Method of treating infectious peritonitis experimentally

FIELD: medicine.

SUBSTANCE: method involves administering intravenously a suspension of allogenic mesenchymal stem cells at 1.5×106 per 100 g of the animal's body weight in a physiological solution 2 ml. Animals' mortality has made 27% in the primary group, and 94% in the reference group.

EFFECT: method of treating infectious peritonitis experimentally opens up new possibilities for using the cell techniques for reducing the rate of patient's death caused by peritonitis.

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The invention relates to medicine, in particular for experimental surgery and cellular technologies.

Today mortality in peritonitis according to many authors is at the level of 19 to 70%, which is slightly different from the data 40 years ago. The analysis of the global statistics showed that the contribution of antibiotic therapy in reducing mortality in patients with peritonitis in nearly 100 years (from 1900 - 1980) does not exceed 20% [1]. It should be noted that the attribute importance of intensive therapy in the improvement of results of treatment of peritonitis about 15%; 15-20% of antibiotic therapy. The remaining 70% - optimization of surgical tactics [2]. But in recent times faced with the resistance of microorganisms to antibacterial preparations, especially in nosocomial infections.

One component of modern comprehensive treatment of peritonitis, which is not included in the standards of treatment is immunotherapy. Today for immunotherapy using drugs, which include complex major classes of immunoglobulins (IgAMG). But existing work on this topic suggests that these drugs should be used with a pathogenetic point of view, in a timely manner, the phase of the first manifestations of the symptoms of peritonitis and organ damage, and their later use as a "therapy of despair" yavl�is pathogenetically and economically unjustified and inefficient [3].

Recently in foreign periodical literature appeared works that open up new possibilities of cellular technologies, namely the properties of multipotent mesenchymal stem cells (MSCS). In particular, it became known that they can reduce systemic inflammation, reduce organ dysfunction, possess immunomodulatory properties; found direct cellular effects with immune cells, directly or indirectly, to simulate the ability of the phagocytes of the host to reduce the bacterial load of the body. In order to justify the use of allogeneic mesenchymal stem cells in peritonitis should be considered research data on the use of MSCS in septic conditions and sepsis. It should also be noted that in the scientific literature the results of experimental studies on the use of cellular technologies in peritonitis are underrepresented.

It is worth emphasizing that the priority in the opening of the MSC belongs to the Soviet scientist A. J. Friedenstein, whose article "Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues" [4] dated 1968 year, up to the present time is one of the most cited. Friedenstein the world's first found that in the bone marrow along with hematopoietic cells, there is a population of stem cells capable d�to ferocious in mesenchymal cells of the germ - fibroblasts, osteocytes, chondrocytes, adipocytes, some years later it was confirmed by many studies performed in foreign countries. Feature of MSCS is not only the potential for their transformation in heterogeneous cells of different organs, called plasticity, but also the ability to independently, or indirectly to induce the production of a number of active mediators diverse processes as inflammation, fibrosis, regeneration - cytokines, growth factors and their inhibitors, enzymes, etc. (paracrine activity) [5]. The processes of accelerated apoptosis, disturbances of microcirculation, endothelial injury, myocardial injury, inhibition of the immune response are those pathogenic mechanisms of sepsis, which could potentially be offset by paracrine and plastic effects of MSCS.

In 2007 JXuetal. reported that MSCS, being injected into mice with bacterial lipopolysaccharide, prevent not only acute lung injury and systemic inflammatory response, significantly reducing serum levels of proinflammatory cytokines IFN-γ, IL-1β, IL-6, MIP-1α and IL-8 [6]. Another experimental study proved the efficacy of MSCS in a model of sepsis in mice, caused by the ligation and puncture of the colon was performed Shirley N. J. Mei and colleagues [7]. In this work set�been not only decrease levels of markers of systemic inflammation under the influence of MSCS but the reduction of bacterial contamination of the spleen and signs of multiorgan failure. In the other study on the model of sepsis in rats proven to reduce the functional depression of the myocardium and inhibition of tissue expression of IL-1β and IL-6 in animals treated MSCS [8]. Further, the same authors found that these effects are more expressed in MSC, donor whom were females [9].

Important, recently installed property MSK explaining their effectiveness in infectious processes, is an independent production of the antimicrobial peptide cathelicidin hCAP-18/LL-37, suppresses the growth of gram-negative microorganisms. Because the inhibition of this protein anti-infective activity of MSCS was decreased by almost 2 times, the authors concluded that the direct antibacterial effect of MSCS in lung infection is comparable in importance with traditionally referred to plasticity and paracrine activity [10].

In addition to the above literature in the U.S. patent office has application for the invention - "Uses of mesenchymal stem cells" (the use of mesenchymal stem cells) No. 20120027730 from 02.2012, in which the authors describe the possibility of using MSCS in systemic inflammatory response and sepsis [11].

