RF Ministry of Health

Rostov Research & Development Institute of Microbiology and Parazitology

(RRDIMP)

Report

On research work “investigations of medical “Biofon” device effect on microorganisms, and evaluation of its efficiency and medical and biological safety”

Deputy Director of RRDIMP
for scientific work, M.D. “15” October, 1996 E.A.Yagovkin

Scientific Director of RRDIMP
Honoured Man of Science of RF,
M.D., Professor, Corresponding
member of AS “15” October, 1996 A.P.Shepelev
R.F.State Prize Laureate

Rostov-on-the Don

1996

List of Executors

Content

Introduction

1. Investigations of “Biofon” apparatus effect on microorganisms.

1.1. Studies of “Biofon” effect on Ch. trachomatis and Ch. psitaci in the experiments on chicken embryo.

1.2. Studies of “Biofon” apparatus effect on Ch. psittaci in the experiments on white mice.

1.3. Studies of “Biofon” apparatus effect on the multiplication of Ch. trachomatis and Ch. psittaci in the cultures of the wound cell line.

3. Investigations of “Biofon” apparatus effect on experimental animals.

4. Investigations of “Biofon” apparatus effect on the human body (a limited group of patients with urogenital chlamydiosis).

5.1. Studies of Physiotherapeutic efficiency of the “Biofon” apparatus effect on patients with urogenital chlamydiosis.

5.2. Studies of “Biofon” apparatus effect on the immune system of the patients with urogenital chlamydiosis.

5.3. Studies of the biochemical shifts in the organisms of patients with urogenital chlamydiosis under the action of the “Biofon” apparatus.

Conclusion.

A list of literature used

An Essay

Report 33pp., 13 tables, 12 references, 1 appendix.

Key words:

“Biofon” apparatus, microorganisms, experimental animals, patients with chlamydiosis, medical and biological efficiency, safety.

The objects of the study were: the “Biofon” apparatus for medical purposes, microorganisms, experimental animals, patients with urogenital chlamydiosis.

The aim of the work was to evaluate medical and biological efficiency and safety of the medical "Biofon" apparatus for treatment of chronic diseases, including urogenital ones (chlamydiosis).

Virological, biological, immunological, microbiological, biochemical and clinical methods of examination have been used in the research work.

In the course of investigations effect of the Biofon apparatus on microorganisms, experimental animals and the humen body (a limited group of patients with urogenital chlamydiosis) hase been studied. As the result of the studies it has been estimated that:

1. Radiation of Ch. trachomatis suspension by the “Biofon” apparatus results in the decrease of infection activity in the cultures of cells and chicken embryos. This effect is less marked in case of infecting white mice. Multiple treatment of Ch. trahomatis suspension and cells cultures, infected by the suspension, by the “Biofon” apparatus inhibites the agent development.

2. The exposure of white mice to IR-radiation of the “Biofon” apparatus does not cause animal death, any changes in their behavior, hair coverage and mucous membranes; refusal in food and water; weight loss or reproductive inability.

Examination of the biochemical parameters of blood, liver and myocardium of the experimental animals has revealed no deviations from the physiological standards.

3. Of 38 patient with urogenital chlamydiosis, exposed to “Biofon” radiation, 60-70% of volunteers have shown positive therapeutical effect, confirmed by the decrease in the number of chlamydia in the smears or by their complete absence.

Application of the apparatus does not result in the negative reactions of cell humoral immunity links in the patients. Biochemical examinations have not revealed any statistically significant deviations from the physiological standards, characterizing functions of the liver, pancreas and kidneys.

But more complete and objective evaluation of clinical and biological erfficiency of the “Biofon” apparatus requires additional investigation of a wider number of patients with urogenital chlamydiosis.

