Ex vivo cytokine production in peripheral blood mononuclear cells after their stimulation with dsRNA of natural origin.

Double-stranded RNA (dsRNA) is a pathogen-associated molecular pattern, known for its ability to induce antiviral response and enhance communication between cells mediating innate and adaptive immune responses. The aim of this study was to characterize the effect of the dsRNA-containing product Larifan on the production of a wide spectrum of cytokines and chemokines in ex vivo cultivated peripheral blood mononuclear cells. Concentrations of 29 different cytokines were detected by a Luminex® 200™ System using three Milliplex MAP Multiplex Assay Kits. Larifan caused strong induction of chemokine macrophage inflammatory protein 1ß, I-309, and TARC, proinflammatory cytokines IL-6, tumor necrosis factor -α, granulocyte macrophage colony-stimulating factor, anti-inflammatory IL-10, and cellular immunity mediating factors IL-23 and interferon-γ. Considerable suppression of IL-16 and chemokine stromal cell-derived factor 1 a+b and interferon gamma-induced protein 10 was also observed. The network of molecules responding to the presence of Larifan revealed the pleiotropic effect this product exerts on immune response.

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

BACKGROUND: Subviral 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. FINDINGS: The 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. CONCLUSIONS: The 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.

Fibronectin-binding nanoparticles for intracellular targeting addressed by B. burgdorferi BBK32 protein fragments.

Virus-like particles (VLPs) are created by the self-assembly of multiple copies of envelope and/or capsid proteins from many viruses, mimicking the conformation of a native virus. Such noninfectious nanostructures are mainly used as antigen-presenting platforms, especially in vaccine research; however, some of them recently were used as scaffolds in biotechnology to produce targeted nanoparticles for intracellular delivery. This study demonstrates the creation of fusion VLPs using hepatitis B core protein-based system maintaining a fibronectin-binding property from B. burgdorferi BBK32 protein, including the evidence of particles' transmission to BHK-21 target cells via caveolae/rafts endocythosis. These results make this construct to be an attractive model in development of HBc-based nanoparticles for cellular targeting applications and highlights the fragment of B. burgdorferi BBK32 as a novel cellular uptake-promoting peptide. FROM THE CLINICAL EDITOR: This paper discusses the nanotechnology-based application of self-assembling viral-like peptides (VLP-s) for targeted delivery using a hepatitis B core protein based system. Creating fusion VLPs may be an attractive model for cellular targeting applications.

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.

Enhancement of the expression of HCV core gene does not enhance core-specific immune response in DNA immunization: advantages of the heterologous DNA prime, protein boost immunization regimen.

BACKGROUND: Hepatitis C core protein is an attractive target for HCV vaccine aimed to exterminate HCV infected cells. However, although highly immunogenic in natural infection, core appears to have low immunogenicity in experimental settings. We aimed to design an HCV vaccine prototype based on core, and devise immunization regimens that would lead to potent anti-core immune responses which circumvent the immunogenicity limitations earlier observed. METHODS: Plasmids encoding core with no translation initiation signal (pCMVcore); with Kozak sequence (pCMVcoreKozak); and with HCV IRES (pCMVcoreIRES) were designed and expressed in a variety of eukaryotic cells. Polyproteins corresponding to HCV 1b amino acids (aa) 1-98 and 1-173 were expressed in E. coli. C57BL/6 mice were immunized with four 25-microg doses of pCMVcoreKozak, or pCMV (I). BALB/c mice were immunized with 100 microg of either pCMVcore, or pCMVcoreKozak, or pCMVcoreIRES, or empty pCMV (II). Lastly, BALB/c mice were immunized with 20 microg of core aa 1-98 in prime and boost, or with 100 microg of pCMVcoreKozak in prime and 20 microg of core aa 1-98 in boost (III). Antibody response, [3H]-T-incorporation, and cytokine secretion by core/core peptide-stimulated splenocytes were assessed after each immunization. RESULTS: Plasmids differed in core-expression capacity: mouse fibroblasts transfected with pCMVcore, pCMVcoreIRES and pCMVcoreKozak expressed 0.22 +/- 0.18, 0.83 +/- 0.5, and 13 +/- 5 ng core per cell, respectively. Single immunization with highly expressing pCMVcoreKozak induced specific IFN-gamma and IL-2, and weak antibody response. Single immunization with plasmids directing low levels of core expression induced similar levels of cytokines, strong T-cell proliferation (pCMVcoreIRES), and antibodies in titer 103(pCMVcore). Boosting with pCMVcoreKozak induced low antibody response, core-specific T-cell proliferation and IFN-gamma secretion that subsided after the 3rd plasmid injection. The latter also led to a decrease in specific IL-2 secretion. The best was the heterologous pCMVcoreKozak prime/protein boost regiment that generated mixed Th1/Th2-cellular response with core-specific antibodies in titer >or= 3 x 10(3). CONCLUSION: Thus, administration of highly expressed HCV core gene, as one large dose or repeated injections of smaller doses, may suppress core-specific immune response. Instead, the latter is induced by a heterologous DNA prime/protein boost regiment that circumvents the negative effects of intracellular core expression.

