Proteolytic theory of pathogenesis of influenza and improvement of its treatment
A new theory of grippe pathogenesis with the use of proteinase-inhibitory system has been offered. It has been established that purification and concentration of grippe viruses by different methods did not release the virus from the cellular enzymes. When the experimental animals have been infected...
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Фізико-хімічний інститут ім. О.В. Богатського НАН України
2011
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| Cite this: | Proteolytic theory of pathogenesis of influenza and improvement of its treatment / V.A. Divocha, A.I. Gozhenko, V.N. Mikhalchuk // Актуальні проблеми транспортної медицини. — 2011. — № 3 (25). — С. 54-64. — Бібліогр.: 27 назв. — англ. |
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| citation_txt | Proteolytic theory of pathogenesis of influenza and improvement of its treatment / V.A. Divocha, A.I. Gozhenko, V.N. Mikhalchuk // Актуальні проблеми транспортної медицини. — 2011. — № 3 (25). — С. 54-64. — Бібліогр.: 27 назв. — англ. |
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| description | A new theory of grippe pathogenesis with the use of proteinase-inhibitory system has been offered. It has been established that purification and concentration of grippe viruses by different methods did not release the virus from the cellular enzymes. When the experimental animals have been infected with grippe virus, we have observed misbalance in enzyme-inhibitory balance took place, especially during first hours after infection. Six isoforms of trypsin-like proteinases were obtained from the lungs of healthy mice. Antiproteinase immune sera have been isolated from these 6 isoforms which served to treat the experimental animals. It was antiserum to the third isoform that has prevented the experimental animals mortality. From the industrial wastes of gamma-globin manufacture the inhibitors of protein-like proteinases have been extracted which prevented the white mice’s mortality in 80% of cases. Endogenous inhibitors of human blood proteinases are prospective in producing anti-influenza drugs for humans.
Запропонована нова теорія патогенеза грипу за участю протеїназно-інгібіторної системи. Встановлено, що очищення та концентрація вірусу грипу різними методами не звільняло вірус від клітинних ферментів. При інфікуванні тварин вірусом грипу відбувалось порушення ферментно-інгібіторної рівноваги, особливо у перші години після інфікування. З легенів дужих мишей одержано шість ізоформ трипсиноподібної протеїнази до яких отримані антипротеїназні імунні сироватки та проведено лікування ними тварин. Тільки антисироватка до третьої ізоформи захистила тварин від смерті. З промислових відходів одержання гамаглобуліну виділено інгібітор трипсиноподібних протеїназ, який захистив білих мишей від смерті на 80%. Ендогенні інгібітори протеїназ крові людини є перспективними антигрипозними препаратами для людини.
Предложена новая теория патогенеза гриппа с участием протеиназно-ингибиторной системы. Установлено, что очистка и концентрация вируса гриппа разными методами не освобождало вирус от клеточных ферментов. При заражении животных вирусом гриппа происходило нарушение ферментно-ингибиторного равновесия, особенно в первые, часы после заражения. С легких здоровых мышей, получено шесть изоформ трипсиноподобной протеиназы к которым, получены антипротеиназные иммунные сыворотки и проведено лечение ими животных. Только антисыворотка к третьей изоформе защитила животных от смерти. С промышленных отходов получения гаммаглобулина выделено ингибитор трипсиноподобных протеиназ, который защитил белых мышей от смерти на 80%. Эндогенные ингибиторы протеиназ крови человека являются перспективными антигрипозными препаратами для человека.
|
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| fulltext |
АКТУАЛЬНЫЕ ПРОБЛЕМЫ ТРАНСПОРТНОЙ МЕДИЦИНЫ � № 3 (22), 2011 г.
54
ACTUAL PROBLEMS OF TRANSPORT MEDICINE �#3 (25), 2011
Резюме
РЕКОМЕНДАЦИИ ПО МЕДИКО�
ПСИХОЛОГИЧЕСКОЙ ПРОФИЛАКТИКЕ
И РЕАБИЛИТАЦИИ МОРЯКОВ В СВЯЗИ
С ПРОБЛЕМОЙ ПИРАТСТВА
Оливер Брюер
В настоящее время пиратство ста�
ло серьёзной проблемой в безопаснос�
ти морского судоходства. Угроза напа�
дения, схватки с пиратами и особенно
пребывание в плену вызывают психо�
эмоциональный стресс, приводят к на�
рушению психосоматического здоровья
и являются угрозой для жизни моряков.
Решению этих важных проблем посвя�
щена настоящая статья.
Ключевые слова: психическое здоровье
моряков, пираты, психоэмоциональный
стресс
Резюме
РЕКОМЕНДАЦІЇ ЩОДО МЕДИКО�
ПСИХОЛОГІЧНОЇ ПРОФІЛАКТИКИ ТА
РЕАБІЛІТАЦІЇ МОРЯКІВ У ЗВ’ЯЗКУ З
ПРОБЛЕМОЮ ПІРАТСТВА
Олівер Брюер
В даний час піратство стало серй�
озною проблемою в безпеці морського�
судноплавства. Загроза нападу, сутички
з піратами і особливо перебування в по�
лоні викликають психоемоційний стрес,
призводять до порушення психосома�
тичного здоров’я і є загрозою для життя�
моряків. Вирішенню цих важливих про�
блем присвячена ця стаття.
