A STING-dependent innate-sensing pathway mediates resistance to corneal HSV-1 infection via upregulation of the antiviral effector tetherin.

Type 1 interferons (IFNs; IFNα/ß) mediate immunological host resistance to numerous viral infections, including herpes simplex virus type 1 (HSV-1). The pathways responsible for IFNα/ß signaling during the innate immune response to acute HSV-1 infection in the cornea are incompletely understood. Using a murine ocular infection model, we hypothesized that the stimulator of IFN genes (STING) mediates resistance to HSV-1 infection at the ocular surface and preserves the structural integrity of this mucosal site. Viral pathogenesis, tissue pathology, and host immune responses during ocular HSV-1 infection were characterized by plaque assay, esthesiometry, pachymetry, immunohistochemistry, flow cytometry, and small interfering RNA transfection in wild-type C57BL/6 (WT), STING-deficient (STING(-/-)), and IFNα/ß receptor-deficient (CD118(-/-)) mice at days 3-5 postinfection. The presence of STING was critical for sustained control of HSV-1 replication in the corneal epithelium and resistance to viral neuroinvasion, but loss of STING had a negligible impact with respect to gross tissue pathology. Auxiliary STING-independent IFNα/ß signaling pathways were responsible for maintenance of corneal integrity. Lymphatic vessels, mast cells, and sensory innervation were compromised in CD118(-/-) mice concurrent with increased tissue edema. STING-dependent signaling led to the upregulation of tetherin, a viral restriction factor we identify is important in containing the spread of HSV-1 in vivo.

Gunshot induced indirect femoral fracture: mechanism of injury and fracture morphology.

INTRODUCTION: Indirect ballistic fractures occur when a projectile passes close to, but not contacting, the bone. The mechanism of how these fractures occur is not yet proven, but recently the acoustic shockwave has been excluded as a cause. The objective of this study is to determine whether the expanding temporary cavity, the collapse of this cavity or its oscillation causes these fractures. In addition, we describe the fracture morphology and biomechanical causes of this injury. METHOD: 40 fresh deer femora were strain gauged and embedded in ballistic gelatin before being shot with four different projectiles with varying distances off the bone. Pressure recordings, chronographs and radar allowed assessment of local pressures and energy transfer. High-speed video allowed the temporal relationship between the temporary cavity and fracture formation to be analysed, while sample dissection allowed the fracture morphology to be described. RESULTS: The fractures produced were consistently wedge-shaped and caused by the expansion of the temporary cavity, flexing the bone beyond its yield point, causing tension failure on the cortex opposite the expanding temporary cavity and a compression wedge on the side of the cavity. Local pressure was not predictive of fracture formation but the energy transfer to the gelatin block was predictive. CONCLUSIONS: Indirect fractures are caused by the expansion of the temporary cavity and relate to the proximity of this cavity to the bone. Fractures occur from flexion of the bone and classically display wedge-shaped fracture patterns with the apex of the wedge pointing away from the expanding cavity.

IFN-α-driven CCL2 production recruits inflammatory monocytes to infection site in mice.

Herpes simplex virus type 1 (HSV-1) is the leading cause of corneal blindness in the developed world due to reactivation of infectious virus and the subsequent immune response. The innate response that facilitates viral control in the cornea is currently unknown. In the present study using a mouse chimera model, we found that a bone marrow component is crucial in inhibiting viral replication and identified inflammatory monocytes (F4/80(+) Gr1(+)) as the responsible cell. CCL2 was critical for recruiting inflammatory monocytes, and a loss of this chemokine in CCL2(-/-) mice resulted in a loss of viral containment and inflammatory monocyte recruitment. To confirm these results, clodronate depletion of inflammatory monocytes resulted in elevated viral titers. Furthermore, siRNA targeting the innate sensor p204/IFI-16 resulted in a loss of CCL2 production. In conclusion, CCL2 expression driven by IFI-16 recognition of HSV-1 facilitates the recruitment of inflammatory monocytes into the cornea proper to control viral replication.

Pretibial injury: key factors and their use in developing laboratory test methods.

