The method of obtaining undifferentiated cells

 

The invention relates to medicine and relates to a method of obtaining undifferentiated cells. The invention includes a direct or indirect participation of more than commitirovannah cells than undifferentiated cell with an agent that causes retrodifferentiation more commitirovannah cells in the undifferentiated. The advantage of the invention is to develop a method of obtaining undifferentiated cells from more commiteeman, 3 S. and 34 C.p. f-crystals, 20 tab., 6 Il.

The present invention relates to a method for undifferentiated cells.

In particular, the present invention relates to a method for producing differentiated cells from more commitirovannah cells.

In addition, the present invention relates to the use of undifferentiated cells according to the invention to obtain a new, more commitirovannah cells, i.e. recommisioning cells.

The present invention also relates to the use of undifferentiated cells according to the invention or recognitional cells according to the invention, which would be influenced (directly or indirectly through the use of received from her illness, or a partial or complete cure from immunological condition or disease.

As an introduction, differentiation is a process in which structure and function of cells progressively switched into more specialized cells; such processes include the production of T-cells or b-cells. Therefore, as cells become more commitirovannah, they become more specialized.

On the contrary, retrodifferentiation is a process in which structure and function of cells progressive change towards less specialized cells.

Undifferentiated cells are able to differentiate into many directions, i.e. they are able to differentiate into two or more specialized cell types. A typical example of undifferentiated cells is a stem cell.

In contrast, the differentiated cells are unable to differentiate into many directions. A typical example of a differentiated cell is a T-cell.

There are many undifferentiated cells and differentiated cells found in vivo, and General science rich General ideas about them.

In ka is nice 45-59) and Roitt et al. (Immunology, 4th Edition, Eds. Roitt, Brostoff and Male, 1996, Publ. Mosby, Chapter 10).

Briefly, however, examples of differentiated cells include lymphohematopoietic precursor cells (LPCs). LPC include pluripotent stem cells (PSC), lymphoid stem cells (LSC) and myeloid stem cells (MSC). LSC and MSC are formed as a result of differentiation of PSC. Therefore, LSC and MSC are more commitirovannah than the PSC.

Examples of differentiated cells include T cells, b cells, eosinophils, basophils, neutrophils, megakaryocytes, monocytes, erythrocytes, granulocytes, mast cells and lymphocytes.

T-cells and b-cells are formed as a result of differentiation LSC. Therefore, T-cells and b-cells are more commitirovannah than LSC.

Eosinophils, basophils, neutrophils, megakaryocytes, monocytes, erythrocytes, granulocytes, mast cells, natural killer cells (NK) and lymphocytes are formed in the differentiation of MSC. Therefore, each of these cells are more commitirovannah than MSC.

Antigens associated with undifferentiated and differentiated cells. The term "associated" means that we are talking about cells expressing or capable of Express or ol the antigen(s).

The most undifferentiated cells and differentiated cells include antigens class I and/or antigens class II major histocompatibility complex (MHC). If these antigens are associated with these cells, they are called the cells of Class+and/or cells Class II+.

Each specific antigen associated with undifferentiated cell or differentiated cell may act as a marker. Therefore, different types of cells can be distinguished from each other based on the associated specific antigen (antigens) or based on a particular combination associated antigens.

Examples of these marker antigens include antigens CD34, CD19 and CD3. If the cell these cells are called antigens, respectively CD34+, CD19+and CD3+. If these antigens are absent, these cells are called CD34-, CD19-and CD3-.

In more detail, the PSC represent cells CD34+. LSC represent entrances DR+, CD34+and Td+. MSC represent cells CD34+DR+, CD13+, CD33+, CD7+and TdT+. B-cells are cells CDl9+, CD21+, CD22+and mposite represent CD4+and CD8+. Activated T-cells are cells DR+. Natural killer cells (NK) are cells CD56+and CDl6+. T-lymphocytes are cells CD7+. Leukocytes are cells of CD45+. Granulocytes represent cells CD13+and CD33+. Monocytes-macrophages are cells CD14+and DR+.

Therefore the above antigenic markers can identify certain types of cells (for example, whether the cell is an undifferentiated cell or differentiated cell) and the specialization of this type of cells (for example, whether the cell T-cell or b-cell).

The General concept of retrodifferentiation not new. Indeed, in 1976, Jose Uriel (Cancer Research, 36, 4269-4275. November 1976) presented an overview on this subject, in which he wrote: "retrodifferentiation seems to be a normal adaptive process to maintain cellular integrity in the fight against the agents of various etiologies (physical, chemical, and viral). Keeping all the information encoded in their genome, cells exposed to retrodifferentiation lose morphological and functional diversity is. which then leads to progressive development of uniformely originally different cellular phenotypes and reduced responsiveness to regulatory signals that act on adult cells. Retrodifferentiation normal balanced oppositely directed process reintegrate, which tends to restore the final phenotype, when it comes to reversion. This explains why retrodifferentiation is invariably associated with cell regeneration and tissue repair." Uriel (ibid) then continued the discussion of the published cases of retrodifferentiation such as work Gurdon related to the nuclei of the cells of the intestinal epithelium of Xenopus tadpoles (Advances in Morphogenesis, 1966, vol. 4, PP. 1-43. New York Academic Press, Eds Abercombie and Bracher), and work Bresnick related to liver regeneration (Methods in Cancer Research, 1971, vol. 6, PP. 347-391).

Uriel (ibid) also reported on the work related to isolated parenchymal liver cells for cultures in vitro. By Uriel: "In contrast to the results obtained with hepatocytes of fetus or newborn, as well as when using hepatocytes from regenerating liver or installed Hepatol, it was difficult to get a permanent line of adult hepatocytes alone."

Uriel take show similar changes reversion to immaturity... during the precancerous stage of carcinogenesis of the liver, is also retrodifferentiation cells."

More recent data on retrodifferentiation include Minoru Fukunda (Cancer Research, 1981, vol. 41, PP. 4621-4628). Fukunda caused specific changes in the profile of cell surface glycoproteins cell line human leukemia K using contributing to the development of tumors forblog ester 12-O-tetradecanoylphorbol-13-acetate (TPA). On Fukunda, TPA, apparently, causes the stage of retrodifferentiation of human leukemia cell C.

Along with this, Hass et al. (Cell Growth & Differentiation, 1991, vol. 2, pp. 541-548) reported long-term culture of differentiated under the action of TPA cell leukemia U-937 in the absence forblog ether within 32-36 days, which resulted in the process retrodifferentiation, and that retrodifferentiation cells were separated from the substrate and re-started proliferation.

Another case of retrodifferentiation contained in the work of Curtin and Snell (Br. J. Cancer, 1983, vol. 48, pp. 495-505). These researchers compared the enzymatic changes during induced diethylnitrosamine of hepatocarcinogenesis and regeneration of the liver after partial hepatectomy, with normal liver differ and similar stepwise elimination of differentiation. According to these researchers obtained their results suggest that the underlying process retrodifferentiation is common for process hepatocarcinogenesis and regeneration of the liver.

Later Chastre et al. (FEBS Letters, 1985, vol. 188, number 2, pp. 2810-2811) reported retrodifferentiation of subclone human colon NT-18.

Still later, Kobayashi et al. (Leukaemia Research, 1994, vol. 18, no. 12, pp. 929-933) reported on the establishment of retrodifferentiation cell line (RD-1) of single cells rat myelomonocytic leukemia, which was differentiated in macrophagecolony cell under the action of lipopolysaccharide (LPS).

In accordance with the modern view is confirmed by the provisions set forth on page 911 of the book Molecular Biology of the Cell (pub. Garland Publishers Inc. , 1983), and later by Levitt and Mertelsman (ibid), a stem cell, such as the PSC has the following four characteristics: i) it is an undifferentiated cell, i.e. it is not completely differentiated; (ii) it is able to share without restriction; (iii) it is able to give the development of differentiated progeny, such as differentiated cells, referred to earlier, and iv) when it divides, each daughter cell has a choice: she Moseley differentiation.

