Cell-based therapy of ischemic tissue
SUBSTANCE: population of mononuclear cells or non-germ stem cells, which is saturated with cells of monocytic lineage containing promonocytes, is used for treatment of ischemia with a patient.
EFFECT: invention allows effective treatment of ischemia with a patient by injection of the above population of therapeutical cells to ischemic tissue of the patient.
13 cl, 8 dwg, 5 tbl, 3 ex
The present invention was made with government support under grant No. RO1NS52839, which was provided by the National Institute of neurological disorders and stroke. The government has rights to this invention.
The scope of the invention
The present invention relates to the treatment of ischemic conditions and diseases, in particular myocardial ischemia, cerebral ischemia and ischemia of the extremities, using monocytes isolated from peripheral blood, umbilical cord blood (HUCB), and bone marrow. The present invention relates to a therapy based on the populations of therapeutic cells, enriched in undifferentiated cells of the monocytic line of differentiation, their compositions and the methods of production. The present invention also relates to methods of using such therapies and compositions for the treatment or prevention of ischemia or otherwise, to stimulate the tissue perfusion and improve the education of collateral blood vessels. According to one object of the present invention, the population of mononuclear cells enriched in undifferentiated cells of the monocytic line of differentiation, is used for treatment of ischemia of the heart and angina. According to another object of the present invention, the disease is stroke, and adifference the bathrooms cells macrophage line differentiation separated from human peripheral blood umbilical cord blood (HUCB), and bone marrow.
The level of technology
Angina is associated with pain and discomfort in the chest that is caused by insufficient blood flow to the heart muscle. Distribution of cases of angina in adults of the U.S. population older than 20 years are estimated to be $ 9,100,000. Stable angina is a angina where pain occurs in a predictable manner during physical effort or impact on the subject of emotional stress. Distribution of cases of stable angina in adults of U.S. residents over the age of 45 (no corresponding myocardial infarction) is 500,000. See Rosamond et al. Circulation 117(4): e25 (2008).
Currently, therapies include aspirin, beta-blockers (e.g. carvedilol, propranolol, atenolol), nitroglycerin (for emergency), vasodilator, such as calcium channel blockers (such as nifedipine (adalat) and amlodipine), Mononitrate from-sorbed and nicorandil inhibitors channel If (for example, ivabradine), ACE inhibitors, statins and ranolazine (Ranexa). However, such therapy, usually only treat the pain, without the prevention of recurring pain, but not all types of pain respond to such treatment. Clinical trials carried out with the use of CD34 stem cells for the treatment of refractory angina, with the aim of research is to improve the possibility of using stem cells to generate new vascularization, that should lead to the prevention of re-emergence of pain. It has not been shown that such treatment is safe and effective. Thus, there is a need in the way of treatment, providing a lasting effect on angina, especially refractory angina.
Care for patients with refractory angina is complex and requires a multifaceted approach. Currently, therapies include aspirin, beta-blockers (e.g. carvedilol, propranolol, atenolol), nitroglycerin (for emergency), vasodilator, such as calcium channel blockers (such as nifedipine (adalat) and amlodipine), isosorbide Mononitrate and nicorandil inhibitors channel If (for example, ivabradine), ACE inhibitors, statins and ranolazine (Ranexa). And such therapy in an attempt to reduce the pain, as a rule, involve complex modes with debilitating schemes taking many different drugs and their combinations. However, such therapy, as a rule, treat pain, without prevent recurring pain, but not all types of pain respond to such treatment.
In addition to pharmaceutical therapies, angina can be treated with interventional techniques, such as percutaneous transluminal coronary angioplasty, or surgery altocor the stationary bypass graft (CABG), but such therapy is not suitable for some people with angina due to a number of factors, such as unfavorable anatomy of the coronary vessels, thin coronary artery, distal or diffusion coronary lesion, etc. Recommendations of the American heart Association on alternative therapies include transmyocardial revascularization laser scalpel (Class 11a), enhanced external counterpulsation, and spinal cord stimulation (Class 11b).
Investigated new methods of treatment, including the use of stem cell populations. A number of studies in the early stages was shown regeneration of cardiac tissue in the case of myocardial infarction when using fractions containing mononuclear stem cells in the bone marrow. Such studies have shown: (i) regeneration of myocardi after a heart attack; (ii) reduction of infarct size; and (iii) de novo expression of cardiac proteins by the cells of human bone marrow. Further extension of these previous studies, several groups have shown the regenerative potential of mesenchymal cells derived from the bone marrow, in different experimental models of heart, and the results were initially based on their myogenic and angiogenic potential.
Clinical trials, mainly in patients with acute myocardial infarction and intrachromosomal stem cells, have already been carried out to study the safety and efficacy of autologous transplantation of cells to improve the recovery of heart. However, these tests have shown controversial results with no apparent reason for the discrepancy, based on the design of clinical research and cell populations or methods of delivery.
In the case of coronary heart disease, some clinical trials have shown that at least the security of mononuclear stem cells derived from bone marrow, with varying degrees of effectiveness. The most common method of delivery in these tests is intramyocardially infusion of stem cells or transendocardial or transapically.
In the case of refractory angina, some groups have conducted clinical trials, mainly using mononuclear bone marrow cells (VMMS), first as a sole treatment or in conjunction with CABG.
For example, Hamano et al. introduced VMS transapically way, together with CABG, five inoperable patients "no option" with ischemic cardiomyopathy. The results showed an objective increase in myocardial perfusion in the injected area three patients. However, the validity of the results of this study violated the accompanying effect of CABG, and therapeutic effects Le is placed VMS remained unclear.
Other researchers have reported on the initial results obtained from endomyocardial injection VMS delivered percutaneous catheter, guided by Electromechanical mapping (NOGA™ system). In General, these non-randomized trials have shown that VMS directly transferred to the ischemic myocardium, leading to reduced symptoms, and also to increase the ability to tolerate physical activity and improved myocardial perfusion and function in patients with refractory angina, however, these results were observed in some but not all patients. In the majority of these trials participated inoperable patients "no option" with ischemic cardiomyopathy.
Recently reported first expected randomized trial in which VMMS endomyocardial were injected with serious coronary heart disease (PROTECT-CAD). This study showed a significant increase in the duration of exercise, ejection fraction of the left ventricle and reduce symptoms caused by stress myocardial ischemia in the group, subject to treatment.
Losordo et al. implemented a double-blind, randomized, placebo-controlled trial phase 1/PA with escalating doses of endomyocardial injection of autologous CD34* stem cells in the case of refractory angina. Parametricity, including the frequency of angina, effectiveness nitroglycerin, duration of physical activity and CCSAC class, showed a trend to improvement of symptoms in patients undergoing treatment with the use of CD34+cells, compared with patients receiving placebo.
Thus, for inoperable patients "no option" with angina pectoris clinical trials with the use of mononuclear stem cells derived from bone marrow, showed some improvement in myocardial perfusion and, but to a lesser extent, improving ventricular function. In most of these studies involved patients with ischemic cardiomyopathy with decreased ejection fraction of the left ventricle from moderate to severe (LVEF). However, no data on treatment based on therapeutic cells (i.e., mononuclear cells or mesoderm stem cells), which could prove a real pain reduction or improvement of perfusion in most or all patients undergoing treatment.
From about 5 to about 15% of patients with chronic disease of the coronary arteries show a strong angina, resulting in a loss of capacity that is not subject to control by a combination of conventional therapeutic tools, including repetitive treatment with the drug therap and, percutaneous transluminal coronary angioplasty (RTSA) and surgery coronary artery bypass graft (CABG) (35, 37). Heavy drinking often leads to a significant deterioration in the quality of life. Reduction of symptoms in inoperable patients "no option" with refractory angina is a complex and demanding process. Alternative therapies according to the recommendations of the American heart Association (11, 14), such as transmyocardial revascularization laser scalpel, enhanced external counterpulsation, and spinal cord stimulation, in the best case, all provide modest results(9, 26, 31, 45). The vast majority of patients with refractory angina (75%) have preserved left ventricular function, and for them, the mortality rate is lower than in patients with coronary heart disease (36, 54); and this group of patients is growing rapidly.
Cell therapy, especially transplantation of autologous bone marrow cells, has emerged as a new possible method of treatment for recovery of the heart. Some hypothetical mechanistic explanations associated with myogenic and angiogenic potential of stem cells, and activation of growth-resident precursor cells through paracrine effects(2, 15-17, 22, 48, 49, 52). Although the regeneration of myocardial tissue and soposto the General restoration of the infarction area have already been shown in experimental animal models, there is uncertainty regarding the transfer of these results to humans (7, 33), the use of VMS transplantation for the regeneration of cardiac tissue is not standard clinical practice.
On the one hand, in patients with refractory angina may improve as a result of therapy on the basis of cells, especially in relation to angiogenesis. Angiogenic effects are considered among researchers, it is very important when studying the effect of cell therapy on patient-man (8).
In some preclinical studies reported angiogenic effects(8, 28, 33, 48). Improvement of angiogenesis was observed in the case of transplantation in the heart of the c-kit cells in the bone marrow (BM), compared with mice that represents the negative control (40). Mobilization of murine BM cells into the bloodstream after acute myocardial infarction leads to regeneration of myocytes and vascular structures (39). A recent study in nonhuman primates using a similar Protocol showed improvement of local microcirculation in the cm-treated animals, which allows to make the assumption that there is a potential angiogenic effects (30). This functional advantage implantation of BM cells is probably due to a paracrine effect, increased angiogenesis through the local release of ones is the R growth factors, such as vascular endothelial growth factor and derived from stromal cell factor 1, among others (8, 48).
In a clinical trial, which involved patients with refractory angina, which was introduced originating from bone marrow stem cells or VMS, there was an improvement in symptoms and ability to endure physical activity, and improved myocardial perfusion(4, 6, 8, 12, 13, 19, 21, 41, 53, 55, 56, 59). Beeres et al. (3) conducted a test using intramyocardial injection of autologous VMMS 25 patients with refractory angina, which showed long-term positive effects exerted on anginalnomu symptoms and myocardial perfusion. Losordo et al. (34) undertook a double-blind, randomized, placebo-controlled trial phase I/IIa with dose escalation using endomyocardial injection of autologous CD34+stem cells in patients with refractory angina, demonstrating increasing duration of physical activity in addition to improving class of angina according to the classification of the Canadian Association of cardiologists (CCSAC) patients entered CD34+cells. Ramshorst et al. (58) conducted a randomized controlled trial intramyocardial injection of bone marrow cells in the case of refractory angina, with short-term n is the following (3-6 months) showing modest improvement in myocardial perfusion, compared with patients receiving placebo.
Watching a minimal improvement in left ventricular and taking into account the clinical response in the previous trial, refractory angina, suggested that the primary action in the transplantation of stem cells in the bone marrow of man is to stimulate myocardial angiogenesis, but not pure migenes. In this case, angiogenesis certainly can improve left ventricular function by the rescue or recovery of hibernating myocardium, but to a limited extent, as was demonstrated in these trials and several meta-analyses(1, 32, 44).
Previous preclinical and clinical studies have confirmed the potential applicability, safety and efficacy of stem cell therapy to restore heart tissue, and participated in the study patients with diseases from acute myocardial infarction to chronic ischemic heart disease (33).
The most significant issue is the applicability of laboratory results in medical practice. Differences in the design of clinical trials, obtaining stem and mononuclear cells, and methods of infusion gave some hope, but in General was on asana reproducible results in different studies (43).
Cerebrovascular disease is considered as one of the five most common non-communicable diseases, sick about 50 million people worldwide, resulting in approximately 5.5 million deaths per year. Of these 50 million stroke occurs in approximately 40 million people.
Stroke is another condition in which ischemia also plays a significant role, as in angina. Stroke is the third leading cause of death in developed countries and is the leading cause of disability in adults. Currently there is only one available method of treatment. Vascular disease affects cognitive and motor function and leads to changes in the immune system. This study focused on the pathophysiology of stroke and the development of new cellular therapy (based on cells derived from umbilical cord blood (HUCB)), which, as has been shown in earlier studies, improves motor dysfunction and reduces the size of the infarct. The role of the immune/inflammatory response in the development of traumatic brain injury after stroke are not clear. After an ischemic event in the immune response leads to the influx of neutrophils, T cells, b cells, natural killer cells, as well as microglia in victims of heart attack hemisphere and the changing profile of these same immune to etok in the peripheral blood. In this study, it was investigated whether the positive effects from the introduction of HUCB to be associated with a specific cell population.
Treatment of stroke can be divided into two categories: prevention and emergency medical care. Warning treatment is currently based on antiplatelet tools, anticoagulation tools, surgical treatment, plastic surgery on vessels, the regulation of lifestyle and medical support. As antiplatelet drugs are usually used aspirin. The use of anticoagulation funds, as it turns out, has no statistical significance. Surgical treatment, as it turns out, is effective for the individual subgroups. Plastic surgery on the blood vessels is still experimental methodology with an insufficient amount of data for analysis. The regulation of lifestyle include Smoking cessation, regular physical training, the regulation of nutrition, limiting salt intake and moderate consumption of alcoholic beverages. Medical support includes drug treatment aimed at lowering blood pressure, lowering cholesterol, controlling diabetes and control problems with blood flow.
Urgent medical care is the use of thrombolytics, arozamena funds therapy on the basis of oxygenated fluorocarbon nutrient emulsion (OFNE), Neuropahty, therapy-based inhibitors of platelet GPIIb/IIIa and rehabilitation/physical therapy.
Thrombolytic means induce or inhibit thrombosis, the most frequently used tool is a tissue plasminogen activator (t-PA). Recombinant t-PA (rt-PA) contributes to the restoration of cerebral circulation by dissolving (decomposition) clots that obstruct blood flow. This treatment is effective, with a very short therapeutic window. Thrombolytic funds must be administered within three hours after the attack. It is also necessary to perform computed tomography before treatment, thus reducing the amount of time available. Genetech Pharmaceuticals produces ACTIV ASE® and is currently the only source of rt-PA.
Neuroprotective funds are medicines that minimize the effects of the ischemic cascade and include, for example, antagonists of glutamate, calcium antagonists, opiate antagonists, GAB A-A agonists, inhibitors of calpain kinase inhibitors and antioxidants. Currently, some clinical trials of acute ischemic stroke. Due to their complementary functions suppress coagula the AI and brain protection, methods for the emergency treatment will be most likely to include a combination of thrombolytic and neuroprotective therapies. However, like thrombolytics most neuroprotective means, to achieve the effect, you must enter within 6 hours after stroke.
Therapy through oxygenated fluorocarbon nutrient emulsion (OFNE) delivers oxygen and nutrients to the brain via the cerebrospinal fluid. Neurophone is an experimental technique in which oxygen-rich blood is redirected through the brain, as a way of minimizing the risk of ischemic stroke. Therapy with inhibitors of the platelet GPIIb/IIIa inhibits the ability of the glycoprotein GPIIb/IIIa receptors on the platelet aggregation or accumulation. Rehabilitation physiotherapy must be timely and must start immediately after a stroke, however, it may not lead to changes in the affected areas of the brain. The goal of rehabilitation is to improve function so that the subject, stroke, could retain the ability to live independently as possible.
Although some methods for the emergency treatment given hope when conducting clinical trials, a study conducted in Cleveland, showed that only 1.8% of patients p is aavso symptoms of a stroke even after t-PA treatment (Katz'an IL, et at., 2000 JAMA, 283:1151-1158). t-PA is currently the most widely used of the above methods for the acute treatment of stroke, however, the number of patients receiving any new "effective" emergency treatment of stroke is estimated that less than 10%. These statistics show a clear need for opportunities for the acute treatment of stroke in a period of time more than 24 hours after a stroke.
For some methods, emergency treatment (i.e. t-PA) time is crucial. Recent studies have shown that 42% of patients who survived a stroke, I waited until 24 hours before being taken to hospital, with an average delivery time is about 6 hours after stroke. t-PA has been shown to increase recovery in 113 patients who underwent treatment, but a recent study conducted on behalf of the FDA (the standard treatment alteplase) showed that about one-third to three-hour treatment was lost, leading to ineffective treatment. Except rehabilitation, other methods for the emergency treatment is still undergoing clinical trials and are not widely available in the United States, especially in rural areas, where there may not be a major medical centers with the necessary expertise in neurology and Department staff MSE of the second means, thus, the availability of any of these new methods of diagnosis of stroke and therapy may be limited for some time.
The cost of stroke care in the United States account for more than 43 billion, including both direct and indirect costs. Direct costs account for about 60% of the total number and include the costs of stay in the hospital, the payment of medical care and the cost of rehabilitation. These costs generally amount to $15,000 per patient in the first three months; however, in about 10% of cases the cost is more than $35,000. Indirect costs make up the remaining part and include compensation for loss of capacity stroke patient and disability guardian stroke patient (see National Institute of Neurological Disorders and Stroke, NIH).
Approximately 750,000 cases of stroke occur each year in the United States, of which 1/3 leads to death. Surviving patients about 1/3 slightly lose capacity, 1/3 becomes incapacitated in a moderate way, and in 1/3 of patients due for a serious loss of capacity. Ischemic stroke accounts for 80% of these cases of stroke.
At that age, which now houses the post-war generation, the total number of strokes, as suggested, is substantially increased. The risk of stroke increases with age. After 55 years, the risk of stroke doubled at key stress is provided every ten years, about 40% of 80-year-old suffered a stroke.
Also over time increases the risk of recurrent stroke. The risk of recurrent stroke is 25-40% of the first five years after the first. As the baby boomers reach their Golden age, this market will grow significantly. Also, the demand for effective treatment will increase up to a critical point.
The inability to effectively attenuate the damaging effects of stroke leads to the need to develop new therapeutic strategies as minimize primary brain injury and recovery of brain lesions immediately after the pathological cascade of stroke went on as usual.
Transplantation of monocytes has been proposed as a treatment for stroke. Due to the complexity of effective treatment of patients after stroke in the art there is a need for ways to improve stroke care.
