Functional immunoassay in vitro

FIELD: medicine.

SUBSTANCE: excitory cells of the immune system are recovered that is followed with primary incubation of the cells with an investigated substance to produce a primary-incubation supernatant or mixed cells and supernatant; secondary incubation of the target cells with the supernatant or mixed cells and supernatant wherein the secondary-incubation target cells are understood as human tumour cells or cell lines of an oncogenetic origins; the target cells are analysed where the analysis is specified in a group including an expression analysis of specific proteins and an apoptosis and/or necrosis analysis.

EFFECT: improvement of the method.

14 dwg

 

This invention relates to a method for in vitro studies of the effects of substances in processes in vivo, as well as to a method for detecting in vitro for identification of immunomodulatory compounds and/or detection steps immunomodulatory compounds, as well as the identification of inducing apoptosis and/or necrosis connections with immune system processes in vivo.

In recent years in the pharmaceutical industry have developed new classes of substances that are prescribed for the treatment of various diseases. Among them are, in particular, is also a means of gene therapy or gene therapy modified homologous substances, such as proteins or DNA constructs.

Because with these brand new classes of substances partly no experience in the pharmaceutical treatment of diseases, there is a need for a method for testing the effectiveness of these tools without the need for simultaneous use of animal experiments and/or clinical trials with patients. Similar experiments with new, unknown substances banned already for ethical reasons. On the contrary, it is recommended to precede this stage the results of in vitro studies, which make possible predictions about the efficacy in vivo of these substances. When the in vitro experiments it is important ascan more closer to the situation in vivo.

In addition, it is important to develop simple methods for dynamic monitoring ("monitoring") of patients before, during and/or after a method of therapy (e.g., immunotherapy or therapy that acts on the immune system), which investigated the reaction of the body or the immune system for the various modes of therapy.

Along with the conventional methods of cancer treatment such as radiation therapy and chemotherapy, which since 1950-ies represent the only possibility of treatment of far advanced and widespread neoplastic diseases, now the goal is to develop therapies that are associated with fewer side effects for patients, but are highly effective in achieving treatment targets.

One approach for this is immunotherapy, which aims to strengthen the natural immune response against neoplastic diseases genetically engineered modifications and, therefore, affect the attention of the immune system to cancer cells and thereby on the defensive so that the tumor can be defeated by the body itself.

Currently, most clinical studies rely on removal of the tumor, subsequent ex vivo transfection of tumor cells therapeutic gene, irradiation of tumorous the x cells and subsequent re-implantation is now modified tumor cells. Through this vaccination tumor cells can enhance the antitumor response depending on transfitsirovannykh therapeutic gene in different degrees.

However, along with the transfection of tumor cells, are also being developed immunomodulating substances that induce the immune system to suppress tumor cells. Through these immunomodulatory substances, the immune system must be deduced to one or "programmed" to tumor cells specifically amazed and, ultimately, destroyed. Thus, the immunomodulatory substances are effective in the treatment of cancer mediated through the immune system on the corresponding tumor or its underlying type of tumor cells.

The method, which allows to investigate the effect in vitro of new substances on the processes in vivo, such as the destruction of tumor cells, would, on the one hand, to avoid experiments in vivo, under large ethical concerns, and, on the other hand, would allow for a short time to test many substances in many different tumor cells. In addition, it would be through such a way to ensure the further course of treatment-induced in vivo effects in the so-called "monitoring therapy."

On the basis of this prior art the problem of danagoulian is the way, which allows you to explore the efficiency of in vitro production processes in vivo in humans or higher mammals.

This problem is solved by the features of independent claims.

In the context of this invention represent:

effector cells of the immune system is a mixture of immune cells, such as, for example, PBMC [peripheral mononuclear cells from the blood (human or higher mammal), spleen cells (animal model) and so on], or obtained FACS-sorting or ACS-sorting of subpopulations, such as, for example, In-, T - and NK-cells, monocytes, dendritic cells, etc.;

CpG-base - demetilirovanie base cytosine-guanine;

dSLIM - immunomodulating oligodeoxyribonucleotide dual structure of the stem-loop, and each loop detects CpG-based, preferably three CpG-base;

One - oligodeoxyribonucleotide;

PBMC - peripheral mononuclear blood cells.

Further, a number of General concepts should be understood as follows.

Under immunomodulatory compounds, in the context of this invention, it should be understood substances that can affect the immune system or also only individual cells, in particular the effector cells. These include, along with chemical compounds, DNA constructs, proteins, antibodies, sugar molecules is whether other substances, who discover the properties that cause the immune system or immune system cells are encouraged to response. This applies in particular to the cells of the immune system, referred to in this invention effector cells, which are able to influence the immune response or to mediate the immune response. This mediating occurs through the release of specific messengers.

Thus, in accordance with this invention features a method that includes the following stages:

a) isolation of cells

b) primary incubation of the cells with the subject of study by the substance,

(C) obtaining a supernatant or a mixture of cells and supernatant primary incubation

d) secondary incubation of target cells with supernatant or a mixture of cells and supernatant,

(e) analysis of the target cells.

In one alternative embodiment proposes a method of detecting in vitro, which provides for the identification of immunomodulatory compounds and/or detection steps immunomodulatory compounds, as well as the identification of inducing apoptosis and/or necrosis connections with immune system processes in vivo, which provides:

a) primary incubation of the effector cells of the immune system with the test on immunomodula is that the relevant action or inducing apoptosis or necrosis substance, with the subsequent;

b) obtaining a supernatant or a mixture of cells and supernatant primary incubation and subsequent;

C) a secondary incubation of target cells with supernatant or a mixture of the cells and the supernatant of the primary incubation and thereafter;

d) analysis of immunomodulating and/or inducing apoptosis and/or necrosis of the action using an appropriate method of detection.

Using the stages of this method it is possible to investigate the in vitro effects of substances in processes in vivo. As a result, new connections can be tested very close to the in vivo situation without risk to animals and/or patients in clinical trials.

In addition, the result already undertaken/conducted therapy may be subjected to follow-up (using analysis of relevant parameters). This application of the methods of this invention are known in the context of this invention also monitoring therapy. This term applies only follow-up in vitro effects of therapy in vivo. These methods of the present invention is not associated with therapy, except that therapy can be monitored or observed (monitored) after therapy.

