The extrachromosomal elements of the Naegleria genus: How little we know.

There are currently 47 characterized species in the Naegleria genus of free-living amoebae. Each amoeba has thousands of extrachromosomal elements that are closed circular structures comprised of a single ribosomal DNA (rDNA) copy and a large non-rDNA sequence. Despite the presence of putative open reading frames and introns, ribosomal RNA is the only established transcript. A single origin of DNA replication (ori) has been mapped within the non-rDNA sequence for one species (N. gruberi), a finding that strongly indicates that these episomes replicate independently of the cell's chromosomal DNA component. This article reviews that which has been published about these interesting DNA elements and by analyzing available sequence data, discusses the possibility that different phylogenetically related clusters of Naegleria species individually conserve ori structures and suggests where the rRNA promoter and termination sites may be located.

Enteroviruses, type 1 diabetes and hygiene: a complex relationship.

Type 1 diabetes (T1D) is an autoimmune disease in which the immune system mounts an attack on the host's insulin-producing beta cells. Because most cases of T1D cannot be attributed only to individual genetics, it is strongly inferred that there is a significant environmental contribution, such as infection, impacting disease development. The human enteroviruses (HEV) are common picornaviruses often implicated as triggers of human T1D, although precisely which of the numerous HEV may be involved in human T1D development is unknown. Experiments using non-obese diabetic (NOD) mice, commonly used to model T1D, show that induction of T1D by HEV infection in NOD mice is a multifactorial process involving both the virus and the host. Interestingly, results demonstrate that HEV infection of NOD mice can also induce long-term protection from T1D under certain conditions, suggesting that a similar mechanism may occur in humans. Based upon both experimental animal and observational human studies, we postulate that HEV have a dual role in T1D development and can either cause or prevent autoimmune disease. Whichever outcome occurs depends upon multiple variables in the host-virus equation, many of which can be deduced from results obtained from NOD mouse studies. We propose that the background to the sharply rising T1D incidences observed in the 20th century correlates with increased levels of hygiene in human societies. Viewing T1D in this perspective suggests that potential preventative options could be developed.

The CVB and etiology of type 1 diabetes.

The origins of type 1 diabetes (T1D) are largely unknown. Fewer than 50% of the cases of the disease are attributable to host genetics, indicating that environmental factors are involved in disease development. The most often cited environmental agents implicated as initiators of T1D are the human enteroviruses, in particular the group B coxsackieviruses (CVB). Although the connection between the CVB and T1D has not been firmly established, significant' evidence supports the role of these pathogens in T1D development.

Evolution of virulence in picornaviruses.

The Picornaviridae encompass many positive-strand RNA viruses, all of which share a generally similar genome design and capsid structure, but which induce quite diverse diseases in humans and other animals. Picornavirus strains of the same serotype have been shown to express different virulence (or pathogenic) phenotypes when studied in animal models, demonstrating that key elements of pathogenesis reside in the viral genome. However, the genetics that determine the virulence phenotype of any picornavirus are poorly understood. Picornaviruses do not have virulence genes per se, but the design ofthe capsid andhow it interacts with the virus receptor expressed on the host cell surface, specific sequences within the nontranslated regions of the viral genome, as well as coding sequences that result in different protein sequences may all have a part in determining the virulence phenotype. Virulence may be better understood as a continuum from an apparent inability to induce disease to the ability to cause severe pathogenic changes. Ultimately, the ability of a picornavirus to induce disease depends upon viral genetics and how they are modulated by the host environment.

Characterization of an infectious cDNA copy of the genome of a naturally occurring, avirulent coxsackievirus B3 clinical isolate.

