Growth-Stimulating Effect of Platelet Preparations Obtained by Different Methods on Human Fibroblast Culture.

We studied the growth-stimulating effect of platelet-based preparations obtained by different methods on cultured human fibroblasts M-22. Platelet lysates prepared from platelet-rich plasma, platelet-poor plasma, concentrated suspension of platelets washed from plasma, and platelet-rich plasma activated with calcium chloride (ActPRP) were used. The volume of the platelet preparations was 10-500 µl per 104 cells. The most effective dose of platelet-rich and platelet-poor plasma in cell culture was 20 µl, whereas for ActPRP, the most effective dose was 500 µl. Lysates of platelet-rich and platelet-poor plasma in doses of 100-500 µl inhibited fibroblast growth and disturbed their structural integrity. At the same time, lysates of washed platelets in doses of 10-500 µl stimulated cell growth and preserved their viability. An inverse correlation was found between the number of cells in culture and the level of proinflammatory cytokines in the platelet preparations.

Experimental Study of the Effect of Biological Matrixes with Stabilized and Non-Stabilized Platelets on Reparative Process in the Wound Equivalent to Deep Burn.

We studied reparative effect of platelet-filled biological matrixes in the treatment of mice with wounds equivalent to deep burn. The wound coatings were based on decellularized dermal matrix without platelets (control), with native platelets, and with platelets stabilized with 2.5 µM nanosilver. In 3 days, the epithelial layer and derma were absent in all groups and extensive scab was formed. Dermal matrix with platelets simulated intensive migration of macrophages and fibroblasts to the wound bottom; in the control group, this migration was absent. In 14 days, granulation tissue appeared in the wound bottom in animals of all groups; in the experimental groups, the number of vessels was 2-4-fold higher than in the control, though the number of inflammatory cells in experimental groups remained high. On day 21, the scab on the most of the wound area was absent in all animals of the experimental groups and epithelialization and hair growth were pronounced, comparing to control. Nevertheless, in experiment dermal layer was not already completed, inflammation reaction remained.

Interaction of Magnesium-Based Materials with Human Blood Cells and Culture of Human Diploid Cells In Vitro.

We studied morphofunctional properties of human blood cells and diploid culture cells exposed to different types of magnesium materials: pure magnesium (Mg), magnesium-yttrium-neodymium-zirconium alloy (Mg-Y-Nd-Zr) and magnesium-zinc-calcium alloy (Mg-Zn-Ca). The materials were incubated with donor blood and mesenchymal multipotent stromal cells over 3 days. The studied materials did not induce massive lysis of human erythrocytes and leukocytes in vitro, but gradually impaired their structural integrity. In all cases, spontaneous platelet aggregation was observed in 6 h. In the presence of pure Mg and Mg-Zn-Ca alloy, this was accompanied by a decrease in the number of platelets with granules. In 24 h, substantial platelet degranulation occurred in all cases and in 72 h, the platelets did not contain granules. In parallel, the formation of large aggregates (60 µ) was observed. In the culture of stromal cells, all Mg-based materials reduced structural integrity of cells in 24 h, but did not significantly inhibit cell proliferation. Structural integrity of stromal cells partially recovered by day 3 in culture. The studied materials (Mg, Mg-Y-Nd-Zr, and Mg-Zn-Ca) seemed to be low-toxic for human cells during short-term contact, but could stimulate platelet aggregation and spontaneous degranulation and reduced the viability of diploid cells in vitro.

Growth-Stimulating Effect of Human Platelets Stabilized with Silver Nanoparticles.

We studied proliferative activity of multipotent mesenchymal stromal cells on collagen matrices containing platelets stabilized with silver nanoparticles. Dose-dependent stimulation of the growth of stromal cells without their structural damage was observed in the presence of stabilized platelets in proportion of 20-120 mln per 105 multipotent mesenchymal stromal cells. The same doses of non-stabilized platelets produced less pronounced stimulation effect. In higher doses (≥180 mln) stabilized platelets inhibited cell proliferation and induced their damage. Stabilized platelets enhanced migration of multipotent mesenchymal stromal cells; after formation of the monolayer, they actively colonized deep layer of the collagen matrix. The formed monolayer of multipotent mesenchymal stromal cells survived over 14 days without appreciable cell damage.

