augmentation of cellular immunity and protection against influenza virus

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Basic nutritional investigation

Augmentation of cellular immunity and protection against influenza virus

infection by bovine late colostrum in mice

Kenji Uchida M.S. a,*, Naoyuki Hiruta B.S. b, Hiroshi Yamaguchi B.S. a, Kousaku Yamashita M.S. a,Katsuyuki Fujimura B.S. a, Hisako Yasui Ph.D. b

a Central R&D Laboratory, Kobayashi Pharmaceutical Co., Ltd., 1-30-3 Toyokawa, Ibaraki, Osaka 567-0057, Japanb Sciences of Functional Foods (Integrated Department), Graduate School of Agriculture, Shinshu University 8304 Minamiminowa, Kamiina, Nagano 399-4598, Japan

a r t i c l e i n f o

Article history:

Received 19 July 2011

Accepted 25 July 2011

Keywords:

Skimmed and concentrated bovine late

colostrum

Influenza virus infection

Natural killer cells

Cytokines product

Mice

a b s t r a c t

Objective: We investigated whether oral administration of skimmed and concentrated bovine late

colostrum (SCBLC) activates the immune system and protects against influenza virus (Flu)

infection.

Methods: Murine Peyer’s patch (PP) cells (2.5 105) were cultured in 0.1 ml RPMI-1640 supple-

mented with SCBLC at a concentration of 0, 0.1 or 1.0 mg/ml. To determine the levels of IL-12 and

IFN-, supernatants were collected on day 3. Mice were orally administered sterile saline solution

(control group), or 400 g/g body weight (SCBLC 400 group) or 2,000 g/g body weight (SCBLC 2,000

group) of SCBLC for three weeks. These mice were measured for natural killer (NK) cells activity on

PP cells, splenocytes and lung cells. Also, these mice in the control and SCBLC 2,000 groups were

infected with Flu and were measured for the accumulated symptom rate.

Results: In PP cells cultured with SCBLC, the levels of IL-12 and IFN- were significantly increased in

vitro. Oral administration of SCBLC to mice significantly increased NK cell activity of PP cells,

splenocytes and lung cells. The accumulated symptom rate of the SCBLC 2,000 group was signif-icantly lower than that of the control group in a mouse model of Flu infection.

Conclusion: These results indicate that oral administration of SCBLC activates not only systemic

cellular immunity but also local cellular immunity, such as in the respiratory tract, and that acti-

vation of cellular immunity is one of the mechanisms of amelioration of Flu infection.

Ó 2012 Elsevier Inc. All rights reserved.

Introduction

Upper respiratory tract infections (URTI) often develop in the

general population. Influenza virus (Flu), one cause of URTI, is an

etiologic agent that causes acute respiratory disease with a high

mortality rate [1]. A large numberof Fluinfection-induced deaths

are reported every year worldwide. Infection with Flu generates

both innate and adaptive immune responses. Both B and T cells

have important roles in Flu clearance [2]. The involvement of

natural killer (NK) cells in the response to Flu infection has also

been indicated [3] and confirmed in vivo [4]. NK cells control

influenza at the early stages of infection through their cytotoxic

activity and cytokine/chemokine production [5–8]. Recent

studies havehighlighteda direct involvementof one of theNK cell

activation receptors, natural cytotoxicity triggering receptor 1

(NCR1), in the recognition of the Flu-derived hemagglutinin

protein [9–11]. Mice expressing an NCR1-dead mutant protein

were more susceptible to Flu infections than wild-type mice [11].

Thus, NK cells play a critical role in the clearance of Flu infection.

Bovine colostrum is the early milk produced by cows during

the first several days postparturition. It is well known that

colostrum, which is a rich source of antiinfection proteins such as

Immunoglobulin G (IgG), secretory Immunoglobulin A lacto-

ferrin, and lysozyme, is important for the growth and health of

mammalian neonates [12–14]. It was recently proposed that

bovine colostrum provides passive protection against URTI and

symptoms of Flu infection in adult humans [15,16], because

IgG-bound viruses and bacteria are inhibited from binding to

host cells. To further clarify the effectof bovine colostrum against

general infection and URTI, we previously analyzed the effects of

complete bovine colostrum, which has a large amount of anti-

bodies against various pathogens [17].

