[Show abstract][Hide abstract] ABSTRACT: There is an emerging need for viral gene specific quantitative PCR (qPCR) assays that validate and complement whole transcriptome level technologies, including microarray and next generation sequencing. Therefore, a compilation of qPCR assays that represented the breadth of the entire Herpes simplex virus type 1 (HSV-1) genome were developed and evaluated. SYBR Green-I-based quantitation of each of the 74 HSV-1 lytic genes enabled accurate and reproducible detection of viral genes using a minimal number of reaction conditions. The amplification specificity of these assays for HSV-1 target genes was confirmed by amplicon size and purity determination on agarose gels, melt temperature dissociation curve analysis, and direct DNA sequencing of amplified products. Analysis of representative target genes demonstrated that these assays accurately and reproducibly quantified target gene expression across a wide and linear range of detection. In addition, minimal intra- and inter-assay variability was observed with significant well-to-well and plate-to-plate/assay-to-assay precision. To evaluate the utility of the developed qPCR assay system, kinetic profiles of viral gene expression were determined for an array of representative genes from all HSV-1 transcriptional gene classes. Collectively, these data demonstrate that the compiled optimized qPCR assays is a scalable and cost-effective method to assess HSV-1 gene expression with broad application potential, including investigation of pathogenesis and antiviral therapies. In addition, they can be employed to validate and complement evolving technologies for genome-wide transcriptome analysis.
[Show abstract][Hide abstract] ABSTRACT: Corneal neovascularization (CNV) is a complication of inflammatory and hypoxic ocular pathologies or immune response to ocular infections. CNV is a manifestation of an imbalance between pro-angiogenic and anti-angiogenic signaling molecules. Despite a magnitude of evidence suggesting angiogenic signaling molecules play a pivotal role in modulating CNV, there are insufficient current pharmacologic treatment options for CNV. Thus, targeting signaling molecules appears to be one promising treatment. Multiple drugs have been used off-label and have shown success in treating CNV, most notably the anti-VEGF medications. In addition, novel drugs are being developed and studied for CNV. This article will describe various molecular mechanisms that induce angiogenesis in the cornea, in addition to potential chemotherapies for CNV that inhibit these molecular mechanisms. This review is pertinent but not all-inclusive. The authors will give examples of publications that have reported novel discoveries related to potential future anti-CNV therapies.
Expert Review of Ophthalmology 01/2014; 8(2). DOI:10.1586/eop.13.8
[Show abstract][Hide abstract] ABSTRACT: The amphoteric C31G solution contains equimolar alkyl dimethlyglycine and alkyl dimethyl amine oxide buffered with citric acid. C31G acts as a broad spectrum antiviral and an antibacterial. No previous in vivo studies have been done to test C31G in an animal model of HSV-1 ocular keratitis. We assessed the anti-herpetic activity of C31G in the rabbit eye model using three treatment groups: (1) 1% trifluorothymidine (TFT); (2) 0.25% C31G plus 0.5% hydroxypropyl methylcellulose (HPMC); and (3) 0.5% HPMC. Scarified rabbit corneas were inoculated with the HSV-1 strain McKrae. On post inoculation (PI) day 3, rabbits were placed in three balanced groups based on slit-lamp examination (SLE) scores. Treatment began on PI day 3, five times a day for five consecutive days. In addition to the daily, masked SLE scoring, the eyes were assessed daily for stromal opacity, scleral inflammation, neovascularization, eyelid inflammation, inflammatory discharge, and epiphora. C31G and TFT were very effective in reducing the lesions and pathogenesis associated with HSV-1 ocular keratitis. The vehicle control SLE scores were significantly higher, proving that the vehicle did not effectively treat HSV-1 keratitis. C31G has valuable potential to treat herpetic keratitis as well as other herpetic topical lesions in humans.