Introduction

Among the variety of infectious pathology of men and animals a vast niche is occupied by chlamydious infections, causing serious problems to healthcare due to a wide spread and significant affection upon the health and reproduction of the population. Causative agents of chlamydious diseases are bacteria of Chlamydia kind, which are pathogenic obligate intracellular parasites, living in the sensitive host cells – in the basal cells of the column and, probably, transitional epithelium. Two main forms of chlamydia are known: elementary body – ET (infectious form), and reticular body – RB (vegetative form). According to the WHO, among the diseases transferred by a sexual way, genital chlamidiosis occupied the 2d place after trichomoniasis, being in the first eight of the leading groups of infections.

Significance of urogenital chlamydiosis in the infectious pathology of the men is determined by the presence both acute diseases and chronicle pocesses, resulting in multifocal damages of the urogenital system, and by their secondaries, affecting the reproductive function of the body. A high incidence of infecting neonatals by chlomydia occurs while they are passing the reproductive ways.

It is also necessary to note the increasing morbidity with urogenital chlamydioses: chlamydious proctitis, pharyngitis, chlamydious pneumonia, chlamydious conjuctivitis, Reiter’s disease.

Though various approaches to the treatment of these diseases have been developed in the recent years, including intensive antibacterial therapy by the antibiotics of the 4^th generation, treatment efficiency remains rather low.

A complex approach to the problem is considered to be the most promising one. It includes the application of antibiotic drug, immunostimulators in combination with physiotherapeutic measures: phonophoresis, laser and ultraviolet radiation of the blood. Application of these methods allows to decrease the drug load and terms of treatment. But these methods, though widely used, have some drawbacks. They are connected with the invasivity of the procedures, which are not always safe enough in respect HIV-infection, gonorrhea, syphilis and AIDS-marking diseases for the group of patients with diseases spread by sexual activity. In this connection, a medicine-oriented "Biofon" apparatus is of great interest as it has been designed for chlamydiosis treatment, including the urogenital tract both without medicamental means and in a combination with chemotherapy. The apparatus effect is based on the electrostatic potential changes, occuring on the bacterium cell membranes, on a decrease of the toxins produced, and on the following activization of the immune system of the man and animals and increase in the rate of bacterium destruction.

The "Biofon” apparatus is a patented device, permitted for export production.

Findings obtained by the R&D Enterprise (RDE) “Bionix”, using lumino-independent chemoluminescence show the evidence of energenic action of the “Biofon” apparatus on Staphylococcus aureus strains.

Observations carried by the Republic Skin and Venereological Dispensary (Izhevsk) revealed that application of the “Biofon” apparatus provided complete cure from urogenital chlamydiosis in 50-60% cases. The findings obtained suggest that it is reasonable to continue more comprehensive studies in order to estimate the safety of the device, evaluate its effect on chlamydia “in vitro” and “in vivo”, and in experiments on patients with urogenital chlamydiosis.

The purpose the present investigation is to evaluate medical and biological efficiency and safety of the medical “Biofon” apparatus for antibacterial treatment of chronic diseases, the following tasks have been set forth:

1. To study the “Biofon” effect on Ch. trichomatis microorganisms;
2. To study the “Biofon” effect on experimental animals;
3. To study the “Biofon” effect on the human body (a limited group of patients with urogenital chlamydiosis). In the course of work virological, biological, immunological, microbiological, biochemical and clinical research methods have been used. The techniques and literature used are given in the corresponding sections of the report.

A complex approach allowed to solve the problems set forth. These investigations have been caried on in cooperation with the collegues from Rostov Medical University (RMU) according to the contract No. 379 of October 01, 1996 with the State Enterprise “Izhevsk Mechanical Plant” and the "Technical Specification" to it.

1. Investigations of “Biofon” effect on Ch. trachomatis microorganisms

The aim of the work:

Materials and Methods:

Chlamydium effect was evaluated according to citopathogenic action on the cell cultures, death and diseases of the animals or embryos with typical morphological changes in tissues and cells. Availability and quantitative evaluation of chlamydium antigen content in the biological material was indentified by immunofluorescence and immunoenzyme analysis, according to the WHO recommendations (ABC on chlamydia. M.Domeica and Mardh, Martiner and Y.Stozz, 1985).

1.1. Investigation of “Biofon” apparatus effect on Ch. trachomatis and Ch. psittaci in the experiments on chicken embryos.

