Peptidemia fraction of spleen mammals possessing immunostimulatory activity and its preparation |
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IPC classes for russian patent Peptidemia fraction of spleen mammals possessing immunostimulatory activity and its preparation (RU 2033796):
  
The method of treatment of purulent and burn wounds / 2016577
The invention relates to medicine, in particular to methods for treating wounds
 Means for stimulation of reparative processes in the liver after surgery on it in terms of the destruction of the body131j / 2005479
The invention relates to medicine, namely to the treatment of liver pathology in terms of the defeat of radionuclides (in particular131I)
 "painkiller "mielopid"" / 2000789
 Method for preventing postsplenectomic syndrome in experiment / 2247567
Embryonic spleen should be sampled, washed in nutritive medium № 199 to be placed into fresh medium № 199 to obtain homogenate in teflon homogenizer followed by centrifuging; then one should isolate the upper, medium and inferior layers, suck off medium layer and the upper part of inferior layer; the cell mixture obtained should be diluted in nutritive medium № 199 to be then introduced by injections into mesentery of small intestine or rectus muscle of abdomen. The present innovation favors the activation of immune system in patients undergone splenectomic operation and in those in case of surgical immunodefficient state due to high functional and regenerating activity of transferred embryonic splenic cells.
 Method for treating the cases of non-operable cardiologic patients / 2250772
Method involves using mononuclear autologic marrow fraction containing 6-9x104 mesenchyma cells per 1 ml or autologic mesenchyma trunk cells. The cells are separated from brain bioptate in the amount of 106 cells/kg of patient body mass. The preparations are intracoronarily introduced in fractions at a rate of 3-5 ml/min into the right coronary artery. The introduction is also carried out in intra-arterial mode in jets or in drops.
Method for treating the cases of allergic diseases / 2250773
Method involves introducing autologic mesenchyma trunk cells as a single intravenous drop dose or in the amount of 1 mln cells per 1 kg of patient body weight.
Method for treatment of suppurative wound / 2257211
Method involves every day covering a wound with an agent comprising of pig spleen homogenate and oxygenated with perfluorane taken in the following amounts: 100 g of pig spleen homogenate and 10 ml of oxygenated perfluorane. Invention promotes to accelerating sanitation of suppurative wounds due to activation of topical immunity and activation of regenerating processes. Invention can be used in treatment of suppurative wounds.
Method for treating noninvasive cancer of urinary bladder / 2257219
The present innovation deals with treating locally spread tumor diseases of urinary bladder due to applying either chemo- and/or radiation therapy. Moreover, one should intravenously once inject by drops autologous mesenchymal stem cells (AMSC) at the quantity of 1 mln cells/kg patient's body weight. Moreover, in case of chemotherapy AMSC should be injected during the period before the 20-th d after the last injection of chemopreparation, in case of radiation therapy - 12-15 d against the day of irradiation. In case of chemoradiation therapy AMSC should be injected after chemotherapy before the course of radiation therapy. This method enables to carry out chemoradiation therapy in patients with severe accompanying diseases that prevent such a therapy, and prevent the development of general toxic complications of chemoradiation therapy and medicinal and radiation-caused cystitis.
Biotransplant and method for treating the cases of osteoporosis / 2265442
Biotransplant has genetically unmodified mesenchyma stem cell culture as active component obtained from fetal donor autologous material. The tissue is subjected to disaggregation and the produced cell suspension is resuspended and cultivated on growth medium containing transferrin, insulin, fibroblast growth factor and heparin to accumulate mature stroma in cell culture. Method involves intravenously dropping mesenchyma stem cell culture in the amount of 50 to 500 mln in 50-100 ml of physiologic saline.
Biotransplant and method for treating the cases of diabetes mellitus of type i and ii / 2265443
Biotransplant has genetically unmodified mesenchyma stem cell culture as active component obtained from fetal donor autologous material and genetically unmodified fetal myoblast culture. Method involves intravenously dropping mesenchyma stem cells in the amount of 50 to 500 mln in 50-100 ml of physiologic saline. The fetal myoblast culture is intramuscularly introduced at a dose of 100 mln of cells per 10 kg.
