Western Blotting-Based Quantitative Measurement of Myosin II Regulatory Light Chain Phosphorylation in Small Amounts of Non-muscle Cells.

Myosin II is the main molecular motor in the actomyosin-dependent motility in cells. Phosphorylation of the myosin regulatory light chain (RLC) at Ser19 is a prerequisite for smooth muscle/non-muscle myosin II activation and serves as a biochemical equivalent of myosin II activity. Simultaneous phosphorylation at Thr18 further promotes the myosin II ATPase activity. A number of methods have been developed to measure myosin RLC phosphorylation at Ser19 or di-phosphorylation at Thr18/Ser19. While these methods are straightforward and robust in myosin-rich muscle tissues, they demonstrate limited applicability in non-muscle cells that have low myosin II content and are usually available in lesser amounts than muscle tissue. Because of this, dynamic analysis of RLC phosphorylation in multiple samples of non-muscle cells is difficult and requires large number of cells. The use of phospho-specific antibodies increases detection sensitivity but allows estimation of only relative levels of RLC phosphorylation at specific residues, which makes it difficult to estimate the physiologic relevancy of the observed changes in RLC phosphorylation. To measure RLC phosphorylation in small amounts of non-muscle cells, we used external calibration standards of non-phosphorylated and in vitro phosphorylated RLC in standard SDS-PAGE and Western blot procedures with phospho-specific RLC antibodies. Here, we describe the method in detail and demonstrate its application for quantitative measurement of myosin RLC phosphorylation in endothelial cells in response to natural agonists (thrombin or insulin) and intact human platelets. We discuss the advantages and limitations of the proposed method vs other approaches for measuring myosin RLC phosphorylation in non-muscle cells.

[Hydrogel microchip as a tool for studying exosomes in human serum].

Exosomes are cell-derived vesicles that are secreted by both normal and cancer cells. Over the last decade, a few studies have revealed that exosomes cross talk and/or influence major tumor-related pathways such as angiogenesis and metastasis involving many cell types within the tumor microenvironment. The protein composition of the membrane of an exosome reflects that of the membrane of the cell of origin. Because of this, tumor-derived exosomes differ from exosomes that are derived from normal cells. The detection of tumor exosomes and analysis of their molecular composition hold promise for diagnosis and prognosis of cancer. Here, we present hydrogel microarrays (biochips), which contain a panel of immobilized antibodies that recognize tetraspanins (CD9, CD63, CD81) and prognostic markers for colorectal cancer (A33, CD147). These biochips make it possible to analyze the surface proteins of either isolated exosomes or exosomes that are present in the serum samples without isolation. These biochips were successfully used to analyze the surface proteins of exosomes from serum that was collected from a colorectal cancer patient and healthy donor. Biochip-guided immunofluorescent analysis of the exosomes has made it possible for us to detect the A33 antigen and CD147 in the serum sample of the colorectal cancer patient with normal levels of CEA and CA19-9.

[The analysis of the effectiveness of the combined treatment of the patients presenting with laryngeal papillomatosis]. / Analiz éffektivnosti kombinirovannogo lecheniia bol'nykh papillomatozom gortani.

The objective of the present work was to evaluate the effectiveness of the application of the inducer of endogenous interferon synthesis in the course of the combined treatment of the adult patients presenting with laryngeal papillomatosis. A total of 50 patients with this pathology were admitted and examined at the ENT Clinics of I.M. Sechenov First Moscow State Medical University and M.F. Vladimirsky Moscow Regional Research Clinical Institute during 24 months. The patients comprising group 1 (n=25) were treated with the use of cold plasma surgery and the inducer of endogenous interferon synthesis while those included in group 2 (n=25) received only surgical treatment. It was shown that the combined treatment including the surgical step (cold plasma surgery) followed by the treatment with the inducer of endogenous interferon synthesis to prevent relapses makes it possible to reduce the number of secondary surgical interventions, increase the duration of the intervals between relapses, and decrease the content of types 6 and 11 human papilloma virus in the saliva.

