RU2284190C1 - Method for treating ischemic cardiac disease, or myocardial infarction and its aftereffects, or cerebral ischemia induced by atherosclerosis or acute dysfunction of cerebral circulation, and its aftereffects, or atherosclerosis-induced ischemia of inferior limbs - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: the present innovation deals with treating diseases associated with affected tissue circulation. It deals with injecting mononuclear cells of donor's umbilical cord blood into the blood. Moreover, the above-mentioned donor's cells have got the set of antigens HLA-A, HLA-B, HLA-DR being distinct against the recipient's set of antigens HLA-A, HLA-B, HLA-DR by not less than four antigens. The quantity of introduced cells ranges 2.5 x 10⁶ up to 10 x 10⁶/kg body weight/d. Injection should be repeated 2-10 times at interval of 10-60 d. The innovation provides substitution of dead and/or recipient's regeneration-impotent cells with the above-mentioned donor's cells and enables to decrease the frequency of immunopathological reactions at decreasing the number of introduced donor's mononuclear cells.

EFFECT: higher efficiency of therapy.

2 cl, 8 ex, 1 tbl

Description

The invention relates to medicine and can be used to treat diseases associated with impaired blood supply to tissues.

Cord blood is the blood of a newborn remaining in the placenta and umbilical cord after childbirth. This blood is rich in mononuclear cells, among which a significant part is made up of so-called stem cells, that is, cells that are in the early stages of differentiation and give rise to the development of many organs and tissues. Isolation and use of these cells in medicine is primarily due to the fact that mononuclear cord blood cells contain a large number of hematopoietic progenitor cells - hematopoietic stem cells (HSCs). The fraction of mononuclear cells isolated from cord blood is used as an alternative source of HSC in bone marrow transplantation. The cells responsible for replacing diseased recipient cells during hematopoietic stem cell transplantation (HSC) are considered the so-called CD 34 - positive cells, that is, cells containing the CD 34 marker antigen on their surface and possessing the greatest clonogenic (colony forming) potential. As you know, cord blood stem cells morphologically belong to the so-called mononuclear cells (MNCs), which in turn are part of a pool of nucleated cells (UCS) of cord blood. Mononuclear cells (lymphocytes and monocytes in total) make up about 1/3 of all nucleated cells (leukocytes) of umbilical cord blood.

Transplantation (transplantation) of cord blood mononuclear cells has been used in medical practice since the 80s of the 20th century. The main indication for cord blood stem cell transplantation is blood diseases caused by a congenital (genetic) defect in poly-, pluro-, or oligopotent bone marrow blood cell cells and / or their malignant transformation, as well as overcoming the consequences of severe myelosuppression of various origins. To achieve repopulation during transplantation of umbilical cord cells of a donor (replacing their diseased cells with healthy strangers), two main methods are used:

- selection of a pair of "donor - recipient" according to the coincidence of the antigens of the main human histocompatibility complex (HLA) and the blood group system AB0 and Rh factor,

- suppression of the immunological response of the recipient himself for the engraftment of foreign transplanted cells.

However, in case of successful repopulation of donor cells, foreign transplanted cells can cause severe immunopathological phenomena, in particular the graft versus host reaction (GVHD).

Thus, the selection of the donor – recipient pair is, on the one hand, determining the success of the engraftment, and, on the other hand, is the most difficult in technical terms, since even people who are identical in HLA type may experience a graft rejection and / or GVHD.

Modern ideas dictate the need for the greatest match between the antigens of the main human histocompatibility complex as the basis for effective allogeneic cell transplantation.

In recent years, methods have been proposed for treating various diseases with umbilical cord blood mononuclear cells, based on the replacement of dead and / or incapable of regenerating recipient cells by these donor cells.

A known method of treating circulatory disorders, including myocardial infarction, by introducing human cord blood cells, US 2004/0197310 A1. The method proposes the introduction of "effective" amounts of mononuclear cells from human umbilical cord blood either directly into the infarction zone or into the systemic circulation. The therapeutic effect in this case is based on the engraftment of the umbilical cord cells of the donor in the host organism and their differentiation into cardiomyocytes. However, this requires a high degree of immunological compatibility of the donor cells with the recipient cells, which makes the method technically unfeasible. According to US 2004/0197310 A1, it is recommended that the administration of umbilical cord cells be suppressed by suppressing the recipient's immune response.

There is also known a method for the treatment of atherosclerosis, neurological diseases, autoimmune diseases, diabetes, amyotrophic lateral sclerosis, injuries and damage to the peripheral and central nervous system by introducing into the blood of cord blood stem cells or cells originating from umbilical cord blood in an amount of not less than 5 × 10 ⁹ nucleated cells, WO 2004/071283. This method is closest to the claimed invention and adopted as its prototype.

When implementing the prototype method, preliminary suppression of the patient’s immune response is not required, preliminary HLA typing (histocompatibility studies) of the donor and recipient is not required. However, its implementation requires extremely large numbers of cells (at least 5 × 10 ⁹ nucleated cells). The number of nucleated cells in a portion of umbilical cord blood from one newborn does not exceed 2.8 × 10 ⁹ , and cells of own cord blood can be taken from a person (donor) only once (at his birth). This amount is not enough to implement the prototype method. Thus, the implementation of the prototype method requires not only the simultaneous introduction into the body of a significant number of foreign cells (carrying foreign antigens), but also cord blood cells from different donors, that is, a complex “cocktail” of foreign antigens. The degree of the ratio of immunogenicity / immunotolerance of the introduced cells in relation to the patient is not taken into account, due to the degree of histocompatibility of the patient and donor tissues. In this case, the risk of allergic, including anaphylactic reactions, as well as the risk of developing other immunopathological conditions, including immunosuppression caused by the attack of other white blood cells against the patient’s own immune system, sharply increases. In the prototype method, in order to achieve a clinical effect using umbilical cord blood stem cells, it is necessary to simultaneously introduce a significant amount of nucleated cells. If we combine stem cells from different donors, then, firstly, the risk of immunopathological reactions to such a complex spectrum of antigens, including “minor” histocompatibility antigens, sharply increases. In addition, the simultaneous immunization with cells (antigens) of various donors, as is expected in the implementation of the prototype method, leads to the impossibility of re-applying the method in the same patient. Attempts to implement the prototype method for the treatment of these diseases in 5 cases out of 10 led to toxic and allergic reactions, with the development of immunosuppression in all patients within 7-30 days after administration. An attempt to re-use the prototype method in patients led to toxic-allergic reactions in most patients in whom the prototype method was previously used.

