Polyphenol-Conjugated Bimetallic Au@AgNPs for Improved Wound Healing.

BACKGROUND: 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.

The synthesis of monodisperse silver nanoparticles with plant extracts.

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.

Antiviral Activity of Tannic Acid Modified Silver Nanoparticles: Potential to Activate Immune Response in Herpes Genitalis.

(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.

Tannic Acid-Modified Silver and Gold Nanoparticles as Novel Stimulators of Dendritic Cells Activation.

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.

Human herpesvirus type 1 and type 2 disrupt mitochondrial dynamics in human keratinocytes.

Mitochondrial movement and distribution throughout the cytoplasm is crucial for maintaining cell homeostasis. Mitochondria are dynamic organelles but can be functionally disrupted during infection. Here, we show that the ubiquitous human pathogens HHV-1 and HHV-2 induce changes in the mitochondrial morphology and distribution in the early and late phases of productive infection in human keratinocytes (HaCaT cells). We observed a decrease in the mitochondrial potential at 2 h postinfection and a decrease in cell vitality at 24 h postinfection. Moreover, we found that mitochondria migrated to the perinuclear area, where HHV-1 and HHV-2 antigens were also observed, mainly in the early stages of infection. Positive results of real-time PCR showed a high level of HHV-1 and HHV-2 DNA in HaCaT cells and culture medium. Our data demonstrate that HHV-1 and HHV-2 cause mitochondrial dysfunction in human keratinocytes.

Tannic acid-modified silver nanoparticles for wound healing: the importance of size.

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-ß and TGF-ß1 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.

Multifunctional Tannic Acid/Silver Nanoparticle-Based Mucoadhesive Hydrogel for Improved Local Treatment of HSV Infection: In Vitro and In Vivo Studies.

Mucoadhesive gelling systems with tannic acid modified silver nanoparticles were developed for effective treatment of herpes virus infections. To increase nanoparticle residence time after local application, semi solid formulations designed from generally regarded as safe (GRAS) excipients were investigated for their rheological and mechanical properties followed with ex vivo mucoadhesive behavior to the porcine vaginal mucosa. Particular effort was made to evaluate the activity of nanoparticle-based hydrogels toward herpes simplex virus (HSV) type 1 and 2 infection in vitro in immortal human keratinocyte cell line and in vivo using murine model of HSV-2 genital infection. The effect of infectivity was determined by real time quantitative polymerase chain reaction, plaque assay, inactivation, attachment, penetration and cell-to-cell assessments. All analyzed nanoparticle-based hydrogels exhibited pseudoplastic and thixotropic properties. Viscosity and mechanical measurements of hydrogels were found to correlate with the mucoadhesive properties. The results confirmed the ability of nanoparticle-based hydrogels to affect viral attachment, impede penetration and cell-to-cell transmission, although profound differences in the activity evoked by tested preparations toward HSV-1 and HSV-2 were noted. In addition, these findings demonstrated the in vivo potential of tannic acid modified silver nanoparticle-based hydrogels for vaginal treatment of HSV-2 genital infection.

A model for the optimization of anti-inflammatory treatment with chemerin.

Routine treatment of mild to moderate pain with a combination of non-steroidal anti-inflammatory drugs such as paracetamol in combination with corticoid opioids can lead to severe complications including death from gastrointestinal injury or to drug dependence. There is a need for the development of new safer drugs. Chemerin is a mediator promoting resolution of inflammation and it is then a promising candidate for a new treatment. A pilot experimental work using the zymosan-induced peritonitis model has found that injecting extra chemerin resulted in an approximately 1% reduction in the total number of inflammatory cells recruited. This paper combines and adapts existing mathematical models of inflammation to reproduce these results and to explore the therapeutic potential of chemerin through simulations. Analysis of the model predicts that the injection of chemerin at a concentration of 2000 ng ml-1 within the first 5 min of inflammation onset leads to maximal inflammation inhibition. The degree of inhibition is shown to be sensitive to data used for the fit with a mean inhibition of 22 ± 3.7% for a series of remove-one bootstrap tests, whereas optimal chemerin injection parameters were not. Overall sensitivity analysis identifies parameters of the model that need to be measured more accurately or with increased sampling rate to improve model robustness and confirm chemerin's therapeutic potential.

The role of tannic acid and sodium citrate in the synthesis of silver nanoparticles.

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.

A mathematical model of cellular swelling in Neuromyelitis optica.

Neuromyelitis Optica (NMO) is a severe neuro-inflammatory disease of the central nervous system characterized by predominant damage to the optic nerve and of the spinal cord. The pathogenic antibody found in the majority of patients targets the AQP4 channels on astrocytic endfeet and causes the cells to swell. Although, the pathophysiology of the disease is broadly known, there are no specific targeted treatments for this process clinically available nor accurate prognostic markers both during attacks and for predicting long term neuronal damage. This lack is, in part, due to the rarity of the disease and its relatively recent pathogenic clarity. Hence, the ability to mathematically model the progress of the condition to test prospective therapies in silico would be a step forward. This paper combines state of the art models of cellular metabolism and cytotoxic oedema in neurons and astrocytes and augments it with a detailed characterization of water transport across the cellular membrane. In particular, we capture the process of perforation of the cell through the human complement cascade and resulting water and ionic fluxes. Simulating NMO by injecting its antibody and human complement into the extracellular space showed a 25% increase of the astrocytic volume after 12 h from onset. Most of the volume change occurred during the first 30 min of simulation with a peak volume change of 38%. The model was further adapted to simulate the therapeutic potential of CD59. It was found that there is a threshold of CD59 concentration that can prevent the swelling of astrocytes. Since the astrocyte volume changes mostly during the first hour, further experimental work should focus on this time scale to provide data for further model refinement and validation.

