Наука и образование для целей биобезопасности: Материалы пятой международной конференции. Пущино 2008 г
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Материалы пятой международной конференции
Наука и образование для целей биобезопасности
131
использовать бытовые, что в целом практически лишает подобные приготовления
заметных маркеров.
Заметным моментом является то, что безаварийное проведение таких работ
возможно только высококвалифицированным специалистом с большим опытом работ по
культивированию конкретного возбудителя. Таких специалистов немного и появление
такого специалиста в неожиданном месте является настораживающим маркером. Таким
образом на вопрос, вынесенный в
заголовок ответ положительный. Представляется
важным провести подробный анализ условий возможности наработки рецептуры в
бытовых условиях для создания лекала маркеров, обеспечивающих своевременное
выявление таких приготовлений.
Материалы пятой международной конференции
Наука и образование для целей биобезопасности
132
ПОЛЕВАЯ ЭКСПРЕСС ДИАГНОСТИКА ОСОБО
ОПАСНЫХ ПАТОГЕНОВ НА ПРИМЕРЕ ВИРУСА
ЭБОЛА
А. Чепурнов
1
, Н. Федосова
2
, И. Егоричева
2
, Ф. Элх
3
1
ГУ НИИ Клинической Иммунологии СО РАМН,
2
ОАО Вектор-БЕСТ,
3
Университет Умеа, Швеция.
Не смотря на широкий спектр надежных методов идентификации вируса Эбола
для их выполнения необходим квалифицированный персонал, специализированные
лаборатории, сложное оборудование и существенные затраты времени. Поэтому
необходим метод, позволяющий проводить идентификацию возбудителя в полевых
условиях или у постели больного специалисту, уровня врач или фельдшер, без
специального оборудования и
в минимальное время. При разработке такого метода себя
хорошо зарекомендовала методика иммуноанализа представляющая собой
иммунофильтрацию на нитроцеллюлозной мембране с использованием фиксированного
антигена (антител) или их иммунозолей. Скорость постановки анализа составляет 7-15
минут.
Материалы пятой международной конференции
Наука и образование для целей биобезопасности
133
ИФА ТЕСТ-СИСТЕМЫ К ОСОБО ОПАСНЫМ
ИНФЕКЦИЯМ
Юсупова Э.Г., Азимова Ш.С.
Институт химии растительных веществ АН РУз, Ташкент, Узбекистан
Заболевания вирусной и бактериальной природы являются чрезвычайно
актуальной проблемой для стран Центральной Азии. СПИД, гепатиты В и С, сифилис
являются инфекциями повышенного риска. Известно, что наиболее эффективными
мерами для борьбы с такими заболеваниями являются их
ранняя диагностика и
выявление источника заражения. Для этого необходимо проводить скрининг населения
на присутствие ВИЧ, вирусов гепатитов В и С, и возбудителя сифилиса. Наиболее
важными группами исследований являются беременные женщины, дети, медицинские
работники, специалисты лабораторных служб и представители групп риска (доноры
крови, пациенты гемодиализа и т.д.). На сегодняшний день наиболее
широко
применяемыми в мире являются тест-системы на основе ИФА.
В лаборатории молекулярной генетики ИХРВ АН РУз разработаны технологии
получения рекомбинантных белков с помощью различных систем экспрессии
(бакуловирусная, дрожжевая, клеточная).
Налажена технология получения гибридом продуцирующих моноклональные
антитела к различным белкам, а также низкомолекулярным веществам. Получены
стабильные гибридомы, продуцирующие моноклональные антитела
к рекомбинантному
HBsAg и эритропоэтину. На основе генно-инженерной и гибридомной технологий
разработаны тест-системы иммуноферментные для выявления различных инфекций
(гепатиты В и С, СПИД).
В рамках международного проекта (STCU 3086 – «Разработка ИФА тест-системы
для диагностики сифилиса и создание референтной панели сывороток для контроля
качества скринирования донорской крови») была завершена разработка
скрининговой
ИФА тест-системы для диагностики сифилиса, а также создана техническая база для
серийного производства ИФА тест-систем.
В настоящее время разработанные нами ИФА тест-системы к ВИЧ, возбудителю
сифилиса, вирусам гепатитов В и С зарегистрированы в Главном управлении по
контролю качества лекарственных средств и медицинской техники при Министерстве
здравоохранения РУз
. Разработки запатентованы (патенты Республики Узбекистан №№
IAP 02994, IAP 03055, IAP 03058, IAP 03126, IAP 03127).
Таким образом, на базе ИХРВ АН РУз может быть налажено производство ИФА
тест-систем для диагностики ВИЧ, сифилиса, гепатитов В и С и других
иммуноферментных диагностикумов в количествах, достаточных для обеспечения служб
крови Республики Узбекистан (с населением более чем 25 млн. чел.) и других стран
Центральноазиатского региона.
