USE OF 5-METHOXYTRYPTOPHAN AS DIAGNOSTIC AGENT OF INFLAMMATORY DISEASES

Abstract

A diagnostic method for inflammatory diseases, and the diagnostic kit used in the method. The diagnostic method includes the use of a novel tryptophan metabolite, 5-methoxytryptophan (5-MTP), as a diagnostic biomarker of inflammation. Specifically, a highly specific competitive ELISA is provided to measure 5-MTP level in human serum for gauging occurrence and severity of inflammatory diseases, including sepsis and systemic lupus.

Claims

1. A diagnostic kit of inflammatory diseases, which comprises an agent for detecting the level of 5-methoxytryptophan (5-MTP) in the serum of a subject.

2. The diagnostic kit of claim 1, wherein the inflammatory disease is sepsis.

3. The diagnostic kit of claim 1, wherein the inflammatory disease is systemic lupus (SLE).

4. The diagnostic kit of claim 1, wherein the detecting agent comprises an anti-5-MTP antibody and a 5-MTP-conjugated HRP compound.

5. The diagnostic kit of claim 1, which comprises an agent for detecting the level of high sensitive-C reactive protein (hs-CRP) in the serum of a subject.

6. The diagnostic kit of claim 1, wherein the detected level of 5-MTP in an inflammatory patient is reduced to about 30-50% of that in a healthy subject.

7. The diagnostic kit of claim 2, wherein the detected level of 5-MTP in a septic patient is reduced to about 30% of that in a healthy subject.

8. The diagnostic kit of claim 3, wherein the detected level of 5-MTP in a SLE patient is reduced to about 30% of that in a healthy subject.

9. The diagnostic kit of claim 6, wherein the detected level of 5-MTP in a septic patient is reduced to about 30% of that in a healthy subject.

10. The diagnostic kit of claim 6, wherein the detected level of 5-MTP in a SLE patient is reduced to about 30% of that in a healthy subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1A shows the serum 5-MTP concentration in septic patients (n=50) and healthy donors (n=30) measured by comparative 5-MTP ELISA. FIG. 1B shows the analysis of the discriminative power 5-MTP, hs-CRP and IL-β in sepsis by ROC curve.

[0013] FIG. 2 shows that 5-MTP is depressed by LPS in a murine sepsis model. Serum 5-MTP concentration were measured by comparative 5-MTP ELISA in mice before and 24 h after LPS infusion (n=8). The solid black line denotes mean.

[0014] FIGS. 3A and 3B show that 5-MTP derived from endothelial cells inhibits LPS-induced COX-2 expression (3A) and cytokine production in macrophages (3B). FIG. 3C shows that 5-MTP neutralizing antibodies dose-dependently abrogated the suppressing effect of HUVEC-CM while a control IgG had no effect.

[0015] FIG. 4 shows the serum 5-MTP concentration in SLE patients (n=135) and healthy donors (n=30) measured by comparative 5-MTP ELISA.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The other characteristics and advantages of the present invention will be further illustrated and described in the following examples. The examples described herein are using for illustrations, not for limitations of the invention.

[0017] Development of 5-MTP Specific Competitive ELISA

[0018] It was unknown whether 5-MTP is present in the circulating blood. In this study, we developed a highly specific comparative ELISA to measure serum 5-MTP levels in human patients, healthy control and mouse. A critical step for performing the 5-MTP specific competitive ELISA is generation of 5-MTP-conjugated HRP. The procedures of 5-MTP-conjugated HRP generation are described below: (1) 0.8˜1.0 mg 5-MTP is completely dissolved in 100 μl 1M sodium bicarbonate buffer (pH=8.3) with 30% DMSO. Add this solution to a tube of NH2-Reactive Peroxidase and pipette to dissolve NH.sub.2—Reactive Peroxidase completely; (2) Pipette and incubate the tube at 37 for 1 h, and then incubated it at 4 for overnight; (3) Add 200 μl PBS to the reaction solution, and transfer the solution to a Filtration Tube; (4) Centrifuge at 8,000×g for 10 min and then discard the filtrate, add 200 μl PBS to the tube; (5) Centrifuge at 8,000×g for 10 min and discard the filtrate. Add 200 μl PBS and centrifuge again; (6) Add 400 μl Storage Buffer (PBS with 30% DMSO), and pipette about 10 times to dissolve the conjugate; (7) Transfer the solution to a microtube, and store at −20 or 80° C.

