CELL CULTURES PURITY CONTROL BY METHODS OF CLINICAL DIAGNOSTICS.

Cell cultures of higher organisms, especially cultures of human cells, are increasingly used in medical, pharmaceutical and scientific research. The main problem of cell cultures ­ non-lethal hidden contamination by mycoplasmas, viruses and outsider cell lines. As an available and reliable method for monitoring the purity of the cell cultures, we offer to use PCR kits designed and officially used in clinical diagnostics. We have tested 50 human cell lines using commercial diagnostic systems for detection of papilloma viruses, herpes viruses, adenoviruses, Mycoplasma hominis and total bacterial mass. Contamination in tested cell lines was not found. In the case of cell lines that contain integrated parts of viral genomes, the presence of the respective DNA sequences was confirmed. The proposed diagnostic systems can be effectively used to control the purity of cell lines, for qualitative detection of possible contamination, as well as for quantitative evaluations with calculation of viral load like it is practiced in clinical diagnostics.

TRANSMERIDIAN JET LAG AND ITS PHARMACOLOGICAL CORRECTION.

The article deals with the development and correction of acute jet lag in a flight across several time zones. The investigation had the purpose to study dynamics of subjective and objective psychophysiological parameters and demonstrate methods of prophylaxis and correction of acute jet lag due to transmerdian flights. Subjects were 8 normal volunteers (experimental group) at the age of 26-55 years flying eastward over 7 times zones. The investigation included 3 stages: baseline, preparatory (21-d course of Euricoma longifolia extraction) and main (intake of donormil, cirkadin and artificial sleep during the flight). Functional diagnostics was performed on the baseline stage, on completion of the preparatory stage and on the next day after the flight (21-22 days from the beginning of the preparatory stage). Objective and subjective methods were used to evaluate the autonomic and cardiovascular systems and mental performance. In the control group (n = 4) functional diagnostics was performed on the same days. The investigations showed the benefit of preparation for transmeridian air travel and experimentally demonstrated positive effects of the proposed pharmacological correction of acute transmeridian jet lag.

[Combined therapy for children and adolescents with Ewing tumors (a 25-year experience)].

A total of 115 children (median age 10.5 years, range 2-17) with Ewing sarcoma family tumors (ESFT) received therapy in N.N. Petrov Institute of Oncology pediatric department from April 1985 till August 2013. These patients were divided into two groups depending on treatment tactics used: patients treated according to modified T9 protocol (n = 64) and patients treated according to EICESS-92 or Euro-Ewing 99 regimens (n = 51). Twenty four patients from the second group with adverse prognostic factors received high-dose chemotherapy with autologous stem cell transplantation. All patients received surgical treatment and/or irradiation for primary tumor local control. Five-year overall and disease-free survival was 39% and 37,9% in the first group. In the second group these values were significantly higher; 55% and 39.5%, accordingly (p = 0.03 and 0.25). All patients from the first group with primary metastatic ESFT died of disease progression, while in the second group OS and DFS reached 45.8% and 28.9%, accordingly. There was a statistically significant correlation between local relapse rate and irradiation dose biological equivalent (in TDF units). The local relapse cumulative rate was minimal (12,6%) in patients receiving 80 TDF.

[Evaluation of ovarian status in women who received anti-tumor therapy in childhood and adolescence].

The paper presents the results of a study of the ovarian reserve in young women who received treatment for malignant tumors in childhood and adolescence and are in complete clinical remission. The function of the reproductive system was evaluated by serum concentrations of gonadotropins, estradiol, anti-Müllerian hormone (AMH) and inhibin B. The results were compared to the treatment, patients' age at the beginning of therapy and at the time of the examination. AMH level in serum was the most informative indicator of ovarian reserve in patients treated for malignant tumors.

[Mechanisms of antioedemic effect of bioflavonoids in experiment].