Aims were to 1 characterize pretibial injuries and evaluate protection offered by garments/fabrics; and 2 develop a laboratory test to determine the potential protection provided by such fabrics. Most (>85%) of 75 patients treated for pretibial injury at Hutt Hospital, New Zealand sustained injury to one site and required surgery. Injuries were typically grade 3 or 4, 10-250 mm wide 30-350 mm long, and at the mid- to lower third of the tibia. The severity grade was lower when at least one fabric layer covered the site, slightly lower again with more than one layer, and when a knitted fabric/garment was worn, and a trouser type garment. Laboratory test methods and their application reflected these known variables. The force transmitted through multiple fabric layers was less then through one layer: thick pantyhouse and either denim or fabrics used in 'sweat pants' would minimize transmitted force and maximize impulse.

The application of a plasmid DNA encoding IFN-alpha 1 postinfection enhances cumulative survival of herpes simplex virus type 2 vaginally infected mice.

Using a hormonally induced susceptibility mouse model to investigate vaginal HSV type 2 (HSV-2) infection, a study was undertaken to determine the efficacy of a plasmid DNA encoding IFN-alpha1 introduced into the vaginal lumen postinfection (PI). Mice infected with HSV-2 intravaginally and treated intravaginally 24 h later with 100 microg DNA encoding IFN-alpha1 showed enhanced survival (10/15) in comparison to mice treated with 100 microg plasmid DNA vector alone (3/10) or vehicle (4/27). In contrast, mice receiving recombinant IFN-alphaA (5-500 U/vagina) 24 h PI showed no significant survival in comparison to the vehicle (saline)-treated group. The protective effect was time dependent in that mice receiving the IFN-alpha1 transgene 48 h PI succumbed at a rate similar to the plasmid DNA vector-treated group. The increase in cumulative survival elicited by the transgene corresponded with a reduction in viral replication and Ag expressed in the vaginal epithelium early (i.e., 3 days PI) during acute infection and replicating virus recovered in the spinal cord day 7 PI. By day 7 PI, HSV-2 glycoprotein B transcript expression was no longer detectable in vaginal tissue from the IFN-alpha1 transgene-treated group (0/8) compared with levels expressed in plasmid vector-treated controls (4/6 mice surveyed were positive). Collectively, these results suggest the application of DNA encoding type I IFN is an effective and alternative approach to currently prescribed therapies in controlling vaginal HSV-2 infection by antagonizing viral replication.

Expression of human and macaque type I IFN transgenes interferes with HSV-1 replication at the transcriptional and translational levels: IFN-beta is more potent than IFN-alpha 2.

A study was undertaken to compare the efficacy of plasmid constructs encoding human IFN-alpha 2 and IFN-beta and macaque IFN-beta against herpes simplex virus type 1 in transfected cells. All type I IFN transgenes significantly reduced viral titers in transfected cells by 3 logs. Human IFN-alpha 2-transfected cells produced significantly more IFN (2274 pg/ml) in comparison to IFN-beta-transfected cells (134-165 pg/ml). Viral lytic gene transcript and viral protein levels were lower in IFN-beta- versus IFN-alpha 2-transfected cells, which coincided with elevated PKR and OAS transcript levels and increased total STAT1 and phosphorylated STAT1 (Y701) protein levels in the IFN-beta-transfected cells. Although comparable viral titers were recovered in IFN-alpha 2 and IFN-beta plasmid-transfected cells, IFN-alpha 2 plasmid-transfected cells exhibited significantly more cytopathic effect compared to the IFN-beta transgene-transfected cells. In addition, IFN-alpha 2 transgene-transfected, infected cells displayed a cell cycle profile similar to that of vector-transfected, infected cells, whereas IFN-beta plasmid-transfected cells displayed a profile similar to uninfected control. Collectively, the results indicate that human IFN-beta is superior to IFN-alpha 2 in antagonizing herpes simplex virus type 1 infection.

Transgenic expression of interleukin-6 in the central nervous system confers protection against acute herpes simplex virus type-1 infection.

