Adjuvant effect and TLR4 mediated activation of macrophages by polysaccharide from Polyporus albicans

7/27/2019 Adjuvant effect and TLR4 mediated activation of macrophages by polysaccharide from Polyporus albicans

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Adjuvant effect and TLR4 mediated activation of

macrophages by polysaccharide from Polyporus albicans

Dr. Antik Kiron Bose1

Senior Scientist and Project Advisor, Fred Hutchinson Cancer Research

Center, 1100 Fairview Avenue N, Seattle, WA 98109.

Abstract:

A water soluble polysaccharide from mycelium of Polyporus albicans has been identified for its

potency in stimulating cellular and humoral immune response. The adjuvant potential of this

polysaccharide was identified and found to be higher than conventional adjuvants. The 37 KDa

polysaccharide was purified using different macroporous absorption resins and Reverse phase

HPLC. Structural determination was done by sugar and methylation analysis, 13C NMR, FTIR,

MALDI-TOF,ESI-IT, MALDI-PSD TOF spectroscopy. The molecular size of the polysaccharide was

determined by Zetasier nanosystem. It showed TLR4 dependent NO, IL-1 and TNF-

production.


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Abbreviation:

HPLC- High Performance Liquid Chromatography

SLS- Static lights scattering

DLS- Dynamic lights scattering

DMSO- Dimethyl sulfoxide

TFA- Trifluoro acetic acid

NMR- Nuclear magnetic resonance

FTIR- Fourier- transform infra red

MALDI-TOF- Matrix assisted laser desorption/ionization on

timeof- flight

ESI-IT- Electro spray ion trap

HPAEC-PAD- high performance anion exchange

chromatography with pulsed amperometric detection

MALDI-PSD- Matrix assisted laser desorption/post- source

decay

TLR- toll- like receptor

NO- Nitric oxide

NOS- Nitric Oxide Synthatase

Introduction:

Vaccination remains the most cost effective

biomedical approach for the control of

infectious disease. The safety of the

traditional vaccines based on live

attenuated or killed microbes is

questionable due to risk of virulence

reversion (H.-X.Sun et al;2009). New

generations of vaccine based on purified

recombinant proteins , synthetic peptides

and plasmid DNA despite their better

tolerability are unfortunately often much

less reactogenic and immunogenic (H.-

X.Sun et al;2009). The majority of these

vaccines require association with adjuvant

capable of increasing the potency or

stimulating the appropriate immune

response.( Y.Lu, D.X Wang , Y.L Hu, X.YHuang, J.M Wang;2008). The benefits

flowing from adjuvant incorporation into

any vaccine formulation have to be

balanced with the risk of adverse reactions

induced by these compounds.

Unfortunately strong adjuvant activity is

often correlated with increased toxicity.

Fruends complete adjuvant (FCA) remains

the most potent of unknown adjuvants and

particularly powerful stimulant of both

cellular and humoral immunities (H.-X.Sun

et al ; 2009). Unfortunately FCA causes

severe reaction and is too toxic for human

use. The unique capacity of the extract Quil

A from bark of Quillaja saponaria and its

purified saponin QS-21 to stimulate both

the TH1 immune response and production

of cytotoxic T-lymphocyte against

exogenous antigens , makes them ideal for

use in subunit vaccines and vaccines

directed against intracellular pathogens as

well as for therapeutic cancer vaccines ( BP

daSilva , G Madeiros de Silva, J.P Parente ;

2009). However , in addition to pain on

injection , severe local reactions and

granulomas, toxicity includes severe

hemolysis (K.M Lima et al ;2004), making

such adjuvants unsuitable for human uses

other than for life threatening diseases,

such as HIV infection and cancer. Although

muramyl dipeptide (MDP) and other

derivatives from Gram negative bacteria,

such as LPS and monophosphoryl lipid A


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have also been used as human adjuvants,

their toxicities remain the single biggest

barrier to the use of such adjuvants for

human prophylactic vaccines (N. Byars et al

; 1990). A major challenge in adjuvantresearch is to increase adjuvant activity

while reducing toxicity ( R.K. Gupta, E.H.

Relyveld, E,B Lindblad, B. Bizzini, Ben-

Efraim, S.Gupta; 1993). Currently aluminium

compounds (Alum) are the only adjuvants

licensed by the Food and Drug

Administration (FDA) for use in human

(D.M. Pascual, R.D Morates ;2006). While

alum is safe ; it is a relatively weak adjuvant

particularly when used with subunit

Antigens. Moreover, the Alum is mild TH2

adjuvant that can effectively enhance IgG1

Antibody responses, but it is rarely

associated with TH1 type immune response

(H.HogenEsch ;2002). Furthermore Alum is

poor at stimulating cell-mediated immune

responses, and may actively block

activation and differentiation of cytotoxic T-

lymphocytes (R. Achirmbeck et al ;1994).

Hence, there is a major unmet need for a

safe and efficacious adjuvant capable of

boosting cellular plus humoral immunity (N.

Petrovsky;2006).

Most polysaccharides from higher plants

are relatively non-toxic and do not cause

significant side effects , which is a major

problem associated with immunemodulatory bacterial polysaccharides and

synthetic compounds.

A water soluble polysaccharide from

mycelium of Polyporus albicans was

identified and characterized. It has a potent

stimulating effect on murine lymphocyte

proliferation induced by mitogen.