However, early (i.e., within 5 d after parturition) colostrum

from cows is not approved for use as a food in Japan. We therefore

* Corresponding author. Tel.: 072-640-0114 (International: þ81-72-640-

0114); fax: 072-640-0137 (International: þ81-72-640-0137).

E-mail address: [email protected] (K. Uchida).

0899-9007/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.doi:10.1016/j.nut.2011.07.021

Contents lists available at ScienceDirect

Nutrition

j o u r n a l h o m e p a g e : w w w . n u t r i t i o n j r n l . c o m

Nutrition 28 (2012) 442–446

mailto:[email protected]

http://dx.doi.org/10.1016/j.nut.2011.07.021

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evaluated the protective ef ficacy of skimmed and concentratedbovine colostrum obtained from healthy lactating cows on days

6-7 after parturition, hereafter referred to as SCBLC, against

infection. This type of colostrum can be used as a food in Japan. The

IgG of SCBLC suppresses infection by rotavirus [18] and adenovirus

(unpublished results) and inhibits binding of norovirus viruslike

particles to human intestinal Caco-2 cells [19]. These studies

suggest that IgG of SCBLC might inhibit URTI and diarrheal virus

infection. Moreover, we previously reported that oral administra-

tion of SCBLC was effective in preventing URTI and in reducing the

duration of URTI in a double-blind clinical trial of 196 infants [20].

We therefore concluded that the IgG of SCBLC was one of the

factors that prevented virus infection and URTI.

To clarify the mechanism by which SCBLC reduces the dura-

tion of URTI, the purpose of the present study was to investigatewhether oral administration of SCBLC to mice activates the

innate immune system of the respiratory tract and whether it

protects against Flu infection.

Materials and methods

Animals

Six-week-old (for in vitro studies) and 12-wk-old (for in vivo studies) female

BALB/c mice were obtained from Japan SLC (Hamamatsu, Japan). These animals

were maintained at 24 Æ 2C under a 12-h light/12-h dark cycle. Commercial

mouse pellets (Oriental Yeast Co., Tokyo, Japan) were continuouslyavailable from

stainless steel feeders, and water was provided ad libitum from drinking bottles.

All procedures were conducted in accordance with the Guidelines for Animal

Experiments at Shinshu University and were approved by the Ethics Committee

at Shinshu University.

SCBLC

SCBLC was prepared from bovine colostrum produced 6 to 7 d after partu-

rition. Briefly, the colostrum was centrifuged to partially remove the fat, filtered

with a membrane to partially remove low molecular weight substances, heat-

treated at 72C for 16 s, and spray-dried. SCBLC consisted of 49.8% protein

including 11.0% IgG, 36.4% lactose, 1.8% fat, 7.8% minerals, and 4.2% moisture.

In fluenza virus

Influenza A/PR/8/34 (PR8, H1N1) virus was grown in the allantoic sacs of 11-

d-old chicken embryos for 2 d at 34C [21]. The allantoic fluid was removed and

stored at À80C. The titer of virus in the allantoic fluid was expressed as the 50%

tissue culture infecting dose (TCID50) [22]. The viral titer of allantoic fluid was

106.9 TCID50/mL.

In vitro experiments

After preliminary breeding for 1 wk, mice were anesthetized with dieth-

ylether, killed by exsanguinations, and intestinal tract Peyer ’s Patch (PPs) were

collected for in vitro assays. PP cells were prepared according to the dispase-treatment method of Frangakis et al. [23] as described by Yasui et al. [24]. PP

cells (2.5Â105) were cultured in0.1 mLRPMI-1640supplemented with 10% heat-

inactivated fetal bovine serum (FBS; Sigma, St. Louis, MO, USA), 100 U/mL peni-

cillin,100 mg/mLstreptomycin,and 2mg/mLConcanavalinA TypeIV (ConA; Sigma)

with SCBLCat a concentration of 0, 0.1, or 1.0 mg/mL at37C i n a 5 % C O2 incubator.

