Method of obtaining stimulated dendrite cells for induction of immune response against human tuberculosis

FIELD: medicine.

SUBSTANCE: invention relates to biotechnology and can be used in treatment of diseases, namely oncologic ones. Method is based on sampling from patient immature dendrite cells, their cultivation ex vivo for maturing and formation of allostimulating activity. Cells are additionally collected with antigen and are introduced to the same patient for formation of adaptive immunity. During cultivation ex vivo of immature dendrite cells fragmented allogenic double-stranded genomic DNA with fragments with size 200-6000 bp is introduced into culture medium in amount 5 mcg/ml of medium.

EFFECT: invention allows to extend field of obtained product application with increase of general immune state of patients.

 

The invention relates to medicine and can be used to obtain stimulated dendritic cells for the induction of immune responses against tuberculosis person.

A method of obtaining vaccines for intravenous, based on immunization combined anthrax vaccine containing antibodies to PA Bacillus anthracis in the titer of at least 1:400 on the results of ELISA of plasma of blood donors [EN 2348429 C2, AC 39/40, 2009.03.10].

The disadvantage of this method is not the individuality of the vaccine, as obtained from the plasma of the donor, and not the patient. This reduces efficiency and may cause undesired disease.

The closest to the essence of the proposed method is based on extraction of bone marrow immature dendritic cells and keeping them in a few days along with the antigens of the pathogen pneumonia - cell lysate or bacterial lipopolysaccharides [www.cbio.ru].

The resulting vaccine - activated dendritic cells were injected intraperitoneally to mice, and the next day infected them with a lethal dose of Klebsiella pneumoniae. From this infection has lost all control group of mice. All vaccinated mice remained alive.

The disadvantage is the closest solution is relatively narrow scope, because the individual VA is of CIN can be used to prevent pneumonia, but cannot be used in other areas of medicine, in particular for the prevention and treatment of cancer with consideration of the peculiarities that arise, for example, when it is used for patients with tuberculosis.

The required technical result is the expansion of the scope.

The desired result is achieved by the fact that, according to the method, based on the fence patient immature dendritic cells, culturing ex vivo for maturation and formation allostimulatory activity for subsequent additional processing of the antigen and reverse the introduction of Mature dendritic cells back into the patient with the purpose of formation of adaptive immunity during culturing ex vivo immature dendritic cells into the culture medium injected fragmented allogeneic double-stranded genomic DNA fragments by size 200-6000 HW / SW components and the complete genome physiologically and genetically healthy donor in an amount of 5 μg/ml of medium.

In addition, the desired result is achieved that, in the method according to claim 1 drug fragmented allogeneic double-stranded genomic DNA fragments by size 200-6000 HW / SW components and the complete genome physiologically and genetically healthy donor are placed in culture medium containing immature dendritic cells of patients with tuberculosis in quantity is as 25 µg/ml of medium.

More broadly, the present invention is the development of sound and experimentally confirmed the effects on isolated from a patient and cultured ex vivo immature dendritic cells fragmented allogeneic double-stranded genomic DNA fragments by size 200-6000 P.N. and components of the complete genome physiologically and genetically healthy donor, so that the impact on DK exogenous DNA is their maturation and induced them allostimulatory activity. After the addition of antigen and the subsequent introduction of these DK back into the body of the same patient should be development of specific adaptive immunity in the patient associated with the activated dendritic cells proliferation trained against these antigens to cytotoxic CD8+ T lymphocytes, that is specific vaccination of the patient.

In modern literature there is no indication in the proposed method obtain stimulated dendritic cells for the induction of immune responses against tuberculosis of the person based on the fence patient immature dendritic cells, culturing ex vivo in the presence of fragmented exogenous allogeneic double-stranded genomic DNA, required for maturation and formation of ALLO is stimulatory activity of these dendritic cells. Subsequent additional processing of these dendritic cells by specific antigen and reverse the introduction of them to the same patient it is necessary for the formation of specific adaptive immunity in these patients.

Therefore, the proposal meets the criteria of novelty inventive step.

Listed below are theoretical and experimental data, confirming that the invention meets the criteria practical (industrial) applications.

The drawing shows:

figure 1 - electron microscopic pictures of DNA preparations, spread method Kleinsmidt and fragmented DNA human placenta used in the experiment, b) resettodobar structure found in the product, (C) nucleosomes on DNA isolated from Hela cells;

figure 2 - electrophoretic characterization of exogenous therapeutic DNA human placenta (M - molecular weight marker - lambda DNA BssT1I digest);

figure 3 - DK man incubated with α32P-labeled human DNA;

figure 4 - evolution of the delivery of labeled material in the inner compartments DK person depending on time of incubation, cell culture and radioactively labeled DNA;

figure 5 - analysis of the correlation of exogenous DNA delivered into the cytoplasm and the nucleus of the three cell types in the point of its maximum presence (peaks [1, 2].

Regarding presented on figure 3 should indicate that after incubation, the cells are sequentially ligated non-ionic detergent Triton X100 and SDS and fractionally on (a) the cytoplasmic fraction, (b) micromemo fraction and (C) chromatin. DNA extracted from each fraction were analyzed by agarose gel electrophoresis. A) the Block of 0.7%agarose, bromide stained with ethidium; C) Radiograph of the same block after drying. The numbers on the blocks indicate the time of incubation of cell culture with labeled extracellular DNA. M - molecular markers - λHindIII DNA and the original labeled DNA added to the culture medium. Digits to the right of block markers of molecular weights in KBP

The proposed method of producing stimulated dendritic cells for the induction of immune responses against tuberculosis human is implemented as follows.

The method of obtaining stimulated dendritic cells for the induction of immune responses against tuberculosis person based on the use of the ripening of incentives in ex vivo culture. Under the proposed method in culture medium containing immature dendritic cells, add fragmented allogeneic double-stranded genomic DNA fragments by size 200-6000 HW / SW components and the complete genome physiologically and genetically healthy donor, which leads to with whom sravani DC and induction they allostimulatory activity defining the development of specific adaptive immunity.

Fragments of exogenous DNA added to the culture medium containing isolated from the body of the patient dendritic cells in an amount of 5 mg/ml

Fragments of exogenous DNA added to the culture medium containing isolated from the body the TB patient dendritic cells in the amount of 25 μg/ml

Ripe and activated ex vivo DC advanced pulsed-specific antigen(s) and put back into the body of the same patient will stimulate development in the body of this patient-specific adaptive immunity.

An example implementation of the method.

