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Piotr Orlowski,1 Magdalena Zmigrodzka,2 Emilia Tomaszewska,3 Katarzyna Ranoszek-Soliwoda,3 Beata Pajak,4 Anna Slonska,5 Joanna Cymerys,5 Grzegorz Celichowski,3 Jaroslaw Grobelny,3 Malgorzata Krzyzowska1 1Laboratory of Nanobiology and Biomaterials, Military Institute of Hygiene and Epidemiology, Warsaw, Poland; 2Department of Pathology and Veterinary Diagnostics, Institute of Veterinary Medicine, Warsaw University of Life Sciences (WULS-SGGW), Warsaw, Poland; 3Department of Materials Technology and Chemistry, Faculty of Chemistry, University of Lodz, Lodz, Poland; 4Laboratory of Genetics and Molecular Biology, Military Institute of Hygiene and Epidemiology, Warsaw, Poland; 5Division of Microbiology, Department of Preclinical Sciences, Institute of Veterinary Medicine, Warsaw University of Life Sciences, Warsaw 02-786, PolandCorrespondence: Malgorzata Krzyzowska Email krzyzowskam@yahoo.comBackground: Polyphenols possess antioxidant, anti-inflammatory and antimicrobial properties and have been used in the treatment of skin wounds and burns. We previously showed that tannic acid-modified AgNPs sized > 26 nm promote wound healing, while tannic acid-modified AgNPs sized 13 nm can elicit strong local inflammatory response. In this study, we tested bimetallic Au@AgNPs sized 30 nm modified with selected flavonoid and non-flavonoid compounds for wound healing applications.Methods: Bimetallic Au@AgNPs were obtained by growing an Ag layer on AuNPs and further modified with selected polyphenols. After toxicity tests and in vitro scratch assay in HaCaT cells, modified lymph node assay as well as the mouse splint wound model were further used to access the wound healing potential of selected non-toxic modifications.Results: Tannic acid, gallic acid, polydatin, resveratrol, catechin, epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate and procyanidin B2 used to modify Au@AgNPs exhibited good toxicological profiles in HaCaT cells. Au@AgNPs modified with 15 μM tannic acid, 200 μM resveratrol, 200 μM epicatechin gallate, 1000 μM gallic acid and 200 μM procyanidin B2 induced wound healing in vivo and did not lead to the local irritation or inflammation. Tannic acid-modified Au@AgNPs induced epithelial-to-mesenchymal transition (EMT) – like re-epithelialization, while other polyphenol modifications of Au@AgNPs acted through proliferation and wound closure.Conclusion: Bimetallic Au@AgNPs can be used as a basis for modification with selected polyphenols for topical uses. In addition, we have demonstrated that particular polyphenols used to modify bimetallic nanoparticles may show different effects upon different stages of wound healing.Keywords: polyphenols, bimetallic nanoparticles, wound healing, mouse model

Piotr Orlowski,1 Magdalena Zmigrodzka,2 Emilia Tomaszewska,3 Katarzyna Ranoszek-Soliwoda,3 Monika Czupryn,1 Malgorzata Antos-Bielska,1 Janusz Szemraj,4 Grzegorz Celichowski,3 Jaroslaw Grobelny,3 Malgorzata Krzyzowska1 1Military Institute of Hygiene and Epidemiology, Warsaw, Poland; 2Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences (WULS-SGGW), Warsaw, Poland; 3Department of Materials Technology and Chemistry, Faculty of Chemistry, University of Lodz, Lodz, Poland; 4Bionanopark, Lodz, Poland Introduction: Silver nanoparticles (AgNPs) have been shown to promote wound healing and to exhibit antimicrobial properties against a broad range of bacteria. In our previous study, we prepared tannic acid (TA)-modified AgNPs showing a good toxicological profile and immunomodulatory properties useful for potential dermal applications.Methods: In this study, in vitro scratch assay, antimicrobial tests, modified lymph node assay as well as a mouse splint wound model were used to access the wound healing potential of TA-modified and unmodified AgNPs.Results: TA-modified but not unmodified AgNPs exhibited effective antibacterial activity against Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli and stimulated migration of keratinocytes in vitro. The tests using the mouse splint wound model showed that TA-modified 33 and 46 nm AgNPs promoted better wound closure, epithelialization, angiogenesis and formation of the granulation tissue. Additionally, AgNPs elicited expression of VEGF-α, PDGF-β and TGF-β1 cytokines involved in wound healing more efficiently in comparison to control and TA-treated wounds. However, both the lymph node assay and the wound model showed that TA-modified AgNPs sized 13 nm can elicit strong inflammatory response not only during wound healing but also when applied to the damaged skin.Conclusion: TA-modified AgNPs sized >26 nm promote wound healing better than TA-modified or unmodified AgNPs. These findings suggest that TA-modified AgNPs sized >26 nm may have a promising application in wound management. Keywords: hydrolyzable tannin, split wound, silver, antimicrobials, inflammation, fibroblasts, monocytes

The overexpression of cyclooxygenase-2 (COX-2) has been documented in many types of cancer occurring in humans and animals. Increasing evidences have shown that the overexpression of COX-2 and increased production of prostaglandin E2 (PGE2) correlate with poor prognosis in human solid tumours and hematological malignancies. Both, in vitro and in vivo studies have demonstrated that increased proliferation of cancer cells as well as an impairment of antitumour immunity are influenced by the overexpression of this enzyme. In leukemia and lymphoma, an increased activity of COX-2 and subsequent increase in prostaglandins (PGs) concentration allow cancer cells to evade immune response and contribute to metastases. Cancer stem cells (CSCs) in tumour microenvironment, suppression of innate and adaptive immunity depends on COX-2/PGE2 axis activity which increases in hematological malignancies. Cyclooxygenases inhibitors block the formation of PGs, consequently inhibiting angiogenesis, and in some malignancies they decrease cancer cells proliferation and tumour invasiveness. They also increase apoptosis of CSCs and cancer cells, decrease their drug resistance as well as enhance the host immune response. Therefore COX-2/PGE2 axis suppressors: selective COX-2 inhibitors or PG receptors antagonists have been considered as promising anticancer drugs. In comparative oncology dogs are increasingly used as a large animal model because they share the same environmental conditions with people and are exposed to the same environmental factors and also due to their relatively short life span. In dogs, spontaneously occurring non-Hodgkin lymphomas and leukemias have a large number of genetic and morphological features that are similar to those of humans' corresponding cancers. This, additionally makes the species a useful model for the study of new therapeutic strategies in human oncology. While the influence of COX-2 activity and PGE2 receptors have been evaluated extensively in human cancer, their role in veterinary oncology still needs to be elucidated. [ABSTRACT FROM AUTHOR]