To determine cytokine levels, supernatants were collected on day 3 and were

stored at À80C until further analysis.

In vivo experiments

Experimental schedule

After preliminary breeding for 1 wk, mice were divided into several groups

that were orally administered sterile saline solution (control group), or 400 mg/g

body weight (SCBLC400 group) or 2000mg/gbody weight(SCBLC 2000group)of

SCBLC dissolved in sterile saline solution. These solutions were administered six

times per week for 3 wk (Fig. 1).

Three weeks after the start of the experiment, mice were anesthetized with

diethylether; blood was collected from the abdominal vena cava, and the mice

were subsequently killed. Mouse serum, prepared from blood collected using

heparin sodium (Mitsubishi Tanabe Pharma Co., Osaka, Japan), was stored at

À80C until analysis of cytokine levels. Intestinal tract PPs spleens and lungs

were also collected from the mice (Fig. 1).

The PPs and lungs were finely minced and incubated for 90 min with RPMI-

1640 (Sigma) supplemented with 0.33 mg/mL collagenase (Sigma) at 37C in a 5%

CO2 incubator. The spleens were mashed in RPMI-1640. To dissociate single cells,

these cells from each tissue were gently tapped and strained through a cell

strainer (BD Biosciences, Franklin Lakes, NJ, USA). These single cells were washed

twice with RPMI-1640 (Sigma) by centrifugation and were resuspended in

medium supplemented with 10% heat-inactivated FBS.

Three weeks after the start of the experiment, the mice in the control and

SCBLC 2000 groups were infected with Flu. Upper respiratory infection was

Fig. 1. Protocol for oral administration of SCBLC and Flu infection.

0.2

0.3

0.4

D

0 5 4 A .

)

1.0

1.5

2.0

A B

**

0.0

0.1

Control SCBLC

0.1mg/ml

SCBLC

1.0mg/ml

. O (

D

0.0

0.5

Control SCBLC

0.1mg/ml

SCBLC

1.0mg/ml

Fig. 2. Effect of SCBLC on the production of IL-12 and IFN-g by PP cells. P cells from mice were cultured in the presence of ConA and SCLBC at a concentration of 0 (control;

black bars; n ¼ 3), 0.1 (gray bars; n ¼ 3), or 1.0 (white bars; n ¼ 3) mg/mL for 3 d. The levels of IL-12 (A) and IFN-g (B) in the supernatants were then determined using an

ELISA. Each bar represents the mean Æ standard deviation (error bar) of triplicate samples. The single asterisks indicate statistically significant difference from the controlusing Dunnett’s test, with P values <0.05, respectively.

K. Uchida et al. / Nutrition 28 (2012) 442–446 443










induced by dropping 1 mL of fluid containing 105.9 TCID50 of PR8 into each nostril

(2 mL per mouse). Three days after induction of upper respiratory infection, 20mL

of phosphate buffered saline (pH 7.4) was administered intranasally to dissem-

inate increased numbers of virus from the nasal cavity to the lower respiratory

tract.The accumulated symptom rates of the mice were then determined (Fig.1).

Thesymptomsmonitored were erect hair anddecreasein body weightaccording

to the method of Hori et al. [25].

Cytokines production and cytotoxicity assay using Yac-1 cells

For analysis of cytokine production from lung cells, 2.5 Â 105 lung cells were

cultured in 0.2 mL RPMI-1640 supplemented with 10% heat-inactivated FBS, 100

U/mL penicillin (Sigma), and 100 mg/mL streptomycin (Sigma) in the presence of

2 mg/mLConAat 37C i n a 5 % C O2 incubator. Supernatants were collected after24

h and were stored at À80C until further analysis.

