Supplementary Information

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Supplementary Information

Early Prediction of Persistent Organ Failure by Soluble CD73 in Patients with Acute Pancreatitis

Maksimow et al.

Supplementary Methods

CD73 ELISA

New mAbs against immunoaffinity-purified human CD73 molecule were generated by immunizing CD73 deficient mice (the mice (1) originally kindly provided by L. Thompson).

The hybridoma generation was performed by fusing the draining lymph node lymphocytes with SP2.0 myeloma cells. The hybridoma supernatants were screened using immunohistochemical stainings of human tissues, immunofluorescence stainings of transfectants expressing CD73 and by immunoblotting, using previously described methodology (2, 3), and the positive hybridomas were subcloned by limiting dilution.

One of the new mabs (clone 118, mouse IgG1) was used together with an old anti- CD73 mAb (clone 4G4 (Ref. 2)) for development of sandwich ELISA using similar approaches as we have previously described for another soluble adhesion molecule sVAP-1 (4). In brief, 4G4 mAb was bound to the bottom of white 96-well Cliniplates, the serum samples were added (1:10 dilution in PBS), and the bound sCD73 was detected with biotinylated-118. Clone 118 was biotinylated with NHS-biotin (Pierce) according to the manufacturer’s instructions. The ELISA reaction was developed using streptavidin- horseradish peroxidase and BM Chemiluminescence ELISA Substrate (Roche).

To validate the ELISA, serial dilutions of recombinant human CD73 (RD Systems) and CD73-depleted sera were used. CD73 was removed from serum by three

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sequential incubations of normal pooled sera with CnBr-activated sepharose beads armed with an anti-CD73 mAb 4G4.

qPCR for CD73 mRNA 

Leukocyte mRNA was isolated from the blood samples collected into PAXgene tubes using PAXgene Blood RNA kit (PreAnalytix) according to the manufacturer’s instructions. mRNA was reverse transcribed into cDNA using iScript^TM cDNA Synthesis kit (BioRad). The CD73 mRNA levels were determined using a TaqMan Gene Expression Assay (Applied Biosystems) for CD73 (NT5E, assay Hs00159686_m1) as the primer/probe set. The PCR reactions were carried out as suggested by the supplier using the Applied Biosystems 7900HT Fast Real-Time PCR System in the Finnish DNA Microarray Center, Turku Center of Biotechnology, Turku, Finland. All samples were run as triplicates and the expression values were normalized using a house-keeping gene (GAPDH) as the endogenous control.

The results were analyzed with the SDS 2.3 software. Changes in cycle threshold levels (ΔCT) were calculated by subtracting the average of GAPDH CT values from the average of target gene CT values. Relative expressionof the gene analyzed was estimated as described (5) using the formula: relative expression = 2^-ΔCT. The quantity of CD73 mRNA was expressed as percentage of GAPDH mRNA after multiplying the relative target gene expression by a factor of 100.

Determination of CRP and creatinine levels

The reagents for immunoturbidimetric measurement of the serum CRP concentration were purchased from Orion Diagnostica (Espoo, Finland). The detection limit was 2 mg/l. The serum creatinine levels were determined in the hospital laboratory.

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Supplementary References

1. Thompson LF, Eltzschig HK, Ibla JC, et al. Crucial role for ecto-5'-nucleotidase (CD73) in vascular leakage during hypoxia. J Exp Med 2004;200:1395-1405.

2. Airas L, Hellman J, Salmi M, et al. CD73 is involved in lymphocyte binding to the endothelium: characterization of lymphocyte-vascular adhesion protein 2 identifies it as CD73. J Exp Med 1995;182:1603-1608.

3. Mikhailov A, Sokolovskaya A, Yegutkin GG, et al. CD73 participates in cellular multiresistance program and protects against TRAIL-induced apoptosis. J Immunol 2008;181:464-475.

4. Kurkijärvi R, Adams DH, Leino R, et al. Circulating form of human vascular adhesion protein-1 (VAP-1): increased serum levels in inflammatory liver diseases. J Immunol 1998;161:1549-1557.

5. Junttila TT, Laato M, Vahlberg T, et al. Identification of patients with transitional cell carcinoma of the bladder overexpressing ErbB2, ErbB3, or specific ErbB4 isoforms:

real-time reverse transcription-PCR analysis in estimation of ErbB receptor status from cancer patients. Clin Cancer Res 2003;9:5346-5357.