Method for treating acute myocardial infarction at st segment lifting / 2266125
One should introduce the suspension of autologous mononuclear medullary cells without preliminary cultivation in to the mouth of coronary artery nourishing infarction area. Cell suspension should be introduced at the quantity of 100-150 mln. cells immediately after stenting coronary artery due to technique of passive passage. The procedure should be performed on the 14th - 21st d against the onset of the disease mentioned. The method provides efficient counterbalance of cardiomyocytes due to applying valuable stem cells at excluding complications induced by arterial occlusion.
Method for production of conditioning medium having colony-promoting activity / 2270022
CBA/CaLac intact mouse spleen is homogenized in medium containing 95 % of RPMI-1640 and 5 % of ethyl silicate; filtered through nylon grid; washed by centrifugation with RPMI-1640. at 1500 rpm for 10-15 min; final concentration of living splenocites is adjusted to 2x106 colony/ml using media containing 10 % of ethyl silicate, 1 % of L-glutamine; 0.2 % of gentamycine, 0.05 % of mercaptoethanol, 89 % of RPMI-1640; obtained product is cultivated at 37°C for 6 days, centrifuged and supernatant containing target product is separated followed by storage at -20°C. Before filtering 1 ml of distilled water is added in sterile spleen homogenate and after 25 sec 10 ml of RPMI-1640 medium is added. Target product is stored for 1 month or less.
Cell elimination by using viruses / 2270685
Invention relates to uses of viruses capable of reducing undesired cells in ex vivo mixtures of normal marrow or peripheral blood cells and tumor cells, such as leucosis or lymphoma cells due to interaction of abovementioned mixture with vesicular stomatitis virus. Invention also relates to method for malignant tumor treatment in mammalian. Claimed method includes sampling of mammalian marrow or peripheral blood cells; interaction of said cells ex vivo with vesicular stomatitis virus; mieloablative therapy; and transplantation of purified hematopoietic cells into mammalian. In another embodiment invention relates to method for malignant tumor treatment in mammalian having transplant of marrow or peripheral blood stem cells. Said method includes interaction ex vivo of collected transplant cell with vesicular stomatitis virus and administration of these purified cells in mammalian. Method of present invention makes it possible to reduce risk of malignant tumor backset in mammalian with transplanted hematopoietic cells.
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(57) Abstract: The invention can be used in medicine, veterinary medicine, specifically in the field of clinical immunology, immunopharmacology, intensive therapy. For fast and effective stimulation of the immune system with combined lesions of the immune system used peptideatlas fraction of the extract of spleen with a molecular weight of 10,000 - the 140,000 daltons. Obtained according to the invention immunostimulant provides distinct and rapid immune-stimulating effect. table 2. The invention relates to the field of immunology, immunopharmacology and can be used in medicine, veterinary medicine, intensive care, intensive therapy. Currently one of the most difficult tasks in the field of resuscitation and intensive therapy remains adequate immunomodulatory therapy in generalized infectious process (sepsis and purulent-resorptive fever), mortality in which, despite the use of modern antibacterial, immunomodulatory and desintoxication funds is quite high. First of all it is connected with the appearance, as the disease progresses, various defects in the system which are virtually all parts of the immune system: the defects of humoral and cellular nonspecific link defects in T - and b-systems of immunity, as well as violations of the mechanisms of immunoregulation. The combined damage of the immune system makes the body virtually defenseless not only to pathogenic and conditionally pathogenic microorganisms. Therefore, one of the main pathogenetic principles of treatment of generalized infectious process remains adequate immunostimulirutuyu therapy. There is currently a wide range of funds, which are hormones or mediators of the immune system, possessing immunostimulatory activity: taktivin, thymopentin, timogen (drugs derived from the Central authority of immunogenesis of the thymus), interleukins 1 and 2, the group of drugs interferon; myeloide (preparations from bone marrow); preparations