Mathematical modelling of the influence of ACE I/D polymorphism on blood pressure and antihypertensive therapy.

The angiotensin-converting enzyme (ACE) gene (ACE) insertion/deletion (I/D) polymorphism raises the possibility of personalising ACE inhibitor therapy to optimise its efficiency and reduce side effects in genetically distinct subgroups. However, the extent of its influence among these subgroups is unknown. Therefore, we extended our computational model of blood pressure regulation to investigate the effect of the ACE I/D polymorphism on haemodynamic parameters in humans undergoing antihypertensive therapy. The model showed that the dependence of blood pressure on serum ACE activity is a function of saturation and therefore, the lack of association between ACE I/D and blood pressure levels may be due to high ACE activity in specific populations. Additionally, in an extended model simulating the effects of different classes of antihypertensive drugs, we explored the relationship between ACE I/D and the efficacy of inhibitors of the renin-angiotensin-aldosterone system. The model predicted that the response of cardiovascular and renal parameters to treatment directly depends on ACE activity. However, significant differences in parameter changes were observed only between groups with high and low ACE levels, while different ACE I/D genotypes within the same group had similar changes in absolute values. We conclude that a single genetic variant is responsible for only a small fraction of heredity in treatment success and its predictive value is limited.

Investigation of Curing Process and Thermal Behavior of Copolymers Based on Polypropylene Glycol Fumarate and Acrylic Acid Using the Methods of DSC and TGA.

In this work, the possibility of preparation of copolymers of three-dimensional crosslinked structure based on polypropylene glycol fumarate and acrylic acid is shown. The initial reagent polypropylene glycol fumarate has been synthesized by polycondensation reaction of fumaric acid and propylene glycol. The curing process of polypropylene glycol fumarate and acrylic acid at various mole concentrations was studied using DSC method at isothermal and dynamic regimens. Curing in isothermal condition was carried out at temperatures of 60 °C, 70 °C, and 80 °C. Residual reactivity was evaluated at a dynamic regimen within the temperature range from 30 °C to 200 °C at a constant heating rate. On the basis of calorimetric studies, the thermal effects and kinetic parameters of the reaction (conversion, reaction rate, activation energy) have been determined. Thermal behavior of cured samples of p-PGF-AA was estimated using dynamic thermogravimetry (TGA). According to TGA data, the process of decomposition of the studied copolymers proceeds in several stages. Based on the results obtained, the activation energies of thermal decomposition were calculated using the iso-conversional methods of Kissinger-Akakhira-Sunose and Friedman.

Flagellin-Fused Protein Targeting M2e and HA2 Induces Innate and T-Cell Responses in Mice of Different Genetic Lines.

Efficient control of influenza A infection can potentially be achieved through the development of broad-spectrum vaccines. Recombinant proteins incorporating conserved influenza A virus peptides are one of the platforms for the development of cross-protective influenza vaccines. We constructed a recombinant protein Flg-HA2-2-4M2ehs, in which the extracellular domain of the M2 protein (M2e) and the sequence (aa76-130) of the second subunit of HA (HA2) were used as target antigens. In this study, we investigated the ability of the Flg-HA2-2-4M2ehs protein to activate innate immunity and stimulate the formation of T-cell response in mice of different genetic lines after intranasal immunization. Our studies showed that the Flg-HA2-2-4M2ehs protein was manifested in an increase in the relative content of neutrophils, monocytes, and interstitial macrophages, against the backdrop of a decrease in the level of dendritic cells and increased expression in the CD86 marker. In the lungs of BALB/c mice, immunization with the Flg-HA2-2-4M2ehs protein induced the formation of antigen-specific CD4+ and CD8+ effector memory T cells, producing TNF-α. In mice C57Bl/6, the formation of antigen-specific effector CD8+ T cells, predominantly producing IFN-γ+, was demonstrated. The data obtained showed the formation of CD8+ and CD4+ effector memory T cells expressing the CD107a.