In the case of selected cells it comes in a preferred embodiment of the method of this invention effector about clickformoney system in accordance with the definition above. Methods according to this invention is suitable, in particular, to explore the effects of substances on the cells, which is mediated by the immune system.

After the cells of the immune system to substances in the primary incubation was able to show them to them, the effects of a substance in vivo find then in the secondary incubation by the fact that supernatant or a mixture of cells and supernatant primary incubation, which, including, contain spin-off products of immune system cells, incubated with the target cells.

As target cells, preferably provided by human cells or cells of higher mammals. In one particularly preferred embodiment of the method according to this invention for the primary incubation selected cells, in particular cells of the immune system, and for the secondary incubation used as target cells tumor cells or cell lines derived from tumor cells. In this arrangement, the method according to this invention, this method is also called "functional immunoassay in vitro".

As tumor cells are considered mostly all types of tumor cells of different origin. The objective functional in vitro immunoassay" is the identification or investigation of substances that is capable of being settled by the immune system to result in apoptosis or necrosis of tumor cells.

But the next target of the method of this invention is also a study of the recognition of tumor cells by the immune system caused by enhanced expression of MHC molecules-I (for example, HLA-ABC) and adhesion molecules (such as ICAM-1) on the surface of tumor cells. The decisive advantage of the method of this invention is that the effect in vivo can be detected without the need for experiments on animals and/or patients in clinical studies with the associated disadvantages.

For the application of the method of this invention for the study of changes in the expression of surface molecules on the basis of induced immunomodulatory substance immune responses are suggested. This set contains prepared to store aliquots of the cells, preferably the effector cells of the immune system, for the primary incubation with the test substances, means for conducting primary and secondary incubation, as well as appropriate means for analyzing the pattern of expression of surface molecules of cells of the secondary incubation. Set according to this invention provides for analysis of the pattern of expression of surface antigens on target cells means second incubation for carrying out RT-PCR, for which this set contains the appropriate primers for amplification of mRNA surface molecules, enzymes for the amp is eficacia and required buffers, and/or means for FACS analysis, for which this set contains fluorescently labeled antibodies directed against surface antigens and markers of apoptosis/necrosis, and, in addition, means for processing the target cells, such as buffers and chemicals.

Methods according to this invention adapted in one advanced version, also to the monitoring of therapy, and as the analyte of interest in the primary incubation of whole blood, blood cells, serum or blood plasma of a patient before, during and/or after therapy (e.g., immunotherapy or therapy that modifies the immune system or affects the immune system).

With this improved version of the method of the present invention can explore, are already actions in vivo therapeutic substances that were injected to the patient and preferably acted stimulus to the immune system. Although this method was investigated, the patient's blood contains cells and/or messengers or part thereof (for example, serum, or plasma, or of a subpopulation of cells), the method of the present invention provides in this embodiment, in the final analysis, the indirect proof of the validity of in vivo substance that was administered to this patient in this therapy, preferably immune therapy.

Because unknown no specific antibodies that could be used in the method of this invention for monitoring therapy", you can see the effect in vivo by virtue of therapeutic substances, or to track changes in the production of specific antibodies due to the reaction of the immune system.

When therapies in which the methods of the present invention are envisioned as monitoring therapy effectiveness input in each case, therapeutic substances, it is preferably of such diseases as cancer, infections, allergies and autoimmune diseases.

Therefore, based on these advantages to the methods of the present invention provides preferably compounds which have immunomodulatory effects or can cause apoptosis or necrosis.

As immunomodulatory compounds in accordance with this invention provides preferably containing CpG motif oligodeoxynucleotide and dSLIM (immunomodulating oligodeoxyribonucleotide dual structure of the stem-loop, see EP 1196178 B1). But in the scope of the present invention is also the use of other biomolecules, such as, for example, natural or modified genetic engineering of antibodies, substances on the basis of DNA or RNA (antisense oligoresorcinol tidy, short interfering RNA (siRNA), and so on), amino acid compounds, messengers or other immunomodulators (such as aluminium salts, imidazolinones, lipopolysaccharides, saponin derivatives, phospholipids, squalene etc).

As inducing apoptosis and/or necrosis of the compounds according to this invention are considered, in particular, such compounds, which are suitable for prolonged disturbances necessary to maintain cell processes. Thus considered, in particular, substances on the basis of DNA or RNA (antisense oligodeoxynucleotide, short interfering RNA (siRNA), and so on), antibodies or chemotherapeutic agents.

Methods according to this invention can be also used to identify messengers that are released due to incubation of the selected cells in the primary incubation with immunomodulatory or inducing apoptosis and/or necrosis substances from these cells. For this purpose, the supernatant of the primary incubation before adding to the target cells pretencious with antibodies that specifically recognize the potential messengers. As a result of interaction between the antibody and the epitope messenger he is no longer able to transmit signals in target cells and, therefore, becomes locked in its functions. This scheme is the way this is about of the invention is important for detection, what specific messengers responsible for the induced action, such as apoptosis.

Within the framework set for the application of the method of the present invention to identify induced release of messengers used preferably hole microtiter tablets with another 24-96 holes, and the surface of each of the wells coated with antibody that is directed against one epitope messenger (for example, IFN-γ), and after incubation the supernatant fractions of the primary incubation with processed so your tablet and subsequent incubation of these fractions with the target cells, there is the possibility of testing many potential messengers for a short time about whether they are really in mediating an immune response or induction of apoptosis.

Thus, the set for the application of the method of the present invention to identify messengers that are released in response incubation of primary cells incubation with the test substance, is also the subject of this invention. This set contains suitable for storage of prepared aliquots of the cells, preferably the effector cells of the immune system, primary and secondary incubation, and, in addition, advance tablets with the number of holes another 24-96, in which the surfaces of these moons is to antibody-coated moreover, the surface of the different holes covered with various antibodies, but is preferably provided by at least 2 wells with the same antibody in each case.

Stage of incubations required when the method of this invention, preferably take place in an incubator with 5% CO2. But there are also other conditions of incubation, which in each case correspond to the requirements inkubiruemykh cells.

