Enhancing radioprotection: A chitosan-based chelating polymer is a versatile radioprotective agent for prophylactic and therapeutic interventions against radionuclide contamination.

To mitigate the risk of radioactive isotope dissemination, the development of preventative and curative measures is of particular interest. For mass treatment, the developed solution must be easily administered, preferably orally, with effective, nontoxic decorporating properties against a wide range of radioactive isotopes. Currently, most orally administered chelation therapy products are quickly absorbed into the blood circulation, where chelation of the radioactive isotope is a race against time due to the short circulation half-life of the therapeutic. This report presents an alternative therapeutic approach by using a functionalized chitosan (chitosan@DOTAGA) with chelating properties that remains within the gastrointestinal tract and is eliminated in feces, that can protect against ingested radioactive isotopes. The polymer shows important in vitro chelation properties towards different metallic cations of importance, including (Cs(I), Ir(III), Th(IV), Tl(I), Sr(II), U(VI) and Co(II)), at different pH (from 1 to 7) representing the different environments in the gastrointestinal tract. An in vivo proof of concept is presented on a rodent model of uranium contamination following an oral administration of Chitosan@DOTAGA. The polymer partially prevents the accumulation of uranium within the kidneys (providing a protective effect) and completely prevents its uptake by the spleen.

Agonists or positive allosteric modulators of α7 nicotinic acetylcholine receptor prevent interaction of SARS-Cov-2 receptor-binding domain with astrocytoma cells.

SARS-Cov-2, the virus causing COVID-19, penetrates host target cells via the receptor of angiotensin-converting enzyme 2 (ACE2). Disrupting the virus interaction with ACE2 affords a plausible mechanism for prevention of cell penetration and inhibiting dissemination of the virus. Our studies demonstrate that ACE2 interaction with the receptor binding domain of SARS-Cov-2 spike protein (RBD) can be impaired by modulating the α7 nicotinic acetylcholine receptor (α7 nAChR) contiguous with ACE2. U373 cells of human astrocytoma origin were shown to bind both ACE2-specific antibody and recombinant RBD in Cell-ELISA. ACE2 was found to interact with α7 nAChR in U373 cell lysates studied by Sandwich ELISA. Our studies demonstrate that inhibition of RBD binding to ACE2-expressing U373 cells were defined with α7 nAChR agonists choline and PNU282987, but not a competitive antagonist methyllicaconitine (MLA). Additionally, the type 2 positive allosteric modulator (PAM2) PNU120596 and hydroxyurea (HU) also inhibited the binding. Our studies demonstrate that activation of α7 AChRs has efficacy in inhibiting the SARS-Cov-2 interaction with the ACE2 receptor and in such a way can prevent virus target cell penetration. These studies also help to clarify the consistent efficacy and positive outcomes for utilizing HU in treating COVID-19.

Ukrainian Consensus on Diagnosis and Management of Vitamin D Deficiency in Adults.

Vitamin D deficiency (VDD) is a global problem, however, there were no Ukrainian guidelines devoted to its screening, prevention, and treatment, which became the reason for the Consensus creation. This article aimed to present the Consensus of Ukrainian experts devoted to VDD management. Following the creation of the multidisciplinary Consensus group, consent on the formation process, drafting and fine-tuning of key recommendations, and two rounds of voting, 14 final recommendations were successfully voted upon. Despite a recent decrease in VDD prevalence in Ukraine, we recommend raising awareness regarding VDD's importance and improving the strategies for its decline. We recommend screening the serum 25-hydroxyvitamin D (25(OH)D) level in risk groups while maintaining a target concentration of 75-125 nmol/L (30-50 ng/mL). We recommend prophylactic cholecalciferol supplementation (800-2000 IU/d for youthful healthy subjects, and 3000-5000 IU/d for subjects from the risk groups). For a VDD treatment, we recommend a short-term administration of increased doses of cholecalciferol (4000-10,000 IU/d) with 25(OH)D levels monitored after 4-12 weeks of treatment, followed by the use of maintenance doses. Additionally, we recommend assessing serum 25(OH)D levels before antiosteoporotic treatment and providing vitamin D and calcium supplementation throughout the full course of the antiosteoporotic therapy.