Herpes simplex virus type 2 (HSV-2) is a sexually transmitted pathogen that causes a persistent infection in sensory ganglia. The infection manifests itself as genital herpes but in rare cases it can cause meningitis. In this study, we used a murine model of HSV-2 meningitis to show that Fas and FasL are induced within the CNS upon HSV-2 infection, both on resident microglia and astrocytes and on infiltrating monocytes and lymphocytes. Mice lacking Fas or FasL had a more severe disease development with significantly higher morbidity, mortality, and an overall higher CNS viral load. In parallel, these Fas/FasL-deficient mice showed a severely impaired infection-induced CNS inflammatory response with lower levels of infiltrating CD4+ T-cells, lower levels of Th1 cytokines and chemokines, and a shift in the balance between M1 and M2 microglia/monocytes. In vitro, we confirmed that Fas and FasL is required for the induction of leucocyte apoptosis, but also show that the Fas/FasL pathway is required for adequate cytokine and chemokine production by glial cells. In summary, our data show that the Fas/FasL cell death receptor pathway is an important defense mechanism in the spinal cord as it down-regulates HSV-2-induced inflammation while at the same time promoting adequate anti-viral immune responses against infection.

Plant polyphenols possess diverse bioactivities, including antiviral activity against a broad spectrum of viruses. Here, we investigated the virucidal properties of an Kalanchoe daigremontiana extract using an in vitro model of human herpesvirus type 1 (HHV-1) infection. Chromatographic analysis indicated that the extract of Kalanchoe daigremontiana is rich in various compounds, among which are polyphenols with virucidal activity confirmed in the literature. We found that Kalanchoe daigremontiana extract shows an ability to prevent HHV-1 infection by direct inhibition of the virus attachment, penetration, and blocking of infection when used in pretreatment or post-entry treatment. Our results indicate that Kalanchoe daigremontiana extract may be a good candidate drug against HHV-1, both as a substance to prevent infection and to treat an already ongoing infection. Our findings illustrate that Kalanchoe daigremontiana could be a potential new candidate for clinical consideration in the treatment of HHV-1 infection alone or in combination with other therapeutics.

(1) Background: Epigallocatechin gallate (EGCG) has been recognized as a flavonoid showing antiviral activity against various types of DNA and RNA viruses. In this work, we tested if EGCG-modified silver nanoparticles (EGCG-AgNPs) can become novel microbicides with additional adjuvant properties to treat herpes infections. (2) Methods: The anti-HSV and cytotoxic activities of EGCG-AgNPs were tested in human HaCaT and VK-2-E6/E7 keratinocytes. HSV-1/2 titers and immune responses after treatment with EGCG-AgNPs were tested in murine models of intranasal HSV-1 infection and genital HSV-2 infection. (3) Results: EGCG-AgNPs inhibited attachment and entry of HSV-1 and HSV-2 in human HaCaT and VK-2-E6/E7 keratinocytes much better than EGCG at the same concentration. Infected mice treated intranasally (HSV-1) or intravaginally (HSV-2) with EGCG-AgNPs showed lower virus titers in comparison to treatment with EGCG alone. After EGCG-AgNPs treatment, mucosal tissues showed a significant infiltration in dendritic cells and monocytes in comparison to NaCl-treated group, followed by significantly better infiltration of CD8+ T cells, NK cells and increased expression of IFN-α, IFN-γ, CXCL9 and CXCL10. (4) Conclusions: Our findings show that EGCG-AgNPs can become an effective novel antiviral microbicide with adjuvant properties to be applied upon the mucosal tissues.

Metallic nanoparticles exhibit broad-spectrum activity against bacteria, fungi, and viruses. The antiviral activity of nanoparticles results from the multivalent interactions of nanoparticles with viral surface components, which result from the nanometer size of the material and the presence of functional compounds adsorbed on the nanomaterial surface. A critical step in the virus infection process is docking and entry of the virus into the host cell. This stage of the infection can be influenced by functional nanomaterials that exhibit high affinity to the virus surface and hence can disrupt the infection process. The affinity of the virus to the nanomaterial surface can be tuned by the specific surface functionalization of the nanomaterial. The main purpose of this work was to determine the influence of the ligand type present on nanomaterial on the antiviral properties against herpes simplex virus type 1 and 2. We investigated the metallic nanoparticles (gold and silver) with different sizes (5 nm and 30 nm), coated either with polyphenol (tannic acid) or sulfonates (ligands with terminated sulfonate groups). We found that the antiviral activity of nano-conjugates depends significantly on the ligand type present on the nanoparticle surface., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Lactoferrin is an iron-binding glycoprotein with multiple functions in the body. Its activity against a broad spectrum of both DNA and RNA viruses as well as the ability to modulate immune responses have made it of interest in the pharmaceutical and food industries. The mechanisms of its antiviral activity include direct binding to the viruses or its receptors or the upregulation of antiviral responses by the immune system. Recently, much effort has been devoted to the use of nanotechnology in the development of new antivirals. In this review, we focus on describing the antiviral mechanisms of lactoferrin and the possible use of nanotechnology to construct safe and effective new antiviral drugs.