Morphofunctional Properties of Human Platelets Treated with Silver Nanoparticles.

We studied structural and functional properties of human platelets in the presence of nanosilver particles. Incubation with 0.05-5 µM silver nanoparticles suppressed platelet adhesion in a dose-dependent manner without affecting internal platelet structure; during adhesion, some granules were not exocytized. Spontaneous platelet activation was observed at nanoparticle concentrations 15-100 µM. Addition of 1-5 µM nanosilver to cells undergoing adhesion blocked massive platelet degranulation, but did not prevent the formation of lamellopodia. The maximum number of preserved granules in platelets was revealed in the presence of 2.5-5 µM silver nanoparticles: 50% after platelet preincubation with silver nanoparticles and 75-77% after stabilization of adherent platelets with silver nanoparticles.

Optimization of the Method of Buccal Epithelial Cell Isolation and Culturing on Collagen Substrate for Ophthalmologic Application.

We performed morphofunctional analysis of cultured rabbit buccal epithelial cells in the primary culture, during passaging, and upon interaction with collagen substrates. Three different morphological types were identified. It was demonstrated that precursors of rabbit buccal epithelium were characterized by high proliferative activity, retain their biological properties throughout long-term culturing, and adhere to various collagen substrates.

Experimental substantiation and clinical use of a combination of dermal matrix with allogenic or autologous cells for the treatment of extensive traumatic wounds.

We carried out a comparative experimental and clinical study of the efficiency of the combination of dermal matrix with autologous and allogenic cells for creation of an optimal biological wound coating. Experiments on outbred mice have demonstrated that the use of dermal matrix in combination with allogeneic or autologous cells shortens the inflammation phase and accelerated maturation of the granulation tissue. Clinical application of biological wound dressing based on dermal matrix in combination with autologous or allogeneic mesenchymal multipotent stromal cells prevented septic complications and shortened the time of preparing extensive traumatic wounds with skin defect to autodermoplasty.

Morphofunctional analysis of human platelets by vital staining.

We developed a method for differential staining of human platelets preserving their functional activity based on vital fl uorochrome stains trypafl avin and acridine orange. Platelets stained with trypafl avin and acridine orange exhibited under a fl uorescent microscope green fl uorescence of the cytoplasm and red-orange fl uorescence of the granules. Morphofunctional analysis of platelets was carried out on the cells from donor blood, donor concentrated platelets, and cells from hematological patients and patients with thromboembolic complications. Populations with low (16 %) and high (2 %) morphofunctional activities of platelets were detected among donors. The morphofunctional parameters of platelets were sharply reduced in hematological patients with the hemorrhagic syndrome and elevated signifi cantly in patients with thromboembolic complications in comparison with donors. The method seemed to be effective for evaluating the platelet quality in donor and patients' blood components.

[The morphologic evaluation of adhesive activity].

The morphologic evaluation of adhesive activity of human thrombocytes using both vital dye of cells and registration of adhesion process with fluorescent microscope is proposed. The regression dependence between adhesive activity of human thrombocytes and morpho-functional and aggregative activity of thrombocytes is established. The blood samples of 100 blood donors, 100 patients with hematologic pathology, 110 patients of traumatology, 50 patients with acute exogenous intoxications and 20 patients with chronic trophic ulcers were analyzed. The drastic decrease of adhesive activity of human thrombocytes and number of adhesively active thrombocytes was established in circulating blood of patients with bleedings of various etiology and hematologic patients. The significant increase of adhesive activity of human thrombocytes and number of adhesively active thrombocytes in patients with thrombosis is established. The morphologic evaluation of quality of thrombocytes is effective in case of analysis of biologic value of all population of circulating cells.

