Method for preparing interleukin-8 from donor blood neutrophiles
FIELD: medicine, immunology, cytokines.
SUBSTANCE: method for preparing interleukin-8 (L-8) from donor blood neutrophiles involves isolating neutrophiles from peripheral blood in double gradient ficol-verografin, bringing about the neutrophile content in cell suspension to the concentration 5 x 106 cells/ml, activation at temperature 37°C for 1 h followed by centrifugation and separation of supernatant containing the end product. For activation the cell suspension of neutrophiles in the concentration 5 x 106/ml of Henx's solution is poured in plastic Petri dishes. Invention provides the development of a simple and available method providing the high level of interleukin-8 (IL-8) from neutrophiles of donor blood not requiring using expensive preparations.
EFFECT: improved preparing method.
The invention relates to medicine, namely to immunology.
Immunocytokine are in the focus of modern immunology. One of the most important cytokines inflammation believe interleukin-8 (IL-8)pertaining to α-the superfamily of chemokines that regulate the activation and motility of cells in the inflammatory focus (Simbirtsev A.S., 1999; Smirnov, V.S., Freidlin, I.S., 2000). The main cells producing IL-8 are monocytes/macrophages and endothelial cells, but it can be produced and many other types of cells: lymphocytes, neutrophilic granulocytes, various epithelial cells, fibroblasts, hepatocytes and other (cetlinski S.A., Kalinin N.M., 1998; Simbirtsev A.S., 1999; Smirnov, V.S., Freidlin, I.S., 2000; Cassatella, M., Bazzoni f, Ceska, M., 1992). Production of IL-8 starts in response to the activation of cells of various biologically active substances, including components of the cell wall of bacteria, viruses, and many cytokines, lectins and other (cetlinski S.A., Kalinin N.M., 1998; Simbirtsev A.S., 1999; Smirnov, V.S., Freidlin, I.S., 2000).
Development of methods for" cytokines including IL-8 is the actual problem in immunology.
A known method for production of IL-8 from peripheral blood leukocytes. It includes a five-fold dilution medium Needle fresh heparinised blood, preparation of solution range of complete the ora of synthesis and secretion of IL-8 (prodigiozana). In a 96-well plate for culturing cells contribute 100 ál of working solution prodigiozana add to the wells, 100 ál of prepared peripheral blood and cultured in the CO2-the incubator for 24 hours at a temperature of 37°With, then carefully gather supernatant and investigated for the presence of IL-8 (cetlinski S.A., Kalinin NM Immunology for the doctor. - St. Petersburg: Hippocrates, 1998. - S-121).
The disadvantage of the described method for production of IL-8 is the use of different populations of cells, secreting cytokines with divergent spectrum of activity that contributes to the inhibition of the activity of the cells and reduce the secretion of cytokines. This method requires the use of expensive drugs and duration takes a lot of time.
The closest to the essential features of the claimed invention is a method for the secretory products of neutrophils (Dolgushin I.I., Bukharin O.V. Neutrophils and homeostasis, Ekaterinburg, 2001. - P.75), consisting in the allocation of leukocyte suspensions from heparinised peripheral blood donors by sedimentation of red blood cells by adding 10% solution of gelatin in a ratio of 10:1 at a temperature of 37°C for 30 minutes, the allocation of neutrophils from the leukocyte suspension on the double gradient is lotnosti ficoll-urografin, bringing neutrophil concentrations up to 5×10 cells/ml of medium 199 with subsequent activation of particles of monodisperse polystyrene latex with a diameter of 1.7 μm, 50 particles on one neutrophil. Then cells and latex particles were removed by centrifugation at a speed of 3000 rpm for 15 minutes and then filtering through Millipore filters with a pore size of 0.24 μm ("Millipore, USA).
The prototype of the invention has the following essential features: to highlight cytokines use the neutrophils of blood donors, which are isolated from the leukocyte suspension, bring neutrophils to a certain concentration and activate these cells.
