Stimulation of mouse hematopoietic stem cells by angiogenin and DNA preparations.

Immature hematopoietic progenitors are a constant source for renewal of hemocyte populations and the basic component of the tissue and cell repair apparatus. A unique property of these cells of internalizing extracellular double-stranded DNA has been previously shown. The leukostimulatory effect demonstrated in our pioneering studies was considered to be due to the feature of this cell. In the present research, we have analyzed the effects of DNA genome reconstructor preparation (DNAgr), DNAmix, and human recombinant angiogenin on both hematopoietic stem cells and multipotent progenitors. Treatment with bone marrow cells of experimental mice with these preparations stimulates colony formation by hematopoietic stem cells and proliferation of multipotent descendants. The main lineage responsible for this is the granulocyte-macrophage hematopoietic lineage. Using fluorescent microscopy as well as FACS assay, co-localization of primitive c-Kit- and Sca-1-positive progenitors and the TAMRA-labeled double-stranded DNA has been shown. Human recombinant angiogenin was used as a reference agent. Cells with specific markers were quantified in intact bone marrow and colonies grown in the presence of inducers. Quantitative analysis revealed that a total of 14,000 fragment copies of 500 bp, which is 0.2% of the haploid genome, can be delivered into early progenitors. Extracellular double-stranded DNA fragments stimulated the colony formation in early hematopoietic progenitors from the bone marrow, which assumed their effect on cells in G0. The observed number of Sca1+/c-Kit+ cells in colonies testifies to the possibility of both symmetrical and asymmetrical division of the initial hematopoietic stem cell and its progeny.

Stimulation of mouse hematopoietic stem cells by angiogenin and DNA preparations

Immature hematopoietic progenitors are a constant source for renewal of hemocyte populations and the basic component of the tissue and cell repair apparatus. A unique property of these cells of internalizing extracellular double-stranded DNA has been previously shown. The leukostimulatory effect demonstrated in our pioneering studies was considered to be due to the feature of this cell. In the present research, we have analyzed the effects of DNA genome reconstructor preparation (DNAgr), DNAmix, and human recombinant angiogenin on both hematopoietic stem cells and multipotent progenitors. Treatment with bone marrow cells of experimental mice with these preparations stimulates colony formation by hematopoietic stem cells and proliferation of multipotent descendants. The main lineage responsible for this is the granulocyte-macrophage hematopoietic lineage. Using fluorescent microscopy as well as FACS assay, co-localization of primitive c-Kit- and Sca-1-positive progenitors and the TAMRA-labeled double-stranded DNA has been shown. Human recombinant angiogenin was used as a reference agent. Cells with specific markers were quantified in intact bone marrow and colonies grown in the presence of inducers. Quantitative analysis revealed that a total of 14,000 fragment copies of 500 bp, which is 0.2% of the haploid genome, can be delivered into early progenitors. Extracellular double-stranded DNA fragments stimulated the colony formation in early hematopoietic progenitors from the bone marrow, which assumed their effect on cells in G0. The observed number of Sca1+/c-Kit+ cells in colonies testifies to the possibility of both symmetrical and asymmetrical division of the initial hematopoietic stem cell and its progeny.

Lithium-Modified Sorbent: Effects on Hemostatic Responses In Vitro.

The effects of a lithium-modified sorbent based on aluminum oxide and polydimethylsiloxane (Al2O3@PDMS/Li) and a lithium-free sorbent (Al2O3@PDMS) on some indicators characterizing blood clotting under hemosorption conditions were compared in vitro. Sorbent Al2O3@PDMS/Li had significantly lower reactogenic effect on the blood passed through the column than the sorbent without lithium. This was seen from the degree of platelet reduction (66×109 vs 19×109/liter) as well as a less pronounced hypercoagulation shift in chronometric indicators. In contrast to lithium-free sorbent, Al2O3@PDMS/Li demonstrated the ability to reduce the concentration of fibrinogen. However, this had no impact on the density characteristics of the blood clot assessed by thromboelastometry such as maximum clot firmness, angle and fibrin clot formation time, amplitudes at 10 and 15 min after clotting time, which are known to depend on the quantity of platelets and the concentration of functionally active fibrinogen.

Phase 3 study of recombinant von Willebrand factor in patients with severe von Willebrand disease who are undergoing elective surgery.