Group B coxsackieviruses (CVB) cause numerous diseases, including myocarditis, pancreatitis, aseptic meningitis and possibly type 1 diabetes. To date, infectious cDNA copies of CVB type 3 (CVB3) genomes have all been derived from pathogenic virus strains. An infectious cDNA copy of the well-characterized, non-pathogenic CVB3 strain GA genome was cloned in order to facilitate mapping of the CVB genes that influence expression of a virulence phenotype. Comparison of the sequence of the parental CVB3/GA population, derived by direct RT-PCR-mediated sequence analysis, to that of the infectious CVB3/GA progeny genome demonstrated that an authentic copy was cloned; numerous differences were observed in coding and non-coding sequences relative to other CVB3 strains. Progeny CVB3/GA replicated similarly to the parental strain in three different cell cultures and was avirulent when inoculated into mice, causing neither pancreatitis nor myocarditis. Inoculation of mice with CVB3/GA protected mice completely against myocarditis and pancreatitis induced by cardiovirulent CVB3 challenge. The secondary structure predicted for the CVB3/GA domain II, a region within the 5' non-translated region that is implicated as a key site affecting the expression of a cardiovirulent phenotype, differs from those predicted for cardiovirulent and pancreovirulent CVB3 strains. This is the first report characterizing a cloned CVB3 genome from an avirulent strain.

Toward testing the hypothesis that group B coxsackieviruses (CVB) trigger insulin-dependent diabetes: inoculating nonobese diabetic mice with CVB markedly lowers diabetes incidence.

Insulin-dependent (type 1) diabetes mellitus (T1D) onset is mediated by individual human genetics as well as undefined environmental influences such as viral infections. The group B coxsackieviruses (CVB) are commonly named as putative T1D-inducing agents. We studied CVB replication in nonobese diabetic (NOD) mice to assess how infection by diverse CVB strains affected T1D incidence in a model of human T1D. Inoculation of 4- or 8-week-old NOD mice with any of nine different CVB strains significantly reduced the incidence of T1D by 2- to 10-fold over a 10-month period relative to T1D incidences in mock-infected control mice. Greater protection was conferred by more-pathogenic CVB strains relative to less-virulent or avirulent strains. Two CVB3 strains were employed to further explore the relationship of CVB virulence phenotypes to T1D onset and incidence: a pathogenic strain (CVB3/M) and a nonvirulent strain (CVB3/GA). CVB3/M replicated to four- to fivefold-higher titers than CVB3/GA in the pancreas and induced widespread pancreatitis, whereas CVB3/GA induced no pancreatitis. Apoptotic nuclei were detected by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay in CVB3/M-infected pancreata but not in CVB3/GA-infected pancreata. In situ hybridization detected CVB3 RNA in acinar tissue but not in pancreatic islets. Although islets demonstrated inflammatory infiltrates in CVB3-protected mice, insulin remained detectable by immunohistochemistry in these islets but not in those from diabetic mice. Enzyme-linked immunosorbent assay-based examination of murine sera for immunoglobulin G1 (IgG1) and IgG2a immunoreactivity against diabetic autoantigens insulin and HSP60 revealed no statistically significant relationship between CVB3-protected mice or diabetic mice and specific autoimmunity. However, when pooled sera from CVB3/M-protected mice were used to probe a Western blot of pancreatic proteins, numerous proteins were detected, whereas only one band was detected by sera from CVB3/GA-protected mice. No proteins were detected by sera from diabetic or normal mice. Cumulatively, these data do not support the hypothesis that CVB are causative agents of T1D. To the contrary, CVB infections provide significant protection from T1D onset in NOD mice. Possible mechanisms by which this virus-induced protection may occur are discussed.

HLA-DQ polymorphism influences progression of demyelination and neurologic deficits in a viral model of multiple sclerosis.