[Kidney allotransplantation from the AB0-incompatible donors].

The experience of 28 kidney allotransplantations from the AB0-incompatible donors was analyzed. The comparative group consisted of 38 patients, who received the AB0-compatible organ. The results were assessed using the following parameters: renal function, morphology of the biopsy samples of the transplanted kidney and actuary survival of the recipients with functioning transplants in both groups. The comparative analysis showed no significant difference between the two groups, giving the right to consider the kidney allotransplantation from the AB0-incompatible donors safe and effective.

[The state of art, problems and future of transfusiology].

The article specifies the main areas of modern transfusiology: blood services, transfusion immunology, clinical and industrial transfusiology. Our research work and the analysis of published data made it possible to specify a new paradigm of transfusion medicine--the transition from the doctrine of blood components transfusion to molecular transfusiology, which comprises medicamentary transfusiology based on the use of gene engineering and biotechnology components as well as blood components preparations, high-tech methods of extracorporeal molecular hemocorrection and highly specific laboratory technology. In view of this concept we develop and introduce modern methods of transfusion medicine and laboratory technologies, aimed at providing the safety of transfusion manipulations and optimizing their use in clinical practice. Transfusion immunology is a part of Transfusion Medicine, studying and implementing in practice the immunological safety and efficacy of transfusions. The article also specifies the main criteria for blood donation and blood components transfusion in patients with hereditary abnormalities of blood; a new approach to evaluation of blood donors potential in endemic foci of inherited abnormalities of blood. The results of the research work formed the basis for protocols of donors examination and the treatment of patients needing hemo transfusion, optimized the tactics of decreed research during the examination of blood donors and patients needing blood transfusion.

[The kidney transplantation from the ABO-incompatible donors].

The experience of 28 allotransplantations of ABO-incompatible kidneys was compared with the treatment results of 38 ABO-compatible renal transplantations. The transplanted kidney function, morphological changes of the transplanted kidney and the comparative analysis of actuary survival in both groups showed no significant difference. The results of the study prove the validity of the kidney transplantation from the ABO-incompatible donors.

Antibodies Against Unusual Forms of Sialylated Glycans.

Previous studies have shown that in the blood of healthy donors (1) there are no natural antibodies against sialylated glycoproteins, which contain Neu5Acα (N-acetylneuraminic acid) as the most widespread form of human sialic acid, and (2) there is a moderate level of antibodies capable of binding unnatural oligosaccharides, where Neu5Ac is beta-linked to a typical mammalian glycan core. In the present study, we investigated antibodies against ßNeu5Ac in more detail and verified the presence of Kdn (2-keto-3-deoxy- D-glycero-D-galacto-nonulosonic acid) as a possible cause behind their appearance in humans, taking into account the expected cross-reactivity to Kdn glycans, which are found in bacterial glycoconjugates in both the α- and ß-forms. We observed the binding of peripheral blood immunoglobulins to sialyllactosamines (where "sialyl" is Kdn or neuraminic acid) in only a very limited number of donors, while the binding to monosaccharide Kdn occurred in all samples, regardless of the configuration of the glycosidic bond of the Kdn moiety. In some individuals, the binding level of some of the immunoglobulins was high. This means that bacterial Kdn glycoconjugates are very unlikely to induce antibodies to ßNeu5Ac glycans in humans. To determine the reason for the presence of these antibodies, we focused on noninfectious pathologies, as well as on a normal state in which a significant change in the immune system occurs: namely, pregnancy. As a result, we found that 2/3 of pregnant women have IgM in the blood against Neu5Acß2-3Galß1-4GlcNAcß. Moreover, IgG class antibodies against Neu5Acß2-3Galß1-4GlcNAcß and Neu5Acß2-6Galß1-4GlcNAcß were also detected in eluates from the placenta. Presumably, these antibodies block fetal antigens.

Effect of Cryo-Processing on Platelet-Rich Autoplasma Preparations.