It should be emphasized last property, namely, that in accordance with the General provisions in this area of expertise, as soon as the undifferentiated cell is differentiated into a more commiteeman the cage, then she can't retrodifferentiation. This view was even supported by the provisions of Uriel (ibid), Fukunda (ibid), Hass et al. (ibid), Curtin and Snell (ibid), Chastre et al. (ibid) and bayashi et al. (ibid); these researchers caused retrodifferentiation certain types of differentiated cells, but these cells remained commitirovannah to the same line, and they didn't retrodifferentiation in undifferentiated cells.

Therefore, in accordance with scientific understanding to the present invention it was believed that it is impossible to obtain undifferentiated cells, such as stem cells, more commiteeman cells. However, the present invention shows that this view is inaccurate and that it is possible to obtain undifferentiated cells from more commiteeman cells.

Thus, in accordance with the first aspect of the present invention provides a method of obtaining undifferentiated cells, and the method includes contacting more ferentiating the cell.

In accordance with the second aspect of the present invention provides a method that includes contacting more commitirovannah cells with an agent that causes retrodifferentiation more commitirovannah cells in an undifferentiated cell, and then commitirovannah undifferentiated cells in recommisioning the cell.

The term "recognitiona cell" means a cell derived from undifferentiated cells, i.e., a new, more commiteeman the cell.

In accordance with a third aspect of the present invention provides for obtaining undifferentiated cells in accordance with the method of the present invention.

In accordance with the fourth aspect of the present invention provides for undifferentiated cells in accordance with the method of the present invention as - or getting - drugs.

In accordance with the fifth aspect of the present invention assumes the use of undifferentiated cells derived in accordance with the method of the present invention, in the manufacture of a medicinal product for the treatment of immunological disorders or diseases.

In accordance with SASO method of the present invention.

In accordance with the seventh aspect of the present invention provides for recommisioning cells in accordance with the method of the present invention as - or getting - drugs.

In accordance with the eighth aspect of the present invention seeks recommisioning cells obtained in accordance with the method of the present invention, in the manufacture of a medicinal product for the treatment of immunological disorders or diseases.

In accordance with the ninth aspect of the present invention provides a more commitirovannah cell having attached thereto an agent that can cause retrodifferentiation more commitirovannah cells in the undifferentiated cell.

In accordance with the tenth aspect of the present invention provides cell CD19+and CD3+.

Thus, in its broadest sense, the present invention is based on the extremely unexpected discovery that it is possible to obtain undifferentiated cell from more commitirovannah cells.

The present invention is of great importance, because now it becomes possible to obtain undifferentiated cells from the more to karstenii funds - either in vitro or in vivo, or their combinations for the treatment of disorders.

The present invention also has the advantage that you can cometravi undifferentiated cell, obtained by retrodifferentiation in recommisioning cell, such as a new differentiated cells, with the purpose of correcting or deleting the original, more commitirovannah, cells or to correct or delete it.

Preferably, if more than commitirovannah cell capable of retrodifferentiation in undifferentiated cell of the I+Class and/or II+Class MNF.

Preferably, when more than commitirovannah cell capable of retrodifferentiation in undifferentiated cell containing the antigen stem cells.

Preferably, if more than commitirovannah cell capable of retrodifferentiation in undifferentiated cell CD34+.

Preferably, if more than commitirovannah cell capable of retrodifferentiation in lymphohematopoietic cell-predecessor.

Preferably, if more than commitirovannah cell capable of retrodifferentiation in pluripotent stem cells.

Undifferentiated cell can contain any kind to the when the undifferentiated cell is a cell+Class and/or II+Class MNF.

Preferably, when the undifferentiated cell contains the stem cell antigen.

Preferably, if the undifferentiated cell is an undifferentiated cell CD34+.

Preferably, if the undifferentiated cell is lymphohematopoietic cell-predecessor.

Preferably, if the undifferentiated cell is a pluripotent stem cell.

More commitirovannah cell can contain any components that are relevant to the presentation of antigen to his capture or recognition. Preferably, the undifferentiated cell was a cell of the I+Class and/or II+Class MNF.

Preferably, when the agent acts extracellular towards more commitirovannah the cell.

Preferably, when more than commitirovannah cell contains the receptor, which may be operatively associated with the agent, and in which the agent promptly binds to a receptor.

It is preferable that the receptor was a receptor on the cell surface.

Preferably, the receptor containedcomponent and/or

Preferably, the receptor contains at least homologous sites-chain of HLA-DR.

Preferably, the receptor contained-chain having homologous sites.

Preferably, the receptor contains at least homologous sites-chain of HLA-DR.

Preferably, when the agent is an antibody to the receptor.

Preferably, the agent was a monoclonal antibody to the receptor.

Preferably, when the agent is an antibody, preferably a monoclonal antibody to the homologous sites-chain of HLA-DR.

Preferably, the agent was an antibody, preferably a monoclonal antibody to the homologous sites-chain of HLA-DR.

Preferably, when the agent is used in combination with biological response modifier.

Preferably, biological response modifier was an alkylating agent.

Preferably, if the alkylating agent represents or includes cyclophosphamide.

In one preferred aspect more commiteeman the years were any of the b-cells or T-cells.

In an alternative preferred aspect more commitirovannah the cell represents a more Mature undifferentiated cell.

In one preferred aspect, when the undifferentiated cell commiteeman in recommisioning cell, recognitiona cell belongs to the same line, the more commitirovannah cell before retrodifferentiation.

In another preferred aspect, when the undifferentiated cell commiteeman in recommisioning cell, recognitiona cell belongs to a different line than commitirovannah cell before retrodifferentiation.

Preferably, recognitiona cell was any of b cells, T cells or granulocytes.

Preferably, when the method is a method in vitro.

Preferably, when the agent modulates the expression of MHC, preferably, if the agent modulates the expression of MHC Class I+and/or MHC Class II+.

Agent operatively engages more commiteeman cage for retrodifferentiation of this cell in an undifferentiated cell. In this respect, the agent for retrodifferentiation more commitirovannah cells in undifferentiated klepalova contact is when more commitirovannah cell has at least one receptor on their surface, such asthe chain, which has homologous sites (sites that usually have the same or similar sequence), those that can be found on b cells and in which the agent comes into direct contact with the receptor on the cell surface. Another example is when more commitirovannah cell has a receptor on their surface, such asthe chain, which has areas homologous to those found on T-cells, and on which the agent comes into direct contact with the receptor on the cell surface.

An example of indirect contact is when more commitirovannah cell has at least two receptor on its cell surface, and the contact of the agent with one of the receptors affects another receptor that causes retrodifferentiation more commitirovannah cells.

Agent for retrodifferentiation more commitirovannah cells in the undifferentiated cell may be a chemical compound or composition. Preferably, however, when the agent is able to come into contact with a receptor on poverhnost from among cyclic adenosine monophosphate (camp), molecules CD4, CD8 molecules, part or all of the receptor T cells, ligand (fixed or free), peptide, receptor T cells (TCR), antibodies, cross-reactive antibodies, monoclonal antibodies or polyclonal antibodies.

If the agent is an antibody cross-reactive antibody, monoclonal antibody or a polyclonal antibody, it is preferable that the agent represented by any one or more of the antibodies, cross-reactive antibodies, monoclonal antibodies or polyclonal antibodies to any one or more of the-chain antigen class II MHC,-chain antigen HLA-DR MHC,-chain antigen class I or class II MHC,-chain antigen HLA-DR MHC,and-chain antigen class II MHC or antigen with class I MHC. An example of a suitable antibody is CR3/43 (supplied by Dako).

More commitirovannah cell is any cell derived from or originate from undifferentiated cells.

Thus, in one preferred aspect more commitirovannah cell represents IGNOU stem cells or myeloid stem cell, and the undifferentiated cell is a pluripotent stem cell.

In another aspect, more commitirovannah cell is a differentiated cell, such as cell CFC-T cell CFC-B cell CFC-Eosin, cell CFC-Bas, cell CFC-GM, cell CFC-MEG, cell BFC-E, cell CFC-E, T-cell, b-cell, eosinophil, basophil, neutrophil, monocyte, megakaryocyte or erythrocyte, and the undifferentiated cell is a myeloid stem cell lymphoid stem cells or pluripotent stem cells.

