S and non-surviving patients NS or the patients who did NI or did not No NI develop nosocomial infections (results not shown).

[0100] The slight increase in the plasma concentration of the soluble form of the I-7 receptor, or sCD127, observed at the onset of septic shock is not statistically significant and returns to normal values over time (FIG. 3A).

[0101] On the other hand, whereas the plasma concentration of sCD127 remained unchanged between the surviving patients S and non-surviving patients NS (FIG. 3B), a marked increase in the plasma concentration of sCD127 was observed in NI patients who developed a nosocomial infection compared with the No NI patients who did not contact a nosocomial infection (FIG. 3C).

[0102] Also it was observed that the plasma concentration of sCD127 does not change over time (between D1-2 and D3-4) in NI patients who developed a nosocomial infection, contrary to the No NI patients who did not contract a nosocomial infection (FIG. 3C).

[0103] By way of comparison, study of the expression of the prior art marker HLA-DR on the same cohort of patients did not allow evidencing of the reduction in expression of this marker between the NI patients who suffered a nosocomial infection and the No NI who did not contract a nosocomial infection (FIG. 4), contrary to what is clearly established in the prior art.

[0104] This difference, probably related to the size of the cohort studied herein which is smaller compared with the cohorts analyzed in prior art studies, allows evidencing of the pertinence of the sCD127 marker compared with HLA-DR. Despite the size of the cohort studied herein the results show that the study of the expression of sCD127 according to the invention is informative with regard to the susceptibility of patients to nosocomial infections.

[0105] It follows that all these results show that measurement of sCD127 expression is a useful immunological marker to determine the susceptibility of a patient to nosocomial infections, and in particular patients hospitalized in intensive care and/or having sustained an insult (surgery, burns, trauma, . . . ) generating a systemic inflammatory response (SIRS), and in particular patients with sepsis, particularly severe sepsis, and preferably patients in septic shock.

[0106] The invention is not limited to the examples described and illustrated since various modifications can be made thereto without departing from the scope of the invention.