Immunomodulatory effect and adjuvant

potential of the polysaccharide on cellular

and humoral immune responses of ICR,C3H/HeN and C3H/HeJ mice were

investigated. It can be a safe and efficacious

adjuvant capable of boosting cellular and

humoral immunity without toxicity.

Adjuvants bared on the Polyporus albicans

polysaccharide have enormous potential for

use in vaccine against both pathogens and

cancer.

Materials Required:

A. Chemicals:AB-8(purchased from Nankai

chemical factory, HPD-450 and HPD-

600( purchased from Hebei

Cangzhou Chem Co.Ltd,

Japan),Macroporous adsorptionresins, Vydac C-18 Reverse Phase

HPLC column (Grace Vydac), ID

SUPERCOSILTM LC-18-D-8 HPLC

column (sigma Aldrich; catalogue

no.195867), Sep-PakR

plus

Environmental C18 cartridge, (55-

105 m pore size product

no.WAT023635, Bridge column), 9%

Ficoll 400 and 15%sodiumdiatrizoate( density

1.13g/ml); Hypaque(sigma), Percoll

(sigma, density 1.064g/ml), Modified

Neuman and Tytells serumiess

medium ( United States Biological,

part no.S1012), Griess Reagent


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system (Promega), MTT assay using

cell titer 96TM non-radioactive cell

proliferation assay kit (Promega),

anti OVA-IgG Ab assat kit(catalogue

# 3011, chondrex assay kit), antiIgG1 Ab [MOPC-21,ab10616, Abcam)

anti IgG2b and antiIgG1bAb

conjugated to HRP(Millipore),

Mouse IgG2b ELISA(cat no. KT-405,

Kamiya Biomedical company),

3,3,5,5 tetramethyl benzidine

(TMB) (sigma), Mouse IgG1b ELISA

(cat no. KT-406, Kamiya Biomedical(

company, seattle, WA), N-G-mono-

methyl-L-arginine (L-NMMA) (sigma)

anti IL-1 and anti TNF- Abs

conjugated to HRP (Millipore), IFN

(1000/ml) (sigma), anti TLR2, anti

TLR4 anti CR3 Abs (sigma), Dead

EndTM colorimetric TUNEL system

(Promega)

B. Inbred Laboratory Mice:Belkin F5L051-ICR retractable

comfort Mouse C3H/HeNCrl(strain code

025,

coat colour Agouti, MHC haplotype H2K,

Agouti, MHC haplocyte H2K, Charles

River products and services), C3H/HeJ(coat colour Agouti, Related genotype

A/A, stock no.000659,strain C3H/HeJ

BirLtJ, JAXR surgical model,carrying a

mutation in toll like receptor 4 gene,

(Tlr4Lps-d)

c. Instruments:

Rotary Evaporator RE-52 type

(Shanghai Sing Pu Haxi instrument

factory). HP1090A HPLC fitted with

vydac C18 reverse phase column ( 2.1

X25 cm) (grace Vydac), Zetasier

nanosystam MRK 577-01 (Malvern

Instrument Ltd, Enigma Business park,

UK), Bruker AvanceTM DRX NMR

spectrometer, Fourier Transform TR

spectroscopy (Shimadzu scientific

instruments) Bruker ( ultraflex) MALDI-

TOF mass spectrometer electrospray ion

trap multi stage (ESI-IT) (1200 series

high throughput MS system, Agilent

Technologies), MALDI-PSD TOF MS

(deep Dyve instruments), ELISA plate

reader (chanel optical system 5sec/24

wells, single wavelength, standard 4

titers, Seeuco Electronics Technology co

Ltd), chemiluminescence NO analyzer (

Seivers instrument , Boulder, CO).

Procedure:

A. Preparation of mycelia culture-Mycelial culture of Polyporus

albicans (Imaz) Teng was used as a

source for water soluble

polysaccharide. Mycelial culture was

prepared on SYP medium containingstarch (16g/l), Fructose (4g/l),

peptone (1.5g/l), formic acid

(0.1g/l), KH2PO4 (1g/l), MgSO4

(0.5g/l), FeSO4 (0.01g/l) and Yeast

extract (1.5g/l). Medium pH was

adjusted to 4.5 before sterilization.


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One 1litre flask containing 400ml of

basal medium with 5% (v/v) of

mycelia of P. albicans was cultured

on a rotary shaking incubator at 110

rpm. Cultivation was done for 10days at 25C (Belinkyet et al, 1994:

Wi Young Lee, Young Ki Park, Tin

Kwon Ahn; 2007).

B. Purification of the polysaccharide-The mycelia of P. albicans were

homogenized in 60%

Ethanol solution at 60C for 2 hrs.

The process is repeated 2 times.

When the concentrated liquid

viscosity is 30 pas, 30% chitosan

solution was added at 60C and pH

5.5 to remove most proteins. The

extract is centrifuged at 10,000 rpm

for 15 mins and the pellet is

discarded. The supernatant was

concentrated using the Rotary

Evaporator RE-52 type (Shanghai

Qing Pu Haxi Instrument Factory) at

oscillations of about 100 times/min

for 24 hours. 1 gm of pre-dry resins

AB-8 (purchased from Nankai

Chemical Factory), HPD-450 and

HPD-600 (purchased from Hebei

Cangzhou Chem Co. Ltd. , Japan)

macroporous adsorption resins were

transferred to chromatographic

columns separately. 20 ml of extract

was added to each of the columns

and eluted with 30%, 50%, 70% and

90% ethanol. Carbohydrate content

of each of the eluted fractions were

measured by phenol sulphuric acid

method (M. Dubiol, K.A. Giller, J.K.