IL-6 is a pro-inflammatory cytokine that has previously been associated with herpes simplex virus type 1 reactivation. To further investigate this relationship during acute infection, ocular HSV-1 infection was studied in transgenic mice homozygous or heterozygous expression of IL-6 by astrocytes in the central nervous system. The virus load in both the eye and trigeminal ganglia was significantly reduced at day 6 but not day 3 post infection in the homozygous IL-6 transgenic mice compared to the wild type and heterozygous littermates. IL-6 protein and mRNA levels in the eye coincided with the level of transgene expression in mice acutely infected with virus (i.e., day 3 post infection). Likewise, IL-6 transcript levels in the TG mirrored the expression of the transgene in the mice throughout the course of the infection into latency. The HSV-1 alpha lytic phase gene ICP27 was rapidly down-regulated by day 6 post infection in the TG of homozygous IL-6 transgenic mice compared to the wild type and heterozygous littermates. The resistance to acute HSV-1 infection in the homozygous IL-6 transgenic mice correlated with a significant elevation in IFN-alpha/beta in the eye compared to the wild type or heterozygous IL-6 transgenic animals. Heterozygous and homozygous IL-6 transgenic mice latently infected with HSV-1 showed elevated anti-HSV-1 antibody titers compared to the latently infected wild type controls. Collectively, the results suggest dose-dependent IL-6 antagonism of acute HSV-1 infection in vivo.

The relationship between interleukin-6 and herpes simplex virus type 1: implications for behavior and immunopathology.

Cytokines are hormones once thought to be restricted to the immune system produced solely by hematopoietic-derived cells and acting on receptors expressed by cells of the immune system. However, it is now clear that many cytokines are produced not only by lymphocytes, monocytes, granulocytes, and dendritic cells but are also synthesized by cells outside the realm of the immune system in response to stimuli that may not be associated with immune homeostasis. In fact, there is evidence supporting a role of selected cytokines modifying behavior and neuroendocrine function. Recently, a potential relationship between the cytokine interleukin (IL)-6 and herpes simplex virus type 1 (HSV-1) reactivation has been found. This article discusses the relevance of these findings and considers the potential impact that HSV-1 infection has on behavior and chronic inflammatory processes that can occur in the nervous system during "latent" virus infection as a result of chronic IL-6 expression.

Morphine reduces mortality in mice following ocular infection with HSV-1.

To determine the effect of chronic opioid treatment on mice infected with herpes simplex virus (HSV-1), female ICR mice were administered saline or morphine (25 mg/kg) subcutaneously (s.c.) 3 X /day and infected five days later via corneal scarification and inoculation with 150-210 plaque forming units of HSV-1. Mice deprived of morphine succumbed following infection faster than morphine- or saline-maintained mice, and morphine-maintained mice had a higher cumulative survival than either saline-maintained or morphine-deprived mice. There was no significant difference in cytokine mRNA or protein levels by RT-PCR and ELISA, respectively, in the eyes, trigeminal ganglia, cerebellum, or brain stem, comparing morphine-maintained to saline-treated mice. Similarly, there were no differences in the viral load in the eyes and trigeminal ganglia during the acute infection comparing these two groups assayed three and six days post-infection. While there were no differences in the expression of viral transcripts in the eyes and trigeminal ganglia during the acute infection, HSV-1 infected cell polypeptide 27 expression was reduced in the brain stem of morphine-maintained mice. Collectively, the results suggest that mice maintained on morphine antagonize the spread of HSV-1 in the central nervous system and thus, reduce the incidence of viral-induced encephalitis.

Push-pull sorbent-based pheresis treatment in an experimental canine endotoxemia model: preliminary report.

The Biologic-DTPF System (DTPF), an extracorporeal blood treatment device with potential to treat sepsis, was tested in a preliminary study using a canine endotoxemia model. Six dogs were used and they formed four treatment groups, as control group (n=1) and three groups based on the type of sorbent present in the plasma filter (PF) system: sham treatment with no sorbent (n=1), charcoal as sorbent (n=2), and charcoal/silica as sorbent ("silica" group, n=2). Cardiodynamic data were recorded before treatment and every 30 minutes, and blood samples were collected to determine blood chemistry and to detect the levels of endotoxin and selected plasma cytokines: interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF). The dogs were given Escherichia coli endotoxin (2 mg/kg) as an intravenous drip (extended over a period of 30 minutes). Thirty minutes after the end of infusion all animals except the control were treated with the DTPF system for four hours. To determine the effect of treatment, data collected at one hour from the initiation of treatment until the end of treatment were compared between control and treated dogs. The endotoxin levels in the control dog were higher (P < 0.05) than other groups. The control dog had lower levels of TNF than other groups. The control dog had similar levels of IL-1 (P > 0.05) and higher levels (P < 0.05) at 4 hours into treatment compared to other groups. The control dog had similar levels of IL-6 as other groups (P > 0.05). In the control dog, the mean arterial pressure (MAP) fell and then remained low but stable at 1-4 hours. The charcoal group had lower MAP than the control dog at 1-4 hours (P < 0.05). The silica group had higher MAP levels similar to the control dog. After treatment, the control dog had higher (P < 0.05) values of hematocrit, hemoglobin, calcium, potassium, and albumin compared to the treated groups. As expected for a system removing plasma during sepsis, the DTPF System had some adverse effects on the physiologic status of the dogs, especially when loaded with charcoal sorbent only. The findings of the present study suggest that the filters are capable of eliminating endotoxin and there is some evidence of cytokine removal. Although the charcoal dogs did poorly, addition of silica to the sorbent offset any negative effects. Further work is underway to improve the efficiency of the system, primarily to enhance the capacity of the sorbents for cytokines. A more realistic canine sepsis model with mortality after several days (the Escherichia coli- infected intraperitoneal clot) will also be considered in future studies.