α7 Nicotinic acetylcholine receptors regulate translocation of HIF-1α to the cell nucleus and mitochondria upon hypoxia.

Nicotinic acetylcholine receptors (nAChRs), initially characterized as ligand-gated ion channels mediating fast synaptic transmission, are now found in many non-excitable cells and mitochondria where they function in ion-independent manner and regulate vital cellular processes like apoptosis, proliferation, cytokine secretion. Here we show that the nAChRs of α7 subtype are present in the nuclei of liver cells and astrocytoma U373 cell line. As shown by lectin ELISA, the nuclear α7 nAChRs are mature glycoproteins that follow the standard rout of post-translational modifications in Golgi; however, their glycosylation profile is non-identical to that of mitochondrial nAChRs. They are exposed on the outer nuclear membrane and are found in combination with lamin B1. The nuclear α7 nAChRs are up-regulated in liver within 1 h after partial hepatectomy and in H2O2-treated U373 cells. As shown both in silico and experimentally, the α7 nAChR interacts with hypoxia-inducible factor HIF-1α and this interaction is impaired by α7-selective agonists PNU282987 and choline or type 2 positive allosteric modulator PNU120596, which prevent HIF-1α accumulation in the nuclei. Similarly, HIF-1α interacts with mitochondrial α7 nAChRs in U373 cells treated with dimethyloxalylglycine. It is concluded that functional α7 nAChRs influence HIF-1α translocation into the nucleus and mitochondria upon hypoxia.

Formation and elimination of soluble fibrin and D-dimer in the bloodstream.

Soluble fibrin is composed mainly of desA fibrin and fibrinogen oligomers consisting of fewer than 16 monomers partially cross-linked by factor XIIIa. Soluble fibrin cannot stimulate Glu-plasminogen activation by tissue plasminogen activator (t-PA); therefore, it may not be a direct predecessor of D-dimer. However, within the microcirculatory system, soluble fibrin oligomers may form microclots. Fibrin microclots stimulate Glu-plasminogen activation by t-PA, a process resulting in the formation of Glu-plasmin. Glu-plasmin dissolves the microclots, forming D-dimer. In normal and pathological blood plasma samples, soluble fibrin levels are substantially higher than those of D-dimer. Their concentrations in the plasma are also regulated by transendothelial transfer, absorption by blood macrophages, and binding and internalization with low-density lipoprotein receptors of the cells of the reticuloendothelial system. Therefore, the exact mechanisms of fibrin clots formation and elimination in normal and pathological conditions remain unclear. In this study, we reviewed findings on the molecular mechanisms of the formation and dissolution of fibrin clots, fibrin-dependent activation of Glu-plasminogen by t-PA, and blood plasma behavior in the microcirculatory system. Finally, we proposed a model that explains the relations of D-dimer and soluble fibrin underlying the common and separate mechanisms of their formation and elimination.

Immunization with 674-685 fragment of SARS-Cov-2 spike protein induces neuroinflammation and impairs episodic memory of mice.

COVID-19 is accompanied by strong inflammatory reaction and is often followed by long-term cognitive disorders. The fragment 674-685 of SARS-Cov-2 spike protein was shown to interact with α7 nicotinic acetylcholine receptor involved in regulating both inflammatory reactions and cognitive functions. Here we show that mice immunized with the peptide corresponding to 674-685 fragment of SARS-Cov-2 spike protein conjugated to hemocyanin (KLH-674-685) demonstrate decreased level of α7 nicotinic acetylcholine receptors, increased levels of IL-1ß and TNFα in the brain and impairment of episodic memory. Choline injections prevented α7 nicotinic receptor decline and memory loss. Mice injected with immunoglobulins obtained from the blood of (KLH-674-685)-immunized mice also demonstrated episodic memory decline. These data allow suggesting that post-COVID memory impairment in humans is related to SARS-Cov-2 spike protein-specific immune reaction. The mechanisms of such effect are being discussed.

The geometry of diphtheria toxoid CRM197 channel assessed by thiazolium salts and nonelectrolytes.