Neovascularization is an integral process of the inflammatory response and subsequent recovery of the cascade in the affected tissue. Monocytes/macrophages play a key role in the inflammatory process, including angiogenesis, as well as protective mechanisms, due to the manifestation of antibacterial and immunomodulating action. The current research has shown the use of monocytes/macrophages in reg the regulation of angiogenesis in ischemic tissue, tumors and chronic inflammation. In respect of neovascularization, accompanied by restoration of the tissue, monocytes/macrophages should be very attractive for therapy on the basis of cells, due to their significant advantages, such as noncoherent, heterogeneity, the absence of ethical disputes about them, numerous functions, including Pro-angiogenic factors and growth factors, and lung failure. Not only the sources of the adult body, such as bone marrow and peripheral blood, and umbilical cord blood (UCB) can be a potential source of autologous or allogeneic monocytes/macrophages. As the first candidate especially UCB monocytes should be considered, due to their rapid suitability, low suppression of the immune system and numerous functions, such as anti-inflammatory reaction, due to the unique immune and inflammatory immaturity, as well as Pro-angiogenic ability. General characteristics and potential monocytes/macrophages presents for therapy on the basis of cells, especially focuses on the neovascularization and UCB-derived monocytes.
One of interest is the function of monocytes/macrophages is increased angiogenesis associated with inflammatory reactions. Angiogenesis or neovascularization) is the main ele is entom inflammatory processes, including subsequent cascades recovery [Sunderkotter, 1994 #4]. During the initial recovery process of circulating blood monocytes invade the tissues [Bosco, 2008 #3]. In the beginning, nearby endothelial and inflammatory cells regulate the passage of monocytes through the wall of the blood vessels through the release of a number of adhesion and chemotactic substances [Baggiolini, 2000 #9; Imhof, 2004 #2; Bosco, 2008 #3]. Along a chemotactic and oxygen gradients between normal and diseased tissue penetrating vessels monocytes move and going in hypoxic and/or necrotic centers unhealthy tissue before differentiation into tissue macrophages. Suitable pathological tissues approached by monocytes/macrophages to accumulate, are as follows: solid tumor, myocardial or cerebral infarction, chronic synovial joint arthritis or atheromatous plaque, bacterial infection and wounds [Baggiolini, 2000 #9; Murdoch, 2004 #1; Bosco, 2008 #3; Mantovani, 2002 #15] (Fig.8).
After differentiation of monocytes into macrophages, the macrophages in tissues known to exist in the form of polarized populations, M1 and M2 subgroups [Mantovani, 2004 #67; Sica, 2006 #16; Mantovani, 2004 #67; Mantovani, 2002 #15]. While M1 polarized macrophages are strong inflammatory cells that produce proinflammatory cytokines and phagocytic pathogens, M2, macropha and modulate inflammatory responses and contribute to angiogenesis and tissue repair [Mantovani, 2004 #67; Sica, 2006 #16; Mantovani, 2004 #67; Mantovani, 2002 #15]. Interestingly, while gene expression of macrophages, the combination of M1 and M2 subgroups in early wound healing turns into domination M2 genes later [Deonarine, 2007 #68]. During the initial stages of the wound-healing process M1 macrophages lead to a direct inflammatory response, which cleans the wound and eliminate germs and diseased tissues, whereas tissue repair and angiogenesis start due to M2 macrophages at the same time. At the last stage, when cleaning M1 macrophages has been almost completed, the prevailing M2 macrophages continue its work, contributing to tissue repair and angiogenesis [Deonarine, 2007 #68]. Accumulation, obviously, assumes that recruited monocytes/macrophages contribute to the modulation and regulation of neovascularization in ischemic tissues, tumors and chronic inflammation, such as victims of arthritis synovial joint and atherosclerosis.
The prerequisite for the creation of the present invention is sensed for a long period of time, the need for methods and compositions for the treatment of ischemia (preferred examples of which are angina and stroke) and to improve perfusion, which eliminate the main causes of ischemia, and likely lead to improved perfusion than pain, is called such States, and enhance the effectiveness of therapy for such conditions on the basis of therapeutic cells.
Monocytes, which originate from monoblasts, precursor cells of hematopoietic stem cells in the bone marrow (BM), circulate in the bloodstream before penetration into the body tissue. In the tissues, monocytes differentiate into different types of resident tissue macrophages depending on their anatomical location, such as Langerhans cells in the skin, copperhouse liver cells, osteoclasts in the bones, of microglia in the Central nervous system, alveolar macrophages in the lungs and synovial cells of type a in synovial joints (Bosco, et al., 2008, Gordon, 2003, Imhof and Aurrand-Lions, 2004, Murdoch, et al., 2004, Sunder-kotter. Et al., 1994) (Fig.8).
Monocytes/macrophages can perform phagocytosis, using mediators, such as antibodies or complement components, which cover the microbes, or by binding to pathogens directly through specific receptors that recognize them (endocytosis). In addition, monocytes/macrophages are able to kill cells of the host that is infected with pathogens, through the response of the immune system, mediated through antibodies cellular cytotoxicity (Nathan, 1987, Sunderkotter et al., 1994). Moreover, they are unique immunoregulatory cells capable of both stimulate and inhibit immune the activity, including the presentation of antigen to T cells and controlled secretion of a wide range of cytokines and growth factors (Bosco, et al., 2008, Murdoch, et al., 2004, Paulnock, et al., 2000). Briefly, monocytes/macrophages play a major role in the innate immune systems due to its ability to kill pathogens, including phagocytosis and cell-mediated cytotoxicity, and the ability to immunomodulation (Bosco, et al., 2008, Paulnock, et al., 2000).
Angina and stroke are examples of ischemic conditions or condition, when the patient is in need of improved perfusion. In this respect, the present invention partially satisfies the need in new and unique ways to treat angina, stroke or other forms of ischemia. In other ischemic conditions or the condition when the patient is in need of improved perfusion, there is also a need to improve with such treatment.
In one embodiment, the present invention method provides an introduction undifferentiated monocytes to a subject in need of treatment, where undifferentiated monocytes are introduced to the subject systemically, in quantity and in time, in a special way for certain treatment.
The object of the present invention is a method of treating ischemia in a subject, containing injection populations of therapeutic cells, enriched nodifference bannymi cells of the monocytic line differentiation, in the ischemic tissue of the subject. In some embodiments, execution of the present invention ischemia is cardiac ischemia, and ischemic tissue is cardiac muscle. Another object of the present invention is a method of improving perfusion of the subject containing the injection populations of therapeutic cells, enriched in undifferentiated cells of the monocytic line differentiation in tissue of a subject in need of improved perfusion. In certain embodiments of the implementation of the present invention, the tissue is cardiac muscle. Another object of the present invention is a method of treating angina pectoris in a subject, containing injection populations of therapeutic cells, enriched in undifferentiated cells of the monocytic line of differentiation in the cardiac muscle of the subject. In embodiments, the execution of any object of the present invention, the methods may include the injection of at least 105undifferentiated cells of the monocytic line differentiation in populations of therapeutic cells, enriched in undifferentiated cells of the monocytic line of differentiation. The following objects and embodiments of the present invention may also include an embodiment of the present invention, when the population of non-differentiated monocytic cells enters the I heart muscle by, at least two separate injections, at least three separate injections, at least four separate injections, at least five separate injections, at least ten separate injections, at least twenty separate injections, at least thirty separate injections, at least forty separate injections, at least fifty separate injections, at least sixty separate injections, at least seventy separate injections, at least eighty separate injections, at least ninety separate injections or at least a hundred separate injections, or, at least, two hundred separate injections. The following objects and embodiments of the present invention may also include an embodiment of the present invention, when the injection is from 0.05 ml to 0.5 ml, or about 0.2 ml. of the Following objects and embodiments of the present invention may also include an embodiment of the present invention, when the population of therapeutic cells, enriched in undifferentiated cells of the monocytic line of differentiation is autologous or allogeneic. The following objects and embodiments of the present invention may also include an embodiment of the present invention, the hen population of therapeutic cells is a population of mononuclear cells, that is, undifferentiated cells of the monocytic line of differentiation or monocytic stem cells. The following objects and embodiments of the present invention may also include an embodiment of the present invention, when the injection should stage selected from the following stages: selection of stem cells from a sample, using the method, which leads to the enrichment of undifferentiated cells of the monocytic line differentiation; culturing the population of therapeutic cells in the enrichment of undifferentiated cells of the monocytic line of differentiation; and adding the undifferentiated cells of the monocytic line of differentiation of a population of therapeutic cells.
Another object of the present invention relates to injectable therapeutic means and contains a device capable of delivering a metered injection of a therapeutic agent in ischemic tissue, where the device includes a reservoir for a therapeutic agent, and therapeutic agent contains a population of therapeutic cells, enriched in undifferentiated cells of the monocytic line of differentiation. In one embodiment, the present invention population of therapeutic cells represents a mononuclear cell population Following objects and embodiments of the present invention may also include an embodiment of the present invention, when the population of therapeutic cells, enriched in undifferentiated cells of the monocytic line differentiation, contains at least 105undifferentiated cells of the monocytic line of differentiation.
Brief description of drawings
In Fig.1 shows a comparison between the number of undifferentiated intramyocardially injected monocytes (number of cells x 10^6, x-axis) and improved myocardial perfusion (%, y-axis), six months after cellular therapy. The correlation between the number entered by the injection of undifferentiated monocytes and improved myocardial perfusion are shown graphically and statistically significant (p<0.05, FD=6).
In Fig.2 shows a comparison between the number intramyocardial injection (x-axis) and improved myocardial perfusion (%, y-axis), six months after cellular therapy. This graph shows that the improvement in perfusion caused by the injection of cells, not the physical impact of the needles or infusion of fluid, as in this case, is not observed significant correlation between improvement in myocardial perfusion and number of injections (p=n.s; FD=6).
In Fig.3 shows the changes in CCSAC in the next 18 months. Each line on the graph represents one patient registered ReACT, and associated improvements in CCSAC cher the C 18 months of control. The x-axis denotes the class CCSAC (number 4 for refractory angina and 0 for the absence of pain). The y-axis indicates the control of the patient from month to month. The table on the left shows a statistical analysis based on one-sided Wilcoxon criterion to improve CCSAC for 3, 6, 12 and 18 months of control. Relative to the baseline; change the class of angina is statistically significant if p<0.0125 (0.05/4) (correction, Bonferroni).
In Fig.4 shows the changes in myocardial ischemic area, measured by the stress scintigraphy with technetium 12 month control. Each line on the graph represents one patient registered ReACT, and the corresponding estimated using scintigraphy improvement of the ischemic region of the myocardium for the 12 months of monitoring. The x-axis denotes the percentage estimated using scintigraphy myocardial ischemic region. The y-axis indicates the control of the patient from month to month (scintigraphic analysis was conducted only for the sixth and twelfth months). The table below on the right shows the statistical analysis on the basis of one-sided Wilcoxon criterion for assessed using scintigraphy improve ischemic region of the myocardium on the sixth and twelfth month control. Relative to the baseline; change estimated using scintigraphy improve coronary heart of the region of the myocardium is statistically significant, if p<0.025 (0.05/2) (correction, Bonferroni).
Fig.5: Monocytes and macrophages are a critical component of cord blood for recovery of the brain after occlusion of the middle cerebral artery (MCAO) in rats. A) After MCAO there is a significant lesion of the ipsilateral hemisphere, especially in the striatum, hippocampus and cerebral cortex. HUCB treatment 48 hours after MCAO reduces the size of the lesion, whereas the removal of CD14+monocytes and macrophages from the fraction of umbilical cord blood (HUCB) reduces this effect. B) deleting CD14+monocytes and macrophages from HUCB, the volume of a heart attack after MCAO returns to the previous volume level of heart attack, i.e. as if untreated rats with MCAO. The volume of infarction in CD14-depleted group was significantly higher than that of HUCB-treated group.
Fig.6: Speed measurement of motor asymmetry. A) After MCAO reduces the number of steps that can make the damaged limb. HUCB introduction improves the runnability of the limb, which again is lost when deleting CD14+cells (monocytes and macrophages) from HUCB faction. C) Injection only CD14+HUCB cells improves digitalnow function povrejdeniy of the forelimb.
Fig.7: Spontaneous activity decreases with the introduction of CD14+HUCB cells. After MCAO, slid the s become hyperactive. Introduction monocytes and macrophages umbilical cord blood reduces the activity to normal levels (baseline) on numerous motion parameters, including (A) horizontal activity, total distance, preodoleem in the cell, (C) vertical activity (rearing) and (D) counterclockwise.
Fig.8: Schematic representation of the ontogeny of monocytes/macrophages. Pluripotent stem cells differentiate into myeloid or lymphoid precursor cells in the bone marrow. Precursor cells of granulocytes-monocytes originate from a common myeloid cells predecessor before differentiation in myeloblast and monoblast. Monocytes are differentiated from monoblast and then move from the bone marrow into the blood. Blood monocytes differentiate into different types of resident macrophages depending on their anatomic location after penetration into the fabric. On the other hand, at the beginning of the inflammatory process replenishment and transendothelial migration of circulating monocytes complemented by a range of adhesive and chemotactic substances, downregulation of inflammatory cells. Recruited monocytes migrate along a chemotactic and oxygen gradients between normal and diseased tissues and accumulate in inflammatory and hypoxic centers and is emii, or solid tumors, or chronic inflammation, before differentiation into macrophages, which have polarization, M1 or M2 subgroups.
Detailed description of preferred embodiments of the present invention
The prerequisite for the creation of the present invention is sensed for a long period of time, the need for methods and compositions for the treatment of ischemia and to improve perfusion in General. The preferred treatment is the treatment of myocardial ischemia and angina. Methods and compositions include, in particular, with the surprising discovery that the enrichment of a population of stem cells undifferentiated cells of the monocytic line of differentiation increases the reliability of the effectiveness of therapies based on stem cells.
Bone marrow and stem cell populations derived from bone marrow, are a natural source of a wide range of cytokines, which are involved in the control of angiogenic and inflammatory processes.
During the various stages of angiogenesis expressed various cytokines, such as tumor necrosis factor-alpha (TNFα), interleukins (ILs), interferon-(IFN-g) and factor stimulation colonies of macrophages (MCSF). These cytokines induce smooth miocic to the expression of interstitial collagenase and stromelysin, which in turn reduce the indicate local collagen, leading to thinning of the blood vessels and their messy bloating. Cytokines expressed to the walls of blood vessels, are potential chemoattractants for induced inflammatory cell expression of adhesion molecules on the endothelium and their controlando on leukocytes contribute to platelet activity and thrombosis, and inhibit thrombosis.
Monocytes, i.e. undifferentiated monocytes, or promonocyte bone marrow can be activated in response to a chemotactic effect and undergo final differentiation into macrophages. Macrophages play a key role in angiogenesis due to their ability to secrete proteases, growth factors, Monokini, and affect each phase of the angiogenic process, as, for example, lead to changes in the local extracellular matrix, induce endothelial cell migration or proliferation and inhibit the growth of blood vessels with formation of differentiated capillaries.
Physiopathological the process of ischemic diseases is to reduce the perfusion of blood in certain areas of the tissue. Cells in hypoperfusion region suffer from a lack of sufficient supply of oxygen and therefore unable to perform their natural functions. In this regard, induction of angiogenesis can improve the function of the tissue by reducing or restoring the population of dormant cells by increasing the supply of oxygen.
Standard patient with refractory angina with viable myocardium and stored or slightly depressed left ventricular function, is the ideal candidate for angiogenic therapy using intramyocardial injection stem cell populations enriched in undifferentiated cells of the monocytic line of differentiation, according to the present invention.
We conducted a non-randomized clinical trials in patients with refractory angina, with preserved or slightly depressed left ventricular function, and intramyocardial injection VMS as the only therapy to strengthen myocardial blood flow through angiogenesis, were important only well-founded results of stem cell therapy bone marrow and unique specific need in the treatment of participating in trials of patients.
As used herein, the term "population of therapeutic cells" can refer to either the population of mononuclear cells, or a population of stem cells capable of differentiating into cells of mesoderm lines of differentiation, or both.
The term "patient", as used herein, includes an animal, preferably a person who receives treatment, the key preventive treatment, with the use of cells according to the present invention. In the case of treatment of conditions and diseases that are specific to a particular animal, such as a patient, the term patient refers to that specific animal. The term "donor" is used to describe the subject (animal, including humans), which take blood from the umbilical cord or umbilical cord blood cells for use on the patient.
The term "cord blood" used herein refers to blood obtained from a newborn or fetus, most preferably in the newborn, and preferably refers to blood, which is obtained from the umbilical cord or placenta of a newborn. Preferably, the umbilical cord blood from a newborn human. The use of umbilical cord blood as a source of mononuclear cells provides advantages due to the ease of selection of cord blood, without injury to the donor. On the contrary, obtaining bone marrow cells from the donor is traumatic. Cells from umbilical cord blood can be used for autologous transplantation or allogeneic transplantation, when and if necessary. Umbilical cord blood is preferably obtained directly outflow from the umbilical cord and/or aspiration needle from the placenta to the root or the bloated veins. As used in this until Amante, the term "umbilical cord blood cells" refers to cells that are present inside the umbilical cord blood. In one embodiment of the present invention, the umbilical cord blood cells are mononuclear cells are isolated from umbilical cord blood using methods known to a person skilled in the art. In the following embodiment of the present invention, the umbilical cord blood cells can further differentiate before the introduction of the patient.
The term "effective amount" is used herein to describe concentrations or amounts of components, such as the differentiation of umbilical cord blood cells, precursor cells or progenitors, specialized cells, such as nerve and/or neural or glial cells, agents that can overcome the blood-brain barrier, and/or other means, effective to obtain a proper result, including the differentiation of stem cells and/or progenitor cells into specialized cells, such as nerve or neural and/or glial cells, or to describe the treatment of neurological disorders or other pathological condition, including the Central nervous system of the patient, such as stroke, heart attack or accident, or for the implementation of the transplantation of these glue is OK for the patient, subject to treatment. The compositions of the present invention can be used to implement the cell transplantation of umbilical cord blood inside the composition, to obtain a favorable changes in the brain or spinal cord or in the disease or condition to be treated, where such a change is an improvement (such as stopping or treatment of macular disease or condition, reduction of neurological disorders or improving neurological response or complete cure of the disease or condition to be treated.