Mononuclear cells (MNCs) were isolated from heparinized venous blood standard in the density gradient ficoll-urografin. Dendritic cells were obtained by culturing adherent fraction of MNCs in 24-hole plates (Nunc, Denmark) for 5 days in medium RPMI-1640 (Sigma-Aldrich, USA), supplemented with 0.3 mg/ml L-glutamine, 5 mm HEPES buffer, 100 μg/ml gentamicin and 5% inactivated serum AB(IV) group, in the presence of GM-CSF (Leucomax, Schering-plough, Switzerland) 1000 U/ml) and INF-a (Roferon-A, Roche, Switzerland) 1000 IU/ml. as the ripening of the stimulus used allogeneic double-stranded genomic DNA fragments by size 200-6000 HW / SW components and full g is nom physiologically and genetically healthy donor in an amount of 5 μg/ml, which was added 24 h before the end of the cultivation DK and in the amount of 25 μg/ml for DK obtained from patients with tuberculosis. Served as a positive control of DC maturation was induced LPS, negative DC generated in the absence of the ripening of the stimulus.

Allostimulatory activity of DC was evaluated in the reaction of the mixed culture of lymphocytes (SCR). As the responding cells were used OLS donors (0,1×106/well), which were cultured in 96-well round-bottom tablets for immunological studies in medium RPMI-1640 and 10% inactivated serum AB(IV) group. Stimulants served as a recreation center in the ratio OOC:DK=10:1. Response was assessed at 5 days radiometrically by the inclusion of3H-thymidine (1 mccu/well), which made for 18 h before the end of the cultivation. Index impact (IV) exogenous DNA allostimulatory activity of DC was calculated as the ratio of intensity include3H-thymidine in the experience to the intensity of including3H-thymidine in control.

The effect of DNA on the phenotype (the expression of CD 14 and CD83) dendritic cells was assessed by the effect of different doses of DNA made 24 hours before the end of the cultivation DK. Served as a positive control of DC maturation was induced LPS, negative DC generated in the absence of the ripening of the stimulus. Because of differentsirovannostj in DC is associated with the loss of monocytes CD 14 molecules, and maturation of immature DC Mature - purchasing a marker of terminal differentiation of CD83, a decrease in the proportion of CD14+ cells and an increase in CD83+ cells was regarded as a sign of differentiation and maturation of DC. Phenotypic testing of cells was performed by the method of flow cytofluorimetry (FACSCalibur, Becton Dickinson)using PE-labeled monoclonal anti-CD14 antibodies and FITC-labeled anti-CD83 antibodies (BD Bioscience Pharmingem, USA).

To confirm that the proposed method can achieve the desired result, will bring his theoretical and experimental validation.

Consider the concept of activation of specific adaptive immunity antigen presenting dendritic cells, Mature and activated ex vivo, which may be optionally pulsed specific antigen(s), and put back into the body of the same patient on the example of a malignant cancer.

It is known that for many malignant diseases associated with the development of tumor-associated immunosuppression, against which tumor cells escape from immune surveillance. Immunoresistance tumors associated with multiple mechanisms that are implemented on the cellular and humoral level, primarily in the microenvironment of a developing tumor tissue. Among them is really: a) shift the cytokine balance towards immunosuppressive mediators, inhibit the functional activity of immune cells infiltrating the tumor; b) associated with this imbalance of effector, cytotoxic and regulatory T-cells; C) the emergence of molecular defects in the systems of cells involved in the recognition and antigen presentation and signaling into the cell [3]; g) forming a tumor protective barrier with increased concentration apokoronou DNA, supporting the genetic stability of the cells of the malignant tissue and exporting of tumor genes in other parts of the body [4-7].

The effectiveness of antitumor immunity is largely dependent upon the activity of immune cells - natural killer cells, cytotoxic T-lymphocytes, macrophages and dendritic cells (DC). These cells are involved in start-up, organization and realization of the reactions of innate and adaptive immunity through a variety of humoral and cell-mediated mechanisms helps eliminate cancer cells. First line anti-defense system is nonspecific resistance (innate immunity), which is comprised of natural killer cells and activated macrophages. In case of insolvency of the nonspecific defense mechanisms associated with the violation of ecosysytem life, genetic characteristics, age of the individual the and, the severity of comorbidity, baseline immunocompatibility etc. in the body include the mechanisms of adaptive immunity [8, 9]. This largely depends on the level of expression in tumor cells of specific tumor-associated antigens that are recognized by immunocompetent cells and against which induced effector reactions aimed at the elimination of the tumor clone. For the effective implementation of adaptive reactions of the immune system are of great importance two stages, associated, first, with processing/presentation of tumor-associated antigens to antigen-presenting cells (mainly DC), and, secondly, with the stimulation of proliferation and maturation of cytotoxic T lymphocytes.

In our previous works [10, 11] has been described antitumor effect fragmented exogenous DNA. In particular, it was found that preparations fragmented genomic DNA have the potential to inhibit the growth of some experimental tumors in mice. At the same time they have a moderate blocking effect on the development of tumor metastases.

In parallel with these research work has been done on the analysis of exogenous DNA into cells adenocarcinoma human breast MCF-7, growing in culture. Analysis proved the fact, in this case there is a change neoplastic cells at the genetic level. It is known that the main factor uncontrolled proliferation of this cell type is a defect in the gene for a key enzyme of apoptosis - caspase 3. Gene esecutore caspase 3 in exon 4 has a deletion of 47 BP, which leads to a shift of the reading frame and the impossibility of synthesis of functionally active enzyme [12]. This fact allowed us to propose the hypothesis that the regression of experimental tumors in mice is associated with direct exposure to fragments of exogenous DNA into the genome of a cancer cell. It was assumed that these effects may in statistical mode to cause substitution sequences, mutations in which have led to retransformation cells on non-defective DNA structures [13]. However, quantitative assessment of DNA delivered into the cells of solid tumors in vivo, testified that, in the used experimental conditions, less than one copy of a fragment size of ~1000 BP internalized in mikromazernom kernel space. It is obvious that the number of exogenous DNA is not able to induce any significant genetic changes in the cell [14].

We assumed that the most probable impact on the development of solid tumors in experiments in vivo [10, 11] is the activation of certain C is egnew immune system, namely adaptive immunity-related activity recreation centre. Although we cannot rule out the possibility of dual anti-cancer actions, based both on direct cytotoxic effect of the immune system against tumors and reverse genetic degeneration of cancer cells in areas of the tumor that are available for internalization of fragments of exogenous DNA in sufficient quantity to modify the genome of cancer cells [15].

In order to answer the question whether the observed regression of experimental tumors to be associated with activation of the immune response induced DK, we conducted an analysis of the impact of fragmented human genomic DNA on DK in the system ex vivo.

It was shown that fragmented human genomic DNA efficiently captured by dendritic cells and internalized mainly in mikromazernom kernel space without concatemeric. A small number of labeled material is detectable in the cytoplasm.

It was found that exogenous DNA stimulates efficient DC maturation, characterized by the change in the ratio of their surface markers of maturity and development allostimulatory activity. The ability DK man, the ripening in the presence of exogenous DNA to stimulate a proliferative response in mixed lymphocyte culture b is La is comparable in strength and in some cases exceeded the standard activator DK - LPS from walls of bacteria. The introduction of DNA in culture DK man was accompanied by an increase in the relative content of CD83+ cells and a decrease in the proportion of CD14+ cells.

