comparative study of biocorrective protein-peptide agent to...

V.M. Gorbatov VNIIMP, 2016 - Moscow

Natural immunostimulators obtained over Sus scrofa

tissue extraction with the use of water with modified

isotope composition (RSF grant No. 15-16-00008)

Ekaterina R. Vasilevskaya

Comparative study of Biocorrective

Protein-Peptide Agent to Improve Quality

and Safety of Livestock Products

Study devoted to analyze water with a modified isotope (D/H) composition (WMIC) influence on Sus Scrofa tissues extracts.

Tasks :

• understand WMIC influence on the protein extractability, peptide and protein profiles;

• Comparative study of complex extracts obtained from Sus scrofa immunocompetent organs prepared with distilled water and WMIC in vivo


WMIC – deuterium depleted water with deuterium concentration 40 ppm DW – standard distilled water with deuterium concentration 140 ppm

RAW MATERIALS CHOICE

BONE MARROW (hematopoietic stem cell)

Predecessor of the myeloid series

Monocyte Macrophage Neutrophil Eosinophils

Basophil Mast cell

Predecessor B-lymphocytes

(immune memory)

B-lymphocytes maturation

Mature B - and T-lymphocytes circulation (CD20/CD14/IgM/IgG)

SPLEEN

cell-mediated immunity (C):

Т-helpers(Th); Т-killers(Тс); Т-suppressors (Ts);

humoral immunity (H): Ig, lymph nodes Migration into blood and lymph

Predecessor T-lymphocytes

(antigen-specific)

THYMUS

Т-cells differentiation

(CD4/CD3/ CD8/TcR)


Main Sus Scrofa immune organs – spleen, thymus and lymph nodes as potential tissues containing compounds with immunnoregulatory properties

EXTRACTION ALGORITHM

• Knives with ceramic coating Grinding

• 4°С, 0,9 % solution NaCl - WMIC, 4 hours

• 4°С, 0,9 % solution NaCl - DW, 4 hours Extraction

• 3500 Rev/min, 8 min,

Plastic tubes Centrifugation

• Pressure 2.5 bar

Polyethersulfone membranes with plastic fittings and tanks Ultrafiltration

• Pressure 3,3 Pa, Т = (-41±1°С)

• Glassware Lyophilic drying


PROTEIN CONCENTRATION CHANGES DURING EXTRACTION

3,77

22,10 23,3 24,4 23,8

25,50 24 24,5

3,05

18 18,1 19,5 20,3 20,4 19,5 20,1

0,00

5,00

10,00

15,00

20,00

25,00

30,00

0 15 30 45 60 75 90 120

Pro

tein

co

nce

ntr

atio

n, g

/l

Extraction time, min

Spleen (WMIC) Spleen (DW)

4,44

17,7 18,9

18,1 17,6 16,5

3,46

10,3 10,8 11,7 11 11,1

0,00

2,00

4,00

6,00

8,00

10,00

12,00

14,00

16,00

18,00

20,00

0 15 30 45 60 90

Pro

tein

co

nce

ntr

atio

n, g

/l


Thymus (WMIC) Thymu (DW)

6,69

16

18,6 18,1 18 19

5,38

11,7 10,9

12,6 11 11,7

0

2

4

6

8

10

12

14

16

18

20

0 15 30 45 60 90

Pro

tein

co

nce

ntr

atio

n, g

/l


Lymph nodes (WMIC) Lymph nodes (DW)

WMIC – water with modified isotope composition DW – distilled water


Increase protein concentration from 15 to 50 %

PROTEIN ANALYSIS

SDS-electrophoresis in 12,5% PAAG.

1. Standard molecular weight (130 kDa– 10 kDa);

2. thymus extract (WMIC);

3. spleen extract (WMIC);

4. lymph nodes extract (WMIC);

5. thymus extract (DW);

6. spleen extract (DW);

7. lymph nodes extract (DW).

Major bands – 13 kDa, 16 kDa, 27 kDa, 43 kDa, 70 kDa, 98 kDa


Leucocyte antigen Cell tumor antigen Interleukins


200150120

10085706050

403025201510

1

2

3

4

5

67

2D ELECTROPHORESIS (O'Farrell) OF EXTRACTS MIXTURE (WMIC)

Identification of protein fractions was performed on DE after trypsinolysis by MALDI-TOF/MS and MS/MS mass spectrometry on Ultraflex MALDI-TOF mass spectrometer

• Marked differences: proteins involved in the immune response

IMMUNOLOGICAL REACTIVITY IN VIVO

• Male rats Wistar (SPF)

• N= 40, m = 390 ± 10 г

ANIMALS

• Intraperitoneal injections of Cyclophosphamide (Sigma) • Dose: 75 мг/кг • Three times every 72 hours, • Model complete: 12 days after first injection

IDF MODEL

• Group А (n=10) – intact animals

• Group B (n=10) – control animals (IDF model)

• Group C (n=10) – treatment for 20 days with DW extract, 2,67 ml/kg

• Group D (n=10) – treatment for 20 days with WMIC extract, 2,62 ml/kg IN VIVO RESEARCH

• Cytometry analysis :

• LYM, MON, GRA; CD4.

• Immunoassay analysis:

• Complement components С1q,, С3,C4, C5

METHODS


Study of immune corrective effect was carried out with:

IMMUNOPHENOTYPING

Guava easyCyte™


Lymphocytes, monocytes, granulocytes content. А – intact; В – control; С – DW extract; D – WMIC extract.

Increase GRA: B group by 87 %, C group by 43 % D group by 21 %


IMMUNOPHENOTYPING

CD4 content (T-helper cells). А – intact; В – control; С – DW extract; D – WMIC extract.

10e0 10e1 10e2 10e3 10e4Yellow Fluorescence (YEL-HLog)

10

e4

10

e3

10

e2

10

e1

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e0

Re

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luo

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M1


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g)

Plot3: Gated by : LYM


10

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10

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А B

C D

CD4 CD4

CD4 CD4

LYM CD4- LYM CD4-

LYM CD4- LYM CD4-

Decrease: B group by 40 %, C group by 20 % D group by 11 %

Immune Recovery

COMPONENT COMPLEMENT IMMUNOASSAY ANALYSIS


35,81

26,34 27,05

31,5

18,897 17,264

16,36 14,96 15,4821

12,4014

15,8127 17,36

16,248 16,467

13,372

17,23

0

5

10

15

20

25

30

35

40

1 2 3 4

Rel

ativ

e co

nte

nt,

ng/

ml

C1q C3 C4 C5

1 – intact; 2 – control; 3 – DW extract; 4 – WMIC extract.

Decrease C1q: B group by 25 %, C group by 22 % D group by 9 %

Adaptive immune response activation by

stimulating C3 and C4 components synthesis

Activated cycle of complementary cascade

CONCLUSION


• Deuterium depleted water intake led to the increase protein concentration during extraction;

• WMIC has an influence on extraction proteins with low molecular weight (by 15 kDa) in animal tissue extracts (spleen, thymus, lymph nodes)

• Use WMIC as solubilizing agent led to increase of proteins and peptides count that are directly or indirectly involved in the immune response

• In vivo research showed immune system recovery, adaptive immune response and functional activity of nonspecific immune defense

This work was supported by Russian Science Foundation (RSF) grant No. 15-16-

00008. “Development of innovative natural adaptogenic stimulants of innate (nonspecific)

immunity based on species and tissue-specific biomolecules”

.


Ekaterina Vasilevskaya [email protected]

comparative study of biocorrective protein-peptide agent to...

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