Neuroinflammation is defined as an inflammatory state within the central nervous system (CNS). Microglia conprise the resident tissue macrophages of the neuronal tissue. Upon viral infection of the CNS, microglia become activated and start to produce inflammatory mediators important for clearance of the virus, but an excessive neuroinflammation can harm nearby neuronal cells. Herpesviruses express several molecular mechanisms, which can modulate apoptosis of infected neurons, astrocytes and microglia but also divert immune response initiated by the infected cells. In this review we also describe the link between virus-related neuroinflammation, and development of neurodegenerative diseases.

Tofacitinib, a Janus kinase inhibitor, is a novel immunosuppressive drug for treatment of rheumatoid arthritis. Herpes simplex virus type 1 (HSV-1) may cause encephalitis during primary infection or following reactivation from a latent state. Long-term tofacitinib treatment may increase the risk of this life-threatening condition. The aim of this study was to investigate the effect of tofacitinib on HSV-1 primary infection using a mouse model. Mice pretreated with tofacitinib were intranasally infected with a clinical strain of HSV-1 and monitored for infection severity and antiviral response. Tofacitinib treatment of HSV-1 primary infection resulted in increased viral loads and worsened clinical outcome. Furthermore, tofacitinib promoted M2 anti-inflammatory phenotype of microglia and infiltrating monocytes, as well as inhibited production of inflammatory and antiviral cytokines by macrophages in vitro. Our findings show that treatment with tofacitinib increases severity of herpes simplex encephalitis in mice, by impairing antiviral response induced by monocytes and microglia., (© The Author(s) 2022. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

(1) Background: Lactoferrin has been recognized as a potent inhibitor of human herpetic viruses, such as herpes simplex type 1 (HSV-1) and 2 (HSV-2). In this work, we tested if silver and gold nanoparticles modified with lactoferrin (LF-Ag/AuNPs) can become novel microbicides with additional adjuvant properties to treat genital herpes infection. (2) Methods: The antiviral and cytotoxic activities of LF-Ag/AuNPs were tested in human skin HaCaT and vaginal VK-2-E6/E7 keratinocytes. Viral titers and immune responses after treatment with LF-Ag/AuNPs were tested in murine vaginal HSV-2 infection. (3) Results: LF-Ag/AuNPs inhibited attachment and entry of HSV-2 in human keratinocytes much better than lactoferrin. Furthermore, pretreatment with LF-AgNPs led to protection from infection. Infected mice treated intravaginally with LF-Ag/AuNPs showed lower virus titers in the vaginal tissues and spinal cords in comparison to treatment with lactoferrin. Following treatment, vaginal tissues showed a significant increase in CD8+/granzyme B + T cells, NK cells and dendritic cells in comparison to NaCl-treated group. LF-Ag/AuNPs-treated animals also showed significantly better expression of IFN-γ, CXCL9, CXCL10, and IL-1β in the vaginal tissues. (4) Conclusions: Our findings show that LF-Ag/AuNPs could become effective novel antiviral microbicides with immune-stimulant properties to be applied upon the mucosal tissues.

The Fas/FasL pathway plays a key role in immune homeostasis and immune surveillance. In the central nervous system (CNS) Fas/FasL is involved in axonal outgrowth and adult neurogenesis. However, little is known about the role of the Fas/FasL pathway in herpes encephalitis. In this study, we used a neuropathogenic clinical strain of herpes simplex virus type 1 (HSV-1) to explore infection-induced inflammation and immune responses in the mouse brain and the role of Fas/FasL in antiviral CNS immunity. HSV-1 CNS infection induced the infiltration of Fas- FasL-bearing monocytes and T cells in the brain and also to an up-regulation of Fas and FasL expression on resident astrocytes and microglia within infected sites. Upon infection, Fas- and FasL-deficient mice (lpr and gld) were partially protected from encephalitis with a decreased morbidity and mortality compared to WT mice. Fas/FasL deficiency promoted cell-mediated immunity within the CNS. Fas receptor stimulation abrogated HSV-1 induced activation and inflammatory reactions in microglia from WT mice, while lack of Fas or FasL led to a more pronounced activation of monocytes and microglia and also to an enhanced differentiation of these cells into a pro-inflammatory M1 phenotype. Furthermore, the specific immune system was more efficient in Fas- and FasL-deficient mice with significantly higher numbers of infiltrating HSV-1-specific cytotoxic T cells in the brain. Our data indicate that the Fas/FasL pathway leads to excessive neuroinflammation during HSV-1 infection, which is associated with a diminished anti-viral response and an excessive neuroinflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Krzyzowska, Kowalczyk, Skulska, Thörn and Eriksson.)

The contribution of virus components to liberation of herpes simplex virus type 2 (HSV-2) progeny virions from the surface of infected cells is poorly understood. We report that the HSV-2 mutant deficient in the expression of a mucin-like membrane-associated glycoprotein G (mgG) exhibited defect in the release of progeny virions from infected cells manifested by ~2 orders of magnitude decreased amount of infectious virus in a culture medium as compared to native HSV-2. Electron microscopy revealed that the mgG deficient virions were produced in infected cells and present at the cell surface. These virions could be forcibly liberated to a nearly native HSV-2 level by the treatment of cells with glycosaminoglycan (GAG)-mimicking oligosaccharides. Comparative assessment of the interaction of mutant and native virions with surface-immobilized chondroitin sulfate GAG chains revealed that while the mutant virions associated with GAGs ~fourfold more extensively, the lateral mobility of bound virions was much poorer than that of native virions. These data indicate that the mgG of HSV-2 balances the virus interaction with GAG chains, a feature critical to prevent trapping of the progeny virions at the surface of infected cells.