Essentials Recombinant von Willebrand factor (rVWF) is effective in von Willebrand disease (VWD). A phase 3 study of rVWF, with/without recombinant factor VIII (rFVIII) before surgery in VWD. Overall rVWF's efficacy was rated excellent/good; rVWF was administered alone in most patients. rVWF was well-tolerated and hemostasis was achieved in patients with severe VWD undergoing surgery. SUMMARY: Background Recombinant von Willebrand factor (rVWF) has demonstrated efficacy for on-demand treatment of bleeding in severe von Willebrand disease (VWD), warranting evaluation in the surgical setting. Objectives This study (NCT02283268) evaluated the hemostatic efficacy/safety profile of rVWF, with/without recombinant factor VIII (rFVIII), in patients with severe VWD undergoing surgery. Patients/Methods Patients received rVWF 40-60 IU kg-1 , VWF ristocetin cofactor activity was measured 12-24 h before surgery. If endogenous FVIII activity (FVIII:C) target levels were achieved 3 h before surgery, rVWF was administered alone 1 h before surgery; rVWF was co-administered with rFVIII if target endogenous FVIII levels were not achieved. rVWF was infused postoperatively to maintain target trough levels. Overall and intraoperative hemostatic efficacy, the pharmacodynamics of rVWF administration and the incidence of adverse events (AEs) were assessed. Results All patients treated with rVWF for major (n = 10), minor (n = 4) and oral (n = 1) surgery had overall and intraoperative hemostatic efficacy ratings of excellent (73.3% and 86.7%) or good (26.7% and 13.3%). Most rVWF infusions (89.4%) were administered alone, resulting in hemostatically effective levels of endogenous FVIII within 6 h, which were sustained for 72-96 h; 70% (n = 7/10) of major surgeries were performed without rFVIII co-administration. Six patients reported 12 treatment-emergent AEs. Two patients each had one serious AE: diverticulitis (not treatment related) and deep vein thrombosis (sponsor-assessed as possibly treatment related). No severe allergic reactions or inhibitory antibodies were reported. Conclusions These data support the efficacy and safety profile of rVWF in patients with severe VWD undergoing elective surgery.

[THROMBOGENIC RISK FACTORS IN PATIENTS WITH EXACERBATION OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE].

We studied activation of hemostatic reactions together with mechanisms of systemic inflammation and genetic factors responsible for thrombogenic risk in 65 patients suffering exacerbation of chronic obstructive pulmonary disease with 2 or 3 positive criteria proposed by N.Anthonisen at al. (1987). The levels of indicators of systemic inflammation, such as C-reactive protein and tumour necrosis factor-alpha, increased concurrently with those of homocysteine, endothelin-1, Willebrand factor plasminogen activator inhibitor type 1, and thrombin-antithrombin complex (marker of thrombinemia). The prevalence of pathological alleles ofprotein genes involved in the hemostasis system and folate cycle remained as in the control group. A relationship between plasma levels of thrombin-antithrombin complex and Willebrand factor and the activity of indicators of systemic inflammation was documented. Treatment of exacerbation resulted in the decrease in the levels of the thrombin- antithrombin complex, inhibitor of the extrinsic pathway of coagulation, and plasminogen activator inhibitor type 1. The treatment failed to normalize parameters of thrombinemia and blood fibrinolytic potential.

[Role of endothelial dysfunction, the interface between hemostatic and system inflammatory responses in the pathogenesis of an infectious inflammation-dependent exacerbation of chronic obstructive pulmonary disease].