Investigations were carried on in two experiment versions. In the first version chlamydium suspension was radiated once during 30sec by the “Biofon” before the embryos were infected. In the second version, the radiation of the chicken embryos, infected with the unradiated chlamydium suspension was made during 4 days for 30 sec/day. In the control group of experimental animals the chlamydium culture introduced was not exposed to radiation, and infected ambryos were not radiated by the “Biofon” either.

Findings of this investigation are presented in Table 1. As it is seen from the table “Biofon” radiation of the suspension with chlamydium virulent cells for 30 seconds resulted in significant inhibition of the infectious activity of chlamydia. Radiation of embryos infected by the untreated syspension during 4 days cut the infectious activity 2 times.

Results of radiation of specific inclusions (EB): Immunofluorescence analysis with monoclonal antibody application showed their presence in the control and experimental samples in the dead animals, which was the evidence of the specific process character.

1.2. Investigations of “Biofon” appartus effect on Ch. psittaci in the experiments on white mice

Intrabrain inoculation of white mice by chlamydium suspension, unexpoesed to “Biofon” radiation caused a disease on the 4^th-5^th day, which resulted in 60% death of the experimented animals. Being infected by the chlamydium suspension, radiated only once by the “Biofon”, mice became ill on the 5^th-6^th day with mortality rate of 50%.

Low efficiency of the apparatus action is evidently connected with a high degree of Ch. psittaci virulence for mice. Therefore, to obtain higher efficiency it is probably neccessary to apply more intensive and prolonged radiation both chlamydium cultures and infected animals, which requires special investigation.

1.3. Investigation of “Biofon” apparatus effect on multiplication of Ch. trachomatis and Ch. psittaci in the wound cell cultures.

A wound L_q2q line of the cells have been used in the work. In 24 hours of cell growth 10% yolk suspension, containing a causative agent was added to the culture. During 2 hours adsorbtion of the agent was carried on, after which monolayer was washed 3 times by 199 medium, and a growth medium (MEM) with 10% bovin serum was added. In 24, 46 and 72 hours cover glasses were taken out the bottles, fixed and stained for immunofluorescence analysis, samples for immunoenzyme test were collected.

In the control group clearly defined citoplasmic inclusions of the agent were seen already in 24 hours. Using an orange acridin dye, dynamics of elementary body maturation from bright red, yellow-green and bright green might be observed depending on the terms.

Experiments on the EMI (electromagnetic radiation) effect on Ch. trachomatis (“LV”) during their multiplication in L_q2q cells culture were carried on in the following modifications:

• a cell monolayer was exposed to EMI for 30 min (control sample of the cells) for toxic effect of EMI;
• Ch. trachomatis suspension was radiated by EMI for 30 min before monolayer inoculation;
• Ch. trachomatis suspension was radiated by EMI for 30 min before inoculation and then for 30 min in 24 and 48 hours after inoculation of the material.

Results:

A signle exposure of Ch. trachomatis suspension resulted also in the decrease of infectious activity of the preparation (8-10 bodies in the field of vision) compared to the control sample. Usually 20-25 fields were examined.

It was stated by the immunoenzyme analysis that multiple treatmnet by EMI gives statistically significant 10-fold decrease in the antigen titer (10, 100, 1000, 10000 suspension delutions).

The effect is probably even greater and requires futher investigations.

Conclusion:

2. Studies of “Biofon” apparatus effect on experimental animals

It has been known that biochemical parameters, characterizing the functions of the body physiological systems are the most important ones in evaluation of the pharmacological substances and preparation harmlessness.

Though the device of the “Biofon” series are not pharmacological preparations, we set the goal to study probable biochemical shifts, occurred in the body of the experimantel animals exposed to the “Biofon-20-006” apparatus radiation, and on the basis of these findings to give an appraisal of its safety.

Materials and Methods:

In the course of the work the following aspects were studied: “Biofon” effect on changes in the content of lipid peroxide oxidation products and antioxidant enzyme activity in the animal organisms, as well as studies of the lipid methabolism after the apparatus application. Investigations were carried out on common white rat males 200-300 g by weight. Animals were divided into 6 groups, 5 rats in each.