Getting supernatants or mixtures of supernatant and cells from the primary incubation is carried out according to the invention by centrifugation. However, according to the invention also valid for all other methods, which are suitable for separating cells from supernatant, such as, for example, filtration cells, if the width of the holes, which allows the passage of only the supernatant, but not found the remnants of the cells. In addition, provides for the separation of cells or sorting cells using specific antibodies and subsequent selection using magnet (ACS) or based on fluorescence selection (FACS).

For analysis of cells suggests ways in accordance with this invention, which can provide changes in the expression of proteins in the target cells. Thus considered, in particular, FACS measurements (sorting cells with activation of fluorescence), Western blot of the gel is istratii or cytospin, get in cytocentrifuge.

Also provided methods for the analysis of changes in expression of certain genes, such as, for example, RT-PCR, real-time PCR analyses with protection from RNase, and Northern-blot and the blot on the Southern.

Finally, in the analysis of in vivo also provides analyses of apoptosis, such as, for example, staining of cells with Annexin V and Tunel analysis (transferase-mediated tagging nick-end dUTP), for example, by using coloring iodide of propecia.

The following examples and the results of the experiments confirm that the application of the method in accordance with this invention is able not only to study the in vitro effects of substances in processes in vivo, but also, in addition, is suitable for confirmation and evidence of the specificity of the detected effects through the improvement of the method of this invention to competitive analysis.

Additional preferred embodiments of this invention are found in the following patent claims and the description. This invention, including the implementation of the method according to the invention will be further described in more detail by means of embodiments and illustrations, not limited to these examples.

Obtaining mononuclear cells

For the implementation of the FPIC of the BA according to this invention has received peripheral mononuclear cells (PBMC) from whole blood or the so-called "leucocytes film". In this case we are talking about the side product, which occurs in the preparation of red cell concentrates from whole blood.

The allocation of PBMC was performed by centrifugation through a gradient ficoll for separation of erythrocytes, granulocytes and dead cells. Ficoll is an uncharged polymer of sucrose, the density of which is positioned so that when layering on him the whole blood or leucocytes film and subsequent centrifugation of the particles with lower density pass through the layer ficoll and collected on the bottom of the tube, while the lymphocytes and the monocytes are collected at the interphase between the plasma (top) and picollo (below).

The interphase, which contains cells after centrifugation, was isolated and washed several times SFR. After that, the selected cells were placed in culture medium to the cells and brought to a concentration of 1-4×106cells per milliliter.

Double-stranded immunomodulatory oligodeoxynucleotide (dSLIM)

Double-stranded immunomodulatory oligodeoxynucleotide are molecules with CpG sequences. For this linear oligodeoxynucleotide (one) covalently closed nucleotide loop, so they are protected from degradation ectonucleoside. This way, handleoversize molecules, which are called dSLIM, "immunomodulators with doubled the second structure of the stem-loop". Immunomodulatory action is based on nonspecific activation of the immune system neetilirovannyj sequences G that bind Toll-like receptors, and, above all, the particular structure of molecules dSLIM. Each loop dSLIM contains three neetilirovannyj CpG-base.

Double-stranded immunomodulators dSLIM type ISS30 (for example, dSLIM-30L1) receive, in accordance with SOP and subsequent quality control in the laboratory class Century For this chain-shaped studs, 5'-phosphorylated oligodeoxyribonucleotide (one) are ligated using T4-DNA-ligase. After splitting the remaining selected substances T7 polymerase, and chromatographic purification of the resulting molecule dSLIM was concentrated by ethanol precipitation with sodium acetate-magnesium and dissolved in SFR. The detailed method is described in WO 01/07055.

Primary incubation of immune cells (PBMC) with dSLIM

Selected cells (PBMC) were sown in advance tablets. The size of the original mixtures and, accordingly, the size of the holes was chosen so that the assembled later, the culture supernatant was exactly the amount that was used for the secondary incubation with the target cells.

In the first initial mixture was estimulando cells (negative control). The second source of the mixture was performed stimulation using 01-10 μm dSLIM-30L1. In two additional original mixes performed stimulation of 0.1-10 microns oligodeoxynucleotides (one), which gives the strongest positive result, to create the possibility of installation of the device and the compensation cell sorting device with excitation fluorescence (FACS). In the following the original mixes were performed stimulation of 0.1-10 microns other one for comparison. The initial mixture were incubated for 48 hours at 37aboutWith CO2thermostat. Supernatant these initial mixtures were obtained by centrifugation and frozen for subsequent work at -80aboutC.

Secondary incubation with target cells (e.g., HT-29)

For the secondary incubation with the target cells have been previously to determine the optimal concentration and the volume in which they will do it. The aim was that after the secondary incubation was present, at least 5×105target cells per well for analysis. This drew attention to the fact that these cells had optimal conditions for growth for 3 days, so after 3 days they were almost confluent. Optimal conditions for growth also lead to necrosis or apoptosis, resulting would be getting false results. As target cells used cells for colon cancer HT-29.

Cells were sown in a pre determined the military optimal densities in the initial mixtures of the appropriate size, and incubated over night at 37 aboutWith CO2-thermostat (for example, 2,4×105cells in 700 µl per well in 24-hole tablet).

The next day was performing a stimulation selection environment of stuck between the cells and adding supernatant from the primary incubation ("indirect stimulation) or by adding specific substances (dSLIM-30L1, lin30L1) directly into the environment ("direct stimulation"). As a negative control to "indirect" source mixture was added only environment. These cells to distinguish from unstimulated cells (Appendix restimulating supernatant from the primary incubation) were designated as untreated cells.

The original mixture of direct and indirect stimulation stimulation - incubated for 48 hours again at 37aboutWith CO2thermostat. Then these cells can be desired in each individual case analyses. To do this, first supernatant cells were collected and cells were washed SFR. The cells were separated from the hole by using a mixture of trypsin/EDTA and after one additional stage of leaching was transferred into centrifuge tubes, and then to determine the number of cells.