In treatment of various-aetiology chronic oedemas, of great importance is a possibility of exerting an influence on the resorptional, transportation and transmission functions of the lymphatic system, as well as on the microcirculatory blood channel, because they all maintain the balance of fluid exchange in tissues. There exist various causes of chronic oedema, which requires a differentiated approach to administration of bioflavonoids possessing different mechanisms of antioedemic action. With this in mind we carried out experimental histomorphometric studies on 24 rats, aimed at determining variously directed effects of agents belonging to the class of bioflavonoids - phlebodia 600 (diosmin) and troxerutin on regeneration ability of the lymphatic endothelium and permeability of blood capillaries. It was determined that phlebodia 600 exerts a direct influence upon the functional state of the lymphatic system, activating proliferation of the lymphatic endothelium by means of gemmation, which leads to the formation of new capillary lymphatic networks with the resulting increase in both the total absorption area of the lymphatic capillary networks and the volume of lymph reabsorption. Troxerutin acts predominantly on the endothelium of blood capillaries, decreasing permeability in the arterial segment of the capillary, thus lowering the total volume of fluid in the interstitial space and accordingly the load on the lymphatic system.

Heat shock protein DnaK--substrate of actin-specific bacterial protease ECP32.

It has been found that actin-specific bacterial protease ECP32 cleaves prokaryotic heat shock protein DnaK, which belongs to the family of heat shock proteins with molecular weight 70 kDa. We propose a new one-step method for DnaK purification using heat treatment. The technique yields ~1 mg of partially purified DnaK from 25 g of wet bacterial biomass. Polyclonal antibodies against DnaK were obtained. The degree of ECP32 catalyzed proteolysis of partially purified DnaK and that of DnaK in initial cell extracts was compared.

[A new method of producing biologically active nanocomplexes by non-covalent conjugation of proteins with viral particles].

Currently, a range of biologically active molecules have been attached to plant and bacterial viras nanoscaffolds, yielding stable nanoparticles that display multiple copies of the desired molecule. In this paper we propose a new method of non-covalent attachment of peptides to the surface of virios. We have demonstrated that this method is efficient in a model system that includes tobacco mosaic virus particles, synthetic polycation (quaternized poly(4-vinylpyridine) carrying ethyl ethyl pendant radicals) and polypeptide of interest. This principle of step-by-step binding to the surface of virions was used for electrostatic association with hydrophilic fragment of influenza virus haemagglutinin.

[Genetic manipulation and studying of differentiation properties of rat induced pluripotent stem cells].

Induced pluripotent stem (iPS) cells are derived from somatic cells reprogrammed to the pluripotent state by the induced expression of defined transcription factors, achieved for the first time by the seminal work of Takahashi and Yamanaka. This new type of pluripotent cells has offered new exciting options in regenerative medicine allowing the replacement of cells and organs with the patient's own cells thereby avoiding immunological complications. In order to develop such technologies in approved animal models, iPS cells were also generated from rodents. Of course, the most important model for studying of different diseases is rat. In this study, we present a method suitable for rat iPS cells genetic modification by stable transfection and show necessary conditions for the first stages of direct differentiation.

[The express-method of obtaining protease ECP32, the unique instrument in actin investigations].

The protease ECP32 is significant in investigations of actin, the basic protein of muscles and the cytoskeleton. The enzyme originates from the natural enterobacteria strain, which accumulates minor amounts of the protease intracellularly at the post-exponential growth phase. The limiting factor for biosynthesis is the amount of oxygen that has entered the medium. The highly effective method of two-phase cultivation with vigorous aeration at the exponential growth phase was recommended. Based upon the enzyme properties studied, there is a decreased potential for success when using either the affinity or one-stage purification methods. In order to overcome obstacles in the aforementioned methods, a simple method for ECP32 preparation and storage was developed, with purity and activity levels satisfying the requirements of actin structure and function investigations.

Effects of aliskiren, a renin inhibitor, on biomarkers of platelet activity, coagulation and fibrinolysis in subjects with multiple risk factors for vascular disease.