Cytokines and endotoxin removal by sorbents and its application in push-pull sorbent-based pheresis: the BioLogic-DTPF System.

The BioLogic-DTPF System (DTPF) combines the Biologic-DT hemodiabsorption system (DT) in series with the Biologic PF push-pull pheresis system (PF) in which PF membranes separate plasma for direct contact between plasma proteins and the sorbents. Preliminary studies conducted in bovine serum albumin (BSA) solution and in bovine plasma allowed charcoal and silica to be evaluated as adsorbents for the PF module. Equilibrium binding experiments in BSA showed a high capacity of cytokine (IL-1 beta, TNF alpha) binding by powdered charcoal, 70-90 ng/g. Kinetic binding studies in bovine plasma revealed relatively quick adsorption of IL-1 beta and IL-6 by charcoal with the capacity range of 1.2-2.0 ng/g for tested cytokines (IL-1 beta and TNF alpha). Further laboratory studies with plasma have shown that powdered silica has an even greater binding capacity, up to 13 ng/g for TNF alpha depending upon particle size, and more rapid binding for all tested cytokines than powdered charcoal. Cholestyramine is a more efficient sorbent for removal of endotoxin than either charcoal or silica. In vitro tests using whole blood have demonstrated that the DTPF, with powdered charcoal as the sorbent, clears cytokines (TNF alpha, IL-1 beta, and IL-6) at 12.6-23.4 ml/min, bilirubin at 17.8-34.7 ml/min, and creatinine at 53.6-82.6 ml/min. The removal of some cytokines during the first clinical trial is also discussed.

Lymphocytes delay kinetics of HSV-1 reactivation from in vitro explants of latent infected trigeminal ganglia.

A persistent immune response to herpes simplex virus type 1 (HSV-1) is evidenced by the expression of cytokine transcripts along with infiltrating mononuclear cells in the ganglia of latently infected mice. In trigeminal ganglion (TG) explant co-cultures, the presence of nonirradiated or irradiated primed splenocytes significantly reduced HSV-1 reactivation as defined by secreted infectious HSV-1 found in the supernatants of TG explant cultures. Primed splenocytes depleted of CD4+ or CD8+ cells did not antagonize HSV-1 reactivation. Cytokines including interleukin (IL)-2, IL-6, IL-10, and IL-12 were all detected in the TG explant cultures containing splenocytes. To further study the role of cytokines in HSV-1 reactivation, dissociated TG cell cultures were treated with exogenous recombinant cytokines including IFN-alpha or -gamma, IL-4, 6, 10, 12 or tumor necrosis factor (TNF)-alpha at concentrations ranging from 2.0 pg to 2.0 ng/culture (or 0.3-300 units/culture for the IFNs). While no cytokines tested at any concentration significantly modified HSV-1 reactivation, neutralizing antibody to IL-6, but not to IFN-alpha/beta, significantly antagonized HSV-1 reactivation. Collectively, the results suggest that IL-6 is directly or indirectly involved in HSV-1 reactivation in TG explant cultures.

Ectopic expression of DNA encoding IFN-alpha 1 in the cornea protects mice from herpes simplex virus type 1-induced encephalitis.

