Elucidating the Role of Interleukin-17A in West Nile Virus Infection

West Nile virus (WNV) is a neurotropic flavivirus of significant public health importance for which no therapeutics and vaccine are currently available. Interleukin-17A (IL-17A) is an inflammatory cytokine that regulates diverse immune functions, while its role is unclear in host’s immune response to WNV. Furthermore, CD8+ T cells are crucial components of immunity and play a vital role in recovery from WNV infection. Here, we report a previously unrecognized function of IL-17A in regulating CD8+ T cell cytotoxicity. We show that WNV induces the expression of IL-17A in both mouse splenocytes and human peripheral blood mononuclear cells cultured in vitro, and in plasma of WNV-infected mice and humans. In a mouse model of WNV infection, we demonstrate that IL-17A deficient mice (Il17a-/-) are more susceptible to WNV and develop a higher viral burden compared to wild-type (WT) mice. Interestingly, the CD8+ T cells isolated from WNV-infected Il17a-/- mice are less cytotoxic and express lower levels of cytotoxic mediator genes, which can be restored by supplying recombinant IL-17A in vitro and in vivo. Moreover, treatment of WNV-infected mice with recombinant IL-17A, as late as day 6 post-infection, significantly reduces viral burden and increases survival, suggesting a therapeutic potential of IL-17A. In conclusion, we demonstrate a novel function of IL-17A in promoting CD8+ T cell cytotoxicity against WNV infection, which may have broad implications in other microbial infections and cancers

Estimation of Pyruvic acid in serum and saliva among healthy and potentially malignant disorder subjects – a stepping stone for cancer screening?

Background: According to Warburg’s effect, the rate of glycolysis increases in cancerous cells. This will increase overall levels of pyruvic acid. The present on-going study was conducted to estimate the levels of pyruvic acid in saliva and serum in normal, oral PMD subjects. Material and Methods: A total of 50 subjects in healthy, PMD of the oral cavity individuals were selected based on clinical and histological criteria. Collected saliva and serum samples were subjected to pyruvic acid level estimation using biochemical analysis. Results: Of the 50 participants 25 (13: Males; 12: Females) & 25 (16: Males; 9: Females) were PMD group. Independent samples t test showed statistically significant difference in serum & salivary pyruvic acid level in between 2 groups ( P < 0.001 respectively) Conclusions: Estimation of pyruvic acid showed sequential increase in the level in PMD group compared to healthy. Hence the study results open new direction in cancer screening

Interleukin-17A Promotes CD8 T Cell Cytotoxicity to Facilitate West Nile Virus Clearance

CD8 T cells are crucial components of immunity and play a vital role in recovery from West Nile virus (WNV) infection. Here, we identify a previously unrecognized function of interleukin-17A (IL-17A) in inducing cytotoxic-mediator gene expression and promoting CD8 T cell cytotoxicity against WNV infection in mice. We find that IL-17A-deficient (Il17a/) mice are more susceptible to WNV infection and develop a higher viral burden than wild-type (WT) mice. Interestingly, the CD8 T cells isolated from Il17a/ mice are less cytotoxic and express lower levels of cytotoxic-mediator genes, which can be restored by supplying recombinant IL-17A in vitro and in vivo. Importantly, treatment of WNV-infected mice with recombinant IL17A, as late as day 6 post infection, significantly reduces the viral burden and increases survival, suggesting a therapeutic potential for IL-17A. In conclusion, we report a novel function of IL-17A in promoting CD8 T cell cytotoxicity, which may have broad implications in other microbial infections and cancers

Loss of Glycosaminoglycan Receptor Binding After Mosquito Cell Passage Reduces Chikungunya Virus Infectivity

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that can cause fever and chronic arthritis in humans. CHIKV that is generated in mosquito or mammalian cells differs in glycosylation patterns of viral proteins, which may affect its replication and virulence. Herein, we compare replication, pathogenicity, and receptor binding of CHIKV generated in Vero cells (mammal) or C6/36 cells (mosquito) through a single passage. We demonstrate that mosquito cell-derived CHIKV (CHIKVmos) has slower replication than mammalian cell-derived CHIKV (CHIKVvero), when tested in both human and murine cell lines. Consistent with this, CHIKVmos infection in both cell lines produce less cytopathic effects and reduced antiviral responses. In addition, infection in mice show that CHIKVmos produces a lower level of viremia and less severe footpad swelling when compared with CHIKVvero. Interestingly, CHIKVmos has impaired ability to bind to glycosaminoglycan (GAG) receptors on mammalian cells. However, sequencing analysis shows that this impairment is not due to a mutation in the CHIKV E2 gene, which encodes for the viral receptor binding protein. Moreover, CHIKVmos progenies can regain GAG receptor binding capability and can replicate similarly to CHIKVvero after a single passage in mammalian cells. Furthermore, CHIKVvero and CHIKVmos no longer differ in replication when N-glycosylation of viral proteins was inhibited by growing these viruses in the presence of tunicamycin. Collectively, these results suggest that N-glycosylation of viral proteins within mosquito cells can result in loss of GAG receptor binding capability of CHIKV and reduction of its infectivity in mammalian cells