Aliskiren, an octanamide, is nonpeptide, low molecular weight, orally active renin inhibitor effectively preventing angiotensin and aldosterone release. This drug has been recently approved for the treatment of hypertension. Considering potential links between hypertension, platelets, the coagulation cascade and fibrinolysis we sought to evaluate the effect of aliskiren on human biomarkers of hemostasis. In vitro effects of whole blood preincubation with escalating concentrations of aliskiren (500, 1,000 and 2,000 ng ml(-1)) were assessed in 20 aspirin-naive volunteers with multiple risk factors for vascular disease. A total of 33 biomarkers were measured, of which 18 are related to platelet function, 12 to coagulation and 3 to fibrinolysis. Pretreatment of blood samples with aliskiren 500 ng ml(-1) resulted in a significant increase of antithrombin-III (AT-III) activity (P=0.003). All other tested biomarkers were not significantly affected. Spiking whole blood with the higher aliskiren doses was associated with various trends in biomarker activity, where 1000 ng ml(-1) concentration mostly decreased (7/33), and 2,000 ng ml(-1) mostly increased (6/33) some biomarkers. In the therapeutic concentration of 500 ng ml(-1) aliskiren does not affect hemostatic biomarkers, except for a moderate but highly significant (P=0.003) increase of AT-III activity. Higher aliskiren doses were associated with more profound biomarker changes, but they are likely not to be clinically relevant since they show diverging (that is, both mild antiplatelet and platelet-activating) trends, and considering the 2- to 4-fold safety margin. It is suggested that antithrombotic properties of aliskiren be explored further in an ex vivo clinical setting.

[Clopidogrel resistance: myth or reality].

The efficacy of clopidogrel, as an established anti-platelet agent for the acute coronary syndrome treatment and for the thrombotic complications prevention after percutaneous coronary angioplasty and coronary artery stenting has been supported by the evidence of several major randomized clinical trials. However, some patients treated with clopidogrel have a minor, but still a risk of coronary artery thrombosis and sudden death. Moreover, clopidogrel was not effective in patients after ischemic stroke, as well as it was not effective for the primary prevention of vascular events. Several authors proposed the theory of " clopidogrel-resistance " . However, this theory is based on a limited number of laboratory findings, and was not supported by the evidence of clinical studies. The phenomena of " clopidogrel-resistance " could be masked by the low patient compliance, while the rate of such patients could exceed 30% after one year of treatment. The offered methods for overcoming clopidogrel-resistance include doubling or tripling of the loading dose (600-900 mg vs. 300mg) or administration one or two more potent antiplatelet agents. However, such approach could not only increase the risk of major and fatal bleedings, but could have a potential to reduce patients compliance, and consequently increase the risk of thrombosis. Only multicenter randomized study with hard outcome or ideally survival endpoint, supported by comprehensive serial platelet assessment, strict compliance rules including measurement of clopidogrel metabolite(s) will determine whether " clopidogrel resistance " is a real danger (as suggested by the platelet biomarkers), or an artificial tool (as suggested by the randomized clinical evidence) introduced to help novel antiplatelet agents to gain the vascular market share.

Platelet inhibition with prasugrel (CS-747) compared with clopidogrel in patients undergoing coronary stenting: the subset from the JUMBO study.

BACKGROUND: Based on the preclinical and phase 1 studies, prasugrel, a novel platelet ADP P2Y12 receptor blocker, may be a more potent platelet inhibitor than clopidogrel. This study compared the antiplatelet properties of prasugrel in a small subset of patients enrolled in the JUMBO trial, and compared with historic clopidogrel treated controls. METHODS AND RESULTS: Nine patients undergoing coronary stenting were randomised to one of three arms of prasugrel (40 mg loading, and 7.5 mg maintenance, n = 1; 60/10 mg, n = 4; or 60/15 mg, n = 2), or clopidogrel (300/75 mg, n = 2). Aspirin and GP IIb/IIIa inhibitors were permitted. Platelet activity was assessed at baseline, at 4, and 24 hours, and at 30 days after stent implantation in substudy participants, and compared with 124 historic controls who received clopidogrel. Independent of the loading, or maintenance dose, patients treated with prasugrel exhibited significantly more potent platelet inhibition as determined by ADP, and collagen induced aggregation, Ultegra Analyser, and surface expression of PECAM-1, GPIIb/IIIa antigen, and activity with PAC-1 antibody, GPIb, P-selectin, CD40-ligand, GP37, and thrombospondin receptor expression when compared with those treated with clopidogrel. There were no differences between antiplatelet agents with regard to vitronectin, LAMP-1, PAR-1 (intact and cleaved epitopes) thrombin receptor expression, or formation of platelet-monocyte microparticles. Expression of GPIIb antigen, vitronectin, and LAMP-3 receptor were not affected by both agents. Two patients treated with prasugrel 10 mg/daily exhibited complete inhibition of collagen induced aggregation at 30 days. CONCLUSION: At the dosing regimens chosen in the JUMBO trial, it seems that prasugrel is a more potent antiplatelet agent than clopidogrel. Two episodes of profound platelet inhibition, which are not seen with clopidogrel, raise the possibility of higher bleeding risks especially during long term prasugrel use. Whether stronger platelet inhibition will yield better clinical outcomes and/or increased bleeding remains to be determined in an ongoing comparative phase 3 superiority trial (TRITON).