Thus, we have shown that exogenous DNA is absorbed to the DC and stimulates their maturation and allostimulatory activity.

The methodology used.

Analysis of the DNA preparation by electron microscopy

The drugs were prepared by the method rasplastyvanija on Kleinsmidt with formamide [16, 17]. The DNA sample (50 µg) in a solution of cytochrome C (Sigma, USA) with the surface of the water was removed on the carbon film, and then spread molecules punctuated by sputtering platinum-palladium.

Analysis of the DNA preparation biochemical method

Biochemical analysis of the sample was performed using the method of fractionating proteins in the system of laemmli's method, followed by double staining of the gel Kumasi-C-silver.

Generation of dendritic cells

Mononuclear cells (MNCs) were isolated from heparinized venous blood standard in the density gradient ficoll-urografin. Dendritic cells were obtained by culturing adherent fraction of MNCs in 24-hole plates (Nunc, Denmark) for 5 days in medium RPMI-1640 (Sigma-Aldrich, USA), supplemented with 0.3 mg/ml L-glutamine, 5 mm HEPES buffer, 100 μg/ml gentamicin and 5% inactivated serum AB(IV) group, in the presence of GM-CSF (Leucomax, Schering-plough Switzerland) 1000 U/ml) and INF-α (Roferon-A, Roche, Switzerland) 1000 IU/ml as the ripening of the stimulus used LPS (10 µg/ml LPS from E. coli 0111:B4, Sigma-Aldrich), which was added for 24 h before the end of the cultivation DC.

Culture treatment DK labeled DNA, the DNA in the cell compartments

8 µg labeled using polymerase maple α32P DNA human placenta was added to the culture DK man (3×106cells), after 0, 15, 30, 60, 120 and 180 min after the cells were treated with trypsin and centrifuged 10 min at 4000 rpm at C the centrifuge. Sediment resuspendable in buffer a with 1% Triton X100 and incubated 10 min on ice. After this suspension was layered on a column of 12%sucrose (3 ml) and centrifuged 20 min at 2000 rpm at C the centrifuge. Besieged DNA from cytoplasmic fraction (supernatant). Sediment cores resuspendable in buffer A, was added sodium chloride to 2M, SDS to 1% and incubated for 15 min at 65°C. were Centrifuged for 30 min at 30°C at 21000 rpm in a centrifuge J2-21 Beckman. Besieged DNA from microsomes fraction, the precipitate chromatin simply dissolved in water. The amount of labeled DNA material in the three cell fractions was determined in a standard way in Eppendorf tubes, placed in counting vials on the counter Rac Betta. Samples were fractionally in 0.7% agarose gel.

Characteristics of blood donors

At work, we have studied DK healthy donors and patients that what herkulesa lungs (TB). Of the 8 patients had 7 people was revealed infiltrative and one patient - fibroproliferative TB. 6 patients were identified active baculoviridae, 5 detected resistance to standard therapy tuberculostatics.

Preincubate DK with exogenous DNA

DNA human placenta DNA enriched in CpG sequences (CpG), was added as the ripening of the stimulus at doses of 1, 5 and 25 μg/ml for 24 h before the end of the cultivation DK. Served as a positive control of DC maturation was induced LPS, negative DC generated in the absence of the ripening of the stimulus.

Assessment allostimulatory activity DK

Allostimulatory activity of DC was evaluated in the reaction of the mixed culture of lymphocytes (SCR). As the responding cells were used OLS donors (0,1×106/well), which were cultured in 96-well round-bottom tablets for immunological studies in medium RPMI-1640 and 10% inactivated serum AB(IV) group. Stimulants served as a recreation center in the ratio OOC:DK=10:1. Response was assessed at 5 days radiometrically by the inclusion of3H-thymidine (1 mccu/well), which made for 18 h before the end of the cultivation. Index impact (IV) exogenous DNA allostimulatory activity of DC was calculated as the ratio of intensity include3H-thymidine in on what yte to intensity include 3H-thymidine in control.

Assessing the impact of DNA preparations on the phenotype DK

The effect of DNA on the phenotype (the expression of CD14 and CD83) dendritic cells was assessed by the effect of different doses of DNA made 24 hours before the end of the cultivation DK. Served as a positive control of DC maturation was induced LPS, negative DC generated in the absence of the ripening of the stimulus. Because the differentiation of monocytes into DC associated with loss of monocytes CD14 molecules and maturation of immature DC Mature - purchasing a marker of terminal differentiation of CD83, a decrease in the proportion of CD14+ cells and an increase in CD83+ cells was regarded as a sign of differentiation and maturation of DC. Phenotypic testing of cells was performed by the method of flow cytofluorimetry (FACSCalibur, Becton Dickinson)using PE-labeled monoclonal anti-CD14 antibodies and FITC-labeled anti-CD83 antibodies (BD Bioscience Pharmingem, USA).

Will focus on the analysis of the molecular characteristics of the drug exogenous allogeneic genomic fragmented double-stranded DNA.

It is known that DNA to form nucleosomes, presented in physiological concentrations, activate myeloid and plasma DK [18, 19]. The DNA preparations used in this study were obtained butfinally way salt extraction and subsequent precipitation with acetone. In order to determine the minimum C the pollution nucleosomal fraction of drugs used DNA we conducted biochemical and electron microscopic analysis of these drugs. In the experiments performed, we did not find any traces of histones by double staining Kumasi-R250-silver proteins fractionated from DNA preparations, the system Lemli or nucleosomes by electron-microscopic examination. At the microscopic level, the DNA preparation was represented by fragments of different lengths and sockets formed, apparently, residual proteins of the nuclear matrix (figure 1).

Let us consider some features of internalization fragmented exogenous allogeneic double-stranded genomic DNA in mikromazernom space cores DK man

In order to assess the effectiveness of the delivery of exogenous DNA into the inner compartments of DK, we incubated generated as described in materials and methods, DK with labeled human DNA fragmented to sizes 1-20 nucleosomal monomers (170-6000 BP) (figure 2).

To quantify delivered into the internal compartments DK fragments of exogenous DNA were determined the following parameters.

Each experimental point was taken 8 µg DNA.

Each experimental point was taken 3×106cells.

In each cell of 6 pg of DNA.

The total amount of DNA released from cells of each experimental point was 18 m is,

The human genome 3×109P.N.

As a result of the analysis identified the following features of internalization of exogenous DNA in DC (table 1).