Ключові слова: психичне здоров’я
моряків, пірати, психоемоційний стрес
Впервые поступила в редакцию 22.08.2011 г.
Рекомендована к печати на заседании
редакционной коллегии после рецензирования
In the pathogenesis of viral diseas�
es the interaction of virus with the cell
has not been sufficiently studied. The
main point here is the virus intervention
into a healthy cell with a mandatory
deproteinization of the virus. However,
deproteinization of viruses is still poorly
understood. This firstly refers to the
УДК 616.921.5
PROTEOLYTIC THEORY OF PATHOGENESIS OF INFLUENZA AND
IMPROVEMENT OF ITS TREATMENT
Valentina A. Divocha, Anatoly I. Gozhenko, Vasily N. Mikhalchuk
Experimental and Clinical Pathology Laboratory, Ukraine Scientific�Research
Institute of Transport Medicine, e�mail: divocha09@ukr.net
A new theory of grippe pathogenesis with the use of proteinase�inhibitory system has
been offered. It has been established that purification and concentration of grippe viruses
by different methods did not release the virus from the cellular enzymes. When the
experimental animals have been infected with grippe virus, we have observed misbalance
in enzyme�inhibitory balance took place, especially during first hours after infection. Six
isoforms of trypsin�like proteinases were obtained from the lungs of healthy mice.
Antiproteinase immune sera have been isolated from these 6 isoforms which served to treat
the experimental animals. It was antiserum to the third isoform that has prevented the
experimental animals mortality. From the industrial wastes of gamma�globin manufacture
the inhibitors of protein�like proteinases have been extracted which prevented the white
mice’s mortality in 80% of cases. Endogenous inhibitors of human blood proteinases are
prospective in producing anti�influenza drugs for humans.
Key words: influenza, trypsin proteinase inhibitors, proteases, antiviral drugs.
ACTUAL PROBLEMS OF TRANSPORT MEDICINE �#3 (25), 2011
АКТУАЛЬНЫЕ ПРОБЛЕМЫ ТРАНСПОРТНОЙ МЕДИЦИНЫ � № 3 (25), 2011 г.
55
mechanism of influenza virus introducing
into cells of mammals, including humans.
In this regard, in 1983 we proposed a new
theory of influenza pathogenesis with
proteinase�inhibitory system participa�
tion [1, 2].
The difficulties in creating products
with selective antiviral action are con�
nected with biology of viral diseases
pathogens. Recent achievements of bio�
chemistry and molecular biology, reveal�
ing the features of virus reproduction,
help to create new generations of drugs
with directed intervention in the cycle of
viral reproduction [3�6].
The purpose of the study � to exam�
ine the state and role of antiproteinase
systems of virus and recipient in the de�
velopment of influenza infection to obtain
and use fundamentally new therapeutic
drugs based on inhibitors of trypsin�like
proteases.
Research objectives:
1. To clear flu virus till homogenous
state.
2. To explore the nature of protease as�
sociated with influenza virus.
3. To examine the role of proteases and
their inhibitors in different, especial�
ly at early stages, of influenza infec�
tion development.
4. To isolated and purified proteinase
and its inhibitor from the lungs of
healthy mice as well as from those
infected with flu virus.
5. To obtain specific antibodies to iso�
forms of trypsin�like proteases and
study their protective effect under
the conditionts of experimental influ�
enza.
6. To study the protective effect of cel�
lular inhibitors on infection of animals
with a lethal dose of influenza virus.
Materials and methods
Materials for the study were:
1. Strains of influenza virus: A/PR/8/34
(H1N1), A/Aichi/2/68 (H3N2), A/
USSR/90/77 (H1N1), A (Extra X�31),
A/WSN/33 (H1N1), A/Phi l/2/82
(H3N2), AO/32 (NON1) grown on a 9�
day chicken embryos, B strain PR�
109, obtained by recombination of
viruses V/Lee/40 and B / USSR /
100/83, and MDCK cells were ob�
tained at the I.D. Ivanovsky Research
Institute of Virology, Academy of
Medical Sciences of Russia and
strain AO/32 (HON1) � from the In�
fluenza Research Institute of St. Pe�
tersburg, Russia;
2. 2358 items of white mice, line BALB/
c and hybrids;
3. 3052 pcs of chicken embryos;
4. 1612 tubes of passaged MDCK cell
culture;
5. 140 items of white rats, Wistar line.
We used the following techniques:
virological, biochemical, immunological,
molecular biological, radioisopotic.
To study the nature of proteolytic
activity associated with influenza virus we
used 10�11�day chicken embryos and in�
fluenza virus AO/32 (HON1) with infec�
tious titers 7 lg EID
50 / 0,2
and HA�1: 256.