Effect of concentration of platelet-derived growth factor on proliferative activity of human fibroblasts.

Serum concentration of platelet-derived growth factor was measured in patients with trophic ulcers and proliferative activity of human fibroblasts was measured in wells with different content of added serum platelet-derived growth factor. In parallel, morphofunctional analysis of platelets from the whole blood of these patients was performed. A close correlation was found between morphofunctional parameters of blood platelets and serum concentration of platelet-derived growth factor. Platelet-derived growth factor in concentrations below 150 pg per well stimulated fibroblast proliferation and preserved their viability. Platelet-derived growth factor in concentrations >200 pg per well suppressed fibroblast proliferation and impaired their viability.

[Selective adsorption of endotoxin in the complex treatment of patients with severe sepsis].

Severe sepsis and septic shock remain the most serious problem of critical care medicine today with a mortality of 25-80%. Bacterial endotoxin is of considerable importance in the pathogenesis of sepsis. A selective hemosorbent in which endotoxin adsorption is accomplished via its binding to a synthetic peptide (LPS-A) has been recently designed in Sweden. The purpose of the study was to evaluate the efficiency of using the LPS-A in the complex treatment of patients with severe sepsis. Eight patients in whom the course of the underlying disease was complicated by the development of gram-negative sepsis in the postoperative period were examined. The patients had an APACHE II score of 20.4 +/- 4.5 LPS-A was performed using the hemosorbent Alteco LPS (Lund, Sweden) and an Aquarius hemoprocessor (Edwards Lifesciences) (U.S.A.). The use of LPS-A caused a significant reduction in endotoxin levels by 2.9 times. The level of procalcitonin was increased in all patients; after a LPS-A session, it was significantly decreased by 1.9 times. The use of LPS-A resulted in diminished tachycardia, elevated blood pressure and could reduce the doses of vasopressor amines. There was an improvement in lung gas exchange parameters. A study of the effect of LPS-A on the immune system showed a significant reduction (36.6%) in neutrophil oxygen-dependent metabolism according to the data of the spontaneous HCT test, a 2.4-fold decrease in large circulating immune complexes, and a rise in the count of mononuclear cells that died both by necrosis and apoptosis, the count of the cells significantly increased by 5.3 times in late apoptosis. A LPS-A session caused a significant drop in the blood level of endotoxin and procalcitonin and it was accompanied by a trend toward better hemodynamics and lung gas exchange and alleviated organ dysfunction. At the same time LPS-A showed the ambiguous effect on immune system parameters, which calls for further study.

[Immunomodulatory effect of extracorporeal hemocorrection in patients with surgical sepsis].

The study was undertaken to evaluate the immunomodulatory effects of various methods of extracorporeal hemocorrection in septic patients. Thirty-two patients in whom the course of the underlying disease was complicated by the development of sepsis were examined. A package of therapeutic measures involved debridement of an infection focus, de-escalation antibacterial therapy, nutritional support, and immunomodulatory therapy. All the patients underwent procedures of plasma filtration and continuous venovenous hemofiltration; granulocytopheresis was carried out in 5 patients. Granulocytopheresis was shown to eliminate dead leukocytes from the bloodstream within a few hours, which contributed to clinical improvement. Plasmafiltration in patients with sepsis resulted to a reduction in the degree of a systemic inflammatory reaction, to normalization of coagulation homeostasis, which indirectly affected immunological parameters. Continuous venovenous hemofiltration made it possible to make continuous elimination of the low and average molecular-weight substances, corrected liquid balance, and well affected gas exchange in the lung.

[Evaluation of severity of immune disorders and prognosis of the development of pyo-septic complications in patients with emergency surgery pathology].