Table 1
Estimated number of fragmented exogenous DNA delivered in micromason space cores DK
time minCPMCorrespond to the total quantity, mcg% from the quantity added-tion in the environment% of the genome, delivering Amy one cellP.N. (×107), delivering units in one cellthe number of fragments of length 1000 BP delivered in one cell
041610,060,730,320,979700
1547640,070,840,371,1111100
30 51340,070,900,401,2012000
6040830,060,720,320,969600
12027310,040,480,210,646400
18059980,081,050,471,4014000

As follows from electrophoretic analysis (Fig 3) and analysis of the accounts of radioactivity captured by dendritic cells DNA directly into micromason space, do not linger in the cytoplasm. In microsomes fraction of the DNA is in a degraded state (200-1000 BP)without concatemeric characteristic of DNA received in micromemo faction culture of MCF-7 cells or embryonic stem cells [1, 2].

The presence of labeled material in the fraction of chromatin associated libosso embedding fragments of exogenous DNA in the chromatin due to recombination processes or by incorporation in synthetic processes, what is happening in the cell, labeled monomers resulting from the degradation of exogenous DNA.

Quantitative evaluation of the obtained results shows that in mikromazernom space cores DK can be free about 10 thousand fragments of length 1000 BP Analysis of the kinetics of delivery of labeled material revealed two peaks - 30 and 180 min (figure 4). It is assumed that the peaks reflect the time of turnover of receptors that bind DNA. The first peak is associated with the capture and delivery of exogenous DNA into the internal compartments of the cell, the decline corresponds to full employment receptors, then the receptors are released and provide the second peak of the labeled delivery of exogenous DNA. As has been shown for cell culture MCF-7, labeled DNA is delivered also in cellular compartments two peaks (at points 30 and 180 min).

When comparing the number of labeled material delivered into the nuclear space and located in the cytoplasm, we have found the following difference between the DC and the other cultures of cells (MCF-7 and embryonic human stem cells), were analyzed in our previous works. It turned out that for DK person number are labeled exogenous DNA in cytoplasmic fractions constantly significantly lower than in the fractions representing nuclear compartments (michrom somna fraction and chromatin) (figure 5). These facts can say that for DK there is another way (in comparison with these types of cells) internalization of fragments of exogenous DNA, oposreduemye specific receptors. Also, apparently, this path is different from that defined for CpG DNA, which shows its internalization in endosome and consistent transport and concentration in tubular the lysosomal compartment of the cytoplasm, where it binds to TLR receptors [20].

One of the most interesting characteristics of the state of fragments of exogenous DNA in mikromazernom kernel space, was the complete absence of the visible concatemeric. According to literature data [21, 22] and our studies [1, 2], implemented on other types of cells (MCF-7 and embryonic stem cells), DNA fragments that fall within the intranuclear space, with the subsequent ligation to one another, forming concatamer. Electrophoretic it looks like this: starting with the incubation time 15 min labeled material loses its mobility in agarose gel, forming sometimes a characteristic ladder [2]. In the case of DK labeled material looks the same, regardless of the time of incubation (figure 3). The analysis of the internalization of fragments of exogenous DNA in DC suggests that there is an active interaction and penetration of exogenous the DNA in the internal compartments of the specified type cells. In mikromazernom kernel space, as well as in the cytoplasm, is deposited (or transit is supposedly to cytoplasm) a large number of fragments of exogenous DNA, which most likely can run multiple processes, activating the DC.

Consider the effect of DNA preparations on the phenotype and functional activity of dendritic cells in vitro.

Immunoactive properties of DNA mammals, and in particular of human origin, particularly in relation DK has not been studied yet, although there are good reasons to assume that fragmented DNA, which can be cells, which was destroyed as a result of apoptosis or necrosis, capable of interacting with antigen presenting cells and to influence the processes of differentiation and maturation [18, 19, 23-26].

In this regard, we assessed the degree of influence of human genomic DNA from human placenta, a mixture of fragmented double-stranded DNA of a healthy person, on the processes of differentiation/maturation, and functional activity of DC. As a comparison, we used samples of human Cotl DNA, representing a mix of repetitive human DNA in double-stranded form, the size of 100-300 BP, not containing nucleosomes, and the standard ind the tor activation DK LPS.

To study the effects of drugs on the human DNA was used DK generated by interferon Protocol (GM-CSF+IFN-α). This type DK has properties of both myeloid and plasmacytoid-like cells of various degrees of maturity, in particular, expresses TLR7 and TLR9 [27-29].

Our previous studies have shown that DK TB patients are characterized by a delay in the differentiation/maturation and a significant portion of patients - reduction allostimulatory activity [30, 31]. So along with a healthy donor evaluation of the effect of DNA on the phenotype and function of DC was also conducted in cultures DK TB. These studies were of interest both in terms of analysis of the effects of DNA on DK, and in terms of potential applications of DNA preparation for correction of violations DK in infectious diseases.

Data describing the influence of DNA on allostimulatory activity DK healthy donors, are presented in Table 2. LPS increased allostimulatory activity DK in 2 of 3 healthy donors (donors 2 and 3). In this case, the DC treated with LPS induced a higher level of proliferative response in SSI than the control of DC cultured without the ripening of the stimulus (in the absence of LPS). The use of DNA as a maturing incentive DK had almost the same effect as the FSC with the greatest stimulating the ith action in the dose of 5 and 25 μg/ml In one case (donor 1) FSC practically not increased allostimulatory activity DK (PE=1,06)that, apparently, is connected with the individual (genetic?) features that provide a very efficient DC maturation under the influence of GM-CSF+IFN-α in the absence of additional, mitogenic stimulation. It is interesting to note that in this case, and DNA had no stimulating effect in any of the analyzed doses. Thus, in General, a healthy donor effect of DNA on allostimulatory activity is similar to the effect of LPS and is manifested in the increased ability of DC to stimulate a proliferative response in SCR.

Table 2
The impact of genomic and Cotl DNA allostimulatory activity DK donors
The ripening stimulus (µg/ml)Donor 1Donor 2Donor 3
The answer in SCRYVESThe answer in SCRYVESThe answer in SCRYVES
034652 3389132716
FSC365831,06399111,18500891,53
DNA 1343050,99380301,12414261,27
DNA 5324550,94515821,52431281,32
DNA 25274430,79475311,40538341,65

Note: the values presented proliferative response (imp/min) in allogeneic SCR. Responsible OLS donors were cultured with DC ratio of 10:1. The positive control (LPS) served as DC maturation was induced LPS, negative control (O) - DK, generated in the absence of the ripening of the stimulus. YVES - index is Liane, the ratio of the response in SCR during stimulation of DC generated with the ripening of the stimulus to the response in SCR, the stimulation of DC generated without the ripening of the stimulus.

