PURPOSE:To investigate the production of monokine induced by gamma-interferon (MIG) during a primary Herpes simplex virus type 1 (HSV-1) infection of the cornea. We hypothesize that multiple CXCR3 ligands are involved in T cell recruitment during HSV-1 corneal infection and that neutrophils have the potential to contribute to their production. MATERIALS AND METHODS:Levels of MIG were evaluated in an in vivo murine model of HSV-1 corneal infection by quantitative ELISA. Cultured murine corneal fibroblast (MCF) cells and purified neutrophils were stimulated in vitro with IFN-γ and IL-1α to determine inducers of MIG. Cellular sources of MIG production in vivo were investigated via cellular depletion studies. Additionally, MIG production resulting from interaction between resident human corneal cells and neutrophils was evaluated in an ex vivo model of human corneal infection. RESULTS:MIG was significantly elevated on days 2-6 and on day 8 following corneal infection. MCF and neutrophils secreted MIG in response to IFN-γ, but not IL-1α stimulation. Co-stimulation with IFN-γ and IL-1α induced a four-fold increase in MIG production by MCF. However, the same combination led to a three-fold decrease in MIG production by neutrophils. In vivo, a 52% reduction in MIG levels was observed in the neutrophil depleted host. In the human ex vivo model, MIG levels were significantly elevated in response to communication between HSV-1 infected corneal tissue and neutrophils. CONCLUSIONS:Here, we report the evidence for the production of MIG, a second CXCR3 ligand, during the primary immune response to HSV-1 corneal infection. Our results support the hypothesis that both neutrophils and resident corneal cells contribute to MIG production in vivo. However, neutrophils produce MIG in response to communication with HSV-1-infected resident corneal cells more efficiently than by direct interaction with virus. In addition, we found that MIG production by neutrophils and resident corneal cells was differentially regulated by IL-1α.

IL-17 has been associated with selected inflammatory and autoimmune diseases. We characterized the expression of this proinflammatory cytokine following HSV-1 corneal infection and investigated whether IL-17R signaling modulated the host response to the viral pathogen at early time-points postinfection. IL-17 was elevated in the murine cornea 24 h after high-dose virus infection and subsequently persisted at low levels during the first week. Immunofluorescent studies showed that the IL-17R was expressed by cultured mouse corneal fibroblasts. Exposure of corneal cells to IL-17 led to production of IL-6 and MIP-2 in vitro and in vivo, indicating that the IL-17R was functional. Mice lacking IL-17R displayed significantly reduced neutrophil infiltration and corneal opacity. However, this effect was transient, as corneal pathology and neutrophil influx resembled that of wild-type (WT) hosts 4 days postinfection. HSV-1 growth and clearance in IL-17R(-/-) hosts were similar to that of the WT controls. Infection of IFN-gamma gene knockout mice was associated with elevated IL-17 levels and accelerated corneal opacity, suggesting that IFN-gamma negatively regulated IL-17 expression. Collectively, our results establish that IL-17 is rapidly produced in the cornea after HSV-1 infection and is regulated at least in part by IFN-gamma. The absence of IL-17 signaling results in a transient decrease in the expression of proinflammatory mediators, neutrophil migration, and corneal pathology, but control of virus growth in the cornea and trigeminal ganglia is not compromised. Thus, IL-17 actively influences early virus-induced corneal inflammation.

The purpose of this study was to characterize the synthesis of alpha-chemokines IP-10, MIG, and I-TAC by human corneal epithelial cells (HCE) following exposure to proinflammatory mediators. Supernatants were collected from HCE cultures stimulated with individual or combinations of TNF-alpha, IL-1alpha, and IFN-gamma, and assayed for alpha-chemokines by ELISA. RT-PCR was used to detect IFN-gamma receptor mRNA. Activation of STAT 1 was determined by Western blotting. Stimulation of HCE with either IL-1alpha or TNF-alpha increased IP-10 protein synthesis up to 6-fold, whereas insignificant levels of MIG and I-TAC were induced. The epithelial cells were found to express IFN-gamma receptors constitutively. Exposure to the ligand resulted in STAT 1 phosphorylation and production of nanogram amounts of IP-10, I-TAC, and MIG. When HCE were stimulated with combinations of TNF-alpha and IFN-gamma, or IL-1alpha and IFN-gamma, the levels of IP-10 and I-TAC secreted were > 150-fold higher than that produced following exposure to a single cytokine. In contrast, MIG protein synthesis was not enhanced upon stimulation with cytokine combinations. The abundant production of ELR(-)alpha -chemokines following appropriate stimulation suggests that HCE may play an important role in the recruitment of effector cells such as activated T-lymphocytes to inflamed corneal tissue. The data also indicate that the synthesis of IP-10, I-TAC, and MIG are differentially regulated in HCE.