Staining of surface antigens

Cells from the original mixes with the stimulation was centrifuged and washed in a special buffer for staining. Immediately after this cell suspension was brought to a close is Tracii 1×10 6cells per milliliter. 500 μl (0,5×106cells of this cell suspension was centrifuged in FACS tubes and after adding 50 μl of buffer for coloring added antibodies (e.g., ICAM-1 (CD54), conjugated with FITC, HLA-ABC, conjugated with phycoerythrin (PE)). For each antibody was injected corresponding control isotype, as well as separately painted a positive test for device installation and compensation. After the stage of incubation, these cells were twice washed SFR and resuspendable to measure in 500-1000 μl SFR. For discrimination of dead cells was added 7-AAD, and incubated for another 10 minutes. After this was performed FACS measurement.

Staining of apoptotic/necrotic cells

Apoptotic cells were stained with a mixture of annexin V-PE that detects apoptotic processes in the cell. To distinguish necrotic cells was painting a contrasting color using 7-AAD.

Cells from the original mixes with the stimulation was centrifuged and washed twice SFR. After that, the cells were diluted in special inexensive buffer and the cell concentration was brought to 1×106cells per milliliter. 100 μl (1×105cells) of this suspension was mixed with 5 μl of a mixture of annexin V-PE and 7-AAF and after thorough mixing for 15 minutes, incubated at room temperature. On the Le of this was added 400 μl of buffer for binding and immediately performed the measurement in FACS.

Flow-cytometrical measurement device FACS

A. Apoptosis/necrosis

Measured fluorescence 2 (annexin V-PE) and fluorescence 3 (7-AAD). The installation of the device was performed with restimulating cells ("direct" initial mixture) or untreated cells ("indirect" initial mixture). In the dot-band FSC (forward scattering=value cells) vs. SSC (scattering in the direction of=the granularity of cells) cell population is established in such a way that it is in the middle. After this was accomplished by the installation of PMT and compensation for fluorescence 2 and 3. Then measured all samples (5000 cells).

C. Surface antigens

Measured fluorescence 1 (ICAM-1-FITC), fluorescence 2 (HLA-ABC-PE) and fluorescence 3 (7-AAD). The installation of the device was performed with lin-30L1-stimulated cells with the appropriate controls on the isotype (double staining) to compensate for nonspecific binding with fluorescently labeled antibodies (at separate times).

In the dot-band FSC versus SSC cell population is established in such a way that it is in the middle. After this was accomplished by the installation PMT for fluorescence 1, 2 and 3 with the controls on the isotype, as well as compensation with individual colouring. After that, all samples were measured (10,000 cells). Thus exclude dead cells (7-AAD-positive cells (fluorescence 3 p is otiv FCS in Dot Blot).

Interpretation of results

A. Apoptosis/necrosis

Form a dot-blot 7-AAD against annexin V. Then set the quadrants using raw cells. Depending on their position in the quadrants in each case, these cells are referred to as apoptotic or necrotic fraction.

• Living cells are annexin-negative and 7-AAD-negative (LL-quadrant)

• Apoptotic cells are annexin-positive and 7-AAD-negative (LR-quadrant)

• Necrotic cells are annexin-positive and 7-AAD-positive (UR-quadrant)

or

annexin-negative and 7-AAD-positive (UL quadrant)

C. Surface markers

Form two dot-blot (fluorescence 1 against fluorescence 2 against FSC) and living cells. Depending on their position in dot-blots in each case, read the fluorescence intensity (fluorescence 1/ICAM-1 or 2/HLA-ABC) cells. After that make comparison with controls in each case.

• Comparison of test initial mixtures with controls in relation to

• the number of positive for the surface markers of cells=number of cells with appropriate surface markers)

• the fluorescence intensity of surface markers (=number of molecules of the surface is of Askerov on the cell surface).

The results of the described examples using methods according to this invention depicted in the figures.

The figures show:

Fig. 1 - schematic representation of the method of the present invention.

Fig. 2 - response analysis in vitro immunomodulator dSLIM through detection of apoptosis and necrosis in tumor cells HT-20.

Fig. 3 - response analysis in vitro immunomodulator dSLIM by detecting the expression of surface markers HLA-ABC in tumor cells HT-29.

Fig. 4 - response analysis in vitro immunomodulator dSLIM through detection of apoptosis and necrosis in the tumor cells of SOME 293.

Fig. 5 - response analysis in vitro immunomodulator dSLIM by detecting the expression of surface markers HLA-ABC in the tumor cells of SOME 293.

Fig. 6 - analysis of the mechanism of action of dSLIM through detection of apoptosis and necrosis in tumor cells HT-29 with the method of the present invention.

Fig. 7 - analysis of the mechanism of action of dSLIM by detecting the expression of surface markers HLA-ABC in tumor cells HT-29 with the method of the present invention.

Fig. 8 - comparison of the effectiveness of dSLIM with linear G-one by detecting the expression of surface markers HLA-ABC in Renca tumor cells.

Fig. 9 - comparison of the effectiveness of dSLIM with linear G on-one through the detection is of apoptosis and necrosis in Renca tumor cells.

Fig. 10 - comparison of the effectiveness of dSLIM with linear G-one by detecting the expression of surface markers HLA-ABC in tumor cells HT-29.

Fig. 11 - comparison of the effectiveness of dSLIM with linear G-one through detection of apoptosis and necrosis in tumor cells HT-29.

Fig. 12 - follow-up (monitoring) in vitro viable tumor cells during cancer therapy of the patient.

Fig. 13 - follow-up (monitoring) in vitro apoptotic/necrotic tumor cells during cancer therapy of the patient.

Fig. 14 - follow-up (monitoring) in vitro surface markers of tumor cells during cancer therapy of the patient.

Figure 1 shows a schematic representation of the sequence of stages of the method in accordance with this invention. On the left, in part, is the image of the sample application in vivo, to the right, in part, depicts the considered method of this invention, in the embodiment, in the form of "functional immunoassay in vitro".

Figure 2 shows the results of the analysis of action in vitro immunomodulator dSLIM when using the method of this invention. The use of supernatants dSLIM-incubated PBMC induces apoptosis and necrosis in tumor cells HT-29 (colon cancer), as can be seen in the right part of the figure. Here you can see the rise of proapoptotic from directly processed dSLIM cells to the treated supernatant of cells from 17% to 46.7%.

In figure 3, we analyzed the effect of in vitro immunomodulator dSLIM in cells HT-29. Use supernatant dSLIM-incubated PBMC causes enhanced expression of surface markers HLA-ABC. You can see the offset of the cell population in the rightmost part of the image.