For cytotoxicity assay of PP cells, the LDH cytotoxicity detection kit (Takara

Bio Inc., Otsu, Japan) was used. PP cells (2 Â 106) were incubated for 4 h at 37C

with 1 Â104 Yac-1 cells in 0.2mL RPMI 1640 supplemented with 10% FBS, in a 5%

CO2 incubator. Supernatants were collected and cytotoxicity was determined

according to the procedure in the kit manual. Cytotoxicity of the splenocytes and

the lung cells was determined using a LIVE/DEAD Cell-Mediated cytotoxicity kit

(LifeTechnologies Inc.,Carlsbad,CA, USA).Cells (2.5Â105) wereincubatedfor 4 h

at 37C with 5 Â 104 Dio-labeled Yac-1 cells in 0.2 mL RPMI 1640 supplemented

with 10% FBS and 0.15 mM/mL propidium iodide (Sigma), in a 5% CO 2 incubator.

Cells were then collected and analyzed using flow cytometry according to the

procedure in the kit manual (Beckman Coulter Inc., Brea, CA, USA).

ELISA

Cytokine levels in the supernatants of the PP cells (in vitro study), in the

serum, and in the supernatants of the lung cells (in vivo study) were determined

using a sandwich enzyme-linked immunosorbent assay (ELISA). In the in vitro

study interleukin-12 (IL-12) and interferon-g (IFN-g) levelswere measured using

monoclonal rat anti-mouse IL-12 (p40/p70) and polyclonal rabbit anti-mouse/rat

IFN-g antibodies, respectively, as the capture antibody, and biotinylated mono-

clonal rat anti-mouse IL-12 and biotinylated rabbit anti-mouse IFN-g antibodies,

respectively, as the detection antibody. All antibodies were purchased from BD

Biosources. The absorbance at 450 nm was measured according to the method of

Segawa et al. [26]. In the in vivo study, IL-12 (p40/p70) levels were assayed using

an Immunoassay kit(BD Biosource) andIFN-g levelswere assayed using a Mouse

IFN-g ELISA kit(eBioscience Inc., SanDiego, CA,USA).The methods used followedthe protocol of each kit.

Statistical analyses

Comparisons of IL-12 and IFN-g production and cytotoxic activity among

control group and two SCBLC groups were performed using Dunnett ’s test for

three-group comparison and Welch’s t test for two-group comparison, respec-

tively. Differences in the accumulated symptom rate were examined by means of

Fisher’s exact test. Probability values of less than 5% were considered significant.

Results

Effect of addition of SCBLC on IL-12 and IFN-g production by

PP cells

When PP cells from mice were cultured with SCBLC for 3 d,

the levels of IL-12 and IFN-g produced in the supernatant were

significantly increased in a dose-dependent manner compared to

controls (Fig. 2A and 2B). Therefore, SCBLC increases IL-12 and

IFN-g production by PP cells.

Effect of oral administration of SCBLC on the immune response

over 3 wk

Effect on the cytotoxic activity of PP cells

The cytotoxic activity of PP cells prepared from the control,

SCBLC 400, and SCBLC 2000 groups of mice differed. The cyto-

toxic activity of PP cells from the SCBLC 400 group was not

significantly higher than that of the control group. However, thecytotoxic activity of PP cells from the SCBLC 2000 group was

significantly higher than that of the control group (Fig. 3).On the

basis of these data, SCBLC wasorally administeredusing a dose of

2000 mg/kg/d for the rest of the study.

Effect on the cytotoxic activity of splenocytes and lung cells

We next investigated the effect of oral administration of

SCBLC to mice for 3 wk on the cytotoxic activity of splenocytes

and lung cells. As shown in Figure 4, the cytotoxic activity of the

30

40

50

60

70 *

0

10

20

30

Control

group

SCBLC

400 group

SCBLC

2000 group

S p e c i f i c l y s i s ( % )

Fig. 3. Effect of oral administration of SCBLC on the cytotoxic activity of PP cells.

Three weeks after the start of the experiment, PP cells were prepared from the

Control group (black bars; n ¼ 7), the SCLBC 400 group (gray bars; n ¼ 7), or the

SCBLC 2000 group (white bars; n ¼ 7). Cytotoxic activity was determined using an

LDH assay. Each bar represents the mean Æ standard deviation (error bar) of trip-

licate samples. The asterisk indicates a statistically significant difference from the

control using Dunnett’s test at a P value <0.05.