It should be noted that in our country there are studies on the use of MSCS in sepsis, �x use in septic conditions is not assigned by the authors questioned. This study is more focused on identifying ways to reduce the level of apoptosis of transplanted cells [12].

The data given above, obtained by foreign authors in the experiment in systemic inflammation and sepsis. We investigated the anti-inflammatory and immunomodulatory effects of MSCS on the model of infectious peritonitis. In the foreign literature there are data Hosoon Choi, Ryang Hwa Lee and co-authors, who suggest that MSCS are activated by inflammatory signals to highlight the anti-inflammatory protein TSG-6) stimulated with TNF-α (TNF-α) and thus creating a negative feedback that reduces inflammation in zymosan (suspension of polysaccharides from yeast cultures) induced sterile peritonitis [13].

The object of the invention is a method of treating infectious peritonitis in the experiment.

The problem is solved by a method for the treatment of infectious peritonitis in the experiment, namely that intravenous injections of a suspension of allogeneic mesenchymal stem cells at the rate of 1.5×106per 100 g weight of the animal.

An example of a specific implementation.

The study was conducted in a surgical unit of the experimental laboratory of the First Moscow state medical University named after I. M. Sich�Nova. All manipulations were performed in compliance with the requirements for humane treatment of animals (Strasbourg, France, 1986) and the manual on experimental (preclinical) study of new pharmacological substances " (Moscow, 2000). Pharmacological immobilization and anesthesia of the animals was carried out by inhalation ether anesthesia.

Work on the isolation of cells and their cultivation was performed in accordance with General principles of cultural studies [4].

Mononuclear fraction cells obtained from the bone marrow aspirate animals. To do this, under ether anesthesia from the medullary canal of the tibia and the femur had received bone marrow cells by aspiration syringe with 18G needle containing environment for the fence (0.5 ml of phosphate-buffer solution containing 50 UNITS/ml of heparin and 0.25 mg/l gentamicin). The cell suspension km centrifuged at 1500 rpm (350g) 5 minutes, the precipitate of cells was resuspended in solution for lysis of red blood cells (114 mm NH4Cl, 7.5 mm JISC3, 100 μm EDTA) for 3 min and re-centrifuged. Demolitionary supernatant was removed by aspiration, and the cell precipitate was resuspended in DMEM (pan Eco, Russia) containing 10% calf fetal serum (Hy Clonegold", USA), 0.4 µm insulin, 0.25 mg/l gentamicin.

These cells presented a primary to�litura, predominantly mononuclear cells km, which are then seeded in an amount of 2.0-2.5 million cells/ml in culture flasks. Then flasks were placed in a CO2incubator with the concentration of CO25%, air 95% and with high humidity. 2 days after isolation of primary culture nepriklausoma of cell suspension was removed, and the remaining cells continued to cultivate. Replacing the culture medium with a fresh carried out every 3-4 days. After the formation of subconfluent monolayer cells washed once with versene solution, then removed with a solution of Versene with 0.25% trypsin, were resuspended in growth medium and poured into new culture dishes.

For 23-24 hours prior to the introduction of fecal suspension into the abdominal cavity, under ether anesthesia, the animals performed the amputation of the distal 1/3-1/5 of the tail, with the aim of creating a hyperresponsiveness of the background and stress in the body of the rat [14]. By overdose of ether anesthesia was carried out the killing of several intact rats. The contents of the cecum were removed, weighed, and prepared a 20% mixture of isotonic sodium chloride. The mixture is then filtered through a double layer of cheesecloth. Then within 15 minutes after preparation of stool suspension was injected from a single puncture (in the centre white line of the abdomen) in right and left hypochondrium, in the right and Leo�Yu iliac region, rate of 0.7 to 0.9 milliliters per 100 grams of animal weight, 25 sexually Mature Wistar rats. After the introduction of suspended animals were placed on standard food and water regime. After 7-8 hours after the introduction of the fecal suspension, the experimental animals were divided randomly into 2 groups. Group 1 major (15 units), which was used for the operation of transplantation of allogenic mesenchymal stem cells in the dose of 1.5×106per 100 g weight of the animal in 2 ml of saline intravenously. Group 2 - control (10 items), which was used for the simulation is the introduction of stem cells injected into the tail vein with 2 ml of saline. Daily was evaluated the General condition of the rats, the degree of weakness, attitude to food and water, hair condition, and the level of mortality on the 3rd day of the experiment.