BACKGROUND:Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection, claiming millions of lives annually. The virus infects various cells of the respiratory tract as well as resident inflammatory cells such as macrophages. Infection activates a variety of cellular factors such as cytokines and the pro-inflammatory transcription factor, NF-kappa B, all of which are important players in the respiratory disease. However, the exact natural route of RSV infection and its etiology remain relatively unknown. In this paper, we test the hypothesis that human corneal epithelial cells, which constitute the outermost layer of the cornea, can be infected with RSV, and that the infection leads to the activation of proinflammatory macromolecules.RESULTS:Corneal swabs obtained from pediatric patients with acute respiratory disease were found to contain RSV at a high frequency (43 positive out of 72 samples, i.e., 60%). Primary corneal epithelial cells in tissue culture supported robust infection and productive growth of RSV. Infection resulted in the activation of TNF-alpha, IL-6 and sixteen chemokines as well as NF-kappa B. Three proinflammatory CXC chemokines (MIG, I-TAC, IP-10) underwent the greatest activation.CONCLUSIONS:The ocular epithelium is readily infected by RSV. The pro-inflammatory cytokines are likely to play critical roles in the etiology of inflammation and conjunctivitis commonly seen in pediatric patients with respiratory infections. RSV-eye interactions have important implications in RSV transmission, immunopathology of RSV disease, and in the management of conjunctivitis.

Human immunodeficiency virus-1 (HIV-1) infection of the brain causes elevation in pro-inflammatory cytokines and inflammatory changes in the striatum. HIV-1-infected individuals who also abuse drugs including the psychostimulant methamphetamine (MA) develop more severe encephalitis and neuronal damage compared to HIV-1-infected patients who do not abuse drugs. In previous studies, we demonstrated that the HIV-1 protein Tat and MA interacted to cause enhanced loss of dopamine in the rat striatum via the destruction of dopaminergic terminals. Since both Tat and MA activate glia and induce cytokine production, we investigated the role of cytokines in the synergistic neurotoxicity induced by Tat and MA using cytokine arrays. Significant increases in monocyte chemotactic protein (MCP-1), interleukin-1 alpha (IL-1α) and tissue inhibitor of metalloproteinase-1 (TIMP-1) levels were noted 4 h following Tat + MA treatment compared to saline, Tat or MA. MCP-1 and TIMP-1 levels remained elevated 16 h after Tat + MA compared to saline or MA but were not different from the Tat-treated group at this time point. Weak, but significant elevations in cytokine-induced neutrophil chemoattractant-3 (CINC-3), ciliary neurotrophic factor (CNTF) and macrophage inflammatory protein-3 alpha (MIP-3α) were also noted with Tat + MA. The interaction of Tat and MA was prevented in mice genetically deficient in MCP-1 with a consequent attenuation of Tat + MA neurotoxicity. Our findings suggest that HIV-1 infection with concurrent drug abuse might profoundly increase chemokine levels in the striatum resulting in enhanced damage to the dopaminergic system.

An intertypic recombinant constructed from HSV-1 x HSV-2 parents was isolated which failed to induce any overt ocular pathology when inoculated onto the sacrificed cornea of four-week-old SJL/J mice. During the 24-48 hour post-infection period there was transient virus replication but by day 3 the infectious titer in the eye had dropped by greater than or equal to 10(4)-fold, and little or no virus could be recovered thereafter. When immunosuppressed (600 r) mice were infected corneally, virus clearance was delayed several days but again no obvious ocular pathology was seen, and no mice died. By contrast, infection of the cornea with either parent was followed by virus replication and development of clinically apparent pathology which could progress to blinding stromal keratitis. The genome of the intertypic recombinant was analyzed by agarose gel electrophoresis of restriction endonuclease digests and found to consist entirely of HSV-1 DNA except for HSV-2 DNA sequences located between map units 0.10-0.16, 0.41-0.43, and 0.77-1.0. Potential explanations for the loss of virulence are discussed.

Abstract The virulence of a herpes simplex virus (HSV) intertypic recombinant possessing HSV-1 DNA sequences from map units 0.31 to 0.44 and HSV-2 sequences from map units 0 to 0.30 and 0.45 to 1.0 were compared with the virulence of the two parental strains. Following ocular inoculation, both the intertypic recombinant and the HSV-1 parent replicated at the infection site and spread to the peripheral and central nervous system (CNS) to produce fatal encephalitis. The HSV-2 parent also replicated at the infection site but failed to progress to the CNS. However, when inoculated intracerebrally, the HSV-2 strain was as lethal as the HSV-1 parent. Furthermore, the HSV-2 strain could produce thymidine kinase at 37 and 39° in levels comparable to the HSV-1 strain. The results indicate that transfer of the HSV-1 DNA sequences imparted to the recombinant virus the necessary genetic information to spread from the cornea into the central nervous system.