Herpes simplex virus type 2 (HSV-2) is a neurotropic virus that can cause meningitis, an inflammation of the meninges in the central nervous system. T cells are key players in viral clearance, and these cells migrate from peripheral blood into the central nervous system upon infection. Several factors contribute to T cell migration, including the expression of chemokines in the inflamed tissue that attract T cells through their expression of chemokine receptors. Here we investigated CD8 +  T cell profile in the spinal cord in a mouse model of herpes simplex virus type 2 neuroinflammation. Mice were infected with HSV-2 and sacrificed when showing signs of neuroinflammation. Cells and/or tissue from spinal cord, spleen, and blood were analyzed for expression of activation markers, chemokine receptors, and chemokines. High numbers of CD8 +  T cells were present in the spinal cord following genital HSV-2-infection. CD8 +  T cells were highly activated and HSV-2 glycoprotein B -specific effector cells, some of which showed signs of recent degranulation. They also expressed high levels of many chemokine receptors, in particular CCR2, CCR4, CCR5, and CXCR3. Investigating corresponding receptor ligands in spinal cord tissue revealed markedly increased expression of the cognate ligands CCL2, CCL5, CCL8, CCL12, and CXCL10. This study shows that during herpesvirus neuroinflammation anti-viral CD8 +  T cells accumulate in the CNS. CD8 +  T cells in the CNS also express chemotactic receptors cognate to the chemotactic gradients in the spinal cord. This indicates that anti-viral CD8 +  T cells may migrate to infected areas in the spinal cord during herpesvirus neuroinflammation in response to chemotactic gradients.

Plant extracts are known for their antihyperglycemic, antioxidant, antimutagenic, antifungal, anti-inflammatory, antiviral and antibacterial properties. These biological properties make plant extracts interesting surface modifiers of nanoparticles (NPs), which are also known for their unique features. Plant extracts can play a multifunctional role in the synthesis of NPs (i.e. can act as a reducing agent, stabiliser and bioactive compound), which gives additional properties to the final hybrid material. The combination of an extract of natural origin with NPs results in bioconjugates with unique final properties that often may not be obvious. The properties of a bioconjugate depend on both the plant extract (chemical composition, amount, a method of conjugation to NP surface, etc.) and the NPs (type, size, shape, polydispersity, etc.). Syntheses of NPs with plant extract usually lead to polydisperse particles with heterogeneous properties that are difficult to control from a biological point of view. In this paper, we present a synthesis protocol to obtain monodisperse silver nanoparticles (AgNPs) with plant extract. Cacao beans and grape seed extracts were selected as natural sources of polyphenols having biomedical importance (e.g. catechin, tannic acid, epicatechin gallate) and used to synthesise using a chemical reduction method. Syntheses were carried out with different molar ratios of reagents to find the best conditions for obtaining AgNPs that are homogeneous in size and shape. The colloids obtained were characterised with ultraviolet-visible (UV-Vis) spectroscopy, scanning transmission electron microscopy (STEM) and dynamic light scattering (DLS). It was found that syntheses carried out only with plant extract resulted in unstable colloids containing polydisperse nanoparticles. Stable colloids containing spherical monomodal particles were obtained by the incorporation of sodium citrate as an additional reagent in the synthesis mixture. The results obtained clearly indicate the crucial role of sodium citrate in the synthesis of spherical AgNPs of controlled size using plant extracts for biological applications., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The overexpression of cyclooxygenase-2 (COX-2) has been documented in many types of cancer occurring in humans and animals. Increasing evidences have shown that the overexpression of COX-2 and increased production of prostaglandin E 2 (PGE 2 ) correlate with poor prognosis in human solid tumours and hematological malignancies. Both, in vitro and in vivo studies have demonstrated that increased proliferation of cancer cells as well as an impairment of anti-tumour immunity are influenced by the overexpression of this enzyme. In leukemia and lymphoma, an increased activity of COX-2 and subsequent increase in prostaglandins (PGs) concentration allow cancer cells to evade immune response and contribute to metastases. Cancer stem cells (CSCs) in tumour microenvironment, suppression of innate and adaptive immunity depends on COX-2/PGE2 2 axis activity which increases in hematological malignancies. Cyclooxygenases inhibitors block the formation of PGs, consequently inhibiting angiogenesis, and in some malignancies they decrease cancer cells proliferation and tumour invasiveness. They also increase apoptosis of CSCs and cancer cells, decrease their drug resistance as well as enhance the host immune response. Therefore COX-2/PGE 2 axis suppressors: selective COX-2 inhibitors or PG receptors antagonists have been considered as promising anticancer drugs. In comparative oncology dogs are increasingly used as a large animal model because they share the same environmental conditions with people and are exposed to the same environmental factors and also due to their relatively short life span. In dogs, spontaneously occurring non-Hodgkin lymphomas and leukemias have a large number of genetic and morphological features that are similar to those of humans' corresponding cancers. This, additionally makes the species a useful model for the study of new therapeutic strategies in human oncology. While the influence of COX-2 activity and PGE 2 receptors have been evaluated extensively in human cancer, their role in veterinary oncology still needs to be elucidated.

(1) Background: Tannic acid is a plant-derived polyphenol showing antiviral activity mainly because of an interference with the viral adsorption. In this work, we tested whether the modification of silver nanoparticles with tannic acid (TA-AgNPs) can provide a microbicide with additional adjuvant properties to treat genital herpes infection. (2) Methods: The mouse model of the vaginal herpes simplex virus 2 (HSV-2) infection was used to test immune responses after treatment of the primary infection with TA-AgNPs, and later, after a re-challenge with the virus. (3) Results: The mice treated intravaginally with TA-AgNPs showed better clinical scores and lower virus titers in the vaginal tissues soon after treatment. Following a re-challenge, the vaginal tissues treated with TA-AgNPs showed a significant increase in the percentages of IFN-gamma+ CD8+ T-cells, activated B cells, and plasma cells, while the spleens contained significantly higher percentages of IFN-gamma+ NK cells and effector-memory CD8+ T cells in comparison to NaCl-treated group. TA-AgNPs-treated animals also showed significantly better titers of anti-HSV-2 neutralization antibodies in sera; and (4) Conclusions: Our findings suggest that TA-AgNPs sized 33 nm can be an effective anti-viral microbicide to be applied upon the mucosal tissues with additional adjuvant properties enhancing an anti-HSV-2 immune response following secondary challenge., Competing Interests: The authors declare no conflict of interest.