The terms "stem cells" or "precursor cells", as used interchangeably herein, refer to stem cells and cells-the precursors derived from umbilical cord blood. The terms "stem cells" and "precursor cells" known from the field of engineering (e.g., Stem Cells: Scientific Progress and Future Research Directions, report prepared by the National Institutes of Health, June, 2001). The term "nerve cells" refers to cells having at least a sign of neural or glial phenotype, as, for example, on the basis of one or more neuronal or glial markers, or cells that will differentiate into cells bearing neuronal or glial markers. Examples of neuronal markers that can be used to identify neural cells is according to the present invention, include, for example, neuron-specific nuclear protein, tyrosinekinase, proteins associated with microtubules, and calbindin, among other things. The term "nerve cells" includes cells that are the precursors of nerve cells, that is, stem cells and/or precursor cells that differentiate into nerve cells or nerve cells, or cells which, ultimately, will show neuronal or glial markers, and includes pluripotent stem cells and/or precursor cells that eventually differentiate into neural and/or glial cells. All of the above cells and their progeny should be understood as the nerve cells for the purposes of the present invention. Neural stem cells are cells capable of proliferation, the expression of self-maintenance or recovery over the lifetime of the organism, and to generate associated clonal progeny of nerve cells. Neural stem cells produce neurons, astrocytes and oligodendrocytes during development and can replace a number of nerve cells in the brain of an adult organism. Neural stem cells are the nerve cells for the purposes of the present invention. The terms "nerve cells and neural cells, in General, apply vtimezone the emo for many objects of the present invention. The preferred nerve cells for use in certain objects of the present invention include those cells that show one or more of the neural/neuronal phenotypic markers, such as Musashi-1, Nestin, NeuN, β-tubulin III, GFAP, NF-L, NF-M protein associated with microtubules (MAR), 8100, CNPase, glypican (especially glypican 4), neural pentraxin II, neural PAS 1, associated with the growth of neurons protein 43 protein enhance axon growth, vimentin, Hu, internexin, 04, basic myelin protein and pleiotrophin, among others.
As used herein, "stem cell" is a cell of an embryo, fetus or adult organism, which has, under certain conditions, the ability to be cultivated in a few divisions without differentiation or death. In addition, stem cells can, among other things, to differentiate into at least two separate cell types.
As used herein, "pluripotent stem cells" have the ability to differentiate into at least two different types of cells belonging to different germ layers (mesoderm, endoderm and ectoderm) from which all cells of the body. Pluripotent cells can be derived from embryos.
"Embryonic stem cells" are stolowicki, which are derived from an embryo, usually from a group of cells called the inner cell mass, which is part of the early embryo (4-5 days), called the blastocyst. After removal of blastocyst cells of the inner cell mass can be cultivated like any other stem cells.
"Adult stem cell" is a stem cell isolated from an adult organism (i.e. not from fetal tissue). Adult stem cells, like all stem cells, capable of creating their identical copies through many cycles of cultivation. This property is called "renewal." Adult stem cells typically generate precursor cells or cells of grandparents under certain conditions, which then differentiate or develop into Mature cell types that have characteristic shapes and specialized functions, such as cells that form the walls of blood vessels. Adult stem cells can be isolated from many tissues, including brain, bone marrow, periosteum, peripheral blood, blood vessels, skeletal muscle, epithelia of the skin and digestive system, cornea, teeth pulp, retina, liver, pancreas and adipose tissue. Preferred sources of the cells are peripheral is Skye blood (using factors stimulate stem cells in the bone marrow, such as granulocyte colony-stimulating factor - GCS-F, or without them) and bone marrow for undifferentiated cells of the monocytic line differentiation, and umbilical cord blood for undifferentiated monocytes and monocytic cell lines differentiation.
The term "introduction" is used herein to describe the process by which cells according to the invention, such as umbilical cord blood cells obtained from umbilical cord blood or derived from these more differentiated cells, are administered to a patient for therapeutic purposes. Cells according to the invention can enter in a number of ways, including, but not limited to, parenteral (this term refers to the intravenous and intraarterial, as well as other appropriate parenteral routes), intrathecal, intraventricular, intraparenchymatous (including introduction to the spinal cord, the brain stem or the motor area of the cerebral cortex), intracisternally, intracranial, intrasternal and intranasally, among other ways, which allow the cells according to the invention to migrate to the target site where they are needed. Cells according to the invention can be administered in the form of intact umbilical cord blood and its fractions (including mononuclear cell fraction or the fraction of mononuclear cells, including high con is entrely stem cells or precursor cells). Compositions according to the present invention can be used without treatment with mobilization or means of differentiation ("raw", i.e. without further processing for differentiation of cells within the cord blood sample) or after treatment ("treated") means differentiation, or another tool that makes stem cells and/or precursor cells within the cord blood sample to differentiate into cells exhibiting a differentiated phenotype, such as neural and/or glial phenotype.
Undifferentiated monocytes can be administered systemically or to a target anatomical site, allowing the cells to differentiate in response to physiological signals received by the cell (for example, site-specific differentiation). Alternatively, cells can be subjected to ex vivo differentiation before the introduction of the patient.
Introduction often depends on the disease or condition to be treated, and may preferably be carried out parenterally, for example intravenously, by injection into the cerebrospinal fluid or by direct injection into the affected brain tissue. For example, in the case of Alzheimer's disease, Huntington's disease and Parkinson's disease, the preferred route of administration will be transplantation directly into striped the body (caudate cutamen) or directly into a black substance (Parkinson's disease). In the case of amyotrophic lateral sclerosis (disease Lou Gehrig's disease (als) and multiple sclerosis, preferably the introduction is carried out through the cerebrospinal fluid. In the case of lysosomal storage disorders, the preferred route of administration is intravenous route or introduction via the cerebrospinal fluid. In the case of stroke, the preferred route of administration will depend on where did the stroke, but the introduction may also be administered directly into the affected tissue (which can easily be determined with the use of MRI or other imaging), or introduction can be carried out systematically. In a preferred embodiment of the present invention, the route of administration for treatment of individual postinsult system will, by intravenous or intraarterial injection.
The term "implantation" and "transplantation" are used as synonyms to describe the process by which cells according to the invention are delivered to the site where cells, as expected, have a favorable effect, such as the restoration of the Central nervous system of the patient (may be a recovery, cognitive or behavioral deficits caused by lesions), treatment of neurodegenerative diseases or eliminate effects ner the aqueous defeat, caused by stroke, cardiovascular disease, heart attack or injury, or trauma, or genetic disorder, or associated with elements and environmental factors adversely affecting the brain and/or spinal cord, which may be caused, for example, an accident or other incident. Cells according to the invention can also be delivered to a remote area of the body by any variant of the method of administration, described above, on the basis of cell migration to the corresponding area. Preferably the cells are administered together with an agent that can overcome the blood-brain barrier.
The term "noncoherent" refers to the fact that cells do not cause tumors or tumors. Stem cells and/or precursor cells preferably are not associated with neoplasia and cancer.
The term "neurodegenerative disease" is used in this invention to describe the disease, which is caused by Central nervous system lesions which may be recovered and/or weakened by transplantation of neural cells according to the invention in the affected area of the brain and/or spinal cord of the patient. Examples of neurodegenerative diseases that can be treated with application of nerve cells and the FPIC of the scale according to the present invention, include Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (disease Lou Gehrig's disease (als), Alzheimer's disease, rett Syndrome, lysosomal storage disease of accumulation ("disease of the white matter or glial disease/demyelination, as described, for example, in Folkerth, J. Neuropath. Exp.Neuro., September 1999, 58:9), including the syndrome Sanfilippo's title, disease, Gaucher disease, and Tay-Sachs (deficit beta hexosaminidase), other genetic diseases, multiple sclerosis, traumatic brain injury or injury caused by ischemia, accidents, environmental factors and so on, spinal injuries, ataxia and alcoholism. In addition, the present invention can be applied to reduce and/or eliminate effects of a stroke or a heart attack on the Central nervous system of the patient, or effects, which are otherwise caused by insufficient blood flow or ischemia in the area of the brain indicated the patient, or which occur as a result of physical injury to the brain and/or spinal cord. Neurodegenerative diseases include, for example, diseases associated with impaired neurological development, including, for example, autism and related neurological disorders, such as schizophrenia, among many others.
The term "gene therapy" is used to describe the transfer and stable is Inoi inserting new genetic information into cells for therapeutic treatment of diseases and disorders. The foreign gene is inserted into the cell, which have proliferated, with the spread of a new gene in the cell population. Thus, umbilical cord blood cells or precursor cells are a target for gene transfer, either before differentiation, or after differentiation to the phenotype of nerve cells. Stem cells from umbilical cord blood or precursor cells of the present invention can be genetically modified heterologous nucleotide sequence and operable linked promoter that controls the expression of the heterologous nucleotide sequence. The nucleotide sequence can encode various proteins and peptides of interest. The gene products produced by genetically modified cells can be assembled in vitro, or cell can be used as vectors for in vivo delivery of gene products (e.g., gene therapy).
Cells of the monocytic line differentiation
Hereinafter in the description are exemplary, but without limiting to this, methods and guidelines for the implementation of many different objects of the present invention.
Various the cell and cell population can be characterized in a number of ways, including, as an example, growth characteristics (e.g., the ability to doubled the th population, the doubling time, passages to senescence), karyotype analysis (e.g., normal karyotype; maternal or neonatal line), flow cyto-metry (e.g., FACS analysis), immunohistochemistry and/or immunocyto-chemistry (for example, for detection of epitopes), profile analysis of gene expression (for example, matrix DNA chips; polymerase chain reaction (e.g., PCR, reverse transcriptase PCR, real-time and conventional PCR)), the protein matrix, the analysis of the secretion of proteins (for example, by assessing the coagulating activity of the plasma or analysis of PDC-air-conditioned environment, for example, by enzyme-linked immunosorbent assay (ELISA)), mixed lymphocytic reaction (e.g., as measured by stimulation RVMS), and/or other methods known in the art.
The selected cell or cell population can be used to start or inoculation of cell cultures for use in the present invention. Such selected cell or cell population can be transferred into sterile vessels for tissue cultures, either uncoated or coated with extracellular matrix or ligands such as laminin, collagen (natural, denatured or cross-linked), gelatin, fibronectin and other proteins of the extracellular matrix. Cells can be cultured in any of culturals the th environment, able to stimulate the growth of cells. Examples of such an environment (which the person skilled in the art can choose the appropriate type of cell or cell population among any other media available to the specialist in the art include DMEM (high or low glucose), advanced DMEM, DMEM/MCDB 201, the basal medium Needle, medium ham's F10 (F10), medium ham's F-12 (F12), Wednesday, Dulbecco 17, modified by way Iscove, environment for the growth of mesenchymal stem cells (MSCGM), DMEM/F12, RPMI 1640 and CELL-GRO-FREE. The culture medium may be supplemented by one or more components suitable for the respective cells or cell populations, including, for example, fetal bovine serum (FBS); equine serum (ES); human serum (HS); beta mercaptoethanol (TOGETHER or 2-ME); one or more growth factors (e.g. platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), growth factor vascular endothelial (VEGF), insulin-like growth factor-1 (IGF-1), factor inhibiting leukemic cells (LIF) and erythropoietin (EPO)); amino acids, including L-glutamine and L-valine; and one or more antibiotics and/or antifungal agents to control bacterial contamination (such as, for example, penicillin g streptomycin sulfate, amphotericin Century, gentamicin, and nystatin, either by themselves, or to whom Muncii). Cells can be seeded in culture vessels with a density that allows the growth of cells.
Methods of selection of the most appropriate culture media, preparation of the environment and is based on cell culture techniques depending on the type of cells and cell populations are known to experts in the art and described in various sources, including Doyle et al., (eds.), 1995, CELL &TISSUE CULTURE: LABORATORY PROCEDURES, John Wiley & Sons, Chichester; and Ho and Wang (eds.), 1991, ANIMAL CELL BIOREACTORS, Butterworth-Heinemann, Boston.
Cells and cell populations suitable for use in the present invention, can be increased by cultivation in a defined growth medium containing at least one factor, which stimulates cell proliferation (suitable for the respective cells or cell populations). At least one factor may include, for example, nicotinamide, members of the TGF-β family, including TGF-β 1, 2, and 3, bone morphogenic proteins (BMP-2, -4, 6, -7, -11, -12, and -13), serum albumin, family members fibroblastic growth factors, platelet-derived growth factor-AA and-BB, platelet rich plasma, insulin growth factor (IGF-I, -II), growth factors, and differentiation (GDF-5, -6, -8, -10, 11), the like peptide-I and-II (GLP-I and II), GLP-I and GLP-II mimetics, the Basis-4, retinoic acid, parathyroid hormone, insulin, progesterone, Aprotinin, GI is Robertson, ethanolamine, betareceptor, epidermal growth factor (EGF), gastrin I and II, copper chelators such as triethylenediamine, Forskolin, sodium butyrate, activin, betacellulin, noggin, a growth factor for neurons, nodal, insulin-transferrin-selenium (ITS), a growth factor for hepatocytes (HGF), keratinocyte growth factor (KGF), bovine pituitary extract, a protein associated with the islet neogenesis (INGAP), proteasome inhibitors, inhibitors of notch road, inhibitors of signaling cascade sonic the hedgehog, or combinations thereof. Alternatively, cells suitable for use according to the invention, can be reproduced by cultivation in air-conditioned environment (i.e., in a particular cell culture medium, in which the population of cells grows, allowing the cells to make soluble factors in the environment, so that air-conditioned environment includes appropriate soluble factors to the cells or cell populations). In accordance with the present invention in certain embodiments of carrying out the invention, the cells are removed from the environment, while soluble factors produced by cells remain.
During hematopoiesis, along with macrophage line differentiation of hematopoietic stem cells first differentiate into common myeloid precursor cells and then myeloblast. After myeloblast the cells and then differentiate in monoblast. Monoblast represents the first cell, which, actually, is part of the cells of the monocytic line of differentiation, which, as has been documented, differentiate into monocyte. Monoblast differentiate in promonocyte, i.e. undifferentiated monocyte. Promonocyte then differentiate into monocyte, that is Mature or differentiated monocyte. Monocyte may then be subjected to monocytopoiesis either macrophages or myeloid dendritic cell. Cells of the monocytic line of differentiation therefore include all types of cells, starting with monoblast to the end of the cell, obtained through monocytes.
Monoblast easily recognized by a person skilled in the art. Monoblast, typically have a length of from twelve to twenty microns. The volume of the nucleus relative to the cytoplasm is from 4:1 to 3:1, and like most myeloid blasts, the core has the shape from round to oval, with delicate chromatin structure. Usually see from one to four cores. The kernel can be Central and eccentric, it can show an obvious depression or folding. The cytoplasm is agranular, moderately colored to pale basophilic and often intensely colored periphery and eye-catching perinuclear zone.
Differentiated or Mature monocytes likewise easily raspoznayutsya expert in this field of technology. The diameter of monoblasts is, as a rule, from thirteen to twenty-five microns. Differentiated monocytes are large circulating phagocytic white blood cells, which have one large, smooth, well-defined nucleus, which has an oval or oval-curved shape. A large area of cytoplasm has many internal vesicles for processing foreign material and includes a thin, Auroville cytoplasmic granules. Differentiated monocytes normally circulate in the blood for from about one to about three days and then by the blood pass into other tissues such as the lungs and liver. After migration to other tissues, differentiated monocytes are monocytopoiesis in different types of macrophages, depending on the type of tissue that move monocytes. Promonocyte similar to monocytes, but the core of promonocytes is more radially symmetric than the core of Mature monocyte, and the volume of the nucleus relative to the cytoplasm of the above, and they are undifferentiated cells of monocytic line of differentiation.
Cells of the monocytic line of differentiation can be selected from multiple sources, applying the methods available to specialists in this field of technology. Examples include bone marrow, peripheral blood and poovinu the blood. In addition to receiving macrophage line differentiation directly from the subject, cells of the monocytic line of differentiation can be obtained by differentiation of stem cells, including, but not limited to, hematopoietic stem cells.
Mononuclear cell population
Mononuclear cell population can be isolated from numerous sources. One example is the allocation of the density gradient (ranging from 1.0 g/l to 1.1 g/l, preferably 1.077 g/l), as shown in the examples below. Other examples include bone marrow, peripheral blood and cord blood. In addition to obtaining mononuclear cells by selection directly from the subject, mononuclear cells can be obtained by differentiation of stem cells, including, but not limited to, hematopoietic stem cells.
The population of stem cells
The population of stem cells suitable for use in the methods of the present invention, can be obtained from any tissue that can provide stem cells capable of differentiating into at least cell types mesoderm lines of differentiation.
Originating from bone marrow stem cells belong to two of the most studied types of adult stem cells. At the present time, these originate from bone marrow stem cells used to restore various blood and immune components in the bone marrow after transplantation. Currently identified two major types of stem cells found in bone marrow: hematopoietic stem cells (HSC, or CD34+cells capable of differentiating into all types of blood and immune cells, and stromal (mesenchymal) stem cells (MSC), which, as a rule, are intended for the formation of bones, cartilage, muscle and fat. However, as has been shown, both types originating from bone marrow stem cells have a more extensive plasticity than expected.
Specialist in the art can apply any means of isolating and culturing stem cells, because there are several well known ways. For example, hematopoietic stem cells can be obtained from umbilical cord blood, which is abundantly supplied with such cells. Hematopoietic cells isolated from umbilical cord blood, and hematopoietic cells isolated from bone marrow or peripheral blood, are essentially identical to the behavior when applying for transplantation. In addition, placenta and bone marrow are excellent sources of mesenchymal stem cells. In this way, art is olavie cells, which can differentiate into cells of mesoderm lines of differentiation that originate from adipose tissue (although, obviously, are not as plastic as mesenchymal stem cells derived from bone marrow), and such stem cells are likely to be present in other tissues.