The importance of genetic susceptibility in determining the progression of demyelination and neurologic deficits is a major focus in neuroscience. We studied the influence of human leukocyte antigen (HLA)-DQ polymorphisms on disease course and neurologic impairment in virus-induced demyelination. HLA-DQ6 or DQ8 was inserted as a transgene into mice lacking endogenous expression of MHC class I (beta(2)m) and class II (H2-A(beta)) molecules. Following Theiler's murine encephalomyelitis virus (TMEV) infection, we assessed survival, virus persistence, demyelination, and clinical disease. Mice lacking expression of endogenous class I and class II molecules (beta(2)m(o) Abeta(o) mice) died 3 to 4 weeks postinfection (p.i.) due to overwhelming virus replication in neurons. beta(2)m(o) Abeta(o) DQ6 and beta(2)m(o) Abeta(o) DQ8 mice had increased survival and decreased gray matter disease and virus replication compared to nontransgenic littermate controls. Both beta(2)m(o) Abeta(o) DQ6 and beta(2)m(o) Abeta(o) DQ8 mice developed chronic virus persistence in glial cells of the white matter of the spinal cord, with greater numbers of virus antigen-positive cells in beta(2)m(o) Abeta(o) DQ8 than in beta(2)m(o) Abeta(o) DQ6 mice. At day 45 p.i., the demyelinating lesions in the spinal cord of beta(2)m(o) Abeta(o) DQ8 were larger than those in the beta(2)m(o) Abeta(o) DQ6 mice. Earlier and more profound neurologic deficits were observed in beta(2)m(o) Abeta (o) DQ8 mice compared to beta(2)m(o) Abeta(o) DQ6 mice, although by 120 days p.i. both strains of mice showed similar extent of demyelination and neurologic deficits. Delayed-type hypersensitivity and antibody responses to TMEV demonstrated that the mice mounted class II-mediated cellular and humoral immune responses. The results are consistent with the hypothesis that rates of progression of demyelination and neurologic deficits are related to the differential ability of DQ6 and DQ8 transgenes to modulate the immune response and control virus.

TGF-beta 2 reduces demyelination, virus antigen expression, and macrophage recruitment in a viral model of multiple sclerosis.

TGF-beta 2 is a potent immunoregulatory mediator that influences B cell, T cell, and macrophage function. To test whether this cytokine alters pathology in a model of virus-induced demyelinating disease, we treated SJL/J mice with TGF-beta 2 either before or after infection with Theiler's murine encephalomyelitis virus. Treatment continued three times weekly through day 35 postinfection. TGF-beta 2 administration resulted in significantly smaller lesions and fewer virus Ag-positive cells in the spinal cords of infected SJL/J mice. Mice treated with TGF-beta 2 had similar levels of virus-specific IgG as infected, control-treated mice. TGF-beta 2 administration significantly increased the level of non-virus-specific activated CTLs, but had no effect on virus-specific CTLs. TUNEL revealed a decrease in the number of apoptotic nuclei in the spinal cord white matter of mice treated in vivo with TGF-beta 2. Immunostaining with an Ab to F4/80 revealed that TGF-beta 2-treated mice had significantly fewer F4/80-positive cells in the white matter of the spinal cord as compared with infected control-treated mice. These data suggest that TGF-beta 2 may control virus-induced demyelination via an immunomodulatory mechanism that reduces macrophage infiltration.

V(beta)8(+) T cells protect from demyelinating disease in a viral model of multiple sclerosis.

Previous studies illustrated the influence of T cell subsets on susceptibility or resistance to demyelination in the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis. Genetic segregation analysis showed a correlation with disease phenotype in this model with particular V(beta) genes. In this study we investigated the contribution of specific V(beta) TCR to the pathogenesis of virus-induced demyelinating disease. Spectratype analysis of cells infiltrating the CNS early in infection demonstrated an over-representation of V(beta)8(+) T cells in mice expressing a susceptible H-2 haplotype. We infected transgenic mice expressing the V(beta)8.2 TCR directed against a non-TMEV antigen and found an increase in demyelinating disease in mice of either susceptible or resistant background compared with littermate controls. In addition, depletion studies with an anti-V(beta)8-specific antibody in both susceptible (B10.Q) and resistant (C57BL/6) mice resulted in increased demyelination. TCR analysis of VP2-specific cytotoxic T cell clones from mice with a resistant genotype identified only the V(beta)8.1 TCR, suggesting that limited T cell diversity is critical to TMEV clearance. Together, these results support a protective role for V(beta)8(+) T cells in virus-induced demyelinating disease.