A novel approach to combat acute herpes simplex virus type 1 (HSV-1) infection has recently been developed by administration with a plasmid DNA construct encoding cytokine genes. Cytokines, especially type I IFNs (IFN-alpha and IFN-beta) play an important role in controlling acute HSV-1 infection. The purpose of the present study was to investigate the potential efficacy of ectopically expressed IFN-alpha 1 against ocular HSV-1 infection following in situ transfection of mouse cornea with a naked IFN-alpha 1-containing plasmid DNA. Topical administration of the IFN-alpha 1 plasmid DNA exerted protection against ocular HSV-1 challenge in a time- and dose-dependent manner and antagonized HSV-1 reactivation. In addition, IFN-alpha 1-transfected eyes expressed a fivefold increase in MHC class I mRNA over vector-treated controls. The protective efficacy of the IFN-alpha 1 transgene antagonized viral replication, as evidenced by the reduction of the viral gene transcripts (infected cell polypeptide 27, thymidine kinase, and viral protein 16) and viral load in eyes and trigeminal ganglia during acute infection. The administration of neutralizing Ab to IFN-alpha beta antagonized the protective effect of the IFN-alpha 1 transgene in mice. Collectively, these findings demonstrate the potential of using naked plasmid DNA transfection in the eye to achieve ectopic gene expression of therapeutically active agents.

Systemic inflammatory response syndrome treatment by powdered sorbent pheresis: the BioLogic-Detoxification Plasma Filtration System.

Systemic inflammatory response syndrome (SIRS) is one of the most common causes of death in intensive care unit patients. The detoxification plasma filtration (DTPF) system (HemoCleanse, Inc., West Lafayette, IN) combines the DT hemodiabsorption system in series with a push-pull pheresis PF system (a suspension of powdered sorbents surrounding 0.5 microm plasma filter membranes). Bidirectional plasma flow (at 80-100 ml/min) across the PF membranes provides direct contact between plasma proteins and powdered sorbents, as well as clearance of cytokines (tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6) at a rate of 15-25 ml/min, without evidence of saturation for 90 minutes. In a U.S. Food and Drug Administration approved study we treated eight patients with SIRS and organ failure with a single DTPF treatment, using powdered charcoal as sorbent in four patients and powdered charcoal and silica in four patients. Treatments proceeded for 6 hours with proper heparin anticoagulation (activated clotting time 250-300 sec) and appeared safe. All patients improved during the treatments and each had increased blood pressure and decreased need for pressor agents. Plasma cytokine levels stabilized or decreased during treatment and were significantly lower the morning after treatment. Multiple organ dysfunction (MOD) and Acute Physiology Chronic Health Evaluation II scores and organ function gradually improved in most patients, and two patients survived for more than 28 days and two for more than 14 days. The DTPF System may prove beneficial in treatment of patients with sepsis.

Astrocyte-targeted expression of IFN-alpha1 protects mice from acute ocular herpes simplex virus type 1 infection.

Type I IFNs (i.e., IFN-alpha and IFN-beta) play a key role in the host's innate defense against viral pathogens. To examine the biologic relevance of IFN-alpha to a viral pathogen within the confines of the nervous system, IFN-alpha1 transgenic mice whose transgene is under the control of the glial fibrillary acidic protein promoter (GFAP-IFN-alpha, astrocyte specific) were examined for resistance to an ocular herpes simplex virus type 1 (HSV-1) infection. GFAP-IFN-alpha mice expressed significantly higher levels of IFN-alphabeta (533 U) in the trigeminal ganglion compared with nontransgenic mice (70 U) 72 h postinfection that corresponded with a significant reduction in the mRNA expression of the HSV-1 immediate early gene infected cell polypeptide 27 and late gene VP16, as well as the chemokines monocyte-chemoattractant protein-1 and cytokine response gene-2 in the eye and trigeminal ganglion. Six days postinfection, the viral load and the expression of infected cell polypeptide 27, CD8, RANTES, IFN-gamma, and IFN-alpha mRNA levels were reduced in the trigeminal ganglion of GFAP-IFN-alpha mice compared with the wild-type mice. Following the establishment of HSV-1 latency (i.e., 30 days postinfection), only one of nine (11%) GFAP-IFN-alpha mice was found to be latent compared with seven of eight (88%) of the wild-type mice, as determined by the expression of the latency-associated transcript RNAs. Likewise, only three of nine GFAP-IFN-alpha mice screened showed seroconversion by day 30 postinfection compared with nine of ten wild-type mice screened. Collectively, the results show that the IFN-alpha1 transgenic mice are less susceptible to acute HSV-1 infection and the establishment of viral latency.

Increased levels of IFN-gamma in the trigeminal ganglion correlate with protection against HSV-1-induced encephalitis following subcutaneous administration with androstenediol.

