Rapid Communication Open Access
Copyright ©2005 Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Dec 7, 2005; 11(45): 7208-7210
Published online Dec 7, 2005. doi: 10.3748/wjg.v11.i45.7208
Effect of liniment levamisole on cellular immune functions of patients with chronic hepatitis B
Ke-Xia Wang, Li-Hua Zhang, Jiang-Long Peng, Yong Liang, Xue-Feng Wang, Hui Zhi, Department of Pathogenic Biology, School of Medicine, Anhui University of Science and Technology, Huainan 232001, Anhui Province, China
Xiang-Xia Wang, Huan-Xiong Geng, Huainan Railway Central Hospital, Huainan 232001, Anhui Province, China
Author contributions: All authors contributed equally to the work.
Correspondence to: Dr. Ke-Xia Wang, Department of Pathogenic Biology, School of Medicine, Anhui University of Science and Technology, Huainan 232001, Anhui Province, China. cpli@aust.edu.cn
Telephone: +86-554-6658770 Fax: +86-554-6662469
Received: September 8, 2004
Revised: October 23, 2005
Accepted: October 26, 2005
Published online: December 7, 2005

Abstract

AIM: To explore the effects of liniment levamisole on cellular immune functions of patients with chronic hepatitis B.

METHODS: The levels of T lymphocyte subsets and mIL-2R in peripheral blood mononuclear cells (PBMCs) were measured by biotin-streptavidin (BSA) technique in patients with chronic hepatitis B before and after the treatment with liniment levamisole.

RESULTS: After one course of treatment with liniment levamisole, the levels of CD3+, CD4+, and the ratio of CD4+/CD8+ increased as compared to those before the treatment but the level of CD8+ decreased. The total expression level of mIL-2R in PBMCs increased before and after the treatment with liniment levamisole.

CONCLUSION: Liniment levamisole may reinforce cellular immune functions of patients with chronic hepatitis B.

Key Words: Liniment levamisole; Chronic hepatitis B; Cellular immune function; T lymphocyte subsets; mIL-2R



INTRODUCTION

It has been reported that cellular immune functions are abnormal in patients with hepatitis B[1]. To explore the effects of liniment levamisole on cellular immune functions of patients with chronic hepatitis B, we measured the levels of CD3+, CD4+, CD8+ and the ratio of CD4+/CD8+ cells as well as the expression level of mIL-2R in PBMC in patients with chronic hepatitis B before and after the treatment with liniment levamisole. The results support that levamisole can improve cellular immune functions of patients with chronic hepatitis B.

MATERIALS AND METHODS
Patients

According to the diagnostic criteria modified during the 10th National Conference on Viral Hepatitis and Hepatopathy 2 000, 76 patients with chronic hepatitis B (45 males and 21 females) aged 25-64 years (average 48.25 years) were enrolled in this study and randomly divided into treatment group (n = 38, 27 males and 11 females, aged 32-64 years and averaged 51.8 years) and control group (n = 38, 18 males and 10 females, aged 25-58 years and averaged 44.7 years).

Treatment methods

Vitamins B and C, silymarin compound and other liver-protecting drugs were used in the 76 patients. The treatment group was primed with liniment levamisole at a dose of 500 mg twice a week for 3 mo (a course of treatment). Five milliliters of liniment levamisole was smeared on the surface of skin inside the four limbs near the body. The area of the skin should be as large as possible and the smeared skin was washed within 24 h to assure absorption of the drug. No antiviral agent or immunomodulator was used in the two groups.

Samples

Five milliliters of peripheral vein blood was collected at 8:00 a.m. from each patient with hepatitis B and 2.5 mL was put into a sterile test tube and 2.5 mL into an anticoagulant test tube with heparin.

Separation of PBMC and detection of T cell subsets, mIL-2R

After the heparinized blood was mixed with an equal volume of Hanks’ liquid without Ca2+ and Mg2+, PBMCs were harvested from heparinized whole blood by centrifugation on Ficoll-Hypaque sedimentation gradient and diluted to (1-3)×106/L cell suspension with RPMI 1 640 culture liquid. Ten microliter suspension of PBMCs was smeared on sheet glass pores so that the cells with CD3+, CD4+, CD8+ and the rest phrase of mIL-2R could be detected. Of the PBMC suspension, 0.5 mL was mixed with RPMI 1640 culture liquid. The cells were grown in continuous culture (37 °C, 50 mL/L CO2 in atmosphere) for 72 h and mIL-2R could be measured by antibodies against the membrane of T cells[1].