Group 1 – experimental animals. Rats were being exposed to the “Biofon” radiation every day during 7 days for 30s.

Group 2 – control animals for the 1^st group. Were not radiated by the “Biofon”.

Group 3 – experimantal animals. Rats were radiated every day during 14 days for 30s.

Group 4 - control group for animals of group 3.

Group 5 – experimental group. Rats were radiated in the same way as in group 1 and 3, but for 28 days.

Group 6 – a control group for the animals of the 5^th group.

During the whole experiment animals were under daily observation: their behavior was noted, as well as use of food and water, condition of the hair coverage and mucous membranes, changes in weight (once a week).

In addition, investigations of the “Biofon” effect on the rat reproductive abilities were started. For this purpose 10 common white female rats and 2 males were radiated by the “Biofon” every day during 26 days. From this group of animals 63 rat were born (130-200 g of weight), of them there were 23 females and 34 males; this generation brought the second one. At present these investigations are going on.

To study the “Biofon” apparatus effect on the animals after the end of the radiation procedure, rats from the experimental and corresponding control group were killed by decapitation (according to the “Order of the RF Ministry of Health No. 755 of August 12, 1977” and the "Rules of working with experimental animals").

Then, blood was collected from these animals (with the addition of 1:10 3% solution of Sodium cictrate) as well as liver and myocardium tissues. Preliminarily, liver perfusion via portal vena and myocardium cavity washing on the ice with a saline solution with addition of 5 mm of trisHCl were performed.

All procedures on biologic material treatment were done at the temperature less than +5° C.

Part of the blood was used for hemolysate preparation, the rest of the blood was centrifuged and utilized for lysate preparation (according to C.Beanchamp, J.Fvidovich, 1972). The remaining erythrocytic mass was used for erythrocyte membrane isolation.

Liver and myocardial tissues after preliminary fine division were homogenized in a glass homogenizer with teflon stick in 10-fold quantity of 0.05M phosphate buffer (pH 7.4) with addition of 0.1 mm EDTA. Obtained homogenates were centrifuged (750 rev./min.) for 10 min in the refrigerating centrifuge, over-precipitate liquid was processed ( to solubilize enzyme membranes) with triton X-100 up to 0.1% final concentration (after albumine identification in the homogenate Albumin was determined in the biological material using Lowyetal method (O.H.Lowvyetal, 1951).

Total lipid content was estimated by phosphovanilin technique (Woodman, C.P.Prise, 1972) in the biological material extracts (Y.Folcheta;, 1957).

Hemoglobincyanide method (M.H. of the USSR No. 950 of Oct., 15.74) using clinical kits, produced by Scientific and Production Enterprises “Biolak” and “Reachim”.

Schiff’s bases in blood plasma were evaluated fluorimetrically (B.Fletchertal, 1973) according to the fluorescent response value of the chloroformal biomaterial extracts.

Diene conjugates content in blood plasma and tissues was calculated spectrophotometrically by the value of optical density of biomaterial extracts.

Antioxidant enzyme activity was determined in hemolysate (glutationreductase and glutation-S-transferase) and erythrocyte lysate (catalase and superoxiddismutase), as well as by tissue homogenates in the over-precipitate liquid. Catalase activity was determined by hydrogen peroxide utilization rate according to D.F.Bcers, I.W.Siver method (1952) in the incubation medium based on 0.05 M phosphate buffer (pH 8.0) containing 10 mm of hydrogen peroxide.

Superoxiddismutase was determined by the value of inhibition of nitro-bene tetrasolium reduction by superoxide anion radial according to M.Nishiorimietal (1972) method. Glutationreductase activity was evaluated by accumulation of oxidized NADP-H in the incubation medium on the base of 0.05 M phosphate buffer (pH 7.4), the medium containing 1 mm EDTA, 0.2 mm NADP-H and 1 mm GSSS (R.E.Pinto, W.Bavtley, 1969).