An object of the present invention is to reduce the risk of toxic and immunopathological complications.

According to the invention, in a method for treating coronary heart disease, or myocardial infarction and its consequences, or cerebral ischemia caused by atherosclerosis or acute cerebrovascular accident, and its consequences, or lower limb ischemia caused by atherosclerosis, comprising introducing donor umbilical cord blood into the blood, said donor cells have a set of antigens HLA-A, HLA-B, HLA-DR different from the set of antigens HLA-A, HLA-B, HLA-DR of the recipient in at least four antigens; the number of introduced cells is from 2.5 x ^{10 6} to ¹⁰ x ^{10 6} cells per kg of patient body weight per day; the introduction of such cells can be repeated from 2 to 10 times with an interval of 10-60 days.

The recognition system "friend or foe" in humans is represented by proteins of the main histocompatibility complex (JAG), which are indicated in the literature by the abbreviation HLA (Human Leukocyte Antigens - a system of leukocyte antigens). The main immunological task of these surface proteins is to participate in the recognition of a foreign antigen by the body's immune system. HLA proteins bind to the antigen and present (“present”) it on the surface of the cell. Only in this form does a person's T-lymphocyte recognize a foreign antigen. In this case, an immune response is induced, which is designed to eliminate the foreign antigen from the body. Part of the JAG proteins (the so-called class I) are single-stranded amino acid chains that attach to the cell surface using a β ₂ -microglobulin molecule. The proteins of this class include the HLA-A, HLA-B and HLA-C proteins. One such protein is encoded respectively by one gene. Another class of proteins (class II) refers to double-stranded dimeric molecules consisting of alpha and beta chains. Thus, 2 separate genes are required to encode each molecule of class II laser. There is also a class of proteins III JAG. Genes encoding proteins of the main histocompatibility complex are closely linked and are located on the short arm of the 6th chromosome.

OGK proteins are not only responsible for the presentation of foreign proteins to the body's protective (immune) system, but they themselves are the strongest antigens against another organism, to which an immune response develops when they are introduced into this other organism. It must be emphasized that the proteins (antigens) of the laser are also produced by the antigen-presenting cells themselves - B-lymphocytes, activated macrophages, dendritic cells.

HLA genes are commonly referred to as HLA antigens. The names of HLA genes and antigens consist of one or more letters and numbers, for example A 3, B 18, DR 13, DQ 4. The letter means the gene, and the number means the allele of this gene.

HLA genes have high polymorphism. The entire set of HLA genes of one individual is often designated as the HLA genotype. The entire set of HLA antigens present on the surface of cells is designated as the HLA phenotype.

Proteins (antigens) of class I OGK - HLA-A, HLA-B and HLA-C - are encoded by three separate pairs of gene loci. Class I proteins are present on the cell surface of cells of almost all body tissues. The product (protein) of the gene of the fourth locus of class I (HLA - G) is produced only by trophoblast cells.

The region of the genome where the genes encoding class II laser proteins are located is called the D region and is divided into DP, DQ, and DR regions, each of which encodes a double-stranded surface protein in the form of alpha and beta chains. Accordingly, the proteins (antigens) encoded by these genes are called HLA-DP, HLA-DQ and HLA-DR and are displayed on the cell surface in the form of DP alpha beta, DQ alpha beta and DR alpha beta proteins.

HLA genes are inherited codominantly (i.e., each of the genes contributes to the appearance of the trait, i.e., a feature of the corresponding protein) and is transmitted to the offspring in two blocks - one from each parent. Such a block is called the HLA haplotype. Thus, all nucleus-containing human cells have four pairs of antigens (A, B, C and D), - 8 HLA antigens (proteins) per cell, of which 4 proteins are inherited from one and 4 from another parent. Loci of HLA genes are located close to each other and are usually inherited from each parent with virtually no recombination (rearrangement) within the block (haplotype). The recombination frequency within the HLA haplotype leaves about 1%, and therefore among the offspring of two parents there are approximately 25% of cases of complete diploid matching of HLA antigens, 50% of cases of one-haplotype similarity of HLA antigens and 25% of cases of complete mismatch of HLA antigens.

Since the set of OGC antigens (proteins) of the main histocompatibility complex is different for different individuals, when cells of one person (donor) are introduced into the body of another (recipient), the incompatibility of donor cells with recipient cells occurs. Overcoming the histocompatibility barrier has always been the main stimulus for the study of genes and proteins of OGKs, and the development of methods for overcoming it is the main key to the problem of rejection of transplanted foreign organs, tissues and cells in human transplantation and cell engineering.

It is clear that it is impossible to ensure full compatibility with all known HLA antigens. However, many years of experience in transplantology showed that the differences in not all antigens (proteins) are of clinical importance, i.e. the mismatch of not all JAG proteins has the same effect on the likelihood of graft engraftment. It turned out that it is sometimes necessary and sufficient for a successful tissue and cell transplant to select a donor and a recipient compatible with 3-4 HLA antigens, despite the fact that other histocompatibility antigens called “minor” can cause a rejection reaction from the recipient’s immune system , although less strong. In case of unsuccessful combination of several such "minor" antigens, an acute rejection reaction may occur. The molecular basis for the mismatch in the “minor” antigens is unknown, but it is believed to be related to the polymorphism of normal non-pathological intrinsic antigens. When transplanting foreign immunocompetent donor cells (or their predecessors) into the immunocompromised recipient organism, another serious problem arises - the so-called “transplantation reaction against the host (GVHD)”. The danger of GVHD is relevant especially in cases where the recipient's body is previously immunosuppressed for successful transplantation. The weakened immune system of the recipient cannot reject the immunocompetent T cells of the donor, which take root and begin to elicit their own immune response against the cells of the recipient, responding to differences in the proteins - antigens of the laser. GVHD may be acute or chronic. The likelihood of developing and course of GVHD depends on the genetic differences between the cells of the donor and the recipient, the number of donor cells and their phenotype. Even if the donor and recipient are siblings, that is, they are completely genotypically HLA - compatible at all defined loci, the frequency of GVHD remains very high (from 30-70%), which is also associated with differences in "minor" antigens.