The virus was accumulated by infecting
chicken embryos in a volume of 0.2 ml,
diluted to 10�3 with infectious material.
Infected chicken embryos were incubat�
ed for 48 hours at +36 ° C. Then they
were cooled for 18 hours at +4 ° C and
then the virus�containing fluid was col�
lected.
Own researches. In the early 80’s,
in purifying and concentrating of various
strains of influenza virus to produce poly�
valent anti flu vaccines, we faced the fact
that we could not release the influenza vi�
rus from proteolytic activity (Fig. 1.) [7].
To resolve this problem we have im�
proved the methods of purification, but
nevertheless failed to release influenza
virus from proteolytic activity.
We analysed purified preparations
of influenza virus for the presence of pro�
teolytic activity and revealed that the
treatment of influenza virus by ultracen�
trifugation techniques does not relieve flu
АКТУАЛЬНЫЕ ПРОБЛЕМЫ ТРАНСПОРТНОЙ МЕДИЦИНЫ � № 3 (22), 2011 г.
56
ACTUAL PROBLEMS OF TRANSPORT MEDICINE �#3 (25), 2011
virus from proteolytic activity. In saccha�
rose gradient (15�60%) proteolytic activ�
ity was clearly separated into several iso�
forms (Table 1) [8].
The results obtained allowed us to
conclude that flu virus is associated with
serin�containing protease of trypsin –like
type of cell origin, which has molecular
heterogeneity. Our further studies (Table
2) showed that all commercial products
contained both inhibitor, and trypsin�like
protease, i.e. the today’s drugs are not
cleared completely of protein impurities,
or it is impossible to separate the viral
proteins from cellular components. Viral
proteins are tightly associated with com�
ponents of the cell, so the structure of
influenza virus should be considered tak�
The original virus-containing allantoic fluid (VCAF)
↓
Centrifugation at 6000 min.-1 30 min. at + 4 ° C
↓ ↓
Supernatant precipitate is discarded
↓
Centrifugation at 10000 min.-1, 20 min. at + 4 ° C
↓ ↓
Supernatant Precipitate is discarded
↓
Centrifugation at 20000 min.-1 2 h at + 4 ° C
↓ ↓
Precipitate (the virus) Supernatant is discarded
↓
Precipitation of purified and concentrated native allantoic virus at 20000 min.-1, through
a 20% sucrose 2 h, t + 4 ° C
↓ ↓
Precipitate (the virus) supernatant is discarded
↓
Purification in the sucrose density gradient at 28.000 min -1, 4h
↓ ↓
supernatant is discarded
Concentration of fractions at 20000 min.-1 ,2 h at + 4 ° C with buffer NTE
↓ ↓
supernatant is discarded
Precipitate / virus / is dissolved in a minimum amount of buffer NTE
↓
Reultratcentrifugation in sucrose density gradient 33000 min.-1, 3h
↓
Precipitate / virus / is dissolved in a minimum amount of buffer NTE
↓
Treatment of virus with 0.1% solution of sodium dodecyl sulphate at +20 ° C, 20 min.
↓
Purification and separation of viral polypeptides with preparative electrophoresis in
polyacrylamide gel
↓ ↓
1/2 of the block for elution 1/2 of block for staining
Figure 1. Scheme of purification of influenza virus.
ACTUAL PROBLEMS OF TRANSPORT MEDICINE �#3 (25), 2011
АКТУАЛЬНЫЕ ПРОБЛЕМЫ ТРАНСПОРТНОЙ МЕДИЦИНЫ � № 3 (25), 2011 г.
57
ing into account the interaction with cel�
lular enzymes and their inhibitors.
In the body the system of protein�
ases and inhibitors is represented by a
large group of proteins. Inhibitors of pro�
teolytic enzymes counterbalance the cor�
responding enzymes in the body, and are
in constant dynamic equilibrium with
them [9�12]. Violation between enzymes
and inhibitors is important for the devel�
opment of pathological processes [13].
Our studies show that in the lungs
and blood serum of uninfected animals
and chicken embryos, the level of pro�
tease activity and protease inhibiting
activity are in equilibrium, and the latter
is violated at influenza virus A infection.
In the infectious process the most
profound changes occur in the first hours
after infection. So, in 6 hours after infec�
tion the number of proteinases in the
lungs and serum of infected animals is
reduced and inhibitory activity is in�
creased [14]. Influenza virus infected
cells induce the appearance of the inhib�
itor both in the lung tissue and blood
serum. Consequently, lung inhibitors are
like the first line of an organ defense
under the action of various strains of in�
fluenza virus.