Silver nanoparticles (AgNPs) are promising new antimicrobial agents against a wide range of skin and mucosal pathogens. However, their interaction with the immune system is currently not fully understood. Dendritic cells (DCs) are crucial during development of T cell-specific responses against bacterial and viral pathogens. We have previously shown that tannic acid-modified silver nanoparticles (TA-AgNPs) consist of a promising microbicide against HSV-2. The aim of this study was to compare the ability of TA-AgNPs or TA-AuNPs of similar sizes (TA-Ag/AuNPs) to induce DCs maturation and activation in the presence of HSV-2 antigens when used at non-toxic doses. First, we used JAWS II DC line to test toxicity, ultrastructure as well as activation markers (MHC I and II, CD40, CD80, CD86, PD-L1) and cytokine production in the presence of TA-Ag/AuNPs. Preparations of HSV-2 treated with nanoparticles (TA-Ag/AuNPs-HSV-2) were further used to investigate HSV-2 antigen uptake, activation markers, TLR9 expression, and cytokine production. Additionally, we accessed proliferation and activation of HSV-2-specific T cells by DCs treated with TA-AgNP/AuNPs-HSV-2. We found that both TA-AgNPs and TA-AuNPs were efficiently internalized by DCs and induced activated ultrastructure. Although TA-AgNPs were more toxic than TA-AuNPs in corresponding sizes, they were also more potent stimulators of DCs maturation and TLR9 expression. TA-Ag/AuNPs-HSV-2 helped to overcome inhibition of DCs maturation by live or inactivated virus through up-regulation of MHC II and CD86 and down-regulation of CD80 expression. Down-regulation of CD40 expression in HSV-2-infected DCs was reversed when HSV-2 was treated with TA-NPs sized >30 nm. On the other hand, small-sized TA-AgNPs helped to better internalize HSV-2 antigens. HSV-2 treated with both types of NPs stimulated activation of JAWS II and memory CD8+ T cells, while TA-AgNPs treatment induced IFN-γ producing CD4+ and CD8+ T cells. Our study shows that TA-AgNPs or TA-AuNPs are good activators of DCs, albeit their final effect upon maturation and activation may be metal and size dependent. We conclude that TA-Ag/AuNPs consist of a novel class of nano-adjuvants, which can help to overcome virus-induced suppression of DCs activation.

Dendritic cells (DCs) are effector cells linking the innate immune system with the adaptive immune response. Many viruses eliminate DCs to prevent host response, induce immunosuppression and to maintain chronic infection. In this study, we examined apoptotic response of dendritic cells during in vitro and in vivo infection with ectromelia virus (ECTV), the causative agent of mousepox. ECTV-infected bone marrow dendritic cells (BMDCs) from BALB/c mice underwent apoptosis through mitochondrial pathway at 48 h post infection, up-regulated FasL and decreased expression of anti-apoptotic Bcl-2 and pro-apoptotic Fas. Similar pattern of Bcl-2, Fas and FasL expression was observed for DCs early during in vivo infection of BALB/c mice. Both BMDCs and DCs from BALB/c mice showed no maturation upon ECTV infection. We conclude that ECTV-infected DCs from BALB/c mouse strain help the virus to spread and to maintain infection., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

We describe herein the significance of a sodium citrate and tannic acid mixture in the synthesis of spherical silver nanoparticles (AgNPs). Monodisperse AgNPs were synthesized via reduction of silver nitrate using a mixture of two chemical agents: sodium citrate and tannic acid. The shape, size and size distribution of silver particles were determined by UV-Vis spectroscopy, dynamic light scattering (DLS) and scanning transmission electron microscopy (STEM). Special attention is given to understanding and experimentally confirming the exact role of the reagents (sodium citrate and tannic acid present in the reaction mixture) in AgNP synthesis. The oxidation and reduction potentials of silver, tannic acid and sodium citrate in their mixtures were determined using cyclic voltammetry. Possible structures of tannic acid and its adducts with citric acid were investigated in aqueous solution by performing computer simulations in conjunction with the semi-empirical PM7 method. The lowest energy structures found from the preliminary conformational search are shown, and the strength of the interaction between the two molecules was calculated. The compounds present on the surface of the AgNPs were identified using FT-IR spectroscopy, and the results are compared with the IR spectrum of tannic acid theoretically calculated using PM6 and PM7 methods. The obtained results clearly indicate that the combined use of sodium citrate and tannic acid produces monodisperse spherical AgNPs, as it allows control of the nucleation, growth and stabilization of the synthesis process. Graphical abstractᅟ.

Hydrolyzable tannins are known to exhibit anti-inflammatory activity, which can be used in combination with silver nanoparticles (AgNPs) for dermal uses. In this study, we investigated the effects of tannic acid-modified 13, 33, 46nm and unmodified 10-65nm AgNPs using the human-derived keratinocyte HaCaT and VK2-E6/E7 cell lines in the form of stationary and spheroids cultures. After exposition to tannic acid-modified AgNPs, VK2-E6/E7 cells showed higher toxicity, increased production of reactive oxygen species (ROS) and activity of JNK stress kinase, while HaCaT cell line demonstrated less ROS production and activation of ERK kinase. AgNPs internalization was detected both in the superficial and internal layers of spheroids prepared from both cell lines. Tannic acid modified AgNPs sized above 30nm did not induce DNA breaks in comet assay performed in both cell lines. Tannic acid-modified but not unmodified AgNPs down-regulated TNF-α and LPS-triggered production of IL-8 in VK2-E6/E7 but not in HaCaT cells. In summary, tannic acid-modified AgNPs sized above 30nm show good toxicological profile both in vitro and possess immunomodulatory properties useful for potential dermal applications in humans., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Mucosal surfaces are exposed to environmental substances and represent a major portal of entry for microorganisms. The innate immune system is responsible for early defense against infections and it is believed that the interferons (IFNs) constitute the first line of defense against viruses. Here we identify an innate antiviral pathway that works at epithelial surfaces before the IFNs. The pathway is activated independently of known innate sensors of viral infections through a mechanism dependent on viral O-linked glycans, which induce CXCR3 chemokines and stimulate antiviral activity in a manner dependent on neutrophils. This study therefore identifies a previously unknown layer of antiviral defense that exerts its action on epithelial surfaces before the classical IFN response is operative.