Androstenediol (AED) is a metabolic product of dehydroepiandrosterone (DHEA), an adrenal steroid known to possess immunomodulatory characteristics. The present study was undertaken to assess the efficacy of AED treatment in mice ocularly infected with herpes simplex virus type 1 (HSV-1). The subcutaneous administration of 320 mg/kg AED 4 h prior to viral inoculation was found to enhance the survival of HSV-1-infected mice while lower doses (32.0-100.0 mg/kg) were without effect. However, there were no apparent differences in the viral load in the eye or trigeminal ganglion (TG) 3 or 6 days post infection (p.i.) in vehicle- or AED (320 mg/kg)-treated mice. Likewise, there were no differences in the expression of cytokine or chemokine mRNAs in the eyes or TG early (i.e., 3 days p.i.) following infection. However, by 6 days p.i., there was a significant increase in the expression of the chemokines IP-10, MCP-1, and RANTES and the cytokines interleukin-6 (IL-6) and interferon-gamma (IFN-gamma) in the AED (320 mg/kg)-treated mice compared to vehicle-treated controls as determined by reverse transcription (RT)-polymerase chain reaction (PCR) and quantitative PCR (for IFN-gamma). Likewise, there was a corresponding increase in IFN-gamma and IL-2 but not IL-12 protein in the TG of AED-treated mice 6 days p.i. AED-treatment also induced a rise in splenic natural killer activity in a dose- and time-dependent fashion. Collectively, these results suggest that the protective effect following subcutaneous administration of AED is associated in a rise in selective type 1 cytokines (IL-2 and IFN-gamma) as well as natural killer activity.

Cytokine and chemokine production in HSV-1 latently infected trigeminal ganglion cell cultures: effects of hyperthermic stress.

The establishment of a primary trigeminal ganglion (TG) cell culture latently infected with herpes simplex virus type 1 (HSV-1) has been useful in studying stress-induced reactivation of the latent virus. However, the immune profile of this culture system prior to and after stress has never been established. In the present manuscript, cytokine and chemokine production were measured in primary cultures of TG cells obtained from uninfected and HSV-1 latently infected mice. Supernates from TG cell cultures contained detectable interleukin (IL)-6 but not IL-1beta, IL-2, IL-10, interferon (IFN)-gamma or tumor necrosis factor (TNF)-alpha as determined by ELISA. The basal level of IL-6 in uninfected TG cell cultures was 20.5 +/- 2.3 ng/ml, whereas latently infected TG cells produced significantly less IL-6 (12.1 +/- 1.9 ng/ml). Supernates from TG cell cultures also contained detectable levels of C-10, MCP-1 and eotaxin but little to no MIP-1alpha, MIP-1beta, or MIP-2. While there were no differences in the basal level of MCP-1 and eotaxin in TG cell cultures from HSV-1-infected and uninfected mice, C10 levels were significantly higher in TG cultures originating from infected mice compared to uninfected ones (5.86 +/- 0.61 ng/ml compared to 1.18 +/- 0.16 ng/ml). Hyperthermic stress (43 degrees C, 180 min), which induces reactivation of latent HSV-1 from TG cell cultures, significantly reduced IL-6 and C-10 levels from both uninfected and latently infected TG cell cultures. However, there was no correlation between cytokine/chemokine levels and HSV-1 reactivation. Immunofluorescent studies showed TG cell cultures contained 10% MAC-3+ staining cells (macrophage specific) but no dendritic cells. By comparison, cells from freshly isolated TG contained 6% positive dendritic cells but < 1% MAC-3 + cells. Both in vivo and in vitro TG consisted of a low percentage of CD3+ and CD8+ cells. Hyperthermic stress (43 degrees C for 3 h) eliminated the lymphocyte population as determined by RT-PCR. Whereas no spontaneous reactivation has been reported in mice, spontaneous reactivation occurred in 4.5% (10/220) of TG cell cultures surveyed over a 20 day period. Collectively, the dichotomy between HSV-1 replication and reactivation comparing the in vitro and in vivo HSV-1 latency models may reside, in part, to the differences in the levels of cytokines, chemokines and immune cell populations within the microenvironment of the in vitro and in vivo TG.

Role of the hypothalamic pituitary adrenal axis and IL-6 in stress-induced reactivation of latent herpes simplex virus type 1.