The geometry of the channel formed by nontoxic derivative of diphtheria toxin CRM197 in lipid bilayer was determined using the dependence of single-channel conductance upon the hydrodynamic radii of different nonelectrolytes. It was found that the cis entrance of CRM197 channel on the side of membrane to which the toxoid was added at pH 4.8 and the trans entrance on the opposite side at pH 6.0 had effective radii of 3.90 and 3.48 Å, respectively. The 3-alkyloxycarbonylmethyl-5-(2-hydroxyethyl)-4-methyl-1,3-thiazolium salts reversibly reduced current via CRM197 channels. The potency of the blockers increased with increasing length of alkyl chain at symmetric pH 6.0 and remained high and stable at pH 4.8 on the cis side. Comparative analysis of CRM197 and amphotericin B pore size with the inhibitory action of thiazolium salts revealed a significant increase in CRM197 pore dimension at pH 6.0. Addition of thiazolium salt with nine carbons alkyl tail increased by ∼30% the viability of human carcinoma cells A431 treated with diphtheria toxin.

SARS-Cov-2 spike protein fragment 674-685 protects mitochondria from releasing cytochrome c in response to apoptogenic influence.

In spite of numerous studies, many details of SARS-Cov-2 interaction with human cells are still poorly understood. The 674-685 fragment of SARS-Cov-2 spike protein is homologous to the fragment of α-cobratoxin underlying its interaction with α7 nicotinic acetylcholine receptors (nAChRs). The interaction of 674-685 peptide with α7 nAChR has been predicted in silico. In the present paper we confirm this prediction experimentally and investigate the effect of SARS-Cov-2 spike protein peptide on mitochondria, which express α7 nAChRs to regulate apoptosis-related events. We demonstrate that SARS-Cov-2 spike protein peptide 674-685 competes with the antibody against 179-190 fragment of α7 nAChR subunit for the binding to α7-expressing cells and mitochondria and prevents the release of cytochrome c from isolated mitochondria in response to 0.5 mM H2O2 but does not protect intact U373 cells against apoptogenic effect of H2O2. Our data suggest that the α7 nAChR-binding portion of SARS-Cov-2 spike protein prevents mitochondria-driven apoptosis when the virus is uncoated inside the cell and, therefore, supports the infected cell viability before the virus replication cycle is complete.

Unique features of brain metastases-targeted AGuIX nanoparticles vs their constituents: A focus on glutamate-/GABA-ergic neurotransmission in cortex nerve terminals.

Gadolinium-based radiosensitizing AGuIX nanoparticles (AGuIX) currently tested two phase 2 clinical trials in association with radiotherapy for the treatment of brain metastases. Here, excitatory/inhibitory neurotransmission was assessed in rat cortex nerve terminals in the presence of AGuIX and their constituents (DOTAGA and DOTAGA/Gd3+) at concentrations used for medical treatment, and those 5-24 times higher. The ambient level, transporter-mediated, tonic and exocytotic release of L-[14C]glutamate and [3H]GABA, the membrane potential of nerve terminals were not changed in the presence of AGuIX at concentrations used for medical treatment ([Gd3+] = 0.25 mM, corresponding to 0.25 g.L-1), and DOTAGA (0.25 mM) and DOTAGA/Gd3+ (0.25 mM/0.01 mM). Difference between AGuIX and the precursors was uncovered, when their concentrations were increased. AGuIX (1.25-6 mM) did not change any transport characteristics of L-[14C]glutamate and [3H]GABA, whereas, DOTAGA (1.25-6 mM) affected the membrane potential, ambient level, and exocytotic release of L-[14C]glutamate and [3H]GABA. Gd3+ did not mask, but even enhanced above effects of DOTAGA. Therefore, AGuIX did not influence glutamate- and GABA-ergic neurotransmission at the presynaptic site. In contrast, DOTAGA and mixture DOTAGA/Gd3+ significantly affected synaptic neurotransmission at high concentrations. AGuIX own structure that overcomes neurotoxic features of their constituents.

Air pollution particulate matter as a potential carrier of SARS-CoV-2 to the nervous system and/or neurological symptom enhancer: arguments in favor.

