[Clinostatting effects on neuronal differentiation of embryonic stem cells from mice strain R1].

The model of embryonic stem cells from R1 mice at the stage of embryoid bodies was used to study effects of slow clinostatting on neuronal differentiation with the help of two markers--beta-III tubulin (early differentiation) and MAP2 (late differentiation). As compared with the control, the number of beta-III tubulin-positive neurons was found increased and of MAP2-positive neurons--decreased. As regards MAP2- positive neurons, it is concluded that the gravity factors have a specific effect on EB. The beta-III tubulin staining makes possible determination of the total number of neuronal cells at different stages of development. The observed increase in the number of beta-III tubulin-positive neurons may evidence a nonspecific mechanic effect of clinostatting at the EB stage. It was shown that EB cells are particularly sensitive to clinostatting.

[Clinical and immunobiochemical study of efficacy and stress-protective properties of thyroliberin at the acute stage of carotid ischemic stroke].

Stress-promoting system is known to be involved in course and outcome of acute stage of ischemic stroke. An important predictor of an unfavorable course of stroke is so-called "low T3-syndrome". Therapy with drugs increasing T3 level on the background of reduced reaction of oxidative stress is one of a perspective direction of neuroprotection. The study aimed at investigating thyroliberin influence on a clinical course and an outcome of ischemic atherothrombotic stroke as well as on thyroid hormones level in 46 patients (27 women and 19 men) aged 55-75 years admitted to the hospital at the first 24 hours of the disease. Twenty-one patients were switched to thyroliberin in dosage 500 mcg twice a day during 5 days. A control group included 25 patients. Neurological status of the patients was evaluated on days 1, 3, 7 and 21 using the Orgogozo scale and functional recovery was assessed on day 21 with the Bartel scale. Radioimmunoassay of TTH level, cT3 and free thyroxine (cT4) in blood plasma was conducted on days 1, 2, 3 and 7 using test-kit IRMA TTG CT (Belorus). Atherosclerotic changes of MAG were measured with USDG on day 1. All the patients underwent MRI of the brain on days 1, 7 and 21 using tomograph Ellips (Russia) 0.15 Tesla. The dynamics of regress of neurological disturbances in patients receiving thyroliberin appeared as the higher total score on the Orgogozo scale on days 3, 7 and 21 especially in a severe course of the diseases compared to the control group (p<0,007 on day 7). The T3 level in these patients was significantly higher (p<0,05) on days 2, 3 and 7 and the thyroxine level was increased significantly on the 3rd day of stroke (p<0,005) as compared to the control group. In patients with moderate severity of the disease, the TTH level was significantly higher on the 2nd day of stroke (p=0,0004). However, in patients with a severe course TTH and T4 concentrations did not differ in both groups. The results of the study suggest that the use of thyroliberin in an acute stage of ischemic stroke prevents development of "low T3 syndrome" that promotes more rapid and essential regress of neurological disturbances.

Different effects of enhanced and reduced expression of pub gene on the formation of embryoid bodies by cultured embryonic mouse stem cell.

The effects of pub gene on proliferation and initial stages of differentiation of embryonic mouse stem cells were studied in vitro. To this end we used enhanced expression of human pub gene (hpub) and suppression of expression of mouse endogenous pub gene with RNA-interference in embryonic stem cells. Proliferative activity of genetically modified polyclonal lines of the embryonic stem cells transfected with plasmids carrying expressing hpub gene or plasmids generating small interference RNA to this gene did not differ from that of the control cells. Inhibition of expression of endogenous pub gene in embryonic stem cells using small interference RNA 2-fold decreased the formation of embryoid bodies, at the same time additional expression of exogenous hpub gene almost 2-fold increased their number in comparison with the control. It was hypothesized that pub gene participates in early stages of differentiation of embryonic stem cells leading to the formation of embryoid bodies.

BFP protein expression in transfected embryonic stem cells.

Cultured mouse embryonic stem cells can be transfected with a reporter gene encoding blue fluorescent protein BFP and regulated by drosophila heat shock protein 70 promoter. This gene is activated after heating and synthesizes matrix RNA. Blue protein is synthesized under these conditions. The system for transfection of stem cells allows us to activate automatically the corresponding transgenes.

[Influence of regulatory genes of type 1 human immunodeficiency virus on proliferation and differentiation of murine embryonic stem cells]. / Vliianie reguliatornykh genov virusa immunodefitsita cheloveka tipa 1 na proliferatsiiu i differentsirovku émbrional'nykh stvolovykh kletok myshi.