Antiviral research 07/2013; 100(1). DOI:10.1016/j.antiviral.2013.07.002 · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The endocervical epithelium is a major reservoir for Chlamydia trachomatis in women, and genital infections are extended in their duration. Epithelial cells act as mucosal sentinels by secreting cytokines and chemokines in response to pathogen challenge and infection. We therefore determined the signature cytokine and chemokine response of primary-like endocervix-derived epithelial cells in response to a common genital serovar (D) of C. trachomatis. For these studies, we used a recently-established polarized, immortalized, endocervical epithelial cell model (polA2EN) that maintains, in vitro, the architectural and functional characteristics of endocervical epithelial cells in vivo including the production of pro-inflammatory cytokines. PolA2EN cells were susceptible to C. trachomatis infection, and chlamydiae in these cells underwent a normal developmental cycle as determined by a one-step growth curve. IL1α protein levels were increased in both apical and basolateral secretions of C. trachomatis infected polA2EN cells, but this response did not occur until 72h after infection. Furthermore, protein levels of the pro-inflammatory cytokines and chemokines IL6, TNFα and CXCL8 were not significantly different between C. trachomatis infected polA2EN cells and mock infected cells at any time during the chlamydial developmental cycle up to 120h post-infection. Intriguingly, C. trachomatis infection resulted in a significant decrease in the constitutive secretion of T cell chemokines IP10 and RANTES, and this required a productive C. trachomatis infection. Examination of anti-inflammatory cytokines revealed a high constitutive apical secretion of IL1ra from polA2EN cells that was not significantly modulated by C. trachomatis infection. IL-11 was induced by C. trachomatis, although only from the basolateral membrane. These results suggest that C. trachomatis can use evasion strategies to circumvent a robust pro-inflammatory cytokine and chemokine response. These evasion strategies, together with the inherent immune repertoire of endocervical epithelial cells, may aid chlamydiae in establishing, and possibly sustaining, an intracellular niche in microenvironments of the endocervix in vivo.
[Show abstract][Hide abstract] ABSTRACT: The exact mechanisms of HSV-1 establishment, maintenance, latency, reactivation, and also the courses of recurrent ocular infections remain a mystery. Comprehensive understanding of the HSV-1 disease process could lead to prevention of HSV-1 acute infection, reactivation, and more effective treatments of recurrent ocular disease. Animal models have been used for over sixty years to investigate our concepts and hypotheses of HSV-1 diseases. In this paper we present descriptions and examples of rabbit and mouse eye models of HSV-1 latency, reactivation, and recurrent diseases. We summarize studies in animal models of spontaneous and induced HSV-1 reactivation and recurrent disease. Numerous stimuli that induce reactivation in mice and rabbits are described, as well as factors that inhibit viral reactivation from latency. The key features, advantages, and disadvantages of the mouse and rabbit models in relation to the study of ocular HSV-1 are discussed. This paper is pertinent but not intended to be all inclusive. We will give examples of key papers that have reported novel discoveries related to the review topics.
BioMed Research International 10/2012; 2012:612316. DOI:10.1155/2012/612316 · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background
Rabbits latent with HSV-1 strain McKrae spontaneously shed infectious virus and viral DNA into their tears and develop recurrent herpetic-specific corneal lesions. The rabbit eye model has been used for many years to assess acute ocular infections and pathogenesis, antiviral efficacy, as well as latency, reactivation, and recurrent eye diseases. This study used real-time PCR to quantify HSV-1 DNA in the saliva and tears of rabbits latent with HSV-1 McKrae.
New Zealand white rabbits used were latent with HSV-1 strain McKrae and had no ocular or oral pathology. Scarified corneas were topically inoculated with HSV-1. Eye swabs and saliva were taken from post inoculation (PI) days 28 through 49 (22 consecutive days). Saliva samples were taken four times each day from each rabbit and the DNA extracted was pooled for each rabbit for each day; one swab was taken daily from each eye and DNA extracted. Real-time PCR was done on the purified DNA samples for quantification of HSV-1 DNA copy numbers. Data are presented as copy numbers for each individual sample, plus all the copy numbers designated as positive, for comparison between left eye (OS), right eye (OD), and saliva.
The saliva and tears were taken from 9 rabbits and from 18 eyes and all tested positive at least once. Saliva was positive for HSV-1 DNA at 43.4% (86/198) and tears were positive at 28.0% (111/396). The saliva positives had 48 episodes and the tears had 75 episodes. The mean copy numbers ± the SEM for HSV-1 DNA in saliva were 3773 ± 2019 and 2294 ± 869 for tears (no statistical difference).
Rabbits latent with strain McKrae shed HSV-1 DNA into their saliva and tears. HSV-1 DNA shedding into the saliva was similar to humans. This is the first evidence that documents HSV-1 DNA in the saliva of latent rabbits.