The next step was the analysis of the influence of DNA on allostimulatory activity DK, obtained from 5 patients with TB (table 3). The level of proliferative response in SCR induced DC patients was lower than in SCR induced DK healthy donors. For example, when using DC treated with LPS, the proliferative response in SSI ranged from 6938 to 18524 pulse/min, whereas in the group of donors - from 36583 to 50089 pulse/min compared with the negative control (DC generated in the absence of the ripening of the stimulus) LPS increased allostimulatory activity DK (YVES>1,2) in 3 of 5 patients. Increased proliferation in SCR after treatment DK preparation DNA was also observed in 3 patients. In two cases (patient 1 and 2) stimulating effect DNA was obvious in the case of DK, which responded to LPS stimulation. In one case (patient 5) stimulating effect DNA was evident in cultures DK, resistant to LPS. It should be noted that, compared with donors in patients with TB were observed, first, dose-dependent effect of DNA in relation to DK, and, secondly, allostimulatory activity of DC induced DNA was lower than when using LPS as Dobreva the corresponding stimulus.

The effect of DNA enriched in repetitive sequences (Cotl), allostimulatory activity of DC was analyzed in cultures DK 4 TB (table 3).

Table 3
The effect of preparations of DNA and DNA enriched in repetitive sequences, allostimulatory activity DK TB
Ripen the overall stimulus (µg/ml)Patient 1Patient 2Patient 3Patient 4Patient 5
The answer in SCRYVESThe answer in SCRYVESThe answer in SCRYVESThe answer in SCRYVESThe answer in SCRYVES
03162547011010 6308139411,0
FSC185245,8690831,66135291,2369381,1145331,0
DNC87022,7561221,12116091,0540060,6180631,3
DNA 565952,0970831,29102070,9360911,096900,7
DNA 25117003,757221,0581790,74 60301,0131550,94
CpG 1--57111,04108750,99--223131,6
CpG 5--66941,2294170,8638680,6144411,0
CpG 25--74161,36107840,9887941,499960,7

Note: the table presents the values of the proliferative response in SCR (imp/min) of mononuclear cells from donors by stimulation DK TB. YVES - index effect, the ratio of the response in SCR during stimulation of DC generated with Dobreva the relevant stimulus, the answer SCR, the stimulation of DC generated without the ripening of the stimulus.

The stimulatory effect of enriched DNA repeats (Cotl) was recorded in 3 of 4 patients. Moreover, if the patient has 2 allostimulatory activity DK amplified as in the processing of FSC and Cotl DNA, patients 4 and 5 strengthening allostimulatory activity of DC were induced only Cotl, but not LPS. This means that the effect of LPS and enriched DNA repeats, obviously, is implemented through different intracellular signaling pathways. It is also important to emphasize that the patient 4 strengthening allostimulatory activity DK has only been observed when handling DK Cotl DNA was not detected in the processing of FSC or DNA, indicating possible differences in the mechanisms of the influence of fragmented DNA and enriched DNA repeats.

Since it is known that more Mature myeloid DC have more pronounced allostimulatory activity the effect of DNA can be associated with the induction of DC maturation. To verify this, an analysis was conducted of the impact of human DNA and DNA enriched in repetitive sequences, the percentage of CD83+and CD14+ DC from healthy donors (table 4). The introduction of culture DK DNA was accompanied by a decrease in the proportion of CD14+ cells and an increase in the relative content of CD83+ cells (donor 4 and donor 5). Most effective when it was used in isawanya DNA in a dose of 5 µg/ml Moreover, if the donor 5 effect of DNA (5 μg/ml) was comparable in intensity and direction with the effect of LPS, the donor 4 LPS had no significant effect on DC maturation and processing of DNA (all three investigated dose) induced their maturation (i.e. led to reduction of the proportion of CD14+ and increase in CD83+ cells).

Table 4
The effect of preparations of DNA and DNA preparations enriched in CpG sequences, phenotypic markers DK donors
The ripening stimulus (µg/ml)Donor 4Donor 5Donor 6
CD14CD83CD14CD83CD14CD83
%YVES%YVES%YVES%YVES% YVES%YVES
01242211157
FSC131,0830,75140,64393,560,418to 2.57
DNA 160,561,5210,95100,9
DNA 56,50,54 71,75120,55514,6
DNA 25100,8361,5231,0590,82
CpG 1191,2711of 1.57
CpG 58 0,5371,0
CpG 25100,6750,71

Note: here and in table 5 IV - index of influence, the ratio of the number of cells grown in the presence of the ripening of the stimulus, the cells grown in the absence of the ripening of the stimulus.

The influence of enriched DNA repeats were tested in cultures DK donor 6, the maturation of which were effectively induced LPS. The increase in the share of CD83+ cells was observed when handling DK Cotl dose of 1 μg/ml, whereas the decrease in the proportion of CD14+ cells require large concentrations of enriched DNA repeats.

In conclusion, we studied the effect of genomic DNA and Cotl on the maturation of DC TB patients (table 5).

Table 5
The effect of preparations of DNA and DNA preparations enriched in repetitive sequences, phenotypic markers DK TB
The ripening stimulus (µg/ml)Patient 6Patient 7Patient 8
% CD83YVES% CD 83YVES% CD 83YVES
0462
FSC102,5122,035of 17.5
DNA 161,561,073,5
DNA 5 51,2550,8373,5
DNA 2582,081,3126,0
CpG 141,050,8373,5
CpG 551,25101,6763,0
CpG 2520,540,6710,5

As you can see, all three patients maturation of DC now and what was dozirovannost FSC as evidenced by the increase in the number of CD83-positive DC. A similar effect, although less pronounced, were recorded using DNA as the ripening of the stimulus. Moreover, if donors are most effective was the dose of 5 µg/ml in patients with TB - 25 µg/ml of Enriched DNA repeats also induced maturation of DC. The optimal effect was observed when handling DK Cotl at a dose of 5 µg/ml, whereas the donor - dose of 1 mg/ml

Consider the phenomenon of activation of the DC when exposed to exogenous allogeneic fragmented double-stranded genomic DNA.

In the work we have identified some features of interaction between the DC and the exogenous DNA in the form of statistical genomic fragments with sizes that are multiples of nucleosomal the monomer (~200 BP) and the components of 1-20 nucleosomes (200-6000 BP).

It is known that in the blood plasma and other interstitial body fluids present extracellular DNA derived from cells that underwent apoptosis (or from any other dead cells, for example, cancer or cell necrotic tissue) [32-36]. To date, little is known about the fate of this DNA. It is obvious that the degradation to monomers and their subsequent participation in the synthetic processes of the cell is the most simple and easily understandable way of utilization of exogenous extracellular DNA. However, with the one with the pile-known that extracellular DNA can internalization in the cell compartments and to participate in the reparative mechanisms of recombination [7, 22, 37]. On the other hand it is shown that the DNA of microorganisms or DNA released into the blood during injury and tissue necrosis can activate the reaction of innate and adaptive immunity [24, 38]. This largely explains the numerous existing evidence that exogenous DNA can act on the tumor, slowing their progression. All these properties of exogenous DNA are directly related to the functioning of immune cells, and in particular DK [39, 40]. In this regard, we conducted a series of experiments aimed at analyzing the effect of exogenous DNA on the activity of the DC person. It turned out that the exogenous DNA enters the nuclear space DK man, almost without stopping in the cytoplasm. In mikromazernom space fragments of exogenous DNA are deposited and at least 6 hours are not subjected to processing (concatemers) (Figure 3), as described for other cell types [2, 21]. The number of internalized in mikromazernom kernel space fragments of exogenous DNA reaches 10000 (assuming an average fragment size of 1000 BP). A characteristic feature of the used DNA was lack of histones and nucleosomal organization, while the outlet of proteins to the nuclei of the second matrix, - associated DNA fragments was observed in sufficient quantities by electron microscopy analysis (Figure 1). Also important characteristics of the DNA preparation was its human origin and size that is a multiple of 1-20 nucleosomal the monomers (200-6000 BP), which corresponds to physiological DNA circulating in the blood (figure 2).

