Journal of Infection (2002) 45: 3238 doi:10.1053/jinf.2002.1007, available online at http://www.idealibrary.

com on

Anti-Septicaemic Effect of Polysaccharide from Panax ginseng by Macrophage Activation

D. S. Lim1, K. G. Bae1, I. S. Jung1, C. H. Kim2, Y. S. Yun1 and J. Y. Song*1
2

Laboratory of Immunology, Korea Cancer Center Hospital, KAERI, Seoul 139-706 and Animal Resources Research Center, KonKuk University, Seoul 143-701, Republic of Korea

The aim of the present research was conducted to elucidate anti-septicaemic effect of a polysaccharide (PS) isolated from Panax ginseng C.A. Meyer (Araliaceae) by nitric oxide production from stimulated macrophage. In vitro assays for the activity measurement of PS, NO production test with Greiss reagent, phagocytic activity test using zymosan and cytokines production test using ELISA kit were also conducted. In vivo anti-septicaemic activity was assessed by using C57BL/6J mice. This was done with Staphylococcus aureus infection test. PS used at 0.025 mg/kg concentration showed a potent anti-septicaemic activity (80%, survival). However, it did not directly inhibit S. aureus in a minimum inhibitory concentration (MIC) test, conducted in vitro (data not shown). Nitric oxide production via macrophage activation showed the highest value of 5.5 nmol/ml at 1 mg/ml PS. In in vitro phagocytic activity test, PS at 10 mg/ml concentration showed a potent phagocytic activity for zymosan with 167% of the control. Production of TNF-a by macrophage activation at 10 mg/ml of PS was 96% lysis of L929. Also production of IL-1 and IL-6 by stimulation of macrophage with 100 mg/ml PS dose increased to 235 pg/ml and 0.47 ng/ml, respectively. The low mortality of PS treated (0.025 mg/kg) infected mice was concurrent with decreased bacterial content in the blood. Nitric oxide production in S. aureus infected mice whose macrophage was stimulated by PS (0.025 mg/kg) increased approximately 4 times than the untreated S. aureus infected group at 24 and 48 h incubation. In the PS treated (0.025 mg/kg) group, the intracellular concentration of S. aureus in macrophages decreased approximately by 50%, compared with the untreated group. Combine treatment with PS (0.025 mg/kg body weight) and vancomycin (10 mg/kg B.W.) resulted in 100% survival of the animals, whereas only 67% or 50% of the animals survived, respectively, when treated with PS or vancomycin alone. These results suggest that PS from Panax ginseng possess a potent anti-septicaemic activity by stimulating macrophage and a potentiality as an immunomodulator against sepsis # 2002 The British Infection Society occurred by Staphylococcus aureus.

Introduction
An individual's reaction to infection is triggered by bacterial toxins or by components of microbial cells, such as cell membrane fragments [1]. The significant morbidity and mortality associated with sepsis have continued to be powerful incentives for attempts to develop novel therapeutic strategies for this disease [2]. Septicemia is an acute invasion of the bloodstream by microorganisms. It can be a serious, rapidly progressing, life-threatening infection that may arise from localized infections of respiratory and gastrointestinal tracts,

* Please address all correspondence to: Jie-Young Song, Laboratory of Immunology, Korea Cancer Center Hospital, KAERI, 215-4 Gongneung-dong, Nowon-ku, Seoul 139-706, Republic of Korea. Tel.: 82-2-970-1309; Fax: 82-2-977-0381; E-mail address: dslim99@hotmail.com or immu@mail.kcch.re.kr (J. Y. Song). 0163-4453/02/$35.00

genitourinary system, or skin. It can also occur concurrently with or be preceded by infections like osteomyelitis, meningitis, or urinary dysfunction. Patients with underlying diseases such as diabetes, cirrhosis, alcoholism, or cancer are at a higher risk for septicemia [3]. The normal reaction to infection involves a series of complex immunologic processes. For example, factors associated with Gram-positive and Gram-negative bacterial infections trigger macrophages to produce cytokines, including tumor necrosis factor (TNF), interleukin (IL)-1 and 6 [4]. This systemic cytokine response appears to represent an uncontrolled and adverse inflammatory response, therefore, it has been proposed that blocking proinflammatory cytokines may improve survival after lethal challenge [5]. Staphylococcus aureus remains a major pathogen that colonizes both hospitalized patients with decreased infectious resistance and healthy, immunologically
# 2002 The British Infection Society