Hyperthermic stress induces reactivation of herpes simplex virus type 1 (HSV-1) in latently infected mice and also stimulates corticosterone release from the adrenals via activation of the hypothalamic pituitary adrenal axis. In the present study, we tested the hypothesis that stress-induced elevation of corticosterone potentiates HSV-1 reactivation in latently infected mice. Because of the putative role of IL-6 in facilitating HSV-1 reactivation in mice, the effect of hyperthermic stress and cyanoketone treatment on IL-6 expression in the trigeminal ganglion was also measured. Preadministration of cyanoketone, a glucocorticoid synthesis inhibitor, blocked the stress-induced elevation of corticosterone in a dose-dependent manner. Furthermore, inhibition of corticosterone synthesis was correlated with reduced levels of HSV-1 reactivation in latently infected mice. Hyperthermic stress elicited a transient rise in IL-6 mRNA levels in the trigeminal ganglion, but not other cytokine transcripts investigated. In addition, there was a significant reduction in MAC-3+, CD8+, and DX5+ (NK cell marker) cells in the trigeminal ganglion of latent HSV-1-infected mice 24 h after stress. Cyanoketone blocked the stress-induced rise in IL-6 mRNA and protein expression in the trigeminal ganglion latently infected with HSV-1. Collectively, the results indicate that the activation of the hypothalamic pituitary adrenal axis plays an important role in stimulating IL-6 expression and HSV-1 reactivation in the trigeminal ganglion following hyperthermic stress of mice.

Androstenediol antagonizes herpes simplex virus type 1-induced encephalitis through the augmentation of type I IFN production.

Dehydroepiandrosterone and androstenediol (AED) have previously been found to protect mice from viral-induced encephalitis resulting in an increased survival rate of the animals. These hormones have been shown to antagonize corticosteroids, which have immunosuppressive effects in vivo and in vitro, suggesting the antiviral effect of DHEA and AED may be linked to the anticorticosteroid action. The present study was undertaken to address the immune response to herpes simplex virus type 1 (HSV-1) during the acute ocular infection with and without AED treatment focusing on the early immune events in the eye and trigeminal ganglion. AED treatment was found to significantly improve the survival of HSV-1-infected mice in a dose-dependent fashion. While AED did not antagonize the elevated serum corticosterone levels following acute infection, AED enhanced the expression of IFN-alpha mRNA and decreased the expression of HSV-1-infected cell polypeptide 27 mRNA in the trigeminal ganglion during the acute (day 6 postinfection) infection of mice, as determined by reverse transcription-PCR. However, there was no change in the viral load from the eye or trigeminal ganglion when comparing the AED-treated with the vehicle-treated mice. Neutralization Abs to IFN-alpha, -beta, or -alpha/beta, but not control Ab, blocked the protective effect following AED exposure, confirming the involvement of type I IFN in the enhancement of survival in AED-treated mice. Collectively, these results identify innate immunity as a key component in augmenting the survival of HSV-1-infected mice following AED treatment.

Acyclovir blocks cytokine gene expression in trigeminal ganglia latently infected with herpes simplex virus type 1.

We have previously found that interleukin (IL)-2, IL-10, interferon (IFN)-gamma, RANTES, and tumor necrosis factor (TNF)-alpha mRNA transcription remain elevated in the trigeminal ganglia (TG) of herpes simplex virus type 1 (HSV-1) latently infected mice up to 120 days postinoculation (p.i.). To determine if this phenomenon was dependent on HSV-1 DNA replication after the establishment of latency (i.e., reactivation), cytokine gene expression was compared in TG of acyclovir-treated and untreated latently infected mice. Oral acyclovir treatment (begun 16 days p.i.) had no effect on serum levels of total anti-HSV-1 antibodies. However, there was a significant reduction in the titer of antibody specific for glycoprotein D and glycoprotein B but not glycoprotein H/L 120 days PI in the acyclovir-treated compared to vehicle-treated mice. These differences were not significant at earlier time points (i.e., days 34 and 60 p.i.). Consistent with these findings, acyclovir had no effect on cytokine gene expression in latently infected TG 35 and 60 days p.i. However, 120 days p.i., IFN-gamma and TNF-alpha mRNA were approaching baseline levels in TG of acyclovir-treated mice, but remained significantly elevated in untreated controls (i.e., IFN-gamma mRNA levels were sixfold higher in TG of untreated mice). Therefore, viral DNA replication appears to provide an antigenic stimulus for persistent cytokine gene expression in latently infected TG.