Platelet-derived growth factor (PDGF) plays an important role in angiogenesis, affects activation of migration and proliferation of mesenchymal stem cells, fibroblasts, smooth muscle cells, osteoblasts; activation of migration of monocytes, macrophages, and neutrophils. The aim of the investigation was to study the effect of cryo-processing on the qualitative properties of platelet-rich autoplasma (PRP) at different time intervals. MATERIALS AND METHODS: Autologous plasma preparations were obtained from the blood of 31 donors. The biological material was prepared by double centrifugation according to the protocol for obtaining P-PRP and L-PRP. Platelet count and the concentration of growth factors (PDGF-AA and PDGF-BB) were studied in fresh PRP preparations. In frozen PRP samples, the concentration of PDGF-AA and PDGF-BB was determined 2 weeks after cryo-processing and 2 months after cryo-processing at -35 °Ð¡. P-PRP and L-PRP samples activated with 10% CaCl2 solution and those non-activated were studied. RESULTS: L-PRP preparations are significantly superior to P-PRP preparations: the concentration of platelets is 1.7 times higher in them. The level of PDGF-AA in non-activated L-PRP is 1.8 times higher than in non-activated P-PRP (p<0.05). The level of PDGF-AA is 1.5 times higher in activated L-PRP than in activated P-PRP (p<0.05). The level of PDGF-BB is 2.9 times higher in non-activated L-PRP than in non-activated P-PRP and 1.8 times higher in activated L-PRP than in activated P-PRP (p<0.05). The concentration of PDGF-BB in non-activated P-PRP sharply increases in the 2nd week after freezing and remains at the same level after 2 months (p<0.05). The concentration of PDGF-BB in activated plasma does not change (p>0.05). CONCLUSION: Cryo-processing of non-activated autologous L-PRP allows preserving and subsequently enhancing the properties of plasma concentrate, which makes it possible to apply it in clinical practice.

[Post-transfusion hemolytic reactions in regions endemic for the hereditary glucose-6-phosphate dehydrogenase deficiency].

This paper deals with the problem of hemocomponent therapy in the use of blood components from donors with hereditary glucose-6-phosphate dehydrogenase (G-6-PD) deficiency and with the matters of family donation in this blood anomaly. Posttransfusion hemolytic reactions during transfusions of packed red blood cells from donors with hereditary blood anomaly are retrospectively analyzed. Recommendations are formulated for recipients who have undergone transfusions of blood components from donors with G-6-PD deficiency.

[Significance of the isoantigen A2 and immune anti-A1 antibodies in transfusiology].

The paper gives data on the frequency of A2 and A2B blood groups in the blood samples of 3590 donors and patients and on the carriage of irregular anti-A1 antibodies. To define the belonging of anti-A1 antibodies to various classes of immunoglobulins, their resistance and temperature optimum, at which the antibodies showed the highest activity, the authors examined the irregular anti-A1 antibodies-containing sera from 43 subjects with A2 and A2B blood groups. The necessity of identifying the carriage of irregular anti-A1 antibodies is determined by the fact that there may be posttransfusion reactions or complications in recipients, the carriers of such antibodies, who are transfused donor red blood cells containing the antigen A1. The risk of the development of such events is particularly great when anti-A1 antibodies are active at 37 degrees C and belong to the IgG class, i.e. they are immune; transfusion to a recipient is carried out during surgery under artificial hypothermia (below 28 degrees C). The red blood cells (A2 or A2B) from a donor having the same group should be selected for recipients having an A2 or A2B blood groups, respectively, and immune anti-A1 antibodies; if the latter are absent, in accordance with the instruction, there may be a selection of other-group donor red blood cells: 0 group red blood cells to recipients with A2 blood group and 0 or B group red blood cells to recipients with A2B blood group. An individual selection should be without fail made for the compatibility of bloods from a donor and a recipient in salt tests (at 4-8, 20-22, and 37 degrees C) and indirect Coombs' test. When plasma components (fresh frozen plasma, native plasma and its concentrate) are transfused to recipients with A2 and A2B blood groups having immune anti-A1 antibodies, one should use the same plasma group--A and AB, respectively.