When studying the dynamics of pro�
teinase and inhibitory activity in the
chicken embryos under the influence of
large and small infecting doses of influ�
enza virus A/RR/8/34 we have found that
there occurred the same changes as in
white mice organisms. During the period
of maximal accumulation of infectious
Table 1
Purification of influenza virus АО/32 in succharose gradient and ultracentrifugation at 28000 min.-1, 4
h
Experiments
1 VCF * 2 VCF * 3 VCF * 4 OAF **
Fr
ac
tio
ns
o
f s
ac
ch
ro
se
g
ra
-
di
en
t
%
s
ac
ch
ar
os
e
HR1
Pr
ot
ei
na
se
, m
g/
m
l
%
s
ac
ch
ar
os
e
HR1
Pr
ot
ei
na
se
, m
g/
m
l
%
s
ac
ch
ar
os
e
HR1
Pr
ot
ei
na
se
, m
g/
m
l
%
s
ac
ch
ar
os
e
HR1
Pr
ot
ei
na
se
, m
g/
m
l
1 5 0 1,6 3 0 1,5 6 0 2,6 6 0 0,58
2 15 1:8 36 11 1:2 13,6 15 1:8 41,3 17 0 1,01
3 32 1:16 9,6 24 1:16 8,2 23 1:16 0,45 27 0 0,37
4 42 1:2048 33,6 24 1:16 10,2 30 1:64 0,9 29 0 0,59
5 49 1:2048 4,4 38 1:512 25,4 37 1:512 13,8 37 0 0,80
6 52 1:64 37 41,5 1:1024 26,2 44 1:16000 38 41 0 1,43
7 55 1:64 37,4 46,5 1:512 12,2 47 1:16000 3,29 49 0 0,64
8 57 1:62 6,8 53 1:16 7,9 51 1:256 0,3 53,5 0 1,28
9 57 1:512 56 0 1,08
Notes: * virus-containing fluid;
** original allantoic fluid; 1 – hemagglutination reaction
АКТУАЛЬНЫЕ ПРОБЛЕМЫ ТРАНСПОРТНОЙ МЕДИЦИНЫ � № 3 (22), 2011 г.
58
ACTUAL PROBLEMS OF TRANSPORT MEDICINE �#3 (25), 2011
and hemagglutinating activity (24 hours)
they did not find either proteinase or in�
hibitory activity [15].
Six isoforms of trypsin�like protein�
ase have been isolated from the lungs of
healthy mice by while 8 isoforms have
been isolated from the lungs of infected
mice. Their specific proteolytic activity
increased sharply compared with the ini�
tial material (Table 2). Proteinase ob�
tained isoforms had a broad substrate
specificity and were capable to hydrolyze
both natural and synthetic substrates
[16]. Antiproteinase hyperimmune rat
sera have been obtained to all isoforms
of trypsin�like proteinases. When study�
ing the protective properties of antipro�
teinase sera and sera of healthy rats on
white mice infected intarnasally with le�
thal dose of influenza virus A/PR/8/34 (IV
passage), it was found that 100% mor�
tality of control mice took place in 4�5
days (Table 3). The animals to which they
administrated six times the healthy rats
serum intranasally died on the 7th day. In
the treatment of mice with the pools of
immune sera I, II, IV, V and VI the animals
mortality rate was reduced and lethality
came much later than in the control
group and 20% of the animals recovered
[17].
The most effective was the fourth
pool of imunne serum to the III isoform.
In its presence 60% of infected mace
survived, and on the 14th post� infection
day in the blood serum and in the lungs,
we did not detect either hemagglutinin,
no infectious virus. Imunne serum to iso�
form VI did not protect mice from death,
although isoform III differed from isoform
VI by only one protein with molecular
mass 32 kDa [18].
From the lungs of healthy mice, we
have isolated an inhibitor of trypsin�like
proteases with molecular mass of 47.5
kDa, with a high degree of purity and
small amount of impurities. We have de�
veloped and patented the method of
obtaining and purification of trypsin pro�
teinase inhibitor [19]. The inhibitor iso�
lated is similar to the α1−proteinase inhib�
itor of human blood serum (48�55 kDa)
and egg white trypsin inhibitor (49 kDa),
but does not have not likeness to trypsin
inhibitor isolated from the lungs of cattle
(an inhibitor of Northrop�Kunitz type),
which had a molecular mass of 65 kDa.
In the study of its effect on the proteolytic
activity of trypsin�like proteinase iso�
forms by the test�tube method it has
been shown that it inhibited the activity
of almost all isoforms, except isoforms IV
(41,8%) and VIII (28.3%). In our studies
[20] we used a cellular inhibitor to sup�
press the development of influenza virus
in chicken embryos. It has been revealed
that it inhibited the development of infec�
tious and hemagglutinating activity and
the formation of total protein. At the
same time, the inhibitor of trypsin�like
proteinase isolated from the lungs of
mice, previously infected with influenza
virus, did not have this ability. In the fur�
ther studies [21] for the treatment of in�
Table 2
Purification DEAE –cellulose-53 of lung trypsin-like proteinase of healthy mice
N of frac-
tion
N of
isoform
Specific proteolic activity
per mg of protein
% of pro-
teinase
outcome
% of purifi-
cation by
protein
33 I 4,285 2,09 96,8
53 II 83,75 5,84 99,07
65 III 22,42 2,703 98,38
75 IV 40,00 6,279 97,92
121-130 V 32,6 136,74 99,98
161-189 VI 0,787 421,74 64,90
ACTUAL PROBLEMS OF TRANSPORT MEDICINE �#3 (25), 2011
АКТУАЛЬНЫЕ ПРОБЛЕМЫ ТРАНСПОРТНОЙ МЕДИЦИНЫ � № 3 (25), 2011 г.
