Cellular and humoral immune response in progressive multifocal ...

Cellular and Humoral Immune Response in Progressive Multifocal Leukoencephalopathy Frank Weber, MD,1 Claudia Goldmann,2 Marcus Kra¨mer,2 Franz Josef Kaup, MD,3 Marcus Pickhardt, PhD,4 Peter Young, MD,5 Harald Petry, PhD,2 Thomas Weber, MD,4 and Wolfgang Lu¨ke, PhD2

Progressive multifocal leukoencephalopathy (PML) is a fatal, demyelinating disease caused by JC virus (JCV) in patients with severe immunosuppression. We studied the JCV-specific cellular and humoral immune response in 7 healthy donors (HD), 6 human immunodeficiency virus-1 (HIV-1)-infected patients without PML (HIV), 4 HIV-1-negative patients with PML (PML), and 8 HIV-1-positive patients with PML (HIV/PML). As antigens, recombinant virus-like particles of the major structural protein VP1 (VP1-VLP) of JCV, tetanus toxoid (TT), or the mitogen phytohemagglutinin (PHA) were used. Proliferation of peripheral blood mononuclear cells (PBMC) after stimulation with the VP1-VLP was significantly suppressed in PML and HIV/PML patients compared to HD. After antigen stimulation the production of interferon-␥ (IFN-␥) was reduced in PML, in HIV/PML, and in HIV patients. The production of interleukin-10 (IL-10), however, was elevated in HIV/PML patients. Neither proliferation nor cytokine production correlated with the presence of JCV DNA in PBMC. The immunoglobulin G serum antibody titer to the VP1-VLP was slightly elevated in HIV, elevated in PML, and highly elevated in HIV/PML patients compared to HD. The development of PML appears to coincide with a general impairment of the Th1-type T-helper cell function of cell-mediated immunity. Ann Neurol 2001;49:636 – 642

Progressive multifocal leukoencephalopathy (PML) was first described in 1958.1 PML is a demyelinating disease of immunocompromised patients caused by the human polyomavirus JC virus (JCV). Once PML was a rare disorder, but the incidence has risen dramatically as a result of the worldwide AIDS pandemic. The acquired immune deficiency syndrome (AIDS) is the underlying immunosuppression in about 85% of PML patients and about 4 – 6% of all patients with AIDS may develop PML.2,3 The disease is also observed in patients with neoplastic diseases and in immunocompromised patients after transplantation or treatment with immunosuppressants.3,4 Very rarely, PML is seen in patients without any signs of immunodeficiency.5 PML results from a lytic infection of oligodendrocytes by JCV, which causes death of oligodendrocytes and demyelination. Clinical symptoms of PML are nonspecific and include motor weakness, cognitive changes, visual deficits, and cranial nerve palsies.6 By magnetic resonance imaging (MRI), multifocal demyelinating lesions are most often found at the gray matter–white matter junction. Detection of JCV DNA in the cerebrospinal fluid (CSF)7 or of an intrathecal JCV-specific antibody synthesis8 is used to confirm diagnosis of PML. From the 1Department of Neurology, University of Go¨ttingen; Departments of 2Virology and Immunology and 3Experimental Pathology, German Primate Center, Go¨ttingen; 4Department of Neurology, Marienkrankenhaus, Hamburg; and 5Department of Neurology, University of Mu¨nster, Germany. Received Dec 15, 1999, and in revised form Aug 11. Accepted for publication Nov 27, 2000.

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© 2001 Wiley-Liss, Inc.

Whereas many studies address the molecular biology of JCV, studies on the cellular and humoral immune response are rare.9,10 This is mainly due to the limited availability of viral antigen based on the difficulties of propagating JCV in cell culture. We describe a systematic analysis of the humoral and cellular immune response to JCV. To obtain sufficient amounts of JCVrelated antigen, we expressed the major structural protein VP1 of JCV as virus-like particles (VP1VLP).11 These VP1-VLP were used to investigate the proliferation of peripheral blood mononuclear cells (PBMC) as well as the cytokine production in seven healthy donors (HD), six HIV-1-infected patients without PML (HIV), eight HIV-1-infected patients with PML (HIV/PML), and four HIV-1-negative patients with PML (PML). In addition, JCV-specific serum immunoglobulin G antibodies were assessed using VP1-VLP as antigen.8,12 Patients and Methods Patients and Controls Up to 40 ml EDTA blood was obtained from 7 HD, 6 HIV, 4 PML, and 8 HIV/PML patients after informed consent was obtained. There was no statistically significant difference