Validation of a VerifyNow-P2Y12 cartridge for monitoring platelet inhibition with clopidogrel.

Despite common use of clopidogrel in patients with vascular disease, monitoring of platelet inhibition is still not conventional in clinical practice. Considering substantial response variability, when some patients may experience inadequate protection, and/or increased risk of bleeding, simple and reliable methods to control adequate antiplatelet regimen is mandatory. We validated a new VerifyNow-P2Y12 assay to measure inhibition of the P2Y12 platelet receptors by clopidogrel by evaluating its receptor specificity, precision, and potential interference with platelet count, hematocrit, age, cholesterol, triglycerides, and other antiplatelet agents. Platelet aggregation induced by ADP or ADP + prostaglandin E1 (ADP + PGE1) in the presence of specific P2Y12 inhibitor 2-methylthio-AMP (2MeSAMP) for the assessment of assay specificity was performed in 10 volunteers. Seventeen medications were used for the VerifyNow-P2Y12 interference testing, and assay interplay with blood constituents was evaluated in a clinical setting in 131 patients with coronary artery disease. In the presence of 2MeSAMP, the average residual aggregation level across the 10 donors was 27% for ADP and 5% for ADP + PGE1. There also was a strong agreement between ADP + PGE1 aggregometry and VerifyNow-P2Y12 assay (93% vs. 95% average inhibition across all donors). The coefficient of variation for the test precision was less than 8%. The VerifyNow-P2Y12 readings were not influenced by age, platelet count, hematocrit, fibrinogen, cholesterol, or triglycerides level. There was an interference with abciximab before P2Y12 inhibition; however, after platelet suppression with cilostazol, the interference with all tested substances was minimal. VerifyNow-P2Y12 is a reliable, simple, and sensitive device suitable for monitoring of P2Y12 platelet receptor inhibitors in the clinical arena.

Criticality of pH for accurate fluorometric measurements of dipyridamole levels in biological fluids.

Extended release dipyridamole (DIP) is widely used in clinical practice as an Aggrenox formulation, which is proven to improve outcomes for secondary stroke prevention in patients after acute vascular events. However, presently established fluorometry techniques are not suitable for trace amount determinations, because of the variable background fluorescence. The authors sought to determine whether biological fluid pH is important for the serial measures of DIP levels in the animal experiments and in patients treated with Aggrenox after ischemic stroke. Post-stroke patient (n = 34) and mice (n = 25) samples were tested to determine DIP levels by established techniques with FluoroMax 3 spectrofluorometer. Both the absorption and emission spectra of DIP were affected by modifications in pH. Fluorescence of DIP was found to be maximal at a wavelength of 490 nm (excitation 420 nm) and the spectral pattern was independent of pH. The intensity of fluorescence, however, was drastically lower at low pH (at pH 2.6, fluorescence was 4% of intensity at pH 9.8). Background plasma fluorescence, however, was completely unaffected by changes in pH. Using these fluorometric characteristics, a regression model that facilitates the efficient and sensitive determination of DIP concentration in biological fluids was formulated. Exploiting pH-dependent characteristics of DIP versus serum fluorescence patterns permits a convenient mathematical model to determine DIP concentration. This relatively inexpensive and time-efficient procedure can quantify drug levels in human/animal plasma/serum, thereby directly determining the level of patient adherence to the prescribed drug regimen, be it in the context of clinical trials or compliance with the animal protocol.

