Ruta Bruvere

Fluorescent Characteristics of Blood Leukocytes of Patients with Malignant and Nonmalignant Diseases

Properties of the naphthalic acid derivative 3-isopropyloxy-6-morpholino-2-phenilphenalen-1-ona (PMPH) as a potential fluorescent probe were investigated. Spectral characteristics of the compound in different solvents as well as their binding to model lipid membranes (liposomes) and human peripheral blood mononuclear cells (lymphocytes) were determined. The fluorescence was found to be sensitive to polarity changes in the environment. Distinctions were observed in the spectral characteristics of the investigated compound when bound to liposomes and peripheral blood mononuclear cells of healthy donors, and patients with malignant and nonmalignant diseases. It was established that spectral characteristics of PMPH in cell suspension qualitatively characterize its structural and functional alterations during pathologic phenomena and correlate directly with the clinical view of disease. PMPH is shown to be a prospective method for screening for various pathologies.

Recombinant Semliki Forest virus vectors encoding hepatitis B virus small surface and pre-S1 antigens induce broadly reactive neutralizing antibodies

Summary.  Most hepatitis B virus (HBV) vaccines consist of viral small surface (S) protein subtype adw2 expressed in yeast cells. In spite of good efficacy, HBV‐genotype and subtype differences, escape mutants and insufficient Th1 activation remain potential problems. To address these problems, we generated recombinant Semliki Forest virus (rSFV) vectors encoding S protein, subtype adw2 or ayw2, or a fragment of the large surface protein, amino acids 1–48 of the pre‐S1 domain, fused to S (pre‐S1.1–48/S). The antigen loop in S protein and the selected pre‐S1 sequences are known targets of neutralizing antibodies. BALB/c mice were immunized intravenously with 107 rSFV particles and 108 rSFV particles 3 weeks later. Antibodies induced by rSFV encoding S proteins reacted preferentially with subtype determinants of yeast‐derived S antigen but equally well with patient‐derived S antigen. Immunization with rSFV encoding pre‐S1.1–48/S resulted in formation of pre‐S1‐ and S‐specific immunoglobulin G (IgG), while immunization with the isogenic mutant without S start codon induced pre‐S1 antibodies only. Neutralizing antibodies were determined by mixing with plasma‐derived HBV/ayw2 and subsequent inoculation of susceptible primary hepatocyte cultures from Tupaia belangeri. S/adw2 antisera neutralized HBV/ayw2 as effectively as antisera raised with S/ayw2. The pre‐S1 antibodies also completely neutralized HBV infectivity. The IgG1/IgG2a ratios ranged from 0.28 to 0.88 in the four immunized groups and were lowest for the pre‐S1.1–48/S vector, indicating the strongest Th1 response. This vector type may induce subtype‐independent and S‐escape‐resistant neutralizing antibodies against HBV.

Structural Changes in Lymphocytes Membrane of Chernobyl Clean-up Workers from Latvia

ABM (3-aminobenzanthrrone derivative) developed at the Riga Technical University, Riga, Latvia) has been previously shown as a potential probe for determination of the immune state of patients with different pathologies .The fist study (using probe ABM) of peripheral blood mononuclear cells (PBMC) membranes of 97 Chernobyl clean-up workers from Latvia was conducted in 1997. Now we repeatedly examine the same (n = 54) individuals in dynamics. ABM spectral parameters in PBMC suspension, fluorescence anisotropy and blood plasma albumin characteristics were recorded. In 1997 screening showed 5 different patterns of fluorescence spectra, from which in 2007 we obtained only two. These patterns of spectra had never been previously seen in healthy individuals or patients with tuberculosis, multiple sclerosis, rheumatoid arthritis, etc., examined by us. Patterns of ABM fluorescence spectra are associated with membrane anisotropy and conformational changes of blood plasma albumin. We observed that in dynamics 1997–2007 the lipid compartment of the membrane became more fluid while the lipid-protein interface became more rigid. The use of probe ANS and albumin auto-fluorescence allowed show conformational alterations in Chernobyl clean-up workers blood plasma. It is necessary to note that all investigated parameters significantly differ in observed groups of patients. These findings reinforce our understanding that that the cell membrane is a significant biological target of radiation. The role of the membrane in the expression and course of cell damage after radiation exposure must be considered. So ten years dynamic of PBMC membrane characteristics by ABM (spectral shift and anisotropy indexes) in Chernobyl clean-up workers reveal progressive trend toward certain resemblance with those of chronic B-cell lymphoid leukemia.