Hamilton, P.A.Robers, F. Smith,

1956). Saccharide content of all four

eluted fractions was also measured

by combining 4 eluted fractions to

calculate the total carbohydratecontent of the extract by the same

method. 70% ethanol extract with

highest carbohydrate content was

further purified by Reverse -Phase

HPLC. 20 l of the extract was

loaded in HP 1090A HPLC fitted with

Vydac C-18 Reverse-Phase

2.1mmx25cmcm column (Grace

Vydac). Separation was achieved

with a linear gradient of 5-50%

acetonitrile containing 0.1%

Trifluoroacetic acid over a period of

60 mins at flow rate of 0.2 ml/ min.

25% acetonitrile elutent with

maximum carbohydrate content

(measured by phenolsulphuric acid

method ) was passed through

2.1mm 1D SUPELCOSIL TM LC-18-D8

HPLC columns ( Sigma Aldrich;

catalogue no. 7195867) using 10-

50% Triethyl amine and acetic acid.

25% Triethyl amine and acetic acid

fraction gave a single peak

containing the polysaccharide.

C. Molecular mass determination ofthe polysaccharide- 25% Triethyl

amine and acetic acid fraction from

ID SUPELCOSILTM

LC-18-D8 HPLC

column was subjected to static light

scattering (SLS) for molecular mass

determination and dynamic light

scattering (DIS) for mean diameter

calculations using Zetasier Nano


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System MRK 577-01 (Malvern

Instruments Ltd., Enigma Business

Park, UK). It combines SLS, DLS and

electrophoretic light scattering

technologies to measure particlesize, Zeta potential and molecular

mass. In SLS, a beam of

monochromatic light is directed

through a sample dissolved in

toluene and intensity of light

scattered at angle of 173 by the

molecule is measured. The intensity

of light scattered over a period of 10

seconds is measured for

0.10,0.11,0.12,0.13,0.14,0.15,0.16,0.

17 and 0.18 gm/ml of the sample

using 633 nm wavelength of laser

beam. MW is given by Rayleigh

equation

KC/R=( 1/M+2A2C)P()

Where

R = Rayleigh ratio = ratio of

scattered light to incident light of

the sample.

M = Molecular weight

A2 = 2nd Virial coefficient

C = concentration

P= Scattering angle

K= optical constant

A plot of KC/ R versus C was made.

From the intercept 1/M was

calculated and the slope was

equivalent to 2nd

virial coefficient

(A2) i.e. Debye plot.

In DLS, the constructive and

destructive interferences between

changing intensities of different

particles lead to fluctuations of total

intensity which is measured by

Autocorrelation function. Mean

diameter of the polysaccharide wasmeasured at pH4.6, 7.6 and 9.6 to

measure degree of aggregation as a

function of pH (J.P. carver, 1991).

D. Structure determination of thepolysaccharide

1. Sugar Analyses1ml of 25% Triethylamine and

acetic acid fraction from ID

SUPELCOSILTM LC-18-D8 HPLC

column (containing 0.2gm/ml

polysaccharide) was transferred

to a 13 x 100 mm Screw cap

tube. 50g of Rha, Fuc, Ara, Rib,

Xyl, Man, Gal, Glc, GlcNAC and

GalNAC (90.5gm/ml) were added

as internal standards. They were

hydrolysed in 0.3ml 2 M TFA at

120C for 2 hrs. The solution was

evaporated to dryness by a

stream of compressed air and

0.5 ml Methanol. The step was

repeated. The solution was

reduced with 0.3 ml Fresh

solution of 50mM NaBH4 in

ammonia for 30 mins at 20C. It

was quenched with 0.5 ml 10%

glacial acetic acid in methanol

and evaporated to dryness. The

step was repeated twice.

Acetylation was done with 0.1ml

10% acetone and 0.1ml of 1N

pyridine at 100C for 20 mins.

50l of water was added and the


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solution was evaporated to

dryness after adding 0.5ml

toluene. The step was repeated.

Partition was done between

0.5ml H2O and 0.5mlethylacetate by stirring fast using

a triangular magnetic rod in

conical vial (Reacti-Vial type) for

3 mins. The upper ethylacetate

phase was transferred to the old

rinsed tube and 0.5 ml

ethylacetate was again added. It

was concentrated to dryness and

transferred into the sample tube

and concentrated to 50l (Per-

Erik Jansson, 1976).

2. Methylation Analyses1mg of dry sample was

transferred to a5ml serum flask.

1cm magnetic rod was added

and the flask was flushed with N2

and then sealed with a rubber

septum. The flask was kept in a

fumehood due to odor and toxic

fumes. 0.5ml of DMSO was

added to the flask using a

syringe while releasing the

pressure in the flask by inserting

another needle through the

septum. The sample was stirred

and sonicated for 30mins. 0.5ml

of 2Mdimsyl sodium was added

and stirred at room temperature

for 5hrs. The sample was frozen

and 0.25ml methylacridone was

added. The sample was melted

for 1 hr by stirring. Excess

pressure was relieved through

the septum and methylacridone

was taken away by pushing a

needle through the septum and

applying vaccum. The Sep-Pak

column (Sep-Pak plusEnvironmental C18 cartridge, 55-

105 m pore size, product no.

