Lack of detection of hepatitis C virus replicative intermediate in abdominal lymph nodes

Hepatology Research 10 (1998) 66 – 73

Lack of detection of hepatitis C virus replicative intermediate in abdominal lymph nodes Michio Sata a,*, Seiji Noguchi a, Tatsuya Ide a, Hiroshi Suzuki a, Ryukichi Kumashiro a, Yumiko Nagao b, Masayoshi Kage c, Hideki Saitsu d, Toshihiko Kurohiji e, Kyuichi Tanikawa a a

Second Department of Medicine, Kurume Uni6ersity School of Medicine, Kurume 830, Japan b Department of Oral Surgery, Kurume Uni6ersity School of Medicine, Kurume 830, Japan c First Department of Pathology, Kurume Uni6ersity School of Medicine, Kurume 830, Japan d First Department of Surgery, Kurume Uni6ersity School of Medicine, Kurume 830, Japan e Second Department of Surgery, Kurume Uni6ersity School of Medicine, Kurume 830, Japan

Received 18 August 1997; received in revised form 31 October 1997; accepted 6 November 1997

Abstract Deep abdominal lymph node enlargement has been detected by ultrasound examination in patients with hepatitis C virus (HCV) infection. To clarify the potential for extrahepatic existence and/or replication of HCV in such abdominal lymph node, positive- and negativestrand HCV RNA were investigated in abdominal lymph node mononuclear cells (MNC) as well as in the serum of patients with HCV. The study population consisted of eight hepatocellular carcinoma (HCC) patients positive for antibody to HCV (anti-HCV). Lymph nodes of hepatoduodenal ligament were resected at surgery for HCC. Both HCV RNA strands were examined using highly strand-specific r Tth reverse transcription-polymerase chain reaction (r Tth RT-PCR) followed by Southern blotting analysis. Positive-strand HCV RNA was detected in three (37.5%) abdominal lymph node MNC and in seven (87.5%) serum samples. Negative-strand HCV RNA was not detected in abdominal lymph node MNC, while it was observed in two (25%) serum samples. Histologically, the lymph nodes taken from subjects showed reactive follicular hyperplasia with or without the presence of HCV RNA. These observations suggest that HCV is located in the abdominal lymph nodes, but does not replicate within them. Negative-strand HCV RNA detected in serum samples

* Corresponding author. Tel.: + 81 942 31 7561; fax: +81 942 34 2623. 1386-6346/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S 1 3 8 6 - 6 3 4 6 ( 9 7 ) 0 0 1 0 3 - 4

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may have been due to contamination with circulating HCV RNA from hepatocytes. © 1998 Elsevier Science Ireland Ltd. Keywords: Hepatitis C virus; Mononuclear cell; Positive- and negative-strand

1. Introduction Deep abdominal lymph node enlargement has been detected by ultrasound examination as well as computed tomography in patients with chronic liver diseases, especially in patients with HCV [1–6], although it must be differentiated from metastatic lymph node enlargement accompanied by malignant disease. In addition to hepatocytes, it is suspected that HCV can replicate in peripheral blood mononuclear cell (PBMC) [7,8]. Moreover, a strong association between HCV infection and mixed cryoglobulinemia and/or B-cell non-Hodgkin’s lymphoma has been reported [9 – 11]. Thus, close interaction between HCV infection and the lymphatic system has been reported. However, the pathophysiological mechanism responsible for these findings is not clear and whether extrahepatic replication of HCV occurs is a critical question. Since HCV RNA may be present because of contamination with plasma and/or adherence of circulating virus, the presence of HCV RNA is not sufficient to demonstrate replication of HCV in extrahepatic tissue [7,8]. It is assumed that HCV uses a negative-strand complementary to the genomic RNA as a template for viral replication. Thus, to demonstrate extrahepatic replication of HCV, it is necessary to detect negative-strand HCV RNA [12–14]. In addition, several reports have indicated that a strand-specific RT method is required to detect negative-strand HCV RNA [15–18]. To investigate the correlation between HCV infection and enlarged abdominal lymph nodes in patients with hepatitis C, this study investigated both strands of HCV RNA in abdominal lymph node MNC as well as in serum samples from HCC patients with HCV using r Tth RT-PCR [17,18]. 2. Methods