containing immunoglobulins; and a wide range of tools, pavlyushina hormones or mediators of the immune system:A. Preparations of lipopolysaccharide of origin: prodigiozan, pyrogenes, zymosan, property etc. Century Produced purines and pyrimidines: oximetry, pentasil, methyluracil, etc. C. Derivatives of 8-oksihinolina: levamisole, diazepan, etc. as well as other funds that are not included in the data group; unithiol, isopr the diversified funds have low therapeutic activity (immunostimulating effect does not appear immediately after taking the drug, and after some time, up to one week or more), as well as a narrow range of immunopotentiating activity. Know the means of having immunostimulatory activity splenin, representing a protein-free extract from bovine spleen. Like other known means splenin does not have a wide range of immunopotentiating activity (stimulates predominantly quantitative and less qualitative indicators In the immune system, and has a moderate demominations effect). In addition, its immunostimulating effect does not appear immediately after injection, and only 8-10 days or more daily injections, even if the drug is administered intravenously. In the scientific literature describes the possibility of obtaining biologically active substances spleen (4). A significant disadvantage of this method is that the resulting product contains very small quantities of biologically active substances of protein nature, and in addition, the drug is not constant in composition, poorly stored (within 4-6 weeks ataboutC) and contains a significant amount of impurities. The prototype of the pre-Christ.>/P>According to this method, the spleen pigs or cattle are washed with water or acetone, crushed, frozen at -5aboutC, extracted with 3% acetic acid at pH 3.0 to 4.0 in the presence of zinc chloride, centrifuged, mixed with acetone, collected precipitate is dissolved with water in the presence of acetic acid followed by filtration and lyophilization of the desired product. However, the product obtained by a known method, is a complex of polypeptides, which does not have a high stimulant and has a significant amount of impurities. The author's task to obtain a drug with a wide range of immunopotentiating activity and little time effect from the beginning of its application. The method of producing drug polyactive X-210 is as follows. Spleen pigs, cattle treated with saline solution with heparin, free from residues of fat and connective tissue, ground frozen at -85aboutC and homogenized in 50 ml of phosphate buffer at pH 7.5. Then the pH of the homogenate was adjusted to 8.0, extracted with the same solution, the m phosphate buffer solution, the eluate is subjected to liquid chromatography high pressure and target product elute 0.01 m solution of phosphate buffer containing 0.01 m sodium chloride, the obtained fraction was diluted with saline to a final concentration of protein in solution 20 mg/m followed by sterilizing filtration, dispensed into ampoules of 5-10 mm and lyophilizers. The method is illustrated as follows. Obedienoe electroshock pigs, in sterile conditions, cut open the abdominal cavity, mobilize the splenic gate and allocate the spleen. Under sterile conditions kanyoro splenic artery and vein and wash the bloodstream sterile saline solution with heparin (to process one spleen using 1000 ml of physiological solution with the addition of 1000 UNITS of heparin). Washed spleen released from capsules, remnants of fat and connective tissue, placed in a sterile flask, pour cooled to +5aboutWith sterile physiological sodium chloride solution and transported to the laboratory. In the laboratory the splenic parenchyma shredded with scissors to size 1,h,h,0 cm, placed on aluminum sheets in a row and placed in a freezer Kama is giving 5 days). Frozen spleen is placed in a homogenizer, add 2-3 volumes of cold (+4-5about(C) 50 mm phosphate buffer solution (pH 7.5) based on 1.0 g of tissue. The mixture is homogenized for 1 minute. Homogenization is repeated, if there are pieces of intact tissue. The homogenate was placed in a vertical cooker IIM-50 and bring the pH of the homogenate to 8.0 1.0 M phosphate buffer solution and then perform extraction for 15-20 minutes at a temperature of +5 +8aboutC. the Extract was centrifuged at 50000 for 60 minutes at a temperature of +2aboutWith poured through the filter gauze or glass wool to remove particles of fat. The extract is subjected to concentration and desalting using ultrafiltration. To this end, apply the system for