WAT023635, Bridge columns

fitted in 10ml syringe was

preconditioned by passing 10ml

ethanol and 2ml H2O. The

sample was applied in

DMSO/H2O (1:1 ratio ) solution

and eluted using 8ml H2O and

8ml 15% CH3CN solution to

separate the methylated

carbohydrates (Per-Erik Jansson,

1976).

2ml CH3CN and 2ml ethanol

were transferred into a

13x100mM screw cap tube and

concentrated to dryness. It is

hydrolysed in 0.3 ml 2M TFA at

120C for 2 hrs. The solvent is

evaporated and 1ml methanol

was added and the solution is

reduced with 0.3 ml fresh

solution of NaBH4 for 1hr at

20C. The solution was quenched

with glacial acetic acid and

evaporated with 0.5ml 10%

glacial acetic acid in methanol.

The solution was acetylated with

100ml acetone and 100l 50mM

pyridine at 100C. for 20 min and

then 50 l water was added.

After cooling, the solvent was

evaporated and 1ml toluene was


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added. The solution was

partitioned between 0.5ml H2O

and 0.5ml ethylacetate and the

organic phase was transferred to

sample tube and concentratedto 0.2 ml.13C- NMR (Bruker AVANCE TM

DRX NMR spectrometer) and

Fourier transform Infrared

spectroscopy (FT-IR) (Shimadzu

Scientific Instruments) of

methylated derivatives were

performed. Methylated and

acetylated derivatives were also

analysed by Matrix-assisted

laser desorption/ionization on

time of flight (MALDI-TOF)

[Bruker (UltraFlex) MALDI-TOF

mass spectrometer] and

electrospray ion trap multi-stage

(ESI-IT) (ESI IT 1200 series high

throughput MS system, Agilent

Technologies) Mass

spectrometer. MALDI-TOF was

employed to determine chain

distribution. The technique was

compared with high

performance-anion-exchange

chromatography with pulsed

amperometric detection

(HPAEC-PAD). Methylated

derivatives were investigated by

MALDI- TOF MS and MALDI post

source decay mass spectrometer

(MALDI-PSD TOF MS) (Deep

Dyve). (Susanna Broberg, 2000).

E. Maintenance of Inbred Mice

C3H/HeNCrl (Strain code 025, coat

color Agouti, MHC haplotype H2K,

Charles River Products and Services)

and C 3H/HeJ (Appearance Agouti,

Related genotype A/A , stock no.000659, strain C3H/HeJ BirLtJ, JAXR

surgical Models, carrying a mutation

in toll-like receptor 4 gene, Tlr$ Lps-

d) mice were fed an atherogenic diet

(1.25% cholesterol, 0.5% cholic acid

and 15% fat) for 2 weeks.A. Belkin

F5LO51-ICR(Imprinting control

region) Retractable comfort mouse

was also fed atherogenic diet for 5

weeks.

F. Study of Adjuvant effect of thewater soluble polysaccharide of

Polyporus albicans

5 weeks old ICR mice weighing 18-

22gm were immunized

subcutaneously with ovalbumin

(0.1mg) alone (control) or

ovalbumin(0.1mg) dissolved in saline

containing alum(0.2mg), or

ovalbumin (0.1mg) +0.5mg

/1mg/2mg of the polysaccharide

separately or with ovalbumin

(0.1mg) + Lipopolysaccharide (LPS)

(0.5mg) on day 1 and 15. On day 18

sera were collected from ICR mice

and IgG1 and IgG2 specific for

ovalbumin were isolated from

different mouses by Affinity

chromatography using ovalbumin as

ligand (W.B. Jakoby, 1974) and

measured by indirect ELISA using

antiisotypic IgG1 and IgG2b


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monoclonal Abs (J.B. Kwapinski,

1972)

G. Isolation of monocytes fromC3H/HeNCrl and C3H/HeNCrl and

C3H/HeJ miceBlood was obtained from both the

mice and immediately mixed with

0.2volume of anticoagulant (0.8%

citric acid) and then diluted 1:1 with

Ca2+

and Mg2+ free phosphate

buffered saline. A mononuclear cell

fraction containing monocytes was

obtained by layering diluted blood

on a cushion consisting of a mixture

of 9% Ficoll 400 and 15%

sodiumdiatrizoate (density=

1.13g/ml) (Hypaque) (Sigma) and

centrifuged at 400xg for 30 min at

room temperature. Cells were

washed and centrifuged in a

gradient of Percoll (Sigma) (density=

1.064g/ml) for 45 min at 800xg. An

isoosmotic percoll was prepared by

mixing 1 volume of NaCl 1.5(M) with

9 volumes of percoll (Pharmacia,

density =1.13g/ml). The percoll

gradient was done by mixing 1:1

(V/V) osmotic percoll with

PBS/citrate (1.49mM NaH2PO4,

9.15mM Na2HPO4, 139.97mM NaCl,

13mM C6H5Na3O7 2H20, pH -7.2).

The monocyte enriched fraction was

washed twice and plotted in 100mm

plastic tissue culture dishes

(3.4x107cells/dish) in modified

Neuman and Tytells serumless

medium supplemented with 2% new

born calf serum, gentamycin

(50g/ml) and Andamphotericin B

(0.25g/ml) (Benett & Breit 1994;

Haskill et al. 1988). After 30mins at

37C, the culture fluid and non-

adherent cells were removed.Adherent mono-layer of spreading

monocytes (>90% pure) was rinsed

twice and reincubated in fresh

culture medium and stimulated with

50 g/ml and 100 g/ml P. albicans

polysaccharide , 10U/ml IFN- or 5

g/ml LPS in both C3H/HeN and

C3H/HeJ isolated monocyte cultures

for 24 hours. A control set was

prepared where C3H/HeN and

C3H/HeJ derived monocyte culture

was stimulated with buffer for 24

hours.

