Peptide Bbeta(15-42) 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

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

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

Molecular genetic analysis of the strain Leningrad-16 used for the production of measles vaccine

Aim. To study the genetic stability of the measles virus strain Leningrad-16 (L-16) used for the production of vaccine at JSC NPO Mikrogen.Materials and methods. A series of production and sowing strains of L-16 (JSC NPO Mikrogen), ready-made series of measles vaccines from various manufacturers, and the strain of measles virus genotype D6 were studied. Molecular genetic study of the strains was performed using RT-PCR followed by restriction analysis and sequencing.Results. The complete genome sequences of the production and sowing strains of L-16 that are used for vaccine production were obtained. The sequence of the vaccine strain was deposited in GenBank. Strain L-16 was confirmed to be genetically stable. The obtained data demonstrated the possibility of using the RT-PCR method with subsequent restriction analysis to confirm the authenticity of the vaccine strain L-16 in finished mono and three component vaccines.Conclusion. The results of the study suggest the applicability of the molecular genetic methods to confirm the authenticity of the studied strains not only at the production stages, but also in the finished series of vaccines

Bacterial Ghosts as an Oral Vaccine: a Single Dose of Escherichia coli O157:H7 Bacterial Ghosts Protects Mice against Lethal Challenge

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

Disease parameters in C57BL/6 mice infected with an adapted strain of DENV-3.

<p>(A) WT mice (<i>n</i> = 6 mice per group) were inoculated with different inoculums of adapted-DENV-3 (i.p) and lethality was evaluated every 12 hours for 14 days. Results are expressed as % of survival. In Figs (B–L) WT mice (n = 6 per group) were inoculated with 10LD₅₀ (1000 PFU) of DENV-3 (i.p) and in the third, fifth or in the seventh day of infection mice were culled and blood and tissues were collected for the following analysis: (B) Change in body weight was expressed as percentage of initial weight loss. (C) Mechanical hypernociception was assessed daily. Results are shown as the difference between the force (g) necessary to induce dorsal flexion of tibio-tarsal joint, followed by paw withdraw, before and after DENV-3 inoculation. In (D), hematocrit was expressed as % volume occupied by red blood cells (left panel) and the number of platelets was shown as platelets ×10³/µl of blood (right panel). (E) Changes in vascular permeability in the liver and lungs are shown as µg Evans blue per 100 mg of tissue (left and right panels, respectively). (F) Shows changes in Systolic blood pressure from baseline until day 7 after infection expressed as Δ of blood pressure in mmHg. (G) AST (left panel) and ALT (right panel) activity determination in plasma of control and DENV-3-infected mice was shown as U/dL of plasma. (H–L) Concentrations of IL-6, TNF-α, IFN-γ IL-12/23p40 and IL-18, quantified by ELISA. Results are shown as pg per mL (serum) or pg per 100 mg (tissue). All results are expressed as mean ± SEM and are representative of at least two experiments. * for P<0.05 when compared to control uninfected mice. 10 LD₅₀ corresponds to 1000 PFU of adapted-DENV-3. ND – not detectable. NA – not assessed. NI- Not-infected. dpi – days post-infection.</p

IFN-γ production is required for host resistance to adapted-DENV-3 primary infection.