Interleukin-17A Facilitates Chikungunya Virus Infection by Inhibiting IFN-α2 Expression

Interferons (IFNs) are the key components of innate immunity and are crucial for host defense against viral infections. Here, we report a novel role of interleukin-17A (IL-17A) in inhibiting IFN-α2 expression thus promoting chikungunya virus (CHIKV) infection. CHIKV infected IL-17A deficient (Il17a−/−) mice expressed a higher level of IFN-α2 and developed diminished viremia and milder footpad swelling in comparison to wild-type (WT) control mice, which was also recapitulated in IL-17A receptor-deficient (Il17ra−/−) mice. Interestingly, IL-17A selectively blocked IFN-α2 production during CHIKV, but not West Nile virus (WNV) or Zika virus (ZIKV), infections. Recombinant IL-17A treatment inhibited CHIKV-induced IFN-α2 expression and enhanced CHIKV replication in both human and mouse cells. We further found that IL-17A inhibited IFN-α2 production by modulating the expression of Interferon Regulatory Factor-5 (IRF-5), IRF-7, IFN-stimulated gene 49 (ISG-49), and Mx1 expression during CHIKV infection. Neutralization of IL-17A in vitro leads to the increase of the expression of these antiviral molecules and decrease of CHIKV replication. Collectively, these results suggest a novel function of IL-17A in inhibiting IFN-α2–mediated antiviral responses during CHIKV infection, which may have broad implications in viral infections and other inflammatory diseases

The Molecular Basis for the Lack of Inflammatory Responses in Mouse Embryonic Stem Cells and Their Differentiated Cells

We reported previously that mouse embryonic stem cells do not have a functional IFN-based antiviral mechanism. The current study extends our investigation to the inflammatory response in mouse embryonic stem cells and mouse embryonic stem cell–differentiated cells. We demonstrate that LPS, TNF-α, and viral infection, all of which induce robust inflammatory responses in naturally differentiated cells, failed to activate NF-κB, the key transcription factor that mediates inflammatory responses, and were unable to induce the expression of inflammatory genes in mouse embryonic stem cells. Similar results were obtained in human embryonic stem cells. In addition to the inactive state of NF-κB, the deficiency in the inflammatory response in mouse embryonic stem cells is also attributed to the lack of functional receptors for LPS and TNF-α. In vitro differentiation can trigger the development of the inflammatory response mechanism, as indicated by the transition of NF-κB from its inactive to active state. However, a limited response to TNF-α and viral infection, but not to LPS, was observed in mouse embryonic stem cell–differentiated fibroblasts. We conclude that the inflammatory response mechanism is not active in mouse embryonic stem cells, and in vitro differentiation promotes only partial development of this mechanism. Together with our previous studies, the findings described in this article demonstrate that embryonic stem cells are fundamentally different from differentiated somatic cells in their innate immunity, which may have important implications in developmental biology, immunology, and embryonic stem cell–based regenerative medicine

Evaluation of holy basil mouthwash as an adjunctive plaque control agent in a four day plaque regrowth model

Objectives: Various antibacterial and antiplaque agents are used in chemical plaque control but none are without their shortcomings. Chlorhexidine considered a gold standard, also has an array of side effects. To overcome these, numerous herbal extracts have been tried and tested and one among them is holy basil. The present study evaluated the antibacterial efficacy of holy basil in vitro against some periodontopathogens and its antiplaque ef fect in vivo. Study Design: Thirty periodontally healthy volunteers were randomly divided into three groups and refrained from all mechanical oral hygiene measures for 4 days and used one of the randomly assigned mouthwash (1- chlor - hexidine; 2- holy basil; and 3- sterile water [placebo]) twice daily. The Plaque Index (PI) was assessed at days 0 and 5. Aqueous extract of holy basil was tested against Prevotella intermedia ( P. intermedia ) and Fusobacterium nucleatum ( F.nucleatum ). Results: Holy basil extract showed inhibition of both the tested periodontopathogens ( P.intermedia and F.nucleatum ) at various concentrations. In all groups, the PI increased from baseline to day 5. There was a statistically significant difference ( p < .05) between the chlorhexidine and placebo rinse and the holy basil and placebo rinse, but no statis - tically significant difference was found between the chlorhexidine and holy basil rinse with respect to PI. Conclusions: These results indicate that the holy basil mouthwash has an antiplaque effect and is efficacious against P. intermedia and F. nucleatum strains in vitro. Hence holy basil mouthwash may have potential as an antiplaque mouthwash with prophylactic benefits