Glutation-S-transferase activity was determined by accumulation of the conjugate of the reduced aglutation according to W.H. Hbigetak (1974) technique.

Phospholipid quantity in biological extracts was calculated by Phosphor quantity according to Ysao Schibuja, Hirado Honda, Bunyi Tarue.

Cholesterol content in biological extracts was determined by modified Levchenko's method.

Biochemical blood analyses were made by routine clinical techniques.

Results and discussion

Investigations on “Biofon” apparatus effect on white rat organisms showed that:

1. “Biofon” apparatus did not cause animal death, changes in their behavior, in their hair and mucous membrane condition, weight loss, refusal to eat or drink, and did not affect their reproduction ability.
2. General mass of the body, liver and myocardium mass (per 100g of the body weight) in experimental animals did not differ from those of the control animals (Table 2.1).
3. Animal blood counts revealed slight differences in the indexes of the experimental group compared to the control one (Table 2.2) though indexes of glucose, amylase, cholesterol, and transaminases in the experimental group appeared to be a little bit higher than for the animals of the control group.
4. Examination of lipid fractions in erythrocyte tissues and membranes showed that the content of total lipids in the liver was higher in the first experimental group, than in the control one, while in the 3^rd and 5^th experimental and 4^th and 6^th control groups significant differences were not noted (Table 2.3). Cholesterol level in the experimental animals was slightly elevated; differences in the phospholipid content in the liver were not found out. In the myocardium of the 1^st experimental group a small reduction of total lipid, cholesterol, phospholipid levels was observed; in other groups no differences were revealed. In the erythrocyte membranes cholesterol count was a bit higher in all experimental groups relative to the control groups. The same trend was noticed in the changes of phospholipid parameters. The changes revealed in some indexes of lipid metabolism in tissues do not have valid confirmation and cannot be explained without additional studies.
5. Data, obtained in the course of studies of the main products of lipid peroxidative oxidation (LPO), diene conjugates and Schiff’s bases in blood plasma, liver and myocardium, as well as activity of the enzymes, participating both in utilization of the active oxygen forms (super-oxiddismutase and catalase) and in the elimination of peroxidative lipid metabolites (glutation-S-transferase, glutation reductase) in the erythrocytes, liver and myocardium in different terms of “Biofon” application to the experimental animals, are presented in Tables 2.4, 2.5, 2.6.

As it followos from the findings presented in Table 2.4, there were no changes in the level of diene conjugates and Shiff’s bases in the blood plasma of all experimental animal groups relative to control ones. No significant differences were registered in the level of diene conjugates in the liver between experimental and control groups of animals. No visible changes were noted in the content of Shiff’s bases in blood plasma, liver and myocardium. The same picture was observed in the myocardium tissue.

Data of Table 3.5 and 3.6 demonstrate that no significant changes in the activity of superoxiddismutase and catalase, as well as glutationreductase and glutation-S-transferase in liver and myocardium erythrocytes in the experimental groups of animals compared to the control ones were observed.

Thus, on the basis of the findings obtained from the studies of “Biofon” apparatus effect on the intricate biochemical processes in the experimental animals it may be suggested that the apparatus does not cause any negative actions on the animals.

Table 2.1

Changes in the blood wight of the body, liver and myocardium in the rats, radiated by the “Biofon” in different terms

Table 2.2

Biochemical parameters of the rat blood after “Biofon” application

Table 2.3

Effect of “Biofon” apparatus action on the content of lipid fractions in the erythrocyte tissues and of lipid fractions in the rat erythrocyte tissues and membranes

Table 2.4

Changes in the content of the products of lipid peroxidative oxidation in blood plasma, tissues of the liver and myocardium after “Biofon” application in different terms

Note: animal number n=5 in all the groups.

Table 2.5

Dynamics of antioxidative enzyme activity in the tissues of liver and myocardium, and in erythrocytes after “Biofon” application to the animals.

Note: animal number n=5 in all the groups.