Anti-Septicaemic Effect of Polysaccharide competent persons in the community [6]. Staphylococcal pathogenicity depends upon the effectiveness of the host defense in dealing with a wide variety of bacterial components such as extracellular toxins, enzymes, and cell wall components [6]. Presently, methicillin, teicoplanin and vancomycin [7,8] are available as the antibiotics for septicaemia. However, those antibiotics result in incurring the antibiotic-resistance of bacteria. Consequently, to resolve this problem, the development of natural products is highly imperative. Thus, the present research was conducted to elucidate anti-septicaemic effect of a polysaccharide (PS) isolated from Panax ginseng by nitric oxide production from stimulated macrophage.

33

centrifuged at 2000 rpm for 15 min, and then the cell pellet was washed twice in phosphate-buffered saline (PBS). The number of cells of this strain was adjusted to 1.0 109 CFU/ml. The mice were infected by intraperitoneal injection of S. aureus (1.0 108) in PBS. Characterization of anti-septicaemic biological activity Acute sepsis models using S. aureus intraperitoneal challenge were developed to evaluate the anti-septicaemic properties of the PS in mice. Female C57BL/6J mice were acclimatized for 7 days after arrival at the test facility. Groups of 12 mice each received 0.1 ml of various concentrations of PS in by intravenous injection. A control group received 0.1 ml of PBS. Mice were returned to their cages, maintained on food and water ad libitum, and were challenged 3 h after the administration of PS by intraperitoneal injection of 0.1 ml (1.0 108 CFU) of S. aureus culture in PBS. Survival was recorded at 2 and 5 days after the challenge. Isolation of peritoneal macrophage Macrophages were isolated from thioglycollate-elicited peritoneal exudates cells as described by Klimetzek et al. [9]. Briefly, the cells were isolated from peritoneal cavity by use of the 5 ml syringe containing Dulbecco's modified Eagle's medium (DMEM) with 10% FBS and resuspended in DMEM containing 10% FBS. Peritoneal exudates cells were seeded at densities of 56105 cells/ cm2 on teflon-coated petri dishes and the macrophages were allowed to adhere for 23 h at 37  C under 5% CO2 humidified atmosphere. After culture, non-adherent cells were removed and the macrophages were harvested by rinsing using a 10 ml syringe. The viability of the detached cells was assessed by trypan blue exclusion. TNF-a, IL-1 and IL-6 bioassay Levels of TNF were determined in a cytotoxicity assay using TNF-sensitive L929 fibroblast (ATCC, Rockville, MD) [10]. One hundred microliters of L929 cells (4 105 cells/ml) in RPMI 1640 medium containing 5% FBS were added to 96 well microtiter plates (Nunc, Denmark). The plates were incubated overnight at 37  C in 5% CO2 humidified incubator. The medium from each well was discarded and 50 ml of supplemented EMEM, 50 ml of the macrophage culture supernatant stimulated by the PS and 50 ml of actinomycin D (2 mg/ml) were added to each well. After 18 h incubation in a humidified CO2 incubator, the supernatants were discarded and the

Materials and Methods

Isolation of polysaccharide Nine hundred grams of Panax ginseng C.A. Meyer (Araliaceae) were extracted in 4 L of distilled water in the cold room (4  C). The extracts were concentrated by use of evaporator and precipitated with ethyl alcohol by adjusting to final concentration of 80% EtOH. The precipitate was dissolved in distilled water and dialyzed (M.W. b 12,000) against distilled water. After removal of insoluble materials in the dialysate, the supernatant was lyophilized to yield 15 g of powder. The PS preparation was purified by Sephacryl S-500 and DEAEA50 column chromatography, and determined to be a(1 3 6) glucopyranoside and b(2 3 6) fructofuranoside at 5 : 2 molar ratio by NMR analysis (M.W. ca. 2000 kD). Mice Female C57BL/6J mice 6 to 8 weeks old were obtained from Jackson Lab. (Boston, USA) and maintained in the animal facility of the department of Immunology, Korea Cancer Center Hospital, Seoul, Korea. Twelve mice were housed to a cage under standard conditions of temperature and light, and were fed standard laboratory chow and water ad libitum. Staphylococcus aureus strain Staphylococcus aureus strain ATCC25923 divided from Korea Culture Center of Microorganisms (KCCM, Seoul, Korea) was subcultured in nutrient agar (Difco) and proliferated in tryptic soy broth (Difco) for 24 h at 37  C. After 24 h incubation, the proliferated strain was