If more commitirovannah cell is a differentiated cell, it is preferable that a differentiated cell was a b-lymphocyte (activated or neaktivirovannye), T-lymphocyte (activated or neaktivirovannye), cell line macrophages-monocytes containing the core cell that can Express the antigens of class I or antigens class II, a cell which may be induced expression of antigens class I or class II, or enableireland.ie cell (i.e. a cell that does not contain the kernel, such as an erythrocyte).

In alternative preferred embodiments, the differentiated cell is selected from any group of cells including preserue receptors on the cell surface CD56 and/or CD16.

The differentiated cell may even be formed by nucleation enableireland.ie cells.

The agent may act intracellularly inside more commitirovannah cells. However, preferably, when the agent acts extracellular towards more commitirovannah the cell.

In a preferred embodiment, the agent promptly takes a receptor present on the surface more commitirovannah cells, and the receptor can be expressed more commitirovannah cell-like receptor, which is able to Express more commiteeman the cell.

Preferably, if the receptor is an antigen Class I or Class II major histocompatibility complex (MHC). In preferred embodiments, the receptor on the cell surface is any one of a number of receptor HLA-DR receptor DM, DP receptor, receptor number, receptor HLA-A, receptor HLA-B receptor HLA-C receptor HLA-E receptor HLA-F or receptor HLA-G.

In the most preferred embodiments of the cell surface receptor is a receptor HLA-DR.

Preferably the step of contacting includes the joining agent in interaction with any one or more of the f">-chain antigens class II, CD4 receptor cell surface receptor CD8 on the cell surface, homologous sites-chain antigen class II in the presence of lymphocytes, homologous sites-chain antigen class I in the presence of lymphocytes or homologous sites-chain antigen class II in the presence of lymphocytes.

Preferably, if the step of contacting occurs in the presence of a biological response modifier.

Preferably, when the biological response modifier is one or more modulators, such as an immunomodulator, a growth factor, a cytokine, a cell surface receptor, hormone, nucleic acid, nucleotide sequence, antigen or peptide.

In a preferred implementation of the present invention undifferentiated cell then commities in recommisioning cell, such as the differentiated cell.

Recognitiona cell can be in the same line, the more commitirovannah cell, from which was derived undifferentiated cell.

Alternative recognitiona cell may refer to a different line is completed with the invention also includes the method according to the invention obtain undifferentiated cells, which includes commitirovannah undifferentiated cells in recommisioning the cage and then merge recommisioning cells with myeloma. This ensures expression in vitro of large quantities of the desired product, such as an antibody or antigen, or a hormone, etc.

Other aspects of the present invention include the following.

The use of any of the agents of the present invention to obtain undifferentiated cells from more commitirovannah cells.

The use of undifferentiated cells derived in accordance with the method of the present invention, to obtain any monoclonal or polyclonal, or specific antibodies from b-lymphocyte or T-lymphocyte; cell line macrophages-monocytes; containing the nucleus of the cell that can Express the antigens of class I or class II; cells capable of inducing the expression of antigens of class I or class II; enableireland.ie cells or apoptotic cells.

The use of undifferentiated cells derived in accordance with the method of the present invention, to obtain effector T-lymphocytes b-lymphocytes and/or Vice versa.

The use of undifferentiated cells obtained in the CE is the ATA, such a drug product comprising or derived from a b-lymphocyte, T-lymphocyte, cell macrophage macrophage-line, containing the nucleus of the cell that can Express the antigens of class I or class II; cells capable of inducing the expression of antigens of class I or class II or enableireland.ie cells.

The present invention also includes methods of using the above applications and the products or compositions derived from such methods.

The present invention also includes a pharmaceutical preparation containing undifferentiated cell in accordance with the present invention or a product derived from it, mixed with a suitable solvent, carrier or excipient.

In one preferred embodiment, the implementation of the drug includes an antibody or antigen derived from undifferentiated cells in accordance with the present invention, mixed with a suitable solvent, carrier or excipient.

Preferably, the drug was intended to treat any of the following conditions: cancer, autoimmune diseases, blood diseases, cell or tissue regenerate In a preferred embodiment implementing the present invention relates to a method for introducing a gene into the genome of cells, moreover, the method includes the introduction of a gene in more commiteeman the cage and then obtaining undifferentiated cells using the method in accordance with the present invention, whereby the gene is present in undifferentiated cell.

In a more preferred embodiment implementing the present invention relates to a method for introducing a gene into the genome of undifferentiated cells, and the method includes inserting a gene into the genome of more commitirovannah cells and then obtaining undifferentiated cells by the method according to the invention, whereby the gene is present in undifferentiated cell.

In an even more preferred embodiment, the implementation of the present invention relates to a method for introducing a gene into an undifferentiated cell, and the method includes inserting a gene into the genome of more commitirovannah cells and then obtaining undifferentiated cells using the method in accordance with the present invention, resulting in a gene present in the genome of the cells.

The present invention encompasses an undifferentiated cell derived using any of these methods of the present invention.

As mentioned previously, this is in any of these ways, mixed with a suitable solvent, carrier or excipient. With this drug undifferentiated cell can be used to obtain predominantly more commitirovannah cells, such as cell having the correct genomic structure, to facilitate any of the symptoms or conditions resulting from or associated with more commitirovannah cell having the wrong genomic structure.

Thus, the present invention also provides a method for elimination of acquired mutations from more commitirovannah cells, and this method includes the formation of undifferentiated cells by the method according to the invention, transforming undifferentiated cell in recommisioning cell, in which the device or reorganization of the genome and/or the nucleus of the cell causes the elimination of mutations.

Preferably the gene is inserted into the immunoglobulin plot or plot TCR genome.

Alternative undifferentiated cell can be used for more commitirovannah cells, which produces an agent that cures the symptoms or condition caused by or associated with more commitirovannah cell having the wrong GE is atarov T cells to the antigen, which is expressed more commitirovannah cell, which was retrodifferentiation in undifferentiated cell. In this respect, the antigen can be phytospecific antigen or cross reactive phytospecific antigen.

The present invention also includes a method of controlling levels of undifferentiated cells and more commiteeman cells. For example, the present invention includes a method, which includes the formation of undifferentiated cells by the method according to the invention, and activation of apoptosis gene for effects on undifferentiated cell so as to cause her death.

In one preferred implementation of the present invention more commitirovannah cell is a cancer cell. In another preferred implementation of the present invention, the agent is neither carcinogenic nor can promote malignant growth.

The present invention also encompasses a method of treating a patient suffering from a disease or disorder associated with defective cell or undesirable cell, and the method includes obtaining undifferentiated cells by contactin the cells in the undifferentiated cell, and then the optional commitirovannah undifferentiated cells in recommisioning cell; in this way undifferentiated cell, or recognitiona cell, the effect on the defective cell or unwanted cell to relieve symptoms of the disease or disorder or for treatment of a patient with the disease or condition.

In the result, the present invention relates to the production of nedifferentsirovannoi cells from more commitirovannah cells.

Now the present invention will be described in the form of an example, which will be done in reference to the following drawings: Fig.1, which is a microscopic image of cells before the application of the method of the present invention; Fig. 2, which is a microscopic image of cells obtained using the method of the present invention; Fig. 3, which is a microscopic image of cells obtained by the method according to the invention, but at a lower magnification; Fig.4, which is a microscopic image of cells before the application of the method of the present invention;
in Fig. 5, which is a microscopic image of cells obtained is current, obtained by the process according to the invention.

A. MATERIALS AND METHODS
PATIENTS
In tubes with a lavender tops containing EDTA were obtained blood samples of patients with b-cell chronic lymphocytic leukemia, patients with deficiency of antibodies (including IgA deficiency and concatenated with the X-chromosome of hypogammaglobulinemia in children), patients with HIV infections and the syndrome of AIDS patients with cytomegalovirus infection (CMV), a patient with Hodgkin's lymphoma, a patient with acute T-cell leukemia, a 6-day baby with Hodgkin's lymphoma, a patient with acute T-cell leukemia, a 6-day baby with blastocytosis, different patients with various diseases and clinical conditions, cord blood, bone marrow and enriched preparations of b-lymphocytes of healthy blood donors.

CLINICAL AND EXPERIMENTAL CONDITION
Clinical and experimental treatment of patients, including various types of processing samples of their blood, are described in table 1. Leukocyte counts (WBC) were obtained using a counter of Colter, and it is included in the same table.