Stem cells can be isolated from many tissues either through the use of antibodies that bind markers specific to stem cells (e.g., SH2, SH3 and SH4 - see U.S. patent No. 5,486,359 and 5,837,539), or by use of antibodies that bind markers that are specific to undesirable cells, such as CD4+and CD8+(T cells), CD45+(panB cells), GR-1 (granulocytes), and lad (differentiated antigen cells). An example of this Protocol can be found in Izaba et al., I. Exp.Med. 176-1693 1702 (1992).
Hematopoietic stem cells can, likewise, be obtained from a variety of sources, including umbilical cord blood, bone marrow and mobilized peripheral blood. Purification of hematopoietic stem cells can be accomplished using techniques based on the affinity of the antibodies (for example, the use of antibodies to bind CD34, which is specific to hematopoietic cells). The technique of affinity column highlight to highlight cells with these antibodies can be detected in But et al., Stem Cells 1 (suppl. 31: 100-105 (1995). See also Brenner, Journal of Hematotherapy 2: 7-17 (1993). Methods of affinity purification and culture expansion of mesenchymal stem cells is also well known (see, for example, U.S. patents 5,486,359 and 5,837,539). Additional examples of such cues can be found in U.S. patent No. 6,087,113, 6,261,549 and 5,914,262, U5,908,782, and US 20040058412.
Populations of therapeutic cells enriched with cells of the monocytic line differentiation
Populations of therapeutic cells can be enriched in any manner available to a person skilled in the art. One example includes the selection of a population of stem cells and then screening those populations that are enriched in undifferentiated monocytes allocation. The essence of this method is shown in the examples below, except that the enrichment was determined after the application of the mononuclear cell population. Examples can be easily modified so that the enrichment of undifferentiated monocytes will be measured before application and then select those populations that have sufficient enrichment and/or those populations that are subject to further enrichment. As the population of stem cells, and populations of mononuclear cells include cells that can be induced to differentiate into cells of monocytic Lin and differentiation, one way of enrichment involves adding one or more relevant factors for the induction of differentiation (which are known to the specialist in the art) to the cell population. Other examples of enrichment include adding one or more cytokines or other growth factors that contribute to the induction of macrophage cells lines of differentiation to more rapid division or growth than cells nemonetarni line of differentiation in cell population and/or adding one or more factors, which selectively inhibit the division or the growth of cells that do not belong to the cells of the monocytic line of differentiation. Finally, cells of the monocytic line of differentiation derived or cultured separately, can be added to a population of therapeutic cells.
The following examples illustrate applications of the methods and compositions disclosed in the present description.
Example 1 - Allocation of mononuclear cell population enriched cells monoethanol line differentiation
Obtaining mononuclear bone marrow cells was performed using Standard density gradient (ranging from 1.0 g/l to 1.1 g/l, preferably 1.077 g/l). Cell population, as disclosed in Example 2 below, have different number is the number of cells of the monocytic line of differentiation.
Getting WMMS was performed using separation by density gradient (in the range of from 1.0 g/l to 1.1 g/l, preferably of 1.077 g/l). 35 ml of blood bone marrow carefully added without stirring to 10 ml of medium Ficoll (the amounts are given per tube), maintaining the surface tension of intact environment Ficoll. This procedure is repeated for nine tubes, which is 10 tubes per person. The tubes are then centrifuged at d without braking, for 40 minutes at 20°C. After separation density mononuclear ring (the boundary plasma/Ficoll) carefully collected from 10 tubes and suspended in four test tubes, adding 0.9% saline solution, to obtain the total volume in the tube - 45 4 ml test tube was centrifuged at g, for 10 minutes at 20°C, to separate the cells from the remaining environment Ficoll. After removal of the supernatant of mononuclear cells from all tubes were collected and combined into one test tube with a total volume of 40 ml 0.9% saline solution. The solution mononuclear cells centrifuged at 400 g for 10 minutes at 20°C. the Supernatant was again removed, and the cell sediment suspended in 10 ml of 0.9% saline solution. After checking cells for compliance with criteria such as sterility, viability and the absence of endotoxin is in, following good medical practice (GMP), and the implementation of automatic counting of cells, cells are suspended in 0.9% saline solution and 20% autologous serum to maintain the viability of the cells (autologous serum was first filtered using 0.22 μm filter to remove contaminating cells), obtaining a final concentration of 1×107cells/ml Obtained mononuclear solution is passed through a 100 μm filter to remove sgruppirovalisj cells.
Example 2 Correlation between the number of injected undifferentiated monocytes and therapeutic result
In this example, the data on patients who were treated using methods and compositions of the present invention.
The purpose of this study was to assess the safety and efficacy of the developed Protocol, Protocol cell therapy refractory angina (ReACT), in which a single series of numerous intramyocardial injection specific composition VMMS carried out in relation to patients as the only surgical therapy.
Therapy ReACT was developed in accordance with the criteria of good medical practice (GMP) and FDA standards.
Patients included in the Protocol were required to show symptoms refractory angina, bladt viable myocardium (diagnosed with stress scintigraphy with technetium), without dysfunction of the left ventricle (ejection fraction at least 45%), and for them it was impossible to myocardial revascularization (either RTS, or CABG).
Eight patients with refractory angina were enrolled in the study from September 2005 to July 2007. All patients had previously undergone surgical revascularization once (4 patients), twice (3 patients) or four times (1 patient), without weakening angina. The baseline characteristics of patients are given in Table 1.
|Baseline characteristics of the patients|
|Patient||Age||Floor||Diabetes||Optimization of drug therapy||The ejection fraction||CABG previously (n)||CCSAC|
Additionally, four patients with refractory angina were enrolled in the study and were subjected to ReACT, but they both needed a bypass coronary artery, and they were excluded from this analysis. Currently, these patients are placed in a separate group.
Patients with refractory angina observed in Sao Paulo Hospital, Sao Paulo, Sao Paulo, Brazil, Federal University hospital coronary heart disease, were included in this study. The study Protocol (ReACT) was approved by local and national ethics Committee (CEP-EPM-0314/05), and all patients gave written consent. Patients with refractory angina classified as functional class IV (angina at rest) according to the classification of angina Canadian Association of cardiologists (CCSAC), despite effective standard therapy, not to be treated by conventional myocardial revascularization and have viable myocardium. The inability to apply revascularization or perk is Tannoy, either surgical - was determined, at least two cardiologists and two cardiovascular surgeons, on the basis of the most recent (six months) coronarography patients. Exclusion criteria were as follows: (1) the ejection fraction of the left ventricle (LVEF) < 45% on the transthoracic echocardiogram; (2) the absence of viable myocardial radionuclide imaging; (3) positive results in serological testing for human immunodeficiency virus (HIV), hepatitis type a, b and C, human T cell lymphotropic virus (HTLV) or Chagas disease; (4) significant heart valve disease (5) chronic renal disease in relation to dialysis; (6) excessive use of alcohol and drugs; (7) any other medical condition, with estimated life expectancy <5 years; (8) participates in previous studies of cell therapy; and (9) pregnancy.
Clinical trial phase 1/PA conducted for a single surgery, in which mononuclear bone marrow cells (obtained according to Example 1) was injected through intramyocardial injection in numerous points of injection in a patient suffering from refractory angina pectoris with normal or slightly poorer left ventricular function. Refractory the angina patients were determined in the usual way and related to functional class IV according to the classification of angina Canadian Association of cardiologists (CCSAC), with a fully optimized pharmacological treatment and without any medical or possible intervention procedures (CABG, RTSA) inoperable patients "option".
Each patient was taken 100 of stem cells in the bone marrow from the iliac crest, and they kept in salt solution with a concentration of 80 U. I. heparin/ml Mononuclear cells were isolated by density gradient and diluted to a final concentration of 107cells/ml, according to ReACT and good medical practice (GMP). Was carried out by differential counting of viable cells, mononuclear cells, white blood cells, and determined the content of CD34+conducted aerobic and anaerobic microbiological tests. The samples were kept for further analysis.
One series of multiple injections in the myocardium of the left ventricle was performed surgically, through a left lateral thoracotomy, as follows: 0.2 ml (2×106cells/injection, the distance between the points of injection is 1 cm, and the depth introduction to epimyocardial fabric is 1 see the Number of injections (40 to 90) for each patient is determined based on the extent of viable ischemic region of the myocardium determined using radionuclide imaging, magnetic resonance imaging (MRI), scintigra-FIDESCO the image and the magnification of the left ventricular chamber.
The course of study
Heart rate of the patient was monitored for 48 hours after surgery. Clinical evaluation according CCSAC was performed at 3, 6, 12 and 18 months after surgery. Echocardiogram was performed at the initial moment of time and at the time 1, 3, 6 and 12 months, and magnetic resonance imaging of the heart was performed in zero time and at the time 6 and 12 months to ensure security. The radionuclide imaging (perfusion stress myocardial scintigraphy with technetium and MRI) was performed at the initial moment of time and at the time 6 and 12 months after the procedure, to assess the proportion of ischemic areas of the myocardium. In relation to objective analysis (stress scintigraphy) ischemic region of the heart it is important to note that all patients, as was believed, had 100% of the ischemic area of viable myocardium on the walls of the left ventricle, which was determined using the stress scintigraphy with technetium, before the injection of the cellular composition. These recoverable ischemic region of the heart was regarded as the baseline, compared with scintigraphic analysis at the time 6 and 12 months. Scintigraphic analysis was performed only at the time 6 and 12 months.
Nonparametric Friedman test was used to atenciosamente class of angina in CCSAC 3, 6 and 12 months and assess changes in the ischemic area of the myocardium after 6 and 12 months. To compare the obtained results were used Wilcoxon test with correction for Bonferroni. The correlation between the results (class of angina and ischemic area of the myocardium after the procedure) and number of injections was evaluated using a test Spearman. For comparison of left ventricular function by echocardiogram before treatment and 12 months after treatments were applied nonparametric Wilcoxon test.
As shown below, the improvement in myocardial perfusion in a patient (or lack thereof) correlates with the dose of cells of the monocytic line of differentiation, i.e. undifferentiated monocytes. Patients who received the greatest number of cells of the monocytic line differentiation, showed a reduction of angina class IV to 0 (CCSAC).
Table 1A below shows the improvement in the class of angina and ischemic areas of the myocardium, and the concentration of the composition, enriched in undifferentiated monocytes VMS, at introduction.
|Age, gender, CCSAC before treatment, CCSAC eighteen months after treatment, the number of undifferentiated monocytes, injected intramyocardially and improve the of myocardial perfusion after 12 months after treatment. Data for patient 1 is not available.|
|Patient||Age||Floor||Class CCSAC (original)||Class CCSAC 18 months after treatment||The number intramyocardial injectable undifferentiated monocytes (cells × 10^6)||Improvement in myocardial perfusion after 12 months (%)|
In Table 1B, below, shows the changes of the class of angina and ischemic areas of the myocardium after administration of a composition enriched in undifferentiated monocytes VMS.
|CCSAC**||% ischemic region|
|Original||3 months||6 months||12 months||18 months||Original***||6 months||12 months|
|p value (potesta Friedman)||<0.001||0.002|
|p value (one-sided Wilcoxon test*)||-||0.008||0.008||0.004||0.004||-||0.063||0.004|
|*** As expected, all patients had 100% ischemic area of viable myocardium on the walls of the left ventricle is determined by using the stress scintigraphy with technetium before the injection of the cellular composition. The analysis conducted at the time 6 and 12 months, has allowed to determine the percentage reduction of the ischemic region of the myocardium on these walls.|
|** according to the classification of angina canadian Association of cardiology|
|&estimated using the stress scintigraphy with technetium|
|* relative to the base is the second line; change class of angina and ischemic areas of the myocardium is statistically significant if p<0.0125 (0.05/4) and p<0.025 (0.05/2) (correction, Bonferroni); accordingly months:months during analysis|
As you can see based on the data shown in table 1A, there is a strict correlation between the number of undifferentiated monocytes, injected intramyocardially, and reducing or eliminating the symptoms of angina. In addition, the correlation is statistically significant (p<0.05), thus demonstrating the effectiveness of the methods and compositions as described in the present invention.
To eliminate the effect caused by the number of injections (which to a certain extent also correlates with the dose of cells of the monocytic line of differentiation), have identified a correlation between the number of injections and improving myocardial perfusion. Fig.2 graphically illustrates the lack of such a correlation. As shown in Fig.2, no statistically significant correlation was not observed.
Subjective improvement myocadial ischemia
After the procedure, myocardial injections according ReACT, it was observed a progressive improvement in median values of angina class, changing from class 4 at the initial moment of time up to 2.5 (p=0.008), 2 (p=0.008), 1 (p=0.004) and 1 (p=0.004) at 3, 6, 12 and 18 months is in analysis, respectively (Fig.3 and table 2).
Table 2 below shows the coefficients of correlation coefficient (rs) between the contents of undifferentiated monocytes VMS (promotion), lymphocytes and CD34+cells, and a better class of angina and ischemic areas of the myocardium during the study. For technical reasons, there was no counting of cells and percent for the first patient that participates in the analysis, and the analysis involved only 7 patients.
|Class angina * and % ischemia||Monocytes||Lymphocytes||CD34+|
|Class angina* - 3 months||rs||-0.759||0.579||-0.318|
|Class angina* - 6 months||rs||-0.759||0.579||-0.458|
|Class angina* 12 months||rs||-0.759||0.579||-0.458|
|Class angina* -18 months||r||-0.759||0.579||-0.458|
|% ischemic region6- 6 months||rs||-0.101||0.339||0.077|
|% chemichemi the612 months||rs||-0.270||0.267||-0.204|
|* according to the classification of angina canadian Association of cardiologists & improvement assessed with stress scintigraphy with technetium months:months during analysis|
Objective improvement of myocardial ischemia
Progressive reduction of the ischemic region of the myocardium was observed with stress scintigraphy with technetium 6 months (decrease of 39.4%, p=0.06) and 12 months (decrease 84.4%, p<0.004), although the difference was statistically significant only at the moment of 12 months the century It is important to note that all patients, as was believed, had 100% of the ischemic area of viable myocardium on specific walls of the left ventricle, which was determined using the stress scintigraphy with technetium, before the injection of the cellular composition. These initial recoverable ischemic region of the heart was considered as the baseline, when compared with the data scintigraphies analysis at the time 6 and 12 months (Fig.4 and table 2).
The correlation between the composition ReACT and improvements
ReACT has a certain percentage of monocytes, and their specific composition is positively correlated with clinical response; especially, to improve class CCSAC (table 3).
|The correlation between the concentration of promonocytes and improvement CCSAC (improving non-class).|
|Patient||Promonocyte (cell × 106)||Improvement class CCSAC|
|* no data available for patient 1|
Other cell types such as lymphocytes or CD34+cells do not show correlation with the improvement of class CCSAC or ischemic region of the myocardium.
The results of this study show that ReACT can help reduce symptoms of refractory angina in patients that do not fit a normal myocardial revascularization, and who show symptoms of angina despite maximal medical therapy. Our results show a decrease symptoms of angina and reduce the extent of myocardial ischemia. Perhaps the causal mechanism of this improvement may be associated with angiogenic properties ReACT composition.
In our study relieving symptoms began 3 months after about the edori and the improvement continued until the 12th of the month with further improvement until the 18th of the month, suggesting that angiogenesis begins earlier and lasts up to 18 month after the procedure (see Table 2 and Fig.3). In addition, because all patients showed improvement associated with the progressive weakening of the symptoms, this suggests that the effect is sustainable, rather than short-term, and the result differs from the results of other studies (6). Accordingly, there has been shown a downward trend in the percentage of myocardial ischemia, the reduction becomes significant to the 12th month. The fact that the reduction of symptoms occurs much earlier than the improvement of perfusion, can be explained by the low spatial resolution and high variability of radionuclide imaging, preventing the detection of small changes, especially in such a small group of patients.
The natural progression of refractory angina shows what can happen spontaneously even serious angina(9, 31, 54, 60). If you look at the group treated with medication and participating in a randomized study shows that from 0 to 19% of patients participating in the trial of percutaneous myocardial laser revascularization, and from 0 to 32% of patients participating in studies of surgical m & e is ardeley laser revascularization, showed improvement in at least two points in the class for CCSAC 12 months, and 0-44% of patients after 3 years (38). Our results showed a much more striking and significant improvement class CCSAC, which was confirmed by a test with increasing load myocardial perfusion.
In studies of refractory angina primarily involved patients with dysfunction of the left ventricle from moderate to severe (13, 41, 57). As a result, our results showed a more significant reduction of symptoms of angina and improve the quality of life, rather than an increase in the ejection fraction of the left ventricle (LVEF). In our study, all patients had LVEF≥45% as the baseline, and, as expected, there was no significant change in LVEF at the time of 12 months (p=0.726). Also suggested that angiogenesis is directly linked to the infusion of stem cells, but not with myocardial puncture, stimulating secondary angiogenesis. On the other hand, an increase in LVEF, as well as improving myocardial perfusion, implies the absence of functional deficit caused by necrosis or fibrosis of the myocardium caused intramyocardial injections ReACT, increasing the safety of the procedure.
Standard group of patients with symptoms refractory angina and viable myocardium would be the ideal Candida is that for angiogenic therapy, using ReACT intramyocardial injection. For obvious reasons, the analysis of LVEF is not the end point for this group. The primary therapeutic goal is to improve myocardial perfusion, using ReACT in the subjective (CCSAC) and objective (tested with increasing load with visualization) study.
In contrast to other studies (12, 57), ReACT in a particular cell composition, which was correlated with clinical and scintigraphic responses; improved classification of angina and reduce ischemic region of the myocardium, respectively. Improvement class CCSAC (subjective evaluation), correlated with the subsequent reduction of the ischemic region of the myocardium (objective assessment), suggests that neoangiogenesis is the main mechanism of action of stem cells (Fig.3 and 4). Hence, a large fraction of undifferentiated monocytes presented in ReACT, as it turns out, is associated with angiogenesis, which restores perfusion to ischemic areas of the myocardium after transplantation of the cells. This mechanism is not yet clarified and will be analyzed in the next phase of this study. Nevertheless, a significant correlation between the number of undifferentiated monocytes and improved clinical response (r=-0.759, p<0.05) effective what about the supports associated with the cells the effect of ReACT in this study.