To confirm the obtained in HT-29 results of the method when using the method of this invention, similar experiments were performed in cells of SOME 293.

Figure 4 shows that dSLIM causes apoptosis (annexin V) and necrosis (7-D). Thus, the number of apoptotic cells increases the supernatant processed dSLIM cells in comparison with the supernatant processed without one of the cells from 12.1% to 21.7%. The number of necrotic cells increased from 9.2% to 16%.

The figure 5 shows the increased influence of surface markers HLA-ABC incubation of target cells (RT-29) with dSLIM-PBMC supernatant. The upper part of the figure shows the displacement (=increased expression of cell populations from cells that were treated one, to cells that were incubated with the supernatant of the treated dSLIM PBMC.

Figure 6 shows the results of the analysis of the mechanism of action of dSLIM in cells HT-29 when using the method of this invention and detection of apoptosis and necrosis. At this stage the primary incubation, PBMC were added to the antibody (anti-IFN-γ, green frame), which is able in order to atalasoft action dSLIM. For comparison conducted methods with antibodies (anti-IFN-α, anti-TNF-α) to prove specificity. You can clearly see (green frame)that using an anti-IFN-γ antibodies minimizing the number of apoptotic and necrotic cells.

In figure 7 the method of this invention corresponds to the use of figure 6, but in the target cells (RT-29) analyzed the expression of surface marker ICAM-1 (CD54). However, these linear CpG-containing oligodeoxynucleotide also have another sequence than dSLIM, and protected against destruction by phosphorothioate.

Figure 8 shows that treatment of target cells dSLIM leads to enhanced expression of surface marker HLA-ABC (upper part), while linear CpG-one has no effect. The table on the right part of the figure maps the numerous differences. Upon the induction of apoptosis and necrosis, figure 9, dSLIM is clearly more efficient than linear CpG-one. In the lower part you can see the difference in induction of apoptosis in percent.

Figures 10 and 11 compare in each case dSLIM with linear CpG-one using the method of this invention in cells HT-29 as target cells. The results of this experiment are consistent with the results that were obtained with Renca tumor cells and are shown on figures 8 and 9. The structure of these Figo is also consistent with figures 8 and 9.

Figures 12, 13 and 14 show the use of the method of this invention for subsequent monitoring) in vitro the number of viable tumor cells (Fig. 12) and apoptotic/necrotic cells (Fig. 13), and changes in the expression of surface markers ICAM-1/HLA-ABC (Fig. 14) during the treatment of the cancer patient.

A patient with colon cancer and liver metastasis were subjected in the first five days of the first week of therapy every time 2.5 mg dSLIM. On the sixth day of the first week served radiation followed by chemotherapy.

For analysis of in vitro in vivo effects in a patient every time in the first six days of the first week took the blood. During chemotherapy at the end of each week also took the blood.

From blood samples was isolated plasma and incubated with the cell line tumor cells HT-29. After it was determined the number of viable cells (Fig. 12) and apoptotic/necrotic cells, and examined the expression of surface markers ICAM-1/HLA-ABC.

Figure 12 shows the results of incubation of cells HT-29 with plasma from eight blood samples. You can see a clear decrease of viable cells on the second day of the introduction of dSLIM. The number of viable cells is reduced on the second day to less than half of the cells of the first day, which is comparable with the number of viable cells of controls.

In the figure 13 and abrajano follow-up (monitoring) in vitro apoptotic/necrotic tumor cells during therapy, cancer patients on days 1, 2, 5 and 20. When this assessment monitoring in vivo effects found that already in the first days after receipt of dSLIM the number of apoptotic/necrotic cells clearly increased.

Figure 14 presents the results of studies on the changes in the expression of surface markers ICAM-1/HLA-ABC during therapy with cancer patients using blood plasma samples 1, 2, 3 and 8. When the sample 1 is taken as a reference index to represent changes in the expression of both surface markers.

ICAM-1 is already on the second day is expressed clearly more strongly, as can be seen in the lower part of the figure to move the position of the fluorescence intensity, which indicates that ICAM-1 is expressed stronger.

In the case of HLA-ABC on the second day there was no displacement of the fluorescence intensity. It is found only on the third day of therapy and also shows stronger expression of HLA-ABC.

The list of used symbols

A=the situation in vivo

In=immunoassay in vitro

1=patient

2=the target tissue, such as tumor

3=immune cells

4=test substance, for example, dSLIM

5=activated immune cells

6=donor

7=immune cells, for example, PBMC

8=test substance, for example, dSLIM

9=activated immune cells, for example, PBMC

10=supernatant

11=the target cells, e.g. tumor cells

12=analysis

1. The way in vitro studies of the effects of substances in processes in vivo, which in the case of the investigated substances we are talking about immunomodulatory, as well as inducing apoptosis or necrosis connections and which provides the following sequence of stages of the method:
a) isolation of effector cells of the immune system that are peripheral mononuclear cells from blood, spleen cells or subjected to FACS or MACS-sorting subpopulations mixtures of cells, such as B-, T - or NK-cells, monocytes or dendritic cells,
b) primary incubation of effector cells with the test substance,
c) obtaining a supernatant or a mixture of cells and supernatant primary incubation,
d) secondary incubation of target cells with supernatant or a mixture of the cells and the supernatant of the primary incubation, which in the case of target cells for the secondary incubation talking about tumor human cells or cell lines of oncogenetic origin
e) analysis of target cells, where the analysis is selected from the group including analysis of the expression of specific proteins and analysis of apoptosis and/or necrosis.

2. The method according to claim 1, in which in the case of target cells secondary incubation it comes to human cells or cells of higher mammals.

3. The method according to claim 1, in which the hold stage, the way the 1d) and 1E) with blood serum or plasma of the patient.