59
fluenza infection in animals, we used the
inhibitor, which was isolated from the
lungs of healthy mice.
The introduction of this inhibitor to
mice previously infected with a lethal
dose of influenza virus reduced the death
rate from this disease due to inhibition of
HA splitting on reproduction of the virus
in the lungs, stopping the generalization
of the process, preventing the increase
of proteolysis in the lungs, as well as
preventing aerohematic barrier and im�
proved some of the reactions of local
protection.
For an antiviral drug preparation
that has the lowest allergic action on
humans, we used wastes of donor blood
which is taken for isolation of gamma �
globulin and albumin.
At the first stage of γ�globulin and
albumin manufacture from fraction (II +III,
by Cohn E.J. ) fibrinogen is precipitated,
which is later recycled. According to our
data, the wastes contain 481.11 mg of
trypsin�like proteinase inhibitor per kg of
weight. This centrifugate contained α
1
�
antitrypsin, which is the main inhibitor of
serine proteases of human blood plasma.
Its share in the norm is 90% of antitripsin
activity of human blood plasma [22].
In the second step of γ�globulin ob�
taining, precipitate containing prothrom�
bin, α�and β�globulins and lipoids is uti�
lized. This precipitate, according to our
Table 3
Influence of antiproteinase immune sera on survival of mice under infection with lethal dose of
influenza virus A/PR/8/34
Terms after infecting , days
N
Gr
Isoform of
proteinase
Se
ra
g
ro
up
6 h
1
2
3
4
5
6
7
8
9
10
14
su
rv
ive
d
%
o
f s
ur
vi
ve
1 I I 2/10 2/10 2/10 2/10 2 20
2 I II 2/10 2/10 4/10 2 20
3 II III 2/10 2/10 2/10 2/10 2 20
4 III IV 2/10 2/10 6 60
5 IV V 2/10 2/10 2/14 2 20
6 V VI 5/10 3/10 2/10 100
7 VI VII 7/10 1/10 1/10 1 10
6 Sodium chlo-
ride solution 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 10 100
7 Serum-free
virus 2/10 6/10 All
died 0
8 Healthy rats
serum 2/10 3/10 5/10 All
died 0
9 Immune se-
rum IVgr., vi-
rus-free (toxic-
ity)
0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 0/10 10 100
Note: 1. numerator – number of mice dies; 2 – denominator – number of mice in the experiment.
АКТУАЛЬНЫЕ ПРОБЛЕМЫ ТРАНСПОРТНОЙ МЕДИЦИНЫ � № 3 (22), 2011 г.
60
ACTUAL PROBLEMS OF TRANSPORT MEDICINE �#3 (25), 2011
data [23], contains 469.87 mg of trypsin
proteinase inhibitor per kg of mass. An�
tithrombin�3 (AT�3) or heparin factor � a
regulator of blood coagulation is includ�
ed in this sediment. According to O.A
Markova et al. [24], normally the content
of AT�3 in donors ranged from 160 to 250
mcgr / ml. α1−antitrypsin and α
2
�macro�
globulin [24, 25] are also in the area of
α�globulins [24, 25].
In the third stage of γ�globulin ob�
taining the sediment containing plasmi�
nogen goes to waste. At this stage
wastes, according to our data, the con�
tent of trypsin proteinase inhibitor was
137.40 mg/kg.
At the fourth stage at і�globulin sed�
imentation centrifugate N 3 is utilized.
According to our records, the material of
centrifuged № 3 contains 166.37 mg of
trypsin – like proteinase inhibitor.
Thus, the waste product of the first
and second stages of the technological
process in which sediment (II +III) is
washing and the allocation of prothrom�
bin takes place may serve as the raw
material for producing an inhibitor of
trypsin�like proteinases. These wastes
contained the highest number
of trypsin�like proteinase in�
hibitor.
To separate trypsin�like
proteinase inhibitor we used
the wastes of the Ith stage (II
+ III) of γ�globulin receipt from
donor blood, which contained
a significant amount of this
inhibitor. We isolated trypsin�
like proteinase inhibitor from
the centrifugate (wastes) of
fraction (II + III) of the first
stage of the і�globulin by ion�
exchange chromatography on
DEAE�cellulose�53 (Watman,
USA).
This method allowed to
obtain five isoforms with in�
hibitory activity. The first two
isoforms, in which there was a
high content of trypsin�like
proteinase inhibitors, have been eluted
from ion�exchange column with 0.1 M
phosphate buffer, pH 7.5. The next three
isoforms containing trypsin�like protein�
ase inhibitor have been eluted by step�
wise NaCl gradient of different molarity:
the third isoform � 0.1 M NaCl, the fourth
isoform � 0.2 M NaCl, the fifth isoform �
0.5 M NaCl. The volumes of isoforms
eluates were, respectively: Ist � 35 ml,
IInd�195ml, IIIrd � 340 ml, IVth � 440 ml,
Vth � 605ml.