The disadvantages of the prototype are:
1. Low secretion of IL-8;
2. The use of expensive drugs (particles of monodisperse polystyrene latex, milliprobe filters).
The claimed invention is directed to solution of the problem consisting in the development of a method for production of IL-8 from neutrophils of blood donors.
The solution of this problem allows us to develop a new simple and accessible way, providing a high level of secretion of IL-8 by neutrophils of blood donors that do not require the use of expensive drugs.
To achieve this result the invention "Method of production of interleukin-8 from neutrophils is rovi donors" includes the use of peripheral blood leukocytes donors, the selection of neutrophils from the leukocyte suspension, breeding them up to a certain concentration and activation of these cells. While neutrophils activate adhesion on polystyrene plastic for 1 hour at a temperature of 37°in Hanks solution without subsequent filtration supernatant through Millipore filters.
In relation to the prototype of the claimed invention has the following distinctive features: activation of neutrophils carry out their adhesion to polystyrene plastic for 1 hour at a temperature of 37°in Hanks solution that provides a high level of secretion of IL-8 and is economically advantageous because it eliminates the use of expensive drugs.
Between the features and the technical result there is a causal relationship: the method provides a high level of secretion of preformed IL-8 and is cost-effective.
For the method of obtaining the IL-8 is used by neutrophils in blood donors, which can activate their adhesion to polystyrene plastic for 1 hour at a temperature of 37°in Hanks solution that provides a high level of secretion of the cytokine in a short period of time without the use of expensive drugs, which is economically advantageous.
According to the author is in the information set of essential features, characterizing the essence of the claimed invention, is not known from the prior art, which allows to make a conclusion about conformity of the invention, the criterion of "novelty".
According to the authors of the essence of the invention is not necessary for the expert in the obvious way from the prior art, since it cannot be detected by the above-mentioned influence on the resulting technical result
- a new property object a set of attributes that differ from the prototype of the claimed invention, which allows to make a conclusion about its compliance with the criterion of "inventive step",
The set of essential features that characterize the invention, in principle, can be repeatedly used in medicine to obtain a technical result, which consists in receiving IL-8 from neutrophils of blood donors, which allows to make a conclusion about conformity of the invention, the criterion of "industrial applicability".
This method is as follows.
Donors make a fence of 20.0 ml of heparinized peripheral venous blood, which is the purpose of sedimentation of red blood cells and obtain a leukocyte suspension defend with the addition of a 10% solution of gelatin in a ratio of 10:1 at a temperature of 37°C for 30 minutes. Selected leukocyte suspension washed once in Hanks solution, centrif is the response at 1500 rpm for 10 minutes. Neutrophils isolated from the leukocyte suspension on a double density gradient sterile solutions ficoll-urografin (Pharmacia, Sweden; Spofa, CSSR). The density of the upper layer of the gradient is 1,075-1,077, and the lower - 1,093-1,095. The volume of each layer of the gradient is equal to 1,5 ml After 30 minutes centrifugation at 1500 rpm at the boundary between the plasma and the upper layer of the gradient is formed a ring, consisting mainly of mononuclear cells (lymphocytes - 45-50%, monocytes - 15-20%, granulocytes 10-15%). In the interphase between two layers of density gradients is a layer of granulocytes with a purity of 96-98%, 2-4% of mononuclear cells. Cells are gently harvested, transferred into sterile centrifuge tubes, washed three times with sterile Hanks solution and diluted with the same solution to a concentration of 5×106cells/ml Poured into 1.0 ml of cell suspension in sterile plastic Petri dishes with a diameter of 40 mm Cultured for 1 hour at a temperature of 37°C. Merge adosados. Neadgezivnye cells are removed from the supernatant by centrifugation at 3000 rpm for 10 minutes. In the selected supernatant of neutrophils donors determine the content of Pro-inflammatory cytokines: IL-1α, IL-1β, IL-8, TNF-α. For this purpose use the appropriate test systems LLP "Cytokine" (Saint-Petersburg). These test systems are based on the "sandwich"method is e solid-phase ELISA using horseradish peroxidase as indicator enzyme (cetlinski S. p., Kalinin K.M. Immunology for the doctor. - St. Petersburg: Hippocrates, 1998. - 156 S.).
