Injection of Multipotent Mesenchymal Stromal Cells as a Cause of Hemorrhages in the Regional Lymph Nodes: Experimental Study.

Hemorrhagic changes after subcutaneous injection of autologous bone marrow multipotent mesenchymal cells with transfected GFP gene and additionally stained cell membranes to WAG rats in the projection of ligated femoral vein were studied by fluorescent microscopy. Hemorrhages in tissues with experimental acute local venous occlusion were caused by a combination of venous hypertension with inflammation around the foreign body - the ligature used for ligation of the vein. Fibrin found in tissues together with erythrocytes in the hemorrhages could stimulate the formation of granulations and new vessels instead of damaged or thrombosed ones. Multipotent mesenchymal stromal cells and their detritus getting into the regional lymph nodes initiated immune reactions morphologically confirmed by stubborn hypertrophy and hyperplasia of the lymphoid nodules, hemorrhages, and manifest diapedesis of erythrocytes to the organ parenchyma and sinus system.

Some Peculiarities of Local Distribution of Multipotent Mesenchymal Stromal Cells after Their Injection into Intact Muscle Tissue in Experiment.

Changes in the muscular tissue after subcutaneous injection of autologous bone marrow multipotent mesenchymal stromal cells transfected with GFP gene and additionally stained with cell membrane dye Vybrant CM-Dil in the projection of ligated femoral vein were studied by light microscopy with luminescence. Stromal cells injected through the skin can appear not only in the damaged tissue where acceleration of regeneration processes is required, but also in intact structures located in superficial or deeper layers. In intact muscular tissue, stromal cells spreading in the perivascular tissue initiate inflammation and migration of macrophages, activate and even trigger sclerotic processes due to differentiation into connective tissue cells (fibroblasts) and stimulation of proliferation and collagen synthesis by host fibroblasts. Injected multipotent mesenchymal stromal cells are gradually phagocytized by macrophages.

Results of Experimental Ligation of the Main Vein with the Use of Cell Technologies.

Autologous multipotent mesenchymal stromal cells (MMSC) of bone marrow origin with transfected GFP gene and additionally stained cell membranes were injected to rats through the skin in the projection of ligated femoral vein. The results were evaluated by fluorescent microscopy. No signs of MMSC incorporation into the wall of ligated vessel or reorganized collaterals were detected. Angiogenesis processes involving MMSC were detected in experimental rats within just 4 days and progressed until week 2 postinjection, mainly in granulations at the site of surgical intervention and the cicatrix forming there. Injected MMSC completely formed all tunics of the new vessels and incorporated in the vessels forming from the recipient cells. MMSC and the objects created from them were gradually eliminated with participation of macrophages and replaced by structures formed from the recipient cells.

Possibility of Aggravation of Tissue Sclerosis after Injection of Multipotent Mesenchymal Stromal Cells Near the Forming Cicatrix in the Experiment.

The peculiarities of tissue sclerosis after injection of autologous bone marrow multipotent mesenchymal stromal cells transfected with GFP gene and stained with Vybrant CM-Dil cell membrane dye were studied by light microscopy with luminescence. The surgical intervention consisting in ligation of the great vein was followed by tissue sclerotic transformation caused by direct damage and chronic inflammation caused by the presence of slowly resorbed ligature. Injection of stromal cells after this intervention led to formation of more extensive scar. This can attest to the possibility of stromal cells differentiation into connective tissue cells, fibroblasts, and stimulation of proliferation and collagen synthesis by host fibroblasts. A decrease in the volume of dense fibrous connective tissue due to scar reorganization at latter terms cannot not excluded.

Initial Stages of Angiogenesis after Acute Experimental Local Venous Outflow Disturbances and Application of Cell Technologies.

The initial stages of angiogenesis in rats after transcutaneous injection of autologous bone marrow multipotent mesenchymal stromal cells transfected with GFP gene and stained cell membranes in the projection of ligated femoral vein were studied by fluorescent light and confocal laser microscopy. Large clusters of brightly fluorescing elongated fibroblast-like cells were seen in the paravasal tissue and in the postoperative scar and signs of angiogenesis were noted as soon as in 4 days. The injected cells not only formed new vessels, but also integrated into vessels formed by host cells. Some injected cells were phagocytizied by macrophages and the latter started to fluoresce due to the presence of the membrane dye. These macrophages within the specified period appeared in the regional inguinal lymph nodes where they formed clusters in the lymphoid parenchyma of the cortical substance.

Possibility of Using Mesenchymal Stromal Cells to Restore Lymph Flow in Experimental Phlebothrombosis.