The highest content of trypsin�like
proteinase inhibitor has been registered
in the fractions of the Vth isoform, which
was the last eluted from the column 0.5
M NaCl, while the lowest content was in
the IVth and IIInd isoforms eluted from
the column 0,2 and 0,1 M NaCl, respec�
tively.
Trypsin�like proteinase plays a key
role in the development of pathological
process in an organism. It splits the out�
er protein of the influenza virus � hemag�
glutinin into two subunits: HA
1
and HA
2
.
Only after the splitting of hemagglutinin
with this protease, the virus enters the
0
10
20
30
40
50
60
70
80
90
100
0 15' 30' 1 6 24 48 72 96 122
hs, post infection
А
0
0,2
0,4
0,6
0,8
1
1,2
1,4
1,6
1,8
in
fe
ct
io
n
tit
re
(L
D
E
ID
5
0)
proteasa inhibitor
total protein activity of hemagglutinin
activity of infection
AI – 1 unit equals 1 mg crystalline trypsin
А – 1 unit activity equals 1 mkg arginin / min.
Figure 2. Changes of proteinase and inhibiting activity in chicken embryos at
a big infecting dose of influenza virus A/PR/8/34
ACTUAL PROBLEMS OF TRANSPORT MEDICINE �#3 (25), 2011
АКТУАЛЬНЫЕ ПРОБЛЕМЫ ТРАНСПОРТНОЙ МЕДИЦИНЫ � № 3 (25), 2011 г.
61
cell and begins to multiply. Inhibitors
block the process of cleavage of viral
proteins by inhibiting the activity of cel�
lular enzymes.
The viral progeny with non�split,
functionally non�active viral proteins is
formed in the presence of inhibitors of
cellular trypsin�like proteinases after a
single reproduction cycle of the primary
virus with split proteins. Subsidiaries vir�
ions are not able to initiate the infectious
process because of the block of the ear�
ly stages of the reproduction cycle �
adsorption and penetration of the virus
[26, 27].
To study the protective action of
trypsin�like proteinase inhibitor on the
survival of mice infected with the lethal
dose of influenza virus A/PR/8/34, we
took 90 white mice of BALB/c line weight�
ing 16�18 g and the fifth isoform of pro�
teinase inhibitor isolated from the wastes
of the first stage of і� globulin production,
as it had the highest indexes of the in�
hibitor activity (132.52 g/l) and the low�
est indexes of trypsin�like proteinase
(0.0027 mmol in the sample).
The mice were divided into 7 groups
containing 15 items in each, with 10
items in control groups (Table 4). The
animals of the first group received lethal
dose of virus (virus control). The virus
was administered intranasally in a volume
of 0.05 ml under Rausch anesthesia. The
second group of animals got a similar
dose of virus, but they were treated with
crystalline trypsin (control of medical
properties of crystalline trypsin) in the
same doses and terms as the animals of
the 3rd group.
The third group of animals was in�
fected with the same dose of virus and
was treated with trypsin – like proteinase
inhibitor obtained from the wastes of і�
globulin.
The fourth group of animals got only
an inhibitor of proteinases from the
wastes of і�globulin production (control
inhibitor’s toxicity).
Crystalline trypsin (trypsin control)
alone was introduced to the animals of
the fifth group, the sixth group got phos�
phate buffer, with which we diluted virus
inhibitor and trypsin. The seventh group
was used as the control of intact animals.
Both trypsin and inhibitor was adminis�
Table 4
Action of cellular inhibitor of trypsin-like proteinase of the survival rate of mice infected
with lethal dose of influenza virus А/PR/8/34
Number of ani-
mals N and name of
group
Number
of ani-
mals
Dose of
influ-
enza vi-
rus
Dose of in-
hibitor on
mice per
protein
Dies Sur-
vived
% of animals
protected
from virus
1. Influenza virus 40 10-3 - 40 - 0
2. Influenza virus
+trypsin, crystal. 40 10-3 18 mkg 40 - 0
3. Influenza virus
+ inhibitor from
healthy lungs
40 10-3 18 mkg 7 33 82,5
4. Cellular inhibi-
tor 40 10-3 18 mkg - 40 100
5. Trypsin, crystal 10 - 18 mkg - 10 100
6.Phosphate
buffer 10 - 0,2 ml - 10 100
АКТУАЛЬНЫЕ ПРОБЛЕМЫ ТРАНСПОРТНОЙ МЕДИЦИНЫ � № 3 (22), 2011 г.
62
ACTUAL PROBLEMS OF TRANSPORT MEDICINE �#3 (25), 2011
tered intranasally under light ether anes�
thesia during seven days. Each mouse
got up to 140 mcg of the inhibitor for the
course of treatment.