Immunocytochemical BSA technique

Different monoclonal antibodies (mAb) against CD3+, CD4+, CD8+ and Tac with biotin and SA-HRP were smeared on different sheet glasses. The smears were left to dry naturally and fixed with acetone for 15-20 min. The cells were incubated in continuous culture (37 °C, 50 mL/L CO2 in atmosphere) for 30 min. The immune sheet glass pores were measured after being stained with color-developing agents and washed with Tris buffer solution (TBS). The total number of PBMCs was counted and the positive cells were analyzed with the help of high power lens.

Reagents and instruments

Antibodies against T-lymphocyte subsets were provided by Shanghai Jing’an Medical Institute; Ficoll-Hypaque sedimentation gradients were offered by Shanghai Second Reagent Factory. Carbon dioxide incubator (MDF-135) was from Japan.

Statistical analysis

Statistical analysis was performed using t test to determine the difference between the two groups.

RESULTS

Before the treatment, there was no significant difference in the levels of CD3+, CD4+, CD8+, mIL-2R and the ratio of CD4+/CD8+ between the two groups. After one course of treatment, the levels of CD3+, CD4+ and the ratio of CD4+/CD8+ increased, while the level of CD8+ decreased. The total expression level of mIL-2R in PBMCs increased before and after the treatment with liniment levamisole.

DISCUSSION

This study demonstrated that levamisole could exert its immunopotentiating activity in patients with chronic hepatitis B. As an immune stimulant, levamisole could be connected with receptors of thymopentin on the surfaces of immunologic cells and induces inhibited T lymphocytes into immunologically competent cells, which take part in cell-mediated immune, induce interferon production and prime the lymphocytes and macrophages[2-11]. According to clinical experiments, levamisole has therapeutic effectiveness and boosts immunity in a variety of infectious diseases and some cancers[11-24]. Krastev et al[25] treated 25 viremic patients with chronic HBV infection with LMS and found that LMS may benefit some patients with chronic ongoing viral replication including patients who are contraindicated for TNF-α. It was reported that LMS acts by resetting the immune balance toward a type 1 response via the induction of IL-18[26].

We have previously shown that decreased CD3+ and CD4+ levels and CD4+/CD8+ ratio as well as increased CD8+ level occur in patients with chronic hepatitis B[1]. We have also shown that the level of mIL-2R in PBMCs is lower than that in normal controls[1]. In the present study, all these parameters including the levels of CD3+, CD4+, CD8+, mIL-2R and the CD4+/CD8+ ratio became normal in the levamisole-treated patients. This reversion of aberrant cellular immunity may improve symptoms of chronic hepatitis B patients.

Liniment levamisole is made from levamisole, which is applied on the surface of skin and can penetrate into the liver, spleen, lungs, kidneys and other internal organs through the skin. We suggest that liniment levamisole should be used in the treatment of patients with chronic hepatitis B.