Entry receptor for SARS-CoV-2 is expressed in nasal epithelial cells, and nasal delivery pathway can be a key feature of transmission. Here, a possibility of interaction of SARS-CoV-2 with air pollution particulate matter (PM) was considered. It was shown in our recent studies that water-suspended plastic and wood smoke aerosol PM and carbon-containing nanoparticles from burning organics can interact with the plasma membrane of brain nerve terminals presumably due to their lipid components. COVID-19 patients have neurological symptoms, viral particles were found in the brain, SARS-CoV-2 enters the cells via fusion of lipid viral envelope with the plasma membranes of infected cells, and so viral envelop can contain lipid components of the host neuronal membranes. Therefore, interaction of SARS-CoV-2 envelope with PM is possible in water surrounding. After drying, PM can serve as a carrier for transmission of SARS-CoV-2 immobilized at their surface. Moreover, PM and SARS-CoV-2 per se can enter human organism during nasal inhalation, and they both use the same nose-to-brain delivery pathways moving along axons directly to the brain, influencing the nervous system and exocytosis/endocytosis in nerve cells. Thus, PM can aggravate neurological symptoms of SARS-CoV-2 and vice versa, due to their identical nose-to-brain delivery mechanism and possible interference of neuronal effects. In addition, different types of PM because of their ability to interact with the plasma membranes of nerve cells can facilitate unspecific SARS-CoV-2 entrance to the cells, and can influence envelope features of SARS-CoV-2. Detailed studies are required to analyze interaction of SARS-CoV-2 with PM.

Different Effects of Nicotine and N-Stearoyl-ethanolamine on Episodic Memory and Brain Mitochondria of α7 Nicotinic Acetylcholine Receptor Knockout Mice.

Nicotinic acetylcholine receptors of α7 subtype (α7 nAChRs) are involved in regulating neuroinflammation and cognitive functions. Correspondingly, α7-/- mice demonstrate pro-inflammatory phenotype and impaired episodic memory. In addition, nAChRs expressed in mitochondria regulate the release of pro-apoptotic factors like cytochrome c. Here we studied whether the cognitive deficiency of α7-/- mice can be cured by oral consumption of either nicotine or N-stearoylethanolamine (NSE), a lipid possessing anti-inflammatory, cannabimimetic and membrane-stabilizing activity. Mice were examined in Novel Object Recognition behavioral test, their blood, brains and brain mitochondria were tested for the levels of interleukin-6, various nAChR subtypes and cytochrome c released by ELISA. The data presented demonstrate that both substances stimulated the raise of interleukin-6 in the blood and improved episodic memory of α7-/- mice. However, NSE improved, while nicotine worsened the brain mitochondria sustainability to apoptogenic stimuli, as shown by either decreased or increased amounts of cytochrome c released. Both nicotine and NSE up-regulated α4ß2 nAChRs in the brain; NSE up-regulated, while nicotine down-regulated α9-containing nAChRs in the brain mitochondria. It is concluded that the level of alternative nAChR subtypes in the brain is critically important for memory and mitochondria sustainability in the absence of α7 nAChRs.

Mesenchymal Stem Cells or Interleukin-6 Improve Episodic Memory of Mice Lacking α7 Nicotinic Acetylcholine Receptors.

Nicotinic acetylcholine receptors of α7 subtype (α7 nAChRs) are involved in regulating cognition, inflammation and cell survival. Neuroinflammation is accompanied by the decrease of α7 nAChRs in the brain and impairment of memory. We show here that α7-/- mice possess pro-inflammatory phenotype and demonstrate worse episodic memory compared to wild-type mice. Previously we reported that mesenchymal stem cells (MSCs) restored episodic memory of lipopolysaccharide-treated wild-type mice. The aim of this study was to examine if MSCs or their soluble factors improve memory of α7-/- mice. The α7-specific signal (ELISA) and α7+ cells (IHC) were found in the brain of α7-/- mice on days 7 and 14 after intravenous injection of α7+ MSCs from either human umbilical cord (hMSCs) or mouse placenta (mMSCs). The intravenously injected MSCs or intraperitoneally injected hMSCs-conditioned medium transiently improved episodic memory of α7-/- mice and decreased cytochrome c release from their brain mitochondria under the effect of Ca2+. Either MSCs or conditioned medium stimulated an IL-6 increase in the brain, which coincided with the improvement of episodic memory. Injections of recombinant IL-6 also improved episodic memory of α7-/- mice accompanied by the up-regulation of α3, α4, ß2 and ß4 nAChR subunits in the brain. It is concluded that MSCs, injected intravenously, penetrate the brain of α7-/- mice and persist there for at least 2 weeks. They improve episodic memory of mice and make their mitochondria more resistant to apoptogenic influence. One of the soluble factors responsible for the memory improvement is IL-6.