The results of our studies showed
that the animals of the first and second
groups died on the 6�7 post�infection
day. In the third group 12 mice survived
(80%). They remained alive for 14 days
after infection (observation period). An�
imals of the 4th, 5th, 6th and 7th groups
remained alive throughout the whole pe�
riod of observation. In addition, newly
acquired inhibitor of trypsin�like protein�
ases, did not cause toxicity, as white
mice of the 4th group remained alive at
the 14th day after introducing of the in�
hibitor.
Thus, the findings of our work indi�
cate that trypsin�like proteinase inhibitor
obtained from the wastes of the first
stage of і – globulin production pos�
sessed antiviral properties. It is not ex�
cluded that it can be used not only for
influenza treatment but for the treatment
of other viral infections in which the split�
ting of viral precursor protein is produced
by cellular tripsin�like proteinases [21].
Conclusions
One of the most important stages of
the development of many viruses in the
host organism is their introduction into
the cell after preliminary deproteinization.
Regulation of this cycle by proteolytic en�
zymes of the host�cell is one of the fun�
damental principles of viral reproduction.
Induction or introduction of inhibitor of
the virus proteolytic activation is one of
the promising ways of viral diseases
treatment, including influenza.
Inhibitor of trypsin�like proteinases
obtained from industrial wastes of і�glob�
ulin manufacture blocked the develop�
ment of grippe infection in white mice
preliminary infected with lethal dose of
influenza virus A/PR/8/34 (A/H1N1).
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Резюме
ПРОТЕОЛІТИЧНА ТЕОРІЯ
ПАТОГЕНЕЗУ ГРИПУ ТА
УДОСКОНАЛЕННЯ ЛІКУВАННЯ ГРИПУ
Дівоча В.П., Гоженко А. І.,
Михальчук В.М.
Український НДІ медицини
транспорту МЗ України, 65039 Одеса
Запропонована нова теорія
патогенеза грипу за участю
протеїназно�інгібіторної системи.
Встановлено, що очищення та
концентрація вірусу грипу різними
методами не звільняло вірус від
клітинних ферментів. При інфікуванні
тварин вірусом грипу відбувалось
порушення ферментно�інгібіторної
рівноваги, особливо у перші години
після інфікування. З легенів дужих
мишей одержано шість ізоформ
трипсиноподібної протеїнази до яких
отримані антипротеїназні імунні
сироватки та проведено лікування
ними тварин. Тільки антисироватка до
третьої ізоформи захистила тварин від
смерті. З промислових відходів
одержання гамаглобуліну виділено
інгібітор трипсиноподібних протеїназ,
який захистив білих мишей від смерті
на 80%. Ендогенні інгібітори протеїназ
крові людини є перспективними
антигрипозними препаратами для
людини.
Ключові слова: грип, трипсиноподібні
протеїнази, інгібітори протеїназ,
очищення віру су, противіру сні
препарати.
Резюме
ПРОТЕОЛИТИЧЕСКАЯ ТЕОРИЯ
ПАТОГЕНЕЗА ГРИППА И
УСОВЕРШЕНСТВОВАНИЕ ЕГО
ЛЕЧЕНИЯ
Дивоча В.А., Гоженко А.И.,
Михальчук В.М.
Предложена новая теория
патогенеза гриппа с участием
протеиназно � ингибиторной системы.
Установлено, что очистка и
концентрация вируса гриппа разными
методами не освобождало вирус от
клеточных ферментов. При заражении
животных вирусом гриппа
происходило нарушение ферментно �
ингибиторного равновесия, особенно в
первые, часы после заражения. С
легких здоровых мышей, получено
шесть изоформ трипсиноподобной
протеиназы к которым, получены
антипротеиназные иммунные
сыворотки и проведено лечение ими
животных. Только антисыворотка к
третьей изоформе защитила животных
от смерти.
С промышленных отходов
получения гаммаглобулина выделено
ингибитор трипсиноподобных
протеиназ, который защитил белых
мышей от смерти на 80%. Эндогенные
ингибиторы протеиназ крови человека
являются перспективными
антигрипозными препаратами для
человека.
Ключевые слова: грипп,
трипсиноподобные протеиназы,
ингибиторы протеиназ,
противовирусные препараты.
Впервые поступила в редакцию 18.07.2011 г.