CD40L is critical for protection from demyelinating disease and development of spontaneous remyelination in a mouse model of multiple sclerosis.

Theiler's murine encephalomyelitis virus (TMEV) induces acute neuronal disease followed by chronic demyelination in susceptible strains of mice. In this study we examined the role of a limited immune defect (deletion or blocking of CD40 ligand [CD40L]) on the extent of brain disease, susceptibility to demyelination, and the ability of demyelinated mice to spontaneously remyelinate following TMEV infection. We demonstrated that CD40L-dependent immune responses participate in pathogenesis in the cerebellum and the spinal cord white matter but protect the striatum of susceptible SJL/J mice. In mice on a background resistant to TMEV-induced demyelination (C57BL/6), the lack of CD40L resulted in increased striatal disease and meningeal inflammation. In addition, CD40L was required to maintain resistance to demyelination and clinical deficits in H-2b mice. CD40L-mediated interactions were also necessary for development of protective H-2b-restricted cytotoxic T cell responses directed against the VP2 region of TMEV as well as for spontaneous remyelination of the spinal cord white matter. The data presented here demonstrated the critical role of this molecule in both antibody- and cell-mediated protective immune responses in distinct phases of TMEV-mediated pathology.

Complementation between specific HLA-DR and HLA-DQ genes in transgenic mice determines susceptibility to experimental autoimmune encephalomyelitis.

To investigate the contribution of human leukocyte antigen (HLA) class II molecules in susceptibility to inflammatory demyelination, we induced experimental autoimmune encephalomyelitis (EAE) in transgenic (tg) mice expressing the HLA-DR3, HLA-DQ8 and HLA-DQ6 molecules in the absence of endogenous class II (Ab(o)). Following immunization with mouse myelin, HLA-DR3 tg mice mounted strong T-cell proliferative responses, and developed inflammatory lesions and demyelination in the central nervous system with mild to moderate clinical symptoms of EAE. HLA-DQ8 and HLA-DQ6 tg mice elicited weak T-cell proliferative responses and did not develop clinical symptoms of EAE. HLA-DR3/DQ6 double tg mice immunized with mouse myelin experienced clinical disease similar to the single tg HLA-DR3 tg mice, indicating that expression of DQ6 in this line had no effect on disease. In contrast, HLA-DR3/DQ8 double tg mice developed severe inflammatory lesions and clinical disease in response to immunization with mouse myelin. Our data suggest that in the presence of two susceptible class II alleles, namely HLA-DR3 and DQ8, there is additional selection and expansion of potential autoreactive T cells, resulting in enhanced severity of disease.

Quantitative assessment of neurologic deficits in a chronic progressive murine model of CNS demyelination.

The precise factors involved in the development of a progressive motor dysfunction, a hallmark of immune-mediated demyelinating diseases such as multiple sclerosis, are not well defined. The ability to identify neurologic deficits that result in impaired motor performance early in disease may allow for the identification of therapeutic interventions that slow or eliminate the progression toward a permanent dysfunction. Here we describe the use of three objective, quantitative functional assays (spontaneous activity box, rotarod, and footprint analysis) to detect early neurologic deficits following the initiation of a demyelinating disease with Theiler's murine encephalomyelitis virus (TMEV). The results show that the assays are capable of revealing neurologic deficits at the early stages of the demyelinating disease process. These findings are the first to objectively characterize neurologic function in an animal model of progressive CNS demyelination.

CNS cell populations are protected from virus-induced pathology by distinct arms of the immune system.