Intravenously Injected Mesenchymal Stem Cells Penetrate the Brain and Treat Inflammation-Induced Brain Damage and Memory Impairment in Mice.

Neuroinflammation is regarded as one of the pathogenic factors of Alzheimer disease (AD). Previously, we showed that mice regularly injected with bacterial lipopolysaccharide (LPS) possessed the AD-like symptoms like episodic memory decline, elevated amounts of amyloid beta (Aß) peptide (1-42), and decreased levels of nicotinic acetylcholine receptors (nAChRs) in the brain. The use of mesenchymal stem cells (MSCs), which can differentiate into multiple cell types, including neurons, is an attractive idea of regenerative medicine, in particular, for neurodegenerative disorders like AD. In the present study, we aimed to investigate whether pathogenic effect of LPS on the brain and behavior of mice can be prevented or treated by injection of MSCs or MSC-produced soluble factors. Fluorescently-labeled MSCs, injected intravenously, were found in the brain blood vessels of LPS-treated mice. Mice co-injected with LPS and MSCs did not demonstrate episodic memory impairment, Aß (1-42) accumulation, and nAChR decrease in the brain and brain mitochondria. Their mitochondria released less cytochrome c under the effect of Ca2+ compared to mitochondria of LPS-only-treated mice. Moreover, MSCs could reverse the pathogenic symptoms developed 3 weeks after LPS injection. Cultured MSCs produced IL-6 in response to LPS and MSCs effect in vivo was accompanied by additional stimulation of both micro- and macroglia. Xenogeneic (human) MSCs were almost as efficient as allogeneic (mouse) ones and regular injections of human MSC-conditioned medium also produced positive effect. These data allow suggesting MSCs as a potential therapeutic tool to cure neuroinflammation-related cognitive pathology.

Dual-use nano-neurotechnology.

The chemical and biological nonproliferation regime stands at a watershed moment, when failure seems a real possibility. After the unsuccessful outcome of the 2016 Eighth Review Conference, the future of the Biological and Toxin Weapons Convention is uncertain. As the Chemical Weapons Convention (CWC) approaches its Fourth Review Conference in 2018, it has almost completed removing the huge stocks of chemical weapons, but it now faces the difficult organizational task of moving its focus to preventing the reemergence of chemical weapons at a time when the international security situation appears to be increasingly more difficult and dangerous. In this article, we assess the current and near-term state (5-10 years) and impact of three related areas of science and technology that could be of dual-use concern: targeted delivery of agents to the central nervous system (CNS), particularly by means of nanotechnology; direct impact of nanomaterials on synaptic functions in the CNS; and neuronal circuits in the brain that might be targeted by those with hostile intent. We attempt to assess the implications of our findings, particularly for the consideration of the problem of state-level interest in so-called nonlethal incapacitating chemical agents for law enforcement at the CWC Review Conference in 2018, but also more generally for the longer-term future of the chemical and biological nonproliferation regime.

Mitochondrial Nicotinic Acetylcholine Receptors Support Liver Cells Viability After Partial Hepatectomy.