Footnotes

Science Editor Wang XL and Li WZ Language Editor Elsevier HK

References
1.  Wang KX, Peng JL, Wang XF, Tian Y, Wang J, Li CP. Detection of T lymphocyte subsets and mIL-2R on surface of PBMC in patients with hepatitis B. World J Gastroenterol. 2003;9:2017-2020.  [PubMed]  [DOI]  [Cited in This Article: ]
2.  Johnkoski JA, Peterson SM, Doerr RJ, Cohen SA. Levamisole regulates the proliferation of murine liver T cells through Kupffer-cell-derived cytokines. Cancer Immunol Immunother. 1996;43:299-306.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 17]  [Cited by in F6Publishing: 14]  [Article Influence: 0.5]  [Reference Citation Analysis (0)]
3.  Daniluk J, Kandefer-Szerszeń M. Isoprinosine and levamisole as stimulators of interferon production in blood leukocytes of patients with alcoholic liver cirrhosis. Arch Immunol Ther Exp (Warsz). 1997;45:183-187.  [PubMed]  [DOI]  [Cited in This Article: ]
4.  Prakash MS, Rao VM, Reddy V. Effect of levamisole on the immune status of malnourished children. J Trop Pediatr. 1998;44:165-166.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 7]  [Cited by in F6Publishing: 7]  [Article Influence: 0.3]  [Reference Citation Analysis (0)]
5.  Hajnzić TF, Kastelan M, Lukac J, Hajnzić T. Immunocompetent cells and lymphocyte reactivity to mitogens in levamisole-treated brain tumor children. Pediatr Hematol Oncol. 1999;16:335-340.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 4]  [Cited by in F6Publishing: 4]  [Article Influence: 0.2]  [Reference Citation Analysis (0)]
6.  Martel F, Ribeiro L, Calhau C, Azevedo I. Inhibition by levamisole of the organic cation transporter rOCT1 in cultured rat hepatocytes. Pharmacol Res. 1999;40:275-279.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 10]  [Cited by in F6Publishing: 10]  [Article Influence: 0.4]  [Reference Citation Analysis (0)]
7.  Artwohl M, Hölzenbein T, Wagner L, Freudenthaler A, Waldhäusl W, Baumgartner-Parzer SM. Levamisole induced apoptosis in cultured vascular endothelial cells. Br J Pharmacol. 2000;131:1577-1583.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 26]  [Cited by in F6Publishing: 27]  [Article Influence: 1.1]  [Reference Citation Analysis (0)]
8.  Kayataş M. Levamisole treatment enhances protective antibody response to hepatitis B vaccination in hemodialysis patients. Artif Organs. 2002;26:492-496.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 29]  [Cited by in F6Publishing: 33]  [Article Influence: 1.5]  [Reference Citation Analysis (0)]
9.  Cuesta A, Esteban MA, Meseguer J. Levamisole is a potent enhancer of gilthead seabream natural cytotoxic activity. Vet Immunol Immunopathol. 2002;89:169-174.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 38]  [Cited by in F6Publishing: 38]  [Article Influence: 1.7]  [Reference Citation Analysis (0)]
10.  Bozić F, Bilić V, Valpotić I. Levamisole mucosal adjuvant activity for a live attenuated Escherichia coli oral vaccine in weaned pigs. J Vet Pharmacol Ther. 2003;26:225-231.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 17]  [Cited by in F6Publishing: 20]  [Article Influence: 1.0]  [Reference Citation Analysis (0)]
11.  Kimball ES, Fisher MC. Levamisole effects on major histocompatibility complex and adhesion molecule expression and on myeloid cell adhesion to human colon tumor cell lines. J Natl Cancer Inst. 1996;88:109-116.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 13]  [Cited by in F6Publishing: 13]  [Article Influence: 0.5]  [Reference Citation Analysis (0)]
12.  Sun A, Chia JS, Chang YF, Chiang CP. Levamisole and Chinese medicinal herbs can modulate the serum interleukin-6 level in patients with recurrent aphthous ulcerations. J Oral Pathol Med. 2003;32:206-214.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 18]  [Cited by in F6Publishing: 22]  [Article Influence: 1.0]  [Reference Citation Analysis (0)]
13.  Holcombe RF, Milovanovic T, Stewart RM, Brodhag TM. Investigating the role of immunomodulation for colon cancer prevention: results of an in vivo dose escalation trial of levamisole with immunologic endpoints. Cancer Detect Prev. 2001;25:183-191.  [PubMed]  [DOI]  [Cited in This Article: ]