BLOOD PROCESSING
After taking the blood samples were processed pure monoclonal antibody to the homologous site

Also used other methods of treatment with the same concentrations, and they included the addition of monoclonal antibodies to the homologous site-chain antigen HLA-DR monoclonal antibodies to the homologous area antigens class I, monoclonal antibody to CD4, monoclonal antibodies to CD8 and immobilized on PE monoclonal antibodies to the homologous site-chain antigen HLA-DR.

Other processing methods include simultaneous addition to the blood samples with monoclonal antibody to the homologous sitesand-chain antigen HLA-DR.

In addition, blood samples were added alkylating agents such as cyclophosphamide in combination with pure monoclonal antibody to the homologous site-chain antigen HLA-DR.

After these processing methods blood samples were stained with a set of labeled monoc the exact cytometry.

Periods of incubation with monoclonal antibodies varied in intervals of 2, 4, 6, 12 and 24 hours.

LABELED ANTIBODIES
The following monoclonal antibodies were used to detect the following markers on the cells using flow cytometry: CD19 and CD3, CD4 and CD8, DR and CD3, CD56&16 and CD3, CD45 and CD14, CD8 and CD3, CD8 and CD28, control with simultaneous samples (IgG1 FITC + IgG2a PE), CD34 and CD2, CD7 and CD13& 33, CD10 and CD25, CD5 and CDIO, CD5 and CD21, CD7 and CD5, CD13 and CD20, CD23 and CD57 and CD25 and CD45 RA (Becton & Dickenson and DAKO).

Analysis of blood samples of each patient both handled and unhandled was performed using most of the components of the above set to determine the nature immunophenotypic changes that accompanied various types of processing, and they were carried out separately at different aliquot of the same sample of blood. Staining and analysis of untreated samples and samples after other control methods of processing carried out at the same time.

FLOW CYTOMETRY
Staining and lizirovania whole blood samples was carried out according to the manufacturer's instructions. Running cytometrics analysis was performed on FACScan@ using either simultaneous test, or computer programs PAINT A GATE (BDIS), which is anili information about 10000-20000 the session handle.

MORPHOLOGY
The morphology was analyzed using microscopy and staining by Wright.

C. RESULTS
SET CD19 and CD3
Treatment of blood samples with monoclonal antibody to the homologous site-chain of HLA-DR was always reduced the relative number of cells CD19+. This token represents the total b-cell antigen (see table). This antigen is present on all In-human lymphocytes at all stages of maturation, but he lost on the final differentiated plasma cells. Therefore, it is an indication that the cells have undergone retrodifferentiation in undifferentiated cells.

The same treatment has caused a sharp increase in the relative number of cells CD3+especially in the blood of patients with b-cell lymphocytic leukemia (B-CLL), which was always accompanied by an increase in the relative number of cells CD3-CD19-. CD3 is present on all Mature T-lymphocytes and 65-85% of thymocytes. This token is always found in Association with-/- or gamma/Delta receptors on T cells (TCR), and together these complexes are important in the transmission of signals into the cell. With EDI and then comitialis in new differentiated cells, namely T cells.

In the treated blood of patients with B-CLL has a new clone of cells, together expressing markers CD19 and CD3-, i.e. cells CD19+and CD3+(see map 1, patient 2, 3 and 4 after 2, 6 and 24 hours after the start of treatment). In other patients with different conditions revealed an increase in the relative number of these cell clones. These cells were extremely large and heavily granulated, and on their cell membrane were expressed extremely high levels of CD19. It seems that the marker CD3 expressed on these cells at levels similar to the levels expressed on normal Mature lymphocytes.

In table 2, patients under the numbers 2, 3 and 4 are actually numbers that represent the same patient, and their designation was simply intended to illustrate the effect of treatment on blood over time (experimental and clinical status of this patient are shown in table 1).

It seems that clones CD19+and CD3+in the treated samples decreased over time, reaching the original levels that were detected in the untreated sample at intervals of 2 , 6 and 24 hours.

Another type cells of the same size is x on their surface, but were negative for the marker CD3 and rich FC receptors. However, it appeared that the relative number of these cells decreased
from time to time. Interestingly, 24 h after treatment, blood samples (2, 3 and 4), a decrease in the relative number of cells CD19-CD3-(in the group of cells, increasing the number which originally was observed after 2 and 6 h after treatment of blood samples. However, there was a decrease in the number of populations of leukocytes counting koltanowski formula in the treatment of blood with monoclonal antibody to the homologous site-chain antigen HLA-DR. These data suggest that this type of treatment causes the development of atypical cells that cannot be detected using a counter Colter (table 1), but can be detected during the measurement using flow cytometry to count cells based on surface markers, size and grit. In addition, these atypical cells were detected by analysis of morphology using dye Wright under the microscope. Graphs of flow cytometry of these phenomena are shown in the graphs (1, 2, 3 and 4), and it seems that the immunophenotypic changes, obtained the ow associated with each other a group of cells, but remain excellent for relative expression of CD19 and CD3 from stem cells.

In table 2 patient number 5 and 6 is the same patient, but the analysis of treated and untreated blood samples were controlled in the dynamics and at the same time (see table 1).

In the blood of patients with malignant b-cell diseases revealed similar trends immunophenotypic changes when compared with blood of patients with B-CLL, but the changes were not expressed to the same extent. However, the relative and absolute number of b-lymphocytes and positive class II MHC cells in the blood of these patients is extremely low compared with that found in the blood of patients with B-CLL.

Two brothers with the concatenated with the X-chromosome child hypogammaglobulinemia, which was a deficiency of b-cells were identified various immunophenotypic changes in the relative number of cells D3+after processing their blood. The younger brother, who was 2 months and has not been sick, after treatment, blood was revealed a slight increase in the relative number of cells CD3+that was accompanied by a decrease in the relative number of cells CD3-and CD19-. the high number of activated T-cells, expressing antigens DR, revealed a decrease in the number of cells CD3+after processing his blood. No other markers were not used to measure other possible immunophenotypic changes, because the blood samples obtained from these two patients were extremely small (table 2, ID43/BD and 04/BD).

The patient 91 table 2 reveals a decline in the relative number of cells CD3+after processing the blood, which was accompanied by an increase in the relative number of cells CD3-and CD19-. However, the analysis of other surface markers such as CD4 and CD8 (see table 3), it was noted that the patient has a high relative number of CD4 cells+CD8+in the blood, it was noted before treatment of blood samples with monoclonal antibody to-chain of the DR antigen, and the number of these double-positive cells was significantly decreased after treatment of blood. In addition, when the analysis was performed on other markers, it was noted that the relative number of cells CD3+increased (see table 4).

In the enriched preparation of b-lymphocytes obtained from healthy blood donors, after treatment of blood samples with monoclonal antibody to

SET CD4 and CD8
The CD4 antigen is a receptor for the human immunodeficiency virus. The CD4 molecule binds to the antigen-class II MHC in B2-domain plot, which is similar to the binding sites with CD8 antigens class I Binding with CD4 antigen class II enhances the reactivity of T cells to antigens, as linking with CD8 antigens class I Antigens present on CD8 subpopulations suppressor/cytotoxic T-lymphocytes person, as well as subpopulations of lymphocytes natural killer cells (NK) and the majority of normal thymocytes. Antigens CD4 and CD8 jointly expressed on thymocytes, and these cells lose all tokens as the maturation of T-lymphocytes.

In the analysis of markers CD4 and CD8 (see below) and in most blood samples are presented in table 2, there is a type of staining to confirm the presence of process retraded CD4+CD8-that are double-positive cells, always appeared after treatment of blood samples with monoclonal antibody to the homologous site-chain, and the number of these cell types was significantly increased in the blood of treated samples of patients with B-CLL, but they are always absent in untreated samples (see table 3 and map 1, 2, 3 and 4). In the same samples was also observed simultaneous increase in the relative number of single positive cells, such as cells CD8+and CD4+. In addition, there was a sharp decrease in the relative number of CD4 cells-CD8-that, at least in the case of B-CLL, corresponds to b-cells in the treated samples compared to untreated samples, which remained at the same level in the dimension in the dynamics. However, determining the relative number of CD4 cells+CD8+in dynamics in the treated samples showed that there was a concomitant increase in the number of single positive cells with a decrease in the relative number of double-positive cells. This type of phenotypic changes characteristic of thymic development of progenitor cells line T-lim is the moat (CD4+CD3+and the majority of normal thymocytes. However, this antigen is present with a low density on the cell surface of monocytes and in the cytoplasm of monocytes and macrophages (CD3-CD4+).