A B

10

15

20

4

6* *

0

5

Control SCBLC

2000 group

S p e c i f i c l y s i s ( % )

0

2

Control SCBLC

2000 group

Fig. 4. Effect of oral administration of SCBLC on the cytotoxic activity of splenocytes and lung cells. Three weeks after the start of the experiment, splenocytes (A) and lung

cells (B) were prepared from the Control group (black bars; n ¼ 5) or the SCBLC 2000 group (white bars; n ¼ 5). Cytotoxic activity was determined using flow cytometry. Each

bar represents the mean Æ standard deviation (error bar) of triplicate samples. The asterisks indicate statistically signi ficant difference from the control using Welch ’s t test, ata P value of <0.05.




splenocytes and lung cells from the SCBLC 2000 group was

significantly higher than that of the control group.

Effect on the levels of IL-12 and IFN-g in serum and on their

production by lung cells

We further investigated the effect of oral administration of

SCBLC to mice for 3 wk on cytokine production by the lungs and

on serum cytokine levels. The mean concentration of IL-12 and

IFN-g in serum did not differ between the control and SCBLC

2000 groups (Fig. 5). However, in the lung cells, although IL-12

production was not detected (data not shown), the mean

concentration of IFN-g produced by the SCBLC 2000 group was

significantly higher than that produced by the control group

(Fig. 6).

Effect of oral administration of SCLBLC on the accumulated

symptom rate of mice nasally infected with FluWe finally investigated whether oral administration of SCBLC

to mice for 3 wk affects the accumulated symptom rate of mice

that were nasally infected with Flu, using an infection method

that disseminates the virus from the nasal cavity to the lower

respiratory tract. The accumulated symptom rate in the SCBLC

2000 group wassignificantly lower (78.6% versus 96.7%; P <0.05)

than that in the control group (Table 1).

Discussion

In this study, we demonstrated that oral administration of

bovine colostrum could modulate NK cell activity and protect

against Flu infection. A clinical study has already shown that oral

administration of SCBLC to infants decreases the duration of URTI

[20]. Moreover, Cesarone et al. reported that oral administration

of skimmed and concentrated bovine fast colostrum to adults

decreased theperiod of Flu symptoms [16]. These studies suggest

that oral administration of bovine colostrum might result in

effective NK cell activity and enhance recovery from infection,

thereby contributing to its protective effects against the early

stages of viral infection.

For NK cell activation it is necessary that the NK cell be

stimulated by IL-12 and/or IFN-a/b, which are produced by cells

such as macrophages [8]. Once activated, NK cells can acquire the

ability to produce IFN-g following contact with cells lacking MHC

class I expression [8]. In this study, cultivation of mouse PP cells

with SCBLC increased the production of IL-12 and IFN-g (Fig. 2).

Furthermore, SCBLC that was orally administered to mice

augmented PP cell cytotoxicity toward Yac-1 cells (Fig. 3). These

findings suggest that oral administration of SCBLC induced

effective NK cell activity by increasing IL-12 production by PP

cells in the intestinal tract. The data in Figure 4 show that SCBLC

that was orally administered to mice also augmented the cyto-

toxicity of splenocytes and lung cells toward Yac-1 cells, sug-gesting that oral administration of SCBLC also induced effective

NK cell activity of mouse spleens and lungs. We therefore

demonstrated in this study that oral administration of SCBLC

effectively induced NK cell activity of cells in PP, the spleens, and

the lungs and transferred innate immunity from the intestinal

tract to the respiratory tract.

Hori et al. reported that NK cell activity is strongly related to

protection against Flu [25]. The results in Table 1 show that the

rate of symptom development in mice orally administered SCBLC

was significantly lower than that in mice administered saline,

using a mouse model of Flu infection in which the virus moved

from the upper respiratory tract to the lower respiratory tract

[25]. These data suggest that a possible mechanism by which oral

administration of SCBLC protects against Flu infection is throughactivationof NK cells in thelocalcellularimmune system of lungs.

B

n.s.

A

n.s.