ultrafiltration of Minuten (Millipore Corporatricn, Bedford, MA 10170), using membranes with nominally limit molecular weight of 150,000. Processing time up to 50 liters of the original extract 3 hours. All of the procedures performed in the cold (+2 +4aboutC) room. For the deposition of protein to 1000 ml of the original extract was added 100 cm3adsorbent (adsorbent used gel phosphate of calcium). The deposition is carried out at constant stirring of the solution at a temperature of +4 +6aboutC. After USAID is it 1 part of the resulting suspension was washed by ultrafiltration cell two parts of 5 mm phosphate buffer pH 7.5 and suck the liquid by vacuum to obtain dry adsorbent. Then dry the adsorbent is suspended in an eluting solution (60 mm phosphate buffer solution with pH 7.5 (two parts of buffer solution to 1 part of the adsorbent (volume/weight). From the obtained protein solution secrete the peptide fraction from M. C. 10000-140000 daltons using liquid chromatography high pressure. In our research system is used for large-scale chromatography high pressure (Biolroeess System, Phormecia, Sweden, order 44-00070) with medium to separate Setapart, izgotovliaemye the same firm. For the identification of proteins in the molecular mass of the column is pre-calibrated by ribonuclease a and human immunoglobulin G. as eluent using 0.01 M phosphate buffer with the addition of 0.01 M sodium chloride. Registration is done by UV absorption at a wavelength of 280 nm. The peptide fraction was diluted with sterile physiological solution of sodium chloride (0.9 per cent) to the concentration of protein in solution 20 mg/ml Then the target product is subjected to sterilizing filtration on membranes, Amicon or Vladipor with a pore diameter of 0.4 nm poured into ampoules (vials) and lyophilizers. The powder of the drug is white with a faint yellowish tinge, odourless and hygroscopic. Constant chromatographic mobility is holding 40 mg protein (biuret method) was diluted to 4.0 ml aqueous buffer and applied 0.1 ml on a steel column, pre-washed aqueous buffer to a constant baseline. The division performed by the method of highly efficient liquid chromatography with speed eluting flow 1.0 ml/min Column pre-calibrated with blue dextran and ribonuclease. Overlooking the peaks detected at a wavelength of 280 nm. On the chromatogram find several peaks: 1 with retention time ranging from 8.5 to 9.5 min is identified as blue dextran: 2nd group of peaks with retention time of 12.8 to 14.5 min identified as peptides that determine the biological activity of the drug, the 3rd with hold time from 16.9 to 18.4 min identified as a ribonuclease. Calculate the constant chromatographic mobility of the peptides that determine the biological activity of the drug (K), which is calculated by the formula: K where Tpthe retention time of ribonuclease A; Tdthe retention time of blue dextran; T the retention time of the peak. Constant chromatographic mobility of the peptides that determine the biological activity of the drug is equal 2,16 0,28. Test specific impurities. The control in the absence of peptides with a molecular weight greater than 100,000 daltons. with a diameter of 18 mm and a height of 500 mm, filled with Sephadex G150. The rate of elution of 2.2 ml for 10 minutes. Registration is done by UV absorption at a wavelength of 280 nm. The chromatogram should not be peaks of UV absorbed material in the exit area of the free volume of the column (the area of the elution of substances with a molecular weight greater than 100,000 daltons). To determine the exit area of the free volume of the chromatographic column is pre-calibrated, causing her mixture of 0.5 ml of 0.05% solution of blue dextran (mol.a lot more than 43,000 daltons) in the buffer for elution. Note: the Preparation of an eluting buffer, 0.2 g of sodium chloride) brand CHP) dissolved in 0.25 l of distilled water and add 0.05 g of sodium azide (mark OFS). Determination of free amino acids. The contents of the vial or ampoule is dissolved in 10.0 ml of distilled water. To 0.01 ml of the prepared solution was added 1 ml of citrate buffer pH 2.2 and 200 μl of the mixture applied to the column amino acid analyzer. Amino acid analysis is performed by a standard method. The content of free amino acids, the drug should not exceed 10 mg. Determination of biological activity. Testing. 