H. Polyporus albicans derived

polysaccharide induced peripheral

macrophage proliferation was measured

by measuring NO production and IL-1

and TNF- production

P. albicans polysaccharide , LPS and

dextran stimulated proliferation of NO

production in both C3H/HeN and

C3H/HeJ mouse, were measured using

Griess Reagent system (Promega). It is

based on chemical diazotization using

50mM sulfanilamide and 50mM N-1-

napthylethylenediamine dihydrochloride

(NED) in presence of phosphoric acid

using the Dead EndTM

colorimetric TUNEL

system to measure NO at 520 nm. (D.S.

Bredt, S.H. Snyder, 1994). The viable cells

were measured by (3-(4,5- dimethyl


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thiazol-2yl)-2,5 diphenyl tetrazolium

bromide) (MTT) assay using a cell titre

96TM non-radioactive cell proliferation

assay kit (Promega) by reading

absorbance at 490nm (Tim, Mossmann,1983). Similarly P. albicans

polysaccharide, LPS and dextran

stimulated IL-1 and TNF- production in

C3H/HeN and C3H/HeJ derived monocyte

culture was measured by ELISA using anti

IL-1 and anti TNF- antibodies (J.B.

Krabinsky, 1972). 50 g/ml P. albicans

polysaccharide and 200 g/ml anti TLR4

were used to stimulate C3H/HeN derived

monocyte culture for 24 hrs and

monocyte proliferation was measured

similarly. Monocyte stimulation of

C3H/HeN mice was also studied in

presence of antiTLR2 and anti CR3

antibodies (Sigma) (200 g/ml).

Results:

Table 1 Carbohydrate content in 30%,

50%, 70% and 90% ethanol elutant of AB-

8, HPD-450 and HPD-600 macroporous

absorption resins (as determined by

phenol-sulphuric acid method) (M. Dubios

et al, 1956).

Percentage

of ethanol

elutant

Carbohydrate

content (g/ml)

Name of resin

AB-8 HPD-

450

HPD-

600

30% 50 48 43

50% 60 63 63

70% 350 360 358

90% 70 73 75

Table-2 Carbohydrate content of 25%

acetonitrile elutant from HP 1090A HPLC

fitted with Vydac C-18 Reverse Phase

column (Grace Vydac) and 25%

triethylamine and acetic acid fraction of ID

SUPELCOSILTM

LC-18-D8 HPLC column

(Sigma-Aldrich)

Carbohydrate content (g/ml)

1. 25% Acetonitrileelutant from

HP1090 A HPLC

fitted with Vydac C-

18 Reverse phasecolumn

300

2. 25% Triethyl amineand acetic acid

fraction of ID

SUPELCOSILTM

LC-

18-D8 HPLC column

250


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Fig 1. Elution profile ofPolyporus albicans

polysaccharide in C-18 Vydac Reverse

phase HPLC (Grace Vydac) (Fig 1a) and ID

SUPELCOSILTM LC-18-D8 HPLC (Fig 1b).

Table-3. Molecular weight (KDa) and 2nd

Virial coefficient (A2) determination of P.

albicans polysaccharide from 25%

triethylamine and acetic acid elution

fraction of IDSUPELCOSILTM

LC-18-D8 HPLC

using Zetasier Nano system MRK 577-01

Character Result

Differential refractive index

increment (dn/dc)

0.14ml/gm

Ray leigh ratio of toluene at

633 nm

1.3522x10 -5

cm-1

Scattering angle 173

Duration of recording

Scattered intensity

10 sec

Molecular weight 37KDa

2nd Virial coefficient (A2) 2.37x10-2

Fig 2. Debye plot where KC/R is plotted

along Y-axis and concentration ( C ) (mg/ml)

along X- axis. The intercept is equivalent to

1/M (M= 37 KDa) and slope equals to 2nd

virial coefficient (A2) = 2.37 x10-2 as

determined by Zetasier Nano System MRK

577-01 (Malvern Instruments) [ Where R =Rayleigh ratio, K- optical constant]


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Fig 3. Mean diameter of the polysaccharide

at pH 4.6, 7.6 and 9.6 were measured by

DLS using Zetasier Nano System MRK 57701

(Malvern Instruments)

At pH 4.6, size distribution by intensity

showed presence of both monomeric

fractions with diameter in range of 10 nm

and aggregates with diameter in range of

100nm in almost equal percentage . A small

peak of highly aggregated portion of the

polysaccharide was also observed in 1000

nm diameter range . At pH 7.6, the amountof aggregation was increased with most

carbohydrate in 100nm range and the

percentage of monomeric fraction with

diameter 10 nm was decreased. A slight

increase in % of 1000 nm diameter range

highly aggregated portion was also

observed. At pH 9.6, almost the entire

polysaccharide was present in 100 nm

diameter range and a very small fraction in10 nm diameter range. Z- mean diameter of

the polysaccharide is steadily increased

from 13.5 nm at pH 4.6, 13.7 nm at pH 7.6

and 14.7 nm at pH 9.6.