4. A method for detecting in vitro, which are suitable for identification of immunomodulatory compounds and/or detection steps immunomodulatory compounds, as well as identify the cause apoptosis and/or necrosis compounds by the immune system processes in vivo, which provides:
a) primary incubation of the effector cells of the immune system that are peripheral mononuclear cells from blood, spleen cells or subjected to FACS or MACS-sorting subpopulations mixtures of cells, such as B-, T - or NK - cells, monocytes or dendritic cells, analyzed by immuno-modulating action or inducing apoptosis or necrosis substance, followed
b) obtaining a supernatant or a mixture of cells and supernatant primary incubation and subsequent
c) a secondary incubation of target cells with supernatant or a mixture of the cells and the supernatant of the primary incubation, which in the case of target cells for the secondary incubation talking about tumor human cells or cell lines of oncogenetic origin and then
d) analysis of immunomodulating and/or inducing apoptosis and/or necrosis of the action using an appropriate method of detection, where the said method of detection selected from the group comprising analsexpics specific proteins and analysis of apoptosis and/or necrosis.

5. The method according to claim 4, in which the cells for the primary incubation, pre-allocate stage method 1A).

6. The method according to claim 4, in which in the case of cells for the primary incubation and target cells for the secondary incubation it comes to human cells or cells of higher mammals.

7. The method according to claim 4, in which the hold stage method 4) and 4d) with blood, serum or plasma of the patient.

8. The method according to any one of claims 1 or 4, in which in the case of immunomodulatory compounds, which are investigated, we are talking about CpG-containing oligodeoxynucleotide or partially double-stranded DNA structures, at least one CpG-base single-stranded region.

9. The method according to any one of claims 1 or 4, in which in the case of inducing apoptosis and/or necrosis of the compounds, the action of which is investigated, it is preferably about the antisense oligodeoxynucleotide, short interfering RNA (siRNA), the antibodies or chemotherapeutic substances.

10. The method according to any one of claims 1 or 4, in which the analyte in the primary incubation of whole blood, blood cells, subpopulations of blood cells, blood serum or blood plasma before, during and/or after treatment.

11. The method according to any one of claims 1 or 4, in which incubation takes place in thermostat.

12. The method according to any one of claims 1 or 4, in which super is of Tanta get by centrifugation.

13. The method according to any one of claims 1 or 4, in which the target cells for the analysis of paint, in particular by annexin V, or spend iodide staining of propecia.

14. The method according to any one of claims 1 or 4, in which the conduct analyses of the cell cycle.

15. The method according to any one of claims 1 or 4, in which when the primary incubation with the test substance add antibodies or other competing agents.

16. A kit for carrying out the detection method in vitro, which are suitable for identification of immunomodulatory compounds and/or detection steps immunomodulatory compounds, as well as the identification of inducing apoptosis and/or necrosis compounds by the immune system in vivo processes that have at least the following components:
- prepared for storage of aliquots of the effector cells of the immune system that are peripheral mononuclear cells from blood, spleen cells or subjected to FACS or MACS-sorting subpopulations mixtures of cells, such as B-, T - or NK-cells, monocytes or dendritic cells,
- means for conducting primary and secondary incubation,
- advance tablets with the number of holes another 24-96, in which the surfaces of these holes are coated with an antibody for detection of messengers that are released in the incubation of cells incubation with primary research is subject to connection, and/or
means for FACS analysis, containing the appropriate fluorescently labeled antibodies, which are directed to the surface antigens, to study changes in the expression of surface molecules caused due to an investigational compound immune response and advanced tools for processing of target cells, such as buffers and chemicals.

17. Set for the image in vitro action of immunomodulatory compounds, as well as inducing apoptosis and/or necrosis connections with the immune system in vivo processes before, during and/or after administration of such compounds having at least the following components:
- prepared for storage of aliquots of target cells for incubation with blood, serum or plasma, as well as
- funds for secondary incubation, and
- advance tablets with the number of holes another 24-96, in which the surfaces of these holes are coated with an antibody for detection of messengers that are released in the incubation of the cells of the primary incubation with the test compound and/or
means for FACS analysis, containing the appropriate fluorescently labeled antibodies, which are directed to the surface antigens to study changes in the expression of surface molecules due to induced investigational compound immune response and more the positive means for processing the target cells, such as buffers and chemicals.

18. Set on 17, in which case the contained target cells we are talking about the tumor cells or cell lines of oncogenetic origin.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, namely to orthopedics. In order to estimate state of bone tissue in case of immobilisation osteoporosis in a laboratory animal examined are homogenates: bone, muscular, bone marrow of any extremity and peripheral blood. Biochemical and integral parametres are determined. Five factor variables F1-F5 are calculated using values of biochemical and integral parametres, constant values of factor coefficients of biochemical parametres and free coefficients. After that calculated is the value of discriminant function, whose value is used to estimate bone state as normal or conclusion about presence of immobilisation osteoporosis is made.

EFFECT: method increases accuracy and efficiency, has high stability of immobilisation osteoporosis recognition.

1 ex, 3 tbl

FIELD: medicine.

SUBSTANCE: blood plasma is analysed for cytochrome oxidase activity, erythrocyte 2,3- biphosphoglycerate and lactic acid concentrations. It is followed by calculating an oxygenation coefficient K by formula: K=(C1+C2):A, where C1 is the erythrocyte 2,3- biphosphoglycerate concentration, mol/l; C2 is the erythrocyte lactic acid concentration, mol/l; A is plasma cytochrome oxidase activity, mol/l. The K value withint 1.0 <K≤3.0 enables to diagnose tissue hypoxia, while in case of the K values being 3.0<K≤5.0 cardiovascular hypoxia is diagnosed. If the oxygenation coefficient is 5.0 and more, blood hypoxia is diagnosed.

EFFECT: use of the method enables the differential diagnostics of blood, tissue and cardiovascular hypoxia.

3 ex

FIELD: medicine.

SUBSTANCE: method for prediction of duration of a recurrence-free interval in patients with rectal cancer consisting in the fact that the surgical removal of a peritumoural recurrence is followed with biochemical analysis of the concentration of polypeptide hormone prolactin, and its specified tissue value enables to determine duration of the recurrence-free interval.

EFFECT: method allows predicting duration of the recurrence-free interval following the removal of the recurrence.

1 tbl

FIELD: medicine.

SUBSTANCE: for an assay, 5-7 cm3 of blood is taken, and before extraction the sample is pre-treated with 2 cm3 of 60% sulphuric acid; the extraction process is executed with 30 cm3 of n-hexane once, and gas chromatography is preceded with single treatment of n-hexane extract with 10 cm3 of concentrated sulphuric acid.