Published online 2 April 2001. Address correspondence to Dr Weber, Section of Neurology, MPI of Psychiatry, Kraepelinstrasse 2–10, 80804 Munich, Germany. Email: [email protected]

with respect to CD4⫹/CD8⫹ ratio and HIV-1 virus load between HIV and HIV/PML patients (Table 1). Among the 4 PML patients, 2 suffered from chronic lymphatic leukemia and 1 from immunosuppression after organ transplantation. In the fourth patient, no underlying immunosuppressive illness could be detected. PML was clinically suspected based on the occurrence of neurological deficits and typical lesions on magnetic resonance imaging (MRI).3,13 Diagnosis was proved by the detection of JCV DNA in cerebrospinal fluid7 or by the demonstration of a JCV-specific intrathecal antibody response.8,12 In 2 HIV-1-negative PML patients, the diagnosis was proved by brain biopsy. PBMC of HD were included in the study, to investigate the proliferative capacity and the cytokine production in healthy volunteers. This study was approved by the ethics committee of the GeorgAugust-University Ga¨ttingen and the institutional review board of the A¨rztekammer, Hamburg, Germany.

Template Preparation From patients who donated 40 ml EDTA blood or more, sufficient amounts of PMBC could be isolated to analyze the presence of JCV DNA in PBMC by polymerase chain reaction (PCR). These were 4 HIV, 3 PML, and 5 HIV/PML patients. In addition, 3 HD with and 3 HD without JCVspecific antibodies were investigated. DNA was isolated from 106 PBMC. Cells were lysed by 1% sodium dodecyl sulfate (SDS) and incubated in the presence of proteinase K (300 ␮g/ml) overnight at 56°C. Thereafter, an equal volume of buffer-saturated phenol was added and mixed, and DNA was extracted by phenol-chloroform extraction. Finally, the DNA was precipitated by the addition of 0.1 volumes of a saturated NaCl solution followed by an equal volume of isopro-

panol for 1 hr at –20°C. DNA was pelleted at 20,000g for 15 minutes; DNA pellets were washed repeatedly with 70% ethanol, dried, and finally suspended in 100 ␮l H2O.

PCR Amplification Nested PCR was carried out using 1 ␮g of purified DNA as previously described,7 with some modifications. The first round of PCR consisted of 10 cycles with the primer pair JC4284/JC4961 flanking a 656-base-pair fragment of the large T gene. The sequence of primer JC4284 in 5⬘ to 3⬘ orientation was from position 4,284 to position 4,527 TTT TTA GGT GGG GTA GAG TGT TG and primer JC4961 from position 4,982 to 4,961 TGG AGC TTA TGG ATT TAT TAG G. The second round of PCR was carried out with 4 ␮l of the first round for 30 cycles using the primer pair JC36/JC39 spanning a 389-base-pair fragment located in the large T gene region.6 In 5⬘ to 3⬘ orientation the primer JC36 has the sequence AGA TCC CTG TAG GGG GGT GTC TCC from position 4,505 to position 4,527 and primer JC 39 from position 4,894 to position 4,874 the sequence AAT GCA AAG AAC TCC ACC CTG ATA AAG GTG G. Each amplification round was initiated at 95°C for 2 minutes 30 seconds. Cycles of first-round PCR consisted of 95°C for 30 seconds, 55°C for 1 minute, and 72°C for 1 minute. Second-round PCR consisted of 95°C for 30 seconds, 60°C for 1 minute, and 72°C for 1 minute. Both rounds were concluded with an extension cycle of 72°C for 5 minutes. By this PCR, at least 10 copies of target JCV DNA molecules were detected in a background of a JCV-negative cell extract from 106 cells of a permanent human T cell line, which was spiked with 106 to 0 JCV DNA molecules.