34

D. S. Lim et al. supernatant was taken and mixed with 100 ml of Greiss reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride and 2.5% phosphoric acid). After 10 min, the concentration of nitric oxide in the supernatant was analysed by absorbance at 540 nm with NaNO2 standard curve. Quantitation of intracellular S. aureus in macrophages from infected mice The quantitation of intracellular killing effect by macrophage stimulated by the PS which was injected intravenously 3 h before S. aureus intraperitoneal challenge in mice was determined in the PS (0.025 mg/kg)-treated group and -untreated control group. The peritoneal macrophages isolated were aliquoted (2105 cells/tube) into 4 ml polystyrene cell culture tubes, and the lysostaphin (Sigma) at 5 mg/ml final concentration was added to each tube, and the tubes were incubated for an additional 20 min to lyse extracellular bacteria. The tubes were centrifuged at 1500 g for 10 min, the pelleted cells were lysed with 1 ml deionized sterile water, and the number of intracellular S. aureus was determined after overnight incubation at 37  C by counting on blood agar plates. Measurement of prophylaxis effect with vancomycin Groups of 12 mice (C57BL/6J, female) each were administered with 0.1 ml of vancomycin (the final concentration, 10 mg/kg), the polysaccharide (PS) (0.025 mg/kg), or the PS together with vancomycin (0.025 10 mg/kg) in PBS by intravenous injection. A control group received 0.1 ml of PBS. The mice were then challenged with intraperitoneal injection of 0.1 ml (1.0108 CFU) S. aureus culture 3 h after the administration of test drugs. Survival was recorded at 2 and 5 days after the challenge. Statistical analysis Statistical evaluation was done by using the Mann Whitney U test for in vivo assays and the Student t-test for in vitro assays with GraphPad Prism (Ver. 3.0) software. Results are presented as means the standard errors of the means (SEMs).

cells were stained for 10 min with 50 ml of 0.05% crystal violet in 20% ethyl alcohol. One hundred microliters of absolute methyl alcohol was added to each well to elute the stain from the cells. The optical density of each well was determined at 595 nm using a Molecular Device microplate reader (Menlo. CA). TNF-a activities were expressed as cytolysis percentage of L929, compared to that of control. The concentrations of cytokines IL-1 and IL-6 in the culture supernatants were determined by the use of ELISA kits (Quantikine, R&D, Minneapols, MN, USA) according to the manufacturer instructions [11]. Evaluation of bacterial growth Growth of staphylococci in blood was evaluated by colony enumeration at 24 h after S. aureus infection. Blood samples of the PS treated (0.025 mg/kg) and untreated groups from infected mice were obtained by retro-orbital sinus bleeding before sacrifice. Ten fold dilutions were made, and 0.2 ml each of blood dilutions were plated on blood agar plates. After incubation for 48 h, colonies were counted and the results were expressed as the number of CFU per milliliter of blood. Nitric oxide production and phagocytic activity The peritoneal macrophages were isolated as above and 2105 cells/well of peritoneal macrophages were incubated in either medium (DMEM containing 10% FBS) alone or medium supplemented with the PS for 24 h and additional 24 h with fresh medium in 96 well microplate. After culture, 50 ml of each supernatant was taken and nitric oxide was measured using Nitric oxide analyzer (Antek Inst., Houston, TX). Peritoneal macrophages were cultured with the PS for 24 h, and zymosan (5106 particles/ml), nitroblue tetrazolium (NBT, 0.6 mg/ml) and fresh medium were added to the cells and incubated for 1 h. Cells were washed and formazan formed was measured at 540 nm using ELISA reader. Nitric oxide production by polysaccharide stimulated macrophage in infected mice Nitric oxide production by macrophages stimulated by the PS which was injected (i.v.) at 0.025 mg/kg 3 h before S. aureus intraperitoneal challenge in mice was determined. The mice were sacrificed 24 h after S. aureus inoculation, the peritoneal macrophages were isolated, and 2105 macrophages were incubated in medium (DMEM containing 10% FBS) in 96 well flat bottom microplate for 24 and 48 h. After culture, 100 ml of each