Materials of the Fifth International Conference on Science and Business
Science and Training for Biosafety
134
BIOCOMPATIBLE COMPOUNDS CONTAINING SILVER
NANO-CLUSTERS USEFUL FOR TREATING BURNS
AND ULCERS: STUDY OF SAFETY AND
BIOACTIVITIES
S. F. Antonov, B.A. Paramonov, V.P. Dobritsa, O.V. Ribalchenko, A.M. Shliakov
Security of antibacterial activity of wound-healing materials determines the favorable
course of the wound process and the soonest recovery of functional integrity and structural
organization of damaged tissues. Due to spreading of numerous strains of pathogenic
microorganisms, resistant to antibiotics, the interest for usage of antibacterial preparations,
based on non-organic junctions (particularly, silver’s junctions) has grown dramatically.
However, molecular and colloidal preparations of silver demand dealing with high
concentrations and their usage as a part of bandaging materials is often followed by numerous
side effects. Using of silver in form of nano particles, due to their high reactivity, provides the
required bactericidal activity while dealing with 2-3 times lower concentrations of silver than
usual, which allows to avoid side-effects and reduce cytotoxicity.
We offer hydrogel dressings, based on chitosan, with silver’s nano-particles. The
complex study of bactericidal properties, toxicology, safety, sanitary and chemical
characteristics and wound-healing activity of the new material was held.
The conditions of generations of silver’s nano-particles in chitosan gel and of formation
of bandaging materials based on it, with properties, staying stable for 3 years were studied. The
bactericidal activity was studied as well. The synergism of chitosan’s and cluster silver’s joint
bactericidal activity in relation to microorganisms was determined. Including cluster silver in
consentrations of 0,1-0,05 mmol into hydrogel led to full suppression of vital activity of such
test cultures as Escherichia coli М-17 and №1337.1, Staphylococcus aureus АТСС-№25923 and
№1169.2 and Pseudomonas aeruginosa АТСС-№27853 , №1321 and others, in dilutions 10
3
-
10
7
CFU/mL after 18 hours of incubation. Efficient silver concentrations in composition with
nano particles turned out to be 1-2 times lower than in case of commercial preparation
“Poviargol”. Minimal inhibiting silver’s concentrations in concentrate of nano particles and
hydrogel with nano particles are 0,05-0,01 mmol, depending on the type of microorganism.
The electronic microscopical study of nano particles’ influence on microorganism cells’
structure when contacting was held. The influence of silver’s nano particles leads to destructive
changes. Enormous quantity of cellular structures can be observed in extracellular space is being
obserbed, and it can be explained by flowing-out of cytoplasm, caused by destruction of
integrity of cell’s membrane by particles. Coagulation and dissection of intracellular structures
are being observed, which finally leads to lysis of cells.
The study of bactericidal activity of silver’s nano particles within hydrogels has shown
high bactericidal efficiency along with very low concentrations of silver, which on the one hand
allows to reduce significantly the risk of side effects, common for silver-containing
preparations, but and the other hand allows to guarantee the requested antibacterial properties of
hydrogel dressings. Polymeric gel structures protect silver’s nano-particles from quick
inactivation by wound biologic environment and prolong antibacterial activity.
According to sanitary, chemical and toxicological factors, lack of irritant, haemolitic
and sensitizing activity chitosan hydrogel dressings with silver meet the safety norms for
medical products, designed to have short and long-term contact with a wound.
135
Chitosan hydrogel dressings with silver clasters were used for local treatment of
patients with trophic ulcers of limbs and for treatment of burns of II, III-a and III-b degree.
Based on received results the conclusion on high efficiency of a wound dressing was made. This
medical product can be widely used in ambulatory and clinic practice. Dressings provide
favorable course of wound process, facilitates regeneration of tissues, leads to reduction of the
healing time. Chitosan hydrogel dressings, containing silver nanoclasters are very perspective
for usage in surgical practice.
Materials of the Fifth International Conference on Science and Business
Science and Training for Biosafety
136
SAFETY AND QUALITY OF AIR CRITERIA IN OBJECTS
WITH ARTIFICIAL HUMAN ENVIRONMENTS
Andrey Beznayev, Alexander Mestcheryakov
Russian Academy of Sciences, Institute of Control Sciences, Moscow, Russia
Issues of determination of air safety and quality criteria in places with
artificial environment are investigated. The main factors of influence of
atmospheric air on human environment are discussed. Risk factors of air security
violation from internal sources of air pollution are reported
The problem of securing of safety and quality of air environment takes on special
significance in the tasks of life-support in artificial environments. The issues of automation of
processes of securing of air safety and quality are among the priority scientific lines of
investigation focused on creation of new technologies of control of air environment
characteristics in various places with artificial environments. Classification of places with an
artificial environment is the following [1]: partially hermetically closed and hermetically closed
places. The air environment in such places can be completely isolated from the external air
environment or have limited connection to the atmospheric air.