Table 1. Characteristics of HIV-1-Infected Patients Patients

CD4 (Cells/␮l)

CD4/CD8 (Ratio)

HIV RO HW LK

660 220 160

0.50 0.60 0.32

5,500 1,600 0

KR

300

nd

0

22 204

0.02 0.38

560,000 24,000

443 373 346

0.50 0.40 0.14

158,000 nd 14,500

BT

60

0.19

600

HA

250

0.24

nd

UM BK

nd nd

nd nd

nd nd

OR

77

0.07

3,700

MM GE HIV/PML MF VP WM

HIV-1 Virus Load (RNA Copies/ml)

Antiretroviral Therapy None Azidothymidin, lamivudin Azidothymidin, lamivudin, saquinavir Azidothymidin, lamivudin, saquinavir None None None IL-2, lamivudin, stavudin Dideoxyinosin, saquinavir, stavudin Azidothymidin, lamivudin, saquinavir Azidothymidin, indinavir, lamivudin Fivefold combination Azidothymidin, indinavir, lamivudin Fivefold combination

HIV ⫽ HIV-1-infected patients without PML; HIV/PML ⫽ HIV-1-infected PML patients. There was no difference between HIV-1-infected patients with or without PML with respect to CD4/CD8 ratio and HIV-1 virus load ( p ⫽ 0.428 and p ⫽ 0.699, respectively).

Weber et al: Immune Response in PML

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Analysis of PCR Products Second-round PCR products were purified by a PCR purification kit according to the manufacturers procedure (Qiagen, Hilden, Germany). For proving JCV specificity, the purified DNA was digested with the restriction endonuclease Rsa I according to the instructions of the manufacturer (Amersham Pharmacia Biotech, Freiburg, Germany) and separated on a 2% agarose gel. In case of JCV specificity, two fragments of about 100 and 300 base pairs will arise after restriction enzyme digestion.

Antigens The major structural protein VP1 of JCV was cloned and expressed in insect cells as previously described.11 Recombinant VP1 spontaneously assembled to virus-like particles resembling empty JCV capsids.11 Tetanus toxoid (TT) was a kind gift of Dr Blackkolb (Behringwerke, Marburg, Germany), and phytohemagglutinin (PHA) was purchased from Sigma (Deisenhofen, Germany).

Preparation of PBMC and Proliferation Assay Peripheral venous blood was separated within 24 hr by Ficoll-Hypaque gradient centrifugation. PBMC were washed twice with phosphate-buffered saline (PBS) and seeded into round-bottomed 96 well culture plates (Nunc, Roskilde, Denmark) at 2 ⫻ 105 cells per well in a final volume of 100 ␮l CG medium (Vitromax, Selters, Germany) supplemented with 2 mM L-glutamine, 103 U/ml penicillin and 103 U/ml streptomycin (Gibco, Eggenstein, Germany). VP1-VLP were added at a final concentration of 10 ␮g/ml. Parallel sets of wells containing TT (10 ␮g/ml), PHA (5 ␮g/ml), or medium alone were used as controls. Cells were maintained at 37°C in a humidified atmosphere (5% CO2). Four days after antigen stimulation, PBMC were labeled with 0.1 ␮Ci [3H]thymidine (Amersham, Braunschweig, Germany) and harvested 18 hr later. [3H]thymidine incorporation was measured with a scintillation counter (Tri-Carb 4000 Series; Packard, Frankfurt, Germany). All determinations were set up at least in triplicate. The stimulation index (SI) was calculated as follows: counts per minute (cpm) of antigen-stimulated culture/cpm of culture with medium.

Determination of Cytokines PBMC cultures were set up as described above for the proliferation assay. After 2, 3, and 4 days the content of 3 wells was pooled and pelleted (2,200g, 5minutes). The supernatant was stored immediately at – 80°C. The Th1-associated cytokine interferon-␥ (IFN-␥) and the Th2-associated cytokine interleukin-10 (IL-10) were determined by a sandwich enzyme-linked immunosorbent assay (ELISA). Antibodies and standard were purchased from Biosource (Ratingen, Germany). The ELISA was performed according to the instructions of the manufacturer, with minor modifications. All samples were determined in duplicate. The detection limits of IFN-␥ and IL-10 were determined at 3.4 pg/ml and 2.4 pg/ml, respectively.