To determine the effectiveness of the proposed method for production of IL-8 was used supernatant unactivated and activated particles monodisperse polystyrene latex with a diameter of 1.7 μm (obtained from the research Institute of synthetic rubber, Saint-Petersburg) neutrophils blood donors. Cultivation of neutrophils without inductor secreting cells and in his presence were carried out in the same mode, i.e. at a temperature of 37°C for 1 hour in Hanks solution.
The results showed that activated adhesion on plastic neutrophils blood donors significantly more compared with nonactivated and activated latex neutrophils secrete preformed Pro-inflammatory cytokines: IL-1β, IL-8, TNF-α. Probably the adhesion of neutrophils to plastic is the most annoying factor that leads to significantly more pronounced secretion of preformed cytokines compared with nonactivated and activated latex neutrophils blood donors. Most high level secretion was noted in relation to IL-8 (table 1).
The content of cytokines in supernatant unactivated and activated n is Trofimov blood donors (M± m).
|IL-1α (PG/ml)||IL-1β (PG/ml)||IL-8 (PG/ml)||TNF-α (PG/ml)|
|Supernatant unactivated neutrophils donors(n=10)||51,3±3,5||12,6±1,4||48,8±and 23.4||4,6±0,27|
|Supernatant activated latex particles neutrophil donors (n=10)||53,5±6,1||24,8±14,1||101,7±42,3||6,2±0,85|
|Supernatant activated adhesion on plastic neutrophil donors (n=10)||63,1±5,3||63,5±16,6*, **||889,5±28,3*, **||to 163.1±43,3*, **|
|Note: * - reliability of differences of performance in relation to the group "Supernatant unactivated neutrophils donors";|
|** - reliability of differences of performance in relation to the group "Supernatant activated latex particles neutrophil donors." Used the Wilcoxon criterion.|
Presents tabular data suggests that the proposed "Method of production of interleukin-8 from neutrophils of blood donors" is the most effective in comparison with used in modern immunology.
The method receiving the Oia interleukin-8 from neutrophils of blood donors, including the allocation of neutrophils from the peripheral blood of donors on the double gradient ficoll-urografin, bringing cell suspension of neutrophils to a concentration of 5·106cells/ml, activating at 37°C for 1 h followed by centrifugation and separation of the supernatant containing the target product, wherein to activate cell suspension of neutrophils at a concentration of 5·106cells/ml Hanks solution is poured into plastic Petri dishes.
FIELD: preparative biochemistry, medicine, pharmacology.
SUBSTANCE: method for purification of interferon proteins is based on using cation-exchange chromatography on a solid matrix. Method is realized at more basic pH value, i. e. at relatively higher pH value corresponding to the isoelectric proteins point, pI, designated for purification. However, at this pH value indicated proteins are remained to be absorbed and therefore method involves using buffer solutions of organic or inorganic salts able to modify the solution ionic strength. Invention provides a simple method for industrial realization of the method and economy availability.
EFFECT: improved purifying method.
8 cl, 1 tbl, 6 ex
FIELD: medicine, gynecology.
SUBSTANCE: one should carry out basic antibioticotherapy and immunocorrection, moreover, as an immunocorrector it is necessary to apply affinoleukin preparation as submucous injections into uterine cervix. A single dosage of affinoleukin corresponds to 1.0 U once during 2 d. Therapy course consists of 10 injections. The innovation provides regional activation of T-lymphocytes' transition from immature forms into mature ones associated with mucous membranes of urogenital tract and, thus, efficient chlamydial elimination.
EFFECT: higher efficiency of therapy.
2 ex, 2 tbl
FIELD: medicine, ophthalmology.
