Биологические науки / 8. Физиология человека и животных

Peklo A.O., Lych I.V.

National University of Food Technologies, Ukraine

IMMUNOMODULATORY PROPERTIES OF THE ABZYMES MILK

Study of antibodies and immunoglobulins, which have their own catalytic (abzymes) activity – one of the new trends in immunology and biotechnology [1]. Today has shown that abzymes can catalyze any type of biochemical reactions. There are even abzymes that are able to catalyze reactions that are not in the nature of the enzymes [2]. There is information about some types abzymes activity directly related to the development immunopathology [1].

Recently, considerable attention attracted by the modern scientific community that the performance of catalytic antibodies – abzymes largely due not only to the type of reactions that need to catalyze and type of pathological processes, but also with all immune processes that occur in the body. Abzymes are bioactive components of milk, which play an important role in the implementation of the functional activity of milk and humoral immunity mukoznoho immunity in general. Because can argue about the effectiveness abzymes in the prevention of viral, bacterial and some cancers [3].

Since the body of healthy people initially abzymes was observed, it was suggested that their products are closely connected with pathological processes. The question of the existence of natural abzymes body normally arose after it was shown that in colostrum and milk of healthy women is secretory immunoglobulin A (sIgA), are able to catalyze the phosphorylation of casein, they were one of the first abzymes that were allocated of colostrum [2]. In this study, we aimed to investigate the effect of a mixture of proteins-abzymes on immunocompetent cells in experiments in vitro.

Materials and methods

Identification and study of immunomodulatory properties of catalytically active antibodies – proteins-abzymes carried out on the basis of experimental data. Abzymes ability to directly influence the immune defense cells in vitro was determined by changes in the functional activity lymphocytes (Lph), neutrophil granulocytes (NG), monocytes (MN), and natural killer cells (NK cells).

A mixture of proteins obtained from milk and colostrum, which is taken from the first day of calving cows within 30 days. Purification scheme, which is shown in fig.1.

Fig.1. The scheme of obtaining and purification of protein mixtures abzymes

Lyophilized preparations of proteins dissolved in saline to a concentration of 1 mg protein/ml. In experiments using mixtures of proteins at concentrations of 1.0 mg/ml and 0.1 mg/ml.

Catalytic properties argued that abzymes can be prepared for catalysis almost all chemical reactions. The potential number of chemical transformations relatively higher than the number of reactions occurring in living systems and the corresponding catalyzed by enzymes. Using abzymes, creating the possibility of their use, both in scientific and inpractical purposes.

Evaluation of the direct effects of mixtures proteins-abzymes on functional activity of immune defense cells in tests performed in vitro. The functional state of neutrophil granulocytes (NG) and monocytes (MC) blood characterized by their ability to absorb latex particles (d = 1,0 – 1,3 mm) with the expectation percentage phagocytosis (PP) – the percentage of phagocytic cells and phagocytic number (PN) – their average activity [4] and the intensity of their oxygen-dependent metabolism through NBTR (Nitro Blue Tetrazolium Reduction) test according to the method [5]. For cytochemical coefficient (CCC) conducted an assessment of peroxidase activity.

Cytotoxic activity of killer cells was determined by non radiometric method based on spectrophotometric count of hemoglobin released from the red blood target cells destroyed by the killers (SE) during their joint incubation [6].

Rosette forming cells (RFC) was defined through the rosette formation method with application of particles coated with monoclonal antibodies [7].

Results and discussion

One of the important criteria for assessing the biological impact of any agent on the immune system is its expression at the cellular level. Therefore, research imunomodulyuvalnyh properties of a mixture of protein abzymes conducted under conditions in vitro, using lymphoid blood cells of healthy donors lymphocytes (LF), neutrophil granulocytes (NG), monocytes (MC), and natural killer cells (NK cells).

We was interesting to investigate the functional activity of phagocytes cells as NG and Mc under different concentrations of a mixture abzymes as a potential immunomodulatory effect on the phagocytic activity of blood cells is one of the necessary tests navnist immunomodulatory effect [8].

To investigate the functional activity of phagocytes cells in the first phase study conducted absorbance NG using latex test.

Figure 1 presents data on the influence of the mixture on phagocytosis abzymes NG expressed in the phagocytes percent (PP) (percentage of cells that have captured the latex particles) (Fig.1a) and phagocytic number (PN) (average number of particles that were swallowed by the phagocytic cell) (Fig. 1b)

a b

Fig.1. Effect of protein on performance abzymes phagocytosis NG

As shown in Fig.1, a mixture at a concentration abzymes 1.0 mg/ml had a marked effect on the level of PP (control – 32% vs. 45%), while the mixture of proteins at a concentration of 0.1 mg / ml slightly increased the rate of PP (32% vs. 39%). Abzymes mixture at a concentration of 0.1 mg/ml reduced the rate of PN (control – 6.5 USD 5.5 USD against), while a mixture of proteins at a concentration of 1.0 mg/ml increased the rate of PN (8.8 against USD 6.5 USD).