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Fig 4. Fourier Transform- IR spectra of P.

albicans polysaccharide measured by FT-IR

spectrophotometer ( Shimadzu Scientific

Instruments) showed presence of OH

groups, weak C-H bond, -d-galactopyranosyl and --d-mannopyranosyl

residues.

Fig 5.13

C- NMR of methylated and

acetylated carbohydrate derivatives of P.

albicans polysaccharide showed (1 3)

linked d- mannopyranosyl, (16) linked

-d-galactopyranosyl residues. (Measured

by Bruker AVANCETM

DRX NMR

Spectrometer at 270 MHz.

Fig 6. MALDI-TOF spectra of methylated

and acetylated carbohydrate derivatives of

P. albicans polysaccharide (measured byBruker Ultra Flex MALDI-TOF mass

spectrometer.


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Fig 7. High performance-anion-exchange

chromatography with pulsed amperometric

detection (HPAEC-PAD) spectra of

methylated and acetylated derivatives ofP.

albicans polysaccharide (measured by 1200

series high throughput MS system, Agilent

Technologies)

Fig 8. MALDI-PSD TOF mass spectra of

methylated and acetylated derivatives of

polysaccharide from P. albicans (measured

by Deep Dyve MALDI-PSD TOF mass

spectrometer)

Combined mass spectra analysis using

MALDI- TOF, ESI-IT, HPAEC- PAD and

MALDI-PSD TOF showed presence of a

backbone of (1 3) linked - d-

mannopyranosyl and (16) linked -d-

galactopyranosyl residues in 3:1:1 ratio and

terminated with single non reducing

terminal (1)-d-mannopyranosyl residues

at C6 position of (13,6)- linked- - d-

mannopyranosyl along the main chain.

Table-4 Amount of IgG1 and IgG2b

monoclonal antibodies formed by

inoculation of different antigen and

adjuvant combinations as measured byIndirect ELISA

Concentration of

Antigen and

adjuvants

inoculated

subcutaneously

Concentration of

Ovalbumin specific

Antibodies produced in

ICR mice

IgG1 IgG2b

1. Ovalbumin

(0.1 mg)

(control)

9mg/ml 2mg/ml

2. Ovalbumin 9.5mg/ml -


15/21

(0.1 mg )

dissolved in

saline containing

alum (0.2 mg)

3. Ovalbumin

(0.1 mg) + 0.5

mg P. albicans

polysaccharide

10.6 mg/ml 3.5 mg/ml

4.Ovalbumin

(0.1mg) + P.

albicans

polysaccharide

(1mg)


5. Ovalbumin

(0.1 mg) + P.

albicans

polysaccharide

(2mg)


6. Ovalbumin

(0.1 mg) + LPS

(0.5 mg)

11 mg/ml 3.5 mg/ml

Mouse serum anti-ovalbumin IgG were

measuered by anti-Ova-IgG Antibody Assay

Kit (Catalog # 3011) (chondrex Assay Kits)

using Mouse anti IgG1 antibody [MOPC-21]

(ab106163, Abcam), anti IgG2b and anti IgG

1b antibodies conjugated to HRP

(Millipore). AntiIgG2b was separately

measured by Mouse IgG2b ELISA (Cat No.

KT-405, Kamiya Biomedical Company).

3,3,5,5 tetramethyl-benzidine (TMB) and

H2O2 measured as the substrate for HRP

and absorbance at 450nm was taken .

Positive control contained 50 l of serum

with 0.1% sodium azide. Serum was

collected by venipuncture. A 1:50,000

dilution of serum sample was done and 100

l of antibodies were used.

Amount of IgG1b production by 0.1mg

ovalbumin dissolved in saline containing 0.2

mg alum was also measured by Mouse

IgG1b ELISA (Cat No. KT 406, Kamiya

Biomedical Company, Seattle, WA) to be 9.5

mg/ ml.

Table 5 : Amount of NO produced by

monocytes derived from C3N/HeN and

C3H/HeT mice as measured by Griess

reagent System (Promega) which were

stimulated by different antigens. The

number of viable monocytes were

measured by MTT assay using 96 TM

nonradioactive cell proliferation assay kit(Promega).

Nature and

Concentration

of Antigen

Concentration of NO

produced (g/ml)

Type of Inbred Mice

C3H/HeN C3H/HeJ

1. Control ( no

Ag)

20 22

2. Dextran (50

g/ml)

80 83

3. P. albicans

polysaccharide

100 22


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(50 g/ml)

4. P. albicans

polysaccharide

(100 g/ml)

140 22

5. IFN-

(10U/ml)

90 93

6. LPS (5 g/ml) 50 22

When the monocyte culture was incubated

with 10mM N-G-monomethyl-L-Arginine (L-

NMMA) (a NOS inhibitor), no NOproduction was observed. As >95% of total

NO released from activated macrophage

was converted to nitrite. NO was evaluated

by measuring nitrite using Griess reagent

using chemiluminescence NO analyzer

(Sievers Instruments, Boulder Co.). 100 l of

sample was injected into a reflux chamber

containing glacial acetic acid and 1% KI,

where nitrite is converted to NO . NO gas

was purged into chemiluminescence NO

analyzer and quantitated by reference to

NaNO2 standards.

Table 6 : Amount of IL-1 and TNF-

production by monocytes stimulated by

different antigens as measured by ELISA

using anti IL-1 and anti TNF- antibodies

conjugated to HRP (Millipore).