At the same time, we found that exogenous DNA in this form induces DC maturation person and enhances their allostimulatory activity with efficiency relevant LPS, often used as a "standard" maturing stimulus [41, 42]. Traditionally DK generated by culturing adherent fraction of mononuclear cells in the presence of GM-CSF and IL-4 followed by the addition of "cocktail" of the ripening of the cytokines IL-1, IL-6, PGE2, TNF-α [43, 44]. However, recently it was shown that partially Mature DC can quickly be generated during culturing monocytes with GM-CSF and IFN-α, and this way generate is more physiological [45]. Obtained in this case DK called "interferon" DK, have a number of advantages - generated faster time, are characterized by a high ability to capture antigen, remain stable in the absence of cytokines, have higher migration activity and effective stimulus is the shape YOU answer [45, 46]. In our experiments, we used such a Protocol generation DK. Analysis of the activation of the DC exogenous DNA reveals the following characteristics of the process. As fragmented genomic DNA, and Cotl human DNA induce DC maturation and increase their allostimulatory activity. To the stimulating effect of DNA sensitive DK as healthy donors and patients with TB. In both analyzed groups (donors and patients) revealed quantitative differences for genomic and Cotl DNA stimulation DK. The intensity of DNA-mediated stimulation was comparable with the effect of LPS. However, we have identified donors who meet one inductor (e.g., DNA) and do not respond to LPS, and Vice versa. This fact is consistent with the induction of DC maturation under the influence of LPS is TLR4 dependent way, whereas for genomic and Cotl DNA this way is not completely understood, and moreover, at least for genomic DNA is not associated with TLR9-CpG induction [42, 47].

It is known that the process of maturation of DC from monocytes of the blood of the person is accompanied by changes in the expression of surface antigens - CD 14 and CD83 [48]. In the population of immature DC share CDH-positive cells is higher than CD83+, and when ripe, the ratio is reversed. It was found that the efficiency of DC maturation induced by both genomic and Cotl, comparable to the action of LPS and has a dose-dependent severity. To the stimulating effect of genomic or Cotl DNA were sensitive DK as healthy donors and patients with TB, although in the latter case, the effect implementation requires large concentrations of DNA/Cotl. The results indicate the ability of the drug to DNA and DNA enriched in repetitive sequences to stimulate DC maturation and enhance their allostimulatory activity. When the similarity of the effect of DNA with LPS, the effect of these inductors is mediated, apparently, different signalling pathways and/or different subpopulations "interferon" DK, for example, myeloid or plasmacytoid-like cells, differing in degree of maturity.

A generalized picture of the received data may indicate the following. Exogenous DNA in the form of fragments of genomic DNA (or Cotl repeats) is absorbed DK and internalities in mikromazernom space of the cell nucleus, do not linger in the cytoplasm. Interaction with the cell, progress in nuclear space and depositing in it fragments of exogenous DNA leads to activation of the DC that accompanied their maturation (changes CD14/CD83 phenotype) and enhanced functional activity (ability to stimulate the proliferation of allogeneic T lymphocytes in SCR).

DK being the most is her "professional" antigen-presenting cells, provide the close relationship between the reactions of innate and acquired immunity. Activation and development of nonspecific and antigen-specific immune response largely depends on the efficiency of binding of the receptors with a specific type agonists. Agonists for activation of innate immunity are the so-called PAMP (pathogen associated molecular patterns), which binds with the receptor PRR (pattern-recognition receptor, induce the synthesis and secretion of cytokines and co-stimulating molecules. Adaptive immune response is associated with antigen-specific receptor complexes of T - and b-lymphocytes (TCR and BCR, respectively). Somatic rearrangement of genes TCR and BCR forms a highly diverse repertoire of molecules with the ability to identify almost all of the antigenic determinants specific manner [38, 49]. The facts obtained in the studies in recent years suggest that nucleic acids are inducers of both the innate and adaptive immunity.

The greatest interest in terms of this problem antitumor activity of the immune system represents a specific interaction of the receptor TLR9 and CpG DNA. This receptor is located in endosomal compartments in the composition of the endoplasmic reticulum [20, 42, 47]. Effect of CpG DNA on the development of the immune response may be direct the output or oposredovanie through the activation of immune cells, constitutive expressing TLR9, such as b-lymphocytes and DC [50]. Thus DK be competent to induce the development of CD8+ and CD4+ T lymphocytes in the direction of the preferential differentiation of cytotoxic CD8+ T cells compared with normal T-Genesis [5 0-53]. TLR9 up to the present time is the only well-studied receptor, which when interacting with CpG DNA generates immune response [24, 39]. It is assumed that TRL9 indirect way of activation of the immune system associated with the presence neetilirovannyj CpG sequences, and any abnormal structure of the oligonucleotide containing, for example, phosphorothioates frame. All ligands TRL9 are polymers with cyclic components of the hydrophobic nature [24].

Recently, it became apparent that the double-stranded DNA of different origin and in different forms and is not dependent on sequence may participate in the regulation of both types of immune response. Moreover, this regulation does not depend on the activation of TLR9 and assumes the availability of alternative molecular mechanisms of activation of immune cells[18, 25, 26, 54, 55]. Characterized by different inductor forms of DNA and determined the effector properties of immune cells induced by this DNA. It is shown that double-stranded DNA to form nucleosomes [18, 19], purified enana DNA mammals, bacteria, viruses [25, 55], double-stranded DNA not containing CpG motifs [55, 56], single-stranded CpG oligonucleotides [57] lead to the maturation and strengthening of the functional activity of DC and macrophages. This process is characterized by the activation of the synthesis of co-stimulatory molecules (MHC class I or class II, CD40, CD54, CD86, CD83) [18, 25, 56, 58-60], cytokines (INF-α/β, TNF-α, IL-6, IL-8, IL-12p40/70) [18, 25, 55], of nitric oxide (NO) [59].

Up to the present time it is not known how such activation, although it is known that this process is essential to the functioning of a certain set of specific genes [56].