VP1-Specific Antibody ELISA The titer of VP1-specific immunoglobulin G antibodies in sera of patients and HD was determined as described previ-

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ously.8 In brief, ELISA plates (Nunc) were coated with 100 ␮l VP1-VLP (1 ␮g/ml) and incubated with serial dilutions of sera. Human IgG was detected by a POD-conjugated goat antihuman IgG (Dianova, Hamburg, Germany). Results were expressed as arbitrary units, which were standardized using a human serum pool.

Statistical Analysis The Mann-Whitney test was used to calculate significance of results. A p value ⬍ 0.05 was regarded as significant.

Results Optimization of PBMC Proliferation In pilot experiments, the dose response of PBMC to increasing amounts of VP1-VLP was investigated in 4 members of the HD group. In the presence of 0.1–10 ␮g/ml VP1-VLP, a dose-dependent proliferation was demonstrated in all HD. A representative result is shown in Figure 1. A similar dose response was obtained with TT as the stimulating antigen (data not shown). In the following experiments, VP1-VLP and TT were used at a concentration of 10 ␮g/ml. Further analysis showed that not only CD4⫹ but also CD8⫹ cells were able to proliferate in response to the VP1VLP (data not shown). Proliferation of PBMC Obtained from HD and HIV, PML, or HIV/PML Patients Figure 2A shows that proliferation after stimulation with the VP1-VLP was highest in the HD group. Compared to the case in the HD group, the proliferation was significantly suppressed in both the PML ( p ⫽ 0.004; Fig 2A) and the HIV/PML groups ( p ⫽ 0.0002; Fig 2A). In the HIV/PML group, the SI values were lower than in the HIV group. However, this difference was not statistically significant ( p ⫽ 0.21; Fig Fig 1. The proliferation of peripheral blood mononuclear cells against VP1-VLP is dose-dependent. A representative result of a healthy donor (HD) is shown. Values represent mean ⫾ SD. All assays were performed in quadruplicate.

PHA, the PBMC proliferation in the HD group was higher than in all other groups (Fig 2C), but differences between the groups were not statistically significant (PML and HD: p ⫽ 0.57; HIV and HIV/PML: p ⫽ 0.6). Cytokine Production of PBMC from HD and HIV, PML, or HIV/PML Patients IFN-␥ secretion after stimulation with VP1-VLP, TT, and PHA was highest in the HD group (Fig 3A–C). A marginal IFN-␥ production was found in all other groups after stimulation with VP1-VLP or TT (Fig 3A,B). After stimulation with PHA, the IFN-␥ production was much lower in the HIV, PML, and HIV/ PML groups compared to the HD group (Fig 3C). Secretion of IL-10 by PBMC obtained from HIV/PML patients in response to VP1-VLP or TT was clearly elevated compared to all other groups (Fig 3D,E). After stimulation with PHA, however, there was a slight difference in favor of the HD and PML groups in comparison to both HIV-1-infected groups (Fig 3F). Fig 3. Peripheral blood mononuclear cells (PBMC) of healthy donors (HD), progressive multifocal leukoencephalopathy (PML) patients without HIV-1 infection (PML), HIV-1infected patients without clinical signs of PML (HIV), and HIV-1-infected PML patients (HIV/PML) were stimulated with VP1-VLP (10 ␮g/ml; A,D), TT (10 ␮g/ml; B,E), and PHA (5 ␮g/ml; C,F). Production of IFN-␥ and IL-10 was determined by ELISA on days 2, 3, and 4 after stimulation. Values are given as mean over 3 days for each patient. In addition, the median of each group is indicated.

Fig 2. Proliferation of peripheral blood mononuclear cells (PBMC) of healthy donors (HD), progressive multifocal leukoencephalopathy (PML) patients without HIV-1 infection (PML), HIV-1-infected patients without clinical signs of PML (HIV), and HIV-1-infected PML patients (HIV/PML) in the presence of VP1-VLP (10 ␮g/ml; A), TT (10 ␮g/ml; B), and PHA (5 ␮g/ml; C) is shown. The difference in proliferation to VP1-VLP (A) and TT (B) between HD and PML is statistically significant ( p ⫽ 0.004 and p ⫽ 0.012, respectively). The difference in proliferation between HIV and HIV/PML is statistically not significant ( p ⫽ 0.21 and p ⫽ 0.19, respectively). Proliferation was determined by [3H]thymidine incorporation. Values are given as stimulation index and median.