Nevertheless, the clinic achieves maximum tissue compatibility by selecting a donor – recipient pair identical in class II laser, in particular, HLA-DR protein, as well as two other class I proteins, namely HLA-A and HLA- B. Such a preliminary determination of the set of JAG proteins in a donor or recipient is called HLA typing. Given that each individual has two varieties of each of these molecules (one from the mother, the other from the father), HLA typing can be represented as determining the characteristics of each of the 6 laser proteins on the cell surface, that is, one pair of species of each of three proteins: HLA-A and HLA-B, and HLA-DR. With the complete coincidence of all 6 donor antigens with 6 recipient antigens, a donor-recipient pair is considered “compatible with 6 antigens”. Accordingly, if we talk about genes encoding these proteins, then in this case 6 alleles of the donor coincide with 6 alleles of the recipient. If the cells of the donor and the recipient coincide in 4 out of 6 (HLA-A and HLA-B, and HLA-DR) possible alleles (and accordingly 4 out of 6 proteins that encode these alleles), then such individuals have “incompatibility in two antigens. " Such a variety of variants of sets of proteins (and, accordingly, genes) of JAG is called JAG polymorphism. Technologically, typing is carried out either by serological methods (by immunological detection using antibodies against a specific protein - JAG antigen) or by molecular methods (by recognition of the gene itself that encodes this protein in the individual’s cell genome). HLA - type for an individual is expressed by a set of letters and numbers. First comes the letter designation (protein) of the gene and then the digital designation of the variant protein (allele of this gene), then the designation of the next protein (gene) and its variants (alleles of the gene), etc. For example, A 2.3 V 5.15 DR 01.15. Improving the methods for recognizing differences between JAG proteins (and, correspondingly, alleles of their genes) leads to further “fragmentation” of allele designations. The allele, originally designated and serologically defined as B 15, subsequently actually turned out to be a combination of two alleles B 62 and B 75.

After the donor stem cells are introduced into the recipient’s organism, on the one hand, there are processes aimed at integrating these cells into the recipient’s tissues with their further reproduction and / or differentiation into functionally mature tissue, and, on the other hand, a process is directed towards eliminating the introduced cells from the body. When transplanting, for example, a foreign donor’s bone marrow containing hematopoietic stem cells (HSC), a completely definite task is set: new cells should take root in a new organism for them and give rise to a hematopoietic germ (repopulate bone marrow). The result of these two processes (repopulation and elimination) determines the fate of the introduced stem cells. The stem cells of the donor themselves, having entered the recipient's body, secrete a huge number of growth factors, hormones and cytokines, the combination of the actions of which leads to certain biological (including therapeutic) effects.

The immunological response of the host organism to foreign cells causes the release of a large number of mediators of the immune response, that is, foreign stem cells themselves are powerful immunomodulators. In addition, donor stem cells can themselves mature to immunocompetent cells and develop their own immune response to host cells, causing, among other things, immunosuppression. The whole complex of multifactor dynamic relationships between donor cells and the recipient organism is determined primarily by the degree of immunological compatibility of the donor cells and the host cells (recipient). On the one hand, the more the donor stem cell is similar to the host cell in terms of JAG antigens, the longer it will circulate in its body and it has more chances of engraftment and further reproduction. On the other hand, the immune response of the recipient's organism to these cells will be small, which leads to the absence of the necessary immunomodulating effect. Thus, the biological effect of introducing foreign stem cells is determined by the parallel development of the following processes:

1. The development of the recipient's immune response to donor cells.

2. Induction by the donor cells of the repopulation of the recipient's own stem cells, either through direct contact or through humoral effects on the recipient's cells.

3. Isolation of biologically active substances, including growth factors, cytokines, tissue hormones, metabolites, by the introduced stem cells.

4. The development of the immune response of donor cells against recipient cells.

5. Repopulation of recipient body tissues by donor cells.

Therapy of a particular disease (with the predominance of a specific pathological component - inflammatory, autoimmune, dystrophic, ischemic or fibrous) using stem cells requires a certain balance of these processes. The determining factors are:

1. The degree of incompatibility of the introduced stem cell of the donor and the recipient cells in the system of OGS;

2. The number of introduced foreign stem cells.

The authors of the present invention found that with a high degree of compatibility of HLA types (coincidence in many HLA antigens) between the cord blood stem cell donor and the recipient, the necessary therapeutic effect in the treatment of diseases associated with circulatory disorders in the tissues cannot be achieved. These diseases include: coronary heart disease, or myocardial infarction and its consequences, or cerebral ischemia caused by atherosclerosis or acute cerebrovascular accident, and its consequences, or lower limb ischemia caused by atherosclerosis.

A well-known compensatory mechanism aimed at improving blood supply to tissues in areas of ischemia is the development of vascular collaterals. Moreover, the immune system (including monocytes and tissue macrophages) plays a huge role in the induction of differentiation of the body's own stem cells into the cells of newly formed vascular collaterals. With the introduction of donor cells compatible with recipient cells for more than 2 antigens (out of 3 pairs of HLA-A, HLA-B, HLA-DR antigens), an adequate immune response to the introduced cells does not occur. When implementing the prototype method without taking into account the ratio of potential immunogenicity / immunotolerance (primarily due to JAG antigens), a huge number of cells are introduced, including progenitor cells of immunocompetent cells. This inevitably leads either to immunosuppression of the recipient's own immune system, or to the occurrence of other undesirable immunopathological reactions.