Nature and

Concentration

of Antigen

Conc. of Cytokines

(pg/ml)

Type of Inbred Mice

C3H/HeN C3H/HeJ

TNF-

IL-

1-

TNF-

1. Control (No

Ag)

20 25 20 21

2. Dextran (50

g/ml

80 83 83 82

3. P. albicans

polysaccharide

(50 g/ml)

160 153 20 21

4. P. albicans

polysaccharide

(100 g/ml)

200 193 20 21

5. IFN-

(10U/ml)

120 130 110 117

6. LPS (5 g/ml) 95 80 20 21


17/21

Table : 7

Number of viable monocytes was

determined using assay using 96TM

non

radioactive cell proliferation assay kit

(Promega) in control and under stimulation

of different Ags.

Nature and

Concentration of

Antigen

No. of viable cells

/l

Type of Inbred

Mice

C3H/HeN C3H/HeJ

1. Control (No Ag) 1.36 X 104 1.36 X

104

2. Dextran (50

g/ml

5.44 X104 5.46 X104

3. P. albicanspolysaccharide

(50 g/ml)

6.8 X104 1.36 X104

4. P. albicans

polysaccharide

(100 g/ml)

13.6 X104 1.36 X104

5. IFN- (10U/ml) 5.44 X104 5.43 X104

6. LPS (5 g/ml) 2.72 X104

1.36 X104

Table-8: Number of viable monocytes was

determined by MTT assay using 96 TM non

radioactive cell proliferation assay kit

(Promega)( and amount NO production (by

Griess Reagent (Using Dead End TMcolorimetric TUNEL system). When

monocytes were incubated with 20g/ml

anti TLR4, anti TLR2 and anti CR3 Abs

(sigma) with 50g/ml P. albicans

polysaccharide separately.

Nature of Antigen and

Abs used C3H/HeN inbred

mice

No. of

viable

cells/

l

Amount

of NO

productio

n (g/ml)

1.50g/ml P. albicans

polysaccharide(contro

l)

6.8

X104

100

2.50/ml P. albicanspolysaccharide+ anti

TLR 4 monoclonal Ab

(20g/ml)

6.8X104

20*

3. 50g/ml P. albicans

polysaccharide + anti

TLR 2 monoclonal Ab

(20g/ml)

6.8

X104

100

4. 50g/ml P. albicans

polysaccharide+

20g/ml anti CR3

monoclonal Ab

6.8

X104

100

*N.B 20g/ml NO produced by

constitrutively active monocytes have been


18/21

observed in TLR4 blocked experimental set.

In other sets, NO produced by constitutively

active monocytes have not been

substracted from 100 g/ml.

Discussion:

A water soluble polysaccharide has been

purified from mycelial culture of Polyporus

albicans (Imaz) Teng grown on SYP medium

(Belinkyet et al;1994, Wi Young et al;2007).

The 60% ethanol extract was purified using

AB-8, HPD-450, HPD-600 macroporus

adsorption resins using 30-90% ethanol.

Carbohydrate content of 70% ethanolelutants from all the columns was found to

be highest (350, 360, 358 g/ml for AB-8,

HPD-450, and HPD-600 columns

respectively) as determined by Phenol-

sulfuric acid method (M.Dubles et al;1956).

The static absorption quantities has been

found to be 80.4, 82.26, 75.96 mg/g for AB-

8, HPD-450 and HPD-600 respectively and

desorption rate was found to be 90.47%,80.88% and 75.84% for AB-8, HPD-450 and

HPD-600 respectively as reported by others

( Xia Li, Lilu Jiao, Liping Zhang;2008). The

70% ethanol elutant was further purified

using Vydac C18 Reverse phase HPLC using

25% acetonitrile and ID SUPERCOSIL TM LC-

18 D8 HPLC using 25% Triethylamine and

acetic acid as a single peak. Carbohydrate

content were found to be 300 and 250g/ml for Vydac reverse phase HPLC and ID

SUPERCOSIL TM LC-18 D8 HPLC respectively.

Molecular weight and 2nd virial coefficient

were determined from Debye plot. The

molecular weight was determined to be 37

KDa using Zetasier nanosystem MRK-577-01

using SLS. Similar reports have been found

by others ( Yong Xu Sun, Shusheng Wang,

Tianboo Li, Xia Li, Lili Jiao, Liping Zhang;

2008). The 2

nd

virial coefficient was foundto be 2.37X10-2 suggesting that the

molecule will stay in solution .Z-Mean

diameter of the polysaccharide was

determined by DLS using Zetasier Nano

System MRK-577-01 to be 13.5 nm at pH

9.6 showing aggregation of the

polysaccharide with increasing pH (4.6-9.6).

FT-IR spectra of the polysaccharide ( as

measured by Shimadzu Scientific

instruments) showed presence of OH

groups , weak C-H bonds ,-d-

galactopyranosyl and -d-mannopyranosyl

residues. 13C-NMR of methylated and

acetylated carbohydrate derivatives of

P.albicans showed (1-3) linked d-

mannopyranosyl, (1-6) linked -d-

galactopyranosyl residues at 270 MHz.

combined Mass Spectra analysis usingMatrix assisted laser desorption /

ionization on time of-flight (MALDI-TOF),

Electrospray ion trap multi-stage MS (ESI-IT

MS), high performance ion exchange

chromatography with pulsed amperometric

detection (HPAEC-PAD) and MALDI post-

source decay (PSD) TOF MS showed

presence of a backbone of (1-3) linked -d-

mannopyranosyl, (1-3,6) linked -d-mannopyranosyl and (1-6)-linked -d-

galactopyranosyl residue in 3:1:1 ratio and

terminated with single non-reducing

terminal (1-) -d-mannopyranosyl residues

at C6 position (1-->3,6) linked -d-

mannopyranosyl along the main chain.