Comparative Immunological Study in Mice of Inactivated Influenza Vaccines Used in the Russian Immunization Program.

A series of commercial inactivated influenza vaccines (IIVs) used in the Russian National Immunization Program were characterized to evaluate their protective properties on an animal model. Standard methods for quantifying immune response, such as hemagglutination inhibition (HAI) assay and virus neutralization (VN) assay, allowed us to distinguish the immunogenic effect of various IIVs from that of placebo. However, these standard approaches are not suitable to determine the role of various vaccine components in immune response maturation. The expanded methodological base including an enzyme-linked immunosorbent assay (ELISA) and a neuraminidase ELISA (NA-ELISA) helped us to get wider characteristics and identify the effectiveness of various commercial vaccines depending on the antigen content. Investigations conducted showed that among the IIVs tested, Ultrix®, Ultrix® Quadri and VAXIGRIP® elicit the most balanced immune response, including a good NA response. For Ultrix®, Ultrix® Quadri, and SOVIGRIPP® (FORT LLC), the whole-virus specific antibody subclass IgG1, measured in ELISA, seriously prevailed over IgG2a, while, for VAXIGRIP® and SOVIGRIPP® (NPO Microgen JSC) preparations, the calculated IgG1/IgG2a ratio was close to 1. So, the immune response varied drastically across different commercial IIVs injected in mice.

Flagellin-fused protein targeting M2e and HA2 induces potent humoral and T-cell responses and protects mice against various influenza viruses a subtypes.

BACKGROUND: Current influenza vaccines are mainly strain-specific and have limited efficacy in preventing new, potentially pandemic, influenza strains. Efficient control of influenza A infection can potentially be achieved through the development of broad-spectrum vaccines based on conserved antigens. A current trend in the design of universal flu vaccines is the construction of recombinant proteins based on combinations of various conserved epitopes of viral proteins (M1, M2, HA2, NP). In this study, we compared the immunogenicity and protective action of two recombinant proteins which feature different designs and which target different antigens. RESULTS: Balb/c mice were immunized subcutaneously with Flg-HA2-2-4M2ehs or FlgSh-HA2-2-4M2ehs; these constructs differ in the location of hemagglutinin's HA2-2(76-130) insertion into flagellin (FliC). The humoral and T-cell immune responses to these constructs were evaluated. The simultaneous expression of different M2e and HA2-2(76-130) in recombinant protein form induces a strong M2e-specific IgG response and CD4+/ CD8+ T-cell response. The insertion of HA2-2(76-130) into the hypervariable domain of flagellin greatly increases antigen-specific T-cell response, as evidenced by the formation of multi-cytokine-secreting CD4+, CD8+ T-cells, Tem, and Tcm. Both proteins provide full protection from lethal challenge with A/H3N2 and A/H7N9. CONCLUSION: Our results show that highly conserved M2e and HA2-2(76-130) can be used as important targets for the development of universal flu vaccines. The location of the HA2-2(76-130) peptide's insertion into the hypervariable domain of flagellin had a significant effect on the T-cell response to influenza antigens, as seen by forming of multi-cytokine-secreting CD4+ and CD8+ T-cells.

The measurement of cutaneous blood flow in healthy volunteers subjected to physical exercise with ultrasound Doppler imaging and laser Doppler flowmetry.

We aimed to evaluate hemodynamic parameters in the cutaneous microcirculation during recovery to graded dynamic exercise performed on a cycloergometer by using two distinctive methods: high-frequency ultrasound Doppler flowmetry (UDF) and laser Doppler fluxmetry (LDF). Regarding UDF, the medium linear blood flow velocity (Vam) was estimated, expressed in absolute units (cm/s) whereas blood flow assessed by LDF was registered in arbitrary perfusion units (PU). We compared the corresponding perfusion values obtained by UDF and LDF, respectively, every 20 s during recovery period to physical exercise. Three types of microvascular blood flow dynamics were recorded: blood flow with an occasional paroxysm, a pulsatile blood flow pattern, and a shunting-type blood flow pattern. In the first type, the reaction time of registration and the direction of changes of UDF and LDF values coincided in 77.7%, exhibiting high correlation (r = 0.77; p≤0.001). In the second type, the direction of changes of UDF and LDF showed 85.0% similarity and a moderate correlation (r = 0.66; p = 0.0015), whereas in the third type, there was also a moderate correlation (r = 0.53, p = 0.0024). Our pilot experiments have shown that UDF could be regarded as a comparable substitutional method to LDF for studying skin microcirculation.