Results
Anti-septicaemic activity of polysaccharide Acute sepsis model by intraperitoneal challenge with S. aureus in mice was developed to evaluate the

Anti-Septicaemic Effect of Polysaccharide

35

Figure 1. Anti-septicaemic activity of polysaccharide on the S. aureus peritoneal sepsis challenge. C57BL/6J mice (n 12 in each group) were challenged by i.p. injection with 1.0108 CFU of S. aureus 3 h after i.v. injection of polysaccharide. Survival was recorded 2 days (a) and 5 days (b) after the challenge. *P ` 0.01, increase vs. control.

anti-septicaemic activity of PS prepared from Panax ginseng. Fig. 1a summarizes representative dose responses observed in this model 2 days after S. aureus intraperitoneal challenge with PS. A single dose of PS (0.025 mg/kg body weight) administered 3 h before the challenge reduced (P ` 0.01) mortality significantly from intraperitoneal sepsis. At high PS doses (b0.5 mg/kg), the protective effects were not detectable, and this was highly enigmatic characteristic of immunomodulators. A single dose of PS (0.025 mg/kg) administered 3 h before challenge maintained the anti-septicaemic effect against S. aureus up to 5 days (Fig. 1b).

Table I. The effect of polysaccharide on nitric oxide production and phagocytic activity by macrophages. Items Concentration of polysaccharide (mg/ml) Control No production (nmol/ml)a Phagocytic activity (% of control)b
a b

1 5.5* 157**

10 3.9 167**

100 2.4 140**

1.7 c

24 h-macrophages culture supernatant. See ``Materials and Methods''. c Phagocytic activity values were calculated as the percentage of the control. *P ` 0.01 compared to the control. **P ` 0.05, significantly different from the control.

Effect on nitric oxide production and phagocytic activity by macrophage To assess the effect of PS on nitric oxide production by macrophages, culture supernatants from macrophages incubated for 24 h and additional 24 h with various concentrations of PS were assayed for the presence of or nitrite ions. After 24 h of incubation, additional NO2 24 h incubated-macrophages which had previously been treated with 1 mg/ml concentration of PS produced the peak nitrite level (5.5 nmol/ml) (P ` 0.01) (Table I). Increasing the concentration of PS higher than 1 mg/ml did not further increase the nitrite level, but rather decreased the level. Macrophages which were pretreated with PS at test concentrations and incubated for 24 h did not produce detectable levels of nitrite. In vitro phagocytic activity test showed that PS had a

potent phagocytic activity (167% of the control) for zymosan at 10 mg/ml concentration (P ` 0.05) (Table I). Effect on cytokine production by macrophage TNF-a activity in the supernatants of macrophages stimulated with PS was assayed by using the murine fibroblast cell line L929. After 24 h-incubation, additional 24 h-incubated macrophages which were pretreated with PS (10 mg/ml concentration) showed 96% cytolysis of L929 (Table II), expressed as % cytolysis of L929 after staining the cells with crystal violet containing 10% formaldehyde. IL-1 and IL-6 production from 24 h-incubated macrophages which were

36

D. S. Lim et al.

Table II. The effect of polysaccharide on TNF-a activity, IL-1, and IL-6 production by macrophages. Items Concentration of polysaccharide (mg/ml) Control TNF-a activity (% lysis of L929)a IL-1b (pg/ml)b IL-6 (ng/ml)c
a b c

1 88 89 0.3

10 96 124 0.41

100 49 235* 0.47**

70 0.17

Additional 24 h-macrophages culture supernatant. 24 h-macrophages culture supernatant. 24 h-macrophages culture supernatant. d TNF-a activity values were calculated as the percentage of the control. *P ` 0.01 compared to the control. **P ` 0.05 compared to the control.