The differences in the main and control groups began to occur immediately after the operation of transplantation of mesenchymal stem cells. Rats in group No. 1 are treated more vigorously, actively moved around the cage, showed interest in food and water, unlike the rats in the control group 2, for 1 days and the total time of the experiment. During the whole time of the experiment the rats of the control group were more adynamic compared with primary, localized PR�property in one corner of the cage, while one group, the coat was ruffled, interest in food was absent, showed moderate interest in the water.

Mortality on the 3rd day in the main group was 27%, and in control of 94%. Surviving animals were removed from the experiment by an overdose of ether anesthesia on the 10th day of the experiment. At audit of all of the species in the abdominal cavity revealed turbid liquid, bowel loops are swollen, hyperemic, edematous, dilated vessels of the mesentery. Anterior abdominal wall, spleen, liver, kidneys, part of the greater omentum were removed for histological examination with the subsequent manufacture of paraffin sections and painting them with hematoxylin-eosin, studying under a light microscope.

The macroscopic comparison of the inflammatory process in the abdominal cavity of rats who died on the 3rd day of the experiment, you should pay attention to the fact that the rats in the treatment group the severity of the inflammatory process, the number of peritoneal exudate, deposition of fibrin on the parietal, the degree of distention of the intestines and visceral peritoneum was less than in rats from the control group (Fig. 1-3).

In the study of stained histological sections showed that all the dead animals from acute peritonitis severity of the inflammatory process was different in the compared groups. In the control gr�PPE histologic picture was characterized by: in the liver hepatocytes in a state of protein malnutrition, with the plethora of Central veins (Fig. 4), and the belly is swollen with diffuse neutrophil infiltration (Fig. 5), observed in renal glomerular ischemia, hyperemia, acute tubular necrosis (Fig. 7), and in the picture of septic spleen spleen with diffuse infiltration of neutrophils (Fig. 6). All this proved picture of acute fibrinous-purulent peritonitis. In the main group the dead animals histologic picture was characterized by a not so bright picture of the inflammatory process in the abdominal cavity, as in the control. We watched resolving peritonitis (Fig. 8-11).

In the study of histological material of animals that were withdrawn from the experiment at 10 days, it was found that the severity of the inflammatory process in rats in the main group was smaller and there was a picture resolves peritonitis. Meanwhile, the rats in the control group was observed macroscopic and histological picture of continuing acute peritonitis.

Thus, the results of the proposed method for the treatment of infectious peritonitis in the experiment provide new solutions to this serious and difficult problems in surgery.

The invention is illustrated by drawings.

Fig. 1 is a perspective view of the opened animal from the control group with common fibrinous-purulent peritonitis (Group �2).

Fig. 2 shows the accumulation of purulent exudate in the lateral channel, and deposition of fibrin on the intestinal loops (Group No. 2).

Fig. 3 shows an inflammatory process in the abdominal cavity of rats from the main group, which is significantly different from the control group (Group # 1).

Fig. 4 shows a micrograph of the liver of a rat from the control group: hepatocytes in a state of protein malnutrition, a sharp hyperemia of the Central veins, neutrophil sludge in the veins, centrolobular necrosis in the spaces of the dissertation of red blood cells, the epithelium of the bile ducts partially sluman into the lumen. Coloring of G+e, magnification ×200.

Fig. 5 shows a micrograph of the parietal peritoneum of the rats from the control group: edema, diffuse neutrophilic infiltration. Coloring of G+e, magnification ×200.

Fig. 6 shows a micrograph of the spleen of the rats from the control group: marked necrosis, diffuse neutrophilic infiltration. Coloring of G+e, magnification ×200.

Fig. 7 shows a micrograph of the kidney of the rat from the control group: in the kidney glomerular ischemia, hyperemia, acute tubular necrosis. Coloring of G+e, magnification ×200.

Fig. 8 shows a micrograph of the liver of the rats of the main group: hepatocytes in a state of protein degeneration, hyperemia of the Central veins, no centrolobular necrosis, èpite�rd bile ducts into the lumen, and there have been no red blood cells in the space of thesis. Coloring of G+e, magnification ×200.

Fig. 9 shows a micrograph of the parietal peritoneum of the rats of the main group: edema, diffuse lymphoplasmacytic infiltration of isolated neutrophils. Coloring of G+e, magnification ×200.

Fig. 10 shows a micrograph of the spleen of rats of the main group: hypoplasia of lymphoid follicles, marked multiple multinucleated giant cell type of megakaryocytes in the red pulp. Coloring of G+e, magnification ×100.

Fig. 11 shows a micrograph of the kidney of the rat from the main group: glomerular ischemia, stenosis, degenerative changes in the epithelium of the proximal convoluted tubules. Coloring of G+e, magnification ×100.