Table 2.6

Dynamics of antioxidant enzyme activity in the erythrocytes of the liver and myocardium after “Biofon” application in different terms

Note: animal number n=5 in all the groups.

3. Studies of “Biofon” apparatus effect on the human body (a limited group of patients with urogenital chlamydiosis).

Global spread of human chlamydiosis, high incidence and a wide spectrum of diseases caused by them make the problem extremely up-to-date. Unfortunately, treatment of patients with chlamydiosis has not been sufficiently enough developed at present. Some of chlamydium features (obligate intracellular development cycle and ability to form spoke-like structures outside the cell) significantly limit antibacterial therapy efficiency.

The aim of this section of the research work is to study the opportunity of “Biofon” apparatus application for treating patients with urogenital chlamydiosis and its safety for the human body.

In the course of the work the following aspects were investigated: physiotherapeutical efficiency of the “Biofon” apparatus action on patient organisms, its effect on their immune status, as well as biochemical shifts in patients under the action of the apparatus.

3.1 Studies of the physiotherapeutical efficiency of “Biofon” apparatus effect on patients with urogenital chlamydiosis.

Patients (volunteers) were selected by clinicists after a complete examination and collection of clinical, physiological, laboratory, immunological and biochemical findings. Clinical manifestations of urogenital chlamydiosis were also taken into consideration: in women – urethritis, cervicitis, paraurethritis; in men – urethritis, prostatitis, Reiter’s syndrome.

53 patients with laboratory confirmed diagnosis of urogenital chlamydiosis were selected. The majority of them (42) complained of a slight tching, burning and unpleasant sensations after water passing or sexual contact. Patients were divided into 3 groups (2 experimental and 1 control) with various course of treatment: 1^st experimental group (12 persons): patients were subjected to “Biofon” apparatus radiation every day 30s during 10 days.

The 2^nd experimental group (26 persons): patients received antibacterial drugs (ezithromycin, doxicycline, erythromycin, rifampicin) together with the radiation by the “Biofon” apparatus (like for the 1^st group).

The 3^rd control group (15 patients): patients were not exposed to the “Biofon” radiation. Patients were treated only by antibiotics.

Chlamydiosis laboratory diagnostics was carried on by serological method using indirect immunofluorescence reaction (IIFR) and “Khlashchiskan” diagnostic kit (Lab diagnostics of Russia), the sensitivity of which, determined by the results of the State trials, was 98.9%, the specificity amounted to 97.1%. Materials for examinations were smears of the scrapes obtained from the urethra mucous membrane in men and women, and from the cervical canal in women. The result was considered to be positive, if a bright-green fluorescence in the form of a point or an oval was registered in the presence of no less than 50 epithelial cells, stained in the red-orange colour.

After the end of the treatment course patients with chlamydiosis were followed-up during 3-4 months with compulsory three control laboratory examinations of the smears (by IIFR-reaction) on chlamidium presence, availability of clinical manifestations and subjective complains.

Criterium of recovery was absence of chlamydia in the smears in control laboratory examinations. Reduction in the number of chlamydia in the smears to single bacteria was considered to be “a positive” physiotherapeutical effect of the “Biofon” apparatus. Findings of the serological control laboratory examination of patients with urogenital chlamydiosis are presented in Table 3.1.1.

As seen from the table, positive effect after “Biofon” apparatus action on the patients with urogenital chlamydiosis was obtained in the 1^st and 2^nd experimental groups in 60-70% of volunteers. This effect is mainly connected with the reduction or complete elimination of chlamydia in the smears, which, probably, influenced the remission of the clinical course of the disease. In the control group of the patients treated only with antibiotics, positive results were obtained only in 26.7% None complaimed of any unpleasant sensations, connected with “Biofon” application.Thus, the apparatus possesses some curative effect and is quite safe for the human body. However, for final solution of the problem of its physiotherapeutical efficiency and safety it is neccessary to carry additional comprehensive studies on larger groups of patients with urogenital chlamydiosis.

