Alena Atrasheuskaya

Essential role of platelet-activating factor receptor in the pathogenesis of Dengue virus infection

Severe dengue infection in humans causes a disease characterized by thrombocytopenia, increased levels of cytokines, increased vascular permeability, hemorrhage, and shock. Treatment is supportive. Activation of platelet-activating factor (PAF) receptor (PAFR) on endothelial cells and leukocytes induces increase in vascular permeability, hypotension, and production of cytokines. We hypothesized that activation of PAFR could account for the major systemic manifestations of dengue infection. Inoculation of adult mice with an adapted strain of Dengue virus caused a systemic disease, with several features of the infection in humans. In PAFR−/− mice, there was decreased thrombocytopenia, hemoconcentration, decreased systemic levels of cytokines, and delay of lethality, when compared with WT infected mice. Treatment with UK-74,505, an orally active PAFR antagonist, prevented the above-mentioned manifestations, as well as hypotension and increased vascular permeability, and decreased lethality, even when started 5 days after virus inoculation. Similar results were obtained with a distinct PAFR antagonist, PCA-4246. Despite decreased disease manifestation, viral loads were similar (PAFR−/−) or lower (PAFR antagonist) than in WT mice. Thus, activation of PAFR plays a major role in the pathogenesis of experimental dengue infection, and its blockade prevents more severe disease manifestation after infection with no increase in systemic viral titers, suggesting that there is no interference in the ability of the murine host to deal with the infection. PAFR antagonists are disease-modifying agents in experimental dengue infection.

Bacterial ghosts as an oral vaccine: a single dose of Escherichia coli O157:H7 bacterial ghosts protects mice against lethal challenge.

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) is a bacterial pathogen that is associated with several life-threatening diseases for humans. The combination of protein E-mediated cell lysis to produce EHEC ghosts and staphylococcal nuclease A to degrade DNA was used for the development of an oral EHEC vaccine. The lack of genetic material in the oral EHEC bacterial-ghost vaccine abolished any hazard of horizontal gene transfer of resistance genes or pathogenic islands to resident gut flora. Intragastric immunization of mice with EHEC ghosts without the addition of any adjuvant induced cellular and humoral immunity. Immunized mice challenged at day 55 showed 86% protection against lethal challenge with a heterologous EHEC strain after single-dose oral immunization and 93.3% protection after one booster at day 28, whereas the controls showed 26.7% and 30% survival, respectively. These results indicate that it is possible to develop an efficacious single-dose oral EHEC bacterial-ghost vaccine.

Peptide Bβ15-42 Preserves Endothelial Barrier Function in Shock

Loss of vascular barrier function causes leak of fluid and proteins into tissues, extensive leak leads to shock and death. Barriers are largely formed by endothelial cell-cell contacts built up by VE-cadherin and are under the control of RhoGTPases. Here we show that a natural plasmin digest product of fibrin, peptide Bß15-42 (also called FX06), significantly reduces vascular leak and mortality in animal models for Dengue shock syndrome. The ability of Bß15-42 to preserve endothelial barriers is confirmed in rats i.v.-injected with LPS. In endothelial cells, Bß15-42 prevents thrombin-induced stress fiber formation, myosin light chain phosphorylation and RhoA activation. The molecular key for the protective effect of Bß15-42 is the src kinase Fyn, which associates with VE-cadherin-containing junctions. Following exposure to Bß15-42 Fyn dissociates from VE-cadherin and associates with p190RhoGAP, a known antagonists of RhoA activation. The role of Fyn in transducing effects of Bß15-42 is confirmed in Fyn−/− mice, where the peptide is unable to reduce LPS-induced lung edema, whereas in wild type littermates the peptide significantly reduces leak. Our results demonstrate a novel function for Bß15-42. Formerly mainly considered as a degradation product occurring after fibrin inactivation, it has now to be considered as a signaling molecule. It stabilizes endothelial barriers and thus could be an attractive adjuvant in the treatment of shock.