The basis for the distinct patterns of brain pathology in individuals experiencing virus-induced encephalitis may be related to either the tropism of the virus or the host's response to virus infection of the central nervous system (CNS). In these studies we used Theiler's murine encephalomyelitis virus (TMEV) and a series of mice deficient in various immune system components (alpha/beta T cells, antibody, Class I MHC, and Class II MHC) to examine the hypothesis that discrete populations of CNS cells are protected differentially from virus infection by distinct arms of the immune response. Here we demonstrate that the Class I-mediated immune response provided more protection from areas of the brain (brainstem, corpus callosum and cerebellum) with abundant white matter as there was significantly more disease in these areas in beta2m -/- (Class I-deficient) mice as compared to A beta(0) (Class II-deficient) mice. In contrast, the striatum, with an abundance of neurons, was protected from virus-induced pathology primarily by antibody. In addition, we determined that antibody and alpha/beta T cells provided protection from severe deficits and death during the acute phase of the disease. The data presented here support the hypothesis that distinct immune system components function to protect discrete areas of the CNS from virus-induced pathology.

Absence of spontaneous central nervous system remyelination in class II-deficient mice infected with Theiler's virus.

We previously showed that Theiler's murine encephalomyelitis virus (TMEV)-infected major histocompatibility complex (MHC) class II-deficient mice develop both demyelination and neurologic deficits, whereas MHC class I-deficient mice develop demyelination but no neurologic deficits. The absence of neurologic deficits in the class I-deficient mice was associated with preserved sodium channel densities in demyelinated lesions, a relative preservation of axons, and extensive spontaneous remyelination. In this study, we investigated whether TMEV-infected class II-deficient mice, which have an identical genetic background (C57BL/6 x 129) as the class I-deficient mice, have preserved axons and spontaneous myelin repair following chronic TMEV-infection. Both class I- and class II-deficient mice showed similar extents of demyelination of the spinal cord white matter 4 months after TMEV infection. However, the class I-deficient mice demonstrated remyelination by oligodendrocytes, whereas class II-deficient mice showed minimal if any myelin repair. Demyelinated lesions, characterized by inflammatory infiltrates in both mutants, revealed disruption of axons in class II- but not class I-deficient mice. Further characterization revealed that even though class II-deficient mice lacked TMEV-specific IgG, they had virus-specific IgM, which, however, did not neutralize TMEV in vitro. In addition, class II-deficient mice developed TMEV-specific cytotoxic T-lymphocytes in the CNS during the acute (7 days) disease, but these cytotoxic lymphocytes were not present in the chronic stage of disease, despite a high titer of infectious virus throughout the disease. We envision that the presence of demyelination, high virus titer, absence of remyelination, and axonal disruption in chronically infected class II-deficient mice contributes to the development of paralytic disease.

Failure of treatment with Linomide or oral myelin tolerization to ameliorate demyelination in a viral model of multiple sclerosis.

Both Linomide (quinoline-3-carboxamide) and tolerization with self-antigens have been demonstrated to successfully ameliorate demyelinating disease in experimental autoimmune encephalomyelitis (EAE). Based on the autoimmune hypothesis of multiple sclerosis (MS), both agents have been tested in clinical trials but have been found to be toxic or not efficacious. We investigated the efficacy of these immunomodulators in an alternative experimental model of MS, a virus-induced demyelinating disease. Oral administration of Linomide to Theiler's virus-infected mice beginning either at time of infection or at day 15 post-infection (p.i.) resulted in an increased percentage of spinal cord quadrants with demyelination. Administration of Linomide beginning at day 15 p.i. increased lesion size as compared to infected control-treated mice. Treatment with 80 mg kg(-1) day(-1) of Linomide beginning at the time of infection significantly increased the number of Theiler's murine encephalomyelitis virus (TMEV)-positive cells mm(-2) of spinal cord white matter. There were no differences in the amount of remyelination between mice treated with Linomide or water. However, chronically infected mice treated with Linomide had severely reduced spontaneous vertical activity as measured using a activity wheel. Oral tolerization of mice with mouse or bovine myelin had no effect on virus-induced demyelination or virus antigen expression. The contrasting results obtained between the TMEV model and the autoimmune model of demyelination do not support recent reports suggesting that the underlying mechanism of demyelination in the Theiler's model is autoimmune.