[The modern concept of the tactics of conservative and combined treatment of limb lymphedema].

The paper is concerned with the general problems of the tactics and stages of conservative and combined treatment of limb lymphedema using compression therapy techniques. The data presented herein cover both our own studies and the reported materials which are grouped so as to understand the basic principle of the concept of combined therapy for lymph edema (CTLE). The treatment presumes the use of two main variants in the form of CTLE as an independent method of conservative treatment of lymphedema and CTLE as a program coupled with surgical operations. The treatment policy for lymphedema involves two basic stages: hospital and prophylactic. The hospital program of the CTLE includes technologies acting on different pathogenetic components of lymphedema. In primary lymphedema, the hospital treatment allows to minimize lymphedema to 36% and in secondary to 4-4% of the initial parameters. During the prophylactic period, edema regression is appreciably delayed or does not take place at all. The next stage aimed at edema decrease is followed by a course of hospital CTLE which, as dependent on lymphedema severity, is carried out 1-4 times a year. The main principle of a current approach to the treatment of lymphedema is based on the concept of multimodality independent or combined treatment. The accuracy of this principle observance predetermines the efficacy of the results obtained.

Uniform platelet activation exists before coronary stent implantation despite aspirin therapy.

BACKGROUND: Platelets play an important role in the natural history of coronary artery disease. Enhanced platelet aggregation and receptor expression unquestionably occur after coronary stent implantation; however, the functional characteristics of platelets before stenting have not been fully elucidated. METHODS: Platelets were assessed before intervention by platelet-rich plasma aggregation (PA) with 5 mmol adenosine diphosphate (ADP) and 1 mg/mL collagen; whole blood aggregation (WBA) by 1 mg/mL collagen; shear-induced closure time (CT); contractile force (CF); and expression of 9 surface receptors by flow cytometry in 126 patients undergoing elective coronary artery stent placement. All patients received aspirin for at least 7 days. The data were compared with those from 64 healthy volunteers. RESULTS: Each test revealed sustained platelet activation in patients undergoing coronary stenting compared with control values. These differences were significant for collagen-induced PA (P =.031); CF (P =.0001); expression of glycoprotein (GP) IIb/IIIa (P =.0001); P-selectin (P =.0008); platelet/endothelial cell adhesion molecule (PECAM)-1 (P =.0001); CD107a (P =.0001); CD107b (P =.0004); and CD63 (P =.009). CONCLUSION: Platelets are indeed activated before coronary stenting despite antecedent therapy with aspirin.

Selective serotonin reuptake inhibitors yield additional antiplatelet protection in patients with congestive heart failure treated with antecedent aspirin.

Clinical depression has been identified as an independent risk factor for increased mortality in patients with coronary artery disease. Enhanced platelet activity has been suggested as the mechanism responsible for this adverse association. Selective serotonin reuptake inhibitors (SSRIs) are known to inhibit platelets in patients undergoing coronary stenting. We sought to determine whether concomitant therapy with SSRIs would yield additional anti-platelet benefit in patients with congestive heart failure (CHF) already treated with antecedent aspirin. A total of 88 patients with left ventricular ejection fraction (LVEF) <40% or CHF symptoms in the setting of preserved systolic function and NYHA Class II-IV were analyzed. Of these, 23 patients (26%) were chronic SSRI users (SSRI+), and 65 patients were free from SSRI therapy (SSRI-). All patients received aspirin (325 mg) for at least 1 month prior to platelet studies. Platelets were assessed by aggregometry, flow cytometry and a rapid analyzer. The SSRI+ group exhibited a substantial decrease in platelet activity when compared with SSRI- patients, as manifested by a significant reduction in ADP- (P=0.001), and collagen-induced (P=0.02) aggregation, and the expression of PECAM-1 (P=0.03), GPIb (P=0.03), GP IIb/IIIa antigen (P=0.02) and GP IIb/IIIa activity with PAC-1 antibody (P=0.04) and P-selectin (P=0.02). Therapy with SSRIs also resulted in the reduced formation of platelet-leukocyte microparticles (P=0.01). Epinephrine-induced aggregation in plasma, collagen-induced whole blood aggregation, closure time and expression of vitronectin receptor, CD63, CD107a, CD107b and CD151 did not differ between groups. In patients with CHF already on aspirin, SSRI therapy was associated with further inhibition of platelet function. This observation may help to explain some of the clinical benefits associated with SSRI therapy. Further clinical trials may help to elucidate the potential outcome benefits of SSRIs in other potential thrombotic circumstances.