2.1. Subjects Eight anti-HCV positive subjects with HCC were chosen for this study, because they were scheduled for resection of HCC (Table 1). Subjects consisted of six males and two females with a mean age of 679 8 years (range 55–76). Six had liver cirrhosis and two had chronic active hepatitis (CAH). None were positive for HBsAg. The maximal size of HCC ranged from 1.0 to 4.0 cm in diameter, except in one patient (patient No. 7; 15.0 cm in diameter). The study was approved by the Medical Ethics Committee of our hospital and informed consent was obtained from all subjects.

76/M 55/M 55/M 63/M 71/M 68/F 74/M 74/F

1 2 3 4 5 6 7 8

LC with HCC CAH with HCC LC with HCC LC with HCC LC with HCC LC with HCC CAH with HCC LC with HCC

Histological diagnosis

— 102 — — 10 104 — —

Positive-strand — — — — — — — —

Negative-strand

— 103 103 10 103 104 103 103

Positive-strand

Abdominal lymph nodes MNCs HCV RNA Serum HCV RNA

— — — — — 103 103 —

Negative-strand

s= ‘‘N N S N’’\— MNCs, mononuclear cells; M, male; F, female; LC with HCC, hepatocellular carcinoma with liver cirrhosis; and CAH with HCC, hepatocellular carcinoma with chronic active hepatitis.

Age (years)/sex

Patient no.

Table 1 Background and detection of both strands of HCV RNA in abdominal lymph nodes MNCs and serum samples from eight anti-HCV positive HCC patients

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2.2. Serum collection and serological tests Sera were collected from each subject on the day before surgery. Serum was separated from the samples and stored at − 20°C until HCV RNA assay. HBsAg was examined by enzyme immunoassay (EIA; Mizuho Medy, Tosu, Japan) and anti-HCV was determined by second-generation passive hemagglutination assay (PHA 2nd Generation, DAINABOT, Tokyo, Japan).

2.3. Sampling of lymph nodes for light microscopic examination and separation of MNC At surgery, enlarged lymph nodes in hepatoduodenal ligament were resected from all subjects. After resection, lymph nodes were immediately divided for microscopic examination and lymph node MNC separation. To prepare specimens for extraction of HCV RNA, lymph nodes were immediately washed with physiological saline, then pressed through a 200-gauge stainless steel mesh and suspended in RPMI 1640. Lymph node tissues were filtered twice through a 200-gauge stainless steel mesh. After washing once with RPMI 1640, MNC were isolated from the filtrate by Ficoll-Isopaque density gradient centrifugation (1450 rpm/20 min). The MNC were washed (1200 rpm/10 min) twice with RPMI 1640 and kept at − 80°C until HCV RNA assay. Finally, RNA was extracted from pelleted MNCs equivalent to 106 – 107 cells/ml. The lymph nodes from all subjects were stained with hematoxlin and eosin and were histologically examined.

2.4. Strand-specific RT-PCR MNC isolated from lymph nodes (100 ml of each) were suspended in 10 vol. of PBS. Then, RNA was extracted from 100 ml of the suspension or sera using the acid guanidium-thiocyanate phenol-chloroform method (Isogen-LS, Nippon Gene, Toyama, Japan), precipitated with isopropanol and resuspended in 20 ml of Rnase-free H2O. To detect HCV RNA by a semi-quantitative method, the RNA preparations were diluted 10-fold serially from which 10 ml aliquots were used for RT-PCR. Primers for RT-PCR (sense primer; HC-1 [5%-CACTCCCCTGTGAGGAACTACTGTC-3% from nt. 21–45] and antisense primer; HC-2 [5%-ATGGTGCACGGTCTACGAGACCTCC-3% from nt. 302–326]) were deduced from the highly conserved 5%untranslated region (5%UTR) of HCV. The presence of positiveand negative-strand HCV RNA in serum and MNC was detected by using the so-called strand specific RT-PCR method [13]. Thus, the primer HC-2 was used as a cDNA primer to specifically prime positive-strand HCV RNA and primer HC-1 was used as a cDNA primer to specifically prime negative-strand HCV RNA. Several studies have indicated that the standard method for detection of positiveand negative-strand HCV RNA lacks sufficient strand specificity [15–18]. Therefore, in this study, RT-PCR was carried out with a modified GeneAmp® Thermostable r Tth Reverse Transcriptase RNA PCR Kit (Perkin-Elmer Cetus, Tokyo, Japan) as described previously [17,18]. Aliquots of 10 ml of each extracted RNA