Nicotinic acetylcholine receptors (nAChRs) expressed on the cell plasma membrane are ligand-gated ion channels mediating fast synaptic transmission, regulating neurotransmitter and cytokine release and supporting the viability of many cell types. The nAChRs expressed in mitochondria regulate the release of pro-apoptotic factors, like cytochrome c, in ion channel-independent manner. Here we show that α3ß2, α7ß2, and α9α10 nAChR subtypes are up-regulated in rat liver mitochondria 3-6 h after partial hepatectomy resulting in increased sustainability of mitochondria to apoptogenic effects of Ca2+ and H2O2. In contrast, laparotomy resulted in down-regulation of all nAChR subunits, except α9, and decreased mitochondria sustainability to apoptogenic effects of Ca2+ and H2O2. Experiments performed in liver mitochondria from α3+/-, α7-/-, ß4-/-, α7ß2-/-, or wild-type C57Bl/6J mice demonstrated that the decrease of α3 or absence of α7 or α7/ß2 subunits in mitochondria is compensated with ß4 and α9 subunits, which could be found in α3ß4, α4ß4, α9ß4, and α9α10 combinations. Mitochondria from knockout mice maintained their sustainability to Ca2+ but were differently regulated by nAChR subtype-specific ligands: PNU-282987, methyllycaconitine, dihydro-ß-erythroidine, α-conotoxin MII, and α-conotoxin PeIA. It is concluded that mitochondrial nAChRs play an important role in supporting the viability of hepatic cells and, therefore, may be a pharmacological target for pro-survival therapy. The concerted action of multiple nAChR subtypes controlling either CaKMII- or Src-dependent signaling pathways in mitochondria ensures a reliable protection against apoptogenic factors of different nature.

Dual-Use Nano-Neurotechnology: An assessment of the implications of trends in science and technology.

The chemical and biological nonproliferation regime stands at a watershed moment, when failure seems a real possibility. After the unsuccessful outcome of the 2016 Eighth Review Conference, the future of the Biological and Toxin Weapons Convention is uncertain. As the Chemical Weapons Convention (CWC) approaches its Fourth Review Conference in 2018, it has almost completed removing the huge stocks of chemical weapons, but it now faces the difficult organizational task of moving its focus to preventing the reemergence of chemical weapons at a time when the international security situation appears to be increasingly more difficult and dangerous. In this article, we assess the current and near-term state (5-10 years) and impact of three related areas of science and technology that could be of dual-use concern: targeted delivery of agents to the central nervous system (CNS), particularly by means of nanotechnology; direct impact of nanomaterials on synaptic functions in the CNS; and neuronal circuits in the brain that might be targeted by those with hostile intent. We attempt to assess the implications of our findings, particularly for the consideration of the problem of state-level interest in so-called nonlethal incapacitating chemical agents for law enforcement at the CWC Review Conference in 2018, but also more generally for the longer-term future of the chemical and biological nonproliferation regime.

N-Stearoylethanolamine protects the brain and improves memory of mice treated with lipopolysaccharide or immunized with the extracellular domain of α7 nicotinic acetylcholine receptor.

Neuroinflammation is an important risk factor for neurodegenerative disorders like Alzheimer's disease. Nicotinic acetylcholine receptors of α7 subtype (α7 nAChRs) regulate inflammatory processes in various tissues, including the brain. N-stearoylethanolamine (NSE) is a biologically active cell membrane component with anti-inflammatory and membrane-protective properties. Previously we found that mice injected with bacterial lipopolysaccharide (LPS) or immunized with recombinant extracellular domain (1-208) of α7 nAChR subunit possessed decreased α7 nAChR levels, accumulated pathogenic amyloid-beta peptide Aß(1-42) in the brain and demonstrated impaired episodic memory compared to non-treated mice. Here we studied the effect of NSE on behavior and brain components of LPS- treated or α7(1-208)-immunized mice. NSE, given per os, non-significantly decreased LPS-stimulated interleukin-6 elevation in the brain, slowed down the α7(1-208)-specific IgG antibody production and prevented the antibody penetration into the brain of mice. NSE prevented the loss of α7 nAChRs and accumulation of α7-bound Aß(1-42) in the brain and brain mitochondria of LPS-treated or α7(1-208)-immunized mice and supported mitochondria resistance to apoptosis by attenuating Ca2+-stimulated cytochrome c release. Finally, NSE significantly improved episodic memory of mice impaired by either LPS treatment or immunization with α7(1-208). The results of our study demonstrate a therapeutic potential of NSE for prevention of cognitive disfunction caused by neuroinflammation or autoimmune reaction that allows suggesting this drug as a candidate for the treatment or prophylaxis of Alzheimer's pathology.

