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ABSTRACT
The results showed that immunomodulin has a moderate regulatory effect on the spontaneous blast transformation
of lymphocytes in the in vitro system. The costimulatory effect of immunomodulin in the reaction of PHA-induced
blast transformation of T-lymphocytes was revealed. The data we obtained on the rather pronounced interferon-
inducing properties of Sanogen developed by us and its combinations with well-known inductors - Cycloferon and
Betaleukin, can be of practical use in the treatment of infectious pathology, especially viral hepatitis, when the
combination of hepatoprotective, anti-inflammatory, antiviral, immunomodulatory and detoxification mechanisms of
action, in the absence of toxicity and side effects, will ensure the development of sanogenetic processes in the
patient's div.
KEYWORDS
Toxicity, side effects, treatment of cancer, cells.
INTRODUCTION
Research Article
INFLUENCE OF MODIFIED PEPTIDES FROM THE FETAL THYMUS ON THE
ACTIVITY OF T-LYMPHOCYTES AND NATURAL KILLERS IN
EXPERIMENTAL VIRAL HEPATITIS
Submission Date:
December 15, 2023,
Accepted Date:
December 20, 2023,
Published Date:
December 25, 2023
Crossref doi:
https://doi.org/10.37547/ajbspi/Volume03Issue12-09
Bolta A. Kahorov
National University of Uzbekistan named after Mirzo Ulugbek, University str., 4, 100174, Tashkent, Uzbekistan
Sevara L. Rasulova
National University of Uzbekistan named after Mirzo Ulugbek, University str., 4, 100174, Tashkent, Uzbekistan
Journal
Website:
https://theusajournals.
com/index.php/ajbspi
Copyright:
Original
content from this work
may be used under the
terms of the creative
commons
attributes
4.0 licence.
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American Journal Of Biomedical Science & Pharmaceutical Innovation
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VOLUME
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48-55
SJIF
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1121105677
Publisher:
Oscar Publishing Services
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Thymus extracts, incl. thymosin is used in the
treatment of cancer, autoimmune diseases, many
chronic infectious processes, etc. [1,2,10, 28, 37]. The
most important mechanism of action of thymic
peptides is the enhancement of the functional activity
of T-lymphocytes, however, the multi-stage process of
developing an immune response includes the
activation of not only cellular but also humoral
immunity factors, contributing to an increase in the
production
of
specific
antibodies,
cytokines,
inflammatory factors, etc. [7, 16, 22 ]. Natural immunity
is largely determined by killer cells (NK), which play a
decisive protective role in the early stages of viral
aggression [26]. Among the known thymic peptides, a
drug obtained from fetal sheep thymus is particularly
interesting. It consists of 15 peptides. Its
immunocorrective effect was shown in experiments
and clinical observations, in connection with which it
received the name "Immunomodulin". It is mass-
produced in the state of emergency "Immunomed"
(Tashkent) and approved for medical use in Uzbekistan
and Kazakhstan [2].
To increase the immunobiological activity of thymic
peptides, we attempted to combine them with metal
ions, as is the case in thymulin, which circulates in the
bloodstream as a nanopeptide combined with zinc.
The aim of the work is to study the effect of zinc-
modified thymus peptides on the functional activity of
T-lymphocytes and human natural killer cells in the in
vitro system and to evaluate the effectiveness of
interferonogenesis and antiviral action under the
influence of Sanogen, Betaleukin, Cycloferon and their
combinations in the experiment.
METHODS
Determination of the effect of drugs on the
proliferative response of T-lymphocytes. The material
for the study of lymphocyte blastogenesis was
peripheral blood mononuclear cells of 32 patients with
chronic viral hepatitis B aged 20-49 years.