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were overlain with mineral oil, heated at 90°C for 1 min, the temperature was lowered to 70°C and 10 ml of pre-heated cDNA reaction mixture was added. The reaction mixture consisted of 10 mM Tris (pH 8.3), 90 mM KCL, 1mM MnCl2, 200 mM each deoxynucleotide triphosphate, 50 ng of cDNA primer and 5U of r Tth. Then, the annealing temperature was lowered to 60°C for 2 min and increased to 70°C for 15 min for the cDNA reaction. To chelate the Mn2 + and inactivate the RT activity of r Tth, the temperature was held at 70°C until the addition of 40 ml of pre-heated buffer consisting of 10% (V/V) glycerol, 20 mM Tris (pH 8.3), 200 mM KC1, 0.1% (W/V) Tween® 20 and 1.5 mM EGTA (ethylene glycol-bis [b-aminoethyl ether]-N,N,N%,N%-tetraacetic acid). Reaction tubes were held at 70°C until the addition of 40 ml of pre-heated PCR mixture (50 ng of each opposite outside primer in 3.75 mM MgCl2). The PCR profile consisted of the following three step reactions. Step 1: 94°C, 5 min; 55°C, 1 min; and 72°C, 2 min, 1 cycle. Step 2: 94°C, 1 min; 55°C, 1 min; and 72°C, 2 min, 35 cycles. Step 3: 72°C, 2 min. 1 cycle. The PCR products were analyzed by 1.2% agarose gel electrophoresis and ethidium bromide staining. These products were used to confirm the 306 bp band and estimate the dilution levels at which this 306 band would disappear. Southern blotting was employed for all samples, when the band prepared by PCR was not detectable. Following electrophoresis, the PCR product was transferred from the gel on to a nitrocellulose filter followed by hybridization with a 32P-labeled probe (5%-AGAGCCATAGTGGTCTGCGGAACCGGTGAGTACACCGGAA-3% from nt. 117 – 155) and autoradiography. It was reported that the sensitivity of single step PCR with 32P-labeled probe methods equals that of nested PCR, which is considered to be the reference assay [19]. Thus, the sensitivity of this RT-PCR assay may be as high as that of a nested PCR.

3. Results Table 1 and Fig. 1a, b Fig. 2. shows the results of analysis of HCV RNA in abdominal lymph node MNCs and serum samples from eight anti-HCV positive HCC patients. In the four specimens (Patient 2, 5 MNC and Patient 3, 4 serum), of which the bands were not detected by PCR, positive bands were detected by Southern blot hybridization. Finally, in abdominal lymph node MNCs, positivestrand HCV RNA was detected in three cases (37.5%), while negative-stranded HCV RNA was not detected (0%). In serum samples, positive-strand HCV RNA was detected in seven cases (87.5%) and negative-strand HCV RNA was detected in two cases (25%). Semi-quantitative analysis showed that positive-strand HCV RNA was present at the same level in abdominal lymph node MNC and serum (10–104). The histological features of the lymph nodes were also investigated. The lymph nodes of all the subjects showed marked reactive follicular hyperplasia with mild sinus histocytosis. None of the subjects had a metastatic lesion in the resected abdominal lymph nodes. There were no histological differences between abdominal lymph node MNC, with or without HCV RNA.