EFFECT: invention provides higher reliability of α-HCCH, γ-HCCH test results and twofold reduced time of sample preparation.

1 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: method includes introduction of sample into sample hole of the device, the device being adjusted for delivering said sample into lateral flow matrix, which has multitude of varieties of IgE antigens, immobilised in respective positions in result window. Method additionally includes possibility of sample movement along lateral flow matrix through immobilised multitude of IgE antigen varieties into the second position lower on flow from the first position, introduction of liquid buffer into lateral flow matrix for mobilisation of labeled reagent, which is adapted for binding aHTH-IgE-antibody and is dried in lateral flow matrix in position higher on flow from introduction of filtered sample into lateral flow matrix, and possibility of movement of labeled reagent, mobilised by liquid buffer, along lateral flow matrix through multitude of immobilised varieties of antigens IgE into position lower on flow from the first position.

EFFECT: simplification of process.

46 cl, 1 ex, 1 tbl, 8 dwg

FIELD: medicine.

SUBSTANCE: in patient's saliva concentration of alfa-interferon is determined. If alfa-interferon concentration is from 78.62 to 120.58 pg/ml, compensated form of carious process course is predicted. If alfa-interferon concentration is from 22.659 to 41.821 pg/ml, decompensated form of carious process course is predicted.

EFFECT: application of method allows to predict carious process course in children reliably.

2 ex

FIELD: medicine.

SUBSTANCE: in patient's saliva concentration of alfa-interferon is determined. If concentration of alfa-interferon from 22.659 to 41.821 pg/ml is determined, increase of caries intensity for not less than two teeth after 6 months is predicted.

EFFECT: application of method allows to predict carious process course in children reliably.

2 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, in particular, to vascular surgery. Phased surgical treatment of frontal arteries in patients with symptomatic bicarotid stenoses, including at the first stage reconstruction of one internal frontal artery, at the second stage - another internal frontal artery, is carried out. In order to predict character of reperfusion syndrome course, carried out are clinical-instrumental examination and determination in blood serum of level of primary, secondary and final isopropanol-soluble (ISPP, ISSP, ISFP), heptane-soluble (GSPP, GSSP, GSFP) products of lipid peroxidation and ascorbate-induced lipid peroxidation (AOA-1, AOA-2) as well as level of biologically active substances - serotonin, nitrites, nitrates in time periods before the second stage of aversion carotid endarterectomy, as well as on 1-3, 4-7 and 8-14 day after its performance. Using values of ISPP, ISSP, ISFP, GSPP, GSSP, GSFP, AOA-1, AOA-2, serotonin, nitrites and nitrates levels with respect to norm in said periods of time, favourable or unfavourable reperfusion syndrome course is predicted.

EFFECT: method increases reliability of reperfusion syndrome prediction after phased surgical treatment of frontal arteries in patients with symptomatic bicarotid stenoses.

4 ex

FIELD: medicine.

SUBSTANCE: invention concerns a method of antibody-dependent cellular cytotoxicity (ADCC) assay.

EFFECT: simplicity and safety as compared with the analysis, higher sensitivity and real-time realisability of the method.

42 cl, 1 ex, 10 dwg

FIELD: medicine.

SUBSTANCE: invention relates to the field of medicine. To forecast resistance of membranes of erythrocytes of peripheral blood in case of exacerbation of herpes-virus infection in expecting mothers, content of lysophosphatidyl choline is determined on silica gel with the help of 2D thin-layer chromatography in phospholipid composition of membranes. Simultaneously, in the same portion of blood in membranes of erythrocytes by method of disk electrophoresis, content of protein of band 4.1 is identified in staged gradient polyacrylamide 10% gel with dodecyl sodium sulfate. Integral assessment of interconnection between examined parametres in healthy expecting mothers and expecting mothers who suffered exacerbation of herpes-virus infection is carried out through determination of discriminant function (D) with the help of discriminant equation according to the formula: D=-6.790 x band 4.1 + {+0.609×Lph}. If D value equals to or is less than -19.01, non-resistance of erythrocytes membranes is forecasted.

EFFECT: method makes it possible to forecast resistance of membranes of erythrocytes of peripheral blood in case of exacerbation of herpes-virus infection of expecting mothers.

1 tbl

FIELD: medicine, hepatology.

SUBSTANCE: one should detect the level of hepato-specific enzymes (HSE) in blood plasma, such as: urokinase (UK), histidase (HIS), fructose-1-phosphataldolase (F-1-P), serine dehydratase (L-SD), threonine dehydratase (L-TD) and products of lipid peroxidation (LP), such as: dienic conjugates (DC), malonic dialdehyde (MDA). Moreover, one should detect the state of inspecific immunity parameters, such as: immunoregulatory index (IRI) as the ratio of T-helpers and T-suppressors, circulating immune complexes (CIC). Additionally, one should evaluate the state of regional circulation by applying rheohepatography (RHG), the system of microhemocirculation with the help of conjunctival biomicroscopy (CB) to detect intravascular index (II). In case of increased UK, HIS levels up to 0.5 mcM/ml/h, F-1-P, L-SD, L-Td, LP products, CIC by 1.5 times, higher IRI up to 2 at the norm being 1.0-1.5, altered values of regional circulation, increased II up to 2 points at the norm being 1 point, not more one should diagnose light degree of process flow. At increased level of UK, HIS up to 0.75 mcM/ml/h, F-1-P, L-SD, L-TD, LP products, CIC by 1.5-2 times, increased IRI up to 2.5, altered values of regional circulation, increased II up to 3-4 points one should diagnose average degree of process flow. At increased level of UK, HIS being above 0.75 mcM/ml/h, F-1-P, L-SD, L-TD, LP products, CIC by 2 and more times, increased IRI being above 2.5, altered values of regional circulation, increased II up to 5 points and more one should diagnose severe degree of process flow.

EFFECT: higher accuracy of diagnostics.

3 ex

FIELD: medicine, infectology, hepatology.