Phytohemagglutinin (PHA) (Sigma) and concanavalin A
(Con A) (Pharmacia) at suboptimal concentrations (10
µg/mL) were used as RBTL activators. Modified and
unmodified peptides were added to the lymphocytes
(1 million/ml) at a final concentration of 0.01 μg/ml in
the test samples. The tablet was incubated at 37ºC for
1 hour, after which the corresponding mitogen was
added to the wells. Only mitogen was added to control
lymphocyte samples. Mitogen was not used in the
study of spontaneous blast transformation of
lymphocytes. After 48 hours, 3H-thymidine was added
to the samples at a concentration of 1 μCi/ml. The
results of the reaction were taken into account 72
hours after the start of cultivation.
To quantify the effect of immunomodulin on the
proliferative response of T-lymphocytes, the impact
index (IV) was used, which was calculated by the
formula:
IV = (Iо –
Ik)/Ik • 10
0%,
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Publisher:
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where: Io - the number of pulses per minute (imp/min)
in the experiment;
Ik - the number of pulses/min in the control.
RESULTS AND DISCUSSION
It was found that the average value of spontaneous
RBTL in patients with hepatitis in control group 1
(without
incubation
with
mitogen
and
immunomodulin) was 280±14 imp/min with a range of
individual fluctuations from 153 to 404 imp/min (Fig. 1).
In the presence of immunomodulin (control 2), the
indicators of spontaneous blastogenesis significantly
increased on average in the group up to 351 ± 26
imp/min with a range of individual fluctuations from
207 to 673 imp/min. The index of drug effect on
spontaneous RBTL was +25.3% (P<0.05). The
introduction of the zinc-modified peptide into the
culture increased the rates of spontaneous
transformation of T-lymphocytes to an average of
415±36 pulses/min. With a range of individual
fluctuations from 248 to 650 imp/min. The index of
drug effect on spontaneous RBTL was +48% (P<0.05
with control 2 and P<0.001 with control 1).
We also studied the functional activity of T-
lymphocytes in terms of their ability to enter the
mitotic cycle under the influence of PHA. It was
established that under the influence of lectin, the blast
transformation of lymphocytes in general in the
control group 1 is (51.4±3.3) x 103 imp/min with
individual fluctuations in indicators from 41 to 74
thousand imp/min.
In control group 2, the mean value of this indicator did
not differ significantly from control 1 and amounted to
57.0 ± 2.4 thousand imp/min with individual values from
42 to 77 thousand imp/min. The impact index of
immunomodulin on average for the group was +11%.
In the main group, the average value of this indicator
significantly differed from control 1 and control 2 and
amounted to 65.0 ± 2.4 thousand imp/min with
individual values from 48 to 86 thousand imp/min. The
impact index of modified immunomodulin averaged
+27% per group (P<0.05 with control 2 and P<0.001 with
control 1).
In the experiment with pre-treatment with the drug
only mononuclear cells, similar results were obtained.
Thus, the incubation of effector cells with
immunomodulin (without target cells) showed a
significant stimulation of the membrane toxic activity
of natural killer cells in all studied groups. In this group
of experiments, 2 controls were used: preliminary
parallel incubation of effector cells only in a nutrient
medium (control 1) and with a peptide (control 2). In
the experimental group, the metallopeptide was
evaluated. Thus, in healthy donors, the EC cytotoxicity
index was 51.2±1.9%; incubation with the modified
peptide increases these values to 65.7±1.6%;
preincubation with immunomodulin activates them up
to 58.3±1.7%. The difference between the experimental
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values and controls was significant (P<0.05 with
control 2 and P<0.001 with control 1).
Quantitative assessment of the induction of 1PM by
Sanogen, Betaleukin and Cycloferon with separate and
combined administration to experimental animals.
It has been established that Betaleukin induces IFN
production within 96 hours (observation period); the
maximum titer was noted after 12 hours and reached
512 units. The average titer was 118±16 units. (Fig.1, 3).
Sanogen also had quite pronounced interferonogenic
properties - the maximum titer was 128 units. after 24
hours with an average value of 47 ± 4 units (Fig. 1, 3).
When using monopreparations, Cycloferon was the
most effective - the maximum IFN was 1024 units. after
48 hours. Under his influence, the average titer during
a 120-hour observation was 295 ± 35 units. (fig.3)
Fig.1. Dynamics of serum interferon activity in mice after a single separate intraperitoneal injection of Sanogen (C),
Betaleukin (B) and Cycloferon (Cy) in effective doses: 2 μg/kg; 10 ng/kg and 4 µg/kg, respectively.