2-3 strips. The supernatant centrifuged 5 min at 400 g (4aboutC). Cellular precipitate resuspended in 0.5 ml of R-re Hanks, add 12 ml of distilled water, and 25 seconds. add 4 ml of 3.6% saline solution of sodium chloride. Then centrifuged (400 g, 4aboutC, 5 min) cellular precipitate resuspended in R-d Hanks to the concentration of cells 2,5x104CL/ml Each portion of the cells are divided into two and placed in scintillation vials (1 ml of the prepared suspension). Two experienced scintillation vial add 1 ml of the prepared solution polyactive, two control vials with 1 ml of a solution HEPES. Experimental and control bottles adapted for 30 min at 37aboutWith PR actinic light. Sequentially added to each vial 20 µl of matrix solution lyuminola, gently stirred for 30 sec. rotating the vial and place it into the counter. In vials sequentially register the level of chemiluminescence (1 minute account) three silver tubes in mode matches. After calculating the control and experimental samples to calculate an annual growth rate of chemiluminescence by the formula: K , where 01, 02, the intensity of chemiluminescence in the experience; K1, K2, the intensity of chemiluminescence in control. yourself, significantly greater immunostimulatory activity has exactly the peptide fraction of the extract of spleen (proposed facility) than protein-free splenin. On the other hand, from literature data it is known that peptides of various animals have species specificity, that is, when repeated in geneticist alien organism develops sensitization, which may occur mainly on the first, second, third or fourth type of immunopathological reactions (according to the classification of Gell et R. A. Coombs) (6). Therefore, the experiments on determination of the part of the protein extract, which has no species specificity. This crude extract spleen shared with gelfiltration on Sephadex into fractions with molecular weight up to 10,000, 10000-20000, 10000-30000, and so on until the faction 10000 250000 daltons. Then these fractions were senzibilizirani laboratory animals: outbred white mice, white rats, rabbits of the Chinchilla breed, purebred dogs. To identify the first type of immunopathological reactions used to define the level of histamine in the peripheral blood and classical test playback of anaphylaxis after administration of the resolving dose of peptide PA and tests of the consumption of complement, antibody-dependent cellular cytotoxicity, and immunoprecipitation agar. To identify a third type of immunopathological reactions used the definition of immune complexes that bind complement, determined their size and activity of complement and the concentration of degradation products of complement components, C-3 and C-4. To identify the fourth type of immunopathological reactions used for the reaction of inhibition of macrophage migration and reaction of besttransport in the presence of the specific antigen. It is noted that with the introduction of all laboratory animals fractions, starting with fractions of 150,000 daltons to faction 250000 with the re-introduction develops immunopathological reaction to the first type (anaphylactic shock) almost 100% of the animals. Thus, these fractions were immediately excluded from further studies. With the introduction of other analyzed fractions with molecular mass less than the 140,000 daltons noted in only one case of anaphylactic shock, upon further study of these fractions anaphylactic shock not occurred, so this one case was regarded by you as an artifact. The development of other types of immunopathological reactions (the second, the third time its development was assessed by the normalization of immunological parameters) used the determination of the immune status of outbred mice with peritonitis (caused by the introduction intraperitoneally culture of Staphylococcus aureus) in 30 minute intervals after a single injection of the studied fractions) neobladder sensitizing activity). The amount of injected substance in terms of protein was equal in all the studied fractions. The immune status was assessed by the following indicators: the number of T, V, D, O-lymphocytes, T helper cells, T suppressor, the reaction besttransport with phytohemagglutinin, the number of classes of immunoglobulins a, M and G phagocytic index, phagocytic number and index bactericides neutrophils; the activity of serum complement; the concentration of circulating immune complexes and medium molecular peptides. As a result of the studies noted normalization of all modified parameters after a single injection of the studied fractions with molecular mass up to the 140,000 daltons. As can be seen from the table.1 the highest