Spontaneous formation of embryoid bodies and subsequent differentiation of some cells into cardiomyocytes were demonstrated on murine embryonic stem cells of R1 line. The lines of embryonic stem cells were obtained that had been transfected with genetic constructs carrying expressing regulatory genes of the human immunodeficiency virus tat and nef and "green protein" gene (GFP). The transfection of embryonic stem cells with the gene tat stimulated their proliferative activity, while this activity decreased in the cells transfected with the gene nef. The time necessary for the formation of embryoid bodies by all lines of transfected cells was similar to that in the control cells. In the cultures of cells transfected with nef and tat, the number of embryoid bodies and the percentage of embryoid bodies with contracting cardiomyocytes were higher and lower than in the control, respectively. Thus, an inverse correlation was observed between the effects of regulatory genes of the human immunodeficiency virus on proliferation and differentiation embryonic stem cells.

[Investigation of mechanisms of neuro-protective effect of semax in acute period of ischemic stroke]. / Izuchenie mekhanizmov neiroprotektivnogo deistviia semaksa v ostrom periode ishemicheskogo insul'ta.

Semax is the first domestic nootropic drug of an unexhausted type from the group of neuropeptides. In experimental studies it showed angioprotective, antihypoxic and neurotrophic activity in the doses 100-150 micrograms/kg. A combined clinical-electrophysiologic study revealed its high efficiency in acute ischemic stroke. A clinical trial was performed of immunobiochemical mechanisms of neuroprotective properties of Semax in acute period of ischemic stroke. A retrospective comparative clinicoimmunobiochemical analysis provided objective data on the molecular level on activating influence of Semax on antiinflammatory postischemic reactions in the brain. Shifting neuromediatory balance toward a prevalence of the antiinflammatory agents (interleukin-10, tumor necrosis factor-alpha) over the factors maintaining the inflammation (interleukin-8, C-reactive protein).

[Clinico-immunobiochemical monitoring of factors of focal inflammation in the acute period of hemispheric ischemic stroke]. / Kliniko-immunobiokhimicheskii monitoring faktorov lokal'nogo vospaleniia v ostrom periode polusharnogo ishemicheskogo insul'ta.

The changes of cytokinis status and C-reactive protein were evaluated in cerebrospinal fluid of 50 patients in the acute period of ischemic hemispheric stroke with consideration of influence of the remote consequences of the ischemia, established experimentally, on the mechanisms of cerebral infarction development as well as on the progression of both atherogenesis and vascular encephalopathy in the period after the stroke. Significance both of a surplus releasing of the proinflammatory cytokines and deficiency of the protective antiinflammatory and trophotropic factors in the development of an inflammatory response was established. Immunobiochemical criteria were proposed for grading of process for stroke course prediction and for recovery of the altered neurologic functions. More favourable prognosis was anticipated in the patients in which a the treatment started within of the "therapeutic window".

[Study of the antigenic structure of human immunoglobulin lambda-chain using monoclonal antibodies]. / Issledovanie antigennoi struktury lambda-tsepei immunoglobulinov cheloveka s pomoshch'iu monoklonal'nykh antitel.

Nine monoclonals against human Ig lambda chains were produced, 4 antibodies react with C-domain, 5--with V-domain of the lambda chain. Anti-C lambda domain antibodies recognize not less than 3 epitopes and one of them is expressed only on the isolated chain. Anti-V lambda antibodies bind both isolated lambda chain and intact IgG, IgM, IgA. Four epitopes are expressed by few lambda Bence Jones proteins of the III subgroup, the immunogen possessing the same isotype. The 4 mentioned epitopes represent private idiotypic determinants. The epitope 3E10 is characteristic of 50% Bence Jones proteins of the II and III V lambda-subgroups thus representing a common idiotypic determinant. Using anti-V lambda antibodies germ line variability of V lambda III proteins was analysed and the similarity of antigenic structure of normal and myeloma human Ig lambda chains was demonstrated.

[Immunoenzyme test system with monoclonal antibodies to human IgG4 in the determination of allergen-specific antibodies in pollinosis]. / Immunofermentnaia test-sistema s monoklonal'nymi antitelami k IgG4 cheloveka dlia vyiavleniia allergenspetsificheskikh antitel pri pollinoze.

ELISA for determination of allergen-specific IgG4 antibodies was developed with the help of monoclonal anti-IgG4 antibodies obtained by classic hybridoma technique. Subclass specificity of antibodies were studied in sera of 108 patients suffering from pollinosis. Antibodies of this isotype were found in the majority of patients with tree pollen allergy but not in patients with grass pollen allergy. The level of IgG4 antibodies correlated with the severity of the disease but not with the intensity of skin tests. Specific hyposensitization resulted in significant increase of IgG4 antibody level in patients with tree pollen allergy. Determination of IgG4 antibodies is proved to be useful to reveal tree pollen allergy and to monitor hyposensitization therapy.