[Show abstract][Hide abstract] ABSTRACT: Type-I interferon (IFN)-mediated responses are a crucial first line of defense against viral infections and are critical for generating both innate and adaptive immunity. Therefore, viruses have necessarily evolved mechanisms to impede the IFN response. HSV-2 was found to completely abolish type-1 IFN-mediated signaling via multiple STAT2-associated mechanisms. Although the extent and kinetics of this inactivation were indistinguishable between the various cell-lines examined, there were distinct differences in the mechanisms HSV-2 employed to subvert IFN-signaling among the cell-lines. These mechanistic differences could be segregated into two categories dependent on the phase of the HSV replicative cycle that was responsible for this inhibition: (1) early phase-inhibited cells which exhibited abrogation of IFN-signaling prior to viral DNA replication; (2) late phase-inhibited cells where early phase inhibition mechanisms were not functional, but viral functions expressed following DNA replication compensated for their ineffectiveness. In early phase-inhibited cells, HSV-2 infection targeted STAT2 protein for proteosomal degradation and prevented de novo expression of STAT2 by degrading its mRNA. In contrast, HSV-2 infected late phase-inhibited cells exhibited no apparent changes in STAT2 transcript or protein levels. However, in these cells STAT2 was not activated by phosphorylation and failed to translocate to the cell nucleus, thereby preventing transactivation of antiviral genes. In primary human fibroblasts, HSV-2 failed to fully degrade STAT2 and therefore, both early and late phase mechanisms functioned cooperatively to subvert IFN-mediated antiviral gene expression. Taken together, these results indicate the importance that HSV-2 has assigned to STAT2, investing significant genomic currency throughout its replicative lifecycle for continuous targeted destruction and inhibition of this protein.
Virus Research 05/2012; 167(2):273-84. DOI:10.1016/j.virusres.2012.05.010 · 2.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Generation and isolation of recombinant herpesviruses by traditional homologous recombination methods can be a tedious, time-consuming process. Therefore, a novel stoplight recombination selection method was developed that facilitated rapid identification and purification of recombinant viruses expressing fusions of immunological epitopes with EGFP. This "traffic-light" approach provided a visual indication of the presence and purity of recombinant HSV-1 isolates by producing three identifying signals: (1) red fluorescence indicates non-recombinant viruses that should be avoided; (2) yellow fluorescence indicates cells co-infected with non-recombinant and recombinant viruses that are chosen with caution; (3) green fluorescence indicates pure recombinant isolates and to proceed with preparation of viral stocks. Adaptability of this system was demonstrated by creating three recombinant viruses that expressed model immunological epitopes. Diagnostic PCR established that the fluorescent stoplight indicators were effective at differentiating between the presence of background virus contamination and pure recombinant viruses specifying immunological epitopes. This enabled isolation of pure recombinant viral stocks that exhibited wildtype-like viral replication and cell-to-cell spread following three rounds of plaque purification. Expression of specific immunological epitopes was confirmed by western analysis, and the utility of these viruses for examining host immune responses to HSV-1 was determined by a functional T cell assay.
[Show abstract][Hide abstract] ABSTRACT: To determine the efficacy of a new formulation of topical dexamethasone 0.1%/povidone-iodine 0.4% (FST-100) in reducing clinical symptoms and infectious viral titers in a rabbit model of adenoviral keratoconjunctivitis.
Rabbit corneas were inoculated bilaterally with 2×10(6) plaque-forming-units (PFU) of adenovirus type 5 (Ad5) after corneal scarification. Animals were randomized 1:1:1:1 (five rabbits per group) to FST-100, 0.5% cidofovir, tobramycin/dexamethasone (Tobradex; Alcon Laboratories, Fort Worth, TX) ophthalmic suspension, and balanced salt solution (BSS; Alcon Laboratories). Treatment began 12 hours after viral inoculation and continued for 7 consecutive days. The eyes were clinically scored daily for scleral inflammation (injection), ocular neovascularization, eyelid inflammation (redness), friability of vasculature, inflammatory discharge (pus), and epiphora (excessive tearing). Eye swabs were collected daily before treatment for the duration of the study. Virus was eluted from the swabs and PFU determined by titration on human A549 cells, according to standard procedures.
The FST-100 treatment resulted in significantly lower clinical scores (P<0.05) than did the other treatments. The 0.5% cidofovir exhibited the most ocular toxicity compared with FST-100, tobramycin/dexamethasone, and balanced salt solution treatments. FST-100 and 0.5% cidofovir significantly (P<0.05) reduced viral titers compared with tobramycin/dexamethasone or balanced salt solution.