<p>(A) WT mice (<i>n</i> = 4 mice per group) were inoculated with 10LD₅₀ (1000 PFU) of DENV-3 (i.p) and seven days later, mice were culled, and splenic cells isolated for assaying IFN-γ production by cellular staining with labeled antibodies and FACS analysis. Results are expressed as % of IFN-γ-positive cells in each population. (B) WT and IFN-γ^−/− mice (n = 8 per group) were inoculated with 1LD₅₀ (100 PFU) of DENV-3 (i.p) and lethality was evaluated every 12 hours during 14 days. Results are expressed as % of survival. In (C–J), WT and IFN-γ^−/− mice (n = 6 per group) were inoculated with 1LD₅₀ (100 PFU) of DENV-3 (i.p) and in the fifth day of infection mice were culled and blood and tissues were collected for the following analysis: (C–D) Viral loads were recovered from the blood (C), spleen and liver (D, left and right panels), respectively. Results are shown as the log of PFU per mL of blood or per g of tissue. (E) Serial sections from each liver were stained with anti-DV NS3 antibody E1D8 (NS3) or an isotype control mouse IgG2a (IgG2a data not shown), and multiple sections of each tissue type were thoroughly examined for staining. Positive staining for NS3 is brown while hematoxylin counterstain is blue. Results are expressed as number of NS3-positive hepatocytes. (F) Mechanical hypernociception was assessed daily. Results are shown as the difference between the force (g) necessary to induce dorsal flexion of tibio-tarsal joint, followed by paw withdraw, before and after DENV-3 inoculation. In (G), hematocrit was shown as % volume occupied by red blood cells (left panel) and the number of platelets was shown as platelets ×10³/µl of blood (right panel). (H) Changes in Systolic blood pressure from baseline until day 5 after infection expressed as Δ of blood pressure in mmHg. In (I), AST activity determination in plasma, shown as U/dL of plasma. (J) shows semi-quantitative analysis of hepatic damage (histopathological analysis performed as modified from Paes et al, 2009) and Hematoxylin & Eosin staining of liver sections of control and WT and IFN-γ^−/− DENV-3-infected mice, five days after infection. Scale bars - 400 µm. The images presented are representative of an animal on the fifth day of infection. All results are expressed as mean ± SEM (except for C–D, expressed as median) and are representative of at least two experiments. * for P<0.05 when compared to control uninfected mice. # fo P,0.05 when compared to WT infected mice. 10 LD₅₀ corresponds to 1000 PFU of adapted-DENV-3. 1LD₅₀ corresponds to 100 PFU of adapted-DENV-3. ND – not detectable. NI- Not-infected. dpi – days post-infection. HS – hepatocyte swelling. N – necrosis. D – degeneration. H – hemorrhage. OS – Overall Score.</p

Characterization of virologic and histopathological parameters in C57BL/6j mice upon adapted-DENV-3 infection.

<p>(A–D) C57BL/6j mice (n = 6 per group) were inoculated with 10LD₅₀ (1000 PFU) of DENV-3 (i.p) and in the third, fifth or in the seventh day of infection, mice were culled and blood and tissues were collected for the following analysis: (A–C) Viral loads were recovered from the spleen, liver and blood, respectively. Results are shown as the log of PFU per g of tissue or per mL of blood. (D) Shows virus NS1 antigen serum levels by ELISA and expressed as O.D. (E–F) C57BL/6j mice (n = 6 per group) were inoculated with 10LD₅₀ (1000 PFU) of DENV-3 (i.p) and in the seventh day of infection mice were culled and liver collected for the following analyses: (E) Liver was collected, formalin-fixed and processed into paraffin sections. Serial sections from each tissue were stained with anti-DV NS3 antibody E1D8 (NS3) or an isotype control mouse IgG2a, and multiple sections of each tissue type were thoroughly examined for staining. Positive staining for NS3 is brown while hematoxylin counterstain is blue. (F) shows semi-quantitative analysis of hepatic damage and Hematoxylin & Eosin staining of liver sections of control and DENV-3-infected mice, seven days after infection (Scale Bar - 400 µm). The images presented are representative of an animal on the seventh day of infection. In (G) Viral inoculum (10LD₅₀ or 1000 PFU) was heat inactivated (Heat, 56°C, 60 min) or treated with UV light (UV, 15 min) before inoculation in C57BL/6j mice. Lethality was evaluated every 12 hours for 14 days. (H) WT mice (<i>n</i> = 6 mice per group) were pretreated i.p with 100 µL of Anti-DENV-3 antiserum or control serum (pre-immune serum) before inoculation of 10LD₅₀ (1000 PFU) of adapted-DENV-3 (i.p). Lethality was evaluated every 12 hours for 14 days. Results are expressed as % of survival. Results are expressed as mean ± SEM (except for A–C, expressed as median) and are representative of at least two experiments. * for P<0.05 when compared to control uninfected mice. 10 LD₅₀ corresponds to 1000 PFU of adapted-DENV-3. ND- not detected. NI- not-infected. dpi- days post-infection. NC – Negative control. HS – hepatocyte swelling. N – necrosis. D – degeneration. H – hemorrhage. OS – Overall Score.</p