[Parenteral nutrition in the treatment of short bowel syndrome].

Relevance of nutritive support problems at patients with SRL is shown in experiences of RSSC members named by B.V. Petrovski RAMS. This article demonstrates feasibility of artificial therapeutic nutrition and role of parenteral nutrition, clinical and social rehabilitation at patients with SRL.

[Nutritional support in surgically treated patients with dilated cardiomyopathy].

The efficiency of nutritional support was studied in patients operated on for dilated cardiomyopathy (DCM). In 36 patients with DCM and signs of circulatory decompensation, preoperative nutritional support with a MD mil Clinipit mixture was carried out in addition to medical treatment. The trophological, laboratory, and functional parameters of the operated patients were assessed on admission to hospital, 14 days after treatment and in the late postoperative period (up to 3 years). Preoperative medical treatment and nutritional support in all the patients during 14 days could achieve circulatory compensation, lean weight gain, improved pool of visceral proteins (prealbumin and albumin), decreased C-reactive protein levels, and increased exercise tolerance. The developed complex protocol provided effective preparation for surgery, rapid activation and recovery of patients with DCM in the postoperative period. In the operated patients with DCM, the three-year actuarial survival rate was 85.6 +/- 7.4% while it was not greater than 12% in the patients receiving medical treatment only. Analysis of long-term results indicated the higher functional activity and improved quality of life in the operated patients.

[Clinical aspects of autologous blood donation in cardiosurgery].

The paper presents auto donation protocols and a procedure for autodonor plasmapheresis used in cardiosurgical patients, complications, and their preventive measures on the basis of an analysis of 308 autodonor plasmapheresis procedures performed at the Russian Surgery Research Center, Russian Academy of Medical Sciences, in 2007. The preoperative autoblood preservation safety concept envisaging the safety of autodonors during blood donation, the correct storage and issue of autocomponents, and the prevention of adverse reactions to blood exfusion in patients was introduced. The rate of the reactions was 6.4% of the total number of auto donations in cardiosurgical high-risk patients, moderate reaction being most common. The findings indicated that exfusion of 450 ml of blood without previous infusion of plasma substitutes was a cause of collaptoid reactions in 14 cases of the collapse-complicated procedures of autodonor plasmapheresis in cardiosurgical patients (in 70% of the total number of complications). No association was found between of the frequency and severity of complications and the nosological entity, age, and body mass index; however, a further retrospective analysis of 1500 fresh frozen autoblood preservation procedures that have recently made at the Russian Surgery Research Center, Russian Academy of Medical Sciences, is required to have statistically significant data.

[Current technologies for blood loss prevention during abdominal hysterectomy].

The paper deals with the development and use of original adequate transfusion-infusion programs to compensate for blood loss in abdominal hysterectomies for uterine myomas of varying sizes, by applying the current blood-supplying technologies that permit avoidance of transfusion of donor blood components. Four blood loss-replenishing programs in abdominal hysterectomy for large-sized myomas were studied for impact on the basic functions in premenopasal women. These included: (1) preoperative blood storage of blood autocomponents; (2) normovolemic hemodilution with hydroxyethyl starch (HES) solution being used as a dilutant; (3) administration of HES solutions as an independent solution compensating for blood loss, and (4) intraoperative reinfusion of autoerythrocytes. The proposed transfusion-infusion programs in abdominal hysterectomy for large-sized myomas by applying blood-preserving methods are effective in avoiding the use of donor blood components and accordingly in reducing the frequency of complications associated with transfusions of donor blood components, and improving the postoperative period.

[The association between the 5-HTTLPR polymorphism of the serotonin transporter gene and the efficacy and tolerability of selective serotonin reuptake inhibitors].