0

50

100

150

Control groupControl group SCBLC

2000 group

I F N -

γ

( p g / m l )

0

50

100

150

SCBLC

2000 group

I L - 1 2 ( p g / m l )

Fig. 5. Effect of oral administration of SCBLC on the serum concentration of IL-12 and IFN-g. Three weeks after the start of the experiment, serum was collected from the

Control group (black bars; n ¼ 5) or the SCBLC 2000 group (white bars; n ¼ 5) and the concentrations of IL-12 (A) and IFN-g (B) were determined using an ELISA. Each bar

represents the mean Æ standard deviation (error bar) of samples. n.s. indicates no significant difference from the control using Welch’s t test.

0

50

100

150

200

250

Control group SCBLC

2000 group

I F N - γ

( p g / m l )

*

Fig. 6. Effect of oral administration of SCBLC on IFN-g production by lung cells.

Three weeks after the start of the experiment, lung cells from the Control group

(black bars; n ¼ 5) or the SCBLC 2000 group (white bars; n ¼ 5) were collected and

cultured in the presence of ConA. The concentration of IFN-g in the supernatants

was determined using an ELISA. Each bar represents the mean Æ standard deviation

(error bar) of samples. The asterisk indicates a statistically significant differencefrom the control using Welch’s t test at a P value <0.05.




We next addressed the mechanismof NK cell activity in lungs.

In the present study, oral administration of SCBLC augmented NK

cell activity in the lungs of mice (Fig. 4). There are three possible

factors that could have mediated the SCBLC-induced augmen-

tation of the NK cell activity of lungs: 1) the milk ingredients of

SCBLC may have reached the lungs and augmented macrophage

or NK cell activity; or cells that were activated via SCBLC in PPs

such as 2) IL-12-producing macrophages or other IL-12-

producing cells, or 3) NK cells, could have homed to the lungs

from PPs. Regarding factor (1), if milk ingredients of SCBLC were

absorbed into the blood and carried to the lungs, the level of IL-

12 and IFN-g in mouse serum should be augmented. However,

oral administration of SCBLC did not augment the levels of IL-12

or IFN-g in mouse serum (Fig. 5), suggesting that the milk

ingredients of SCBLC might not be absorbed into the blood.

Regarding factor (2), if homing of IL-12-producing cells such as

macrophages from PPs to the lungs through the bloodstream

occurred as a result of SCBLC administration, such homing would

augment the level of IL-12 in mouse serum. However, oral

administration of SCBLC did not augment the level of IL-12 in

mouse serum (Fig. 5), suggesting that homing of IL-12-producing

cells did not occur. The data shown in Figure 5 therefore appear

to rule out factors (1) and (2), leaving the hypothesis that NK

cells, activated in PPs as a result of SCBLC administration, homed

to thespleen and the lungs. Ourdata provide evidence to supportthis hypothesis. Thus, activated NK cells produce IFN-g when the

NK cells contact cells that have lost MHC class 1 expression [8].

We showed that, whereas the level of serum IFN-g in mice did

not change following oral administration of SCBLC compared to

control mice, the lung cells of mice showed strongly enhanced

IFN-g production, but no change in IL-12 production (Fig. 6).

In addition, oral administration of SCBLC protected against Flu

infection (Table 1). One mechanism by which oral administration

of SCBLC might enhance the protective effect of NK cells against

Flu is by inducing increased expression of the Flu receptor NCR1,

which is up-regulated in NK cells during Flu infection [11].

However, further study is needed to confirm this possibility.

Conclusion

In this study, we demonstrated that administration of SCBLC

to adult mice could modulate NK cell activity on systemic and

local cellular immunity. This is one of the mechanisms to protect

against Flu infection by oral administration of SCBLC to animals

in other species as well, including humans.

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Table 1

Assessment of the accumulated symptoms of mice infected with Flu

Symptoms* No symptoms* P valuey

Control group 29 1

0.0483

SCBLC 2000 group 22 6

* The development of accumulated symptoms was determined based on the

condition of each mouse’s hair and decrease of each mouse

’s body weight.y The level of symptoms in the control and SCBLC 2000 groups were compared

using Fisher’s exact test.


augmentation of cellular immunity and protection against influenza virus

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