Viral Infections MxA expression in patients with viral infection ... - Nature

Bone Marrow Transplantation (2003) 32, 313–316 & 2003 Nature Publishing Group All rights reserved 0268-3369/03 $25.00

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Viral Infections MxA expression in patients with viral infection after allogeneic stem cell transplantation T Yoshimasu1,5, A Manabe1, Y Ebihara1, R Tanaka1, J Ooi2, T Iseki2, N Shirafuji3, T Maekawa4, S Asano2, N Yoshikawa5 and K Tsuji1 1 Department of Pediatric Hematology-Oncology, Institute of Medical Science, University of Tokyo, Japan; 2Department of Hemotology-Oncology, Institute of Medical Science, University of Tokyo, Japan; 3Department of Internal Medicine, Teikyo University, Japan; 4Cell Transfusion, Kyoto University, Japan; and 5Department of Pediatrics, Wakayama Medical College, Japan

Summary: Many patients suffer febrile diseases soon after allogeneic stem cell transplantation (SCT). Some of the symptoms of viral infections and acute GVHD are often difficult to distinguish. However, an accurate diagnosis is important since the treatments for these conditions are different. It is known that MxA protein is specifically induced in patients with several viral infections. We investigated the cytoplasmic expression of MxA in the peripheral blood mononuclear cells (PBMCs) of patients with fever after allogeneic SCT using a newly generated monoclonal antibody (KM1135) and flow cytometry. The level of MxA expression was significantly higher in patients diagnosed with viral infections (n ¼ 6, cytomegalovirus in three, Epstein–Barr virus in one, human herpesvirus-6 in one, adenovirus in one) than control individuals (n ¼ 9) (Po0.05, Mann–Whitney test). The level of MxA in patients with aGVHD (n ¼ 7) was identical to that in controls. The level of MxA correlated well with the amount of the cytomegalovirus antigen-positive cells in the presence of acute GVHD in two patients. The measurement of MxA is simple and useful in distinguishing viral disease from acute GVHD after allogeneic SCT. Bone Marrow Transplantation (2003) 32, 313–316. doi:10.1038/sj.bmt.1704128 Keywords: MxA; allogeneic stem cell transplantation; viral infection; acute graft-versus-host disease

Many patients suffer febrile diseases soon after allogeneic stem cell transplantation (SCT). The clinical symptoms of various viral infectious diseases mimic those of acute graftversus-host disease (aGVHD), and it is difficult to discriminate between the two based upon symptoms.1–7 However, it is important to do so because the treatment of these conditions is very different.

Correspondence: Dr A Manabe, Department of Pediatric HematologyOncology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan Received 6 January 2003; accepted 28 February 2003

Interferon (IFN) a and b, type I IFNs, are predominantly produced in response to viral infections and contribute to host defense by establishing an antiviral state in target cells.8 In contrast, levels of certain cytokines such as IFNg, tumor necrosis factor a, and several interleukins are elevated in the serum of patients with aGVHD.9–14 MxA protein is specifically induced in a dose-dependent manner by type I IFNs15,16 and accumulates in the cytoplasm of stimulated cells. It has been reported that MxA protein measurements are useful for the diagnosis of viral infections.17–20 In this study, we investigated the expression of MxA protein in peripheral blood mononuclear cells (PBMCs) of febrile patients who underwent allogeneic SCT using a newly generated monoclonal antibody.

Materials and methods Patients A total of 21 febrile patients 11–81 days after allogeneic SCT were consecutively enrolled in the study after providing written informed consent. The age ranged from 8 to 48 (median 22) years. Of the patients 16 were male and five were female. A total of 25 blood samples from patients who had a fever of over 37.51C were collected (Table 1). The diagnosis of aGVHD was based on the histological findings of specimens obtained by biopsy. The degree of aGVHD was graded according to the criteria of the 1994 Consensus Conference on aGVHD grading.21 The description ‘aGVHD suspected’ was used when patients had symptoms similar to aGVHD without histological examination and the symptoms improved with continuing or increased treatment with immunosuppressants. The diagnosis of viral infections was based on the detection of virus genome or antigen in blood or stool samples regardless of other symptoms. Nine control blood samples were collected from seven patients who had no fever or clinical symptoms of infection of aGVHD 17–92 days after allogeneic SCT (Table 2).

Analysis of MxA protein expression in PBMCs A total volume of 1 ml of heparinized venous blood was collected from the patients. PBMCs, separated on a Ficoll-

MxA in viral infection after allo-SCT T Yoshimasu et al

314 Characteristics of the patients

Table 1 No.