AIM: To analyze the systemic manifestations of vascular endothelial damage, the activation of hemostatic and inflammatory responses in patients with an infectious inflammation-dependent exacerbation of chronic obstructive pulmonary disease (COPD). SUBJECTS AND METHODS: The paper provides the data of examinations of 111 patients with the clinical signs of an infectious inflammation-dependent exacerbation of COPD who had 2 or 3 positive criteria elaborated by N. Anthonisen et al. (1987). The patients were divided into 2 phenotypically different subgroups: 1) 92 (82.9%) COPD patients without clinical manifestations of bronchoectasis; 2) 19 (17.1%) patients with COPD concurrent with documented bronchiectasis. The patient subgroups were matched for smoking status and the characteristics of COPD and respiratory failure. The investigators assessed the time course of changes in the serum level of endothelin-1 (ET-1), the aggregation function of platelets, and the plasma concentrations of D-dimers and homocysteine in patients with COPD compared to healthy, never smokers (n = 35) and smokers (n = 27). RESULTS: An increase in the levels of the endothelial dysfunction markers ET-1 and homocysteine was found in patients with COPD, which was comparable with the changes in these indicators in the group of smokers. In both subgroups, the rise in plasma D-dimer levels was more pronounced in the patients with a COPD exacerbation than in the smokers. Its therapy with systemic and inhaled glucocorticosteroids reduced C-reactive protein and ET-1 levels in both patient subgroups and in D-dimers in subgroup 1. Elevated D-dimer levels remained when achieving remission, which points to the risk of thrombogenic and thromboembolic events in the patients with an infectious inflammation-dependent exacerbation of COPD and concomitant circulatory system diseases. CONCLUSION: The patients with an infectious inflammation-dependent exacerbation of COPD are observed to have elevated peripheral blood markers of endothelial dysfunction and thrombinemia. These changes are pathogenetically caused by smoking or neutrophilic inflammation and associated with a higher risk of thrombogenic events.

[Use of recombinant factor VIIa to reduce transfusional load in hemorrhaging patients after surgery for malignant tumors].

Often expanded surgical interventions in patients with malignant tumors are accompanied by huge bleeding and require transfusion of big mass of medications prepared from donor blood that may develop different complications. It is showed that with the aim of treatment of hemorrhagic complications the use of recombination factor VIIa allows reducing the surgical blood loss, as well as diminishing hemotransfusional load after surgery especially in patients with malignancies of urogenital system and malignant tumors of the colon and lung.

[The preanalytical stage of studying the hemostatic system].

The efficiency of applying precision devices and multiple laboratory methods devised to reveal the causes of bleeding and thromboses significantly depends on whether a wide range of rules of the preanalytical stage of the study is observed. The paper gives detailed information on conditions for taking blood samples, their transport, and preparation for examination of the hemostatic system. The given information is based on the worldwide recommendations and many years' experience gained by the authors at the laboratory of hemostasis.

[Laboratory monitoring of recombinant factor VIIA effects].

Recombinant activated factor Vll originally proposed to prevent and arrest bleedings in patients with the inhibitory form of hemophilia has been shown to be a universal hemostatic agent that is effective in arresting and preventing a broad spectrum of spontaneous and postoperative hemorrhages. Procedures for monitoring the action of this agent on the hemostatic system have not been studied so far. The paper presents the results of a study of various parameters of the hemostatic system in patients with profuse hemorrhages before and after the use of recombinant activated factor VII. It has been ascertained that monitoring of the pharmacokinetics of the agent can be achieved when the prothrombin test with bovine thromboplastin is carried out.

[Diagnostic procedure for thrombophilia due to factor VIII hyperproduction].

The range of factor Vlll (FVlll) is known to be very wide and its elevated concentration is a risk factor of thromboses. This paper describes a simple and available laboratory method for determining the high level of coagulation FVlll. Moreover, the results of determination of FVlll levels in 102 patients were used to study the incidence of thrombophilia due to FVlll hyperproduction in a region of Western Siberia.

[A new method for determination of antithrombin III and its diagnostic significance]. / Novyi metod opredeleniia antitrombina III i ego diagnosticheskoe znachenie.

A new assay based on using an original chromogenic substrate (z-Ala-Ala-Arg-pNa) and designed for the determination of antithrombin III is described in the paper. It was used in examinations of patients with malignancy after surgery. The content of antithrombin III was detected to be lower in 12% of cases in 2 days after surgery. Additionally, patients with DIC, as complications after surgery, were examined to find out that the antithrombin III content was decreasing in them.

[Immunostimulating and anticoagulating activity of fucoidan from brown algae Fucus evanescens of Okhotskoe sea]. / Immunostimuliruiushchaia i antikoaguliantnaia aktivnost' fukoidana iz buroi vodorosli Okhotskogo moria Fucus evanescens.