The DNA of varicella-zoster virus (VZV) has been characterized by sucrose gradient sedimentation, restriction enzyme cleavage with either EcoRI or HindIII site-specific endonucleases, and by isopycnic banding in caesium chloride. Comparisons of the DNAs from different clinical isolates have been made. DNAs from VZVs isolated from either varicella or herpes zoster are indistinguishable on the basis of size and restriction enzyme cleavage pattern. The buoyant density in caesium chloride of the DNA of VZV isolated from varicella was reproducibly slightly lower than that of the DNA of VZV isolated from herpes zoster.

Human cells transformed by cytomegalovirus and transplanted to athymic nude mice yielded a cytopathic virus, Hershey Medical Center virus, following prolonged in vitro passage of the tumor cells. The virus is a double-enveloped herpesvirus, is sensitive to ether, and is inhibited by iododeoxyuridine. No significant antigenic relationship to herpes simplex virus was detected using herpes simplex virus-immune sera in neutralization and immunofluorescence tests, but indirect immunofluorescence tests revealed cytomegalovirus-related antigenicity. Further immunological tests revealed that Hershey Medical Center virus is antigenically indistinguishable from infectious bovine rhinotracheitis virus. Thus, it appears that Hershey Medical Center virus is infectious bovine rhinotracheitis virus, which presumably appeared in the cell culture as a contaminant from fetal calf serum.

The DNA of varicella-zoster virus (VZV) has been characterized by sucrose gradient sedimentation and by isopycnic banding in CsCl. Comparisons of the DNAs from different clinical isolates have been made by mixing radiolabeled DNAs prior to centrifugation. VZV-DNA sedimented just behind T4 DNA on neutral sucrose gradients. Thus VZV-DNA has a molecular weight of about 100 x 106. There were no distinguishable differences in the sedimentation behavior in neutral sucrose gradients of the DNAs from several clinical VZV isolates. The buoyant density in CsCl of the DNA of VZV isolated from varicella was reproducibly slightly lighter than the buoyant density of the DNA of VZV isolated from herpes zoster.

The DNAs from three varicella virus isolates and two herpes zoster virus isolates were digested with either Eco RI or Hin D III site-specific endonucleases. The restriction patterns of the DNAs obtained from the five isolates were then compared following separation of the specific fragments by electrophoresis through 0.5% agarose gels. The number and mobilities of all DNA bands were found to be indistinguishable for all five isolates. Further analysis of Eco RI-digested varicella-zoster virus DNA revealed the presence of both molar and submolar DNA fragments as found previously for other human herpesvirus DNAs.

Herpes zoster virus DNA was isolated from infected human embryo lung cells because it is released into the "Hirt supernatant". The buoyant density of the virus DNA was found to be slightly higher than that of cellular DNA. The molecular weight of this DNA was estimated to be 92 x 10(6) daltons by cosedimentation with T4 DNA in neutral sucrose gradients. As with the DNA of other herpesviruses, the DNA of herpes zoster virus is fragmented when denatured under alkaline conditions and is therefore "alkali-labile". Studies of virus DNA using reassociation kinetics reveal that the kinetic complexity of the DNA correlates with the genome size as estimated by sucrose gradient sedimentation.

Herpes simplex virus (HSV) DNA sediments as a homogeneous band just behind T4 DNA on neutral sucrose gradients but sediments heterogeneously on alkaline sucrose gradients. If the HSV DNA is pretreated with DNA polymerase and DNA ligase, then at least half the fragmentation seen on an alkaline sucrose gradient is prevented. Thus, it is concluded that there are preexisting nicks and gaps in HSV DNA which are repairable in vitro by enzyme treatment. To determine whether the preexisting gaps are located at unique positions or are randomly located, HSV DNA was repaired in vitro using [α-32P]dGTP as one substrate. Restriction enzyme analysis of the repaired HSV DNA suggests that the preexisting gaps are located at random on the genome.

The genome location of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) polyadenylated RNA has been studied by hybridization of herpesvirus-specific nuclear and cytoplasmic polyadenylated RNA to the Eco R, restriction enzyme fragments of herpesvirus DNA, which have been separated by agarose gel electrophoresis and transferred to nitrocellulose paper. For both HSV-1 and HSV-2 DNA, the nuclear and the cytoplasmic polyadenylated RNA are distributed on all the Eco RI fragments of the homologous DNA. In a heterologous system, total polyadenylated RNA from HSV-1-infected cells was annealed to the Eco RI fragments of HSV-2 DNA and vice versa. HSV-2 polyadenylated RNA hybridized to all fragments of HSV-1 DNA, and HSV-1 polyadenylated RNA hybridized to all but three of the fragments of HSV-2 DNA. These data indicate that the DNA base sequence homology between the two herpesvirus strains is dispersed throughout the genome and is not located in a single contiguous block.