This positive correlation between the number of undifferentiated monocytes to ReACT and stable improvements in angina class may indicate the importance of these cells for the mechanism of action of stem cells VMS and stable angiogenesis in the myocardium. Also important is that these results provide the hypothesis that the results of this study due to the cellular effect ReACT composition rather than nonspecific effects techniques ReACT. These controlled studies are explained next.
Bone marrow is a natural source of a wide range of titoki-new, which are involved in the control of angiogenic and inflammatory processes. The leukocyte bone marrow play an important role in the angiogenic mechanism, and neutrophile and monocytes play a key role in this process(10, 24, 29, 47).
Undifferentiated monocytes and promonocytes bone marrow can be activated in response to the chemotactic boosters and undergo final differentiation into macrophages. Macrophages play a key role in angiogenesis due to their ability to secrete proteases, growth factors and Monokini, and affect each phase of the angiogenic process, including changes in the local extracellular matrix, induction of endothelial cell migration or proliferation, and the inhibition of the growth of vessels with formed the eat differentiated capillaries (5, 10, 46, 51).
The optimal number or optimal composition of stem cells to promote regeneration of the myocardium remains controversial among various researchers, most of them do not show a dose-dependent effect (18,20). Iwasaki et al. (25) conducted one of the studies that demonstrate a positive correlation between the number of injected stem cells and myocardial regeneration in rats with experimental acute myocardial infarction. Henning et al. (23) were injected with stem cells from bone marrow derived from umbilical cord blood of humans, rats, suffered a heart attack, comparing different doses and routes of administration (intramyocardially, intracoronary or intravenous), demonstrating higher efficiency intramyocardial injection. In one study, we conducted ReACT with one series of multiple injections of 2×106VMS song/myocardial puncture. No correlation was observed between the number of injections and change class of angina over time, however, the sample was probably too small for any significant assumptions, and because it was decided to continue the application of the Protocol ReACT, the following data can confirm or not to confirm it.
Intramyocardially the delivery path of the cellular composition was selected on the basis of experimental data, is the quiet show higher myocardial uptake of stem cells in the process intramyocardially infusion, compared to other ways, such as intracoronary (either anterograde or retrograde injection) or transcoronary (33, 42, 50). Also, chronic clinical sign of refractory angina provides a much safer profile in case intramyocardial approach, without serious complications, like intramyocardial injection in the case of acute disease (acute myocardial infarction). Analysis Perin et al. (42), assessed the safety and feasibility of endomyocardial injections in the case of acute myocardial infarction. Was not observed life-threatening complications, and the way it turned out, was safe. Although we cannot exclude a significant risk of arrhythmia, if we use direct intramyocardial injection in the acute infarction unstable.
It is important to note that, despite the favorable forecasts, our study had some limitations. Despite the fact that our study included a higher number of patients and the duration of the study was considerably longer in comparison with most published studies of refractory angina, small group, consisting of 8 patients complicates the definition of efficiency, but allows you to demonstrate safety. In connection with the ethical aspects and the inability Oprah is to provide a surgical intramyocardial introduction placebo for this group, it was a non-randomized open study; therefore, the placebo effect could not be ruled out. However, one should emphasize the fact that the objective to increase myocardial perfusion was assessed and this increase was maintained over time.
Further clinical studies are still needed to confirm our results. However, the effectiveness of this technique, ReACT, used in accordance with the technical description and proper medical practice, as well as obtaining specific cells according ReACT, as it turned out, lead to better results than the results previously reported by other studies.
In conclusion, the surgical transplantation of the drug, based on autologous cells derived from the bone marrow, according to the standard Protocol may be a safe and effective method of achieving a progressive and sustained improvement in patients with refractory angina.
The following references are referred to in example 2, and the technical level.
1. Abdel-Latif, A.; Bolli, R.; TIeyjeh, I. M.; Montori, V. M.; Perin, E. C.; Hornung, C. A.; Zuba-Surma, E. K.; AI-Mallah, M.; Dawn, B. Adult bone marrow-derived cells for cardiac repair. A systematic review and meta-analysis. Arch. Intern. Med. 167: 989-997; 2007.
2. Anversa, P; Leri, A; Rota, M; Hosoda, T; Bearzi, C; et al. Concise review: stem cells, myocardial regeneration, and methodological artifacts. Stem Cells 2007:25:589-601.<> 3. Beeres, SLMA; Bax, JJ; Dibbets-Schneider P, Stokkel, MPM; Fibbe, WE; et al. Sustained effect of autologous bone marrow mononuclear cell injection in patients with refractory angina pectoris and chronic myocardial ischemia: twelve-month follow-up results. Am Heart J 2006; 152: 684. e11-684. e16.
4. Beeres, SLMA; Bax, JJ; Kaandorp TA; Zeppenfeld, K; Lamb, HJ; et al. The Usefulness of intramyocardial injecton of autologous bone marrow-derived mono-nuclear cells in patients with severe angina pectoris and stress-induced myocardial ischemia. Am. J. Cardiol. 2006; 97: 1326-31.
5. Benelli, R; Albini, A; Noonan, D. Neutrophils and angiogenesis: potential initiators of the angiogenic cascade. Cassatella MA (ed): The Neutrophil. Chem Immunol Allergy. Basel, Karger, 2003, vol. 83, pp.167-181.
6. Briguori C; Reimers, B; Sarais, C; Napodano, M; Pascotto, P; et al. Direct percutaneous intramyocardial delivery of autologous bone marrow in patients with refractory myocardial angina. Am Heart J 2006; 151: 674-80.
7. Charwat, S.; as well as the gyöngyösi, M; Lang, I; Graf, S; Beran, G; et al. Role of adult bone marrow stem cells in the repair of ischemic myocardium: current state of the art. Experimental Hematology 2008; 36: 672-80.
8. Cogle, CR; Madlambayan, GJ; Hubsher, G; Beckman, C; Speisman, R; et al. Marrow cell therapies for cardiovascular diseases. Experimental Hematology 2008; 36: 687-94.
9. DeJongste, MJL; Tio, RA; Foreman, RD. Chronic, therapeutically refractory angina pectoris. Heart 2004; 90: 225-30.
10. Dirkx, AEM; oude Egbrink, MGA; Wagstaff, J; Griffioen, AW. Monocyte/macrophage infiltration in tumors: modulators of angiogenesis. J Leukoc Biol 2006; 80: 1183-96.
11. Fraker, Jr, TD; et al. 2007 Chronic Angina Focused Update of the ACC/AHA 2002 Guidelines for the Management of Patients With Chronic Stable Angina: A Report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines Writing Group to Develop the Focused Update of the 2002 Guidelines for the Management of Patients With Chronic Stable Angina. Circulation, 2007; 116: 2762-72.
12. Fuchs, S; Kornowski, R.; Weisz, G; Satler, LF; Smits, PC; et al. Safety and feasibility of transendocardial autologous bone marrow cell transplantation in patients with advanced heart disease. Am J Cardiol 2006; 97: 823-9.
13. Fuchs, S; Satler, LF; Kornowski, R.; Okubagzi, P; Weisz, G; et al. Catheter-based autologous bone marrow myocardial injection in no-option patients with advanced coronary artery disease. A feasibility study. J. Am. Coll. Cardiol. 2003; 41: 1721-4.
14. Gibbons, RJ; et al. ACC/AHA 2002 Guideline Update for the Management of Patients With Chronic Stable Angina - Summary Article. A Report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Comittee on the Management of Patients With Chronic Stable Angina). Circulation 2003; 107: 149-58.
15. Gordon, MY. Stem cells for regenerative medicine - Biological attributes and clinical application. Experimental Hematology 2008; 36: 726-32.
16. Guan, K; Hasenfuss, G. Do stem cells in the heart truly differentiate into cardiomyocites? J Molecular and Cellular Cardiology 2007; 43: 377-83.
17. Haider, H Kh; Ashraf, M. Bone marrow stem cell transplantation for cardiac repair. Am J Physiol Heart Circ Physiol 2005; 288: 2557-67.
18. Hale SL; Dai, W; Dow, JS; Kloner RA. Mesenchymal stem cell administration at coronary artery reperfusion in the rat by two delivery routes: a quantitative assessment. Life Sciences 2008; 83: 511-5.
19. Hamano, K; Nishida, M; Hirata, K; Mikamo, A; Li, T-S; et al. Local implantation of autologous bone marrow cells for therapeutic angiogenesis in patients with ischemic heart disease - Clinical trial and preliminary results. Jpn Circ J 2001:65:845-7.
20. Hashemi, SM; Ghods, S; Kolodgie FD; Parcham-Azad, K; Keane, M; et al. A placebo controlled, dose-ranging, safety study of allogenic mesenchymal stem cells injected by endomyocardial delivery after an acute myocardial infarction. European Heart J 2008; 29: 251-9.
21. Hattori, R; Matsubara, H. Therapeutic angiogenesis forsvere ischemic heart diseases by autologous bone marrow cells transplantation. Molecular Cellular Biochemistry 2004; 264: 151-5.
22. Henning, RJ; Abu-Ali, H; Balis, JU; Morgan, MB; Willing, AE; et al. Human Umbilical cord blood mononuclear cells for the treatment of acute myocardial infarction. Cell Transplantation 2004; 13: 729-39.
23. Henning, RJ; Burgos, JD; Vasko, M; Alvarado, F; Sanberg, CD, et al. Human cord blood cells and myocardial infarction: effect of dose and route of administration on infarct dementia sze. Cell Transpl 2007; 16 (9): 907-17.
24. Hoefer, IE; Grundmann, S; van Royen, N; Voskuil, M; Schirmer, SH; et al. Leukocyte subpopulations and arteriogenesis: specific role of monocytes, lymphocytes and granulocytes. Atherosclerosis 2005; 181: 285-93.
25. Iwasaki, H; Kawamoto, A; Ishikawa, M; Oyamada, A; Nakamori, S; et al. Dose-dependent contribution of CD34-positive cell transplantation to concurrent vasculogenesis and cardiomyogenesis for functional regenerative recovery after myocardial infarction. Circulation 2006; 113: 1311-25.
26. Jolicoeur, EM; Granger, CB; Henry, TD; Holmes, DJ; Pepine CJ; et al. Clinical and research issues regarding advanced chronic coronary artery disease: Part I: Contemporary and emerging therapies. Am Heart J 2008; 155: 418-34.
27. Kang, S; Yang, Y-j; Li, C-j; Gao, R-I. Effects of intracoronary autologous bone marrow cells on left ventricular function in acute myocardial infarction: a systematic review and meta-analysis for randomized controlled trials. Coronary artery disease 2008; 19: 327-35.
28. Kinnaird T; Stabile E, Burnett MS; Epstein SE. Bone marrow-derived cells for enhancing collateral development; mechanisms, animal data, and initial clinical experiences. Circ Res 2004; 95: 354-63.
29. Kusumanto, YH; Dam, WA; Hospers, GAP; Meijer, C; Mulder, NH. Platelets and granulocytes, in particular the neutrophils, form important compartments for circulating vascular endothelial growth factor. Angiogenesis 2003; 6: 283-7.
30. Laflamme, MA; Zbinden, S; Epstein SE; Murry CE. Cell-based therapy for myocardial ischemia and infarction: pathofhysiological mechanisms. Ann Rev Pathol Mech Dis 2007; 2: 307-39.
31. Leon, MB; Kornowski, R; Downey, WE; Weisz, G; Bairn, DS; et al. A blinded, randomized, placebo-controlled trial of percutaneous laser myocardial revascularization to improve angina symptoms in patients with severe coronary disease. J Am Coil Cardiol. 2005; 46: 1812-9.
32. Lipinski, MJ; Biondi-Zoccai, GGL; 'abbate, A; Khianey, R; Sheiban, I; et al. Impact of intracoronary cell therapy on left ventricular function in the setting of acute myocardial infarction: A collaborative systematic review and meta-analysis of controlled clinical trials. J Am Coil Cardiol 2007; 50: 1761-7.
33. Losordo, DW; Renault, M-A. Therapeutic myocardial angiogenesis. Mi-crovacular Research 2007; 74: 159-71.
34. Losordo, DW; Schatz, RA; White, CJ; Udelson, JE; Veereshwarayya, V; et al. Intramyocardial transplantation of autologous CD34+stem cells for intractable angina: A phase I/IIa double-blind, randomized controlled trial. Circulation 2007; 115: 3165-72.
35. Mannheimer, C; Camici, P; Chester, MR; Collins, A; DeJongste, M; et al. The problem of chronic refractory angina. Report from de ESC joint study group on the treatment of refractory angina. Eur Heart J 2002; 23: 355-70.
36. Moore, RK; Groves, D.; Bateson, S.; Barlow, P; Hammond, C; et al. Health related quality of life of patients with refractory angina before and one year after enrolment onto a refractory angina program. European Journal of Pain 2005; 9: 305-10.
37. Mukherjee, D; Bhatt, D; Roe, MT; Patel, V; Ellis, SG. Direct myocardial revascularization and angiogenesis: how many patients might be eligible? Am J Cardiol 1999; 84: 598-600.
38. Nordrehaug, JE; Salem, M. Treatment of chronic refractory angina pectoris - light at the end of the tunnel ? European Heart J 2006; 27: 1007-9.
39. Norol F; Merlet P, Isnard R; et al. Influence of mobilized stem cells on myocardial infarct dementia repair in a nonhuman primate model. Blood 2003; 102: 4361-68.
40. Orlic D; Kajstura J; Chimenti S; et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001; 410: 701-5.
41. Perin EC; Dohmann, HFR; Borojevic, R; Silva, SA; Sousa, ALS; et al. Transendocardial, autologous bone marrow cell transplantation for severe, chronic ischemic heart failure. Circulation 2003; 107: 2294-2302.
42. Perin EC; Silva, GV; Assad, JAR; Vela, D; Buja, LM; et al. Comparison of intracoronary and transendocardial delivery of allogeneic mesenchymal cells in a canine model of acute myocardial infarction. J Molecular Cellular Cardiology 2008; 44: 486-95.
43. Rana, JS; Mannam, A; Donnell-Fink, L; Gervino EV; Selike, FW; et al. Longevity of the placebo effect in therapeutic angiogenesis and laser myocardial revascularization trials in patients with coronary heart disease. Am J Cardiol 2005;95:1456-9.
44. Rosenzweig, A. Cardiac cell therapy - Mixed results from mixed cells. N Eng J Med 2006; 355 (12): 1274-7.
45. Saririan, M; Eisenberg, MJ. Myocardial laser revascularization for the treatment of end-stage coronary arterydisease. J Am Coil Cardiol 2003; 41: 173-83.
46. Scapini, P; Morini, M; Tecchio, C; Minghelli, S; Di Carlo, E; et al. CXCLI/Macrophage inflammatory protein-2-induced angiogenesis in vivo is mediated by neutrophil-derived vascular endothelial growth factor-A. The Journal of Immunology 2004; 172: 5034-40.
47. Schruefer, R; Lutze, N; Schymeinsky, J; Walzog, B. Human neutrophils promote angiogenesis by a paracrine feedforward mechanism involving endothelial interleukin-8. Am J Physiol Heart Circ Physiol 2005; 288: H1186-H1192.
48. Schuldt, AJT; Rosen, MR; Gaudette, GR; Cohen, IS. Repairing damaged myocardium: evaluating cells used for cardiac regeneration. Current Treatment Options Cardiovascular Medicine 2008; 10: 59-72.
49. Steinhoff, G; Choi, Y-H; Stamm, C. Intramyocardial bone marrow stem cell treatment for myocardial regeneration. European Heart J Supplements 2006; 8 (Sup. H): H32-H39.
50. Sun, Z; Wu, J; Fujii, H; Wu, J; Li, S-H; Porozov S, Belleli A, Fulga V, Porat Y, Li RK. Human angiogenic cell precursors restore function in the infarcted heart: a comparison of cell delivery routes. European J Heart Failure 2008; 10: 523-33.
51. Sunderkotter C; Steinbrink, K; Goebeler, M; Bhardwaj, R; Sorg, C. Mac-rophages and angiogenesis. J Leukoc Biol 1994; 55: 410-22.
52. Ting, AE; Mays, RW; Frey, MR; Hof, WV; Medicetty, S; et al. Therapeutic pathways of adult stem cell repair. Critical Reviews Oncology/Hematology 2008; 65: 81-93.
53. Tse, H-F; Kwong, Y-L; Chan, JKF; Lo, G; Ho, C-L; et al. Angiogenesis in is-chaemic myocardium by intramyocardial autologous bone marrow mononuclear cells implantation. Lancet, 2003; 361: 47-49.
54. Tse, H-F; Siu, C-W; Zhu, S-G; Songyan, L; Zhang, Q-Y; et al. Paracrine effects of direct intramyocardial implantation of bone marrow derived cells to enhance neovascularization in chronic ischaemic myocardium. European J Heart Failure 2007; 9: 747-53.
55. Tse, H-F; Thambar, S.; Kwong, Y-L; Rowlings, P; Bellamy, G; McCrohon J, Bastian B, Chan JK, Lo G, Ho CL, Lau CP. Safety of catheter-based intramyocardial autologous bone marrow cells implantation for therapeutic angiogenesis. Am J Cardiol 2006; 98: 60-2.
56. Tse, H-F; Thambar, S.; Kwong, Y-L; Rowlings, P; Bellamy, G; McCrohon J, Bastian B, Chan JK, Lo G, Ho CL, ParkerA, HauserTH, Lau CP. Comparative evaluation oflong-term clinical efficacy with catheter-based percutaneous intramyocardial autologous bone marrow cell implantation versus laser myocardial revascularization in patients with severe coronary artery disease. Am Heart J 2007; 154: 982.e1-982.e6.