The basic criteria of air quality in such places are chemical and physical characteristics
of air, presence of various pollutants and microorganisms which are pathogenic for the human
being. Physical characteristics of the air such as temperature, humidity, quantity of the positive
and negative air ions having various speeds of mobility are of decisive importance for air safety
in partially hermetically closed and hermetically closed places. Air ion composition of the air is
qualitative and quantitative indicator of its purity and is able to have a strong physiological
influence on human organism [2].
The air is considered physiologically favorable when it secures the comfort of human
staying in the environment and reduces risk factors of occurrence and development of various
pathological changes in an organism.
By now in Russia there are no standards which define the quality of the air in the places
with an artificial environment. There are no criteria of air environment safety also.
The existing approaches to improvement of air quality in artificial environments do not
take into consideration the impact of natural, man-caused and anthropogenic factors which
influence on air safety and quality in places, and also the impact of internal elements of the
place on air pollution processes.
The distinctive feature of the current approach to the decision of a problem of securing
air safety and quality is the use of the system approach and of the modern knowledge acquired
in the information technologies area, during investigation and simulation of methods of control
of air quality in different places with an artificial environment, perceptions of air quality by an
individual, own investigations, concerning the problem of control of physical characteristics of
air environment in hermetically closed and partially hermetically closed places.
Various aspects of new technologies of control of air quality and safety in hermetically
closed and partially hermetically closed places are investigated in this work.
It is shown that solving these fundamental scientific problems allows to determine new
ways of solving practical problems in the field of automation of processes of securing air quality
and safety in various places.
137
References.
1. Mestcheryakov, S. Osipov. New Technologies of Control of Air Quality and Safety in
Objects with Environment. // Information Technologies and Computing Systems, – 2008. –
№ 2. – P. 20-26.A.
2. Mestcheryakov, S. Osipov. Investigation of Therapy by Negative-Unipolar Air Ions in
Objects with an Artificial Environment. // Works of Institute of System Analysis of Russian
Academy of Sciences: Vol. 19. – Moscow: ComKniga. – 2006. – P. 171-181.
Materials of the Fifth International Conference on Science and Business
Science and Training for Biosafety
138
CHANGE IN UNDERSTANDING OF BIOHAZARDS AND
NEW THREATS OF BIOLOGICAL WAR AND
BIOTERRORISM
Borovick R.V.,
Remnev J.V., Kolombet L.V.
State Federal Enterprise for Science "Research Centre for Toxicology and Hygienic Regulation
of Biopreparations" at Federal Medico-Biological Agency of Russian Federation, Serpukhov,
Moscow region
Sense of biohazard in animals and humans is an instinct developed in evolutionary
process. Humans express it in the form of disgust to practically all biological products of
foreign nature, in animals – in the form of a series of ingrained reflexes (e.g., animal habit to
bury excrements).
Bioterrorism is distinguished as independent component in biohazard problem, and
ordinary hooliganism, banditry and other antisocial phenomena are often related to bioterrorism.
We believe terrorism can doubtfully be addressed in cases when no anti-governmental social
and/or religious objectives affecting interests of all social layers are pursued. Unfortunately
etiology of terrorism on the whole is insufficiently explored. As a rule, terrorism attracts
attention after commitment of crime. Of much importance is to understand the motives drawing
an individual to act of aggression prior to commitment of crime.
Understanding of reasons will allow finding the means of terrorism prevention by
influencing certain personality attributes. These personality attributes are to a variable extent
affected “from the outside”. For better understanding of these peculiarities we propose “atomic
model” of effects on human personality in an assumption that not all individual’s principles,
habits, and views can be equally changed. “Atomic model” demonstrates that an individual can
be comparatively easily convinced to shift his political views (how easily State Duma members
change one party for another!); on the contrary, religious beliefs are very hard to change.
Likewise, electron can be easily detached from the outer shell of an atom, but it is hard to detach
being in the inner shell.
We would like to dwell upon the generally accepted understanding of biological
weapons as of “weapons for poor countries”, “the simplest and cheapest weapons”. This thought
is fallacious; biological weapons are more complicated, vulnerable, and less well understood
than, for example, nuclear weaponry. My teacher, Professor Fedor Z. Amfiteatrov, often said
that in biology 2x2 sometimes can be 5.
The assertion of apparent simplicity was introduced purposely to distract attention from
highly dangerous perspectives of new generation biological weapons. Over ten years ago
academician A.S. Spirin warned about change in biological war paradigm. He drew attention to
unique possibilities of regulatory functions of lesser messenger RNA and the consequences of
manipulations with them. Half a step remains to constructing ethnic weapon.