OBJECTIVE: To search for the association between the 5-HTTLPR polymorphism of the serotonin transporter gene and the efficacy and tolerability of selective serotonin reuptake inhibitors (SSRIs). MATERIAL AND METHODS: The complex clinical/psychopathological and genetic study of 38 patients with depression treated with SSRIs was carried out. RESULTS AND CONCLUSION: The 5-HTTLPR polymorphism was associated with the rate of achievement of remission and tolerability of treatment: carriers of the SS genotype achieved remission less frequently and more frequently experienced side-effects.

[Pulmonary tuberculosis in patients with hereditary glucose-6-phosphate dehydrogenase deficiency]. / Tuberkulez legkikh u bol'nykh s nasledstvennym defitsitom gliukozo-6-fosfatdegidrogenazy.

A study was made of the occurrence of glucose-6-phosphate dehydrogenase (G-6-PDH) deficiency among patients with lung tuberculosis including those suffering from mental diseases (alcoholism or schizophrenia). In Azerbaijani patients, the rate of G-6-PDH demonstration was higher as compared to that among the healthy population. On combined lung tuberculosis and alcoholism the rate of that abnormality demonstration increased whereas on associated lung tuberculosis and schizophrenia, it slightly decreased. Among patients with hereditary G-6-PDH deficiency, the portion of chronic destructive forms of pulmonary tuberculosis is high, the tuberculous process is accompanied more often by isolation of M. tuberculosis. The etiological role of G-6-PDH as a genetic marker is evaluated as 14%; in associated lung tuberculosis and alcoholism, it grows to 18%.

[Use of plasmapheresis in the treatment of drug-resistant cardiac arrhythmias]. / Ispol'zovanie plazmafereza pri lechenii narushenii ritma serdtsa, rezistentnykh k lekarstvennoi terapii.

AIM: To study the therapeutical efficiency of plasmapheresis (PA) in patients with drug-resistant cardiac arrhythmias (CA) and its mechanisms. MATERIALS AND METHODS: Discrete PA sessions were carried out in 56 patients with drug-resistant CA: paroxysmal atrial arrhythmia (AA), ventricular and supraventricular premature contractions, supraventricular tachycardia of various etiology. Biochemical blood values, coagulographic parameters, lipid peroxidation (LPO), the spectrum of nonesterified fatty acids (NEFA), the level of medium-sized molecules were determined, ECG monitoring, EchoCG, and left ventricular radioisotope computed tomography were performed before and after a PA session. RESULTS: PA was effective in 50% of cases. The duration of its effect averaged 3.0 (1.25-5.0) months. PA was more beneficial for patients with IHD, AA with normal left atrial dimensions, and hyperlipidemia. The duration of the effect was significantly higher when antiarrhythmic drug therapy was continued after PA. Due to PA, there were significant decreases in the blood concentrations of cholesterol, medium-sized molecules, malonic dialdehyde (MDA) and in the proportion of polyunsaturated NEFA. The antiarrhythmic effect was associated with the decreases in MDA and NEFA, with a tendency for a reduction in the rate of chemiluminescence. CONCLUSION: PA may be used in the treatment of drug-resistant CA. The most significant mechanism of its antiarrhythmic activity is to recover sensitivity to antiarrhythmics. The intrinsic antiarrhythmic activity may be associated with its effect on NEFA metabolism and LPO; however, its mechanisms await further studies.

[Surgery without donor's blood]. / Khirurgiia bez donorskoi krovi.

Complex program "Surgery without blood" was developed and introduced into a broad clinical practice. The main elements of this program are stimulation of erythropoesis, preparing in autoserum and autoblood before surgery with acute normovolemic hemodilution method, precise surgical technique, reinfusion of blood from wound and drainages, adequate anesthesia and correction of hemostasis system. This program permits one to minimize infusion of donor's blood components (DBC) in elective surgery (cardiosurgery, orthopedic surgery, neurosurgery, oncology, general surgery) and reduce significantly transfusion of DBC in urgent surgery. Rejection of DBC transfusion decreases number of postoperative complications and hospital stay, improves results of treatment and is cost-effective.