As it turned out, in order to achieve a significant clinical effect in these diseases, donor cord blood stem cells must differ from the recipient cells in at least 4 antigens from 6 antigens (3 pairs: HLA-A and HLA-B, and HLA-DR). That is, the donor and the recipient must be incompatible in at least four antigens. A series of tests in a mixed culture of lymphocytes, which is widely used in identifying the compatibility of donor and recipient tissues, showed that only with the indicated antigenic difference in the cultivation medium does a sufficient number of trophic and growth factors, biologically active substances capable of affecting the formation and growth of blood vessels, develop. The recipient's lymphocytes and donor umbilical cord blood mononuclear cells were cultured together at 37 degrees Celsius for 5 days, the cells were separated by centrifugation and the in vitro angiogenic activity of the culture fluid was determined using a model of cultured bull aortic cells embedded in a fibrin gel supported by a nylon membrane. Samples of the culture fluid were introduced into the culture medium of the aortic cells, and the growth rate of the vascular disc (vascular stain) in the fibrin gel was determined. The angiogenic activity of the culture fluid of a mixed lymphocyte culture was manifested only if the donor and recipient cells differed in at least 4 antigens from 6 antigens (3 pairs: HLA-A and HLA-B, and HLA-DR). If the donor cells differed from the recipient cells only by 3, 2 or 1 antigen out of 6, the angiogenic effect was not manifested.

In the clinical use of umbilical cord blood mononuclear cells, the therapeutic effect for the above ischemic conditions and their consequences was manifested only if the donor and recipient cells differed in at least 4 antigens from 6 antigens. The authors of the present invention found that if the donor and recipient were compatible with more than 2 antigens (i.e., 3, 4, 5 or 6 antigens of 3 pairs: HLA-A and HLA-B, and HLA-DR), then the introduction of cord blood stem cells did not lead to a therapeutic effect in these diseases.

When using the proposed method, the effective dose of umbilical cord blood mononuclear cells is when introduced into the bloodstream from 2.5 × 10 ⁶ to 10 × 10 ⁶ MNCs per 1 kg of human weight, which corresponds to not more than 1 × 10 ⁹ MNCs or not more than 3 × 10 ⁹ YSC per patient. Thus, 3 times fewer cells are used than in the prototype method. This makes it possible to use only one donor for the treatment of cord blood cells, since the amount of cord blood of one donor and, accordingly, the number of stem cells of cord blood of one donor is limited.

When applying the proposed method in no case did not cause toxic or immunopathological reactions

The present invention is illustrated by the following examples.

EXAMPLE 1. Treatment of coronary heart disease.

Patient P., 68 years old, was admitted with complaints of paroxysmal compressive pain behind the sternum, stopping with nitroglycerin, as well as weakness and insomnia. He suffers from coronary heart disease (angina pectoris) for 8 years. About 3 months ago, despite the treatment (vasodilators, antiplatelet agents, anti-lipid drugs), he suffered an acute myocardial infarction. Pain behind the sternum appeared even when climbing to the 1-2 floor and when walking to a distance of 50-100 meters. Sometimes pains appear at night, at rest. The pain stopped after taking nitroglycerin. During the last 2 weeks before admission to the hospital, the pain became more frequent, the patient also noted weakness, tinnitus, memory loss, and insomnia. The number of angina attacks was 5-7 per day. Also, the patient complained of regular rises in blood pressure up to 200/100 mm Hg.

Upon admission to the hospital, the condition is satisfactory. The position in bed is active, but preference is lying. HELL - 176/95 mm RT. Art., heart rate 72 beats / min, NPV 24 / min, body temperature 36.6 ° C.

Auscultation of the lungs: hard breathing, dry whistling rales are heard over the entire surface of the lungs mainly in the area of the projection of the bronchi. Examination in the test on a bicycle ergometer revealed a sharp decrease in exercise tolerance, the reason for stopping the test was the occurrence of chest pains with the simultaneous occurrence of ST segment depression on the electrocardiogram. The threshold load power was 55 W, the test execution time was 6.5 minutes. An angiographic examination revealed occlusion of the main left coronary artery by more than 60%. Clinical and biochemical parameters of blood are normal. Urine analysis without features. When electrocardiography revealed focal cardiosclerosis in the posterior septal region, local intraventricular block, moderate changes in the myocardium of the upper and lateral walls of the left ventricle. Echocardiography revealed focal changes in the apex, dilatation of the upper chambers of the heart with a slight decrease in contractile function of the left ventricular myocardium, fibrosis of the interventricular septum, segmental fibrosis of the walls of the aortic root, structures of the aortic and mitral valves, mitral insufficiency of 1 tbsp. Despite the ongoing drug therapy, the symptoms of coronary heart disease did not regress. Based on angiographic data (significant stenosis of the main left coronary artery), the patient was offered coronary artery bypass grafting, which the patient refused. With the consent of the patient, he was treated with the claimed method. The established HLA is the type of donor A 1.25 B 37.44 DR 01.15, and the HLA is the type of patient A 1.25 B 18.14 DR 08.13. Thus, the set of antigens of HLA-A, HLA-B, HLA-DR of donor cells differed from the set of antigens of HLA-A, HLA-B, HLA-DR of the patient (recipient) in 4 antigens from 6 antigens. Mononuclear cells of umbilical cord blood were introduced in an amount of 2.5 × 10 ⁶ cells / kg of body weight once, by intravenous infusion for 2 hours at a speed of 8 ml / hour. Over the next 2 months after the treatment, the patient's condition improved, the number of angina attacks by the end of the 2nd month decreased to 2-3 per day, shortness of breath decreased, nightly attacks of chest pains almost stopped. During bicycle ergometric research, the power of the threshold load was 85 W, the test execution time increased to 9 minutes. At the patient's insistence, the cell infusion was repeated (60 days after the first injection). Cord blood cells of the same donor were introduced in an amount of 5.0 × 10 ⁶ / kg body weight. 1.5 months after the second injection of cells, the patient was able to climb 3-4 floors without pain, there were almost no angina attacks at night, with a bicycle ergometry study, the power of the threshold load was 110 W, the test execution time increased to 14 minutes.

Clinical analysis of blood and urine, biochemical blood tests unchanged. The immunogram is normal.

EXAMPLE 2. Treatment of acute myocardial infarction.