Thus, during normal phagocytic activity (32% ± 0,9%) NG, PP values studied compounds differ from each other. The percentage of cells that have captured the latex particles during processing of cells with a mixture of proteins at a concentration of 1.0 mg/ml of white control and greater relative concentration of 0.1 mg/ml. As for PN, this index increased significantly also for processing the cells in mixture abzymes concentration of 1.0 mg/ml.

The second stage of this research was to study the digestive ability of NG in NBTR-test. Results of change in activity of oxygen dependent metabolism after interaction of NG with study drugs and comparison preparations are shown in Fig. 2. Definition of oxygen dependent metabolism of NG was performed by evaluating the percentage of NBTR-positive cells (Fig. 2a) and cytochemical coefficient (CCC) (Fig. 2b).

a b

Fig.2. Effect of protein on performance abzymes spontaneous NBTR-test of NG

Data from this test indicate that the mixture of proteins at a concentration of 0.1 mg/ml was not significantly influenced not by the number of NBTR-positive cells than their functional activity (Fig. 2). A mixture abzymes at a concentration of 1.0 mg/ml had a stimulating effect on NBTR-positive cells by 31.8% and 20% CCC. This may suggests that this concentration mixture abzymes positive effect both on the functional activity of NG and on the activity of oxygen dependent metabolism of NG.

Similar studies of protein-abzymiv impact on functional activity of phagocytic cells was performed on a model of monocytes (MC). First, the absorbance capacity of MN was also studied by means of latex test (Fig. 3).

The data presented in Fig. 3a determine absorption activity of MN before and after interaction with the studied drugs and show that MC and rydostyn significantly increase the phagocytic activity of MN both absorption and digesting one, unlike the NG.

a b

Fig. 3. The impact on performance of phagocytosis MC

As shown in Fig. 3 none of the studied mixtures in concentrations abzymiv 1.0 mg/ml and 0.1 mg/ml did not affect the percentage phagocytosis and phagocytic number. Thus, PP and PN cells monocytes treated with different concentrations of proteins remained at control.

Thus, cells monocytes were not sensitive to the activation of the studied mixtures, even when changing the concentration.

Summarizing the results obtained, we can note that proved a direct impact on abzymes cells and phagocytes Lph. This is confirmed by the increasing number of fahotsytuvalnyh cells of monocyte-macrophage population increased cytotoxic activity of PP cells and cells responsible for antibody-dependent cytotoxic activity. So abzymy exhibit in vitro immunomodulatory properties. It shows promise for further study its effects on the level of microorganism.

References

1. Berger T., Rubner P., Schautzer F. et al. Antimyelin antibodies as a predictor of clinically definite multiple sclerosis after a first demyelinating event // N Engl J Med. – 2003. – № 10. – V. 349(2) – P. 139 – 145.

2. Albrechtsen M., Massaro A., Bock E. Enzyme-linked immunosorbent assay for human glial fibrillary acidic protein using a mouse monoclonal antibody // J, Neurochem. – 2001. – № 44. – P. 560 – 565.

3. Cuello A.C., Priestley J.V., Milstein C. Immunocytochemistry with internally labeled monoclonal antibodies // Proc Natl Acad Sci U S A. 2000 – V. 79(2) – P. 65 – 96.

4. Унифицированные иммунологические методы обследования больных на стационарном и амбулаторном этапах лечения: Метод. рекомендации / Киевский НИИ фтизиатрии и пульмонологии. – К.: 1988. – 18 с.

5. Нарциссов Р.П. Цитохимия ферментов лейкоцитов в педиатрии. Цитохимический метод. Дисс. Докт. Мед. Наук. М. – 200. – 378 с.

6. Lotze, Michael T. Natural Killer Cells: Basic Science and Clinical Application // London: Elsevier. – 2010. – 678 p.

7. Новиков Д.К., Новиков П.Д. Метод определения Т- и В- лимфоцитов диагностикумами на основе моноклональных антител // Иммунол. – 2000. – №2. – С. 31 – 33.

8. Доклінічні дослідження лікарських засобів: Метод. рекомендації / За ред. О.В. Стефанова. Вивчення імунотоксичної дії лікарських засобів. – К.: МОЗ України, 2001. С. 102 – 113.