The Immunogenicity in Mice of HCV Core Delivered as DNA Is Modulated by Its Capacity to Induce Oxidative Stress and Oxidative Stress Response.

HCV core is an attractive HCV vaccine target, however, clinical or preclinical trials of core-based vaccines showed little success. We aimed to delineate what restricts its immunogenicity and improve immunogenic performance in mice. We designed plasmids encoding full-length HCV 1b core and its variants truncated after amino acids (aa) 60, 98, 152, 173, or up to aa 36 using virus-derived or synthetic polynucleotides (core191/60/98/152/173/36_191v or core152s DNA, respectively). We assessed their level of expression, route of degradation, ability to trigger the production of reactive oxygen species/ROS, and to activate the components of the Nrf2/ARE antioxidant defense pathway heme oxygenase 1/HO-1 and NAD(P)H: quinone oxidoreductase/Nqo-1. All core variants with the intact N-terminus induced production of ROS, and up-regulated expression of HO-1 and Nqo-1. The capacity of core variants to induce ROS and up-regulate HO-1 and Nqo-1 expression predetermined their immunogenicity in DNA-immunized BALB/c and C57BL/6 mice. The most immunogenic was core 152s, expressed at a modest level and inducing moderate oxidative stress and oxidative stress response. Thus, immunogenicity of HCV core is shaped by its ability to induce ROS and oxidative stress response. These considerations are important in understanding the mechanisms of viral suppression of cellular immune response and in HCV vaccine design.

Proteasomal degradation of core protein variants from chronic hepatitis B patients.

The accumulation of complex hepatitis B virus (HBV) variants with internal in-frame deletions in the C gene in immunosuppressed renal transplant recipients is associated with a severe course of the infection leading to end-stage liver disease (ESLD). A set of six HBV C genes with internal in-frame deletions corresponding to the pattern of HBV population in immunosuppressed patients has been expressed in two different eukaryotic cell lines. Synthesis and proteasomal degradation of HBV core (HBc) protein variants were compared with those of the wild-type HBc. In all cases, the steady-state level of internally deleted HBc proteins, predominantly with longer deletions, were considerably lower and turnover was significantly higher in comparison with those of the wild-type HBc, since all deletion variants were degraded rapidly via the proteasome pathway. Involvement and consequences of the proteasomal degradation machinery in the HBc protein turnover during HBV infection with complex HBV variants in the immunosuppressed patients are discussed.

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.

Fluorescent characteristics of rheumatoid arthritis patients blood lymphocytes.

A fluorescent probe, ABM, aminoderivative of benzanthrone, synthesized in the Department of Organic Chemistry of the Riga Technical University (Latvia), has been successfully used to characterize changes in the structural and functional properties of cell membranes during different pathologies. In the present study the physicochemical properties and the functional activity of the peripheral blood mononuclear cells (lymphocytes-Ly) in patients with rheumatoid arthritis (RA) were studied using the ABM probe. Intensity of the ABM fluorescence in the celi suspension, functional activity of the ly anisotropy of the membranes differ patients with different titres of rheumatoid factor in blood. Patients with seropositive RA had decreased proliferative activity and lower number of iy in blood plasma indicating greater alterations of the immunoregulating processes in these patients as compared to patients with seronegative RA. In the latter the Ly deficiency is compensated to some extent by increased proliferation activity of these cells. The ABM fluorescence intensity correlated not only with membrane anisotropy (r = 0.97, but also with the proliferation activity of the Ly (r = 0.98). The above parameters correlated with the clinical manifestations of the disease. The results indicate that the fluorescent probe ABM is useful for screening the physicochemical status of Ly membranes and the proliferation activity of these cells in RA patients.

Translation of hepatitis B virus (HBV) surface proteins from the HBV pregenome and precore RNAs in Semliki Forest virus-driven expression.

Hepatitis B virus (HBV) pregenome RNA (pgRNA) serves as a translation template for the HBV core (HBc) protein and viral polymerase (Pol). HBV precore RNA (pcRNA) directs the synthesis of the precore (preC) protein, a precursor of the hepatitis B e antigen (HBeAg). pgRNA and pcRNA were expressed in the Semliki Forest virus (SFV) expression system. Besides the HBc and preC proteins, there was revealed the synthesis of all three forms of HBV surface (HBs) proteins: long (LHBs), middle (MHBs) and short (SHBs), the start codons of which are located more than 1000 nt downstream of the HBc and preC start codons. Moreover, other HBV templates, such as 3'-truncated pgRNA lacking 3' direct repeat and Pol mRNA, both carrying internally the HBs sequences, provided the synthesis of three HBs protein forms in the SFV-driven expression system. Maximal production of the HBs was provided by Pol mRNA, while HBc- and preC-producing templates showed relatively low internal translation of the HBs. These data allow the proposal of a ribosome leaky scanning model of internal translation initiation for HBs proteins. The putative functional role of such exceptional synthesis of the HBs proteins from the pgRNA and pcRNA templates in the natural HBV infection process needs further evaluation.