Рекомендована к печати на заседании
редакционной коллегии после рецензирования
|
| id | nasplib_isofts_kiev_ua-123456789-136563 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 1818-9385 |
| language | English |
| last_indexed | 2025-12-07T17:14:30Z |
| publishDate | 2011 |
| publisher | Фізико-хімічний інститут ім. О.В. Богатського НАН України |
| record_format | dspace |
| spelling | Divocha, V.A. Gozhenko, A.I. Mikhalchuk, V.N. 2018-06-16T13:52:07Z 2018-06-16T13:52:07Z 2011 Proteolytic theory of pathogenesis of influenza and improvement of its treatment / V.A. Divocha, A.I. Gozhenko, V.N. Mikhalchuk // Актуальні проблеми транспортної медицини. — 2011. — № 3 (25). — С. 54-64. — Бібліогр.: 27 назв. — англ. 1818-9385 https://nasplib.isofts.kiev.ua/handle/123456789/136563 616.921.5 A new theory of grippe pathogenesis with the use of proteinase-inhibitory system has been offered. It has been established that purification and concentration of grippe viruses by different methods did not release the virus from the cellular enzymes. When the experimental animals have been infected with grippe virus, we have observed misbalance in enzyme-inhibitory balance took place, especially during first hours after infection. Six isoforms of trypsin-like proteinases were obtained from the lungs of healthy mice. Antiproteinase immune sera have been isolated from these 6 isoforms which served to treat the experimental animals. It was antiserum to the third isoform that has prevented the experimental animals mortality. From the industrial wastes of gamma-globin manufacture the inhibitors of protein-like proteinases have been extracted which prevented the white mice’s mortality in 80% of cases. Endogenous inhibitors of human blood proteinases are prospective in producing anti-influenza drugs for humans. Запропонована нова теорія патогенеза грипу за участю протеїназно-інгібіторної системи. Встановлено, що очищення та концентрація вірусу грипу різними методами не звільняло вірус від клітинних ферментів. При інфікуванні тварин вірусом грипу відбувалось порушення ферментно-інгібіторної рівноваги, особливо у перші години після інфікування. З легенів дужих мишей одержано шість ізоформ трипсиноподібної протеїнази до яких отримані антипротеїназні імунні сироватки та проведено лікування ними тварин. Тільки антисироватка до третьої ізоформи захистила тварин від смерті. З промислових відходів одержання гамаглобуліну виділено інгібітор трипсиноподібних протеїназ, який захистив білих мишей від смерті на 80%. Ендогенні інгібітори протеїназ крові людини є перспективними антигрипозними препаратами для людини. Предложена новая теория патогенеза гриппа с участием протеиназно-ингибиторной системы. Установлено, что очистка и концентрация вируса гриппа разными методами не освобождало вирус от клеточных ферментов. При заражении животных вирусом гриппа происходило нарушение ферментно-ингибиторного равновесия, особенно в первые, часы после заражения. С легких здоровых мышей, получено шесть изоформ трипсиноподобной протеиназы к которым, получены антипротеиназные иммунные сыворотки и проведено лечение ими животных. Только антисыворотка к третьей изоформе защитила животных от смерти. С промышленных отходов получения гаммаглобулина выделено ингибитор трипсиноподобных протеиназ, который защитил белых мышей от смерти на 80%. Эндогенные ингибиторы протеиназ крови человека являются перспективными антигрипозными препаратами для человека. en Фізико-хімічний інститут ім. О.В. Богатського НАН України Актуальні проблеми транспортної медицини Материалы 11 Международного симпозиума по морской медицине Proteolytic theory of pathogenesis of influenza and improvement of its treatment Протеолітична теорія патогенезу грипу та удосконалення лікування грипу Протеолитическая теория патогенеза гриппа и усовершенствование его лечения Article published earlier |
| spellingShingle | Proteolytic theory of pathogenesis of influenza and improvement of its treatment Divocha, V.A. Gozhenko, A.I. Mikhalchuk, V.N. Материалы 11 Международного симпозиума по морской медицине |
| title | Proteolytic theory of pathogenesis of influenza and improvement of its treatment |
| title_alt | Протеолітична теорія патогенезу грипу та удосконалення лікування грипу Протеолитическая теория патогенеза гриппа и усовершенствование его лечения |
| title_full | Proteolytic theory of pathogenesis of influenza and improvement of its treatment |
| title_fullStr | Proteolytic theory of pathogenesis of influenza and improvement of its treatment |
| title_full_unstemmed | Proteolytic theory of pathogenesis of influenza and improvement of its treatment |
| title_short | Proteolytic theory of pathogenesis of influenza and improvement of its treatment |
| title_sort | proteolytic theory of pathogenesis of influenza and improvement of its treatment |
| topic | Материалы 11 Международного симпозиума по морской медицине |
| topic_facet | Материалы 11 Международного симпозиума по морской медицине |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/136563 |
| work_keys_str_mv | AT divochava proteolytictheoryofpathogenesisofinfluenzaandimprovementofitstreatment AT gozhenkoai proteolytictheoryofpathogenesisofinfluenzaandimprovementofitstreatment AT mikhalchukvn proteolytictheoryofpathogenesisofinfluenzaandimprovementofitstreatment AT divochava proteolítičnateoríâpatogenezugriputaudoskonalennâlíkuvannâgripu AT gozhenkoai proteolítičnateoríâpatogenezugriputaudoskonalennâlíkuvannâgripu AT mikhalchukvn proteolítičnateoríâpatogenezugriputaudoskonalennâlíkuvannâgripu AT divochava proteolitičeskaâteoriâpatogenezagrippaiusoveršenstvovanieegolečeniâ AT gozhenkoai proteolitičeskaâteoriâpatogenezagrippaiusoveršenstvovanieegolečeniâ AT mikhalchukvn proteolitičeskaâteoriâpatogenezagrippaiusoveršenstvovanieegolečeniâ |