FST-100 was the most efficacious in minimizing the clinical symptoms of adenovirus infection in rabbit eyes. FST-100 and 0.5% cidofovir were both equally effective in reducing viral titers and decreasing the duration of viral shedding. By providing symptomatic relief in addition to reducing infectious virus titers, FST-100 should be a valuable addition to treatment of epidemic adenoviral keratoconjunctivitis.
[Show abstract][Hide abstract] ABSTRACT: To determine host response by gene expression in HSV-1 latent trigeminal ganglia (TG) after sodium butyrate (NaBu) treatment.
Corneas of 6-week-old female BALB/c mice were scarified and inoculated with HSV-1 17Syn(+) (high phenotypic reactivator) or its mutant 17ΔPst(LAT(-)) (low phenotypic reactivator) at 10(4) plaque-forming units/eye. NaBu-induced viral reactivation was by intraperitoneal (IP) administration at postinfection (PI) day 28, followed by euthanasia after 1 hour. NaBu-treated, uninfected mice served as the control. The resultant labeled cRNA from TG isolated total RNA was hybridized to gene microarray chips containing 14,000 mouse genes. Quantitative real-time PCR was performed to confirm gene expression.
Differential induction of gene expression between 17Syn(+) and its mutant 17ΔPst(LAT(-)) was designated as NaBu-induced gene expression and yielded significant upregulation of 2- to 16-fold of 0.4% (56/14,000) host genes probed, comprising mainly nucleosome assembly and binding, central nervous system structural activity, hormonal activity, and signaling activity. Approximately 0.2% (24/14,000) of the host genes, mainly of the same functional categories were downregulated 3- to 11-fold. Immune activity was minor in comparison to our reports on gene expression during latency and heat stress induction. Euchromatin analysis revealed that the LAT-ICP0 locus is amenable to the effects of NaBu. Histone activity was detected by early transcription of histone cluster 2 H2be (Hist2h2be). CONCLUSIONS NaBu-induced reactivation of HSV-1 is twofold: drug action involving significant moderation of specific host epigenetic changes and failure to elicit or suppress immune activity at the early time point of 1 hour.
[Show abstract][Hide abstract] ABSTRACT: The sustained virological response to interferon-alpha (IFN-alpha) in individuals infected with hepatitis C virus (HCV) genotype 1 is only 50%, but is about 80% in patients infected with genotype 2-6 viruses. The molecular mechanisms explaining the differences in IFN-alpha responsiveness between HCV 1 and other genotypes have not been elucidated.
Virus and host cellular factors contributing to IFN responsiveness were analyzed using a green fluorescence protein (GFP) based replication system of HCV 2a and Huh-7 cell clones that either possesses or lack a functional Jak-Stat pathway. The GFP gene was inserted into the C-terminal non-structural protein 5A of HCV 2a full-length and sub-genomic clones. Both HCV clones replicated to a high level in Huh-7 cells and could be visualized by either fluorescence microscopy or flow cytometric analysis. Huh-7 cells transfected with the GFP tagged HCV 2a genome produced infectious virus particles and the replication of fluorescence virus particles was demonstrated in naïve Huh-7.5 cells after infection. IFN-alpha effectively inhibited the replication of full-length as well as sub-genomic HCV 2a clones in Huh-7 cells with a functional Jak-Stat pathway. However, the antiviral effect of IFN-alpha against HCV 2a virus was not observed in Huh-7 cell clones with a defect in Jak-Stat signaling. HCV infection or replication did not alter IFN-alpha induced Stat phosphorylation or ISRE promoter-luciferase activity in both the sensitive and resistant Huh-7 cell clones.
The cellular Jak-Stat pathway is critical for a successful IFN-alpha antiviral response against HCV 2a. HCV infection or replication did not alter signaling by the Jak-Stat pathway. GFP labeled JFH1 2a replicon based stable cell lines with IFN sensitive and IFN resistant phenotypes can be used to develop new strategies to overcome IFN-resistance against hepatitis C.