Any lesion in organism, causing the development of inflammation, is followed by changed work of the immune system that is extremely important for prognosis of the course of the pathological process. The adequate compensation-defense reaction was estimated using well-known immunological tests and normal reaction of the immune system was determined to be different from the pathological reaction, especially in patients in critical state. The adequate reaction of the immune system is characterized by mild leukocytosis, two-three fold increase of the spontaneous HCT test, normal or moderately pronounced indices of the cellular and humoral links of immunity. The markers of the formation of secondary immune incompetence are not only decreased but also extremely increased indices of immunogram at early terms of the disease.

[Immunological aspects of the early application of extracorporeal hemocorrection techniques in the complex therapy of purulent mediastinitis].

Acute purulent mediastinitis (APM) is one of the most complicated forms of surgical infection, showing a high incidence of sepsis--from 45 to 100%, mortality rates of 17 to 80%. Sixty-eight patients with APM admitted to the N. V. Sklifosovsky Research Institute of Emergency Care in October 2002 to March 2007 were examined. Postoperatively, all the patients received extracorporeal hemocorrection techniques (EHT): plasmapheresis (PA) via filtration and continuous venovenous hemofiltration (CVVHF). According to the time of initiation of EHT, the patients were divided into 2 groups: 1) 34 patients in whom EHT was initiated within the first 24 hours after surgery; 2) 34 patients in whom it was started on postoperative day 2. The efficiency of early use of EHT in the complex therapy of APM was evaluated. The early initiation of EHT (PA and CVVHF) caused a reduction in endogenous intoxication and the magnitude of a systemic inflammatory reaction, which resulted in the rapidest restoration of the size of major populations and subpopulations of lymphocytes and prevented the development of immune system incompetence. The early use of EHT caused a significant reduction in hospital mortality (11.8 and 35.3% in the early and late EHT use groups, respectively).

[The level of dead blood leukocytes in sepsis and the significance of their elimination by extracorporeal hemocorrection techniques].

Sepsis with 18-to-33% mortality also remains the most serious problem of modem medicine today. Forty-five patients treated at the N. V. Sklifosovsky Research Institute of Emergency Care in June 2006 to February 2008 were examined. Twenty-seven primary blood donors were examined to determine the physiological concentration of dead leukocytes. Two groups of patients were formed according to the signs of a systemic inflammatory reaction (SIR). A study group included 29 patients in whom the course of the underlying disease was complicated by the development of sepsis. All the patients from the study group underwent extracorporeal hemocorrection techniques (EHT): plasma filtration (PF) and continuous venovenous hemofiltration (CVVHF). A granulocytopheresis procedure was performed in 4 patients. A control group comprised 16 patients without clinical signs of SIR. The blood taken from patients with sepsis showed the significant increase in the levels of dead leukocytes as compared with both the physiological normal values and the values obtained in patients without signs of systemic inflammation, which significantly correlates with the severity of organ dysfunction. The elevated content of dead leukocytes is an independent risk factor for a poor outcome. Plasma filtration used in sepsis patients facilitates a significant reduction in the blood concentration of dead leukocytes. The first experience with granulocytopheresis in patients with severe sepsis, a leukemoid reaction and high absolute concentrations of dead leukocytes is indicative of its clinical effectiveness, which is manifested itself by the elimination of dead leukocytes.

[Clinical assessment of immune parameters at surgical patients with syndrome of systemic inflammatory reaction].

Results of immunological examination of 423 patients with syndrome of systemic inflammatory reaction of different ethiology (mediastinitis--78 patients, peritonitis--85, severe acute pancreatitis--91, trauma of thorax and abdomen complicated with massive hemorrhage--111, severe burn trauma--40 patients) are analyzed. For complex and objective assessment of immune reaction the system of scores has been developed. This scale permitted to detect the immune disorders, to diagnose the degree of immune insufficiency at early stages of disease, to predict the purulent complications and to start timely immune therapy.

[Complex of immunological characteristics for the diagnostics of pancreatogenic immunodeficiency].