Patient, L., 71 years old. He suffers from hypertension for about 12 years, angina pectoris - about 10 years. 5-7 years ago, attacks of paroxysmal tachycardia began to appear, which stopped on their own or after taking verapamil. A history of several hypertensive crises over the past 7 years (maximum blood pressure - 210/100 mm Hg). Was admitted to the hospital with complaints of burning chest pains, which first occurred 2 days before hospitalization after significant physical exertion. The pain was first stopped by nitroglycerin 30-40 minutes after the occurrence, repeating several times a day. On the day of hospitalization, the pain became especially severe, lasted more than 3 hours, did not stop with nitroglycerin, radiated to the left arm, shoulder and lower jaw, was accompanied by sharp weakness. Severe headaches, dizziness, veil, fog before the eyes, sensation of pulsation of blood vessels in the occipital and temporal areas, nausea and vomiting were added. All this forced the patient to call emergency care. Upon admission to the hospital HELL 170/100 mm Hg, heart rate 75, the patient is pale, slightly inhibited, pronounced cyanosis of the lips and feet, a sharp expansion of the borders of the heart to the left, systolic murmur at the apex, conducted to the Botkin-Erb point. When electrocardiography: QRS - 0.16; RR 0.46; QT - 0.36; PQ - 0.20; Heart rate - 70 bpm; sinus rhythm. Focal changes in the posterior wall of the left ventricle, acute stage. Ultrasound examination revealed dilatation of the left heart, moderately severe left ventricular myocardial hypertrophy, myocardial contractility was moderately reduced, and complete blockade of the right bundle branch block was revealed. A clinical blood test revealed an increase in ESR - 20 mm / h, in a biochemical blood test revealed an increase in blood enzymes: lactate dehydrogenase (LDH) - 250 nmol / l (at a rate of 89-221 nmol / l), creatine phosphate kinase (CPK) - 575 nmol / l (with a norm of 26-174 nmol / l). Diagnosis: coronary heart disease, acute myocardial infarction of the posterior wall of the left ventricle, acute stage, coronary atherosclerosis. Blockade of the right bundle branch block. Hypertension stage III.

The patient's treatment included the relief of a pain attack (Promedol), vasodilators (nitrates), lowering blood pressure (intravenous magnesium sulfate solution, verapamil), antiplatelet agents (aspirin), anticoagulants, glucose solution.

Over the next week, there was a pronounced positive dynamics: the cessation of sternal pain, discomfort in the heart, a decrease in blood pressure, a marked decrease in weakness. The deterioration of the patient's condition was observed on the 8th day of treatment, when blood pressure again increased to 170/100 mm Hg, pulse 75 beats / min, headache, nausea and vomiting appeared. An ECG revealed ischemic changes in the anterior wall of the left ventricle. In addition to the ongoing drug therapy, the patient was treated with the claimed method. The established HLA is donor type A 1.25 B 18.13 DR 01.08, and HLA is patient type A 3.28 B 7.35 DR 01.08. Thus, the set of antigens of HLA-A, HLA-B, HLA-DR of donor cells differed from the set of antigens of HLA-A, HLA-B, HLA-DR of the patient (recipient) in 4 antigens from 6 antigens. Mononuclear cells were injected at a dose of 5.5 × 10 ⁶ cells / kg body weight once, by intravenous infusion for 2 hours at a rate of 10 ml / hour. Over the next 2 weeks, the patient's condition improved. Pain in the heart did not bother. An electrocardiographic study revealed an acceleration (more than 1.5 times) in the dynamics of ECG indicators characteristic of the course of acute myocardial infarction. Normalization of the levels of enzymatic activity of the blood also occurred at an accelerated pace. The introduction of cord blood mononuclear cells was repeated 10 days after the first injection. Established HLA is donor type A 1.34 B 40.56 DR 15.13. Thus, the set of antigens of HLA-A, HLA-B, HLA-DR of donor cells differed from the set of antigens of HLA-A, HLA-B, HLA-DR of the patient (recipient) in 6 antigens from 6 antigens. Mononuclear cells were injected at a dose of 5.5 × 10 ⁶ cells / kg body weight once, by intravenous infusion for 2 hours at a rate of 10 ml / hour. An ultrasound examination of the heart on day 20 after the second injection of cells revealed a significant improvement in the contractile activity of the myocardium, a decrease in the degree of dilatation of the heart. Side effects from the application of the claimed method was not observed.

EXAMPLE 3. Treatment of the consequences of acute myocardial infarction.

Patient N., 58 years old. At the age of 50, he suffered the first left ventricular myocardial infarction, about which he was treated in a hospital and then outpatient. Therapy in the hospital was carried out with heparin, nitroglycerin, corinfarum, aspirin. After the extract was recommended constant application hood and propranolol in _^1/2 tablets morning and evening. The patient followed the recommendations, but eight months before this hospitalization he suffered a second myocardial infarction of the posterolateral wall of the left ventricle. It was treated stationary (antiplatelet agents, vasodilators, anticoagulants). After discharge, the patient appeared and began to grow short of breath. Shortness of breath first occurred during physical exertion, and then began to occur at rest. First there was pastility, and then swelling of the lower extremities. Upon admission to the hospital with an echocardiographic study, a significant decrease in the contractility of the myocardium was established, with areas of physical inactivity in places of scarred myocardial tissue. Diagnosis: coronary heart disease (painless form), post-infarction cardiosclerosis, complete blockade of the left bundle branch block. Circulatory failure 2B Art., Ischemic cardiomyopathy. The patient was prescribed furosemide, veroshpiron, propranolol and enalapril. The patient was treated with the claimed method. The established HLA is the type of donor A 1.2 V 40.56 DR 15.13, and the HLA is the type of patient A 1.2 V 18.14 DR 01.08. Thus, the set of antigens of HLA-A, HLA-B, HLA-DR of donor cells differed from the set of antigens of HLA-A, HLA-B, HLA-DR of the patient (recipient) in 4 antigens from 6 antigens. Mononuclear cells of umbilical cord blood were injected in an amount of 8 × 10 ⁶ cells / kg body weight by intravenous infusion. 30 days after the introduction of cells, the patient noted an improvement, shortness of breath at rest disappeared, the dose of diuretics was halved. According to the data of an ultrasound scan of the heart (echocardiography), performed a month after the introduction of the cells, the final diastolic volume of the left ventricle decreased from 210 to 186 ml, the ejection fraction increased from 35 to 45%. The functional class of heart failure according to the classification of the New York Heart Association (NYHA) has changed from III-IV to II-III.