[Show abstract][Hide abstract] ABSTRACT: Ocular infection with HSV-1 continues to be a serious clinical problem despite the availability of effective antivirals. Primary infection with HSV-1 can involve ocular and adenaxial sites and can manifest as blepharitis, conjunctivitis, or corneal epithelial keratitis. After initial ocular infection, HSV-1 can establish latent infection in the trigeminal ganglia for the lifetime of the host. During latency, the viral genome is retained in the neuron without producing viral proteins. However, abundant transcription occurs at the region encoding the latency-associated transcript, which may play significant roles in the maintenance of latency as well as neuronal reactivation. Many host and viral factors are involved in HSV-1 reactivation from latency. HSV-1 DNA is shed into tears and saliva of most adults, but in most cases this does not result in lesions. Recurrent disease occurs as HSV-1 is carried by anterograde transport to the original site of infection, or any other site innervated by the latently infected ganglia, and can reinfect the ocular tissues. Recurrent corneal disease can lead to corneal scarring, thinning, stromal opacity and neovascularization and, eventually, blindness. In spite of intensive antiviral and anti-inflammatory therapy, a significant percentage of patients do not respond to chemotherapy for herpetic necrotizing stromal keratitis. Therefore, the development of therapies that would reduce asymptomatic viral shedding and lower the risks of recurrent disease and transmission of the virus is key to decreasing the morbidity of ocular herpetic disease. This review will highlight basic HSV-1 virology, and will compare the animal models of latency, reactivation, and recurrent ocular disease to the current clinical data.
Seminars in Ophthalmology 07/2009; 23(4):249-73. DOI:10.1080/08820530802111085 · 0.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Epstein-Barr Nuclear Antigen 1 (EBNA1) is essential for Epstein-Barr virus to immortalize naïve B-cells. Upon binding a cluster of 20 cognate binding-sites termed the family of repeats, EBNA1 transactivates promoters for EBV genes that are required for immortalization. A small domain, termed UR1, that is 25 amino-acids in length, has been identified previously as essential for EBNA1 to activate transcription. In this study, we have elucidated how UR1 contributes to EBNA1's ability to transactivate. We show that zinc is necessary for EBNA1 to activate transcription, and that UR1 coordinates zinc through a pair of essential cysteines contained within it. UR1 dimerizes upon coordinating zinc, indicating that EBNA1 contains a second dimerization interface in its amino-terminus. There is a strong correlation between UR1-mediated dimerization and EBNA1's ability to transactivate cooperatively. Point mutants of EBNA1 that disrupt zinc coordination also prevent self-association, and do not activate transcription cooperatively. Further, we demonstrate that UR1 acts as a molecular sensor that regulates the ability of EBNA1 to activate transcription in response to changes in redox and oxygen partial pressure (pO(2)). Mild oxidative stress mimicking such environmental changes decreases EBNA1-dependent transcription in a lymphoblastoid cell-line. Coincident with a reduction in EBNA1-dependent transcription, reductions are observed in EBNA2 and LMP1 protein levels. Although these changes do not affect LCL survival, treated cells accumulate in G0/G1. These findings are discussed in the context of EBV latency in body compartments that differ strikingly in their pO(2) and redox potential.
[Show abstract][Hide abstract] ABSTRACT: Herpes simplex virus type 1 glycoprotein K (gK) and the UL20 protein (UL20p) are coordinately transported to the trans-Golgi network (TGN) and cell surfaces and are required for cytoplasmic virion envelopment at the TGN. In addition, cell surface expression of gK and UL20p is required for virus-induced cell fusion. Previously, confocal microscopy colocalization and intracellular transport experiments strongly suggested direct protein-protein interactions between gK and UL20p. Direct protein-protein interactions between gK and UL20p were demonstrated through reciprocal coimmunoprecipitation experiments, as well as with glutathione S-transferase (GST) pull-down experiments. A fusion protein consisting of the amino-terminal 66 amino acids of UL20p fused in-frame with GST was expressed in Escherichia coli and purified via glutathione column chromatography. Precipitation of GST-UL20p from mixtures of GST-UL20p fusion protein with cellular extracts containing gK specifically coprecipitated gK but not other viral glycoproteins. The purified UL20p-GST fusion protein reacted with all gK-associated protein species. It was concluded that the amino terminus of UL20p, most likely, interacted with gK domain III, which is predicted to lie intracellularly. UL20p-gK domain-specific interactions must serve important functions in the coordinate transport of UL20p and gK to the TGN, because retention of UL20p in the endoplasmic reticulum (ER) via the addition of an ER retention signal at the carboxyl terminus of UL20p forced the ER retention of gK and drastically inhibited intracellular virion envelopment and virus-induced cell fusion.