Fas receptor-Fas ligand (FasL) signalling is involved in apoptosis of immune cells as well as of the virus infected target cells but increasing evidence accumulates on Fas as a mediator of apoptosis-independent processes such as induction of activating and proinflammatory signals. In this study, we examined the role of Fas/FasL pathway in inflammatory and antiviral response in lungs using a mousepox model applied to C57BL6/J, B6. MRL-Faslpr/J, and B6Smn.C3-Faslgld/J mice. Ectromelia virus (ECTV) infection of Fas- and FasL-deficient mice led to increased virus titers in lungs and decreased migration of IFN-γ expressing NK cells, CD4+ T cells, CD8+ T cells, and decreased IL-15 expression. The lungs of ECTV-infected Fas- and FasL-deficient mice showed significant inflammation during later phases of infection accompanied by decreased expression of anti-inflammatory IL-10 and TGF-β1 cytokines and disturbances in CXCL1 and CXCL9 expression. Experiments in vitro demonstrated that ECTV-infected cultures of epithelial cells, but not macrophages, upregulate Fas and FasL and are susceptible to Fas-induced apoptosis. Our study demonstrates that Fas/FasL pathway during ECTV infection of the lungs plays an important role in controlling local inflammatory response and mounting of antiviral response.

Fas receptor-Fas ligand (FasL) signaling is involved in apoptosis of virus-infected cells but increasing evidence accumulates on Fas receptor as a mediator of apoptosis-independent processes such as induction of activating and pro-inflammatory signals. In this study, we examined the role of Fas/FasL pathway in regulation of anti-viral response to genital HSV-2 infection using a murine model of HSV-2 infection applied to C57BL6/J, B6. MRL-Faslpr/J and B6Smn.C3-Faslgld/J mice. HSV-2 infection of Fas- and FasL-deficient mice led to decreased migration of IFN-γ expressing NK cells and CD4+ T cells, but not of γδ T cells, into the vaginal tissue. The vaginal tissues of HSV-2 infected Fas- and FasL-deficient mice showed increased production of IL-10, followed by low expression of the early CD69 activation marker on CD4+ and CD8+ T cells and increased numbers of regulatory T cells (Tregs). Experiments in co-cultures of CD4+ T cells and bone marrow derived dendritic cells showed that lack of bilateral Fas-FasL signaling led to expansion of Tregs and increased production of IL-10 and TGF-β1. Our results demonstrate that Fas/FasL can regulate development of tolerogenic dendritic cells and expansion of Tregs early during HSV-2 infection, which further influences effective anti-viral response., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

The interaction between silver nanoparticles and herpesviruses is attracting great interest due to their antiviral activity and possibility to use as microbicides for oral and anogenital herpes. In this work, we demonstrate that tannic acid modified silver nanoparticles sized 13 nm, 33 nm and 46 nm are capable of reducing HSV-2 infectivity both in vitro and in vivo. The antiviral activity of tannic acid modified silver nanoparticles was size-related, required direct interaction and blocked virus attachment, penetration and further spread. All tested tannic acid modified silver nanoparticles reduced both infection and inflammatory reaction in the mouse model of HSV-2 infection when used at infection or for a post-infection treatment. Smaller-sized nanoparticles induced production of cytokines and chemokines important for anti-viral response. The corresponding control buffers with tannic acid showed inferior antiviral effects in vitro and were ineffective in blocking in vivo infection. Our results show that tannic acid modified silver nanoparticles are good candidates for microbicides used in treatment of herpesvirus infections.

Apoptotic cell death is critical for maintaining integrity of the epithelia as well as for removal of the virus infected cells. We assessed the role of Fas/FasL-dependent pathway in apoptosis of genital epithelium during HSV-2 infection using a murine model of HSV-2 infection applied to C57BL6, MRL-Fas(lpr)/J (Fas-/-) and C3-Fasl(gld)/J (FasL-/-) mice and an in vitro model of HSV-2 infection in monocyte RAW 264.7 and keratinocyte 291.03C cell cultures and peritoneal macrophages. In contrast to keratinocyte in vitro cultures, HSV-2 infection of the monocytic cell cultures led to early induction of apoptosis. HSV-2 infection of peritoneal macrophages isolated from Fas- and FasL-deficient mice showed decreased activation of apoptosis, which could be further blocked by caspase-9 inhibitor. Infection of Fas and FasL-deficient mice increased the percentage of apoptotic cells and activation of caspase-9 in the vaginal tissue in comparison to C57BL6 wild type strain. Furthermore, Fas and FasL-deficient mice showed increased infiltration of neutrophiles in the vaginal mucosal epithelium at 3 and 7 day of infection in contrast to HSV-2 infected wild-type mice. Our results show that while the Fas/FasL pathway during HSV-2 infection of the vaginal epithelium plays an important role in controlling early local inflammatory response, mitochondrial apoptotic pathway also becomes activated by the inflammatory reaction., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Hydrolyzable tannins are known to exhibit diverse biological effects, which can be used in combination with silver nanoparticles (AgNPs). In this study, we tested toxic and inflammatory properties of tannic-acid modified 13, 33, 46 nm and unmodified 10-65 nm AgNPs using murine 291.03C keratinocyte and RAW 264.7 monocyte cell lines. Both cell lines exposed for 24h to 1-10 μg/ml of 13 nm, 33 nm, 46 nm and unmodified AgNPs showed dose-dependent toxicity and decreased cell proliferation. Only small-sized AgNPs induced production of ROS by monocytes, but not keratinocytes. Monocytes internalized large aggregates of 33, 46 nm and 10-65 nm AgNPs in cytoplasmic vacuoles, whereas keratinocytes accumulated less particles. AgNPs of 13 nm were localized ubiquitously within both cell types. The tested AgNPs strongly down-regulated production of tumor necrosis factor-α (TNF-α) by monocytes, whereas keratinocytes exposed to AgNPs showed an opposite effect. Unmodified but not tannic acid-modified AgNPs increased production of the pro-inflammatory MCP-1 by monocytes and keratinocytes. In summary, low inflammatory potential and lack of ROS production by tannic-acid modified AgNPs sized above 30 nm suggests that tannic acid modification of large silver nanoparticles may help to increase AgNPs biosafety., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Monocytic cells represent important cellular elements of the innate and adaptive immune responses in viral infections. We assessed the role of Fas/FasL in promoting monocyte apoptosis during HSV-2 infection by using an in vitro model based on the murine RAW 264.7 monocytic cell line and an in vivo murine model of HSV-2 infection applied to C57BL6, MRL-Fas(lpr)/J (Fas-/-) and C3-Fasl(gld)/J (FasL-/-) mice. HSV-2 infection of the monocytic cell line led to early induction of apoptosis, with no protective expression of anti-apoptotic Bcl-2. HSV-2 infected monocytes up-regulated Fas and FasL expression early during in vitro infection but were susceptible to Fas induced apoptosis. The vaginal monocytes in the HSV-2 murine model of infection up-regulated FasL expression and were susceptible to Fas induced apoptosis. HSV-2 infection of Fas and FasL- deficient mice led to decreased apoptosis of monocytes and impaired recruitment of NK, CD4+ and CD8+ T cells within the infection sites. The vaginal lavages of HSV-2 infected Fas and FasL- deficient showed decreased production of CXCL9, CXCL10 and TNF-α in comparison to HSV-2 infected wild-type mice strain. The decreased recruitment of immune competent cells was accompanied by delayed virus clearance from the infected tissue. Triggering of the Fas receptor on HSV-2 infected monocytes in vitro up-regulated the expression of CXCL9 chemokines and the cytokine TNF-α. Our study provides novel insights on the role of Fas/FasL pathway not only in apoptosis of monocytes but also in regulating local immune response by monocytes during HSV-2 infection.