After processing in different blood samples marked with a variety of effects on cells with a relatively low number of CD4+. It appears that the treatment does not affect the relative amount of this type of cells in blood samples of patients with B-CLL compared with untreated samples. Such low levels of expression of CD4 was detected on monocytes and on thymocytes at very early stages of development.

The HIV patient+25 after treatment revealed a significant increase in the number of double-positive cells simultaneously expressing CD4 and CD8. On the other hand, the patient 91 after treatment revealed a decrease in the number of this subtype of cells, and the observation of such a phenomenon depends on the time. Observed increase in the relative number of cells CD8+in untreated blood samples of patients with B-CLL in the measurement of the dynamics, whereas in the same periods, a decrease in the relative number of CD4+cells and cells with low CD4+(table 3, patieni T-lymphocytes.

In the treated and untreated samples analyzed using this kit, immunophenotyping revealed changes similar to the changes obtained when blood samples were analyzed using markers CD19 and CD3 (see table 2), and these antigens, as indicated previously, are respectively In - and T-cell markers.

It seems that treatment of blood with monoclonal antibodies affects the relative amount of b-lymphocytes DR+so the level of cells DR+reduced. In contrast, the relative number of cells CD3+(T-cells) is significantly increased (see table 4 and map). In addition, the relative number of activated T-cells were increased in the majority of treated blood samples of patients with B-CLL, and these cell types was different effects in the treated patient samples with other States. In addition, the relative amount of DR high DR positive cells appeared in significant quantities processed samples of patients with B-CLL and a 6-day baby with an increased content of blasts DR+CD34+in the blood. However, it should be noted that the blasts, which were present in the blood of this patient were from eloigne line after treatment. The relative number of cells DR-CD34-increased in the majority of treated blood samples, and was proportional to the increase in the relative number of cells CD3+(T-cells) and inversely proportional to the decline in the relative number of cells DR+(B-cells).

SET CD56&16 And CD3
Markers CD56& CD16 detected on a heterogeneous group of cells, lymphocyte subpopulations, known generally as large granular lymphocytes and lymphocytes - natural killer cells (NK). The CD16 antigen expressed on virtually all resting NK lymphocytes and weakly expressed on some T-lymphocytes CD3+from certain persons. This antigen is found on granulocytes in fewer and associated with lymphocytes containing large azurophil granules. The CD16 antigen is a receptor III IgG FC.

A different number of lymphocytes CD16+expresses either the CD57 antigen, or low-density antigen CD8, or both antigen. In most people there is virtually no overlap with other antigens to T-lymphocytes, such as antigens CD5, CD4 or CD3. The CD56 antigen is present in essentially all resting and activated lymphocytes and CD16+NK, and these subpopulations cleto is the processed and unprocessed blood samples of patients with B-CLL and some other patients with other States showed an increase in the relative number of cells, together expressing the CD56 antigens& CD16, which were very granular and had a medium size (see table 5 and map 1, 2, 3 and 4). These observations were accompanied by a pronounced increase in the relative number of cells expressing only the CD3 antigen (without the expression of markers CD56 and CD16), and cells, together expressing markers CD56&CD16 and CD3.

Table 5 patient rooms 2, 3 and 4 represent the same blood sample, but are analyzed, respectively, by 2, 6 and 24 hours (before and after treatment). This trial shows that treatment of blood with monoclonal antibody to the homologous site-chain of the DR antigen seems to spontaneous production of cells CD56+and CD16+cells CD3+and CD56+and cells CD16+CD3-and these observations were always accompanied by the disappearance of markers of b-cells (CD19, DR, CD56, CD16-CD3-).

Continuing in the dynamics analysis of the blood samples before and after treatment showed that the levels of cells CD56+and CD16+decreased and the level of cells CD3+over time, increased.

In the blood samples of the patient 7 with B-CLL did not reveal any changes in the number of cells expressing antigenic samples and this is because the number of added monoclonal antibodies was very low compared with the number of b-lymphocytes. However, processing the blood samples of the patient in a separate case, the appropriate number of monoclonal antibodies showed a significant increase in the relative number of cells D3+, D56+&CD16+and CD56+and CDl6+CD3+.

Samples of blood of other patients in other States showed different changes in the level of these cells, and it appears to be dependent on the number of b-lymphocytes present in the blood before treatment, duration of treatment and, probably, from the clinical condition of the patients.

SET CD45 and CD14
Antigen CD45 is present on all human leukocytes, including lymphocytes, monocytes, polymorphonuclear cells, eosinophils and basophils in the peripheral blood, thymus, spleen and tonsils, and the precursor cells in the bone marrow.

CD14 is present in 70-93% of normal peripheral blood monocytes, 77-90% of phagocytes pleural or peritoneal fluid. This antigen is weakly expressed on granulocytes and does not exist on unstimulated lymphocytes, mitogen-activated T-lymphocytes, lekule interacts with external stimuli (antigens) and affects the transmission of the signal through family members Scr, leading to the regulation of growth and differentiation of cells.

Part-chain of the DR antigens in the treated blood samples, especially in samples obtained from patients with B-CLL suggests that this treatment affects the level of antigens CD45 in B-lymphocytes. It seems that the immunophenotypic changes that occur during stimulation-chain of the DR antigen are responsible for the emergence of different types of cells that can be separated on the basis of the level of expression of CD45 and CD14, and morphology according to the definition of direct scattering and side scattering (respectively the size and the grain size), and these results are presented in table 6 and maps (1, 2, 3, 4 and 5).

When processing the relative number of cells with low levels of CD45 (when compared to untreated samples) was significantly increased, as well as the relative number of cells, together expressing antigens CD45 and CD14. This type immunophenotypic changes coincided with a decrease in the relative number of cells with high levels of CD45 (compared to untreated samples). However, this latter population of cells can be further divided on the basis of morphology and extent of the lines from the other cells, presented in maps (see maps 1, 2, 3 and 4). The analysis of this set after processing in the dynamics (see table, patient 2, 3 and 4, and map) relative number of cells CD45+originally declined sharply over time, causing the cells with low levels of CD45. However, a blood test after 24 hours showed the opposite situation.

Samples 5 and 7 reveal immunophenotypic changes opposite to those obtained with other samples taken from other patients with B-CLL, and this is due to the fact that the samples were analysed at a much earlier stages of incubation with a monoclonal antibody. In fact, the serial analysis of blood samples after treatment suggests that immunophenotypic changes c In lymphocytes, are time-dependent, because they reflect the stage of development, and immunophenotypic changes identified during X will not be the same as during X-plus (being induced, they are not the same). However, these types of changes should be more strictly defined way in the body, kind of immunopathology. The influence of processing blood samples from other patients with malignant diseases, not Holy is city present in smaller quantities. However, processing of enriched fractions of b-lymphocytes obtained from healthy blood donors, shows immunophenotypic changes similar to the changes obtained with B-CLL with a large number of b-lymphocytes.

SET CD8 And CD3
Antigenic determinants of CD8 interacts with molecules of class I MHC, which leads to increased adhesion between T lymphocytes CD8+and target cells. This type of communication enhances the activation of resting lymphocytes. The CD8 antigen is connected with protein protein kinase (p56ick), and in turn the complex CD8/p56ick may play a role in the activation of T-lymphocytes.

Processing of blood samples obtained from patients with B-CLL, a monoclonal antibody to the b-chain, resulting in a significant increase in the relative number of cells positive for CD3CD8 and CD3 (very likely positive for CD4CD3) that more clearly indicates that the double positive cells generated initially undergo development in Mature T-lymphocytes. It is a process that can be directly measured using antigens CD19 and DR and indirectly using antigens CD8-CD3-. It seems that the dynamic serial evaluation of samples processed blood of the same patient is consistent with processdoc CD8+increases over time in treated and untreated samples, but to a greater extent in untreated samples. On the other hand, the relative number of cells CD8+CD3+decreases over time in untreated samples. However, the relative number of cells CD3+increases in the treated blood samples when measuring in the dynamics, and these types of cells are highly consistent single positive CD4 cells+CD3+more Mature form of thymocytes. In addition, since these samples were also immunophenotypically other sets (mentioned above in tables 3, 4, 5 and 6), the overall change actively involve b cells in the generation of predecessors and descendants of T-lymphocytes.