Figure 2. Evaluation of bacterial growth in blood from orbital sinus or plexus of infected mice. Blood samples were obtained from three mice from each group 1 day after S. aureus peritoneal challenge.

previously stimulated by PS (100 mg/ml) increased up to 235 pg/ml (P ` 0.01) and 0.47 ng/ml (P ` 0.05), respectively (Table II). Bacteriologic findings The preferential colonization of staphylococci from blood in the early stage of infection in host was assessed. Infected mice without the prior PS treatment had rapid bacterial spread (8106 CFU/ml). In contrast, approximately 90% lower bacterial counts (7105 CFU/ml) were found in blood from the PS treated group (0.025 mg/kg, i.v.) than the untreated group (Fig. 2). Effect of polysaccharide on nitrite levels in macrophages stimulated in infected mice In S. aureus-infected mice treated with PS (0.025 mg/ kg), nitric oxide production was found to increase (P ` 0.05) approximately 4 times compared with that of the untreated group at 24 and 48 h incubation (Fig. 3). Intracellular killing of S. aureus The quantitation of intracellular killing effect of macrophage stimulation by PS injected (i.v.) 3 h prior to S. aureus challenge was determined. In the PS treated (0.025 mg/kg) group, the intracellular concentrations of S. aureus in macrophages from infected mice decreased approximately by half, compared with the untreated group (Fig. 4), showing good agreement with the above result.

Figure 3. The effect of polysaccharide on nitric oxide production by macrophages from infected mice. C57BL/6J mice (n 6 in each group) were challenged by i.p. injection with 1.0108 CFU of S. aureus 3 h after i.v. injection (0.025 mg/kg) of the polysaccharide. Peritoneal macrophages were isolated 24 h after the challenge, and were cultured for 24 and 48 h. *P ` 0.05, **P ` 0.05, increase vs. control.

Prophylaxis with polysaccharide and vancomycin As shown in Table III, the prophylaxis effect with combined administration of PS and vancomycin indicated 100% survival (P ` 0.05), and the PS or vancomycin alone groups showed 67% and 50% survival, respectively. More detailed studies on prophylaxis effect with vancomycin are in progress.

Anti-Septicaemic Effect of Polysaccharide

37

Figure 4. The effect of polysaccharide on the intracellular killing of S. aureus in macrophages from infected mice. C57BL/6J mice (n 6 in each group) were challenged by i.p. injection with 1.0108 CFU of S. aureus 3 h after i.v. injection (0.025 mg/kg) of the polysaccharide. Peritoneal macrophages were isolated 24 h after the challenge, they were lysed, and the number of intracellular bacteria in CFU was determined by plating on blood agar plates.

Table III. Prophylaxis with polysaccharide and vancomycin. Treatment Dose (mg/kg/body weight) PS Saline PS Vancomycin PS Vancomycin
a

% Survivala Survivors (Survival at 10 days at 10 days)

Vancomycin 0 0 10 10 25 (8) 67 (50) 50 (50) 100* (92) 1/12 6/12 6/12 10/12

0 0.025 0 0.025

Survival was recorded 2 days after the challenge. *P ` 0.05 compared to the control.

Discussion
In a number of studies, the polysaccharide from Panax ginseng C.A. Meyer has been shown to be a potent possible biological response modifier (BRM), particularly for the proliferation of lymphocytes, generation of Lymphokine activated killer (LAK) cells, increase of Granulocyte macrophage-colony forming unit (GM-CFU) and production of cytokines [1214]. However, studies on the anti-septicaemic activity of the polysaccharide using S. aureus had hardly been reported previously, although some publications with other pathogens appeared [1517]. In the present study, the polysaccharide from Panax ginseng was shown to possess a potent anti-septicaemic activity through nitric oxide via cytokine production in