Expression of the human histocompatibility leukocyte antigen DR3 transgene reduces the severity of demyelination in a murine model of multiple sclerosis.

The role of various MHC genes in determining the progression of multiple sclerosis (MS) remains controversial. The HLA-DR3 gene has been associated with benign relapsing MS in some genetic epidemiologic studies, but with disease progression in others. We induced demyelination in highly susceptible B10.M and B10.Q mice expressing the DR3 (HLA-DRB1*0301) transgene to determine directly the effects of a human transgene by infecting them with Theiler's murine encephalomyelitis virus (TMEV). DR3+ mice experienced a dramatic reduction in the extent and severity of demyelination compared with DR3- littermate controls, whereas anti-TMEV antibody titers, delayed-type hypersensitivity responses, and levels of infectious virus, virus antigen, and virus RNA were similar in both groups. To address a possible mechanism of how the human transgene is reducing virus-induced demyelination, we analyzed cytokine expression in the lesions and also determined whether B10.M mice can respond to peptides derived from the DR3 molecule. Intense staining for IFN-gamma and IL-4, T helper (TH) 1 and TH2 cytokines, respectively, was found in the lesions of TMEV-infected DR3- mice but not in the DR3+ transgenic mice at day 21 after infection. DR3 peptides elicited strong proliferative responses in B10.M mice but not in B10.M (DR3+) mice. These experiments are the first to demonstrate that a human class II DR gene can alter the severity of demyelination in an animal model of MS without influencing viral load. These experiments are consistent with a mechanism by which DR3 reduces demyelination by altering the cytokine expression in the lesions, possibly by deleting T cells involved in virus-induced pathology.

Chromosome 14 contains determinants that regulate susceptibility to Theiler's virus-induced demyelination in the mouse.

Theiler's murine encephalomyelitis virus causes a chronic demyelinating disease in susceptible strains of mice that is similar to human multiple sclerosis. Several nonmajor histocompatibility complex-linked genes have been implicated as determinants of susceptibility or resistance to either demyelination or virus persistence. In this study, we used linkage analysis of major histocompatibility complex identical H-2d (DBA/2J x B10.D2) F2 intercross mice to identify loci associated with susceptibility to virus-induced demyelinating disease. In a 20-cM region on chromosome 14, we identified four markers, D14Mit54, D14Mit60, D14Mit61, and D14Mit90 that are significantly associated with demyelination. Because two peaks were identified, one near D14Mit54 and one near D14Mit90, it is possible that two loci in this region are involved in controlling demyelination.

Evidence for induction of interferon-alpha and interferon-beta in retinal glial cells of Müller.

Studies were performed to determine if retinal glial cells of Müller transcribe the genes for interferon-alpha (IFNalpha) or IFNbeta upon exposure to virus. Responses to herpes simplex virus type 1 (HSV-1) were tested with cultured murine Müller cells and, in vivo, with retinas obtained after bilateral injection of either HSV-1 or buffer into the anterior chamber of the eyes of BALB/c mice. Induction of IFN transcription and relative temporal changes in transcript levels occurred over time after either in vitro or in vivo exposure to HSV-1. Transcription of both IFN genes was induced in cultured glia within 1 hr after exposure to virus. IFN transcripts were detected in retinas by 24 hr postinfection and these were maximal at 3 days. By in situ hybridization (ISH), IFNalpha2 mRNA localized to focal areas in the intact retinas of virus-injected eyes and was consistent with our previous report of a transient, focal appearance of viral antigens in those retinas. Uninfected cells and ocular tissues were negative for IFN transcripts. Combined ISH and immunohistochemistry on retinal impression smears confirmed that glial fibrillary acidic protein-positive Müller cells are an intraretinal source of IFNalpha and IFNbeta transcripts after ocular exposure to HSV-1. Our results support a role for Muller cells as participants in intraretinal antiviral or immunomodulatory responses via type 1 IFN production and may have implications for future therapeutic interventions.