To assess the role of mannosylated lipoarabinomannan (ManLAM) in the inflammatory and apoptotic response of mycobacteria-infected and uninfected, bystander cells we applied a mouse macrophage model of infection with avirulent strains--Mycobacterium bovis BCG, Mycobacterium tuberculosis (MTB) H37Ra and compared with a virulent MTB H37Rv strain infection. ManLAM contributed to the infection of macrophages by protection from apoptosis with stabilized Bcl-2 expression and down-regulated Bax expression for infected cells (BCG) or with stabilized Bcl-2 expression for uninfected bystander target cells (H37Ra). Additionally, ManLAM up-regulated FasL expression on the infected cells. Active extracellular signal-regulated kinase (ERK1/2) in BCG and H37Rv infection provided an anti-apoptotic effect by stabilization of anti-apoptotic Bcl-2 expression in the infected cells. Inhibitors specific for c-Jun-NH2-terminal kinase or stress-activated kinase (JNK) and p38 kinase decreased apoptosis of infected cells (BCG, H37Ra) and of uninfected bystanders (H37Ra) by down-regulating Bax. ManLAM significantly down-regulated production of pro-inflammatory IL-12 and TNF-alpha and activation of JNK by both avirulent strains. We conclude that by stabilization of Bcl-2 expression, down-regulation of JNK activity and down-regulation of pro-inflammatory cytokines production ManLAM can contribute to suppression of apoptosis and inflammatory reaction of uninfected, bystander cells., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Newborn higher vertebrates are largely immuno-incompetent and generally survive infections--including poxviruses--by maternal antibody protection. Here, we show that mice survived epidemics as adults only if exposed to lethal orthopoxvirus infections during infancy under the umbrella of maternal protective antibodies. This implies that both the absence of exposure to infection during early infancy or of effective vaccination renders the population highly susceptible to new or old re-emerging pathogens.

The mannosylated lipoarabinomanan (ManLAM) from mycobacterial species possesses strong anti-apoptotic action. Here we examined the ability of ManLAM isolated from Mycobacterium tuberculosis H37Rv to alter expression profiles of apoptosis-related genes in mouse macrophages infected with Mycobacterium bovis BCG Danish strain. ManLAM suppressed BCG-induced apoptosis and activities of caspase-1, -3, -8 and 9. Mouse Apoptosis Gene Array showed that ManLAM significantly down-regulated pro-apoptotic and proinflammatory genes: caspase-1, -3, -7, -8 and -9, TNF-alpha/TNFSF2, Fas/TNFRSF6, Bax-alpha, as well as IL-12 p35 and iNOS simultaneously up-regulating anti-apoptotic genes such as Bcl-2 and Mcl-1. The effect of ManLAM was contrary to BCG-induced up-regulation of proapoptotic and pro-inflammatory genes and consistent with the functional data.

Background: The guidelines approved by the American Thoracic Society in 2002 definitely recognize the six-minute walk test (6MWT) as a useful tool for the evaluation of physical efficiency in individuals with at least moderate chronic obstructive pulmonary disease, heart failure and intermittent dysbasia. So far, the American Thoracic Society has not approved the use of a treadmill to determine the six-minute walking distance (6MWD) because patients are unable to pace themselves on a treadmill. The purpose of our work was to prove that these problems could be avoided if physical efficiency is evaluated with the use of a modified treadmill., Methods: The work evaluates the function of a treadmill able to adjust its speed to the walking speed of healthy volunteers. The evaluation is based on a comparison of the distance covered by the healthy volunteers and the comfort of the test on the treadmill during six minutes with the distance covered and comfort during the same period in a 22-metre-long hallway in 29 healthy volunteers. Non-invasive blood pressure and pulse measurements were taken immediately before and after the test., Results: The average distance covered during the six-minute period on the treadmill was 57.1 m longer than in the hallway. The comfort of the treadmill test was indicated to be better by 18 subjects, worse by 4 subjects and identical by 7 subjects., Conclusions: The tests confirm that the speed of the modified treadmill adjusts properly to the walking speed of the healthy volunteers. The hemodynamic effects were identical for the healthy volunteers both in the hallway and treadmill tests. The distance differences were caused by turnarounds in the corridor test. The results obtained with the special treadmill allow us to develop a new method and, at present, provide a basis for a second stage of research comprising subjects with diagnosed heart failure. (Cardiol J 2007; 14: 447-452).