Conducted in this paper suggests that fragmented exogenous allogeneic DNA activates the maturation of the DC, which is manifested by an increase in the number D83+ cells and reducing D14-positive cells. Exogenous DNA increases allostimulatory activity DKK SCR, which indicates the ability of the DC under the influence of DNA effectively recognize alloantigen and in response to secrete into the environment of various cytokines (IL-12, IFN-γ), stimulating the proliferation of the responding T cells. The presence of exogenous DNA for a long time in mikromazernom space cores DK can be one of the factors determining the shift of the molecular processes of DK in the direction of their activation. It is known that exogenous DNA double-stranded ends which activates the hierarchical system of kinases and arrests the cell cycle to complete removal of the stimulus [61-63]. It can be assumed that if DK is the same situation that led to the launch of reparative molecular processes and, ultimately, to activate genes immunocompetent DC.

1. The method of obtaining stimulated dendritic cells for the induction of immune responses against tuberculosis of the person based on the fence patient immature dendritic cells, culturing ex vivo for maturation and formation allostimulatory activity for subsequent additional processing of the antigen and reverse the introduction of Mature dendritic cells back into the patient with the purpose of formation of adaptive immunity, characterized in that during culturing ex vivo immature dendritic cells into the culture medium injected fragmented allogeneic double-stranded genomic DNA fragments by size 200-6000 softwares that make up the complete genome physiologically and genetically healthy donor in an amount of 5 μg/ml of medium.

2. The method according to claim 1, characterized in that the drug fragmented allogeneic double-stranded genomic DNA fragments by size 200-6000 softwares that make up the complete genome of F. zoologichesky and genetically healthy donor, enter into the culture medium containing immature dendritic cells of patients with tuberculosis, in the amount of 25 μg/ml of medium.



 

Same patents:

FIELD: medicine.

SUBSTANCE: there is produced a new CT-B1/F8 hybrid cell clone producing monoclonal cholera toxin antibody (MCAB) in the environment of cell culture and abdominal cavity of syngeneic animals. The clone is produced by the fusion of mouse myeloma SP-2/0 cells with popliteal lymph node cells of BALB/c mice immunised with a commercial preparation of cholera toxin (SIGMA) in posterior pads. A fusing agent is polyethylene glycol of molecular weight 4000. Hybridoma selection has been performed on Dulbecco modified Eagle's medium with bovine foetal serum and hypoxantine-aminopterin-thymidine added. Hybridoma synthesises MCAB specifically interacting with cholera toxin and not interacting with thermolabile E.coli toxin. The antibody titre reaches 1:20000 in the cultural fluid, 1:2000000 in ascitic.

EFFECT: antibodies can be used for designing immunobiological systems of cholera toxin detection exceeding available analogues in sensitivity.

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FIELD: medicine.

SUBSTANCE: there is offered a method of precursor recovery from a human body, including all cells having stem cell characteristics or similar, in particular closely active or multiple active precursors, and such cells are recovered either directly, or indirectly from human breast secretion sampled from the specified human body. This secret can be colostrum, ripened milk or secretions in males and females in a lactation interval, during at least one of the following periods: absence of pregnancy, pregnancy period, lactation period, involution period. Further, the present invention refers to preferable applications of such recovered cells.

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24 cl, 8 dwg

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to methods of obtaining deposited lymphokine-activated killer cells (LAKC) for treatment of oncologic diseases. Claimed is method, according to which mononuclear lymphocytes (MNL) are isolated from peripheral blood of malignant effusion by centrifugation on one-step gradient of ficoll with density 1.06-1.08 g/cm3, LAKC are generated and concentrated by successive precipitation of MNL by centrifugation, re-slurring it in RPMI 1640 or DMEM medium with addition of 2-10% AB human serum, interleikin-2 in concentration 0.5-1.0 mlnIU/ml and incubation in CO2 incubator for 48-72 hours, after that suspension of obtained LAKC is deposited in culture medium in amount 2 mln LAKC per 200 ml of medium on sterilised and washed with culture medium porous titanium carriers with 55-60% porosity.

EFFECT: invention allows to increase efficiency of obtained medication for local and local-regional immunotherapy of oncologic patients.

1 ex, 8 dwg, 2 tbl

FIELD: medicine.

SUBSTANCE: diploid cell cultures are reduced and collected by propagation in a monolayer in an Eagle's growth medium. The conditioned growth medium is separated from the formed cell layer by sterile bottling. The cell layer is removed, washed by centrifugation. The prepared cell suspension is reduced to the required and bottled in the sterile environment. The conditioned growth medium bottles and cell suspension bottles are consistently single frozen to temperature -20°C and higher and thawed at temperature +25°C and lower, and bottled in the sterile environment. The conditioned growth medium and cell suspension are frozen in open bottles at temperature minus 50°C and lower, kept in the frozen environment for at least 48 hours and then lyophilised in two stages. The first stage involves desorption at temperature minus 50°C to 0°C for at least 20 hours; and the second stage requires sublimation for at least 20 hours at temperature 0°C to 30°C; the finished lyophilised bottles are closed in the sterile environment. The invention allows preparing the lyophilised preparations effective in treatment of deep vast burning wounds, frostbites, degrees III (a, b) and IV subdermal burns, treatment of oral and nasopharyngeal mucosa, are storage-stable at temperature +4°C for at least 12 months.

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1 dwg, 3 tbl, 2 cl

FIELD: chemistry; biochemistry.

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8 cl, 10 dwg, 14 ex

FIELD: chemistry; biochemistry.

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7 cl, 5 dwg, 4 tbl, 6 ex

FIELD: medicine, veterinary science.

SUBSTANCE: invention refers to veterinary science. A culture medium for pre-implantation embryos contains DMEM medium, inactivated blood serum of 6-7-month nanny-goats with blood content of IGF1 and IGF2 making 54.73 and 231.50 nmol/l respectively, glucose, streptomycin and penicillin, in the following ratio, wt %: inactivated blood serum of 6-7-month nanny-goats - 15.0-20.0; glucose - 0.40-0.45; streptomycin - 0.004-0.005; penicillin - 0.003-0.0035; DMEM medium - the rest.

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1 tbl, 6 ex

FIELD: chemistry; biochemistry.

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5 dwg, 7 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to genetic engineering and can be used to produce mammal nucleolar protein SURF-6. The NIH/3T3-174 cell line is obtained through genetic modification of mouse fibroblast line NIH/3T3. Modification is carried out using two successive transfections when the cells reach 70% confluence. The first transfection is carried out with 5 mcg of the pUHrT62-1 plasmid which codes the cell neomycin resistance gene (G-418) and the tetracycline-dependant transactivator, and 15 mcl of a liposomal transfection reagent in 500 mcl of DMEM medium. The second transfection is carried out with a mixture of 2.5 mcg of the pBI-SURF6 plasmid which bears the EGFP gene (green fluorescent protein) and the SURF-6 protein, and 2.5 mcg of a linear DNA fragment which codes puromycin resistance gene, and 15 mcl of a liposomal transfection reagent in 500 mcl of DMEM medium. Expression of EGFP and SURF-6 genes is controlled by a promoter which is activated by addition of an inducing substance - doxycycline antibiotic to the culture medium.