Blood samples from a patient with B-CLL (number 2, 3 and 4, table 1, 2, 3, 4, 5, 6, 7) in a separate aliquot was not treated with anything processed associated with PE monoclonal antibody to the homologous site-chain of the DR antigen and unbound form of the same monoclonal antibodies. When the comparison processing by antigen associated with PE, does not indicate a change in the relative number of cells positive for CD3, and related markers, such as CD4, which were observed in significant was cricetulus increase in the number of cells, positive for CD45, without the expression of DR antigen on their surface (see table 8). These data were similar to data obtained in untreated samples when immunophenotyping in the dynamics (table 6). In addition, the relative number of cells expressing low levels of CD45 has decreased, a phenomenon that was also observed in untreated samples (when measured in the dynamics) of the same patient (see map 1A).

C. COMPARISON of the influence of OTHER MONOCLONAL ANTIBODIES WITH DIFFERENT SPECIFICITY ON T-LYMPHOPOIESIS
SET CD19 And CD3
Treatment of blood samples with monoclonal antibody to the homologous site-chain of the DR antigen and homologous site antigens Class I MHC reduced the number of cells CD3+and increased the number of cells CD19+. Processing the same blood with monoclonal antibody to the homologous site-chain of the DR antigen reduced the number of cells CD19+and increased the number of cells CD3+. The treatment of the latter monoclonal antibody with cyclophosphamide showed the same effect (table 14, the patient 5/6 with B-CLL after 2 h after treatment).

Ongoing analysis of cells CD19+and CD3+in the same obrasocial antibody to the homologous site-chain of the DR antigen (table 14, the patient 5/6 with B-CLL, 24 h after treatment). However, ongoing analysis (after 24 hours, the patient 5/6, table 14) blood samples treated with cyclophosphamide plus monoclonal antibody to-chain of the DR antigen, revealed elimination of changes in the relative number of cells CD19+and CD3+compared with the number observed after an incubation period of 2 hours in the same conditions.

In General, the processing of blood samples of the same patient a monoclonal antibody to the homologous site-chain of the DR antigen or monoclonal antibody to the homologous site-chain antigen class I shows the increase in the relative number of cells CD19+(In General-token) when compared to untreated sample. The relative number of cells CD19-CD3-slightly decreased in blood samples treated with monoclonal antibody to the homologous site-chain of the DR antigen or monoclonal antibody to antigens of class I (see table 14 and map 2, 3 and 4). Processing of blood samples of the patient 09 monoclonal antibody to antigens class I increased D3-. However, processing of enriched preparation of b-lymphocytes obtained from healthy blood donors, a monoclonal antibody to the homologous site-circuit or-chain of the DR antigen immunophenotype showed the same changes as obtained from a patient with B-CLL.

The treatment of the patient with HIV+and deficiency of IgA monoclonal antibody to-chain of the DR antigen increased the relative number of cells CD3+and slightly reduced the relative number of cells CD19+. However, the treatment of the same sample of blood with monoclonal antibody to the homologous site of antigen class I did not give the same effect. Processing of blood samples obtained from patients (34/BD and 04/BD) deficiency of b-cells showed different immunophenotypic changes in the processing of a monoclonal antibody to the homologous site-chain of the DR antigen, antigens class I and antigen CD4.

SET CD4 And CD8
Blood samples, the analysis was performed using a set of CD19 and CD3 (table 14), was also immunotherapy set CD4 and CD8 (table 15). It seems that both sets are consistent and confirm each other. Incubation for oginome site-chain of the DR antigen or monoclonal antibody plus cyclophosphamide increased the relative number of cells CD8+and CD4+and cells expressing both markers. On the other hand, the processing of the same samples with monoclonal antibody to the homologous site-chain of the DR antigen or to the homologous site-chain antigen class I, did not cause the same effects.

Comparison of immunophenotype trends obtained after incubation periods 2 and 24 hours with a monoclonal antibody to the homologous site-chain of the DR antigen plus cyclophosphamide, revealed an inverse change in the relative number of cells positive for CD4 and CD8 (table 15, the patient 5/6 with B-CLL after 2 and 24 hours), and these changes were consistent with the changes obtained in the analysis of the same blood sample with a set of CD19 and CD3 (table 14, the same patient). More recent data show that the subsequent differentiation of reversible as undifferentiated cells can differentiate into T-lymphocytes or b-lymphocytes.

SET DR And CD3
Immunophenotypic changes obtained with a set of DR and CD3 (table 16), confirming the data obtained with a set of what itelli homologous site of beta - or alpha-fragment of the DR antigen or monoclonal antibody to antigens class I or a monoclonal antibody to-chain of the DR antigen plus cyclophosphamide in the analysis of 2 hours.

Based on these results it seems that the monoclonal antibody to the homologous site-chain of the DR antigen has a high ability to start the production of cells positive for CD3, cells DR+.

In addition, the processing similar to that in which involved-chain of DR antigens orthe fragment molecule in combination with cyclophosphamide (long incubation), contributed to the increase in the relative number of cells CD19+or cells DR+.

SET CD56&16 And CD3
Processing of blood samples, especially samples of patients with B-CLL with high levels of b-lymphocytes, monoclonal antibody to the homologous site-chain of the DR antigen increased the relative number of cells positive for CD56&16.

In these patients, the relative number of cells CD3+and CD56+and CD16+CD3-also increased after treatment of blood samples with monoclonal antibody to-circuit, confirming previous observations marked with the same effect, when the same samples were analyzed �://img.russianpatents.com/chr/946.gif">and-chain of DR antigen or-chains plus cyclophosphamide or to antigens class I, analyzed a set of CD45 and CD14 (table 18). The definition of low CD45, high CD45 and average CD45 is arbitrary. Processing of the blood sample 5/6 (2 hours) monoclonal antibodies to-chain of the DR antigen or monoclonal antibody plus cyclophosphamide generated cells, low CD45+and increased the relative number of cells with intermediate levels of CD45+. However, the first of these treatments increased the relative number of cells with high levels of CD45+and the last of these treatments reduced the relative number of cells with intermediate levels of CD45+and these changes, as it turned out, depended on the time.

In blood samples from the patient 5/6 and 10 (B-CLL) after treatment with monoclonal antibody to antigens class I detected a decline in the relative number of cells with intermediate levels of CD45+and similar data were observed in blood samples of 09 and HIV+after the same treatment as compared with untreated samples. Processing of blood samples of patients with HIV+and IgA/D monoclonal the p> compared to untreated samples or samples treated with monoclonal antibody to-chain of the DR antigen. However, in blood samples of these patients revealed a decrease in the relative number of cells with intermediate levels of CD45+after treatment with monoclonal antibody to the homologous sites-chain of the DR antigen. The number of cells with intermediate levels of CD45+in the blood samples of the patient IgA/D after treatment with monoclonal antibody to the antigen class I grew. Cells, which were very large, rich granules and expressed intense levels of antigen CD45 was observed in blood samples treated with monoclonal antibody to the homologous site-chain of the DR antigen antigen class II MHC (see maps 1,2, 3, 4 and 5).

SET CD8 And CD28
The CD8 antigen is present in approximately 60-80% of T (CD3+)-peripheral blood lymphocytes, 50% of T-lymphocytes and CD8+and 5% of immature thymocytes CD3-. During maturation of thymocytes expression of CD28 antigen increases from low density on the majority of Mature thymocytes CD4+CD8+to a higher density on virtually all Mature thymocytes CD3+, CD4+or CD8< + two functional groups. Lymphocytes CD8+CD28+mediates alloantigen-specific cytotoxicity, which is limited to class I major histocompatibility complex (MHC). Suppression of cell proliferation mediated by a subpopulation of CD8+CD28+. The CD28 antigen is a cell adhesion molecule and functions as a ligand for antigen B7/BB-1, which is present on activated b-lymphocytes.