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Supplementary Figure 1

Supplementary Figure 1. Development of sCD73 ELISA. (A) FACS analyses of Jurkat cells (Control) and Jurkat cells transfected with CD73 (CD73 transf) stained with a negative control (grey histograms) and anti-CD73 mAbs 4G4 and 118 (open histograms). (B) Immunohistochemical stainings of human tonsil with a negative control (3G6) and anti-CD73 mAbs 4G4 and 118. Brown reactivity in the vessels indicates positivity. (C) The linearity of the CD73 ELISA assay with purified CD73. The relative light units (RLU) obtained at the indicated concentrations of purified CD73 (mean ± sem) are shown (n = 5). (D) Normal values for sCD73 protein concentration and activity in sera of 20 healthy volunteers (mean ± sd). (E) Plotting of sCD73 protein concentrations (x-axis) and activity (y-axis) against each other in the healthy persons.

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Supplementary Figure 2  

Supplementary Figure 2. (A) ROC curves for sCD73 activity, CRP and creatinine to predict the development of severe form of AP among the male patients who presented with moderately severe or severe AP. (B) ROC curves for sCD73 activity, CRP and creatinine to predict the development of severe form of AP among the male patients who presented with Modified Marshall score (MMS) < 2 on admission.

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Supplementary Figure 3

Supplementary Figure 3. (A) ROC curves for sCD73 activity, sCD73 protein and CD73 mRNA (n=158) to predict the development of severe AP in the whole AP patient cohort. (B) ROC curves for sCD73 activity, CRP and creatinine to predict the development of severe AP in the whole AP patient cohort.

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Supplementary Table 1 sCD73 Levels in Male AP Patients

    sCD73 Activity

(nmol/ml/h) 

sCD73 ELISA (ng/ml) 

CD73 mRNA

(Relative expression)  

Severity of AP Mild (n=74)

Moderately severe (n=20) Severe (n=24) 

339.1 (110-2127) 233.7 (88.5-1187) 157.2 (80.2-1417) p ^a< 0.001 

48.6 (12.4-414) 40.4 (9,6-256) 45.3 (9.8-293) p ^a= 0.076 

0.23 (0.01-1.90) 0.12 (0.01-0.43) 0.09 (0.01-1.53) p ^a= 0.001  Etiology of AP

Alcohol (n=95) Biliary (n=13) Other (n=10) 

250.6 (80.2-2294) 787.8 (130-2562) 342.5 (124-957) p ^b < 0.001 

43.4 (9.6-293) 127.0 (29.6-918) 47.8 (23.7-299) p^b < 0.001 

0.16 (0.01-1.90) 0.20 (0.01-0.49) 0.44 (0.01-1.56) p ^b= 0.215 

Median (range) are shown

a Jonkheere-Terpstra test

b Kruskall-Wallis test  

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Supplementary Table 2 Performance of Laboratory Markers and Modified Marshall Scoring System to Predict Severe AP in Male AP Patients

Parameter/Score  Cut-off  Sensitivity  Specificity  AUC 

Moderately severe and severe AP patients (male)

sCD73 activity(nmol/ml/h) CRP (mg/l)

Creatinine (µmol/l) sCD73+CRP sCD73+Creatinine 

≤ 126

≥ 224

≥ 164

≤126, ≥224

≤126, ≥164 

0.33 0.417 0.417 0.542 0.625 

0.900 0.650 0.850 0.650 0.750 

0.658 (0.495-0.822) 0.523 (0.345-0.701) 0.629 (0.464-0.794) NA

NA  AP patients (male) with Modified

Marshall score < 2 on admission

≤ 126

≥ 224

≥ 164

≤126, ≥224

≤126, ≥164 

0.250 0.083 0.083 0.333 0.333 

0.967 0.944 1.00 0.922 0.967 

0.754 (0.595-0.913) 0.520 (0.348-0.692) 0.526 (0.357-0.695) NA

NA 

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Supplementaray Table 3 Performance of Laboratory Markers to Predict Severe AP in the Whole AP Patient Cohort

Parameter/Score  Cut-off  Sensitivity  Specificity  AUC  All AP patients

≤138

≥227

≥164

≤138, ≥227

≤138, ≥164 

0.40 0.40 0.40 0.60 0.60 

0.933 0.933 0.971 0.90 0.90 

0.789 (0.689-0.888) 0.694 (0.569-0.820) 0.748 (0.636-0.959) NA

NA   