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4. Discussion Deep abdominal lymph node enlargement, especially of the hepatoduodenal ligament, has been detected by ultrasound examination as well as by computed tomography in patients with chronic liver diseases, especially patients with HCV [5,6]. Maeda et al. [6] reported that among 75 patients with enlarged lymph nodes of benign disease, 63 (84%) had liver dysfunction and 42 (65%) of these subjects were positive for anti-HCV. From these findings, it was hypothesized that HCV might be present and/or replicate in the abdominal lymph nodes. In addition, it was also suspected that HCV can replicate in PBMC [7,8] due to the detection of not only positive-strand but also negative-strand HCV RNA, an intermediate of HCV replication. Other researchers suggested that negative-strand HCV RNA detected in extrahepatic sites may be due to contamination with plasma and/or adherence of circulating virus because methods for detecting negative-strand HCV RNA lack sufficient strand specificity [15 – 18]. Therefore, we attempted to detect both strands of HCV RNA using highly strand-specific r Tth RT-PCR [17,18]. Positive-stranded

Fig. 1. (a) Positive-stranded HCV RNA in MNCs detected by PCR. Lane M: Marker (ø × 174 HaeIII); Lanes 1, 2, 3, 4, 5: Patient 6 MNC diluted RNA ( × 10, 102, 103, 104, 105); Lane 6: Patient 7 undiluted MNC; Lane 7: negative control. Detection of negative-stranded HCV RNA in MNCs by PCR. (b) Lane M: Markers (ø × 174 HaeIII); Lanes 1, 2, 3, 4, 5, 6, 7, 8: Patients 1, 2, 3, 4, 5, 6, 7, 8, MNC undiluted RNA, respectively; Lane 9: negative control.

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Fig. 2. Positive-stranded HCV RNA in MNCs and serum detected by Southern blot hybridization of PCR products. Lane M: Markers (ø ×174 HaeIII); Lane 1: Patient 5 MNC diluted RNA ( × 10); Lane 2: Patient 2 MNC diluted RNA (× 102); Lane 3: Patient 3 serum diluted RNA (× 103); Lane 4: Patient 4 serum diluted RNA ( × 10); Lane 5: positive control; Lane 6: negative control; Lane 7: positive control.

HCV RNA was detected in three (37.5%) abdominal lymph node MNCs and in six (75%) serum samples. However, negative-strand HCV RNA was not found in any abdominal lymph node MNC samples. Negative-strand HCV RNA was detected in two (25%) of the serum samples and might have been due to contamination and/or adherence of circulating negative-strand HCV RNA. These findings indicate that HCV infection in abdominal lymph node MNC may be transient rather than replicative. In Patients 6 and 7, negative-strand HCV RNA was detected in the serum but not in MNC. In the study, to evaluate HCV RNA in the lymph nodes avoiding contamination of HCV RNA from serum and another cells except MNC, only MNC were carefully separated and washed from lymph nodes of the subjects. Thus, HCV RNA that was isolated was thought to solely be from MNC. It is not yet clear whether HCV RNA can exist and/or replicate in other cells except MNC in lymph nodes and result in lymphadenopathy and awaits further study. On the basis of histological studies of enlarged abdominal lymph nodes with chronic hepatitis C [1,2], it has been reported that enlargement is due to reactive hyperplasia caused by hepatic inflammation. In the present study, marked reactive hyperplasia was observed in lymph nodes with or without HCV RNA. These findings suggest that enlarged lymph nodes are associated with hepatic inflammation but not always associated with HCV and/or replication in these lymph nodes. It was also reported that patients with chronic hepatitis C frequently have autoantibodies and/or concurrent autoimmune diseases [20–22]. Further studies are needed whether lymph node enlargements correlate with autoantibodies of these patients. Additionally, further studies are necessary to determine whether HCV is present and/or replicates in abdominal lymph nodes of patients with chronic hepatitis C, using recently developed in situ hybridization [23].

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