The results of the immunological study of blood in 89 patients with severe acute pancreatitis (SAP) were analyzed. Immune reactions in SAP were characterized by the presence of moderate leukocytosis, an increase in the phagocytic and metabolic activity of neutrophils, relative lymphopenia, a decrease in the number of B-lymphocytes. The average values of the parameters of the immunogram of patients with the favorable course of the disease on days 2 - 3 were analogous to the average population values in 89 examinees. The direction and the degree of manifestation were slightly variable and could be registered within one standard quadratic deviation. Such heterogeneity in the character of immune reactions made it possible to regard the average values of the immunogram parameters of these patients as "the norm". For the objective evaluation of the immune status of SAP patients a scale for the evaluation, in points, of disturbances in immune reactions to the destructive process in the pancreas was developed. This scale for the evaluation, in points, of the severity of disturbances in the immune status of SAP patients made it possible to prognosticate the development of complications.

[Immunological estimation of the degree and prognosis of acute pancreatitis].

An analysis of data of immunological investigation of blood of 89 patients with a severe acute pancreatitis (SAP) on the 2-3 day of the disease has revealed changes in immunogram characteristic also of other pathological conditions and pointing to a systemic inflammatory reaction. Among the patients who did not have purulent complications against the background of therapy of SAP, the character and degree of changes in immunogram were in line with average values, and so they could be considered as "a norm of pathology". A system of scores of impairments of the immune response to the destructive process was developed for an objective evaluation of the immune status of patients with SAP. In patients with a normal physiological immune response (sum of scores 0-5) the prognosis of the disease is considered to be good. No immunocorrectors should be included in the complex of treatment measures. In patients with an inadequate pathological immune response (sum of scores 9 and more) the prognosis is unfavorable. The complex of treatment measures for such patients should include immunocorrectors.

[Propranolol treatment of effort angina in patients with arterial hypotension]. / Pripranolol pri lecheniii stenocardii napriazheniia u bol'nykh arterial'noi gipotoniei.

AIM: To compare antianginal efficacy and tolerability of propranolol therapy in patients with stable angina pectoris and chronic hypotension (Hpts) and normotensive patients with angina of effort (Npts). MATERIAL AND METHODS: A randomized, single-blind, placebo-controlled study was made in 35 Hpts and 38 Npts was made using bicycle exercise tests, echocardiography, stress myocardial scintigraphy with 77-199. RESULTS: Acute bicycle exercise tests showed high anti-ischemic activity of propranolol in 86% Hpts and 65% Npts. Stable antianginal propranolol effect in 57% Hpts was accompanied with a decrease of myocardial perfusion defect. Secondary resistance or pseudotolerance to an antianginal effect of propranolol was observed in 43% Hpts in 4-12 weeks (vs 0 of Npts; p < 0.01) as evidenced by T-199 stress myocardial scintigraphy. Hpts with secondary resistance and pseudotolerance to propranolol had lower control hypotension and bradicardia (p < 0.05), more anginal attacks (p < 0.001). CONCLUSION: Hpts had rapidly developing secondary resistance and pseudotolerance to propranolol antianginal effect, bad tolerability of the drug.

[Diltiazem and verapamil therapy in patients with angina pectoris associated with arterial hypotension]. / Terapiia diltiazemom i verapamilom u bol'nykh stenokardiei na fone arterial'noi gipotonii.

A randomized blind cross-over study with placebo lead-in compared efficacy of calcium antagonists diltiazem and verapamil in 71 patients with stable angina concurrent with arterial hypotension (group 1) and 38 normotensive patients with ischemic heart disease (group 2). By acute bicycle exercise test evidence, verapamil was effective in 80% and 82% patients of group 1 and 2, respectively, dilitiazem--in 67 and 77%, respectively. Cumulation of the effect (p < 0.01) to the third month of verapamil course was comparable in both groups. Tolerance to an antianginal effect of dilitazem developed in 53% patients of group 1 (against 30% in group 2, p < 001) in 2-4 weeks of therapy (against 4-12 weeks in group 2, p < 0.05). By stress 199-T1 scintigraphy of the myocardium, administration of effective doses of diltiazem reduced the number of hypoperfused segments by at least 30%.