Nobel award given to Christine Meyer, the University of Massachusetts – is a
convincing proof of rightness of these fears.
New biological war can begin without a cannonade, warplane patrolling, or missile
launches and explosions …it might have already been started! That is why of deep regret is our
government’s lack of understanding of the need in funding the studies of means for protection
from biological weapons of both old and new generation.
The USSR possessed fine biosafety and biosecurity system (SSM – special safety
measures) and strong anti-plague system, which were inherited by Russian Federation almost in
full. Today we have the possibility to compare how the two ways of biosafety and biosecurity,
Russian and foreign ones, were “evolving”. Recent changes in the world, processes of mutual
139
integration in science, particularly in biotechnology, require harmonization of our approaches
both in the field of biosafety and biosecurity and in terrorism combating.
Materials of the Fifth International Conference on Science and Business
Science and Training for Biosafety
140
USE OF DYES TO DISTINGUISH SPORES B. CEREUS BY
A PRINCIPLE ALIVE / DEAD
V.V. Buyanov
1
, Chmyr I.A.
1
, Semenov А.М.
2
, Nikolskaya V.P.
1
, Pudova О.B.
1
1
Federal State Unitary Enterprise State Scientific Centre «Scientific Research Institute of
Biological Engineering» Federal Agency for Medicine and Biology, Moscow , Russia,
2
Department of Microbiology, Biological Faculty, Moscow State University, Moscow , Russia,
Quickly to receive the answer about quality of the lead disinfection, works
on selection of dye for differentiation of spores by a principle alive / dead are
conducted. The technique of application of dye L-7012 LIVE/DEAD BacLight
Bacterial Viability Kit which allows to differentiate and take into account alive and
dead cells in one drug after processing by dry hydrogen dioxide is fulfilled.
The complex of researches has been lead with the purpose of search of ways of fast
differentiation of viable and dead spores. B. cereus var. anthracoides 250. For this purpose it
was offered to use dyes L-7012 LIVE/DEAD BacLight Bacterial Viability Kit which allow to
differentiate and take into account alive and dead cells in one drug (Molecular probes, 2001).
With this purpose there was a suspension of spores В. cereus on 5 test tubes with the
oblique culture medium of the following composition: the Solution A, in g/0,5 л distilled water
(sterilization at +1 atmosphere): К2НРО4 - 5,0; NaCl - 1,0; MnCl2*4H20 - 0,04; FeSO4*7H2O
- 0,0025; molybdate of ammonium - 0,002; an agar - 17 g; рН-7,0. A solution В, in g/0,5 л
distilled water (sterilization at +0,5 atmosphere): glucose - 30; hydrolysate of casein (Difko) -
2,5; MgSO4*7 H2O - 0,5; CaCl2 * 2 H20 - 0,3. Solutions A and В also would seat in an
autoclave separately and mix equal volumes after cooling up to 50оС (Methods of the
general{common} bacteriology under Gerhard's edition, т. 1.1983). Cultivation carried out
within 1 day in a thermostat at 30оС. A biomass of diurnal culture washed off in distilled water
with a loop and have transferred on 1 ml to microtest tubes. Suspension has been mixed with
use of the device for agitating (Elmi V-3). One of test tubes has been warmed up during 4 mines
in a boiling water bath.
Dye L-7012 LIVE/DEAD BacLight Bacterial Viability Kit stored{kept} at a minus
20оС, defroze at ambient temperature in darkness and centrifuged at 15000 rpm during 1 mines,
according to the instruction on use of dye. After experiment, dye again has been frozen. With
micropipet have added in each test tube on 1,5 mcl of a component of dye A and 1,5 mcl of a
component В of dye In from L-7012 LIVE/DEAD BacLight Bacterial Viability Kit. Contents
has been mixed of each test tube and has been sustained at ambient temperature in darkness
within 15 minutes. On 5 mcl pigmented bacterial cloud observed in a fluorescent microscope,
light filters (ФС1-6 - filtering and ЖС 18-2 cutting), an objective х100 with oil immersion.
In the drugs prepared from not heated up suspension of cells, containing alive
vegetative cells, in a plenty the extended cells luminous by brightly green light, rods are
distinctly visible. There are luminous brightly green light, shallow spherical forms which,
undoubtedly, are spores "which have "left" cells. There was a set of vegetative cells containing
spores, thus the spores which are taking place in cells, shine saturated green light and is
brighter, than cells bearing them. Besides the extended which according to the instruction
applied to dye should be attributed to impractical were sometimes found out luminous yellow -
orange, sometimes with points of faintly green color.
In the drugs prepared from heated up suspension of cells, the lengthy rods, in part in the
chains, luminous yellow - orange, sometimes with points of faintly green color are found out.
Also there are spherical forms luminous by brightly green light - spores which were not lost at