The Soluble Heparin-Binding EGF-Like Growth Factor Stimulates EGF Receptor Trafficking to the Nucleus.

Most ligands of epidermal growth factor receptor (EGFR) have the ability to induce EGFR translocation into the nucleus, where EGFR acts as an important transcriptional regulator. Soluble form of heparin-binding EGF-like growth factor (sHB-EGF) is one of the ligands for EGFR in many cell types; however, there is no evidence for the ability of sHB-EGF to induce EGFR nuclear importation. Here, we demonstrated that treatment of A431 cells with sHB-EGF resulted in nuclear localization of EGFR and such translocation occurs via retrograde pathway. It was shown by confocal microscopy and co-immunoprecipitation assay that the translocation complex consisted of both ligand and receptor. The chromatin immunoprecipitation assay showed the association of sHB-EGF-EGFR complex with promoter region of cyclin D1 in the cell nucleus and this association was prevented by application of EGFR kinase inhibitor AG-1478. The obtained data suggest that sHB-EGF acts similarly to other EGFR ligands and is capable to induce EGFR nuclear translocation as a part of ligand-receptor complex in a tyrosine phosphorylation-dependent manner.

Cellobiose-coated poly(lactide-co-glycolide) particles loaded with diphtheria toxoid for per os immunization.

AIM: To evaluate the dose-dependent immunogenic properties of poly(lactide-co-glycolide) (PLGA) particles coated with cellobiose as antigen carriers for oral immunization. METHODS: Two types of PLGA-cellobiose particles (PLGA-cellobiose-1, ~ 0.8 µm and PLGA-cellobiose-2, ~ 1.2 µm) containing non-toxic recombinant subunit B (SbB) of diphtheria toxin fused with enhanced green fluorescent protein were characterized in vitro for their size, shape, antigen loading, and ability to induce phagocytosis. Different doses of antigen, immobilized on the particles (2.5 µg, 25 µg, 250 µg, and 2500 µg per 1 kg of body weight), were administered per os 3 times with intervals of 2 weeks to BALB/c female mice. The antigen-specific IgG and IgA antibodies were estimated in serum by ELISA. RESULTS: After the first immunization, increase in concentration of blood antitoxic antibodies was detected. Antigen dose 250 µg/kg was the most immunogenic for IgG antibodies induction for both types of PLGA-cellobiose particles. Antigen doses 25 µg/kg and 2.5 µg/kg were the most immunogenic for IgA antibodies induction by PLGA-cellobiose 1 and 2 particles, respectively. The second and the third treatment had no significant effect on the immune response or even reduced it, which could be explained by immune tolerance induction by the antigens delivered per os. CONCLUSION: Our results suggest that the correct dose of PLGA-cellobiose particles loaded with antigen could significantly increase the humoral immune response against the introduced antigen already after the first immunization. Thus, PLGA particles can be considered as a potent component of oral vaccines.

Fibrin(ogen)olytic and platelet modulating activity of a novel protease from the Echis multisquamatis snake venom.

The variety of enzymes including serine proteases that possess fibrin(ogen)olytic and platelet modulating activity have been discovered in different snake venoms. In our work the fibrin(ogen)olytic and platelet modulating activity of a new protease from Echis multisquamatis snake venom was studied. It was shown that purified enzyme cleaved the ВßR42-A43 bond of fibrinogen during first contact with the substrate following much slower hydrolysis of C-terminus of fibrinogen Aα-chain. Protease hydrolysed fibrin clot too, but at much slower rate and cleaved both C-terminus of Aα-chain and ВßR42-A43 bond of Bß-chain simultaneously. Preincubation of fibrinogen with protease dramatically elongated thrombin clotting time and the clot formed from a mixture of native fibrinogen and fibrinogen desВß(1-42)2 digested by plasmin much faster than a native fibrin clot. The protease did not activate platelets nor cause changes in their shape and granularity, but it reduced platelets aggregation induced by ADP.