2A). Stimulation of PBMC with TT yielded similar results. The HD group showed the highest proliferation (Fig 2B). Whereas the response to TT in the PML group was significantly lower than in the HD group ( p ⫽ 0.012), the difference between the HIV group and the HIV/PML group was small and not statistically significant ( p ⫽ 0.19). After stimulation with


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JCV DNA Detection in PBMC and JCV-Specific Humoral Immune Response PBMC JCV DNA was detected in 50% of HD, in 75% of HIV patients, in 66% of PML patients, and in 60% of HIV/PML patients (Table 2). All PCR products were demonstrated to be JCV-specific by restriction endonuclease digestion (data not shown). In the HD group, the presence of JCV DNA in PBMC correlated with the occurrence of JCV-specific antibodies. No correlation, however, was found in any other group (Table 2). The highest VP1-specific antibody titers were found for HIV/PML patients, who showed a very low proliferation in response to VP1-VLP. No correlation of the SI values after PBMC stimulation with VP1-VLP, TT, or PHA and the presence of JCV DNA in the PBMC was found in any of the groups. The high VP1-specific antibody titer in the HIV/PML group is in agreement with the IL-10 production found in this group. However, there was no significant difference between the antibody titers of the PML and the HD groups or between the HIV and the HIV/ PML groups ( p ⫽ 0.11 and p ⫽ 0.06, respectively).

response to purified natural JCV particles in a PML patient compared to HD.16 This observation is in line with the findings reported here for VP1-VLP, which appeared to be a very useful antigen for the assessment of the JCV-specific cellular and humoral immune response. The present study demonstrates that a pronounced JCV-specific proliferative response in HD is obviously one of the important mechanisms to control JCV. A significantly lower JCV-specific proliferation of PBMC compared to HD was found in PML and HIV/PML patients. Furthermore, the proliferative response of PBMC to TT and PHA was also impaired in PML patients compared to HD. Our observation of a reduced lymphocyte proliferation in response to PHA stimulation is in line with earlier reports10,16 and the previous description of the absence of a delayed-type hypersensitivity to various antigens in PML patients.10,15 In parallel with the suppressed proliferation, the production of the Th1-type cytokine IFN-␥, which is associated with an antiviral cellular immune response, was strongly reduced in PML, HIV/PML, and HIV patients after stimulation with VP1-VLP, TT, and PHA. In contrast, the production of the Th2-type cytokine IL-10, which is associated with an antiviral humoral immune response, was elevated in response to VP1-VLP and TT in HIV/PML. Neither the proliferative capacity nor the cytokine production was correlated to the JCV DNA detected in the PBMC. The frequency of JCV DNA was in line with several studies.17 The relatively high percentage of JCV DNA detected in the PBMC of patients in the HIV group was similarly correlated with the low CD4⫹ T cell count, as previously reported.18 The elevated titer of VP1-specific IgG antibodies in PML and HIV/PML patients compared to HD and HIV patients provides further evidence for an antiviral humoral immune response. These findings support the

Discussion Investigations of the immunopathogenesis of PML are limited to humans, because no animal model for PML is known.14 Only a few studies have addressed the cellular and humoral immune response to JCV in PML.9,10,15 We analyzed the JCV-specific cellular and humoral immune response in HIV-1-infected patients without or with PML and in PML patients without HIV-1 infection. HD were included in the study to analyze the normal situation at baseline. The present investigation became feasible with the availability of sufficient amounts of the recombinant major structural protein VP1 of JCV.11 The recombinant VP1 formed VP1-VLP, which exhibits the same structural and functional properties as natural JCV capsids.11 In an earlier study, we demonstrated a highly reduced proliferative

Table 2. JCV-Specific Humoral Immune Response and Detection of JCV DNA in PBMC by PCR HD

Median:

PML

HIV

HIV/PML

Donor

Titers

PCR

Donor

Titers

PCR

Donor

Titers

PCR

Donor

WL MK SZ SP EM CG KL

4,800 1,664 0 0 4,500 0 17,500

nd ⫹ ⫺ ⫺ ⫹ ⫺ ⫹

ED GH PW

0 60,000 3,500

⫹ ⫺ ⫹

RO HW LK KR MM GE

6,800 0 44,000 43,200 0 0

⫹ ⫺ nd ⫹ nd ⫹

MF VP WM BT HA UM BK 0

OR

1,664

33,550

3,400

Titers 8,400 316,000 7,584,000 353,600 2,480 87,000 150,000 118,700

PCR nd nd ⫺ ⫹ ⫺ ⫹ nd ⫹

HD ⫽ healthy donors; PML ⫽ PML patients without HIV-1 infection; HIV ⫽ HIV-1-infected patients without PML; HIV/PML ⫽ HIV1-infected PML patients; nd ⫽ not done; PCR ⫽ polymerase chain reaction; ⫹ ⫽ ⬎10 JCV molecules; ⫺ ⫽ ⬍10 JCV molecules; PBMC ⫽ peripheral blood mononuclear cells. Antibody titers were expressed as arbitrary units.

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hypothesis of an impaired cellular immune response leading to the reactivation of JCV and the development of PML. Although Willoughby et al.10 described a specific deficiency in the cellular immune response to JCV in PML patients, our more detailed study does not support a JCV-specific immunodeficiency. This is corroborated by the observation of a reduced PBMC proliferation and suppressed production of IFN-␥ after stimulation with TT, PHA, and VP1-VLP. Our results indicate a general loss of Th1-type T-helper functions in PML patients as evidenced by the reduction of proliferation and IFN-␥ production in the presence of all three stimulants. However, to support this observation, more Th1-type cytokines (interleukin-2, tumor-necrosisfactor-␣, and lymphotoxin) should be investigated in further studies. This hypothesis is in agreement with several reports emphasizing the importance of IFN-␥ in the control of a number of virus infections.19 In addition, the effect of highly active antiretroviral therapy (HAART) argues against a selective JCV-specific immunodeficiency. It has recently been shown that HAART significantly prolongs the survival of HIV-1-infected PML patients.12,20 –27 The positive effect of HAART appears to be due to the reconstitution of the immune system and correlates with an increase of the number of CD4⫹ T cells.23–25 In contrast to the diminished proliferation of PBMC, JCV-specific serum antibodies are elevated in most PML patients, in particular in the HIV/PML patients. Obviously, the B cell response against JCV is not impaired in most PML patients and may be T cellindependent. Such a T cell-independent humoral immune response occurs after infection with the mouse polyomavirus28 and provides resistance to acute mortality.29 The finding of a high antibody titer in HIV/ PML patients together with an elevated IL-10 production after stimulation with VP1-VLP supports a functional Th2-type T-helper response. It may reflect a shift from a Th1-type to a Th2-type immune response in these patients. Recent evidence also suggests an association of prolonged survival of PML patients receiving HAART with the development of a VP1-specific intrathecal humoral immune response.12 The demonstration of a JCV-neutralizing capacity of serum or intrathecal VP1-specific antibodies would elucidate the role of the humoral immune response in the development of PML. Because the VP1-VLP carry authentic neutralizing epitopes of JCV,11 they represent an ideal antigen to perform such a detailed study for JCVspecific neutralizing antibodies. Furthermore, recent findings of a comparable incidence of PML in patients receiving HAART and historical controls indicate the need for a further detailed and systematic study of the cellular and humoral immune response in these patients.19,20 This study should include the analysis of more Th1- and Th2-type cyto-

kines to elucidate further the importance of the Th1and Th2-type response in controlling JCV. Such a study with a higher number of patients appears now to be feasible, with a median survival of more than 46 weeks in PML patients receiving HAART19,20 and the availability of a useful JCV-authentic antigen in sufficient amounts.11 The study was supported by the Deutsche Forschungsgemeinschaft (We1297/3–1 and Lu397/5–1) and by a grant from the Werner Otto Stiftung to T.W. The work of M. Pickhardt is supported by the Werner Otto Stiftung, Hamburg, Germany. We thank Drs A. Argyrakis and C. Trebst for providing blood samples and case histories of patients with PML. W.L. and T.W. contributed equally to this work.

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