SUBSTANCE: in hepatic bioptate one should detect products of lipid peroxidation (LP), such as: dienic conjugates (DC), activity of antioxidant enzymes, such as: catalase (CAT)and superoxide dismutase (SOD). One should calculate by the following formula: C = DC/(SOD x CAT)x100, where DC - the content of dienic conjugates, SOD - activity of superoxide dismutase, CAT - activity of catalase. At coefficient (C) values being above 65 one should predict high possibility for appearance of cirrhosis, at 46-645 - moderate possibility and at 14-45 -low possibility for appearance of cirrhosis.

EFFECT: higher accuracy of prediction.

3 ex

FIELD: medicine, clinical toxicology.

SUBSTANCE: at patient's hospitalization one should gather the data of clinical and laboratory values: on the type of chemical substance, patient's age, data of clinical survey and laboratory values: body temperature, the presence or absence of dysphonia, oliguria being below 30 ml/h, hemoglobinuria, erythrocytic hemolysis, exotoxic shock, glucose level in blood, fibrinogen and creatinine concentration in blood serum, general bilirubin, prothrombin index (PTI), Ph-plasma, the state of blood clotting system. The state of every sign should be evaluated in points to be then summed up and at exceeding the sum of points being above "+20" one should predict unfavorable result. At the sum of "-13" prediction should be stated upon as favorable and at "-13" up to "+20" - prediction is considered to be doubtful.

EFFECT: higher accuracy of prediction.

2 ex, 3 tbl

FIELD: medicine, juvenile clinical nephrology.

SUBSTANCE: disease duration in case of obstructive pyelonephritis should be detected by two ways: either by detecting the value of NADPH-diaphorase activity, as the marker of nitroxide synthase activity in different renal department and comparing it to established norm, or by detecting clinico-laboratory values, such as: hemoglobin, leukocytes, eosinophils, urea, beta-lipoproteides, lymphocytes, neutrophils, the level of glomerular filtration, that of canalicular reabsorption, urinary specific weight, daily excretion of oxalates, arterial pressure, and estimating their deviation against average statistical values by taking into account a child's age.

EFFECT: higher efficiency of detection.

7 dwg, 1 ex, 6 tbl

FIELD: clinical medicine, pulmonology.

SUBSTANCE: one should carry out complex estimation of interleukin-1β) concentration in blood, saliva, bronchoalveolar liquid. Moreover, one should detect distribution coefficient (DC) for IL-1β as the ratio of IL-1β blood content to IL-1β salivary content. At increased IL-1β blood content by 10 times and more, by 2 times in saliva, unchanged level of bronchoalveolar IL-1β, at DC for IL-1β being above 1.0 one should predict bronchial obstruction. The method enables to conduct diagnostics of the above-mentioned disease at its earlier stages.

EFFECT: higher efficiency of prediction.

2 tbl

FIELD: medicine, diagnostics.

SUBSTANCE: the present innovation deals with genetic trials, with diagnostic field of oncological diseases due to analyzing DNA by altered status of gene methylation that take part in intracellular regulation of division, differentiating, apoptosis and detoxication processes. One should measure the status of methylation in three genes: p16, E-cadherine and GSTP1 in any human biological samples taken out of blood plasma, urine, lymph nodes, tumor tissue, inter-tissue liquid, ascitic liquid, blood cells and buccal epithelium and other; one should analyze DNA in which modified genes of tumor origin or their components are present that contain defective genes, moreover, analysis should be performed due to extracting and purifying DNA out of biological samples followed by bisulfite treatment of this DNA for modifying unprotected cytosine foundations at keeping 5-methyl cytosine being a protected cytosine foundation followed by PCR assay of bisulfite-treated and bisulfite-untreated genes under investigation and at detecting alterations obtained according to electrophoretic result of PCR amplificates, due to detecting the difference in the number and electrophoretic mobility of corresponding fractions at comparing with control methylated and unmethylated samples containing normal and hypermethylated forms of genes one should diagnose oncological diseases. The method provides higher reliability in detecting tumors, detection of remained tumor cells after operation.

EFFECT: higher efficiency of therapy.

1 cl, 3 dwg, 4 ex

FIELD: medicine, gastroenterology.

SUBSTANCE: one should carry out diagnostic studying, moreover, on the 5th -6th d against the onset of exacerbation in case of gastric and duodenal ulcerous disease one should detect the content serotonin, histamine and acetylcholine in blood, then during 2-3 wk one should conduct medicinal therapy to detect serotonin, histamine and acetylcholine level in blood again and at serotonin content being by 2-3 times above the norm, histamine - by 1.15-1.4 times above the norm and acetylcholine - by 20-45% being below the norm one should predict the flow of gastric and duodenal ulcerous disease as a non-scarring ulcer.

EFFECT: higher accuracy of prediction.

3 ex

FIELD: medicine.

SUBSTANCE: method involves taking blood from ulnar vein (systemic blood circulation) and from large vein of the injured extremity proximal with respect to lesion focus (regional blood circulation). Spontaneous NST-test value is determined and difference is calculated in systemic and regional blood circulation as regional-to-systemic difference. The difference value is used for predicting clinical course of pyo-inflammatory disease in extremities.

EFFECT: high accuracy of diagnosis.

4 cl, 2 tbl

FIELD: medicine, gastroenterology.

SUBSTANCE: one should introduce biologically active substance, moreover, in patient's blood serum one should detect the content of acetyl choline and choline esterase activity followed by 2-h-long intragastric pH-metry at loading with biologically active substance as warm 40-45%-honey water solution at 35-40 C, and at increased content of acetyl choline being above 1.0 mM/l, choline esterase being above 0.5 mM/l/30 min and pH level being 6.0-6.9 it is possible to consider apitherapy to be useful for treating ulcerous duodenal disease.

EFFECT: higher efficiency and accuracy of detection.

3 ex

FIELD: medicine, gastroenterology.

SUBSTANCE: it has been suggested a new method to detect pharmacological sensitivity to preparations as acidosuppressors. After the intake of the preparation a patient should undergo fibrogastroduodenoscopy 3 h later, then, through endoscopic catheter one should introduce 0.3%-Congo red solution intragastrically and the test is considered to be positive at keeping red color that indicates good sensitivity to the given preparation, and in case of dark-blue or black color the test is considered to be negative that indicates resistance to this preparation. The suggested innovation widens the number of diagnostic techniques of mentioned indication.

EFFECT: higher efficiency of diagnostics.

2 ex

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