The possibility of formation of lymphatic vessels after introduction of autologous bone marrow-derived multipotent mesenchymal stromal cells transfected with GFP gene into thrombosed femoral vein was studied by fluorescent microscopy. Vascular thrombosis caused by ligation of the great vein with subsequent injection of thrombin solution was accompanied by blockade of regional lymph flow. The cells injected into thrombosed vein directly participate in the formation of new lymphatic vessels in the paravasal tissue surrounding the vein, its tissue region, and around regional lymph nodes. This is seen from bright specific fluorescence of individual cells in the walls of lymphatic vessels and all vascular layers and valves in UV light.

Acceleration of Angiogenesis after Paravasal Injection of Mesenchymal Stem Cells at the Site of Modeled Venous Thrombosis.

The results of transplantation of autologous bone marrow multipotent mesenchymal stem cells carrying GFP gene and labeled with cell nucleus-specific dye DAPI near the thrombosed vein in rat hind limb were studied by methods of luminescent microscopy. It was demonstrated that autologous multipotent mesenchymal stem cells participate in the formation of granulations at the site of surgery. The blood fl ow in the thrombosed great vein was always restored through thrombolysis. We observed no signs of incorporation of the transplanted cells into the wall of the great vessel, clot recanalization, or formation of collaterals. Small branches of the great vein in the affected region were also thrombosed. The blood fl ow in these branches was always restored with participation of the transplanted cells or through clot recanalization or through obliteration of the thrombosed vessels and formation of new vessels. The transplanted cells and structures formed by them were gradually replaced by the recipient cells.

[The rat submandibular lymph node after introduction in mandibular bone defect ultipotent mesenchymal cells adsorbed on polyhydroxyalkanoate scaffold].

The reactions of rat regional lymph nodes, caused by implantation of the autologous multipotent mesenchymal stromal cells of a bone marrow origin (AMMSCBM) for acceleration of bone defect regeneration in bottom jaw experiment were studied by methods of fluorescent light microscopy. After introduction in an injury site of a bottom jaw bone of polyhydroxyalkanoate with adsorbed AMMSCBM with a transfected GFP gene the numerous large macrophages with a set of oval fluorescent inclusions in cytoplasm appear in lymph nodules of submandibular lymph nodes. The number of such macrophages increases within 2 weeks after operation, and further starts decreasing. Probably, entered via such way the AMMSCBM partially are phagocytized by macrophages. At destruction of the structures created from AMMSCBM, debris also are phagocytized by macrophages. In that and other case these macrophages appear in the germinative centers of lymph nodules in lymph nodes where initiation of immunity reactions against DNA and same GFP isn't excluded.

Antibodies against benzo[a]pyrene in immunized mouse and in lung cancer patients.

AIM: To evaluate the production of antibodies against benzo[a]pyrene (BP) (Ab1) and corresponding antiidiotypic antibodies (Ab2) in mice after immunization with BP-protein conjugate and in lung cancer patients. MATERIALS AND METHODS: The Ab1 and Ab2 levels were measured by non-competitive ELISA in blood serum of 10 mice immunized with BP-protein conjugate, and in blood serum of 288 healthy persons and 165 lung cancer patients. RESULTS: The Ab1 level of was 2-fold higher than Ab2 level in blood serum of BP-immunized mice. In lung cancer patients the Ab1 level was almost 3 times higher and the Ab2 level was by 30% higher than these indexes in healthy individuals. The Ab1/Ab2 ratio was 2 in BP-immunized mice and healthy individuals and 1 in lung cancer patients. CONCLUSION: Our data have shown that the Ab1/Ab2 ratio in lung cancer patients differ from that in healthy individuals and is close to the Ab1/Ab2 ratio in BP-immunized mouse.

[Peculiarities of angiogenesis after the implantation of polyhydroxyalkanoate films with the adsorbed multipotent stromal stem cells of a bone marrow origin].

The processes developing in various rat tissues after implantation of polymeric polyhydroxyalkanoate (PHA) film fragments with adsorbed autologous multipotent stromal (mesenchymal) cells of bone marrow origin (AMMSCBM), were studied by methods of light microscopy. After the implantation of PHA film with AMMSCBM, the number of blood vessels in the surrounding tissues was found to increase as a result of neoangiogenesis. In this case,AMMSCBM did not migrate and were not destroyed at the place of injection, but differentiated into the cells forming blood vessel structures. The processes of angiogenesis in the tissues around PHA implant, in turn, lead to development of a larger number of blood vessels in the granulations formed around the implanted foreign body, higher volume of granulations proper and subsequent development of a thicker capsule delimiting polymer implant.

Reaction of the rat tissues to implantation of polyhydroxyalkanoate films and ultrafine fibers.