[0019] Serum 5-MTP is measured by a modified competitive ELISA in a 96-well microtiter plate as described below: (1) 96-well microtiter plate is coated with polyclonal rabbit anti-5-MTP antibodies in a coating buffer, 0.05 M carbonate-bicarbonate (pH 9.6) at 4° C. overnight; (2) After PBST washing and treatment with blocking buffer, a mixture of 5-MTP-conjugated HRP and 5-MTP standards or serum samples is added to the wells and incubated at 4° C. overnight; (3) The wells are washed with PBST, and treated with a substrate tetramethylbeuzidine at room temperature for 20˜30 minutes; (4) After incubation, 0.1 N H.sub.2SO.sub.4 stop solution is added to each well; and (5) Within 30 min, the product is analyzed at 450 nm. The calibration curve is established by using pure 5-MTP at concentrations of 0.1-500 μM.

[0020] Determinant of Serum 5-MTP Level in Sepsis Patients by Competitive ELISA

[0021] To determine the clinical relevance of 5-MTP, we measured serum 5-MTP in patients with clinical evidence of sepsis and normal healthy controls. Serum 5-MTP was measured by a modified competitive ELISA in a 96-well microtiter plate coated with polyclonal rabbit anti-5-MTP antibodies (Abcam) using a coated buffer (0.05 M carbonate-bicarbonate, pH 9.6) at 4° C. overnight. After PBST washing and treatment with blocking buffer, mixture of 5-MTP-conjugated HRP generated by using an NH.sub.2 peroxidase labeling kit (Abnova) and 5-MTP standards or serum samples were added to the wells and incubated at 4° C. overnight. The wells were washed and treated with a substrate tetramethylbeuzidine at room temperature for 20˜30 minutes. After then, 0.1 N H.sub.2SO.sub.4 stop solution was added to each well. Within 30 min, the product was analyzed at 450 nm. The calibration curve was established by using pure 5-MTP at concentrations of 0.1-500 μM.

[0022] Fifty sepsis patients (twenty-nine male and 21 female with a mean age of 64.0±19.6 years old) were enrolled. Average normal serum 5-MTP level from 30 healthy controls was 1.05 μM (Table1). 5-MTP concentration in sepsis patients was markedly reduced with a mean value of 0.37 μM (FIG. 1A and Table 1). Patients with sepsis had a significant increase in high sensitive-C reactive protein (hs-CRP) (98.2±74.3 mg/L versus 0.5±0.5 mg/L, P<0.001) and IL-1β (1327±2445 pg/ml versus 526±175 pg/ml, P=0.025) level compared with those of healthy subjects (Table I).

TABLE-US-00001 TABLE I Serum level of 5-MTP, hs-CRP and IL-1β in septic patients and healthy donors Septic patient Healthy donors n = 50 n = 30 P value 5-MTP (μM) 0.37 ± 0.15 1.05 ± 0.39 <0.0001 hs-CRP (mg/L) 98.2 ± 74.3 0.5 ± 0.5 <0.001 IL-1β (pg/ml) 1327 ± 2445 526 ± 175 = 0.025

[0023] As serum hs-CRP and IL-1β levels were reported to be a diagnostic marker of sepsis severity, we used area under the receiver operating characteristic curve (AUROC) to determine the discriminative power of 5-MTP, hs-CRP and IL-1β in this group of patients (FIG. 1B and Table II).

TABLE-US-00002 TABLE II Sensitivity and specificity of 5-MTP, hsCRP and IL-1β at optimum diagnostic cut-off values for sepsis Cut-off Sensitivity (%) Specificity (%) 5-MTP (μM) 0.63 83 94 hs-CRP (mg/L) 2.73 100 93.9 IL-1β (pg/ml) 467.29 53.3 78

[0024] The AUCROC value of 5-MTP (0.958, 95% CI 0.919-0.997) was comparable with the value of hs-CRP (0.995, 95% CI 0.987-1.000) and significantly greater than that of IL-1β (0.696, 95% CI 0.581-0.810). Cutoff level of hs-CRP and IL-1β with the optimum diagnostic efficiency derived from the AUROC curves were 2.73 mg/L (sensitivity 100%, specificity 93.9%) and 467.29 pg/ml (sensitivity 53.3%, specificity 78%), respectively. Using serum 5-MTP level of 0.63 μM as a cutoff, the sensitivity was 83% and the specificity was 94% (FIG. 1B). These results suggest that 5-MTP is a predictive biomarker of sepsis severity. More importantly, serum 5-MTP in septic patients was inversely correlated with inflammatory markers such as hs-CRP and IL-1β. In addition, serum 5-MTP has a high discrimination power to predict severity of sepsis. Our results suggest that concurrent measurement of hs-CRP and 5-MTP will provide a better prediction of sepsis severity than measurement of hs-CRP or IL-1β alone.