Journal of Virology 08/2008; 82(13):6310-23. DOI:10.1128/JVI.00147-08 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to determine the presence and copy numbers of herpes simplex virus type 1 (HSV-1) DNA in human
trigeminal ganglia (TG) with respect to age, gender, and postmortem interval (PMI). Human TG (n = 174, obtained from the Oregon Brain Bank, with data on age, gender, and PMI) were analyzed for HSV-1 DNA copies (HSV-1
DNA polymerase gene) by using real-time PCR. We found that 89.1% (131/147) of subjects and 90.1% (155/174) of TG contained
HSV-1 DNA. The copy numbers of HSV-1 DNA in the positives ranged from very high (>106) to very low (5). These data confirm and strengthen our previous findings that subjects were positive for HSV-1 DNA in tears
(46/50; 92%) and saliva (47/50; 94%). These TG data and tear and saliva data demonstrated considerable variability in copy
numbers of HSV-1 DNA per subject. Statistical analysis showed no significant relationship between gender and copy number,
age and copy number, or PMI and copy number for each pair of variables. A factorial analysis of gender, age, and PMI with
respect to copy number also showed no statistical significance. This is the first study that provides statistical analysis
that documents that the prevalence of HSV-1 DNA in the human TG is not a function of either gender or age.
Journal of Virology 06/2008; 82(16):8230-4. DOI:10.1128/JVI.00686-08 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To determine the role of herpes simplex virus-1 (HSV-1) glycoprotein K(gK) in corneal infection, neuroinvasion, and virus latency in trigeminal ganglia of mice.
The recombinant virus HSV-1 (McKrae) Delta gK (MKDelta gK) carrying a deletion of the gK gene was constructed by insertional/deletion mutagenesis and replaced by a gene cassette constitutively expressing the enhanced green fluorescence protein. The gK deletion of the MKDelta gK virus was rescued to produce the wild-type-like virus MKgK. Balb/c mice were infected ocularly with either virus, and the infection pattern in the eye, clinical disease progression, and establishment of viral latency was monitored.
Mice infected with the MKDelta gK strain produced in a gK complementing cell line did not exhibit clinical signs when compared with mice infected with the MKgK virus. Direct visualization of infected eyes revealed that the MKDelta gK virus was unable to spread in mouse corneas, while the MKgK rescued virus spread efficiently. Nineteen of 20 scarified and 5/12 unscarified mice infected with the MKgK virus produced infectious virus after coculture with permissive cells, while 0/20 scarified and 0/12 unscarified mice infected with the MKDelta gK virus produced infectious virus. HSV DNA was detected in trigeminal ganglia by PCR in 19/20 scarified and 9/12 unscarified mice inoculated with MKgK, while HSV DNA was detected in the trigeminal ganglia of 3/20 scarified and 0/12 unscarified mice inoculated with MKDelta gK.
The results show that HSV-1 gK is essential for efficient replication and spread in the corneal epithelium and trigeminal ganglia neuroinvasion in MKDelta gK inoculated mice.
Current eye research 06/2008; 33(5):455-67. DOI:10.1080/02713680802130362 · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To evaluate efficacy of the small apolipoprotein E (apoE) mimetic dimer peptide (apoEdp) in the treatment of herpetic stromal keratitis in a mouse ocular model and determine its therapeutic effects against HSV-1-induced inflammatory cytokines.
Female C57Bl/6 mice were corneally infected with HSV-1 strain KOS-GFP; topical treatment was initiated 24 hours after infection and continued for 10 consecutive days. Treatment groups were 1% apoEdp, 1% trifluorothymidine (TFT), and phosphate-buffered saline (PBS). The incidence and severity of stromal keratitis were monitored by slit lamp examination in a masked fashion. Infectious HSV-1 titer in eye swabs and alteration in inflammatory cytokines were determined in the early postinfection period by real-time RT-PCR.
One percent apoEdp treatment, which significantly reduced the incidence and severity of HSK, was equal to the effect of 1% TFT; both groups had significantly lower incidence and severity than the placebo treatment group. The in vivo mouse ocular model results of apoEdp therapy correlated with accelerated clearance of virus from eye swabs. Topical 1% apoEdp treatment in mice significantly downregulated gene expression of mouse proinflammatory cytokines.
These results suggest that topical treatment with apoE peptide has efficacy against HSK through anti-HSV-1 and anti-inflammatory activities.