EXAMPLE 4. Treatment of cerebral ischemia caused by atherosclerosis.

Patient N., 71 years old, was admitted to the hospital with complaints of headaches that occur more often in the evening or at night, of different localization (sometimes half the head, sometimes the whole head), which the patient removed with analgesics (analgin, baralgin); dizziness. For many years suffers from orthostatic hypotension.

A complex of laboratory and instrumental studies was carried out. On the ECG: semi-horizontal position of the electrical axis of the heart, slowing of the atrial conduction, local disturbances of the intraventricular conduction.

An ultrasound examination of the vessels revealed multiple atherosclerotic lesions of the brachiocephalic vessels, stenosis of more than 60% of the lumen of the left internal carotid artery, circulatory failure in the vertebrobasilar basin due to extra- and intracranial atherosclerotic lesions of the vertebral artery (mainly left) and the main artery. Computed tomography of the brain also revealed aneurysm of the right internal carotid artery. In the hospital, conservative therapy was carried out: sibazon, antiplatelet agents, analgin. Taking vasodilator drugs due to the presence of hypotension was contraindicated.

As a result of the above treatment, the state of health improved slightly, dizziness and fainting continued.

With the consent of the patient, he was treated with the claimed method. Established HLA is a donor type A of 3.28 B, 18.13 DR 01.15, and HLA is a patient type A of 3.30 B, 7.40 DR 03.07. Thus, the set of antigens of HLA-A, HLA-B, HLA-DR of donor cells differed from the set of antigens of HLA-A, HLA-B, HLA-DR of the patient (recipient) for 5 antigens from 6 antigens. Mononuclear cells of cord blood were introduced in an amount of 5 × 10 ⁶ cells / kg body weight by intravenous infusion. Over the next three weeks, the patient's condition gradually improved. Dizziness persisted, but fainting ceased. Sleep improved, headaches disappeared. Laboratory tests of blood and urine did not reveal any deviations.

EXAMPLE 5. Treatment of cerebral ischemia caused by acute cerebrovascular accident.

Patient K., 80 years old, during the usual physical patient felt severe headache, dizziness. Involuntary urination occurred, ceased to understand speech, lost the ability to move in the left half of the body. During hospitalization, the general condition of the patient was difficult, the position was passive, his mood was depressed, his face was indifferent, his muscle tone was reduced. Speech contact was difficult due to motor aphasia, the patient was busy, slowed down, thinking was slow, quickly tired during the conversation, spoke monosyllables, her voice was quiet, delirium and hallucinations were not observed. A defeat of the third pair of nerves was noted: the width of the palpebral fissures is not the same S> D. The left pupil has been dilated. The pupils reacted to the light. There was no doubling of objects in front of my eyes. Pupil convergence was impaired. The defeat of the VII pair was noted: the patient could not wrinkle her forehead, while squinting the eyes, the eyelashes did not hide in the skin folds. Nasolabial folds were weak. Examination of the circular muscle of the mouth revealed weakness on the left. The defeat of the XII pair of nerves was noted: when the tongue is protruded, deviation to the left is noted. The presence of symptoms of Babinsky and Zhukovsky on the left was noted, in addition, a decrease in surface, temperature and tactile sensitivity in the left half of the body. Active movements were restricted in the left limbs. Muscle tone was reduced in the left arm and left leg. The patient was not stable in the Romberg position. The patient could not perform a palatine and knee-calcaneal test. High tendon reflexes with a left-sided prevalence were noted. There was a proboscis reflex on both sides. Neutrophilic leukocytosis and an increase in ESR were noted in the blood. No blood was found in the cerebrospinal fluid, hemorrhagic foci in the brain were not detected by examination on a computed tomograph. Diagnosis: cerebrovascular accident according to ischemic type in the pool of the right middle cerebral artery with left-side hemiparesis and hemihyposthesia. Hypertension of the III degree. Atherosclerosis of the cerebral vessels.

Within 2 weeks in the hospital, the patient received antiplatelet agents, anticoagulants, antihypertensive, nootropic drugs, as well as vitamin therapy and drugs that improve cerebral circulation, but there was almost no positive dynamics. The patient was treated with the claimed method. Established HLA is donor type A 1.34 B 40.56 DR 01.08, and HLA is patient type A 3.28 B 7.40 DR 01.03. Thus, the set of antigens of HLA-A, HLA-B, HLA-DR of donor cells differed from the set of antigens of HLA-A, HLA-B, HLA-DR of the patient (recipient) in 4 antigens from 6 antigens. Mononuclear cells of the cord blood of the donor were introduced in an amount of 10 × 10 ⁶ mononuclear cells / kg of body weight by intravenous infusion. Over the next month, the patient's condition improved significantly. Sensitivity was restored in the left half of the body, signs of damage to the II and III pairs of nerves regressed. Finger and heel tests began to be performed. The patient began to easily engage in verbal contact, lethargy was not observed.

EXAMPLE 6. Treatment of the consequences of cerebral ischemia caused by acute cerebrovascular insufficiency.