Fucoidan--nontoxic sulfated polysaccharide was isolated from brown algae Fucus evanescens in Okhotskoe Sea. Chemical analysis of the compound was performed, it was shown that fucoidan is freely soluble in water and acid solutions. Immunotropic and anticoagulating properties of the compound were evaluated in comparison with heparin. It was demonstrated that fucoidan in wide range of doses stimulated phagocytic and bactericidic activity at leucocytes of mice peritoneal exudate. Heparin on the contrary demonstrated depressive effect on these functions at high dose. It was shown that fucoidan has dose-dependent anticoagulating activity in vitro and in vivo comparable with heparin activity. The results of investigation demonstrated possibility of fucoidan application as immunomodulating and anticoagulating agent of plant origin.

Observation of spatial modulation instability in intracavity second-harmonic generation.

We report the observation of spatial instabilities in singly resonant intracavity second-harmonic generation. Self-focusing as well as spatially ordered near- and far-field structures are observed. Experimental results are compared with mean-field analysis as well as three-dimensional numerical simulations.

Anticoagulant activity of fucoidan from brown algae Fucus evanescens of the Okhotsk Sea.

In vitro and in vivo experiments showed that anticoagulant activity of sulfated polysaccharide from Fucus evanescens (brown algae of the Okhotsk Sea) was similar to that of heparin. Anticoagulant properties of fucoidan are determined by thrombin inhibition mediated via plasma antithrombin III.

[A method for determining plasminogen with a Russian chromogenic substrate and its diagnostic significance]. / Metod opredeleniia plazminogena s otechestvennym khromogennym substratom i ego diagnosticheskoe znachenie.

Measurement of plasminogen, the key component of fibrinolysis system, is one of the basic methods for estimation of fibrinolysis. Methods based on the use of chromogenic substrates are often used in diagnosis. Plasminogen measurements are important for laboratory diagnosis of thrombophilia caused by deficiency or abnormalities of this fiber, for detection and evaluation of the DIC syndrome, and for monitoring the treatment by fibrinolytic preparations (streptokinase, t-PA, urokinase, etc.). An original chromogenic substrate having no foreign analogs has been created at Institute of Genetics and Selection of Industrial Microorganisms and Research Center of Hematology (Moscow). Unlike previously described plasmin substrates, pNa has been obtained by microbiological methods with Russian commercial enzymes subtilisine 72 and megaterine. This paper presents the results of plasminogen measurements in patients with DIC with the use of the original chromogenic substrate. The results were compared with those of tests with Berihrom-Plasminogen diagnostic kit (Behringwerke AG).

Spontaneous hexagon formation in photorefractive crystal with a single pump wave.

A new scheme for spontaneous formation of hexagonal optical patterns in photorefractive crystals with dominant reflection gratings and a single pump wave is proposed and realized experimentally. It is shown theoretically that spontaneous hexagon formation results from random small-angle scattering of the pump wave inside the crystal. The threshold of spontaneous hexagon formation is found.

[Use of elimination of plasma phospholipid membranes for detection of "lupus anticoagulant" effects]. / Ispol'zovanie eliminatsii fosfolipidnykh membran plazmy dlia vyiavleniia effektov "volchanochnogo anticoagulianta".

The coagulation activity of plasma phospholipid membranes (PM) was measured in plasma depleted for platelets (PDP) in 50 normal subjects and 33 patients with the antiphospholipid syndrome (APS) and the "lupus anticoagulant" in the plasma. The normal value for phospholipid activation of clotting was 99.8 +/- 3.5% (from 75 to 125%), whereas in patients with APS and lupus anticoagulant it was 42.1 +/- 8.2% (p < 0.001). Addition of PM from patients' PDP to normal plasma free from PM did not normalize clotting. Addition of PM from normal plasma to patients' PDP normalized the clotting time. Therapy with discrete plasmapheresis increased the phospholipid activation value in the patients from 42.1 to 73.3% (p < 0.01), which was due to removal of the PM-antiphospholipid antibody complex from PDP. The proposed microfiltration method can be used in the complex of tests for detecting the lupus anticoagulant in patient's plasma.

Modulational instability and pattern formation in the field of noncollinear pump beams.

We study pattern formation that is due to modulational instability of noncollinear counterpropagating beams. Angular misalignment of the pumps over a broad range leads to the generation of lines oriented perpendicular to the plane of the pump beams. A theoretical expression for the angular separation of the lines is in close agreement with observations. Outside this range, for both small and large misalignments, we observe squeezed hexagonal patterns.