14.  Creagan ET, Rowland KM, Suman VJ, Kardinal CG, Marschke RF, Marks RS, Maples WJ. Phase II study of combined levamisole with recombinant interleukin-2 in patients with advanced malignant melanoma. Am J Clin Oncol. 1997;20:490-492.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 8]  [Cited by in F6Publishing: 8]  [Article Influence: 0.3]  [Reference Citation Analysis (0)]
15.  Lai WH, Lu SY, Eng HL. Levamisole aids in treatment of refractory oral candidiasis in two patients with thymoma associated with myasthenia gravis: report of two cases. Chang Gung Med J. 2002;25:606-611.  [PubMed]  [DOI]  [Cited in This Article: ]
16.  Aksoy H, Baltaci S, Türkölmez K, Seçkiner I, Bedük Y. Combined interferon alpha with levamisole in patients with metastatic renal cell carcinoma. Int Urol Nephrol. 2001;33:457-459.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 4]  [Cited by in F6Publishing: 4]  [Article Influence: 0.2]  [Reference Citation Analysis (0)]
17.  Lam P, Yuen AP, Ho CM, Ho WK, Wei WI. Prospective randomized study of post-operative chemotherapy with levamisole and UFT for head and neck carcinoma. Eur J Surg Oncol. 2001;27:750-753.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 24]  [Cited by in F6Publishing: 24]  [Article Influence: 1.0]  [Reference Citation Analysis (0)]
18.  Cafiero F, Gipponi M, Peressini A, Bertoglio S, Lionetto R. Preliminary analysis of a randomized clinical trial of adjuvant postoperative RT vs. postoperative RT plus 5-FU and levamisole in patients with TNM stage II-III resectable rectal cancer. J Surg Oncol. 2000;75:80-88.  [PubMed]  [DOI]  [Cited in This Article: ]
19.  Fu LS, Chi CS. Levamisole in steroid-sensitive nephrotic syndrome children with steroid-dependency and/or frequent relapses. Acta Paediatr Taiwan. 2000;41:80-84.  [PubMed]  [DOI]  [Cited in This Article: ]
20.  Sun A, Chia JS, Chang YF, Chiang CP. Serum interleukin-6 level is a useful marker in evaluating therapeutic effects of levamisole and Chinese medicinal herbs on patients with oral lichen planus. J Oral Pathol Med. 2002;31:196-203.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 46]  [Cited by in F6Publishing: 49]  [Article Influence: 2.2]  [Reference Citation Analysis (0)]
21.  Donia AF, Amer GM, Ahmed HA, Gazareen SH, Moustafa FE, Shoeib AA, Ismail AM, Khamis S, Sobh MA. Levamisole: adjunctive therapy in steroid dependent minimal change nephrotic children. Pediatr Nephrol. 2002;17:355-358.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 19]  [Cited by in F6Publishing: 22]  [Article Influence: 1.0]  [Reference Citation Analysis (0)]
22.  Sun A, Chiang CP. Levamisole and/or Chinese medicinal herbs can modulate the serum level of squamous cell carcinoma associated antigen in patients with erosive oral lichen planus. J Oral Pathol Med. 2001;30:542-548.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 14]  [Cited by in F6Publishing: 16]  [Article Influence: 0.7]  [Reference Citation Analysis (0)]
23.  Wolmark N, Rockette H, Mamounas E, Jones J, Wieand S, Wickerham DL, Bear HD, Atkins JN, Dimitrov NV, Glass AG. Clinical trial to assess the relative efficacy of fluorouracil and leucovorin, fluorouracil and levamisole, and fluorouracil, leucovorin, and levamisole in patients with Dukes' B and C carcinoma of the colon: results from National Surgical Adjuvant Breast and Bowel Project C-04. J Clin Oncol. 1999;17:3553-3559.  [PubMed]  [DOI]  [Cited in This Article: ]
24.  Burch PA, Keppen MD, Schroeder G, Rubin J, Krook JE, Dalton RJ, Gerstner JB, Jancewicz MT, Ebbert LP. North Central Cancer Treatment Group Phase II study of 5-fluorouracil and high-dose levamisole for gastric and gastroesophageal cancer using survival as the primary endpoint of efficacy. Am J Clin Oncol. 1999;22:505-508.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 2]  [Cited by in F6Publishing: 3]  [Article Influence: 0.1]  [Reference Citation Analysis (0)]
25.  Krastev Z, Jelev D, Antonov K, Alagozian V, Kotzev I. Chronic HBV infection. Immunomodulation with levamisole in viremic HBeAg positive or anti-HBe positive patients--a pilot study. Hepatogastroenterology. 1999;46:3184-3188.  [PubMed]  [DOI]  [Cited in This Article: ]
26.  Szeto C, Gillespie KM, Mathieson PW. Levamisole induces interleukin-18 and shifts type 1/type 2 cytokine balance. Immunology. 2000;100:217-224.  [PubMed]  [DOI]  [Cited in This Article: ]  [Cited by in Crossref: 73]  [Cited by in F6Publishing: 76]  [Article Influence: 3.2]  [Reference Citation Analysis (0)]