Sex/age (year)

Disease

SCT source/donor

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

M/34 M/23 M/23 F/28 M/22 M/8 M/32 M/48 F/41 M/30 M/41 M/12 M/17 M/9 M/14 M/34 M/48 F/16 F/19 M/17 M/23 F/20 M/30 M/14 M/16

RAEB-t RA AML RA ALL ALL CML RA ALL AML AML Kostmann syndrome CML ALL NHL RAEB-t AML SAA AML CML RA NHL AML SAA NHL

CB/unrelated BM/unrelated CB/unrelated BM/unrelated BM/sibling CB/unrelated BM/sibling CB/unrelated CB/unrelated BM/unrelated PB/sibling PB/father BM/unrelated CB/unrelated BM/sibling CB/unrelated CB/unrelated BM/unrelated PB/cousin BM/unrelated BM/unrelated BM/sibling BM/unrelated BM/sibling CB/unrelated

Days after SCT 67 25 67 38 17 81 17 67 27 55 39 20 45 11 19 74 27 78 71 53 11 34 22 26 40

Clinical symptoms

Diagnosis

FI of MxA

Skin eruption Skin eruption, Skin eruption, Skin eruption Skin eruption, Skin eruption, Skin eruption Skin eruption Skin eruption Skin eruption Skin eruption Skin eruption, Skin eruption Skin eruption Skin eruption Skin eruption Skin eruption Jaundice Hematochezia Skin eruption No symptoms No symptoms No symptoms No symptoms No symptoms

aGVHD I aGVHD II aGVHD II aGVHD I aGVHD II aGVHD II aGVHD I aGVHD suspected aGVHD suspected aGVHD suspected aGVHD suspected aGVHD suspected aGVHD suspected aGVHD suspected HHV-6 infection CMV infection CMV infection EBV infection Adenovirus infection CMV infection Bacteremia FUO FUO FUO FUO

2.9 2.1 1.7 1.3 1.2 1 1 3.3 2 1.7 1.7 1.6 1.3 1 38.9 9 8.3 5 3.2 2.6 1.3 4 3.7 3.1 2.5

diarrhea diarrhea diarrhea diarrhea

diarrhea

Nos. 1 and 16, 2 and 21, 10 and 23, and 13 and 20 were the same patients. SCT, stem cell transplantation; FI, fluorescence index; RA, refractory anemia; RAEB-t, RA with excess of blasts in transformation; AML, acute myelogenous leukemia; ALL, acute lymphoblastic leukemia; CML, chronic myelogenous leukemia; NHL, non-Hodgkin’s lymphoma; SAA, severe aplastic anemia; CB, cord blood; BM, bone marrow; PB, peripheral blood; HHV6, human herpesvirus-6; CMV, cytomegalovirus; EBV, Epstein–Barr virus; FUO, fever of unknown origin.

Table 2

Characteristics of the controls

No.

Sex/age (years)

Disease

SCT source/donor

1 2 3 4 5 6 7 8 9

M/14 M/41 M/17 M/17 F/28 F/28 M/21 F/49 M/30

NHL AML CML CML RA RA RA RAEB-t ALL

BM/sibling PB/sibling BM/unrelated donor BM/unrelated donor BM/unrelated donor BM/unrelated donor CB/unrelated donor CB/unrelated donor BM/sibling

Days after SCT

FI of MxA

64 92 70 77 17 31 28 73 43

2.0 2.0 1.5 1.2 1.2 1.2 1.2 1.0 0.9

Nos. 3 and 4, and 5 and 6 were the same patients.

Metrizamide (density ¼ 1.077 g/ml, Lymphoprep; Nyegaad, Oslo, Norway) density gradient, were washed and resuspended in alpha-MEM (Gibco, NY, USA) supplemented with 10% FCS (HyClone, Logan, UT, USA). The cytoplasmic staining technique was previously described.22 The cells (1 105–1 106) were transferred to tubes, washed once with PBS supplemented with 0.2% BSA and 0.2% sodium azide (PBS-AA), pelleted, and resuspended in 0.1 ml of PBS-AA. They were then fixed for 15 min by adding 0.1 ml of Fix & Perm MEDIUM A (An Der Grub, Austria). Subsequently, cells were washed and resuspended in PBS-AA at a final volume of 0.1 ml. The cell membrane was permeabilized for 10 min by adding 0.1ml of Fix & Perm MEDIUM B and stained for MxA protein simultaneously with anti-human MxA antibody (KM1135, a Bone Marrow Transplantation

mouse monoclonal antibody, IgG1 isotype). The antibody was a gift from Kyowa Co, Tokyo, Japan. Mouse IgG1 (DAKO) was used as a control. After being washed, cells were resuspended in 0.1 ml of PBS-AA. As a secondary antibody, PE-labeled goat anti-mouse IgG (DAKO) was used at an optimal dilution of 1:25. Cells were washed, resuspended in 0.1 ml of PBS-AA to which 0.3 ml of a 0.5% paraformaldehyde solution was then added, and stored at 41C in the dark until analyzed. The expression of MxA protein in PBMCs was assessed by flow cytometry using a FACScan (Becton Dickinson, Mountain View, CA, USA). As a measure of the intensity of staining, a fluorescence index (FI) was used, which represents the ratio between the fluorescence intensity of cells stained with the specific antibody (anti-human MxA antibody) and that of cells stained with the isotype-matched control.

Statistical analysis For statistical comparison of MxA levels in PBMCs of patients with aGVHD and viral infections, Mann–Whitney test was applied. Differences with a P value of less than 0.05 were considered to be statistically significant.