SUBSTANCE: at the background of anti-tuberculosis therapy one should additionally introduce Betaleukin parabulbarly per 0.33 ng every other day, therapy course - 5 procedures. The innovation provides local immunostimulating action of Betaleukin.
EFFECT: higher efficiency of therapy.
1 ex, 1 tbl
SUBSTANCE: method involves carrying out surgical intervention at the first stage. Micro irrigators are withdrawn through the posterior fornix. Chemotherapy begins in two weeks. Autoplasma chemotherapy is applied with Cyclophophsphan at a dose of 1000 mg/m2 and Carboplatin at a dose of 150 mg/m2 on plasma cleared from cellular elements. Lymphocytes isolated from blood plasma are incubated with 1,2 mg of Proleukin for 24 h and introduced into the abdominal cavity via a micro irrigator at the second day. The specified procedure sequence is repeated every 3 weeks in 4 courses and later on as 6 courses every 6 weeks.
EFFECT: accelerated treatment course; reduced adverse toxic side effects.
SUBSTANCE: invention relates to composition containing as the first component serogroup B N.meningitis outer membrane vesicle, and as the second one treatment immunogenic component associated with brain fever or other Nisseria infections.
EFFECT: immunotherapy for treatment of Nisseria infections with increased effectiveness.
9 cl, 5 tbl
SUBSTANCE: method involves applying radiation therapy and two regional intra-arterial chemotherapy courses with Cysplatin given at a dose of 90 mg/m2 with hyperglycemia combined with microwave frequency hyperthermia 4-6 weeks later with 4 weeks long pause. Cyclophosphan is additionally introduced concurrently with radiation therapy for 10 weeks once a week at a dose of 200 mg. Roncoleukin is intravesicularly introduced after the second regional intra-arterial chemotherapy course at a dose of 2.0 mg dissolved in 50 ml of 0.9% NaCl solution twice a week during at least 8 weeks.
EFFECT: reduced risk of complications; increased clinical response frequency.
FIELD: medicine, pediatrics, infection diseases.
SUBSTANCE: method involves subcutaneous administration of roncoleukine in the dose 250000 U by the following schedule: course is 4 injections, at two days intervals on the third day, and this course is repeated if necessary up to disappearance of clinical symptoms. Invention promotes to more complete body sanitation due to inhibition of blood virus replication, decrease and in some cases elimination of symptoms of extrapyramid syndrome, diminishing hepatomegalia and normalization of the immune function system. Invention can be used in treatment of early age children with cytomegalovirus (CMV) infection.
EFFECT: improved treatment method.
FIELD: medicine, therapy.
SUBSTANCE: invention relates to a method for prognosis of effectiveness in treatment of infectious and inflammatory diseases with recombinant cytokines. Method involves using assay of functional polymorphism of cytokine gene as a prognostic criterion as the effectiveness degree and by-side effect of therapy with recombinant cytokine analog. The advantage of invention involves the possibility for prognosis of effectiveness of treatment with recombinant cytokine regulating inflammation and depending on genetic specificity. Invention can be used in immunotherapy, immune correction, immune rehabilitation or immune prophylaxis of different diseases, in particular, in diseases of otorhinolaryngological organs, chronic hepatitis C and pulmonary tuberculosis.
EFFECT: improved prognosis method of treatment.
3 cl, 4 dwg, 3 ex
FIELD: medicine, immunology.
SUBSTANCE: invention relates to compositions and method for immunosuppression achievement. Claimed compositions contain two main agents: namely the first agent targeted to interleukin-15 receptor (IL-15R), and the second agent which inhibits costimulating signal transferred between T-cell and antigen-presenting cell (APC).
EFFECT: diagnosis and therapy of immune deceases, in particular autoimmune deceases with improved effectiveness.
45 cl, 3 dwg
FIELD: medicine, infectious diseases, psychotherapy.