Processing of blood samples of patients (table 19 patients 5/6 and 8) with B-CLL monoclonal antibody to the homologous site-chain of the DR antigen increased the relative number of cells CD8+, CD28+and CD8+CD28+and all other treatments did not increase.

SET CD34 And CD2
The CD34 antigen is present on immature hematopoietic cells predecessors and all of hematopoietic colony-forming cells in the bone marrow, including unipotent (CFU-GM, BFU-E) and pluripotent precursors (CFU-GEMM, CFU-Mix and CFU-blast). CD34 is also expressed on stromal cells-precursors. B - and T-lymphoid precursors with terminal deoxynucleotidyltransferase (TdT) in normal bone marrow are CD34-. Antigen CD34 Pris is nih erythroid cells, which Express CD71 antigen and weakly Express the antigen CD45. The CD34 antigen is also found on capillary endothelial cells and approximately 1% of human thymocytes. Normal lymphocytes, monocytes, granulocytes and platelets in the peripheral blood does not Express the CD34 antigen. The density of CD34 antigen is highest in early hematopoietic cells predecessors and decreases with maturation cells. The antigen is absent in the fully differentiated hematopoietic cells.

Committeename cells-the precursors of CD34+are CD38-DR-and they do not have a specific line of antigens, such as CD71, CD33, CD10 and CD5, whereas cells CD34+that are linearly commitirovannah, with high density Express the antigen CD38.

Most cells CD34-reciprocal Express or antigen CD45RO or CD45RA antigen. Approximately 60% of the cells of acute B-lymphoid leukemia and acute myeloid leukemia Express the CD34 antigen. The antigen is not expressed on the cell chronic lymphocytic leukemia (B - or T-line) or lymphomas. The CD2 antigen is present on T-lymphocytes and subpopulations of lymphocytes - natural killer cells (NK).

Achiev the following processing of monoclonal antibodies to the-chain of the DR antigen or-chain of the same antigen revealed a pronounced increase in the relative number of cells CD34+and CD34-CD2-after processing the first of these antibodies. Because the same blood samples were immunotherapy the above sets (see tables 14 to 19) on other markers, observed here, the increase in the relative number of cells CD34+and CD34-CD2-as it turned out , coincides with the increase in the relative number of single positive (SP) CD4+CD8+, CD8+CD3+and CD4+CD3+. Moreover, the data that are likely to be exceptional in terms of involvement-chain antigen HLA-DR, confirm that the process runs T-lymphopoiesis through regression of b-lymphocytes.

When analyzing the same treatment after 24 hours, the levels of cells CD34+seems declined to provide further increasing the relative number of T-lymphocytes. The process of retrodifferentiation, which was originally provided T-lymphopoiesis, may be cancelled to ensure the b-lymphopoiesis. The first of these phenomena were observed after 2-hour incubation period with monoclonal antitail noted after 24 hours of incubation period at the same treatment in the same sample (see map 2).

Processing of blood samples of HIV patient+(table 20, the HIV patient+) a monoclonal antibody to-chain antigen HLA-DR was significantly increased relative number of cells CD34+and CD2+CD34+and it also gave a joint processing of the same sample of blood with monoclonal antibody to-chain antigen HLA-DR monoclonal antibody to-chain of the same antigen. However, processing of blood samples with monoclonal antibody to-chain antigen HLA-DR did not affect the level of cells CD34+. Processing of blood samples from a 6-day baby (BB/ST table 20), which was examined in the time to detect leukemia and who had a very large number of atypical cells (blasts) in the blood, a monoclonal antibody to-chain antigen HLA-DR or a monoclonal antibody to-chain of the same antigen or both of these monoclonal antibodies, added together, have led to the following immunophenotype changes.

In the analysis of unprocessed blood samples relative number of cells CD34+and DR+markedly increased, and further, when processing monoclonal the observed decrease of this indicator after treatment with monoclonal antibody to-chain antigen HLA-DR or processing of monoclonal antibodies to theand-chain molecules by their joint Appendix. However, the latter treatment increased the relative number of cells CD34+CD2+, and was the opposite, when the same blood sample was processed only monoclonal antibody to-chain antigen HLA-DR. In the analysis of treated and untreated aliquot of blood of the same patient after 24 hours, the relative number of cells CD34+decreased in all of the above methods, except that it was maintained at a much higher level when processing a monoclonal antibody to-chain antigen HLA-DR. The last processing continued to reduce the relative number of cells CD34+CD2+after 24 hours.

These results show that the involvement of antigen HLA-DR throughchain produces more cells CD34+from the pool CD2+CD34-or more Mature cell types, such as b-lymphocytes of patients with B-CLL, and these results show that this type of treatment causes retrodifferentiation. However, the scrap belong to another line, and in this case, cells, apparently, belong or, more likely, by themselves commiserate in myeloid line that was observed in the analysis of blood samples processed by the set CD7 and CD13&33.

Morphology changes immunophenotypic characteristics of b-lymphocytes (B-CLL and enriched fractions of healthy individuals (using CD19 beads) after processing of monoclonal antibodies to the homologous sites-chain antigen-class II MHC. They were accompanied by morphological changes In lymphocytes. It has been observed that b-lymphocytes, colonizing the glass cover, raw smears of blood was replaced by granulocytes, monocytes, large quantities of primitive-looking cells had nuclei of erythrocytes. Neither treated nor untreated blood smears were observed pattern of mitosis or significant cell death.

The results presented in table 20 also shows another important discovery, namely, that in accordance with the methods of the present invention can be obtained undifferentiated cell by retrodifferentiation more Mature cells.

D. PHOTOMICROGRAPH
In addition to the test antigen, as described above, the pic is on the micrograph of differentiated b-cells before the application of the method according to the invention. In Fig.2 shows the micrograph of undifferentiated cells obtained by retrodifferentiation b cells In accordance with the present invention, in which the agent was a monoclonal antibody to the homologous sites-chain antigen HLA-DR. Undifferentiated cells are dark-colored groups of cells. In Fig. 3 shows a micrograph of the same undifferentiated cells, but at a lower magnification.

Thus, Fig.1-3 illustrate retrodifferentiation b cells In undifferentiated stem cells using the method of the present invention.

In Fig.4 shows a micrograph of differentiated b-cells prior to use of the method of the present invention. In Fig.5 shows a micrograph of undifferentiated cells obtained by retrodifferentiation b cells In accordance with the present invention, and the agent used was a monoclonal antibody to the homologous sites-chain antigen HLA-DR. And in this case, the undifferentiated cells are darkly stained cell groups. In Fig.6 shows a micrograph of education differentiated granulocytopenia b cells In undifferentiated stem cells using the method of the present invention with the subsequent transformation of undifferentiated cells in new and differentiated cells related to lines other than the source of differentiated cells.

Retrodifferentiation T cells in undifferentiated stem cells using the method of the present invention with the subsequent transformation of undifferentiated cells in new and differentiated cells belonging to a line other than the source of differentiated cells were observed by microscopy.

E. SUMMARY
In short, the examples describe the in vitro experiments, which revealed a very interesting phenomena associated with the ontogeny and development of T - and b-lymphocytes, which can be used to generate stem cells to influence lymphohematopoietic in samples of peripheral blood for several hours.

Processing samples of peripheral blood obtained from patients with b-cell chronic lymphocytic leukemia (B-CLL) with high levels of b-lymphocytes, monoclonal antibody to the homologous site-chain antigen class II caused a pronounced increase in the relative number of single positive (SP) T-lymphocytes and their precursors, which were double-positive for a marker antigens CD4 and CD8 thymocytes, and these cells expressibility odnovremenno was not observed, when the same blood samples were treated with monoclonal antibodies to the homologous site-chain antigen class II or homologous to the site of antigen class I.

As it turned out, the processing of whole blood obtained from patients with b-cell chronic lymphocytic leukemia (CLL), a monoclonal antibody to the homologous area of the b-chain antigen HLA-DR was caused by the development of T-lymphopoiesis. This process was marked by the appearance of double-positive cells, together expressing markers CD4 and CD8, the emergence of cells expressing CD34, and a simultaneous increase in the number of single positive CD4 lymphocytes+CD3+and CD8+CD3+. Moreover, immunophenotypic changes that took place during the generation of these cells were identical to the changes characteristic for the development of thymocytes, especially in the study of dynamics.