Development of a candidate influenza vaccine based on virus-like particles displaying influenza M2e peptide into the immunodominant loop region of hepatitis B core antigen: Insertion of multiple copies of M2e increases immunogenicity and protective efficiency.

The extracellular domain of the transmembrane protein M2 (M2e) of influenza A virus is a promising target for the development of "universal" vaccines against influenza. M2e is a poor immunogen by itself; however, when M2e is linked to an appropriate carrier, such as hepatitis B virus core (HBc) particles, it becomes highly immunogenic. Insertions of target peptides into the surface-exposed major immunodominant loop region (MIR) of the HBc antigen are especially immunogenic, but such insertions often affect the protein folding and formation of recombinant virus-like particles. To facilitate an appropriate conformation of the M2e insert, we introduced flexible linkers at the junction points between the insert and flanking HBc sequences. This approach allowed the construction of recombinant HBc particles carrying 1, 2 and 4 copies of M2e in the MIR region. These particles were produced in Escherichia coli and purified to homogeneity. The immune response and protective activity of hybrid HBc particles in mice correlated with the number of inserted M2e peptides: the highest immunogenicity and complete protection of mice against the lethal challenge by influenza virus was observed with particles carrying four copies of M2e. The possibility of the simultaneous presentation of M2e peptides from several important influenza strains on a single HBc particle could also facilitate the development of a broad-specificity vaccine efficient not only against influenza A strains of human origin but also for newly emerging strains of animal origin, such as the avian influenza.

Protection against multiple influenza A virus strains induced by candidate recombinant vaccine based on heterologous M2e peptides linked to flagellin.

Matrix 2 protein ectodomain (M2e) is considered a promising candidate for a broadly protective influenza vaccine. M2e-based vaccines against human influenza A provide only partial protection against avian influenza viruses because of differences in the M2e sequences. In this work, we evaluated the possibility of obtaining equal protection and immune response by using recombinant protein on the basis of flagellin as a carrier of the M2e peptides of human and avian influenza A viruses. Recombinant protein was generated by the fusion of two tandem copies of consensus M2e sequence from human influenza A and two copies of M2e from avian A/H5N1 viruses to flagellin (Flg-2M2eh2M2ek). Intranasal immunisation of Balb/c mice with recombinant protein significantly elicited anti-M2e IgG in serum, IgG and sIgA in BAL. Antibodies induced by the fusion protein Flg-2M2eh2M2ek bound efficiently to synthetic peptides corresponding to the human consensus M2e sequence as well as to the M2e sequence of A/Chicken/Kurgan/05/05 RG (H5N1) and recognised native M2e epitopes exposed on the surface of the MDCK cells infected with A/PR/8/34 (H1N1) and A/Chicken/Kurgan/05/05 RG (H5N1) to an equal degree. Immunisation led to both anti-M2e IgG1 and IgG2a response with IgG1 prevalence. We observed a significant intracellular production of IL-4, but not IFN-γ, by CD4+ T-cells in spleen of mice following immunisation with Flg-2M2eh2M2ek. Immunisation with the Flg-2M2eh2M2ek fusion protein provided similar protection from lethal challenge with human influenza A viruses (H1N1, H3N2) and avian influenza virus (H5N1). Immunised mice experienced significantly less weight loss and decreased lung viral titres compared to control mice. The data obtained show the potential for the development of an M2e-flagellin candidate influenza vaccine with broad spectrum protection against influenza A viruses of various origins.