Literature

1. Wittmann, D. H. Intraabdominal infections // Pathophysiology anoireannent. 1991. P. 84.

2. Saveliev B. C., Gelfand B. R. Abdominal surgical infection // Russian national recommendations. 2011.

3. Briskin B. S. Immune disorders and immunotherapy with intra-abdominal infection / B. S. Briskin, N. N. Khachatryan, 3. I. Savchenko // Surgery. - 2004. - No. 2. - Pp. 24-27.

4. Friedenstein AJ, Petrakova KV, Kurolesova AI, Frolova GP. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation. 1968; 6(2): 230-247.

5. Garcia-Gomez I, Elvira G, Zapata AG, et al Mesenchymal stem cells: biological properties and clinical applications. ExpertOpinBiolTher. 2010 Oct; 10(10): 1453-68.

6. Xu J, Woods CR, Mora AL, et al. Prevention of endotoxin-induced systemic response b-bone marrow-derived mesenchymal stem cells in mice. Am.J. Physiol. LungCellMol. Physiol. 2007;. 293: L131-41.

7. Mei SH, Haitsma JJ, Dos Santos CC, et al. Mesenchymal Stem Cells Reduce Inflammation while Enhancing Bacterial Clearance and Improving Survival in Sepsis Am J RespirCrit Care Med. 2010 182 (8) 1047-57.

8. Weil BR, Manukyan MC, Herrmann JL, et al. Mesenchymal stem cells attenuate myocardial functional depression and reduce systemic and myocardial inflammation during endotoxemia Surgery. 2010; 148(2): 444-52.

9. Manukyan MC, Weil BR, Wang Y, et al. Female stem cells are superiortomalesmpreseivingmyocardialfunctionfollowingendotoxemiaamjphysiolregulintegrcompphysioljune 2011 300:(6) R1506-R1514.

10. Krasnodembskaya A, Song Y, Fang X et al, Antibacterial Effect of Human Mesenchymal Stem Cells Is also been other ideas where in Part from Secretion of the Antimicrobial Peptide LL-37 Stem Cells 2010; 28:2229-2238.

11. Delgado; Mario; et al. Uses of mesenchymal stem cells. United States Patent Application. No. 20120027730, February 2, 2012.

12. Averianov, A. V., konopliannikov A. G. et al. Effects of combined treatment of allogeneic mesenchymal stem cells of bone marrow and erythropoietin in experimental models of sepsis / Infection in surgery 2012. - №4. - 43-48.

13. Hosoon Choi, RyangHwa Lee, Nikolay Bazhanov, JooYoun Oh and Darwin J. Prockop // Anti-inflammatory protein TSG-6 secreted by activated MSCs attenuates zymosan-induced mouse peritonitis by decreasing TLR2/NF-B signaling in resident macrophages. Blood. 2011 118: 330-338.

14. Simonyan K. C. Peritonitis // M.: Medicine. 1971. - 216 c.

A method for the treatment of infectious peritonitis in the experiment, namely that rats intravenously injected with a suspension of allogeneic mesenchymal stem cells at the rate of 1.5×106per 100 g weight of the animal.



 

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SUBSTANCE: invention relates to novel pyridin-2-one and pyridazin-3-one derivatives, having Btk inhibiting activity. In formulae I-IV:

,

R denotes -R1-R2-R3 or -R2-R3; R1 denotes a heteroaryl containing 6 ring atoms, including one N heteroatom; R2 denotes -C(=O), -C(=O)N(R2'), where R2' denotes H; R3 denotes R4; where R4 is a lower alkyl, heterocycloalkyl, (lower alkyl) heterocycloalkyl or heterocycloalkyl (lower alkyl), where the heterocycloalkyl contains 6 ring atoms, including two heteroatoms selected from N and O; and where R4 can be substituted with one or more substitutes selected from lower alkyl, oxo group and lower alkoxy group; X denotes CH or N; Y1 denotes lower alkyl; n and m are equal to 0; values of radicals Y2, Y4 are given in the claim.

EFFECT: improved properties of compounds.

6 cl, 2 tbl, 42 ex

FIELD: medicine.

SUBSTANCE: invention relates to the field of biotechnology, in particular to the lentiviral delivery of apoptin into tumour cells, and can be used in medicine. The method includes obtaining a lentiviral construct, expressing modified apoptin, fused with a sectretory signal of lactotransferrin and a transduction signal (ST-CTP-apoptin), with the further obtaining of recombinant lentiviral particles, defective by replication and carrying the modified apoptin, which are later introduced into T-lymphocytes (TILs), obtained in the surgical ablation of the tumour or in the process of obtaining a biopsy, possessing the ability to penetrate into tumour cells. After that, obtained TILs are autotransplanted to the said patient.

EFFECT: invention makes it possible to increase the ability of apoptin to penetrate into tumour cells and produce an oncolytic effect with respect to all the tumour cells.

2 dwg, 3 ex

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