IFN-γ Production Depends on IL-12 and IL-18 Combined Action and Mediates Host Resistance to Dengue Virus Infection in a Nitric Oxide-Dependent Manner

Dengue is a mosquito-borne disease caused by one of four serotypes of Dengue virus (DENV-1–4). Severe dengue infection in humans is characterized by thrombocytopenia, increased vascular permeability, hemorrhage and shock. However, there is little information about host response to DENV infection. Here, mechanisms accounting for IFN-γ production and effector function during dengue disease were investigated in a murine model of DENV-2 infection. IFN-γ expression was greatly increased after infection of mice and its production was preceded by increase in IL-12 and IL-18 levels. In IFN-γ−/− mice, DENV-2-associated lethality, viral loads, thrombocytopenia, hemoconcentration, and liver injury were enhanced, when compared with wild type-infected mice. IL-12p40−/− and IL-18−/− infected-mice showed decreased IFN-γ production, which was accompanied by increased disease severity, higher viral loads and enhanced lethality. Blockade of IL-18 in infected IL-12p40−/− mice resulted in complete inhibition of IFN-γ production, greater DENV-2 replication, and enhanced disease manifestation, resembling the response seen in DENV-2-infected IFN-γ−/− mice. Reduced IFN-γ production was associated with diminished Nitric Oxide-synthase 2 (NOS2) expression and NOS2−/− mice had elevated lethality, more severe disease evolution and increased viral load after DENV-2 infection. Therefore, IL-12/IL-18-induced IFN-γ production and consequent NOS2 induction are of major importance to host resistance against DENV infection.

Changes in immune parameters and their correction in human cases of tick‐borne encephalitis

Tick‐Borne Encephalitis virus (TBEV) causes dangerous central nervous system diseases in humans. General infection leads to the development of meningitis or encephalitis, which is characterized by swelling of the brain due to inflammation. Tetracyclines may act locally to moderate inflammation in the CNS. In this study, we investigated the potential clinical benefits of administering tetracycline hydrochloride to patients hospitalized due to suspected TBEV infection presenting with fever and evidence of a recent tick bite. We also characterized an acute immune response to TBEV by profiling certain cytokines and soluble receptors in Tetracycline‐treated and untreated patients. Increased serum levels of TNF‐α, IL‐1α and IL‐6 were found in all patients at admission.

A model of DENV-3 infection that recapitulates severe disease and highlights the importance of IFN-γ in host resistance to infection.

There are few animal models of dengue infection, especially in immunocompetent mice. Here, we describe alterations found in adult immunocompetent mice inoculated with an adapted Dengue virus (DENV-3) strain. Infection of mice with the adapted DENV-3 caused inoculum-dependent lethality that was preceded by several hematological and biochemical changes and increased virus dissemination, features consistent with severe disease manifestation in humans. IFN-γ expression increased after DENV-3 infection of WT mice and this was preceded by increase in expression of IL-12 and IL-18. In DENV-3-inoculated IFN-γ−/− mice, there was enhanced lethality, which was preceded by severe disease manifestation and virus replication. Lack of IFN-γ production was associated with diminished NO-synthase 2 (NOS2) expression and higher susceptibility of NOS2−/− mice to DENV-3 infection. Therefore, mechanisms of protection to DENV-3 infection rely on IFN-γ-NOS2-NO-dependent control of viral replication and of disease severity, a pathway showed to be relevant for resistance to DENV infection in other experimental and clinical settings. Thus, the model of DENV-3 infection in immunocompetent mice described here represents a significant advance in animal models of severe dengue disease and may provide an important tool to the elucidation of immunopathogenesis of disease and of protective mechanisms associated with infection.