The percentage of double positive cells (DP) generated after a 2-hour period of incubation of whole blood with monoclonal antibody to the homologous sites-chain of the DR antigen, decreased over time, and these effects were accompanied by an increase in the percentage of single positive CD4+CD3+and f">-chain TCR were also expressed in these cell types.

Watched a permanent loss of b-cell markers such as CD19, CD21, CD23, IgM and DR, and this coincided with the appearance of cells CD34+and CD34+CD2+by increasing the number of cells CD7+by increasing the number of cells CD8+CD28-and CD28+by increasing the number of cells CD25+the advent of cell CD10-and CD34+and CD34+and cells CD19+by increasing the number of cells CD5+and cells expressing low levels of antigen CD45. These changes were the result of treatment of blood with monoclonal antibody to the homologous site-chain antigen HLA-DR.

Immunophenotypic changes associated with such processing, consistent with retrodifferentiation and subsequent commituranium (i.e. recommisioned) b-lymphocytes, because the majority of leukocytes in the blood of patients with B-CLL before the processing were In lymphocytes. In addition, b-lymphocytes of patients with B-CLL, turning them into T-lymphocytes was induced after treatment with cyclophosphamide and a monoclonal antibody to-chain antigen HLA-DR, it was possible again to return to In-lymphocytoma phenotype after a long time the src="https://img.russianpatents.com/chr/946.gif">-chain antigen HLA-DR using sets CD16&56 and CD3 and CD8 and CD3 relative number of cells expressing these markers is constantly increasing, and the growth of the cells is consistent with that defined in the application of such sets as CD19 and CD3 and DR and CD3. The study supernatant treated and untreated blood samples of patients with HIV infection using nephelometry and immunoelectrophoresis helped to identify elevated levels of IgG, indicating that b cells must go through a stage of plasma cells. The increase in the relative number of all of the above cells was also accompanied by the appearance of cells of medium size, rich granules, expressing the CD56 antigens&16 in extremely large quantities. Observed transient appearance of other cells, which were very large and full of pellets, and they were positive for CD34 and double-positive for the markers CD4 CD8. There was also other transient cells, and they were large and grainy and positive receptors CD3 and CD19. CD25, who was present on the majority of b-lymphocytes, was lost, and began to be expressed newly formed T-lymphocytes, increasing the number of which was su is oclonal antibody to the homologous area of the b-chain of the DR antigen. These data are obtained from the processing of blood antibody to the homologous site-chain antigen HLA-DR.

T-lymphopoiesis generated thereby was also observed in the peripheral blood of healthy blood donors, umbilical cord blood, bone marrow of patients with various infections, including persons with HIV+and AIDS patients, in enriched fractions of b-lymphocytes obtained from blood samples of healthy blood donors, patients with IgA deficiency and other patients with a variety of other conditions. In addition, analysis of myeloid markers in blood samples of two patients with B-CLL treated with antibody to the homologous site-chain antigen HLA-DR showed a significant increase in the relative number of cells expressing myeloid markers such as CD13 and CD33. These markers together expressibility with CD56 antigens&16 or CD7. However, populations of cells were observed in the relative number of cells CD7+with markers of T-lymphocytes and without myeloid antigens. These changes were not observed in untreated samples or in samples treated with monoclonal antibodies to antigens of class I or homologous sitetime running through-chain antigen HLA-DR, able not only to reverse the development of precursor cells T-lymphocytes, but also can exist in the framework of the myeloid and erythroid lineages.

It should be noted that stem cells obtained using the method of the present invention may be stem cells of any tissue and is not necessarily limited lymphohematopoietic cells predecessors.

Other modifications of the present invention will be obvious to specialists in this field.


Claims

1. The method of obtaining undifferentiated cells, including direct or indirect contacting cells, more commitirovannah than undifferentiated cell with an agent that causes retrodifferentiation more commitirovannah cells in an undifferentiated cell, and the specified agent comprises a modulator of the expression of MHC gene and optionally a biological response modifier used in conjunction with the specified modulator of gene expression MNF.

2. The method according to p. 1, characterized in that more commitirovannah cell capable of retrodifferentiation in undifferentiated cell Class I+MTL and/or cle to retrodifferentiation in undifferentiated cell, containing the antigen stem cells.

4. The method according to any of paragraphs. 1-3, characterized in that more commitirovannah cell capable of retrodifferentiation in undifferentiated cell CD34+.

5. The method according to any of paragraphs. 1-4, characterized in that more commitirovannah cell capable of retrodifferentiation in lymphohematopoietic cell-predecessor.

6. The method according to any of paragraphs. 1-5, characterized in that more commitirovannah cell capable of retrodifferentiation in pluripotent stem cells.

7. The method according to any of paragraphs. 1-6, wherein the undifferentiated cell is a cell Class I+MTL and/or Class II+MNF.

8. The method according to any of paragraphs. 1-7, wherein the undifferentiated cell contains the stem cell antigen.

9. The method according to any of paragraphs. 1-8, wherein the undifferentiated cell is an undifferentiated cell CD34+.

10. The method according to any of paragraphs. 1-9, wherein the undifferentiated cell is lymphohematopoietic cell-predecessor.

11. The method according to any of paragraphs. 1-10, wherein the undifferentiated cell is an onion skin is etka is a cell Class I+MTL and/or cell Class II+MNF.

13. The method according to any of paragraphs. 1-12, characterized in that the agent acts extracellular towards more commitirovannah the cell.

14. The method according to any of paragraphs. 1-13, characterized in that more commitirovannah cell contains the receptor, which operatively interacts with the agent, and the agent operatively interacts with the receptor.

15. The method according to p. 14, wherein the receptor is a receptor found on the cell surface.

16. The method according to p. 14 or 15, characterized in that the receptor containscomponent and/orcomponent.

17. The method according to p. 16, characterized in that the receptor contains-chain having homologous sites.

18. The method according to p. 17, characterized in that the receptor contains at least homologous sites-chain of HLA-DR.

19. The method according to p. 16, characterized in that the receptor contains-chain having homologous sites.

20. The method according to p. 19, characterized in that the receptor contains at least homologous sites-chain of HLA-DR.

21. The method according to any of paragraphs. 14-20, wherein the agent predstavlenie antibody to the receptor.

23. The method according to p. 21, wherein the agent is an antibody, preferably a monoclonal antibody to the homologous sites-chain of HLA-DR.

24. The method according to p. 21, wherein the agent is an antibody, preferably a monoclonal antibody to the homologous sites-chain of HLA-DR.

25. The method according to any of paragraphs. 1-24, wherein the agent modulates the expression of MHC gene, and preferably, the agent is modulated gene expression of Class I+MTL and/or Class II+MHC.

26. The method according to any of paragraphs. 1-25, characterized in that the agent is used in combination with biological response modifier.

27. The method according to p. 25, wherein the biological response modifier is an alkylating agent, preferably, when the alkylating agent represents or includes cyclophosphamide.

28. The method according to any of paragraphs. 1-27, wherein more commitirovannah cell is a differentiated cell.

29. The method according to p. 28, characterized in that more commitirovannah cell is a B-cell or T-cell.

30. The method according to any of paragraphs. 1-27, featuring the 31. The method according to any of paragraphs. 1-30, wherein the undifferentiated cell commiteeman in recommisioning the cell.

32. The method according to p. 31, characterized in that recognitiona cell refers to cells of the same row (or direction) of differentiation, the more commitirovannah cell before retrodifferentiation.

33. The method according to p. 31, characterized in that recognitiona cell refers to the row (or direction) of differentiation that is different from that of more commitirovannah cells before retrodifferentiation.

34. The method according to any of paragraphs. 31-33, wherein recognitiona cell is a B-cell, T-cell or granulocyte.

35. The method according to any of paragraphs. 1-34, wherein the method is a method in vitro.

36. Drug containing undifferentiated cell, obtained in accordance with the method according to any of paragraphs. 1-35 produced for the treatment of immunological disorders or diseases.

37. Drug containing recommisioning cell obtained in accordance with the method according to any of paragraphs. 31-35 produced for the treatment of immunological disorders or diseases.

 

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