The effect of feeding mice during gestation and nursing with Rhodiola kirilowii extracts or epigallocatechin on CD4 and CD8 cells number and distribution in the spleen of their progeny.

Rhodiola kirilowii, a member of Crassulaceae family, grows wildly in Asiatic mountains and is also cultivated in some European countries. Its underground parts traditionally are used for enhance physical and mental performance of the body. In our previous papers we reported immuno- and angio-modulatory effects of aqueous and hydro-alcoholic extracts of radix and rhizome of this plant in mice. In the present work we evaluated the effect of Rhodiola kirilowii water- (RKW) or hydro-alcoholic (RKW-A) extracts and epigallocatechin (one of the polyphenols present in these extracts) given to mice, during pregnancy and nursing period, on the number and localization of CD4+ and CD8+ cells in spleens of adult progeny mice. Previously, we observed several abnormalities in functionality of spleen cells collected from these mice. No differences in CD4+ T cells localisation or numbers were found between all tested mice groups. In contrast, CD8+ T cells localisation and staining were altered in progeny of water or alcohol extract-fed mice. CD8+ T cells were found not only in the PALS but also in the B cell follicle and in the red pulp. Furthermore, CD8+ T cells from T cell zones in the progeny of extract-fed mice showed much intensive staining for CD8 antigen and significantly higher numbers per area in comparison to control mice.

Dendritic cells during mousepox: The role of delayed apoptosis in the pathogenesis of infection.

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.

Involvement of Fas/FasL pathway in the murine model of atopic dermatitis.

OBJECTIVE AND DESIGN: The aim of this study was to elucidate the role of apoptosis mediated through Fas/FasL pathway using the mouse model of atopic dermatitis (AD). MATERIALS AND TREATMENT: AD was induced by epicutaneous application of ovalbumin (OVA) in wild-type C57BL/6, B6. MRL-Faslpr/J (Fas-) and B6Smn.C3-Faslgld/J (FasL-) mouse strains. METHODS: Skin samples were subjected to staining for Fas/FasL expression, M30 epitope and assessment of inflammatory response via immunohistochemical staining. Cytokine and chemokine production was assessed by real-time PCR. RESULTS: In comparison to wild-type mice, OVA sensitization of Fas- and FasL-deficient mice led to increased epidermal and dermal thickness, collagen deposition and local inflammation consisting of macrophages, neutrophils and CD4+ T cells. Fas- and FasL-deficient mice showed increased total counts of regulatory T cells (Tregs) and IgE levels in blood as well as increased expression of IL-1ß, IL-4, IL-5, IL-13 and TGF-1ß mRNA in comparison to wild-type mice. On the other hand, expression of CXCL9 and CXCL10, IL-17 mRNAs in the skin samples in Fas- and FasL-deficient mice was decreased. CONCLUSIONS: Our results show that lack of the Fas-induced apoptosis leads to exacerbation of AD characteristics such as Th2 inflammation and dermal thickening. Therefore, Fas receptor can play an important role in AD pathogenesis by controlling development of the local inflammation.

Toxicity of tannic acid-modified silver nanoparticles in keratinocytes: potential for immunomodulatory applications.

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.

Hydrogen storage materials: room-temperature wet-chemistry approach toward mixed-metal borohydrides.

The poor kinetics of hydrogen evolution and the irreversibility of the hydrogen discharge hamper the use of transition metal borohydrides as hydrogen storage materials, and the drawbacks of current synthetic methods obstruct the exploration of these systems. A wet-chemistry approach, which is based on solvent-mediated metathesis reactions of precursors containing bulky organic cations and weakly coordinating anions, leads to mixed-metal borohydrides that contain only a small amount of "dead mass". The applicability of this method is exemplified by Li[Zn2(BH4)5] and M[Zn(BH4)3] salts (M=Na, K), and its extension to other systems is discussed.

Tannic acid modified silver nanoparticles show antiviral activity in herpes simplex virus type 2 infection.

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.

Role of Fas/FasL signaling in regulation of anti-viral response during HSV-2 vaginal infection in mice.

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-ß1. 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.

An approach to the symbolic representation of brain arteriovenous malformations for management and treatment planning.

INTRODUCTION: There is currently no standardised approach to arteriovenous malformation (AVM) reporting. Existing AVM classification systems focuses on angioarchitectural features and omit haemodynamic, anatomical and topological parameters intuitively used by therapists. METHODS: We introduce a symbolic vocabulary to represent the state of an AVM of the brain at different stages of treatment. The vocabulary encompasses the main anatomic and haemodynamic features of interest in treatment planning and provides shorthand symbols to represent the interventions themselves in a schematic representation. RESULTS: The method was presented to 50 neuroradiologists from 14 countries during a workshop and graded 7.34 ± 1.92 out of ten for its usefulness as means of standardising and facilitating communication between clinicians and allowing comparisons between AVM cases. Feedback from the survey was used to revise the method and improve its completeness. For an AVM test case, participants were asked to produce a conventional written report and subsequently a diagrammatic report. The two required, on average, 6.19 ± 2.05 and 5.09 ± 3.01 min, respectively. Eighteen participants said that producing the diagram changed the way they thought about the AVM test case. CONCLUSION: Introduced into routine practice, the diagrams would represent a step towards a standardised approach to AVM reporting with consequent benefits for comparative analysis and communication as well as for identifying best treatment strategies.

Fas/FasL pathway participates in resolution of mucosal inflammatory response early during HSV-2 infection.

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.