immunostimulating activity had a fraction with a molecular weight of 10000 140000 Dalton, time normalization of the indicators in the application of this fraction was also minimal (about 1.0-1.5 hours), when applying the fraction with molecular weight less than 10,000 daltons time parameters normalization was applied the same as the splenin. P R I m m e R. Used immunomodulation in treatment of dogs with sepsis (54 pieces) using splenin and proposed facilities. Sepsi the s with an interval of 2 days. On the fourth day after the last injection condition of the dogs was heavy, from the blood were inoculated to 130-160 microbial colonies. Clinically recorded pronounced weakness, dryness of the mucous membranes of the skin, shortness of breath, tachycardia and hypotension. In both groups of dogs treatment was started 5 days after the last injection of microbes. In both groups of dogs were used the same antibiotic therapy, detoxification and parenteral nutrition; correction of water-electrolyte metabolism and the treatment of DIC. The immunomodulation in group 1 dogs was performed by injection of 5 ml intramuscularly daily offered funds in the 2nd group of 5 ml splenin. Indicators of immunity was assessed before treatment, after one day and after 5 days after the start of treatment. By the end of 5 days in the second group survived only 6 dogs in the first group after 5 days died only one dog. Clinically in the first group behavior of animals did not differ from healthy individuals, dogs were active, eating alone; when bakposev the blood of these dogs was sterile. In the second group, the condition of the dogs was moderate, clinically observed weakness, dogs were not fed, from the blood were inoculated by 20-45 colonies is Oh continued. After 14 days in the first group, all dogs were alive in the second group, the last dog died on the 14th day. Dynamics of the immunological parameters presented in table 2. As can be seen from the table before the start of therapy dogs has been a sharp decrease in the number of T-lymphocytes mainly due to the reduction of T-helper cells, a sharp decline rbtl with lymphocyte mitogen, reduced levels of immunoglobulins M and G, reduced phagocytic number and index bactericides neutrophils, high content of circulating immune complexes. Therefore, the observed defects by qualitative and quantitative indicators of the T-system of immunity, the quality of the immune system, as well as indicators of phagocytosis. In the first group of dogs after the first day of therapy almost all the studied parameters significantly from control values did not differ in the second group remained significantly reduced the total number of T-lymphocytes, T-helper cells, immunoglobulins M and G, rbcl and phagocytosis indices and concentration of the CEC. After 5 days in the first group of dogs, some indicators even exceeded the reference level. The second group is the first day of therapy. Thus, the application for immunostimulation of the proposed tools is more pronounced and rapid immune-stimulating effect, which significantly affects the effectiveness of the combined therapy. The proposed remedy for immunostimulation, also used in treatment of 109 dogs with distemper and 98 dogs with viral enteritis. The dogs were taken for treatment after they were wypracowanie vet in terms of inefficiency further treatment. When applying the proposed drug of 109 dogs with distemper died 7 dogs (mortality of 6.4%), 98 dogs with enteritis, only one (case fatality rate of 1%), indicating a high therapeutic activity of the drug. The proposed tool can be used for correction of immune disorders of various etiologies, as well as a component in the treatment of severe forms of chronic cautionsly infection of any etiology in veterinary medicine and medicine. 1. Peptidemia fraction of spleen mammals, mol.m.10000 - 140000 D, which has immunostimulating activity. 2. The method of obtaining peptidase fraction of spleen mammals, including the washing of the body, grinding, freezing, extragenomic fact, the spleen was washed with a saline solution after freezing homogenized in a solution of 50 mm phosphate buffer, the homogenate is extracted with a solution of 1.0 M phosphate buffer after centrifugation the supernatant is subjected to ultrafiltration, adsorption on halfspace calcium handling sorbent solution of 60 mm phosphate buffer with subsequent liquid chromatography and elution of the target product solution in 0.01 M phosphate buffer.
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