Antibody-Dependent Enhancement Activity of a Plant-Made Vaccine Against West Nile Virus

West Nile virus (WNV) causes annual outbreaks globally and is the leading cause of mosquito-borne disease in Unite States. In the absence of licensed therapeutics, there is an urgent need to develop effective and safe human vaccines against WNV. One of the major safety concerns for WNV vaccine development is the risk of increasing infection by related flaviviruses in vaccinated subjects via antibody-dependent enhancement of infection (ADE). Herein, we report the development of a plant-based vaccine candidate that provides protective immunity against a lethal WNV challenge mice, while minimizes the risk of ADE for infection by Zika (ZIKV) and dengue (DENV) virus. Specifically, a plant-produced virus-like particle (VLP) that displays the WNV Envelope protein domain III (wDIII) elicited both high neutralizing antibody titers and antigen-specific cellular immune responses in mice. Passive transfer of serum from VLP-vaccinated mice protected recipient mice from a lethal challenge of WNV infection. Notably, VLP-induced antibodies did not enhance the infection of Fc gamma receptor-expressing K562 cells by ZIKV or DENV through ADE. Thus, a plant-made wDIII-displaying VLP presents a promising WNV vaccine candidate that induces protective immunity and minimizes the concern of inducing ADE-prone antibodies to predispose vaccinees to severe infection by DENV or ZIKV

Antiviral Responses In Mouse Embryonic Stem Cells: Differential Development of Cellular Mechanisms In Type I Interferon Production and Response

We have recently reported that mouse embryonic stem cells (mESCs) are deficient in expressing type I interferons (IFNs) in response to viral infection and synthetic viral RNA analogs (Wang, R., Wang, J., Paul, A. M., Acharya, D., Bai, F., Huang, F., and Guo, Y. L. (2013) J. Biol. Chem. 288, 15926–15936). Here, we report that mESCs are able to respond to type I IFNs, express IFN-stimulated genes, and mediate the antiviral effect of type I IFNs against La Crosse virus and chikungunya virus. The major signaling components in the IFN pathway are expressed in mESCs. Therefore, the basic molecular mechanisms that mediate the effects of type I IFNs are functional in mESCs; however, these mechanisms may not yet be fully developed as mESCs express lower levels of IFN-stimulated genes and display weaker antiviral activity in response to type I IFNs when compared with fibroblasts. Further analysis demonstrated that type I IFNs do not affect the stem cell state of mESCs. We conclude that mESCs are deficient in type I IFN expression, but they can respond to and mediate the cellular effects of type I IFNs. These findings represent unique and uncharacterized properties of mESCs and are important for understanding innate immunity development and ESC physiology

Congenital Zika Virus Infection in Immunocompetent Mice Causes Postnatal Growth Impediment and Neurobehavioral Deficits

A small percentage of babies born to Zika virus (ZIKV)-infected mothers\u27 manifest severe defects at birth, including microcephaly. Among those who appeared healthy at birth, there are increasing reports of postnatal growth or developmental defects. However, the impact of congenital ZIKV infection in postnatal development is poorly understood. Here, we report that a mild congenital ZIKV-infection in pups born to immunocompetent pregnant mice did not display apparent defects at birth, but manifested postnatal growth impediments and neurobehavioral deficits, which include reduced locomotor and cognitive deficits that persisted into adulthood. We found that the brains of these pups were smaller, had a thinner cortical layer 1, displayed increased astrogliosis, decreased expression of microcephaly- and neuron development- related genes, and increased pathology as compared to mock-infected controls. In summary, our results showed that even a mild congenital ZIKV infection in immunocompetent mice could lead to postnatal deficits, providing definitive experimental evidence for a necessity to closely monitor postnatal growth and development of presumably healthy human infants, whose mothers were exposed to ZIKV infection during pregnancy