Paradoxical activation of major platelet receptors in the methadone-maintained patients after single pill of aspirin.

BACKGROUND: Chronic drug abuse is an established cause of acute coronary events and sudden death. The association between use of narcotics and platelet abnormalities is well described. However, it is still not clear, how aspirin affects expression of major platelet receptors in chronic drug users with coronary artery disease, especially those recovering in the methadone clinic maintenance program. We sought to compare how a single pill of non-enteric coated aspirin (325-mg) affects human platelets in patients with coronary artery disease dependent on methadone use. METHODS: Data from 30 subjects were analyzed, eight of them were the chronic drug addicts, and participated in a methadone recovery program. Platelets were assessed twice at baseline (pre-aspirin), and after 3-24 hours (post-aspirin). The expression of platelet receptors was determined by using the following monoclonal antibodies: CD31 (PECAM-1), CD41 (GPIIb), CD42b (GPIb), CD51/CD61 (vitronectin receptor), CD62p (P-selectin), CD63 (LIMP or LAMP-3), CD107a (LAMP-1), CD151 (PETA-3), and PAC-1 for fibrinogen-platelet binding determination (PharMingen, San Diego, CA). Platelet-leukocyte interactions were assessed by using dual antibodies for a pan-platelet marker (CD151), together with CD14, a monocyte/macrophage marker. RESULTS: In a drug free group, digestion of a single tablet of aspirin resulted in a significantly (p<0.05) diminished expression of PECAM-1, GP IIb, fibrinogen binding with PAC-1 antibody, GP Ib, P-selectin, and CD151. In contrast, patients receiving methadone exhibited opposite trends, resultant in a paradoxical activation of major platelet receptors after digestion of aspirin. These differences reached statistical significance (p<0.05) for PECAM-1, GPIIb, and P-selectin expression. CONCLUSION: There are some fundamental differences in the responsiveness to aspirin in chronic methadone users when compared with drug-free patients. Suspecting narcotics abuse may be critical in patients with limited aspirin efficacy, or those who developed an aspirin resistance. Unexpected platelet activation may indeed represent a missing link between use of narcotics and enhanced incidence of vascular death in this high-risk population.

Platelet activation as a universal trigger in the pathogenesis of acute coronary events after cocaine abuse.

Numerous reports document the widespread use of cocaine in most parts of the world, which confers an increased risk of vascular morbidity and mortality. The mechanisms responsible for this association are multifactorial, but platelet activation might play a substantial role linking these events. Contradictory data exist regarding the cellular mechanisms of cocaine's effects on thrombocytes. In terms of therapeutic interventions, a possible activation of platelets would conceptually require more aggressive anti-platelet therapy with aspirin, clopidogrel or other compounds, however, no data exist to date to support this approach. Further studies elucidating these issues are warranted. This review summarizes the latest and often confusing data on the interaction between cocaine and platelets in certain in vitro, animal and clinical scenarios.

[Properties of thermostable HtpR-mutant of Escherichia coli]. / Svoistva termoustoichivogo HtpR-mutanta Escherichia coli.

A thermoresistant htpR mutant having a decreased level of proteolytic activity has been selected in E. coli strain K802 after the directed mutagenesis in vivo. The mutation results in the bacteriophage T7 RNA-polymerase stability, aminoglycosidephosphotransferase stability as well as in the decrease in the rate of proteolytic degradation of cytoplasmic proteins during the heat shock. The obtained mutant strain can, probably be used as a host for alien polypeptides production.