Patient F., 60 years old, was admitted to the hospital in the order of planned hospitalization with complaints of weakness and restriction of movements in the right arm and leg, swelling in them. Sick for about a year, when after taking a significant amount of alcohol, waking up in the morning, he found that he could not move his right arm and leg, he could not carry out the movements of the facial muscles of the right half of his face, and speech was difficult. The patient was urgently hospitalized in a neurological hospital, where he was diagnosed with ischemic stroke in the basin of the left middle cerebral artery with right-sided hemiplegia. After the treatment (vasodilators, antiplatelet agents), the phenomena of right-sided hemilegia significantly decreased, and the patient was discharged in satisfactory condition with residual effects in the form of right-sided hemiparesis. The patient did not notice any deterioration or improvement in the condition prior to the time of this hospitalization. A neurological examination revealed a decrease in strength in the right hand and right leg, a feeling of numbness in the right hand and a decrease in skin sensitivity on both hands. On the paresis side, deep reflexes are sharply increased, there is a clonus of the foot and patella. Strength of the hands: 27 kg on the right, 42 kg on the left. Muscular strength of the legs: 2 points on the left, 3 points on the right with a maximum of 5 points (the higher the score, the greater the strength). The patient was treated with the claimed method. Established HLA is the type of donor A 1.25 V 7.35 DR 15.13, and HLA is the type of patient A 1.34 V 37.41 DR 02.07. Thus, the set of antigens of HLA-A, HLA-B, HLA-DR of donor cells differed from the set of antigens of HLA-A, HLA-B, HLA-DR of the patient (recipient) for 5 antigens from 6 antigens. Mononuclear cells of umbilical cord blood were injected in an amount of 10 × 10 ⁶ cells / kg body weight by intravenous infusion. A study of neurological status 3 weeks after this showed a decrease in neurological deficit. The numbness in the right hand disappeared, the coordination of movements improved, the sensitivity of the skin of the hands increased. Clonus of the foot and patella is not noted, deep reflexes are close to normal. The strength of the hands after treatment by the claimed method: 42 kg on the right, 42 kg on the left, and leg muscle strength: 3 points on the left, 4 points on the right.

EXAMPLE 7. Treatment of lower limb ischemia caused by atherosclerosis.

Patient A., 71 years old. Symptoms of the disease appeared about 2 years ago, when pain appeared in the calf muscles when walking. The treatment (trental, parmidin) was carried out with an insignificant effect: the pain slightly decreased, but did not disappear. At the same time, he suffered a myocardial infarction. After 2 years, against the background of the same treatment, the pain began to increase again. By the time of admission to the hospital, pain in the left calf muscle occurred during the passage of about 50 meters. He took trental constantly, parmidin.

When examining the pulse on the radial arteries of good filling, the rhythm is correct, heart rate 92 beats / min., Blood pressure 160/95 mm Hg. Ripple on the common carotid, temporal, brachial arteries, abdominal aorta is preserved. With auscultation of the carotid arteries and the abdominal aorta, vascular murmurs are not detected. Pulsations on the right femoral, right popliteal, right and left posterior tibial arteries, right dorsal artery of the foot are not determined. On the left popliteal artery and the left dorsal artery of the foot, the pulsation is sharply weakened. In the left inguinal region in the projection of the left femoral artery, a rough systolic murmur is auscultated. Samples of Goldflam, Samuels, Panchenko's knee phenomenon, symptom of compression of the nail bed and symptom of plantar ischemia (Oppel) are positive. Gomans symptom is negative. An angiographic examination revealed impaired patency of the superficial femoral artery on the left.

The skin of the legs and feet is thinned, cyanotic, dry, flaky, there are cracks. On the skin of the feet - severe hyperkeratosis. There is a deformation of the nail plates of the toes, violation of the hair of the skin of the feet and legs. The subcutaneous fatty tissue of the lower extremities is thinned. The muscles of both thighs, legs and feet are atrophic;

muscle tone and muscle strength of the lower extremities are reduced.

An angiographic examination was performed, as well as laser dopplerography of the tissue blood flow of the lower extremities. To study muscle blood flow under aseptic conditions, a disposable needle was inserted into the medial head of the gastrocnemius muscle at the level of the border of the middle and lower third of the left calf by means of a puncture, a laser fluometer sensor was passed through its inner lumen. Laser dopplerography revealed a decrease in tissue blood flow in the muscle of the left leg to 0.72 ml / min / 100 g of muscle tissue (at a rate of 6.5 ml / min / 100 g of muscle tissue).

In connection with the exhaustion of the possibility of conservative treatment (vasodilators, antiplatelet agents, vitamin therapy), the patient was offered surgical treatment. The patient refused from reconstructive-plastic (shunting) surgical treatment. With the consent of the patient, he was treated with the claimed method. The established HLA type of donor A is 1.2 V 37.41 DR 01.03, and HLA is the type of patient A 3.28 V 7.35 DR 08.13. Thus, the set of antigens of HLA-A, HLA-B, HLA-DR of donor cells differed from the set of antigens of HLA-A, HLA-B, HLA-DR of the patient (recipient) in 6 antigens from 6 antigens. Mononuclear cells were introduced at a dose of 7.5 × 10 ⁶ cells / kg body weight once, by intravenous infusion for 2 hours at a rate of 10 ml / hour. 3 months after the therapy, the distance the patient passed without pain in the calf muscles increased 3 times. The indicator of tissue blood flow according to laser flowmetry increased to 3.5 ml / min / 100 g of tissue. The trophic indices of the lower extremities improved - peeling of the skin of the legs disappeared, the tone of the calf muscles improved. Blood counts before treatment with the claimed method: hemoglobin - 156.0 g / l; white blood cells - 4.7 × 10%; neutrophils: stab - 3%, segmented - 54%, eosinophils - 4%; lymphocytes - 39%; monocytes - 6%; ESR - 2 mm / h. General analysis of urine before exposure to the treatment method: straw-yellow color, amount - 240 ml, relative density - 1015; alkaline reaction; protein - traces; no glucose; leukocytes - 4-5 in the field of view, red blood cells - single in the field of view; cylinders are hyaline. General analysis of blood after therapy by the proposed method: hemoglobin - 148 g / l; white blood cells - 4.8 × 10 ⁹ / l; neutrophils: stab - 13%, segmented - 59%; lymphocytes - 27%; monocytes - 4%; ESR - 7 mm / h. Biochemical analysis of blood before treatment: total protein 69 g / l, urea 5.5 mmol / l, creatinine 68 mmol / l, total bilirubin 10.5 μmol / l, ALT 85 nmol / s × l, ACT 82 nmol / s × l, α-amylase 14 μg / s × l, alkaline phosphatase 2.3 nmol / s × l, glucose 4.61 mmol / l, thymol sample 2. Biochemical analysis of blood after treatment: total protein 70 g / l, urea 4.2 mmol / L, creatinine 65 mmol / L, total bilirubin 41.3 μmol / L, direct bilirubin 15.4 μmol / L, ALT 72 nmol / s × L, ACT 104 nmol / s × L, α-amylase 7 μg / s × L, glucose 5.52 mmol / L, thymol sample 2. ECG before treatment: sinus rhythm, blue owl tachycardia (heart rate 105 beats / min); single ventricular extrasystoles. The vertical position of the electrical axis of the heart. Cicatricial changes on the posterior and anterior walls of the left ventricle. Left ventricular hypertrophy. ECG after treatment: sinus rhythm, sinus tachycardia (heart rate of 95 beats / min). Single ventricular extrasystoles. The vertical position of the electrical axis of the heart. Cicatricial changes on the posterior and anterior walls of the left ventricle. Left ventricular hypertrophy.