[Show abstract][Hide abstract] ABSTRACT: The isoform-specific role of human apolipoprotein E (apoE) has been assessed in a mouse model of ocular herpes. Female, age-matched transgenic mice knocked-in for the human allele apoE3 or apoE4 and their parent C57Bl/6 mice were inoculated corneally with HSV-1 strain KOS. Ocular HSV-1 pathogenesis was monitored through viral replication and clinical progression of stromal opacity and neovascularization by slit-lamp examination. Establishment of latency was determined by analysis of HSV-1 DNA (copy number) by specific real-time PCR in the cornea, trigeminal ganglia (TG), and brain. Representative groups of transgenic mice were sacrificed for the analysis of gene expression of vascular endothelial growth factor (VEGF) by reverse-transcription PCR, and apoE expression by Western blot analysis. At 6days post-infection (P.I.), the ocular infectious HSV-1 titer was significantly higher (p<0.05) in apoE4 mice compared with apoE3 and C57Bl/6 mice. Corneal neovascularization in apoE4 mice was significantly higher (p<0.05) than apoE3 and C57Bl/6 mice. The onset of corneal opacity in apoE4 mice was accelerated during days 9-11 P.I.; however, no significant difference in severity was seen on P.I. days 15 and beyond. At 28 days P.I., infected mice of all genotypes had no significant differences in copy numbers (range 0-15) of HSV-1 DNA in their corneas, indicating that HSV-1 DNA copy numbers in cornea are independent of apoE isoform regulation. At 28 days P.I., both apoE4 and C57Bl/6 mice had a significantly higher (p=0.001) number of copies of HSV-1 DNA in TG compared with apoE3. ApoE4 mice also had significantly higher (p=0.001) copies of HSV-1 DNA in their TGs compared with C57Bl/6 mice. In brain, both apoE4 and C57Bl/6 mice had significantly higher numbers (p<or=0.03) of copies of HSV-1 DNA compared with apoE3 mice. However, the number of HSV-1 DNA copies in the brain of C57Bl/6 mice was not significantly different than that of apoE4 (p=0.1). Comparative molecular analysis between apoE3 and apoE4 mice on selected days between 7 and 28 P.I., inclusive, revealed that the corneas of apoE4 mice expressed VEGF. None of the corneas in the apoE3 mice expressed VEGF during this time. Western blot analysis showed proteolytic cleavage of the apoE protein in the corneas of the apoE4 mice. Through days 14-28 P.I., a approximately 29 kDa C-terminal truncated apoE fragment was present in the corneas of apoE4 mice, but not in apoE3 mice. ApoE4 is a risk factor for ocular herpes, in part, through increased replication of virus in the eye, an earlier onset in clinical opacity, significantly higher neovascularization, and increased HSV-1 DNA load in TG and brain than that of apoE3. Increased pathogenesis of ocular herpes in apoE4 mice was also mediated, in part through up-regulated expression of VEGF and apoE proteolysis in the cornea. This is the first report linking a human gene, apoE4, as a risk factor for ocular herpes pathogenesis in a transgenic mouse model.
Experimental Eye Research 06/2008; 87(2):122-30. DOI:10.1016/j.exer.2008.05.007 · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Glycoprotein K (gK) is a virion envelope component of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), which plays an important role in virion morphogenesis and egress. We previously demonstrated that immunization of mice with gK, but not with any of the 10 other HSV-1 glycoproteins, resulted in exacerbation of corneal scarring and herpetic dermatitis following ocular HSV-1 infection. However, little is known about the gK epitope(s) that is (are) involved in T cell activities in vitro or in vivo. Thus, epitope mapping of gK was performed using a panel of 15-mer peptides with five-amino acid overlaps spanning the full-length gK, and four expressed gK recombinant proteins representing different regions of gK. Epitope mapping within the gK polypeptide defined the amino acid sequence STVVLITAYGLVLVW as the predominant CD4(+) and CD8(+) T cell stimulatory region both in vitro and in vivo. IFN-gamma expression by CD4(+) T cells was CD8(+) T cells-dependent. This immunodominant epitope is located within the signal sequence of the gK polypeptide and is highly conserved in HSV-1 and HSV-2 strains. Using prediction algorithms, the peptide is predicted to bind to numerous MHC class I and class II molecules.
Virus Research 10/2007; 128(1-2):71-80. DOI:10.1016/j.virusres.2007.04.007 · 2.32 Impact Factor