57. Tuma-Mubarak, J; Fernández-Vina, R; Carrasco-Yalan, A; Castillo-Aguirre, J; Rios-Diaz, H; et al. Refractory angina treatment by percutaneous retrograde sinus technique transplantation of unselected autologous bone marrow mono-nuclear cells: long-term follow-up.Abstracts, Cardiovasc Revascularization Med 2007; 8: 153-4.
58. van Ramshorst, J.; Bax, J. J.; Beeres, S. L.; Dibbets-Schneider, P.; Roes, S. D.; Stokkel, M. P.; de Roos, A.; Fibbe, W. E.; Zwaginga, J. J.; Boersma, E.; Schalij, M. J.; Atsma, D. E. Intramyocardial bone marrow cell injection for chronic myocardial ischemia: a randomized controlled trial. JAMA 301 (19); 1997-2004; 2009.
59. Vicario, J; Campo, S; Piva, J; Faccio, F; Gerardo, L; et al. One-year follow-up of transcoronary sinus administration of autologous bone marrow in patients with chronic refractory angina. Cardiovasc. Revascularization Med. 2005; 6: 99-107.
60. Yang, EH; Barsness, GW; Gersh, BJ; Chandrasekaran, K; Lerman, A. Current and future treatment strategies for refractory angina. Mayo Clin. Proc. 2004; 79 (10): 1284-92.
Example 3 - Monocytes for cerebral ischemia Methods in molecular biology
Standard molecular biology techniques known in the technical field and are not specifically described in this application, in General, follow from Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), and in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989). Polymerase chain reaction (PCR) in General is carried out according to PCR protocols: A Guide to Methods and Applications, Academic Press, San Diego, California (1990). Reactions and methodologies, including other methods based on nucleic acid, if other is not established, carry out as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory Press, and the methodology outlined in atento U.S. No. 4,666,828; 4,683,202; 4,801,531; 5,192,659; and 5,272,057, and incorporated herein by reference. In situ PCR in combination with flow cytometry can be used for detection of cells containing specific DNA sequences and mRNA (see, for example, Testoni et al., Blood, 1996, 87:3822).
Standard methods in immunology known from the technical field and are not specifically described in this application, in General, follow from Stites et al. (Eds.), Basic And Clinical Immunology, 8thEd., Appleton & Lange, Norwalk, CT (1994); Mishell and Shigi (Eds.), Selected Methods in Cellular Immunology, W. H. Freeman and Co., New York (1980).
In General, immunoassays are used for analysis of the sample, such as cell surface markers or the like. Immunocytochemical analyses of well-known specialists in this field of technology. In the analysis can be used as polyclonal and monoclonal antibodies. If necessary, other immunoassays, such as enzyme binding immunosorbent assay (ELISAs) and radioimmunoassays (RIA) can be used, as is well known to specialists in this field of technology. Available immunoassays are widely described in the patent and scientific literature. See, for example, U.S. patents№3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771; and 5,281,521, and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor, New York, 1989. Other links so the e may be referred to for the purposes of the present invention.
Antibodies may be monoclonal, polyclonal or recombinant. Antibodies can be raised against the immunogen or immunogenic parts, such as, for example, a synthetic peptide based on the sequence, or received recombinante using cloning techniques or the natural gene product and/or parts thereof can be selected and used as immunogen. Immunogen can be used to generate antibodies by standard methods of obtaining antibodies are well known in the art and generally described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Springs Harbor, New York (1988) and Borrebaeck, Antibody Engineering - A Practical Guide by W. H. Freeman and Co. (1992). Antibody fragments can also be obtained from the antibody by using methods known in the art, and include Fab and F(ab')2. To obtain polyclonal antibodies media, such as a rabbit or goat, subjected to immunization with immunogen or immunogenic fragment, in General, together with the adjuvant and, if necessary, with the carrier, antibody to the immunogen are harvested from the serum. Further, the polyclonal antibody can be absorbed from the condition that it is monospecific. That is, the serum is exposed related immunogenic, so that cross-reactive antibodies are removed from the serum, demonstrating their Monos ecificity.
To obtain monoclonal antibodies, an appropriate donor, usually a mouse, hyperimmunizing the immunogen, and produce splenic cells producing antibodies. These cells merge with termed cells, such as myeloma cells, to obtain the fused cell hybrid, which is immortal and secretes the required antibodies. The cells are then cultivated, and monoclonal antibodies are harvested from the culture medium.
To obtain recombinant antibodies informational RNA from producing antibodies of b-lymphocytes of animals, or hybridoma back-Transcriber, obtaining complementary DNA (cDNA). cDNA of the antibody, which may be full or partial length, amplificates and cloned in phage or plasmid. cDNA may be partial length heavy and light chain cDNA, separated or connected by a linker. The antibody or antibody fragment is expressed, using a suitable expression system. cDNA antibodies can also be obtained by screening appropriate expression libraries. The antibody may be associated with the solid substrate carrier or conjugated with a detectable component, or may be connected, and conjugated, as is well known in the art (Conjugation of fluorescent or enzymatic components in General is discussed in Johnstone & Thorpe, Immunochemistry in Practice, Blackwell Scientific Publications, Oxford, 1982). The binding of the antibody with the solid substrate carrier is also well known in the art (see Harlow &Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Publications, New York, 1988 and Borrebaeck, Antibody Engineering - A Practical Guide, W. H. Freeman and Co., 1992). Find components that are suitable according to the present invention, may include, but not limited to, fluorescent, metallic, enzymatic and radioactive markers. Examples include Biotin, gold, ferritin, alkaline phosphate, galactosidase, peroxidase, urease, fluorescein, rhodamine, tritium, 14, iodized and green fluorescent protein.
Gene therapy used according to the present invention, associated with the transfer of genetic material (DNA or RNA) of interest into the body of the recipient, to treat or prevent a genetic or acquired disease or condition. Representing the genetic material of interest encodes a product (e.g., protein, polypeptide and peptide, functional RNA, antisense), the production of which in vivo is desirable. For example, representing the genetic material of interest encodes a hormone, a receptor, enzyme polypeptide or peptide therapeutic value. Alternatively, the gene of interest materialgirl suicide gene. See "Gene Therapy" in Advances in Pharmacology, Academic Press, San Diego, California, 1997.
The introduction of cells for transplantation
Undifferentiated monocytes according to the present invention can be administered and dosed according to good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, mode of administration, patient age, sex, body weight and other factors known in the medical practice. So pharmaceutically effective amount" for purposes of the present invention is determined from considerations known in the art. The amount must be effective to achieve improvement including but without limiting to this, the increased survival rate or more rapid recovery, or reduction or elimination of symptoms and other factors that are selected when evaluated by an expert in the field of technology.
In the method according to the present invention undifferentiated monocytes according to the present invention can be administered in various ways, which are suitable for implantation in the Central nervous system, including, but not limited to, intravenous and intraarterial introduction, intrathecal, intraventricular introduction, intraparenchymatous introduction, intracranial introduction, intracisternally introduction, II andstc the veins and intranigral introduction. If desired, the umbilical cord blood cells injected with immunosuppressive agent.
Pharmaceutical compositions containing an effective amount of cells in umbilical cord blood, also provided by the present invention. These compositions contain an effective amount of cells, optionally, in combination with a pharmaceutically acceptable carrier, additive or excipient. In accordance with certain objects of the present invention, the cells are administered to a patient in need of transplantation, in sterile physiological solution. According to another object of the present invention, the cells are introduced in a balanced salt solution Hanks (HBSS) or Isolyte S, pH 7.4. Can also be used other approaches, including the use of cell-free environment from the whey. Systemic injection of cells, the patient may be preferable under certain signs, whereas the direct introduction into place or near the patient and/or diseased tissue, it may be preferable to other signs.
The pharmaceutical compositions according to the present invention preferably contain an effective amount of cells in solution, in General, in the range of from about 1.0×104cells to about 1.0×109cells, more preferably from about 1×105to about 1×107cells, even more preferably from about 2×105to about 8×0 6cells, optionally with a pharmaceutically acceptable carrier, additive or excipient.
Preferably undifferentiated monocytes are introduced together with the agent, allowing to overcome the blood-brain barrier. In one embodiment, the cells are combined with an agent that can overcome the blood-brain barrier, before the introduction of the patient. In another embodiment, cells are administered to the patient separately from the agent, allowing to overcome the blood-brain barrier. Optional, if cells are administered to the patient separately from the agent, allowing to overcome the blood-brain barrier, there is a time interval between the introduction of cells and agent that can overcome the blood-brain barrier. The time interval can last from less than a minute to hours or days. The optimal timing and order of introduction of the easy, standard way, determined by a person skilled in the technical field.
In the present description are referred to various publications. All the mentioned documents included in this application in full by reference, for a more complete description of the prior art, relates to the present invention. The following examples are not intended to limit the scope and essence of the present invention, but rather the purpose is s to illustrate certain embodiments of the invention. Any variation in the approximate methods, which are carried out by a person skilled in the art are included in the scope of the present invention.
One of interest is the function of Mature monocytes/macrophages is to promote angiogenesis associated with inflammatory reactions. Angiogenesis or neovascularization) is a key element of inflammatory processes, including subsequent cascades recovery [Sunderkotter, 1994 #4]. During the earlier process of inflammation circulating monocytes penetrate the blood vessels in the tissue [Bosco, 2008 #3]. Initially, nearby endothelial and inflammatory cells regulate the passage of monocytes through the walls of blood vessels through the allocation of a number of adhesive and chemotactic substances [Baggiolini, 2000 #9; Imhof, 2004 #2; Bosco, 2008 #3]. Infiltrated monocytes move along a chemotactic and oxygen gradients between normal and diseased tissues are hypoxic and/or necrotic centers tissues of patients before differentiation into tissue macrophages. Examples of pathological tissues approached by monocytes/macrophages to accumulate, are the following: solid tumors, myocardial and cerebral infarction, synovial joints of chronic arthritis or atherosclerotic plaque, bacterial infection and healing wounds [Baggiolini, 2000 #; Murdoch, 2004 #1; Bosco, 2008 #3; Mantovani, 2002 #15] (Fig.8).
After differentiation of monocytes macrophages known to exist in tissues in the form of polarized populations, M1 and M2 subgroups [Mantovani, 2004 #67; Sica, 2006 #16; Mantovani, 2004 #67; Mantovani, 2002 #15].
While M1 polarized macrophages are potent inflammatory cells that produce proinflammatory cytokines and phagocytic pathogens, M2 macrophages modulate inflammatory responses and contribute to angiogenesis and tissue repair [Mantovani, 2004 #67; Sica, 2006 #16; Mantovani, 2004 #67; Mantovani, 2002 #15]. Relative gene expression of macrophages interesting to note that in the healing of wounds first involved the combination of M1 and M2 subgroups, and then M2 genes become dominant [Deonarine, 2007 #68]. During the early stages of the wound-healing process M1 macrophages lead to inflammatory reactions, which helps cleanse the wound by removing residues of microbes and/or diseased tissue, whereas tissue repair and angiogenesis induced by M2 macrophages at the same time. At the last stage, when the purification of M1 macrophages was nearly complete, the prevailing M2 macrophages continue its work related to tissue regeneration and induction of angiogenesis [Deonarine, 2007 #68]. Accumulation clearly suggests that arrived monocytes/macrophages contribute to the modulation and regulation of neovascularization is ischemic tissue, tumors and chronic inflammation, such as arthritic joints and atherosclerosis.
Angiogenesis during ischemia
Undifferentiated monocytes or promonocyte give rise to Mature or differentiated monocytes/macrophages in the circulating blood through a process called differentiation.
In recent times the value of circulating Mature monocytes/macrophages for neovascularization has been shown in relation to coronary heart disease [Shireman, 2007 #23; Herold, 2004 #33; Capoccia, 2008 #32]. Arteriogenic, structural growth of pre-existing arteriolar networks in effective collateral arteries, as it turns out, is initiated by the increase of the tangential stress in the fluid, which is the result of arterial obstruction inside the developing collateral arteries, and not induced tissue hypoxia and ischemia [Ito, 1997 #41; Heil, 2006 #19]. In contrast, angiogenesis, formation of new capillaries from preexisting blood vessels, is induced by hypoxia, and the density of capillaries provides proof in the areas of serious and acute ischemia [Scholz, 2002 #20; Ito, 1997 #41].
Whereas arteriogenic, and angiogenesis cause neovascularization through various mechanisms, Mature monocytes/macrophages are a significant contribution in both cases. When arteriogenesis sudden obstruction of the inflow of blood is rovi occurs as a result of the presence of the pitch, or progressive stenosis increases the tangential stress in the fluid in the arteriolar network, and, consequently, the number of adhesion molecules and chemokines, such as endothelial adhesion molecules [Nagel, 1994 #21] and macrophage chemotactic protein-1 (MCP-1) [Eischen, 1991 #22], increases significantly. Mature blood monocytes are activated and drawn in collateral artery with MCP-1. Then they immediately pass through the walls of the blood vessels by binding to adhesion molecules and/or differentiate into tissue macrophages before production of many growth factors and cytokines [Sunderkotter, 1994 #4], which may promote proliferation of endothelial cells and smooth muscle cells [Heil, 2006 #19; Shireman, 2007 #23].
Angiogenesis is a combination of more complex processes, many of which are regulated by vascular endothelial growth factor (VEGF) and its receptors (VEGFR), which are known to initiate angiogenesis [Shireman, 2007 #23]. Recent data suggest that some subgroups angiopoietins (Ang-1 and 2) and their receptors (Tie) play a crucial role for the secondary stages of the angiogenic process, such as maturation, stabilization and vascular remodeling [Thurston, 2003 #57]. Hypoxia and necrosis have a significant impact on the production of VEGF/VEGFR and angiopoietin/Tie receptor [Milkiewiz, 2006 #24; Zhang, 2005 #56; Beck, 2000 #58;Murdoch, 2007 #59]. In turn, VEGF and angiopoietin induce the inflow of endothelial precursor cells and Mature monocytes/macrophages [Tammela, 2005 #25; Murdoch, 2007 #59].
Recruited Mature monocytes/macrophages promote angiogenesis through several potential mechanisms. First, macrophages destroy the extracellular matrix using the matrix metalloproteinases and proteolytic enzymes, leading to migration of endothelial cells [Moldovan, 2005 #26]. By passage through the extracellular matrix, growth factors and endothelial cells are moved from the formed vessels, with the formation of new capillaries [Shireman, 2007 #23].
Secondly, Mature monocytes/macrophages release a lot of Pro-angiogenic cytokines such as basic fibroblast growth factor (bFGF), VEGF, interleukin-8 (IL-8), substance P, tumor necrosis factor-α (TNF-α), transforming growth factor (TGF)-α and-β, and prostaglandins [Sun-derkotter, 1994 #4; Moldovan, 2005 #26], which have a direct or indirect effect on the stimulation of proliferation of endothelial cells, their migration and the formation of tubules [Shireman, 2007 #23; Sunderkotter, 1994 #4]. Although, as well as Pro-angiogenic factors, Mature monocytes/macrophages are also able to release anti-angiogenic cytokines, such as thrombospondin 1, interferon-α and-γ [Sunderkotter, 1994 #4], producing them and hibitory cytokines is regulated by proangiogenic factors. For example, IL-12 inhibited the increase in Ang-2 [Murdoch, 2007 #59].
Thirdly, Mature monocytes/macrophages can differentiate into endothelial cells, which directly contribute to the formation of blood vessels [Moldovan, 2005 #26; Moldovan, 2000 #28; Anghelina, 2006 #27]. During stimulation of the relevant Pro-angiogenic factors, precursors of Mature monocytes/macrophages can transdifferentiate in cells, like endothelial integrated directly into new blood vessels [Schmeisser, 2003 #30; Schmeisser, 2002 #29; Shireman, 2007 #23; Hoenig, 2008 #31].
Fourth, under the influence of VEGF or hypoxia, endothelial cells produce MCP-1 [Marumo, 1999 #34; Lakshminarayanan, 2001 #35], as well as VEGF [Moldovan, 2005 #26], and angiopoietin [Murdoch, 2007 #59], all of which activate and attract Mature monocytes/macrophages [Shireman, 2007 #23]. On the contrary, Mature monocytes/macrophages not only polysoude regulate Tie-2 receptor angiopoetin) [Murdoch, 2007 #59], but also secrete MCP-1 and VEGF, when activated by hypoxia, which affect endothelial cells and even themselves by autodate and paracrine actions, which, consequently, leads to a doubling effect on angiogenic processes [Ferrara, 2004 #60].
Angiogenesis in tumors and chronic inflammation
Over the last decade were collected evidence that in addition to the tumor cells themselves, Mature the monocytes/macrophages also play an important role in angiogenesis and progression of tumors [Sunderkotter, 1994 #4], as neoplastic tumors show neovascularization in the presence of macrophages [Mostafa, 1980 #64], and animals depleted monocytes showed a significant decrease in tumor angiogenesis [Evans, 1977 #63]. The number of macrophages in the tumor tissue more than in most normal tissues [Gouon-Evans, 2002 #79]. Enhanced mobilization and differentiation of circulating Mature monocytes are more likely leads to an increased number of macrophages in the tumor tissue compared to proliferation of macrophages tissue itself [Mantovani, 2002 #15; Schmid, 2007 #13]. Proinflammatory cytokines that are induced by tumor cells and the Central hypoxia, attract Mature monocytes/macrophages to sites of neoplastic necrosis and growth [Pugh-Humphreys, 1992 #44; Coussens, 2002 #78].
As it turned out, in most cases, Mature monocytes/macrophages are recruited with increased neovascularization, which is a critical factor for the growth and development of tumors. In most tumors larger fraction of tumor-associated macrophages (TAMs) M2 is a subset of macrophages that enhances angiogenesis, compared to M1 subgroup, which kills tumor cells [Sironi, 2006 #17; Mantovani, 2002 #15; Sica, 2006 #16]. M2 THERE secrete many Pro-angiogenic factors such as VEGF, TNF-α, IL-8, TGF-β and bFGF [Mantovani, 2002 #15; Sica, 2006 #16; Murdoch, 2004 #1; Nozawa, 2006 #18; Schmid, 2007 #13; Mantovani, 204 #67], and a wide range of proteolytic enzymes [Nozawa, 2006 #18], which can destroy the extracellular matrix and, in turn, lead to migration of endothelial cells for angiogenesis [Moldovan, 2005 #26; Schmid, 2007 #13]. It is important to note that between THERE and vascular densities showed a significant correlation in the case of colon cancer [Oosterling, 2005 #10], breast cancer [Leek, 2002 #12] and pancreatic cancer [Esposito, 2004 #11], suggesting that THERE enhance tumor angiogenesis [Schmid, 2007 #13]. In addition, the strong inflow THERE is significantly associated with poor prognosis of some types of tumors [Leek, 2002 #12; Oosterling, 2005 #10].