[Antigenic determinants of the constant region of human immunoglobin kappa-chain detected by monoclonal antibodies]. / Antigennye determinanty postoiannogo domena kappa-tsepei immunoglobulinov cheloveka, vyiavliaemye s pomoshch'iu monoklonal'nykh antitel.

Ten different monoclonal antibody (monAB) preparations reacting with human IgL chains of the kappa type have been obtained. Nine of the monAB interacted with the kappa-chain C domain, whereas only one monAB reacted with the V domain. It has been determined that monAB against the C domain react with three different epitopes. One epitope is expressed on intact Ig molecules as well as on isolated kappa-chains, whereas the other two epitopes are found only on isolated kappa-chains. The expression of these epitopes in 40 different myeloma kappa-chain preparations belonging to four various subgroups was studied. The level of this C domain epitope expression has been shown to depend on the variable subgroups of kappa-chains indicating a close association between V and C domains. This association leads to the alteration of antigenic activity of some C domain epitopes. The alterations are thought to be local because, as a rule, they involve only one of the three epitopes.

A new approach to studies of cell-antibody interactions using fluorescent lipid probes.

The interaction of poly- and monoclonal antibodies against the L-chain of human Ig with Burkitt lymphoma EB-3 cells was studied using a fluorescent lipid probe, anthrylvinyl-labelled sphingomyelin, incorporated into the cell plasma membrane. Binding of the antibodies to Ig receptors on the surface was shown to induce changes in the fluorescence polarization of the probe. The high sensitivity of the method allows one to detect less than 100 antibody molecules per cell. The possibility of using cells or liposomes carrying antigens and fluorescent lipids for the determination of antibodies in solution is discussed.

[Selective cytotoxicity of antibody-ricin-A-chain conjugate for human tumor B cells. II. Comparison of activity of immunotoxins conjugated with poly- and monoclonal antibodies]. / Izbiratel'naia tsitotoksichnost' kon''iugata antitelo-A-tsep' ritsina dlia opukholevykh B-kletok cheloveka. II. Sravnenie aktivnosti immunotoksinov, poluchennykh na osnove poli- i monoklonal'nykh antitel.

The A-chain of a plant toxin ricin has been coupled to poly- and monoclonal antibodies specific to the L-chains of human IgG. The inhibitory effect of the conjugates has been compared with the ability of the antibodies to bind to target cells. Cytotoxicity of the conjugates has been monitored following incorporation of 14C-leucine radioactivity into Burkitt lymphoma cells with surface Ig. The 50% inhibition of protein synthesis is observed 18 h after treatment of cells with immunotoxins, when the concentration of the conjugates with poly- and monoclonal antibodies is 1.2.10(-9) M and 0.7.10(-9) M, respectively. The data take into account that only part of the polyclonal antibodies molecules is able to react with target cells. The control conjugates containing either monoclonal antibodies that do not react with the lymphoma cells surface L-chains or nonimmune serum IgG proved to have no effect on target cells even at the level of 10(-7) M. The immunotoxins with poly- and monoclonal antibodies produce almost the same kinetics of protein synthesis inhibition, when incubated with lymphoma cells for 60 min. However, a 30 min treatment reveals a considerably higher cytotoxicity of the conjugate with monoclonal antibodies.

[Selective interaction of immunoglobulin polypeptide chains. Quantitative evaluation of the idiotypic and antigen binding activity of reassociated immunoglobulin molecules]. / Izbiratel'noe vzaimodeistvie polipeptidnykh tsepei immunoglobilinov.Kolichestvennaia otsenka idiotipicheskoi i antigensviazyvaiushchei aktivnosti reassotsiirovannykh molekul immunoglobulinov.

We have analysed reassociated Ig molecules, containing heavy (H) or light (L) chains of Ig-B2 monoclonal antibody with human fibronectin binding activity and L of H chains of normal mouse serum immunoglobulin (Ig-NM). Examination of Ig-B2 idiotype expression in reassociated Ig indicated that 0.4% L-NM and 0.8% H-NM were able to restore Ig-B2 idiotype. The analysis of antigen binding capacity of reassociated Ig demonstrated, that only 4% H-NM created antigen binding site in complex with L-B2. We have determined the leading role of L-chain in creation of idiotype and binding site of Ig-B2. Selectivity of interaction between H and L chains is discussed. The results indicate, that not more than 4-6% of random H--L combinations produce functional Ig.