[0025] 5-MTP is Depressed by LPS in a Murine Sepsis Model

[0026] We suspected that reduction of serum 5-MTP in sepsis patients is due to endotoxemia. To provide evidence for this issue, we analyzed serum 5-MTP in a murine sepsis model, in which mice were infused with LPS. C57BL/6 mice (6-8 wks old) were treated intraperitoneally with saline or with different concentrations of 5-MTP (23.4 or 100 mg/kg) for 30 min before intraperitoneal administration of LPS (60 mg/kg). Animals were monitored for survival and other clinical signs including ruffled fur, lethargy, diarrhea, and body weight loss. Some animals were sacrificed at different times after LPS injection. Blood samples, peritoneal exudates, lungs, and spleens were collected. All mouse experiments were approved by the Institutional Animal Care and Use Committee, National Health Research Institutes.

[0027] Average serum 5-MTP level in mice prior to LPS treatment was 0.187 μM which is about ⅙ of the mean value in humans. Serum 5-MTP was significantly reduced to 0.036 μM after LPS infusion (FIG. 2). Serum 5-MTP became undetectable or dramatically decreased after LPS infusion in 6 of the eight mice, depressed in 1 and unchanged in the remaining one (FIG. 2). These results suggest that endotoxemia inhibits 5-MTP which contributes to clinical severity.

[0028] 5-MTP Derived from Endothelial Cells Inhibits LPS-Induced COX-2 Expression and Cytokine Production in Macrophages

[0029] Endothelial cells are considered to play a key role in controlling progression of inflammatory tissue damage in sepsis (Deanfield et al., 2007). We hypothesize that endothelial cells produce soluble factors such as 5-MTP to modulate inflammatory responses. To test this hypothesis, we cultured mouse macrophage RAW264.7 cells with conditioned medium (CM) from HUVECs in the presence or absence of LPS for 24 hours and COX-2 expression and IL-6 production were measured. COX-2 expression and IL-6 production induced by LPS in RAW264.7 cells were attenuated by addition of HUVEC CM (FIG. 3A, B).

[0030] As 5-MTP has been identified to be a fibroblast-releasing factor which plays an important role in suppressing proinflammatory mediator induced COX-2 expression (Cheng et al., 2012), we determined whether 5-MTP is a soluble factor in the HUVEC-CM responsible for suppression of LPS-induced COX-2 expression and cytokine production by using 5-MTP neutralizing antibodies. 5-MTP neutralizing antibodies dose-dependently abrogated the suppressing effect of HUVEC-CM while a control IgG had no effect (FIG. 3C).

[0031] SLE Patients have a Lower Level of 5-MTP

[0032] It was unknown whether serum 5-MTP level is associated with SLE pathology. In this study, we also measured serum 5-MTP levels in SLE patients by a highly specific ELISA as described above. 135 patients were enrolled. It is of interests to observe that serum 5-MTP concentration in SLE patients was markedly reduced with a mean value about 0.45 μM (FIG. 4), suggesting that reduction of 5-MTP may compromise the defense against excessive inflammatory responses in SLE.

[0033] In summary, it is observed that serum 5-MTP concentrations in septic patients and SLE patients were significantly reduced to about 30% of that of healthy subjects. More importantly, serum 5-MTP in septic patients was inversely correlated with inflammatory markers such as hs-CRP and IL-1β. In addition, serum 5-MTP has a high discrimination power to predict severity of sepsis. Our results suggest that concurrent measurement of hs-CRP and 5-MTP will provide a better prediction of sepsis severity than measurement of hs-CRP or IL-1β alone. In addition, we also claim that 5-MTP is a novel diagnostic biomarker for predicting sepsis, SLE and inflammatory diseases. Further, the highly specific 5-MTP competitive ELISA acts as a diagnostic kit for gauging occurrence and severity of inflammatory diseases.