Any undesirable, including delayed, phenomena when applying the claimed method was not observed. The immunogram is normal.

EXAMPLE 8. Treatment of ischemia caused by atherosclerosis, in particular ischemia of the lower extremities with multiple course introduction of mononuclear cells.

Patient F., 52 years old, is in the hospital, was admitted with complaints of alternating pains in the lower extremities that occur when walking, chills and tingling in the toes, pain in the right half of the foot, worse when walking. The nature of the pain was characterized as stitching and pulling, without irradiation. Sick for 5 years, when pain first appeared when walking, passing at rest. It was observed by a therapist. Periodically took vasodilators and anticoagulants (trental). An examination of the patient revealed ulcerative necrotic changes in the distal parts of the fifth toe of the right foot, revealed a violation of nail growth. Violation of the hair on the legs. The skin is cyanotic, there is a violation of pigmentation ("marble" skin or ivory skin. Hypotrophy of the leg muscles was observed). The feet are cold, there is no ripple on the arteries of the foot and the popliteal artery of the right leg. On the femoral artery of the right leg, the pulsation is weakened; satisfactory on the femoral, popliteal, and arteries of the left foot. Dopplerographic examination revealed pronounced blood flow disorders in the femoral and iliac arteries, more to the right. Clinical and biochemical blood and urine tests did not reveal abnormalities, with the exception of elevated levels of low density lipoproteins. The distance of painless walking on the treadmill was 82 meters. On the basis of instrumental and physical examinations, a diagnosis was made of obliterating atherosclerosis of the lower extremities, ischemia IIIb, occlusion of the femoral-popliteal segment on the right of the third century, Lerish’s syndrome, ulcerative necrotic changes of the 5th toe of the right foot. The patient was prescribed a dairy and vegetable diet with restriction of fatty, salty and spicy foods, barotherapy (8 sessions), reopoliglyukin, xanthinol nicotinate, trental, topical use of alcoholic solution of furatsilina for foot ulceration. Despite the treatment carried out for 2 weeks, the patient's condition did not improve. Tolerance to walking did not increase, and the depth of the ulcerative lesion of the foot tended to increase. With the consent of the patient he was given a course of treatment of the claimed method. Established HLA - Patient Type A 1.3 B 7.8 DR 01.02. The introduction of cells was repeated several times as follows (see table):

Table Dose of cells (× 10 ⁶ / kg body weight) HLA - type of donor cells Interval after previous administration, days The number of excellent antigens HLA-A, HLA-B, HLA-DR of the donor and recipient 7.5 A 3.11 V 14.44 DR 02.07 - 4 from 6 2,5 A 2.11 V 08.13 DR 02.15 10 4 from 6 5,6 A 2.3 V 39.44 DR 08.15 fourteen 4 from 6 5.2 A 1.2V 2.14 DR 07.15 fourteen 5 from 6 6.3 A 11.30 V 2.17 DR 02.07 twenty 5 from 6 10.0 A 1.3V 13.35 DR 01.07 32 5 from 6 10.0 A 1.2 V 13.14 DR 01.07 60 4 from 6 9.1 A 1.2 V 14.44 DR 01.15 fourteen 4 from 6 5.3 A 2.11 V 17.27 DR 08.15 fourteen 6 from 6 4.7 A 2.11 V 14.44 DR 02.07 27 5 from 6

A suspension of cord blood mononuclear cells was administered by intravenous infusion.

After 2 weeks from the start of therapy, the patient noted a decrease in the intensity of intermittent claudication. This was confirmed during the treadmill test and Doppler study. The distance of painless walking increased to 112 m. The speed of the main blood flow in the arteries of the feet and legs also increased: the average speed of blood flow from 0.77 cm / s (before treatment) to 1.11 cm / s, and the speed of linear blood flow from 5.43 cm / s to 5.96 cm / s. The level of systolic and diastolic blood pressure has not changed. At the 8th week of treatment, the foot ulcer almost completely epithelized. After 10 weeks of treatment, the positive dynamics in the clinical picture became even more pronounced: the distance of painless walking continued to increase, reaching 119 m, the average blood flow velocity and linear velocity of blood flow increased. The final assessment of the effectiveness of treatment of patients after 2 weeks at the end of the course. The average blood flow velocity increased to 1.59 cm / s, the linear blood flow velocity to 6.12 cm / s, which practically makes up the indicators characteristic of healthy individuals. The distance of painless walking reached 150 m. Biochemical and clinical laboratory parameters were within the age norm. Thus, the claimed method is highly effective in its version of the course treatment of ischemic disorders caused by atherosclerosis, in particular ischemic disorders in the lower extremities.

Claims (3)

1. A method of treating coronary heart disease, or myocardial infarction and its consequences, or cerebral ischemia caused by atherosclerosis or acute cerebrovascular accident, and its consequences, or lower limb ischemia caused by atherosclerosis, comprising introducing donor umbilical cord blood into the blood, characterized the fact that these donor cells have a set of antigens HLA-A, HLA-B, HLA-DR, different from a set of antigens HLA-A, HLA-B, HLA-DR of the recipient in at least four antigens. 2. The method according to claim 1, characterized in that the number of introduced cells is from 2.5 · 10 ⁶ to 10 · 10 ⁶ per kg of body weight per day. 3. The method according to claim 1, characterized in that the introduction of mononuclear cells of cord blood of a donor is repeated from 2 to 10 times with an interval of 10-60 days.