Angiogenesis also contributes to the pathology of chronic inflammation. The state of chronic inflammation can be maintained due to the formation of new blood vessels, which continuously deliver inflammatory cells to areas of inflammation and supply it with oxygen and nutrients [Jackson, 1997 #37]. Mechanism and characteristic of neovascularization associated with chronic inflammation, do not differ from the angiogenesis induced by ischemia or tumor. The majority of cytokines and growth factors known to be produced Mature monocytes/macrophages to regulate angiogenesis [Sun-derkotter, 1994 #4]. During the formation of the surface diffuse vascular keratitis in rheumato the bottom arthritis and formation of atherosclerotic plaques in atherosclerosis, proliferative inflammatory tissue contains a number of inflammatory cells, especially monocytes/macrophages, forming new blood vessels, and obtained by the differentiation of inflammatory mediators [Jackson, 1997 #37]. The end result is that an increased number of Mature monocytes/macrophages can be observed for most of the inflammatory areas where angiogenesis is in an abnormal environment, including pathological conditions such as ischemia, tumor, and chronic inflammatory disease, and wound healing [Wagner, 2008 #38; Jackson, 1997 #37; Sunderkotter, 1994 #4; Hunt, 1984 #39].
Transplantation of Mature monocytes compared with stem cell transplantation
A number of studies have been conducted to study therapeutic potential of Mature monocytes/macrophages to arteriogenesis and/or angiogenesis, primarily on models of ischemic disease [Buschmann, 2001 #42; Heil, 2002 #43; Herold, 2004 #33; Ito, 1997 #45; Hirose, 2008 #65]. For arteriogenesis and angiogenesis Mature monocytes may be new and to serve as target cells for cell therapy designed to enhance the growth of collateral vessels, with subsequent tissue regeneration, which may reduce local tissue ischemia and to improve clinical outcomes [Sasayama, 1992 #40; Krupinski, 1994 #54]. Neovascularization of the endogenous Mature monocytes can be induced directly the infusion of MCP-1 [Ito, 1997 #45] or granulocyte-macrophage colony-stimulating factor (GM-CSF) [Buschmann, 2001 #42], or indirectly, through the effect of the resumption of symptoms after administration of 5-fluorouracil [Heil, 2002 #43]; all these substances can contribute to return to collateral arteries and accumulation around them, or proliferation of endogenous monocytes.
In addition, neovascularization can be achieved by transplantation of exogenous autologous or allogeneic Mature monocytes, using ex vivo engineering or without it. Through the development of effective allocation method of the Mature monocytes from peripheral blood [Herold, 2006 #46; Gonzalez-Barderas, 2004 #47; de Almeida, 2000 #48; Rep-nik, 2003 #49], can be collected the required number of autologous Mature monocytes. Although peripheral blood gives a limited number of Mature monocytes, the number of Mature monocytes can be increased up to the number of cells needed for later use, by re-collecting autologous Mature monocytes from the peripheral blood of a subject using leukapheresis [Herold, 2006 #46]. Development of ex vivo tissue engineering leads to a new strategy for the application of Mature monocytes as vectors for therapeutic gene transfection, for example, delivery of GM-CSF to enhance neovascularization [Herold, 2004 #33], as well as for the immediate implementation of cell transplantation. Atarrabia, from the selection of cells to the application, must make Mature monocytes more valuable for regenerative medicine, especially from the point of view of strengthening arteriogenesis and angiogenesis.
The transplantation of Mature monocytes, compared with transplantation of stem cells, you can find several advantages (table 4). First, unlike progenitor cells/stem cells are not capable of self-renewal and proliferation, thus reducing the possibility of formation of tumors from cell transplant. Secondly, they can differentiate into cells of other lines of differentiation that may allow you to avoid unwanted effects in specific tissues when transplanted them there or they migrate there. Third, in contrast to the tissues of the embryo and fetus, they are not associated with ethical and moral aspects related to the retrieval and transplantation, because they can be easily collected from the peripheral blood or bone marrow of an adult organism or in the perinatal period. Fourth, the transplantation of Mature monocytes can prevent immune responses such as graft-versus-host (GvHD), which often occurs after transfusion of allogeneic leukocytes or BM transplantation master mismatched human leukocyte antigen (HLA). GvHD represents the t of an immune response, which is the result of activation of T cells in the graft (donor cells) after detection of tissues of the host (recipient cells) due to antigenic differences [Brichard, 2001 #75]. Activated donor T-cells produce an abundance of cytotoxic and inflammatory cytokines and attack host tissue. As for GvHD, regardless of HLA match, the transplant fraction of Mature monocytes in itself should be more secure than transplantation of mononuclear fraction of BM or GM-CSF mobilized peripheral blood, which certainly includes lymphocytes. Finally, Mature monocytes/macrophages can secrete a number of cytokines, growth factors and trophic substances, which can directly contribute to the regeneration of other tissues, as well as angiogenesis [Sunderkotter, 1994 #4]. For example, VEGF is known to be able to stimulate the development of nerve cells [Jin, 2002 #55; Teng, 2008 #61].
|Advantages and disadvantages of monocytes/macrophages to cell transplantation|
|- no self-renewal||no effect of cell
|- no samorazrusheniya||substitution|
|- no transdifferentiation||- may increase harmful|
|no induction of tumor||the inflammatory response|
|no ethical and moral problems||- possible assistance angio Genesis tumor|
|- possible re-autologous collection||- additional allocation processes and extensions|
|- secretory function of several||relatively narrow, applied|
|cytokines and growth factors||criterion violations|
|- possible construction of the fabric as a vector for gene transplantation||such as coronary heart disease|
Monocytes from cord blood
Mature monocytes constitute about 5-10% of peripheral blood leukocytes [Gordon, 2005 #72; Mielcarek, 1997 #74], whereas undifferentiated monocytes or promonocyte contain 2% BM mononuclear cells [Mielcarek, 1997 #74]. On Rotel, not reported no difference between the contents of monocytes in umbilical cord blood (UCB) and peripheral blood of an adult organism [Sorg, 2001 #73; Mills, 1996 #71]. Therefore, BM and UCB, as well as peripheral blood, may be potential candidates as a source of autologous or allogeneic undifferentiated monocytes or Mature monocytes/macrophages. Actually, BM and UCB are currently the standard sources of hematopoietic precursor cells used to reconstitute the lines of differentiation in the blood of cynomolgus monkeys) after treatment for malignant and benign disease of the blood. However, the potential of this population of stem cells for cell therapy was also demonstrated in the case of other degenerative diseases, especially coronary heart disease. Gain evidence that the delivery of stem cells bone marrow or stem cells of umbilical cord blood to the ischemic area by direct local transplantation or injection into the blood can improve the condition of the pathological changes and the deterioration of the functions [Henning, 2007 #70; Chen, 2003 #51; Willing, 2003 #52; Newcomb, 2007 #26; Chang, 2007 #143]. It is believed that this improvement is partly the result of angiogenesis induced stem cells in ischemic and/or priesininka center [Chen, 2003 #51; Taguchi, 2004 #53; Chang, 2007 #143] even however is the fact/that the mechanisms by which these cells exert their angiogenic effects are not fully understood.
Essentially, the monocytes in UCB are unique compared to monocytes originating from bone marrow (BM) and peripheral blood of an adult organism [Newcomb, 2007 #26]. Only monocytes adult organism are activated hepatocyte growth factor, which is essential for the normal functions of monocytes, such as antigen presentation [Jiang, 2001 #154]. UCB monocytes Express less leukocyte antigen-DR man than the cells of an adult, so their cytotoxic ability below [Theilgaard-Monch, 2001 #155]. Moreover, UCB monocytes do not differentiate into Mature dendritic cells to the extent to which differentiate Mature monocytes, even upon stimulation with IL-4 and GM-CSF [Liu, 2001 #66]; dendritic cells play an important role in the activation of natural T-cells. Secretory function also differs for UCB monocytes and monocytes in the blood of an adult organism. Less secretion of IL-1R and TNF-α, both of which stimulate inflammation, but also play an important role in immune reactions, such as GvHD [Hill, 1997 #76], UCB monocytes after exposure to recombinant interferon-γ is most likely related to the differences in the expression of macrophage antigens, such as CD64, CD14, CD33 and CD45RO, compared with blood monocytes adult [Brichard, 2001 #75]. CD4 +monocytes/macrophages in uterine decidual tissue and blood women with normal pregnancy primarily represents M2 subgroup, which modulates the immune response in the mother-fetus and promote tissue remodeling and angiogenesis to sustain a successful pregnancy [Gus-tafsson, 2008 #69]. These results suggest that the majority of UCB monocytes can also be M2 polarized macrophages, which are less inflammatory and more angiogenic because of the decidual macrophages may partly differentiate from UCB monocytes.
Immaturity functions, stimulating immune response and inflammation, UCB monocytes may contribute to a lower incidence associated with immune suppression, including GvHD, and/or inhibition of dangerous inflammatory response after transplantation, even if they come from an allogeneic source. Although transplantation of autologous monocytes from VM or autologous peripheral blood can prevent immune rejection and GvHD, collection of bone marrow, itself, needs more time, costs and creates physical stress for the patient. Repeated collection of monocytes and the allocation of their own peripheral blood of a patient also requires additional time and cost. Nab is otiv, from the point of view of cell therapy, one of the best benefits of UCB monocytes, compared with autologous monocytes adult organism, is the rapid availability. For example, for patients with stroke, "available time" is a critical factor, and recovery, intervention and process manipulation of cells can be feasible in therapeutic window, according to a state of good medical practice today (cGMP). Because they are treated and feasibility, and safety, UCB monocytes are the most valuable allogeneic cells, which can advance to fully manipulate, without harm to the donor or the recipient prior to transplantation, even if the cells immunological not coincide with the cells of the recipient. Table 5 shows the advantages of UCB monocytes for cell therapy.
|Advantages for cell transplantation of monocytes/macrophages cord blood compared with Mature|
|- Poor immunoregulatory function|
|- Poor inflammatory response||- Possible anti-inflammatory response|
|- Large polarization to a subset of M2 macrophages, which contributes to tissue remodeling and angiogenesis|
|Low incident GvHD compared with bone marrow and peripheral|
|the blood of an adult organism|
|- Relatively easy getting compared to bone marrow|
|- No load for donors|
This superiority UCB monocytes compared with monocytes bone marrow and peripheral blood leads to a more careful study of their promising role for cell therapy. Recently, our group showed in a rat model of occlusion of the middle cerebral artery that UCB transplantation of human cells depleted subpopulation of monocytes (CD14+), does not improve neurological outcome to the extent that transplantation of other UCB cells (depleted of T-cells, depleted In cells depleted CD133+and the price is Oh mononuclear fraction) (Womble, et al., 2008). In relation to angiogenesis, the results suggest that transplantation depleted monocytes UCB human cells could not induce angiogenesis properly and, in turn, would lead to the improvement of neurological dysfunction. At least a subpopulation of monocytes UCB should be critical to UCB-induced recovery after a stroke.
In addition, murine models for syndrome Sanfilippo's title type b (MPS III b), we also showed that musculoskeletal dysfunction and memory will improve after intravenous transplantation of monocytes/macrophages from UCB person (Garbuzova-Davis et al., 2008). When MPS III deficiency of the enzyme α-N-acetylglucosaminidase (Naglu) leads to accumulation of heparan sulfate (HS), glycosaminoglycans and collagen inside the cells and, finally, leads to progressive cerebral and systemic anomalies of organs. Although glycosaminoglycans are known as molecules of the extracellular matrix, which affect the phagocytic capacity of macrophages, monocytes/macrophages in pathological condition with an excess of HS, such as the syndrome Sanfilippo's title, is not clear. In our study, in addition to neurological improvement, histopathological study showed that the number of microglia (macrophages in the brain) increased in all hippocampal regions of mice with Mut is corruption in the Naglu gene, treated monocytes, and HS levels decreased in the liver of treated mice. Moreover, the swelling of the bladder, usually a serious problem for older mice also decreased in treated mice. These results suggest that the introduction of UCB monocytes/macrophages person leads to improvements in mouse models of MPS III, possibly due to the influence of the transplanted cells on the mechanisms of phagocytosis in terms of the accumulation of HS in this disease (Garbuzova-Davis et al., 2008).
Of course, the effectiveness of monocytes in cell transplantation has also been carefully evaluated before clinical therapeutic use because of their specific role and function in inflammation and immunomodulation are not yet fully understood. Their destructive potential in the development of tumor vascularization, diabetic retinopathy, artricheskogo surface diffuse vascular keratitis and atherosclerotic plaques may also need to be studied [Herold, 2006 #46]. However, these dangerous side effects can be prevented by careful assessment of whether the proposed recipient previously existing malignancy, uncontrolled diabetes, arthritis, atherosclerosis or not. If during the pre-treatment evaluation of patients showed the presence of pre-existing disease that may about Ostritsa by transplantation of monocytes, cell transplantation will not be performed for these patients. This is unlikely, because, as we know to the best of our knowledge, no reported complications associated with monocytes, as, for example, about the progression of pre-existing tumors or chronic inflammatory diseases, after BM or UCB transplantation in patients with haematological malignancy or benign diseases, even if the monocytes contain a significant part of the BM and UCB.
On the contrary, the use of UCB monocytes can prevent possible harmful effects or to exert anti-inflammatory effects. Were collected evidence that UCB mononuclear cells provide an anti-inflammatory response in some painful conditions. We have previously shown that transplantation of UCB mononuclear cells significantly reduces the number of CD45+/CD11b+(microglia/macrophages) and CD45+/B220+(In-cell) cells in the brain of rats with occlusion of the middle cerebral artery [Vendrame, 2005 #106]. In addition, UCB transplantation reduces Pro-inflammatory cytokines, such as TNF-α and IL-1β [Vendrame, 2005 #106]. Recently, we also found that transplantation of UCB mononuclear cells in man by intravenous injection of older rats can significantly reduce the number of activated microglia and improve development is the development of nerve cells [Bach-stetter, 2008 #5]. Chronic microglia indicates chronic inflammatory response in the brain tissue and is involved in the nervous structure of the ischemic lesion, as well as other neurode-generative diseases, such as Parkinson's and Alzheimer's disease [Streit, 1999 #9]. Therefore, the results suggest that UCB mononuclear cells can improve the adverse environment of the hippocampus in elderly patients by anti-inflammatory reaction and, then, the regenerative potential of neural stem cells/precursor cells in the body of an elderly patient. Based on these studies we can conclude that macrophage fraction, which contains UCB mononuclear cells to a significant degree, must have the potential to initiate inflammatory reactions, which may be partially responsible for the functional improvement observed in animal models of injury, including stroke.
In conclusion, it should be noted that undifferentiated monocytes or promonocyte are a great alternative to transplantation of stem cells for therapeutic purposes due to their ability to enhance arteriogenesis and angiogenesis in various diseases. These undifferentiated monocytes should be the first candidates due to their is Idumaea efficiency of use, security and numerous functions, such as antitumor response, and angiogenesis.
The present invention has been described in an illustrative manner, and it should be understood that the terminology used is intended only to describe the present invention but not to limit. Obviously, numerous possible variations and modifications of the present invention, and a specialist in the art can generate additional embodiments of the present invention and its modifications, without deviating from the essence and scope of the claimed invention. Therefore, it is necessary to understand that in the framework of the appended claims, the invention can be implemented in a manner different from those described. Accordingly, it is necessary to understand that the information provided in this application drawings and description proposed as an example to facilitate understanding of the present invention, and should not be construed as limiting the scope of the present invention.
1. The use of populations of therapeutic cells for the treatment of ischemia in a subject, comprising the injection of a population of therapeutic cells in ischemic tissue of a subject, where the population of therapeutic cells includes (i) a population of mononuclear cells enriched with cells of the monocytic line differencer the Cai, or (ii) the population embrionalnych stem cells enriched with cells of the monocytic line differentiation by culturing the population embrionalnych stem cells under conditions that enrich the cells of the monocytic line differentiation, or adding cells of the monocytic line of differentiation of a population embrionalnych stem cells, where the cells of the monocytic line differentiation contain promonocyte.
2. Application under item 1, where ischemia is cardiac ischemia, and ischemic tissue is ischemic myocardium.
3. Application under item 1, where the population of therapeutic cells is a population of mononuclear cells and contains 107cells of the monocytic line of differentiation.
4. Application under item 1, where the population of therapeutic cells injected into the myocardium through at least two separate injections, at least three separate injections, at least four separate injections, at least five separate injections, at least ten separate injections, at least twenty separate injections, at least thirty separate injections or at least forty separate injection.
5. Application under item 1, where the injection is from 0.05 ml to 0.3 ml
6. Application under item 5, where the injection is about 0.2 ml.
7. When is the change in p. 1, where the population of therapeutic cells is autologous with respect to the subject.
8. Application under item 1, where the population of therapeutic cell is allogeneic relative to the subject.
9. Application under item 1, where the population of therapeutic cells is a mononuclear cell population, and mononuclear cell population was obtained from bone marrow, umbilical cord blood or mobilized peripheral blood.
10. Application under item 1, where the population of therapeutic cells is a mononuclear cell population, and mononuclear cell population was obtained from the bone marrow.
11. Application under item 1, where the population of therapeutic cells is a mononuclear cell population, and enrichment of cells of the monocytic line differentiation enables the selection of therapeutic cells from the sample, using the method, which leads to the enrichment of cells of the monocytic line differentiation; culturing the population of therapeutic cells in the enrichment of cells of the monocytic line of differentiation; and/or adding cells of the monocytic line of differentiation of a population of therapeutic cells.