Table 3.1.1

Findings of serological examinations (in IIFR) of the patients with urogenital chlamydiosis on chlamydia presence

3.2. Investigations of “Biofon” apparatus effect on the immune system of the patients with urogenital chlamydiosis.

Materials and Methods:

While studying “Biofon” apparatus effect on the immune systems 38 patients with the diagnosis of urogenital chlamydiosis were examined. Blood collection was made twice: before the beginning and after the end of treatment. Immunity status was examined by 7 parameters:

leukogram – complete blood count was made in the smear, stained according to Romanovsky, leurocyte and erythrocyte number was calculated in the Goryaev chamber, ESR was counted using Panchenko’s apparatus;

relative and absolute number of T-lymphocytes – this parameter was determined by rosette-forming method with ram erythrocytes;

relative and absolute number of B-lymhosytes – was determined by reaction of rosette-forming with mice erythrocytes;

phagocytic activity of neutrofils was measured by incubation with latex particles with the following counting of the number of cells actively ingesting latex;circulating immune complexes in blood serum were determined by the method of precipitation with polyethylenglycol with subsequent photocolometry;

subpopulation compositions of T-lymphocytes was determined in the reaction with commercially produced monoclonal antibodies containing T4 and T8 subpopulations, and with staining by luminescent serum;

general immunoglobulines of A, M, G-classes were determined by Manchini method in radial immunodiffusion reaction in gel.

Results and Discussion:

Studies of blood cell composition have revealed that by the end of the “Biofon” treatment normalization of the general leurocyte index, as the most mobile parameter, has been observed. In the course of treatment alongside with disappearing or decreasing of clinical signs reduction in leukocyte level occurred, which is the evidence of a favourable outcome of the inflammatory process. Other cell parameters of the blood formula remained without changes (Table 3.2.1).

Table 3.2.1

Blood count parameters

2. Indexes, characterizing T- and B-cell immunity link, are given in Table 3.2.2. As it is shown in the Table, no significant differences in the number of T- and B-rosette-forming cells before and after treatment have been observed.

Table 3.2.2.

Condition of T- and B-cell link

3. Examination of phagocytic neutrophils activity has not revealed any deviations from the physiological norm before chlamydiosis treatment, as well as after it.

4. Immune complex formation is one of the protective reactions of the body which helps to fight antigens and microorganisms. Content of the circulating immune complexes in the group under examination is showed in Table 3.2.3.

Table 3.2.3.

Content of the circulating immune complexes (CTC) in the blood serum

Obtained data demonstrate no increase in CIC over the physiological norm before treating by the “Biofon”, and absence of the parameter changes during its application.

5. Studies of subpopulation composition have not revealed deviations from the norm in the content of T-helpers (T4) and T-supressors (T8) before and after “Biofon” treatment. T4/T8 relation corresponded to physiological norm deviations.

6. Humoral immunity link condition has been investigated by the content of immunoglobuline A, M, G classes in the blood serum. Results are included into Table 3.2.4.

Table 3.2.4.

As it is seen from the table, immunoglobuline level has not changed greatly, no significant deviations from the physiological norm have been observed.

Thus, summing all the findings connected with immunological safety of “Biofon” apparatus application, one can conclude, that its action does not result in negative reactions of cell and humoral immunity links in patients with urogenital chlamydiosis.

3.3 Studies of biochemical shifts in the organism of patients with urogenital chlamydiosis under the action of the “Biofon” apparatus.

Biochemical parameters, characterizing functions of the body physiological systems are very important indexes of the safe action of the “Biofon” on the patients with urogenital chlamydiosis.

Studying the apparatus effect on the biochemical shifts in the body, 38 patients with urogenital chlamydiosis undergone complete course of treatment by the “Biofon” apparatus were examined. The biochemical parameters studied were connected with the functional liver condition:

determination of the total albumin level, by biuret reaction, developed by the All-union Scientific and Methodological Center for Laboratory Affairs of the Ministry of Health;

albumin fractions – by neutrophil layer precipitation;

determination of transaminase activity according to Reitman and Freinkel (1957), alcaline phosphatase according to Bodansky (1933);

determination of the total unconjugated and conjugated bilirubin level by Brayshaw Braind method (1971);

thymol test according to Hyori and Popper (1944).