Experimental study on the possibility of treatment of some hemorrhagic fevers

After intracerebral challenge with 100 PFU of Lassa virus (strain Josiah), all infected mice (CBA/calac) died (control group). Production of pro-inflammatory cytokines (IL-1beta, TNF-alpha) significantly increased in the blood of these mice during the infection. For neutralization of increasing concentrations of these cytokines recombinant IL-1RA was used intraperitonealy at a dose 100 microg kg(-1), everyday, within 5 days from the third day after the challenge. Injections of IL-1RA decreased the concentration of IL-1beta and TNF-alpha and resulted in survival of all infected mice (treatment group). Marburg fever (strain Popp) caused in guinea pigs by 5 LD(50) of virus lead to the significant increase of TNF-alpha in the animals blood and caused a lethal outcome (control group). Treatment of infected guinea pigs by IL-1RA or anti-TNF serum decreased the concentration of TNF-alpha and resulted in survival of half of the animals (treatment group). For the treatment recombinant IL-1RA was used at a dose 100 microg kg(-1), intramuscularly, everyday, within 6 days from the third day after the challenge or anti-TNF serum, intramuscularly 0.5 ml (2000 U ml(-1); 1 U of the antiserum neutralises 0.03 ng of TNF-alpha), everyday, within 6 days from the third day after the challenge.

Measles cases in highly vaccinated population of Novosibirsk, Russia, 2000-2005.

While the proportion of measles cases in vaccinees is expected to increase as vaccine coverage increases, such cases must be carefully investigated. The present study was conducted to examine possible contributions to vaccine failures (VFs) and to genetically characterize measles virus (MV) strains circulating in Novosibirsk, Russia during 2000-2005. Totally, 27 adult measles patients admitted to a regional hospital were prospectively enrolled in our study. Genetic characterization of the MV strains revealed circulation of genotypes A, D4 and D6 between 2000 and 2003 years; a genotype D6 MV was associated with the 2005 measles outbreak. Based on IgG avidity testing, half of the vaccinated patients demonstrated evidence of secondary vaccine failure (SVF). Patients, representing both levels of vaccine failure in our study were characterized by the lack of protective titers of neutralizing antibodies against circulating MVs, despite high IgG levels in many cases and high IgG avidity in SVF cases.

Rectal single dose immunization of mice with Escherichia coli O157:H7 bacterial ghosts induces efficient humoral and cellular immune responses and protects against the lethal heterologous challenge

Bacterial ghosts (BGs) have been applied through oral, aerogenic, intraocular or intranasal routes for mucosal immunization using a wide range of experimental animals. All these applications required a booster after primary immunization to achieve protective immunity against the lethal challenge. Here we report for the first time that a single rectal dose of BGs produced from enterohaemorrhagic Escherichia coli (EHEC) O157:H7 fully protects mice against a 50% lethal challenge with a heterologous EHEC strain given at day 55. BGs from EHEC O157:H7 were prepared by a combination of protein E‐mediated cell lysis and expression of staphylococcal nuclease A guaranteeing the complete degradation of pathogen residual DNA. The lack of genetic material in the EHEC BGs vaccine abolished any potential hazard for horizontal gene transfer of plasmid encoded antibiotic resistance genes or pathogenic islands to the recipients gut flora. Single rectal immunization using EHEC O157:H7 BGs without any addition of adjuvant significantly stimulated efficient humoral and cellular immune responses, and was equally protective as two immunizations, which indicates the possibility to develop a novel efficacious single dose mucosal EHEC O157:H7 BGs vaccine using a simplified immunization regimen.

Protective effect of exogenous recombinant mouse interferon-gamma and tumour necrosis factor-alpha on ectromelia virus infection in susceptible BALB/c mice.

The resistance to mousepox is correlated with the production of type I cytokines: interleukin (IL)‐2, IL‐12, interferon (IFN)‐gamma and tumour necrosis factor (TNF)‐alpha. We intend to describe the modulation of generalized ectromelia virus (EV) infection with exogenous administration of mrIFN‐γ and mrTNF‐α separately and in combination using susceptible BALB/c mice. The treatment schemes presented resulted in the localization of the generalized EV infection and its development into non‐fatal sloughing of the infected limb. This was accompanied by low virus titres in the treated mice due to control of systemic virus replication and virus clearance. The balance of type I versus type II cytokines was dominated by a type I response in the treated groups. The group treated with the combination of IFN‐γ and TNF‐α exhibited the best survival with Th1‐dominant (IFN‐γ and IL‐12) cytokine profiles, whereas the TNF‐α‐treated group of mice was less successful in clearance of virus and demonstrated the lowest survival rate. The successful cytokine treatment schemes in this orthopoxvirus model system may have important implications in the treatment of viral diseases in humans and, in particular, of variola virus infection.