SUBSTANCE: method involves antiviral therapy, immune correction with thymus hormones and interferon inductors. Since the first day the relapse symptom method involves prescription of antiox+ (1 capsule per a day) and detox+ (1 capsule, 2 times per a day) for 30 days, profluzak (20 mg, 3 times per a day for 5 days) and then in the dose 20 mg, 1 time per a day for 20 days. Derinate is prescribed topically as installation into urethra in the dose 3-5 ml or with tampon into vagina and with simultaneous prescription of microenemas in the dose 10-40 ml for 10 days. Since 10-14 day in exacerbation period in the proliferative stage of an antiherpetic immune response derinate is prescribed by intramuscular injections in the dose 5 ml, 1 time in a day, 10 injections in total number. Then since 6-th day of exacerbation and intake of profluzak psychotherapy seances are carried out. The first seance of rational psychotherapy involves explanation to a patient in available form mechanism of the disease, the necessity of prolonged treatment and motivation for treatment is enhanced by suggestion. The second psychotherapy seance involves neurolinguistic programming wherein a patient colorful and detailed description of desirable function when he imagines achievement of the desire result, and positive emotional and vegetative symptoms are notes and the conditional-reflect association is formed by tactile contact. Under psychotherapist control a patient imagines "part of person" responsible for achievement of the desire result the patient attention is accented for the desire result and arisen physiological responses are fixed by using tactile contact. Also, new behavior methods are proposed to take for a patient that are directed for achievement of the desire result - avoiding sexual contacts during exacerbation of genital herpes in one of partner and during every month hormonal cycles, avoiding stress situations, and in case of each stress situation significant for patient profluzak has to be intake in a single dose 40 mg, using a condom in sexual contact in the exacerbation period. Patient analyzes the proposed new behavior methods that help avoiding relapses, provide good state of health, promotes to recovery process of genitals recovery and selects at least three the most rationally available for him behavior methods. In the case of the positive response that is controlled by physiological symptoms the result is fixed by tactile contact. The third seance involves the suggestive psychotherapy directed for fixing the attained result. The suggestive therapy seance is carried out once per a week for 6 months. Method provides declining the treatment time, to reduce relapse frequency of genital herpes and to recover the emotional state of patient.
EFFECT: improved treatment method.
2 cl, 3 tbl, 1 ex
SUBSTANCE: method involves applying basic therapy until stroke type is unclear and differentiated therapy after having determined stroke type. Recombinant interleukine-2-roncoleukine as subcutaneous injections at a dose of 500 000 MU into external surface of intact arm at the fourth-fifth day after stroke beginning. Roncoleukine is administered as a course of 1-3 injections given with two-three days pause on the background of traditional stroke treatment.
EFFECT: enhanced effectiveness of treatment.
2 cl, 3 tbl
SUBSTANCE: method involves mechanically cleaning biological connective tissue and treating animal or human connective tissue with solutions. After mechanical cleaning, the animal or human connective tissue is hold in 3-6% hydrogen peroxide solution 2-4 h long, frozen at temperature below 0°C in wet state, thawed and treated with alkaline detergent solution of pH 7.0-14.0. Then, it is treated with 3-6% hydrogen peroxide solution 2-4 h long , heat treated at 40-90°C, shaped as required and sterilized.
EFFECT: improved quality of allergen-free biomaterial.
FIELD: medicine, surgery.
SUBSTANCE: in an experiment in rats it is necessary to replant an intact fragment of thyroid tissue or its cellular suspension into the strand of greater omentum due to its wrapping. Moreover, one should introduce once 500000 autologous adhesive hemogenic cells into the intact fragment of thyroid tissue under replanting or its cellular suspension after omental fixation. The innovation enables to restore functional activity of thyroid gland.
EFFECT: higher efficiency of restoration.
4 dwg, 2 ex
FIELD: medicine, gynecology.
SUBSTANCE: invention relates to a method for treatment of uterus myoma. Method involves carrying out the selective catheterization and the following administration of actovegin in the dose 100 mg in 2.5 ml into both uterus arteries followed by immediate administration of embolyzate. Method provides the painless course of post-embolization period and decreasing symptoms of post-embolization syndrome.