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5 dwg, 7 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, particularly to a method of producing three-dimensional matrices for tissue-like structures from animal cells. The method involves covalent bonding of histons with the surface of pre-activated biocompatible polymer microspheres made from crystalline dextran. The microspheres with covalently bonded histons are then deposited by centrifuging. Microspheres containing 160-200 mcg protein per 1.0 g are deposited on a substrate surface in amount of 0.5-1.0 mg per 1.0 cm2 and then dried at room temperature. Further, the substrate is washed with a solution at pH 7.5 to remove material which is not bonded to the substrate. The layer of microspheres obtained on the surface of the substrate on which cells are deposited is used as a base for obtaining tissue-like cell structures.

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6 cl, 11 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and veterinary science. There is offered a method of treating tuberculosis including administration of Siofor 500 3 times a day, 1 tablet of Delagyl 3 times a day, acetylsalicylic acid three times a day. The 1-year follow-up has shown no recurrence.

EFFECT: method allows intensifying tuberculosis immunity, suppressing tuberculosis and accompanying infection.

1 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine and pharmaceutical industry, namely to method of obtaining poly-DL-lactide-co-glycolide nanoparticles with antituberculosis medications, incapsulated in them, which includes: obtaining lyophilised particles of stable medications, including: stage 1: obtaining lyophilised particles of stable medications, including: stage 2: obtaining lyophilised particles of unstable medications, including: stage 3 combination of lyophilised particles obtained at stages 1 and 2 with obtaining said composition.

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14 cl, 1 tbl

FIELD: medicine.

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EFFECT: method allows to activate processes of microcirculation, trophism and cellular metabolism in tissues, which contributes to reduction of spastic and edema components, eliminbation of pain, inflammation and stagnation in kidneys and prostate.

1 dwg, 2 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention describes novel benzothiazinone derivatives of formula (I) and their use as antibacterial agents in infectious diseases caused by bacteria, especially mycobacterium tuberculosis (TB) and leprosy, in which R1 and R2 independently denote NO2, CN, CONR7R8, COOR9 CHO, halogen, SO2NR7R8, OCF3, trifluromethyl; R3 and R4 independently denote H or methyl; R5 and R6 independently denote a straight or branched aliphatic radical having 1-8 members in the chain, or R5 and R6 together denote a divalent radical -(CR92)m- or R5 and R6 together denote a divalent radical: R7, R8 and R9 independently denote H or a straight or branched aliphatic radical having 1-7 members in the chain, or phenyl.

EFFECT: design of an efficient method of obtaining benzothiazinone derivatives, a pharmaceutical composition having anti-mycobacterial activity.

12 cl, 6 tbl, 12 ex

FIELD: medicine.

SUBSTANCE: invention belongs to microbiology and biotechnology, notably to tuberculosis anatoxin manufacture for specific prevention of tuberculosis. Tuberculosis anatoxin is made by detoxication of tuberculosis exo- and endotoxins with two detoxifiers - 0.2% formalin solution during 7-9 days at 42-45 C° and 0.5% aethonium solution during 7-9 days at 42-45 C°. Then tuberculosis anatoxin is sorbed on 1-3 mg/ml of aluminium hydroxide, concentration of inactivated toxic allergens is doubled by decantation of 50±5% of supernatant.

EFFECT: usage of anatoxin manufactured by this method gives vaccinated animals immunity to induced by virulent tuberculosis mycobacterium infection due to immune reorganisation of organism; animals acquire moderate sensibility to tuberculin.

4 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of cell biotechnology, veterinary and medicine. Method of treating experimental tuberculosis in mice includes introduction of suspension of bone marrow cells into affected mice. Bone marrow cells are isolated from femoral bone of inbred mice line AKR, resistant to tuberculosis. Introduction is performed into caudal vein once a week in dose 6 mln cells per a mouse during two months. Method allows to reduce number of mycobacteria in organism of experimental animals considerably and guarantees elongation of affected animals life.

EFFECT: invention can be used in veterinary and medicine.

2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely to a preparation with antituberculous action. The method for making the preparation with antituberculous action, characterised by that a dried blastema of sort Cladonia is mechanically treated with solid sodium alkali added. The prepared dry mixture is drawn in an aqueous-alcoholic mixture under certain conditions. The solution is separated from sediment by centrifugation and neutralised with citric acid.

EFFECT: produced preparation shows improved antituberculous action.

1 dwg

FIELD: medicine.

SUBSTANCE: invention is related to composition with high tuberculostatic activity.

EFFECT: reduced toxicity and by-effects of its components.

1 cl, 1 ex, 4 tbl

FIELD: medicine.

SUBSTANCE: for treatment of pulmonary tuberculosis in HIV-infected patients carried out is anti-tuberculosis therapy according to standard schedules. Additionally since first day of treatment galavit is administered intramuscularly in dose 100 mg one time per 3 days. On the whole 15 injections are made. Addition of galavit to treatment, with given schedule and duration of introduction, produces normalising effect on imbalance of cellular and humoral immune response in patients with HIV-associated tuberculosis.

EFFECT: increase of treatment efficiency due to reduction of abacillation terms and closing of disintegration cavities, resolution of infiltrative changes in lungs.

2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to phthisiology, and can be used for treating pulmonary tuberculosis in HIV-patients. That is ensured by performed tuberculous therapy according to standard conditions. In addition, from the first day and upon termination of the therapeutic course, Wobenzym in dose 3 tablets 3 times a day 30 minutes before meals daily is prescribed.

EFFECT: therapeutic addition of Wobenzym in a specially developed dose and schedule makes a normalising effect on imbalanced cell and antibody response in the patients of the given category that allows improving clinical effectiveness ensured by shorter abacilliation and closure of destruction cavities, resorption of infiltrative changes in lungs.

2 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to anti-M-CSF-specific antibodies based on RX1 or originating from RX1, and which more than 785% compete with monoclonal antibodies RX1, MC1 and/or MC3 for bonding with M-CSF (macrophagal colony-stimulating factor). The non-mouse antibody is two-stranded, contains a certain amino acid sequence (given in the formula of invention and list of sequences) and retains high affinity towards M-CSF. The invention discloses an isolated nucleic acid which codes the said antibody, an expression vector, a host cell and a method of producing the anti-M-CSF-antibody using a host cell or hybridome, particularly ATCC PTA-6263 or ATCC PTA-6264 hybridome. The invention describes a pharmaceutical composition containing said antibodies, sets containing pharmaceutical compositions and methods of preventing and treating osteoporosis in a person suffering from an osteolytic disease.

EFFECT: disclosed antibodies can inhibit osteoclast differentiation, which facilitates their use as highly effective preparations for treating osteolysis, cancer with metastases and osteoporosis associated with cancer metastases.

131 cl, 44 dwg, 12 tbl, 16 ex

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