12. Injecting a therapeutic agent for introduction into the ischemic tissue by means of a device capable of delivering a metered injection terapeutiche what someone means in ischemic tissue, where the device includes a reservoir for a therapeutic agent, and therapeutic agent contains a population of therapeutic cells, where the population of therapeutic cells includes (i) a population of mononuclear cells enriched with cells of the monocytic line differentiation, or (ii) the population embrionalnych stem cells enriched with cells of the monocytic line differentiation by culturing the population embrionalnych stem cells under conditions that enrich the cells of the monocytic line differentiation, or adding cells of the monocytic line of differentiation of a population embrionalnych stem cells, where the cells of the monocytic line differentiation contain promonocyte.
13. Injecting a therapeutic agent for p. 12, where the population of therapeutic cells is a population of mononuclear cells and contains at least 107cells of the monocytic line of differentiation.
SUBSTANCE: invention relates to the field of biotechnology and cell technology. The claimed invention is aimed at the creation of pluripotent, multipotent and/or self-renewing cells, which are able to start differentiating in a culture into various types of cells and are capable of further differentiation in vivo. The claimed invention is also aimed at the creation of populations of the required differentiating cells, which can be transplanted to patients, genetic modification of endogenic cells and treatment of patients, suffering from diseases, intensity of which can be reduced by means of the said methods.
EFFECT: invention also claims methods of prevention, treatment or retardation of a disease, associated with an infection of immunodeficiency virus.
17 cl, 1 dwg, 13 ex
SUBSTANCE: method provides placing a sperm cell drop and a culture medium drop in the Petri dish at a distance from each other of no more than 5 cm, coupling the drops with a viscous medium strip having a viscosity of 1-4 Pa·s, incubating the dish with its content for 30-90 min in the environment simulating natural environment of the female cervical canal. Before placing into the Petri dish, the culture medium and the viscous medium is incubated until pH value of 7.2-7.6 is achieved.
EFFECT: method enables higher quality of sperm cells selection possessing the highest fertility ability for extracorporeal fertilisation.
11 cl, 7 dwg, 2 tbl
SUBSTANCE: invention refers to medicine, biotechnology and cell technologies. A method for differentiating pluripotent stem cells presenting a human cell line in cells expressing markers specific for a formed endoderm line involves preparing the pluripotent stem cells in a medium differing by the fact that it is free from activin A and contains GDF-8 for the period of time adequate to differentiate the pluripotent stem cells in the cells expressing the markers specific for a formed endoderm line.
EFFECT: invention can be used in medicine for transplantation applications.
14 cl, 19 tbl, 27 dwg, 24 ex
SUBSTANCE: invention proposes oligopeptide versions extracted from protein RAB6KIFL (KIFL20A), which can induce cytotoxic T lymphocytes (CTL) consisting of a complex with molecule HLA-A*0201. Besides, the following has been considered: a pharmaceutical composition and an elimination method of cells expressing RAB6KIFL and HLA-A*0201, an exosome, an extracted antigen-presenting cell and a method for its induction, a pharmaceutical composition and a method of CTL induction, use of active ingredients as per this invention to obtain a pharmaceutical composition for cancer treatment, as well as a polynucleotide coding the oligopeptide as per this invention.
EFFECT: invention can be further used in therapy of diseases associated with RAB6KIFL.
13 cl, 6 dwg, 1 tbl
FIELD: food industry.
SUBSTANCE: invention relates to the field of food industry and represents a brewage method involving thermostable protease addition to the wort after the latter filtration but before cooking; protease thermostability means that such protease activity accounts for at least 70% of its activity measured in the following way: protease is diluted till concentration equal to 1 mg/ml in an analytic buffer (containing 100 mmol of succinic acid, 100 mmol of HEPES, 100 mmol of CHES, 100 mmol of CABS, 1 mmol of CaCl2, 150 mmol of KCl, 0.01% Triton X-100) with pH conditioned to 5.5 with the help of NaOH; the protease is pre-incubated i) in ice and ii) for 10 minutes at a temperature of 70°C; the substrate in relation whereto protease displays activity is suspended in 0.01% Triton X-100: for reaction beginning protease is added in an amount of 20 mcl into a test tube and incubated in an Eppendorf thermomixer at 70°C, 1400 rpm during 15 minutes; the reaction is stopped by way of the test tubes placement into ice; the samples are centrifuged in a cold condition at 14000 g during 3 minutes; the supernatant optic density OD590 is measured; the obtained OD590 value of samples without protease is subtracted from the obtained OD590 value of samples treated with protease; protease thermal stability is determined by way of calculation of protease percentage activity in the samples pre-incubated at a temperature of 70°C relative to protease activity in the samples incubated in ice as 100%-activity.
EFFECT: invention allows to enhance colloidal stability of wort and beer as well as preserve the level of total nitrogen in wort and beer due to protease addition to filtered wort.
16 cl, 1 dwg, 17 tbl, 7 ex
SUBSTANCE: invention refers to cell biology, cell transplantology and tissue engineering. A method for increasing the angiogenic activity of stromal cells of the fatty tissue in tissues and organs involves recovering the stromal cells of the fatty tissue, culturing the recovered cells in the presence of tumour necrosis factor-alpha in amounts of 5 or 100 ng/ml for 24-72 hours, and transplanting into the tissues or organs.
EFFECT: invention can be used for repairing the injured tissues and arresting an ischemia-related developing pathology.
3 cl, 11 dwg
FIELD: medicine, pharmaceutics.
SUBSTANCE: inventions deal with a membrane, used as a substrate for growing cells of retinal pigment epithelium, its application for supporting cells and a method of inoculating the cells on such a membrane. The characterised membrane is non-biodegradable and porous, covered from at least one side with a glycoprotein-containing coating, with pores with a diameter approximately from 0.2 mcm to 0.5 mcm, with a density of membrane pores constituting approximately from 1×107 to 3×108 pores per 1 cm2, and hydraulic conductivity of the membrane higher than 50×10-10 m sec-1 Pa-1,and having the maximal thickness of 11 mm.
EFFECT: claimed inventions make it possible to obtain a transplant for treatment of age-related macular degeneration.
19 cl, 4 dwg, 6 ex, 3 tbl
SUBSTANCE: invention relates to biotechnology, cell technologies and tissue surgery. A method for preparing a smooth muscle cell culture consists in cutting a blood vessel fragment, grinding it to a piece size of no more than 2 mm in any dimension, and incubating the pieces in a culture flask having its bottom preliminarily scratched and containing a culture medium containing 10% embyo foetal serum for at least ten days, but no more than 24 days at 37°C in the CO2 incubator environment; the method differs by the fact that the above blood vessel fragment is an ascending thoracic aorta fragment cut during the coronary artery bypass surgery; before the incubation, the above pieces of the ascending thoracic aorta fragment are kept in the culture medium containing 0.1% collagenase for at least 30 minutes, but no more than 60 minutes at 37°C and then washed in the cell culture medium.
EFFECT: invention enables preparing the cells of the aortic tissue directly from the patient's vital tissues for transplantology applications.
SUBSTANCE: method involves photodynamic exposure on a Vero cell culture of HSV-1 and HSV-2 infected African green monkey's kidney, on samples containing HSV-1 and HSV-2 and on a virus progeny; if observing a decrease of virus titre from a reference by 2 and more of lg TCD50/0.1 ml in the cell culture, samples containing the viruses, and in the virus progeny, the anti-herpetic action is considered to be effective.
EFFECT: using declared method enables more accurate assessment in vitro of the efficacy of the photodynamic therapy to study the mechanisms of various PDT options for the purpose of the further prediction of the PDT efficacy in the herpes-infected patients.
FIELD: medicine, pharmaceutics.
SUBSTANCE: presented solutions relate to field of immunology. Claimed are: pharmaceutical, containing peptide, obtained from HIG2 or URLC10, capable of inducing cytotoxic T-lymphocytes (CTL) by formation of antigen-presenting complex with antigen HLA-A0206. Described are isolated antigen-presenting cell and CTL and methods for their induction. Antigen-presenting cell is induced by contact of cell, expressing antigen HLA-A0206, with peptide, obtained from HIG2 or URLC10. Cytotoxic lymphocyte is induced by contact of CD8-positive T-cell with antigen-presenting cell, presenting on its surface complex of antigen HLA-A0206 and peptide, obtained from HIG2 or URLC10. Characterised are method and means of immune anti-tumour response induction by introduction to patient of medication, which contains peptide, capable of inducing CTL.
EFFECT: claimed inventions can be used in treatment of cancer disease, characterised by higher expression of HIG2 or URLC10.
12 cl, 8 dwg
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to novel compounds of formula
where values A, R1-R6 are given in i.1 of the invention formula. Methods of obtaining the formula (I) compound are described.
EFFECT: compounds demonstrate an inhibiting activity of the cathepsin enzyme, which makes it possible to use them for the preparation of a pharmaceutical composition and for the preparation of a medication.
38 cl, 12 dwg, 495 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to novel compounds of general formula  or their pharmaceutically acceptable salts, which possess properties of an inhibitor of the JAK2 thyrokinase activity. In general formula radicals are selected from group (I) or (II). In group (I) X represents CH or N; R1 represents a halogen atom and R2 represents H, a halogen atom, CN, or is selected from the groups of formulas
or a group -ORP or 5-6-membered heteroaryl, containing 1-4 nitrogen atoms and optionally additionally containing an oxygen or sulphur atom or containing an oxygen atom as a heteroatom, optionally substituted; or (II) X represents -CRA; and RA represents a group of formula , RB represents (a) amino, optionally substituted with one or two groups, selected from the group, consisting of C1-6alkyl, C3-6cycloalkyl, (C3-6cycloalkyl)C1-6alkyl and C1-3alcoxyC1-3alkyl, (b) C1-3alcoxy, (c) hydroxy or (d) a 5-6-membered saturated cyclic amino group, which additionally can contain a heteroatom, selected from an oxygen atom; R1 represents a halogen atom and R2 represents H; R3 -R5 have values given above. Other values of the radicals are given in the invention formula.
EFFECT: compounds can be applied for the prevention or treatment of cancer, for instance hematologic cancer disease or a solid form of cancer, inflammatory disorder, for instance, rheumatoid arthritis, inflammatory intestinal disease, osteoporosis or multiple sclerosis and angiopathy, for instance, pulmonary hypertension, arteriosclerosis, aneurism or varicose veins.
14 cl, 19 tbl, 234 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to medicine, pharmacy and pharmacology. What is presented is using 1-(β-phenylethyl)-4-amino-1,2,4-triazolium bromide (Hypertril) as an active base of drug preparations for correcting dysfunctions of the nitroxydergic system of target organs accompanying homocysteinemia and acute cerebrovascular disorders.
EFFECT: what has been shown is achieving the declared application that is normalising the nitroxydergic function irrespective of whether the NO deficiency or hyperproduction is observed; Hypertril is three times more effective to reduce the homocystein production in comparison to the similar known preparation L-arginine, and has no side effect.
2 tbl, 2 ex
SUBSTANCE: invention relates to a method of obtaining a polymer conjugate of an indolocarbazole compound of formula (I), where R1, R2, R3, W1 and W2 represent hydrogen, X represents methoxy-polyethyleneglycol. The method includes the interaction of a polymer compound of formula (II) with an indolocarbazole compound of formula (III), where Y stands for a methoxygroup. The nvention also relates to a polymer conjugate of formula (I), a pharmaceutical composition, containing the conjugate of formula (I) as an active ingredient, and to the application of the polymer conjugate of formula (I).
EFFECT: obtaining the polymer conjugate of the formula with a high output, the polymer conjugate of the formula for treatment of skin pathologies and HMGB1-associated pathologies.
48 cl, 7 dwg, 7 tbl, 15 ex
SUBSTANCE: invention relates to the field of organic chemistry, namely to benzoimidazole derivatives of formula (I), as well as to their enantiomers, diastereoisomers, racemates and pharmaceutically acceptable salts, where n equals from 2 to 4, each of R1 substituents is independently selected from H, halogen, -C1-4alkyl, -C1-4pergaloalkyl, trifluoro-C1-4alkoxy, -NO2, -CN, CO2H, -OC1-4alkyl, -SC1-4alkyl, -S(C1-4alkyl)-Rc, -S(O)2(C1-4alkyl)-Rc, -S(O)-C1-4alkyl, -SO2-C1-4alkyl, -S-Rc, -S(O)-Rc, -SO2-Rc, -SO2-NH-Rc, -O-Rc, -CH2-O-Rc, -C(O)NH-Rc, -NRaRb, benzyloxy, phenyl, optionally substituted with one-two Rd, cyanobiphenyl-4-ylmethylsulpfanyl, cyanobiphenyl-4-ylmethanesulphonyl, or -S-(CH2)2-morpholine and two adjacent groups R1 can bind with formation of an aromatic 5-6-membered ring, optionally substituted with one methyl group or two atoms of halogen, optionally containing one or two S or N; Ra and Rb each independently represents C1-4alkyl, -C(O)C1-4alkyl, -C(O)-Rc, -C(O)CH2-Re, C1-4alkyl-Re, -SO2-Rc, -SO2-C1-4alkyl, phenyl, benzyl; or Ra and Rb together with a nitrogen atom, which they are bound with, form a monocyclic 5-6- membered heterocycloalkyl ring, optionally containing one heteroatom, selected from O; Rc represents -C3-8cycloalkyl, phenyl, optionally substituted with one-two Rd, benzyl, optionally substituted with one-three Rd; morpholine; Rd independently represents halogen, -OH, -C1-4alkyl or -C1-4perhalogenalkyl, trifluorine C1-4alcoxy, -OC1-4alkyl, or -O-benzyl optionally substituted with halogen, Re represents -C6heterocycloalkyl, optionally containing one or two of O or N atoms, optionally substituted with a methyl group; R2 and R3 both represent H, -CF3 or C1-3alkyl; each of Z represents a C or N atom, on condition that simultaneously not more than two Z represent N. The invention also relates to particular compounds, a pharmaceutical composition, based on formula (I) compound or a particular said compound, a method of treating diseases, mediated by propyl hydroxylase activity.
EFFECT: novel derivatives of benzimidazole, possessing an inhibiting activity with respect to PHD are obtained.
11 cl, 1 tbl, 186 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to new compounds of formula I: cis-COOR-XCH-(CH2)a-CH=CH-(CH2)b-CH3, wherein (a) and (b) can take any value from 0 to 14, (X) is specified in: OH, NH2, CH3, F, F3C, HS, O-CH3, PO4(CH2-CH3)2 and CH3COO, and (R) represents sodium (Na) applicable for preventing and/or treating obesity, hypertension and/or cancer. Also, the invention refers to using the compounds of formula I for preparing a pharmaceutical and/or nutrient composition, to the pharmaceutical and/or nutrient composition based on the compounds of formula I, to a cosmetic, non-therapeutic method for improving skin manifestations and to a method for preventing and/or treating the diseases in humans and animals with using the compounds of formula I.
EFFECT: preparing the new compounds.
18 cl, 22 dwg, 5 tbl, 9 ex
SUBSTANCE: invention relates to medicine, in particular to treatment of cardiovascular disease, and deals with the reduction of cholesterol level in blood plasma. The method includes introduction of an efficient quantity of the first composition, which contains quercetin, vitamin C and vitamin B3, and an efficient quantity of the second composition, containing statin, to a patient in need. In the first composition the weight ratio of quercetin, vitamin C and vitamin B3 equals to 1:0.2-2.5:0.02-1. The efficient quantity of the first composition represents the quantity which provides 1000 mg of quercetin per day.
EFFECT: method provides substantial reduction of cholesterol level, including patients, who have already received therapy by statins.
30 cl, 2 tbl
SUBSTANCE: invention relates to medicine, namely to neurology and cardiology, and deals with treatment or prevention of thrombosis. For this purpose dabigatran etexilate is introduced in a dose from 150 to 300 mg twice per day.
EFFECT: method provides prevention of thromboembolic complications and development of stroke in patients with atrial fibrillation, who have creatinine clearance less than 80 ml/min with an absence of additional factors of risk of massive bleeding.
7 cl, 7 tbl, 4 ex, 3 dwg
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to a method of preparing a drug form of trimetazidine dihydrochloride with the modified release. According to the method modifiers of release - Kollidon SR and hydroxypropylmethylcellulose are mixed, trimetazidine dihydrochloride is added to the mixture, microcrystalline cellulose is mixed with an aerosol in a separate vessel and sifted to obtain a homogenous mass, two mixtures are mixed with each other, powdered with sliding substances, the obtained mass is pressed with the following application of a film coating Opadry II on tablet cores, with taking 10-40% of trimetazidine dihydrochloride, 10-70% of the said modifier of release, 10-80% of the said filler, 0.1-1.0 % of sliding substances and 2-6% of the said film coating of a pill weight.
EFFECT: method of preparing the solid drug form of trimetazidine dihydrochloride is simple and ensures less time and labour consumption.
2 tbl, 5 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to quinazolinone compounds of formula (I) and its pharmaceutically acceptable salts, wherein n is equal to 0 to 3, and R1 is defined as stated in the patent claim. The above compounds are prolyl hydroxylase inhibitors and can be used in pharmaceutical compositions and methods of treating pathological conditions, disorders and conditions mediated by prolyl hydroxylase activity.
EFFECT: compounds can be administered into the patient for treating, eg anaemia, vascular diseases, metabolic disorders, as well as for wound healing.
22 cl, 2 tbl, 211 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology and medicine. What is described is an active immunostimulating vaccine containing at least one RNA, preferentially iRNA coding at least two antigens evoking the immune response in a mammal and used for treating lung cancer, first non-small cells lung cancer (NSCLC), preferentially specified among three primary subtypes, squamous cell carcinoma, adenocarcinoma and large-cell lung carcinoma, or NSCLS-related disorders.
EFFECT: there are produced kits, first containing the active immunostimulating vaccine.
21 cl, 34 dwg, 1 tbl, 8 ex