Jurnal s. Aureus Zymosan

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Journal of Infection (2002) 45: 3238doi: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

1Laboratory of Immunology, Korea Cancer Center Hospital, KAERI, Seoul 139-706 and2Animal 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) isolatedfrom Panax ginseng C.A. Meyer (Araliaceae) by nitric oxide production from stimulated macrophage. In vitro assaysfor the activity measurement of PS, NO production test with Greiss reagent, phagocytic activity test using zymosan andcytokines production test using ELISA kit were also conducted. In vivo anti-septicaemic activity was assessed by usingC57BL/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 minimuminhibitory concentration (MIC) test, conducted in vitro (data not shown). Nitric oxide production via macrophageactivation showed the highest value of 5.5 nmol/ml at 1 mg/ml PS. In in vitro phagocytic activity test, PS at 10 mg/mlconcentration showed a potent phagocytic activity for zymosan with 167% of the control. Production of TNF-a bymacrophage activation at 10 mg/ml of PS was 96% lysis of L929. Also production of IL-1 and IL-6 by stimulation ofmacrophage with 100 mg/ml PS dose increased to 235 pg/ml and 0.47 ng/ml, respectively. The low mortality ofPS treated (0.025 mg/kg) infected mice was concurrent with decreased bacterial content in the blood. Nitric oxideproduction in S. aureus infected mice whose macrophage was stimulated by PS (0.025 mg/kg) increasedapproximately 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 by50%, 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 apotent anti-septicaemic activity by stimulating macrophage and a potentiality as an immunomodulator against sepsisoccurred by Staphylococcus aureus. # 2002 The British Infection Society

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,

genitourinary system, or skin. It can also occur con-

currently with or be preceded by infections like osteo-

myelitis, 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 bac-

terial infections trigger macrophages to produce cyto-kines, 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

* 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:

[email protected] or [email protected] (J. Y. Song).

0163-4453/02/$35.00 # 2002 The British Infection Society


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competent persons in the community [6]. Staphylococcal

pathogenicity depends upon the effectiveness of the host

defense in dealing with a wide variety of bacterial com-

ponents 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 anti-

biotic-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.

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.b12,000) against distilled water. After removal

of insoluble materials in the dialysate, the supernatant

was lyophilized to yield 15 g of powder. The PS pre-

paration was purified by Sephacryl S-500 and DEAE-A50 column chromatography, and determined to be

a(136) glucopyranoside and b(236) 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 tem-

perature and light, and were fed standard laboratorychow 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

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

1.0109 CFU/ml. The mice were infected by intraper-

itoneal injection of S. aureus (1.0108) in PBS.

Characterization of anti-septicaemic biological activity

Acute sepsis models using S. aureus intraperitoneal

challenge were developed to evaluate the anti-septi-

caemic 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 var-

ious 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 administra-

tion of PS by intraperitoneal injection of 0.1 ml(1.0108 CFU) ofS. 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 mod-

ified Eagle's medium (DMEM) with 10% FBS and resus-

pended 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% CO2humidified 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 (4105

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

Anti-Septicaemic Effect of Polysaccharide 33


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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 wereexpressed 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 byretro-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 for48 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 incu-

bated in either medium (DMEM containing 10% FBS)

alone or medium supplemented with the PS for 24 h andadditional 24 h with fresh medium in 96 well micro-

plate. After culture, 50 ml of each supernatant was taken

and nitric oxide was measured using Nitric oxide ana-

lyzer (Antek Inst., Houston, TX). Peritoneal macro-

phages 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

supernatant was taken and mixed with 100 ml of Greiss

reagent (1% sulfanilamide, 0.1% naphthylethylenedia-

mine dihydrochloride and 2.5% phosphoric acid). After

10 min, the concentration of nitric oxide in the super-

natant 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 macro-

phage stimulated by the PS which was injected intrave-

nously 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 lysosta-

phin (Sigma) at 5 mg/ml final concentration was added to

each tube, and the tubes were incubated for an additional20 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 admi-

nistered with 0.1 ml of vancomycin (the final con-

centration, 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 chal-

lenged 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) soft-

ware. Results are presented as means

the standarderrors of the means (SEMs).

Results

Anti-septicaemic activity of polysaccharide

Acute sepsis model by intraperitoneal challenge with

S. aureus in mice was developed to evaluate the

34 D. S. Lim et al.


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anti-septicaemic activity of PS prepared from Panax

ginseng. Fig. 1a summarizes representative dose respon-

ses observed in this model 2 days after S. aureus intra-

peritoneal challenge with PS. A single dose of PS (0.025

mg/kg body weight) administered 3 h before the chal-

lenge 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).