EFFECT: improved method of treatment.
SUBSTANCE: method involves applying gel Pantonic over mucous membrane of nasal passages and over superior cervical sympathetic nodes. Each of four fields is sequentially exposed to ultrasonic frequency current action. Action power is 1-2 kV. Action time is equal to 3-4 min.
EFFECT: improved optic nerves excitability and conductivity.
FIELD: chemical-pharmaceutical industry, pharmacy.
SUBSTANCE: invention relates to a pantohematogen-base medicinal formulation for external using that comprises the following components, wt.-%: dry pantohematogen, 0.01-6; polymeric gel-forming composition, 0.1-20, and water, the balance. Proposed medicinal formulation provides using pantohematogen an aqueous solution without addition of alcohol and therefore it can be used in cases wherein alcohol-containing formulations are contraindicative - in burns of different degree, small skin damages and others and can be used in treatment of patients showing intolerance to alcohol-containing preparations. Method for preparing the pantohematogen medicinal formulation for external using involves preparing "dry" pantohematogen aqueous solution in the ratio limits pantohematogen : water = (1:50)-(1:200) by weight and solution is kept under normal conditions for 1 h and filtered. Additionally an aqueous solution of rare-linked polymer is prepared in the ratio = (1:10)-(1:200) and an aqueous solution of neutralizing agent in the amount necessary for providing slightly alkaline medium of final solution. In the volume ratio of pantohematogen aqueous solution and rare-linked polymer in the ranges = (3:1)-(1:10) components are mixed and stirred to obtain the homogenous solution. Then neutralizing agent aqueous solution is added to a mixture and stirred to obtain homogenous solution with slightly alkaline reaction (pH = 6-8). The advantage of proposed technology in preparing the medicinal formulation involves its simplicity and providing prolonged fitness period.
EFFECT: improved preparing method.
6 cl, 1 ex
FIELD: medicine, cardiosurgery.
SUBSTANCE: the present innovation deals with complex procedures for carrying out open-heart operations associated with high blood losses and considerable requirement for blood-substitution therapy. It is necessary to carry out preoperational autoblood sampling in cardiosurgical patients due to a single exfusion of 5-10% volume of patient's circulating blood and its conservation, moreover, 1-2 d before autoblood sampling it is necessary to conduct total hypoxic sample, detect the values of oxygen balance and lactate concentration in blood serum and at its values being SpO2>85%, SvO2>60%, O2EI<30 and the absence of lactate concentration growth at the background of 40-min-long respiration with hypoxic gaseous mixture with 10%-content of oxygen one should consider tolerance to hypoxia to be high and sample autoblood at the quantity of 10% against total volume of circulating blood; at physiological ability of a patient to breathe for 40 min with hypoxic gaseous mixture with 12%-content of oxygen it is possible to consider tolerance to hypoxia to be average and sample autoblood at the quantity being not more than 7% against the volume of circulating blood, and at physiological ability of a patient to breathe with hypoxic gaseous mixture only at the content of oxygen being under 14% tolerance to hypoxia should be stated to be low and, thus, one should sample not more than 5% against the volume of circulating blood. The innovation decreases postoperational complications, increases efficiency of hemotransfusional therapy due to individualized approach for detecting optimal quantity of autoblood sampled.
EFFECT: higher efficiency of sampling.
SUBSTANCE: the suggested hemostatic material contains collagen and thrombocytic mass dried due to freeze drying and structured in formaldehyde vapors, moreover, it contains transfusion-unsuitable thrombocytes at the following ratio: collagen : thrombocytes = 100:(0.5-2.5). Additionally, this material may contain antiseptics or antibiotics of wide spectrum of action. The innovation enables to apply thrombocytes after cryodestruction, and, also, transfusion-unsuitable thrombocytes (overdue storage period), that is apply the material which is considered to be useless. The material suggested is of high hemostatic activity.
EFFECT: higher efficiency of application.