stimulated macrophage (Fig. 1), in agreement with the mechanism(s) observed by many others [1821]. These results suggested that the polysaccharide from Panax ginseng augment the production of these cytokines (TNFa, IL-1, IL-6 and IFN- ). Since cytokines such as tumor necrosis factor-a, interleukin-1, 6 and interferon- are known to be potent macrophage activators as well as immunomodulating agents, it was, therefore, possible that the Panax ginseng polysaccharide activated macrophages by upregulating the synthesis and production of these cytokines [22]. When activated by cytokines, macrophages show enhanced ability to kill both invading extracellular as well as intracellular pathogens residing within these cells [22]. One of the primary and important pathways by which intracellular killing may be achieved, at least in murine macrophages, is the production of reactive nitrogen intermediates (RNIs), including nitric oxide [23]. Nitric oxide produced by activated macrophages is a potent effector molecule and it is highly cytotoxic to invading microorganisms [23]. Nitric oxide has also been shown to be involved in the destruction of a number of intracellular parasites, including S. aureus [24]. Mycobacterium spp. [25] and Listeria monocytogenes [26]. Interestingly enough, the cytokines which are capable of inducing nitric oxide production by murine macrophages include tumour necrosis factor-a, interleukin-1 and interleukin-6, all of which were increased by the Panax ginseng polysaccharide in the present study. Cytokine-activated macrophages elevated nitric oxide levels, and the nitric oxide decreased after being persisted for a few days [27]. The clinical relevance of the present observation that the polysaccharide from Panax ginseng induced nitric oxide production in murine macrophages is not clear. Although human mononuclear phagocytes do not produce nitric oxide in response to specific cytokines which induce nitric oxide production in murine macrophages, they may respond to stimulation with different combinations of cytokines [28]. The ability of the Panax ginseng polysaccharide to modulate phagocyte functions might offer obvious therapeutic benefits for bacterial infections, since phagocytes play an essential role in the host's defense against infections by ingesting invading microorganisms and by mediating inflammation process. In addition, combined with vancomycin, the Panax ginseng polysaccharide showed the excellent anti-septicaemic effect (Table III). These results propose that the Panax ginseng polysaccharide may be applied to the clinical trials. Taken together, the present study suggests that the anti-septicaemic effect of the Panax ginseng polysaccharide against sepsis (incurred by S. aureus) is due to

38

D. S. Lim et al.
14 Kim KH, Lee YS, Jung IS, Park SY, Chung HY, Lee IR, Yun YS. Acidic polysaccharide from Panax ginseng, Ginsan, induces Th1 cell and macrophage cytokines and generates LAK cells in synergy with rIL-2. Planta Med 1998; 64: 110115. 15 Matsuda H, Kubo M, Tani T, Kitagawa I, Mizuno M. Pharmacological study on Panax ginseng C.A. Meyer (XI) Protective effect of red ginseng on infection (2) On phagocytic activity of mouse reticuloendothelial system. Shoyakugaku Zasshi 1987; 41: 135141. 16 Matsuda H, Hasegawa T, Kubo M. Pharmacological study on Panax ginseng C.A. Meyer. VII. Protective effect of red ginseng on infection (1) On phagocytic activity of mouse reticuloendotherial system. Yakugaku Zasshi 1985; 105: 948954. 17 Park SH, Jo JS. The effects of Korean ginseng (Panax ginseng C.A. Meyer) extracts and their fractions on the growth of Escherichia coli. Korean J Ginseng Sci 1993; 17: 203209. 18 Walley KR, Lukacs NW, Standiford TJ. Balance of inflammatory cytokines related to severity and mortality of murine sepsis. Infect Immun 1996; 64: 47334738. 19 Zhao YX, Abdelnour A, Kalland T, Tarkowski A. Overexpression of the T-cell receptor Vb3 in transgenic mice increase mortality during infection by enterotoxin A-producing Staphylococcus aureus. Infect Immun 1995; 63: 44634469. 20 Cohen J. Meningococcal disease as a model to evaluate novel antisepsis strategies. Crit Care Med 2000; 28: S64S67. 21 Muller Kobold AC, Tulleken JE, Zijlstra JG, Sluiter W, Hermans J, Kallenberg CGM, Cohen Tervaert JW. Leukocyte activation in sepsis; correlations with disease state and mortality. Intensive Care Med 2000; 26: 883892. 22 Corradin SB, Buchmuller-Rouiller YB, Mauel J. Phagocytosis enhances murine macrophages activation by interferon- and tumor necrosis factor-a. Eur J Immunol 1991; 21: 25532558. 23 Fortier AH, Polsinelli T, Green SJ, Nacy CA. Activation of macrophages for destruction of Francisella tularensis: identification of cytokines, effector cells and effector molecules. Infect Immun 1992; 60: 817825. 24 Malawista SE, Montgomery RR, van Blaricom G. Evidence of reactive nitrogen intermediates in killing of staphylococci by human neutrophil cytoplasts. J Clin Invest 1992; 90: 631636. 25 Denis M. Tumor necrosis factor and granulocyte-macrophage colony-stimulating factor stimulate human macrophages to restrict growth of virulent Mycobacterium avium and to kill avirulent M. avium: Killing effector mechanism depends on the generation of reactive nitrogen intermediates. J Leuko Biol 1991; 49: 380387. 26 Beckerman KP, Rogers HW, Corbett JA, Schreiber RD, Mcdaniel ML, Unanue ER. Release of nitric oxide during the T-cell-independent pathway of macrophage function: its role in resistance to Listeria monocytogenes. J Immunol 1993; 150: 888895. 27 Green SJ, Nacy CA. Antimicrobial and immunopathologic effects of cytokine-induced nitric oxide synthesis. Cur Opin Infect Dis 1993; 6: 384396. 28 Eizirik E. Nitric oxide synthase and antimicrobial armature of human macrophages. J Infect Dis 1994; 170: 744745.