Functional state of the pancreas was tested by amylase activity using Smith method and ESR (1949). Functional condition of kidneys was checked by the urea level in blood serum using color reaction with diaceton monooxide.

Results of the biochemical examinations are presented in Table 3.3.1.).

The data shows that the biochemical examinations of patients with chlamydiosis after “Biofon” radiation have revealed no statisticakly significant deviations from standard physiological parameters, characterizing liver, pancreas and kidneys functions. This is an indication of the safe effect of the “Biofon” apparatus.

Table 3.3

Biochemical parameters of blood for patients with urogenital chlamydiosis after “Biofon” application.

Summary

As the result of the conducted studies of “Biofon-2C-006” apparatus effect on Ch. trachomatis microorganisms, experimental animals and patients with urogenital chlamydiosis, it has been estimated that:

1. “Biofon” treatment of Ch. trachomatis suspension results in reduction of infection activity in L_q2q cell and chicken embryo culture. This effect is less marked in infected white mice.

2. Essentially less is the effect of the apparatus on Ch. trachomatis when embryos and cell cultures inoculated with unexposed suspension were radiated by the “Biofon”.

3. Multifold exposure of Ch. trachomatis suspension and cell culture, inoculated by this suspension, to the “Biofon” apparatus inhibits agent development.

4. “Biofon” apparatus action on white mice does not cause animal death, changes in their behavior, condition of hair and mucous membranes, refusing to eat or drink, weight loss, and reproductive ability.

Studies of biochemical parameters showed no deviations from physiological norms. Under “Biofon” effect on patients with urogenital chlamydiosis, 60-70% of volunteers showed positive curative results, confirmed by decrease or complete absence of chlamydia in the smears, which is likely to have influenced the remission of the disease clinical course. Apparatus application does not induce negative reactions of cell and humoral immune link in the patients.

Fulfilled biochemical tests in patients after the apparatus application revealed no statistically significant deviations from physiological parameter norms, characterizing liver, pancreas and kidneys functions. Thus, radiation of Ch. trachomatis microorganisms by the “Biofon” apparatus results in inhibiting their infectious activity. The apparatus was harmless when applied to the experimental animals and patients with urogenital chlamydiosis, and possessed a marked curative effect in treating the latters. However, for complete and more objective evaluation of medical and biological efficiency and safety of the apparatus it is necessary to carry on additional comprehensive studies on wider groups of patinets with urogenital chlamydiosis.

List of References used

1. Delektorsky B.B., Yashin G.I. “Current ideas on the role of chlamydia in the urogenital tract pathology (clinical picture, diagnostics, treatment) – M., 1992.
2. Diagnostics and treatment of urogenital infections//Methodological recommendations. – M., 1991.
3. Ilyin I.I., Kovalyov Y.N. Considerations on urogenital chlamydiosis treatment//Vestnik of Dermatology. No.4, 1994.
4. Krotov S.A., Krotova B.A., Yuryev S.Yu. Chlamydioses: epidemiology, laboratory diagnostic methods, genital chlamydiosis treatment. – Koltsovo, 1995.
5. Domeika M. Mardin, Martiner and Y.Stozz. ABC on Chlamydia, 1985.
6. Kolb V.G., Kamushnikov V.S. A reference book on clinical chemestry. – Minsk, 1982.
7. Clinical immunology and allergology... Edited under I.M.Gandgi. – “Health”, 1985.
8. Lazareva D.N., Alyokhin E.K., Immunity stimulatord. – “Medicine”, 1985.
9. Lrbrdyev K.A., Ponyatkina I.D. Immunogram in clinical practice. – “Science”, 1990.
10. Reference book “Laboratory examination methods in clinics”. Edited under V.V.Menshikov. – “Medicine”, 1987.
11. Bray shaw Braind. Manual and automated techniques for total and conjugated bilirubin//Med.lab.technol., 28, 2, 1971.
12. Crocker C.L//Amer. J.med.technol., 33, 361, 1967.