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

NO2

or nitrite ions. After 24 h of incubation, additional24 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, addi-

tional 24 h-incubated macrophages which were pre-

treated 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 produc-

tion from 24 h-incubated macrophages which were

Figure 1. Anti-septicaemic activity of polysaccharide on the S. aureus peritoneal sepsis challenge. C57BL/6J mice (n12 in each group) werechallenged by i.p. injection with 1.0108 CFU ofS. 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.

Table I. The effect of polysaccharide on nitric oxide production andphagocytic activity by macrophages.

Items Concentration of polysaccharide(mg/ml)

Control 1 10 100

No production (nmol/ml)a 1.7 5.5* 3.9 2.4Phagocytic activity

(% of control)b c 157** 167** 140**

a24 h-macrophages culture supernatant.bSee ``Materials and Methods''.cPhagocytic activity values were calculated as the percentage of thecontrol.*P`0.01 compared to the control.**P`0.05, significantly different from the control.



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previously stimulated by PS (100 mg/ml) increased upto 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, approxi-

mately 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 macro-

phage 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.

Prophylaxis with polysaccharide

and vancomycin

As shown in Table III, the prophylaxis effect with com-

bined administration of PS and vancomycin indicated

100% survival (P`0.05), and the PS or vancomycin

alone groups showed 67% and 50% survival, respec-

tively. More detailed studies on prophylaxis effect with

vancomycin are in progress.

Table II. The effect of polysaccharide on TNF-a activity, IL-1, and IL-6production by macrophages.

Items Concentration of polysaccharide (mg/ml)

Control 1 10 100

TNF-a activity(% lysis of L929)a

d 88 96 49

IL-1b (pg/ml)b 70 89 124 235*IL-6 (ng/ml)c 0.17 0.3 0.41 0.47**

aAdditional 24 h-macrophages culture supernatant.b24 h-macrophages culture supernatant.c24 h-macrophages culture supernatant.d TNF-a activity values were calculated as the percentage of thecontrol.*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 orplexus of infected mice. Blood samples were obtained from three micefrom each group 1 day after S. aureus peritoneal challenge.

Figure 3. The effect of polysaccharide on nitric oxide productionby macrophages from infected mice. C57BL/6J mice (n6 in eachgroup) were challenged by i.p. injection with 1.0108 CFU ofS. aureus3 h after i.v. injection (0.025 mg/kg) of the polysaccharide. Peritonealmacrophages were isolated 24 h after the challenge, and were culturedfor 24 and 48 h. *P`0.05, **P`0.05, increase vs. control.

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Discussion

In a number of studies, the polysaccharide from Panax

ginseng C.A. Meyer has been shown to be a potent pos-

sible biological response modifier (BRM), particularly

for the proliferation of lymphocytes, generation of

Lymphokine activated killer (LAK) cells, increase ofGranulocyte 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 (TNF-

a, IL-1, IL-6 and IFN-). Since cytokines such as tumor

necrosis factor-a, interleukin-1, 6 and interferon- areknown to be potent macrophage activators as well as

immunomodulating agents, it was, therefore, possible

that the Panax ginseng polysaccharide activated macro-

phages by upregulating the synthesis and production of

these cytokines [22]. When activated by cytokines,

macrophages show enhanced ability to kill both invad-

ing 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 byactivated 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 poly-

saccharide 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 pro-

duce nitric oxide in response to specific cytokines which

induce nitric oxide production in murine macrophages,

they may respond to stimulation with different combi-

nations of cytokines [28].

The ability of the Panax ginseng polysaccharide to

modulate phagocyte functions might offer obvious

therapeutic benefits for bacterial infections, since pha-

gocytes play an essential role in the host's defenseagainst infections by ingesting invading microorganisms

and by mediating inflammation process. In addition,

combined with vancomycin, the Panax ginseng poly-

saccharide 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 poly-

saccharide against sepsis (incurred by S. aureus) is due to

Figure 4. The effect of polysaccharide on the intracellular killing ofS. aureus in macrophages from infected mice. C57BL/6J mice (n6 ineach group) were challenged by i.p. injection with 1.0108 CFU ofS. aureus 3 h after i.v. injection (0.025 mg/kg) of the polysaccharide.

Peritoneal macrophages were isolated 24 h after the challenge, theywere lysed, and the number of intracellular bacteria in CFU wasdetermined by plating on blood agar plates.

Table III. Prophylaxis with polysaccharide and vancomycin.

Treatment Dose(mg/kg/body weight)

% Survivala

(Survivalat 10 days)

Survivorsat 10 days

PS Vancomycin

Saline 0 0 25 (8) 1/12PS 0.025 0 67 (50) 6/12

Vancomycin 0 10 50 (50) 6/12PSVancomycin 0.025 10 100* (92) 10/12

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



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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 poly-

saccharide. Studies on anti-septicaemic effect against

methicillin-resistant S. aureus (MRSA) or vancomycin-resistant S. aureus (VRSA) by the Panax ginseng poly-

saccharide are underway.

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Jurnal s. Aureus Zymosan