the production of nitric oxide by the macrophage activation. The macrophage activation by the cytokines such as TNF-a, IL-1 and IL-6 was implied as a key factor in the anti-septicaemic activity of Panax ginseng polysaccharide. Studies on anti-septicaemic effect against methicillin-resistant S. aureus (MRSA) or vancomycinresistant S. aureus (VRSA) by the Panax ginseng polysaccharide are underway.

References
1 Murphy K, Haudek SB, Thompson M. Molecular biology of septic shock. New Horiz 1998; 6: 181193. 2 Rangel-Frausto MS. The epidemiology of bacterial sepsis. Infect Dis Clin North Am 1999; 13: 299312. 3 Limjoco CM, Youmans K. Reviewing the details of coding septicemia. J AHIMA 2000; 71: 7980. 4 Van der Poll T, van Deventer JH. Cytokines and anticytokines in the pathogenesis of sepsis. Infect Dis Clin North Am 1999; 13: 413426. 5 Michel P, Glauser MD. Pathophysiologic basis of sepsis: Considerations for future strategies of intervention. Crit Care Med 2000; 28: S4S8. 6 Nilsson IM, Lee JC, Bremell T, Ryden C, Tarkowski A. The role of Staphylococcal polysaccharide microcapsule expression in septicemia and septic arthritis. Infect Immun 1997; 65: 42164221. 7 Hiramatsu K, Hanaki H, Ino T, Yabuta K, Oguri T, Tenover FC. Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. J Antimicrob Chemother 1997; 40: 135136. 8 Hanaki H, Kuwahara-Arai K, Boyle-Vavra S, Daum RS, Labischinski H, Hiramatsu K. Activated cell-wall synthesis is associated with vancomycin resistance in methicillin-resistant Staphylococcus aureus clinical strains Mu3 and Mu50. J Antimicrob Chemother 1998; 42: 199200. 9 Klimetzek V, Remold HG. The murine bone marrow macrophage, a sensitive indicator cell for murine migration inhibitory factor and a new method for their harvest. Cell Immunol 1980; 53: 257. 10 Flick DA, Gifford GE. Comparison of in vitro cell cytotoxicity assays for tumor necrosis factor. J Immunol 1984; 68: 167175. 11 Nordan PR. Measurement of human and murine IL-6. In: Coligan JE, (eds) Current Protocols in Immunology, 2nd edn. NY: Greene publishing and Wiley-Interscience, 1994, 6.6.16.6.5. 12 Yun YS, Lee YS, Jo SK. Inhibition of autochthonous tumor by ethanol insoluble fraction from Panax ginseng as an Immunomodulator. Planta Med 1993; 59: 521524. 13 Lee YS, Chung IS, Lee IR, Kim KH, Hong YS, Yun YS. Activation of multiple effector pathways of immune system by the antineoplastic immunostimulator acidic polysaccharide Ginsan isolated from Panax ginseng. Anticancer Res 1997; 17: 323332.