The introduction of Betaleukin with Cycloferon sharply
increased the activity of serum IFN to an average of
1060 ± 80 units. The expected increase was to be 395
units. (100 units of Betaleukin + 295 units of
Cycloferon), i.e. it turned out to be 2.7 times higher
than the additive value. Synergy was also manifested in
the accumulation of the maximum titer up to 2048
units, which is 1.3 times higher than expected (512 units
+ 1024 units = 1536 units). There was also a shift in the
peak of IFN production for a period of 48 hours. Even
4 hours after the combined administration of inducers,
the IFN activity was high and amounted to 256 units,
while the serum activity remained significant and after
120 hours was 512 units. The synergistic effect for this
period was 32 times higher than the additive one.
0
200
400
600
800
1000
1200
4
12
24
48
72
96
120
Activity titer IFN, units
B
C
Cy
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The simultaneous administration of Sanogen and
Cycloferon sharply increased the activity of serum IFN
to an average of 805 ± 75 units, since the expected
average increase was to be 323 units. (28 Sanogen units
+ 295 Cycloferon units), it turned out to be 2.5 times
higher than the additive value (Fig. 3). Synergy also
manifested itself in the accumulation of the maximum
IFN titer up to 2048 units, which is 1.8 times higher than
expected (128 units + 1024 units). The peak of IFN
production was recorded for a period of 48 hours,
which corresponded to the period of maximum serum
activity with the introduction of Cycloferon. It should
be noted that already 4 hours after the combined
administration of these inducers, the IFN activity was
the highest - 256 units. (Fig. 2). Despite the early and
very pronounced induction, serum activity remained
significant throughout the entire observation period,
and even after 120 hours it was 256 units. (Fig. 2),
whereas with the separate administration of Sanogen
or Cycloferon, the titers were only 2 units each. and 16
units. (Fig. 1), i.e. the synergistic effect was 14 times
higher than the additive one.
Therefore, with the simultaneous administration of
Sanogen and Cycloferon, a pronounced synergism was
found in the induction of endogenous IFN, since IFN
synthesis was noted, the high activity of which is
recorded in the circulating blood from 4 to 120 hours
with a maximum of 2048 units 48 hours after injection,
1.8 times higher than additive action when used
separately.
To quantify the activity of interferonogenesis under
the influence of monodrugs and their combinations,
we calculated the average values for the duration of
the study for 5 days for each option (Fig. 3). It has been
established that Cycloferon (295±36 units) is the most
active
among
monopreparations.
All
used
combinations of drugs were more powerful
interferonogens compared to it. Under the influence of
Sanogen with Betaleukin, the average activity of
interferonogenesis was 457±45 units, Sanogen with
Cycloferon - 805±56 units, and Betaleukin with
Cycloferon -1060±52 units.
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Fig.
3. Average values of serum interferon titers for 5 days after separate and combined
administration of Sanogen (C), Betaleukin (B), and Cycloferon (Cy) to mice.
CONCLUSIONS
The fundamental feature of the action of fetal thymus
peptides is the dependence of the severity and
direction of their effects on the initial state of
regulated cells, which contributes to the normalization
of processes that are out of balance.
-thymic peptides combined with zinc have a regulatory
effect on the proliferative activity of T-lymphocytes
through the interaction of their cell receptors with
mitogen and thymus peptides, which ultimately leads
to a cascade synthesis of cytokines, which in turn
modulates the proliferation of T-cells and cytotoxic
activity of natural killers.
- pronounced interferon-inducing properties of
Sanogen developed by us and its combinations with
well-known inductors - Cycloferon and Betaleukin, can
have practical application in the treatment of
infectious pathology, especially viral hepatitis, when
the
combination
of
hepatoprotective,
anti-
inflammatory, antiviral, immunomodulatory and
detoxification mechanisms of action
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