The reaction of various tissues of rats to implantation of polyhydroxyalkanoate films and ultrafine fibers was studied by optic microscopy. Implantation of polyhydroxyalkanoate films into the abdominal cavity caused a peritoneal reaction, leading after 1 month to the formation of fibrous adhesions between polyhydroxyalkanoate and intestinal loops. Under the skin and in the muscle tissue polyhydroxyalkanoate films were encapsulated in a thick fibrous capsule. Implantation of polyhydroxyalkanoate ultrathin fibers led to formation of foreign body granulomas in all tissues with perifocal inflammation and sclerosis of the adjacent tissues. The polymer was fragmented in these granulomas and phagocytosed by macrophages with the formation of giant foreign body cells. Hence, polyhydroxyalkanoate materials implanted in vivo caused chronic granulomatous inflammatory reaction and were very slowly destroyed by macrophages.

[Morphological results of stromal stem cells of bone marrow origin into the thrombosed vein in experiment].

Using the methods of luminescent microscopy, the results of injection of autologous multipotent stromal (mesenchymal) stem cells of bone marrow origin (SSCBMO) containing GFP gene, into thrombosed hindlimb vein were studied in 226 male Wag rats. It was found that the restoration of blood flow through the thrombosed main vein was not always the result of thrombolysis. No signs of incorporation of injected SSCBMO into the wall of thrombosed vessel, clot recanalization or collateral formation were detected. In experimental thrombosis model with thrombin administration and main vein ligation, the thrombosis of its small branches also took place. The restoration of blood flow occured via either blood clot recanalization or obliteration of thrombosed vessels and the outgrowth of the new ones. SSCBMO were found to participate in both of these processes resulting in faster restoration of a blood flow in the tissue microregion of thrombosed vein. Gradually the injected SSCBMO and the structures formed with their participation, were replaced by the own cells of a recipient organism.

[Morphological tissue changes after the implantation of elastic lamellar foreign bodies in the experiment].

The reaction of rat tissues was studied using the methods of light microscopy 4, 12, 18 days, 1, 2, 6 and 12 months after hypodermic implantation of polymeric films made of polyhydroxyalkanoates (PHA). It was found that polymer, like any foreign matter in an organism, become immediately covered by fibrin. By day 4, there the deformation and destruction of polymeric films were observed due to fibrin contraction. Further, the foreign body was covered by a connective tissue capsule. Under the action of myofibroblasts, the capsule around PHA contracted, thus further deforming and breaking the polymer. Small particles of polymer were covered by macrophages, after some time the cytoplasm of macrophages fused forming the giant cells of foreign body type. After the prolonged period, small fragments of polymeric films were almost completely degraded by macrophages. Large polymeric fragments that were not deformed or crushed, became encapsulated by fibrous tissue and remained unchanged for long time periods.

Regeneration of red bone marrow in rat lower jaw after transplantation of mesenchymal stem cells into the site of injury.

Regeneration processes in rat mandibular bone after transplantation of a suspension of autologous BM MSC in culture medium were studied by methods of light microscopy and X-ray densitometry. It was found that the structures of red BM in the callus after transplantation of autologous BM MSC formed earlier than in natural reparation. The formation of cavities containing BM determines lower tissue density at the site of injury after transplantation of autologous BM MSC on weeks 4 and 5 of observation than during spontaneous healing. These changes progressed throughout the observation period and attested to accelerated bone tissue reparation.

[The effect of autologous mesenchymal stem cells of bone marrow origin on regeneration of damaged rat bottom jaw bone].

The regeneration processes after introduction in site of damaged bone of rat bottom jaw of autologous mesenchymal stem cells of bone marrow origin (AMSCBMO) in suspension were studied by methods of light microscopy and x-ray densitometry. Morphological methods showed that formation of red bone marrow in bone callosity occurs much earlier after use AMSCBMO than in the time of natural course of reparation. Formation of cavities with a marrow decreases tissue density after application AMSCBMO in a site of damage in 4 and 5 weeks of supervision. The specified changes progress during all time of observation and are the certificate of accelerated development of regenerative processes in bone tissue.

[The peculiarities of rat tissue reactions to intraperitoneal implants made out of biodegradable polyhydroxyalkanoates].

The reaction of rat tissues to intraperitoneal placement of implants made out of biodegradable polyhydroxyalkanoates (PHA), was studied at different time points after the operation by the methods of light microscopy. It was found that after intraperitoneal PHA implant placement, the active adhesive process started leading to formation of fibrous adhesions between PHA implants and intestinal loops. When PHA implants were used in the form of films, they became surrounded by a thick fibrous capsule. As a result of PHA implant placement in the form of ultrathin fibers, the extensive foreign body granulomas with perifocal inflammation and sclerosis of surrounding tissues were formed. In these granulomas the polymer fragmentation and phagocytosis by macrophages with formation of giant cells of foreign body occured. It is concluded that PHA implants are not biodegradable and induce tissue reactions similar to those caused by other foreign bodies.