The mannosylated lipoarabinomannan (ManLAM) from mycobacterial species possesses strong immunomodulatory effects. Here we examined the ability of Mycobacterium tuberculosis ManLAM to interfere with the apoptotic response of mouse monocyte cell line, RAW 264.7 infected with Mycobacterium bovis BCG Danish strain. Incubation of BCG-infected monocytes with ManLAM decreased production of NO and the numbers of apoptotic cells which synergized with the polarization of mitochondrial membrane. Activities of caspase-1, -3, -8 and 9 followed pattern of apoptosis suppression by ManLAM, except for caspase-1, which showed no significant change in activity. ManLAM also stabilized anti-apoptotic ratio of bcl-2/bax expression in BCG-infected cells and blocked activation of Fas/FasL-induced pathway of apoptosis. Thus, ManLAM, apart from blocking mitochondrial pathway of apoptosis, may induce several other pathways regulating apoptotic response in BCG-infected mouse monocytes.

In this study we showed that the virulent Moscow strain of Ectromelia virus (ECTV-MOS) infection leads to induction of apoptosis in the BALB/c mouse central nervous system. ECTV-MOS-infected cells and inflammation sites were found in brain parenchyma between 5 and 15 days after footpad infection with ECTV-MOS. Infected cells consisted of microglia and monocytes, astrocytes and oligodendrocytes and these type of cells underwent apoptosis within 5-15 days post infection (d.p.i.). The highest number of apoptotic cells was found at 5 and 10 d.p.i. and represented mainly microglia (61.4% and 38.6% of apoptotic cells, respectively) and astrocytes (21% and 8.9%, respectively). The number of apoptotic oligodendrocytes was 5.4% and 4.5%, respectively. Fluorometric assays demonstrated involvement of caspase-1, -3 and -8 but not caspase-9 in apoptosis in ECTV-MOS-infected mouse brains. Expression of Fas/FasL was significantly increased on ECTV-MOS-infected cells between 5 and 15 d.p.i., whereas Fas was up-regulated also on the surrounding, non-infected cells. Taking together we may conclude that ECTV-MOS infection of microglia and astrocytes leads to local inflammation resulting in Fas/FasL up-regulation and apoptosis, which limits mouse central nervous system infection with ECTV-MOS.

Mastitis remains a major cause of economic losses in dairy herds. It is believed, that the enhancement of natural defense mechanisms in mammary gland may be helpful in the treatment of bovine mastitis. Our study was designed to assess the apoptosis of leukocytes isolated from bovine milk during subclinical staphylococcal mastitis. Milk samples were collected from cows naturally infected with one pathogen--Staphylococcus aureus and from animals with mastitis caused by several pathogens, including S. aureus. It has been determined that the rate of apoptosis was lower in mastitic milk, as compared with control samples, although varied significantly between groups. High percentage of apoptotic cells was detected in milk with high counts of pathogenic bacteria. In all groups the rate of apoptosis was dependent on the bacterial load, although various correlations were identified. Thus, it is postulated, that the rate of apoptosis of somatic cells in mastitic milk is related to the etiology of infection and is determined by the bacterial load.

Objective: Elevated levels of interleukin (IL)-7 are present in the blood of HIV-positive patients and it is known that IL-7 receptor (IL-7R)alpha expression decreases on T cells during HIV infection. The subset(s) of T cells with low IL-7Ralpha and the consequence of low IL-7Ralpha expression for T-cell survival are poorly characterized., Design: The frequency of IL-7Ralpha-negative T cells in HIV-positive patients was studied in relation to CD4 T-cell counts, IL-7 concentration and survival in culture. We analysed IL-7Ralpha expression in different T-cell populations and in relation to Bcl-2 expression., Methods: Specimens from 38 HIV-1 patients and 17 controls were examined. IL-7Ralpha and Bcl-2 expression in different T-cell populations was studied by flow cytometry. The influence of IL-7Ralpha expression on T-cell survival was studied by culturing T cells in the presence of IL-7., Results: Down-regulation of IL-7Ralpha on T cells correlated with depletion of CD4 T cells (P < 0.001) and also with increased concentration of serum IL-7 (P < 0.05). The decreased IL-7Ralpha expression was associated with low Bcl-2 expression and with the reduced survival capacity of T cells in the presence of IL-7 in vitro. Particularly, T cells with memory phenotype showed a decreased IL-7Ralpha expression in association with CD28 down-regulation., Conclusions: The positive effects of IL-7 on survival and homeostatic proliferation of T cells might be severely impaired in HIV-infected individuals due to IL-7Ralpha down-regulation. Differentiation towards a CD28-negative memory phenotype in response to chronic activation may lead to an overall decrease of IL-7 mediated survival within the peripheral T-cell pool.

BALB/c mice infected with the Moscow strain of Ectromelia virus (ECTV-MOS) show a large number of apoptotic cells, and an influx of lymphoid cells in the epithelium and substantia propria of conjunctivae, respectively. The presence of ECTV-MOS antigens in the epithelium of conjunctivae significantly upregulates Fas in the epithelial layer and FasL in the suprabasal layer of conjunctiva. Inhibition of FasL with blocking antibodies in cultures of conjunctival cells isolated from ECTV-MOS-infected BALB/c mice showed that the Fas/FasL pathway is important in apoptosis of ECTV-MOS-infected cells. The results also showed that the presence of cytokines, in particular interferon (IFN)-gamma, upregulated expression of Fas. Interleukin (IL) 2, 4, 10 and IFN-gamma were produced at the peak of conjunctivitis (at day 15 of infection) with a predominance of IFN-gamma and a small, but significant, production of IL4 and IL10 compared with non-infected animals. These results suggest that not only is Fas/FasL expression in conjunctiva involved in elimination of migrating Fas+ cells but also plays an important role in the turnover of conjunctival epithelium and thus may be crucial for ECTV spreading to the surrounding environment.