Results In virus-infected patients, the FI of MxA ranged from 2. 6 to 38.9 (median 6.7), compared to 0.9 to 2.0 (median 1.2) in control individuals (Po0.05, Mann–Whitney test,


315 40

a P < 0.05

P < 0.05 NS

5

Control (n=9)

aGVHD (n=7)

10 9 8 7 6 5 4 3 2 1 0 day 67 aGVHD I grade

4 3.5

FI of MxA

FI of MxA

10

0

The number of CMV antigen positive cells/1.5×105 leukocytes 4.5

Viral infection (n=6)

Figure 1 Fluorescence index (FI) of MxA protein expression in subgroups of patients. The mean of each group is represented as a horizontal bar. NS: not significant.

FI of MxA

3

CMV antigen positive

2.5 2 1.5 1 0.5 0

74 II

78 IV

81 IV

88 II

Discussion Generally, a quick and definitive diagnosis of viral infection is difficult because of technical limitations. Measuring MxA protein can be done rapidly. Our method to detect MxA is a standard cytoplasmic staining technique followed by flow cytometric analysis, and is less complicated than other techniques employing enzyme-linked immunosorbent assay (ELISA)17 or measuring luminescence units of antibodyconjugated substance.19,20

3

FI of MxA

99 II

The number of CMV antigen positive cells/1.5×105 leukocytes 25

b

FI of MxA CMV antigen positive cells

2.5

Figure 1). In patients with aGVHD, the FI ranged from 1.0 to 2.9 (median 1.3), which was identical to that in controls (Figure 1). The FI of MxA in patients with ‘aGVHD suspected’ ranged from 1.0 to 3.3 (median 1.7), similar to that in patients with aGVHD. The results demonstrate the feasibility of using MxA protein in diagnostics. The protein level was significantly higher in PBMCs of patients with viral infections rather than aGVHD (Po0.05, Figure 1). In two patients (nos. 1/16 and 13/20), cytomegalovirus (CMV) antigen and MxA protein levels were simultaneously measured several times. In both cases, MxA protein levels correlated well with the number of CMV antigen-positive cells (Figure 2a, b). In addition, grade IV aGVHD developed in patient no. 1/16 later and the level of MxA decreased at the same time. Also in patient no. 13/20, grade II aGVHD occurred before CMV infection and the MxA level was low at that time. Thus, MxA expression correlated with the emergence of viral infections in patients who had aGVHD simultaneously. Four of five patients (nos. 21–25) with no symptoms except fever had a relatively high FI for MxA. The Creactive protein was negative in these patients. Accordingly, they were suspected of having a viral infection although no virus was detected. The other patient (no. 21) whose FI was not elevated was diagnosed with a bacterial infection based on the results of a blood culture (Table1). This was compatible with a previous report.18

92 II

2

20 15

1.5 10 1 5

0.5 0 day aGVHD grade

0 38 II

45 I

52 I

56 I

59 I

66 0

70 0

Figure 2 Time course of simultaneous analyses of MxA levels and the number of CMV antigen-positive cells in patient 1/16 (a) and in patient 13/ 20 (b). The number of CMV antigen-positive cells was below the detection limits on days 67, 78, 81, 88, and 99 in patient 1/16 (a). The number of CMV antigen-positive cells was also below the detection limits on days 38, 45, 59, 66, and 70 in patient 13/20 (b).

The rapid differential diagnosis of viral infections and aGVHD is vital because immunosuppressive therapy for aGVHD may harm those with viral infections. It has been reported that elevated serum levels of soluble interleukin-2 receptor (sIL-2R) are useful in diagnosing aGVHD. These results should be interpreted with caution because sIL-2R levels may be elevated in CMV infections.11 It is known that the 20 –50 oligoadenylate synthetase (20 –50 OAS) level is elevated in patients with viral infections. However, IFNg also induces the expression of 20 –50 OAS as well as p68.23,24 The expression of these proteins is induced not only by viral infections but also by aGVHD. In fact, it has been reported that basal 20 –50 OAS activity was elevated in PBMNCs of patients with aGVHD.25 MxA protein is specifically expressed in response to type I IFNs.15,16 The biological half-life of IFNa is very short (1–2 h) whereas that of MxA is rather long (2.3–2.5 days),20,26 suggesting that the elevated expression of MxA a few days after the onset of a viral infection may still be diagnosed. Our results clearly show that the measurement of MxA protein is appropriate for distinguishing viral infections from aGVHD after allogeneic SCT. Bone Marrow Transplantation


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In addition, viral infections including CMV infection often occurs concurrently with aGVHD. We demonstrated that the level of MxA correlated with the amount of the CMV antigen-positive cells in the blood even in the presence of aGVHD; therefore, the MxA detection would be also useful in such occasions. However, as the number of patients in this study is small, further investigation is needed to establish the validity of this assay.

Acknowledgements We thank Kyosuke Nagata and Akiko Furuya for providing the KM1135 antibody and Daniel Mrozek for proofreading the manuscript. We also thank the physicians and nurses who cared for the patients in this study.

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