[Preparation and characterization of the recombinant protein containing immunomimetic peptide of benzo[a]pyrene].

Two recombinant plasmids were constructed. The first plasmid contained the hybrid gene composed of immunomimetic peptide of benzo[a]pyrene, of the protein pIII of bacteriophage M13 and of cellulose binding domain encoding sequences. The second plasmid contained the hybrid gene composed of the signal peptide of the protein pIII of bacteriophage M13, of immunomimetic peptide of benzo[a]pyrene, of the protein pill of bacteriophage M13 and of cellulose binding domain sequences. The obtained recombinant plasmids were used in expression of chimeric protein containing immunomimetic peptide ofbenzo[a]pyrene based on strain E. coli M15. The lack of the recombinant protein expression using first plasmid was demonstrated. In the same time, it was shown that accumulation of recombinant protein contained immunomimetic peptide with signal peptide of the protein pIIIl of bacteriophage was present. This chimeric protein was produced in "mature" (without signal peptide) and "unprocessing" (with signal peptide) forms. Using the Western-blot analysis, it was shown that the "mature" form only specifically bound to the B2 monoclonal antibody against benzo[a]pyrene. Thus, we expressed, purified, and characterized the recombinant protein containing immunomimetic peptide of benzo[a]pyrene.

[Regeneration of the damaged mandibular bone in rat after the injection of autologous mesenchymal stem cells of bone marrow origin adsorbed on the fibrin clot].

The processes of the repair of the damaged mandibular bone in rats were studied using light microscopy and x-ray densitometry at various time intervals after the local injection of the platelet-rich fibrin clot (PRFC), autologous mesenchymal (stromal) stem cells of bone marrow origin (AMSCBMO) or AMSCBMO, adsorbed on PRFC, into the damaged site. The best results were obtained after the application of PRFC with AMSCBMO. One week after the operation, the mandibular bone defect was largely filled with the newly formed bone tissue. It seems most probable that in this case the effects of fibrin and stem cells on the damaged bone were summarized or even amplified. Bone formation in these cases appeared to begin in the center, but not at the edges, of the defect. AMSCBMO were distributed over the whole volume of PRFC, filling all the defect more or less uniformly. As a result, maximally fast and successful restoration of bone tissue was reached in the area of the defect.

[Features of interaction of monoclonal antibody B2 with polycyclicaromatic hydrocarbons and peptide-mimotope of benzo[a]pyrene].

To define of possible mechanism of monoclonal antibody B2 interaction with haptens and peptide-mimotope of benzo[a]pyrene Fab-fragment structure was modeled. Antibody active centre structure was defined using docking with a number of different polyclic aromatic hydrocarbons. It was shown that active center of monoclonal antibody B2 composed of two binding pockets. Correlation was revealed between experimental and calculated data on relative free energies of monoclonal antibody B2 binding with different ligands. We found that synthetic peptide-mimotope of benzo[a]pyrene weakly competeswith benzo[a]pyrene conjugate in monoclonal antibody B2 binding. Immunization of mice with peptide-mimotope conjugate did not revealed antibodies to benzo[a]pyrene. To define structural features of peptide-mimotope of benzo[a]pyrene preliminary model of peptide-mimotope in the frame of pIII protein was calculated. It was shown that tryptophan located inside of peptide can be exhibited on a surface and accessible to antibody. Obtained modeling results could be applied for following optimizations ofbenzo[a]pyrene peptide-mimotope and active center of monoclonal antibody B2.

A novel approach to the development of anticarcinogenic vaccines.

Human exposure to chemical carcinogens is an important etiological factor in cancer diseases. In this article, we will discuss a new approach to the development of anticarcinogenic vaccines. The main task in our research was to select a benzo[a]pyrene immunomimetic peptide considered as a hapten-specific component. For this purpose, we synthesized carcinogen-protein conjugates and prepared mono- and polyclonal antibodies to benzo[a]pyrene. Phage display technology was used to select the benzo[a]pyrene immunomimetic peptide, followed by an evaluation of the immunological properties of the obtained peptide. The obtained benzo[a]pyrene immunomimetic peptide could only simulate chemical carcinogens in the frame of the pIII protein. As a result, we prepared a recombinant protein composed of the benzo[a]pyrene immunomimetic peptide and pIII-encoding sequences. Using ELISA, we demonstrated that the recombinant protein specifically interacts with the anti-benzo[a]pyrene monoclonal antibody (mAB B2). Using molecular modeling, we predicted the 3-D structure of the mAB B2 active center and analyzed the characteristics of its interaction with different polycyclic aromatic hydrocarbons, as well as with the benzo[a]pyrene immunomimetic peptide. Thus, a comprehensive analysis of the results of the obtainment of hapten-specific components of anticarcinogenic vaccines allowed us to outline a strategy for future development in this direction.