Antiviral immune responses modulate the nature of central nervous system (CNS) disease in a murine model of multiple sclerosis.

The spectrum of disease is influenced by factors related to both the pathogen and the host, as well as the end points used in defining disease. In this article, the issue of disease resistance versus susceptibility will be examined in the framework that genetic manipulation of either the pathogen or the host immune response alters the balance from disease protection towards pathogenesis. The response of the host may trigger both a protective and a pathogenic immune response. The failure to mount a protective immune response predisposes the pathogen to persistence, which then becomes the target for immunopathology. This review will examine the factors involved both in virus-mediated pathogenesis and in disease protection in the Theiler's model of human multiple sclerosis. By manipulating the character of the virus pathogen and the specificity of the immune response, the entire spectrum of human demyelinating disease is reproduced.

Herpes simplex virus type 1 alters transcript levels of tumor necrosis factor-alpha and interleukin-6 in retinal glial cells.

PURPOSE: Studies were performed to determine whether retinal Müller cells transcribe genes for the proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF alpha). Isolated murine retinas were used to test whether these cytokines were upregulated in the retina in vivo after anterior chamber inoculation of herpes simplex virus type 1 (HSV-1). The effects of exposure to HSV-1 or interferon-gamma (IFN gamma) on transcript levels of these cytokines in cultured retinal glia also were examined. METHODS: In situ hybridization (ISH) using digoxigenin (DIG)-labeled RNA probes was used to localize mRNA for IL-6 and TNF alpha in cultured retinal glial cells. Changes in IL-6 and TNF alpha relative transcript levels were assessed in cultured retinal glial cells using a semiquantitative approach comprised of reverse transcription-polymerase chain reaction (RT-PCR) assay at low amplification cycle number followed by slot blotting and hybridization with DIG-labeled internal sequence probes. In the murine model of herpetic retinitis, the same methods were used to compare temporal changes in relative cytokine transcript levels in retinas isolated from eyes 1 to 7 days after anterior chamber injection of live HSV-1 (KOS strain; 2 x 10(4) pfu/eye) or buffer with levels in retinas isolated from normal, uninjected eyes. Densitometry was used to quantify relative signal changes obtained with serial diluted samples in slot blot assays. Cytokine signal was normalized to hypoxanthine phosphoribosyl transferase signal obtained from the same cDNA samples. RESULTS: Under baseline culture conditions, ISH and RT-PCR indicated that both IL-6 and TNF alpha were transcribed by cultured retinal glia. In vitro exposure to either viral (HSV-1) or inflammatory (IFN gamma) stimulants increased levels of these transcripts in a time-dependent manner. Peak TNF alpha mRNA levels were detected 4 hours after exposure to HSV, whereas IL-6 peaked 4 hours later (increases of 10.3 and 8.7 times over baseline, respectively). Differential increases in TNF alpha and IL-6 transcript levels were detected in retinas isolated from BALB/c mice that received anterior chamber injections of either HSV-1 or Hanks' balanced salt solution (HBSS). By day 3 after HSV-1 injection, increases of 4.5-fold in TNF alpha and 17-fold in IL-6 were detected, whereas substantially smaller changes in TNF alpha and IL-6 (1.5-fold and 6.3-fold, respectively) were observed in HBSS-injected eyes Virus-induced changes in TNF alpha mRNA levels occurred slightly earlier than for IL-6 because maximal levels of TNF alpha were detected 2 to 3 days after infection, but IL-6 peaked at day 3. CONCLUSIONS: Cultured retinal glial cells exhibit upregulated TNF alpha and IL-6 transcript levels after exposure to virus or inflammatory mediators. HSV-1 infection of the anterior segment of the mouse eye markedly upregulates TNF alpha and IL-6 mRNA levels compared to smaller responses to nonspecific inflammation. Taken together, these results identify retinal Müller cells as an intraretinal source of TNF alpha and IL-6 and support the potential of these resident cells to act as intraretinal modulators of immune and inflammatory responses.