19/21

Similar reports have been found by others

in P.albicans (Yong Xu Sun et al ,2008 and in

P. umbellatus ( Xingqun Li, Wen Xu;2011).

5 week old Belkin F5L051-ICR mice was

immunized with ovalbumin alone (0.1mg)

(control) or ovalbumin (0.1 mg) + 0.2 mg

alum or ovalbumin (0.1mg) + P. albicans

polysaccharide (0.5/1/2 mg) or ovalbumin

(0.1 mg) + 0.5 mg LPS on day 1 and 15. On

day 28, ovalbumin specific IgG1 and IgG2b

Abs in sera were measured by indirect

ELISA. Alum as an adjuvant with ovalbumin

showed 5.2% increase in OVA-specific IgG1b

Ab production in comparison with control

(0.1 mg ovalbumin) but no IgG2b Ab

production, showing that it is a weak

adjuvant. Bacterial LPS (0.5 mg) as an

adjuvant with 0.1mg ovalbumin showed

15.7% increase in OVA specific IgG Ab

production and 75% increase in OVA

specific IgG2b Ab production. 0.5 or 1 or 2

mg P. albicans used as an adjuvant with

0.1mg ovalbumin showed 16.8%, 36.84%,and 64.21% increase in OVA specific IgG1

Ab production respectively while it showed

75%,166.67% and 266.67% increase in

OVA-specific IgG2b Ab production with

0.5/1/2 mg P. albicans polysaccharide

respectively. It does not cause any toxicity

even at 2 mg. so it could be a safe and

efficacious adjuvant capable of boosting

humoral immunity without toxicity.

P. albicans polysaccharide induced

monocyte proliferation was measured in

C3H/HeN and C3H/HeJ mice by measuring

NO production. (Griess Reagent system

using Dead EndTM

colorimetric TUNEL

System), number of viable monocytes

(measured by MTT assay using 96TM non

radioactive cell proliferation assay kit) and

IL-1 and TNF- production (using ELISA).

50g/ml dextran induced 80g/ml and83g/ml NO production (300% and 315%

increase) in C3H/HeN and C3H/HeJ mice

respectively. In control ( no Ag) 20 g/ml

and 22 g/ml NO production was observed

by constitutively activated monocytes in

C3H/HeN and C3H/HeJ mice respectively.

300% and 301.47% increase in number of

monocytes was observed using 50g/ml

dextran in 24 hours. 10U/ml IFN- (Sigma)

induced 350% and 331.8% increase in NO

production and 300% and 299.26% increase

in monocyte production in C3H/HeN and

C3H/HeJ mice, respectively in 24 hours.

5g/ml bacterial LPS induced 150% increase

in NO production and 100% increase in

monocyte number in C3H/HeN and

C3H/HeJ mice in 24 hours but no increase in

NO production and no increase in monocyte

number in C3H/HeN and C3H/HeJ mice

suggesting that TLR4 ( toll like receptor) is

required for LPS induced NO production

and monocyte proliferation. 50g/ml P.

albicans polysaccharide induced 400%

increase in NO production and 544%

increase in monocyte number in C3H/HeN

and C3H/HeJ mice in 24 hours but no

increase in NO production and no increase

in monocyte number in C3H/HeN and

C3H/HeJ mice. Similarly, 100 g/ml P.

albicans polysaccharide induced 600%

increase in NO production and 900%

increase in number of monocyte in

C3H/HeN mice but no increase NO


20/21

production and increase in monocyte

number in C3H/HeJ mice suggesting that

TLR4 is required for PAP induced NO

production and monocyte proliferation.

Similar results have been observed byothers ( Xingqun Li, Wen Xu;2011) in P.

umbellatus polysaccharide. 50g/ml PAP

induced 700% and 512% increase in IL-1

and TNF- production in C3H/HeN mice

respectively in 24 hours but no increase in

IL-1 and TNF- production in C3H/HeJ

mice. 100g/ml PAP showed 900% and

672% increase in production of IL-1 and

TNF- in C3H/HeN mice respectively but no

increase in IL-1 and TNF- production in

C3H/HeJ mice suggesting that TLR4 is

required for PAP induced IL-1 and TNF-

production by monocytes. No increase in

monocyte number and NO production was

observed when monocyte cultures were

incubated with 50g/ml PAP and 20g/ml

anti TLR4 Ab for 24 hours suggesting that

TLR4 is required for PAP induced NO

production and monocyte proliferation but

when anti TLR2 and antiCR3 Ab were used

normal NO production was observed. It

suggests that even in PAP induced

monocyte cultures, anti TLR4 Ab can inhibit

induced monocyte NO production by

blocking signaling through TLR4 receptor.

Similar reports have been observed by

others (Xingqun Li et al;2011 and Yongxu

Sun et al;2008). Contitutively activated

monocyte produce NO indepecdent of TLR4

receptor but PAP induced monocytes

require TLR4 signaling. N-G-monomethyl L-

arginine (L-NMMA), an inhibitor of Nitric

oxide synthetase can completely inhibit NO

production by monocyte suggesting that

NOS is the main enzyme in induced

monocytes for NO production.

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Adjuvant effect and TLR4 mediated activation of macrophages by polysaccharide from Polyporus albicans