Long-term effects of low doses of ionising radiation on Chernobyl clean-up workers from Latvia

Around 6000 inhabitants (20-49 years old in 1986) of Latvia took part in clean-up work in Chernobyl from 1986 till 1991. Most of them were officially documented as recipients of ionising radiation exposure (1-50 cGy). ABM (a 3-aminobenzanthrone derivative developed at the Riga Technical University, Riga, Latvia) has been previously shown to be a potential probe for determining the immune state of patients with different pathologies. The first study (using ABM) of Peripheral Blood Mononuclear Cell (PBMC) membranes of Chernobyl clean-up workers (n-97) from Latvia was conducted in 1996-1997. In 2006-2007 we examined the same (n = 54) individuals. Lipid peroxidation, ABM and ANS spectral parameters in PBMC suspension, fluorescence anisotropy and blood plasma albumin characteristics were recorded. In 1997 screening showed five different patterns of fluorescence spectra, but in 2007 we obtained only four. These patterns of spectra had never been seen previously in healthy individuals or patients with tuberculosis, multiple sclerosis, rheumatoid arthritis, etc., examined by us. The patterns of ABM fluorescence spectra were associated with membrane anisotropy and conformational changes of blood plasma albumin. It is necessary to note that all investigated parameters significantly differed in the observed groups of patients. These findings reinforce our understanding that the cell membrane is a significant biological target of radiation. These studies reveals a progressive trend towards certain resemblances to PBMC membranes of chronic B-cell lymphoid leukaemia and protein coformational alterations.

Posttranslational modifications and secretion efficiency of immunogenic hepatitis B virus L protein deletion variants

BackgroundSubviral particles of hepatitis B virus (HBV) composed of L protein deletion variants with the 48 N-terminal amino acids of preS joined to the N-terminus of S protein (1-48preS/S) induced broadly neutralizing antibodies after immunization of mice with a Semliki Forest virus vector. A practical limitation for use as vaccine is the suboptimal secretion of such particles. The role of the N-terminal preS myristoylation in the cellular retention of full-length L protein is described controversially in the literature and the relation of these data to the truncated L protein was unknown. Thus, we studied the effect of preS myristoylation signal suppression on 1-48preS/S secretion efficiency, glycosylation and subcellular distribution.FindingsThe findings are that 1-48preS/S is secreted, and that removal of the N-terminal myristoylation signal in its G2A variant reduced secretion slightly, but significantly. The glycosylation pattern of 1-48preS/S was not affected by the removal of the myristoylation signal (G2A mutant) but was different than natural L protein, whereby N4 of the preS and N3 of the S domain were ectopically glycosylated. This suggested cotranslational translocation of 1-48preS in contrast to natural L protein. The 1-48preS/S bearing a myristoylation signal was localized in a compact, perinuclear pattern with strong colocalization of preS and S epitopes, while the non-myristoylated mutants demonstrated a dispersed, granular cytoplasmic distribution with weaker colocalization.ConclusionsThe large deletion in 1-48preS/S in presence of the myristoylation site facilitated formation and secretion of protein particles with neutralizing preS1 epitopes at their surface and could be a useful feature for future hepatitis B vaccines.

Fluorescent Probe ABM and Estimation of Immune State in Patients with Different Pathologies (Review Article)

The fluorescent probe ABM (3-aminobenzanthrone derivative) one of the fluorescent probes synthesized in Riga Technical University proved to be an excellent, independent model for studying cell membranes. In our work we have investigated the possibility of using the fluorescent probe ABM for detection of immune state in patients with different pathologies. There is a strong correlation among all studied ABM spectral parameters, immunological characteristics, clinical and laboratory investigations of the all observed patients groups. The obtained results suggest that ABM spectral parameters in cell suspension reflect the alterations of the cellular mechanisms of immunity. Therefore fluorescent method could be used as preliminary screening test in immune diagnostics instead of more expensive, time consuming methods (subset detection, radioisotope method etc.) used as routine in clinics. Spectral parameters of ABM reflect a wide range of interrelated (interdependent) characteristics of cells (physico-chemical state and microviscosity of membrane, proliferating and lipid metabolic activity of cells, distribution of cells among subsets). The observed change of the studied parameters reflects alterations of the cellular mechanisms of immunity which is a main focus for its application as preliminary screening test in immune diagnostics. The fluorescence based method is sensitive, less expensive and time consuming, technically simple and convenient.

Refractive properties of separate erythrocytes of Chernobyl clean-up workers at different pH

This study is focused on the modifications in erythrocytes of Chernobyl nuclear power plant accident clean-up workers as a late health effect of short-term impact of high level radioactive contamination. As a result, a new method based on erythrocyte refractive index properties at different pH has been elaborated.