Intraocular lymphoma: a series of 14 patients with clinicopathological features and treatment outcomes.

AIMS: To assess the clinical features, pathology, mortality (systemic outcome) and ocular complications (visual outcome) of a cohort of patients treated for intraocular lymphoma. METHODS: Retrospective case analysis of medical records and review of pathology of a consecutive series of patients presenting with intraocular lymphoma in Melbourne over 11 years between 1990 and 2000. Categorical factors influencing survival were examined by the Kaplan-Meier estimator and groups compared with the log rank test. RESULTS: A total of 14 patients were included. The median age of onset of symptoms was 62.5 years. Most were male (64%) and had bilateral eye involvement (64%). The commonest presentation was vitritis in 12 patients, with a median delay of 4 months before diagnosis. In all, 10 patients had B-cell lymphoma, three patients T-cell lymphoma and one null-cell. Four patients had prior systemic lymphoma. Eight patients had primary central nervous system non-Hodgkin's lymphoma (PCNSL). Treatment included combined radiation to the eye and chemotherapy in 10 patients. Complications of radiotherapy included cataract in five (50%), dry eyes in four (40%), punctate keratopathy in two (20%), radiation retinopathy in two (20%), and optic atrophy in one (10%). A total of 11 patients died of lymphoma (79%). One has residual ocular disease, while two have survived for more than 5 years from initial presentation. Although currently disease free, one of these has a poor visual outcome with acuity less than 6/60 secondary to ocular complications of treatment. CONCLUSIONS: Our study had 29% with prior systemic lymphoma, 57% associated with PCNSL and 14% with intraocular disease only. Overall survival is low (21%) and relapses common in those surviving beyond 12 months. Visual outcome in survivors is very poor due, in large part, to significant complications from radiotherapy.

Evidence for oxidative damage in a murine leukemia virus-induced neurodegeneration.

Vacuolation in cellular organelles within the central nervous system is a common manifestation of oxidative injury. We found that the spongiform vacuolation observed in PVC-211 murine leukemia virus (PVC-MuLV) neurodegeneration was associated with oxidative damage as detected by immunoreactivity for 3-nitrotyrosine and protein carbonyl groups. This oxidative injury was present in brain before or concomitant with the appearance of activated microglia, vacuolation, and gliosis that characterize PVC-MuLV neuropathology. Treatment of infected F344 rat pups with the antioxidant vitamin E transiently protected and prolonged the latency of PVC-MuLV neurodegeneration. Taken together, these findings implicate oxidative damage and lipid peroxidation in the pathogenesis of PVC-MuLV neurodegeneration. This animal model may be useful for studies of mechanisms and potential therapies for progressive neurodegeneration following a well-defined insult.

Analysis of receptor usage by ecotropic murine retroviruses, using green fluorescent protein-tagged cationic amino acid transporters.

Entry of ecotropic murine leukemia virus (MuLV) into host cells is initiated by interaction between the receptor-binding domain of the viral SU protein and the third extracellular domain (TED) of the receptor, cationic amino acid transporter 1 (CAT1). To study the molecular basis for the retrovirus-receptor interaction, mouse CAT1 (mCAT1) was expressed in human 293 cells as a fusion protein with jellyfish green fluorescent protein (GFP). Easily detected by fluorescence microscopy and immunoblot analysis with anti-GFP antibodies, the mCAT1-GFP fusion protein was expressed in an N-glycosylated form on the cell surface and in the Golgi apparatus, retaining the ecotropic receptor function. The system was applied to compare Friend MuLV (F-MuLV) and its neuropathogenic variant, PVC-211 MuLV, which exhibits a unique cellular tropism and host range, for the ability to use various CAT family members as a receptor. The results indicated that F-MuLV and PVC-211 MuLV could infect the cells expressing wild-type mCAT1 at comparable efficiencies and that rat CAT3, but not mCAT2, conferred a low but detectable level of susceptibility to F-MuLV and PVC-211 MuLV. The data also suggested that CAT proteins might be expressed in an oligomeric form. Further application of the system developed in this study may provide useful insights into the entry mechanism of ecotropic MuLV.

Capillary endothelial cell tropism of PVC-211 murine leukemia virus and its application for gene transduction.

PVC-211 murine leukemia virus (MuLV) causes neurodegenerative disease following inoculation of neonatal, but not adult, mice and rats. It was previously shown that tropism for brain capillary endothelial cells (CEC) was a determinant of the viral neuropathogenicity. In this study, we demonstrate that host age-dependent replication of PVC-211 MuLV in vivo occurs in CEC in the brain as well as in other organs, such as the liver, kidney, and heart. In contrast, primary explant cultures of CEC derived from brains and livers of adult and neonatal rats could be infected by PVC-211 MuLV, suggesting that the age-dependent susceptibility was abrogated in vitro. Although CEC were generally less susceptible to MuLV-mediated gene transduction than fibroblasts, treatment of CEC with 2-deoxyglucose followed by inoculation of a PVC-211 MuLV-pseudotyped vector in the absence of heparin improved the transduction efficiency. These observations support the possibility that PVC-211 MuLV may be useful for establishing models of CEC gene transduction.

Molecular mechanism for retroviral neuropathogenesis: possible involvement of capillary endothelial cells.

A neuropathogenic variant of Friend MuLV, PVC-211, causes rapidly progressive spongiform neurodegeneration in susceptible rats and mice. Major targets of PVC-211 MuLV infection are brain capillary endothelial cells (BCEC), suggesting that virus-infected BCEC may play crucial roles in neurological disease induction. Consistent with this possibility, studies using chimeric viruses constructed between PVC-211 MuLV and non-neuropathogenic Friend MuLV have revealed that the BCEC tropism of the virus correlates with its neuropathogenicity. Possible involvement of cytokine expression by PVC-211 MuLV-infected BCEC in the induction of neuropathological changes will be discussed.

Analysis of the unique hamster cell tropism of ecotropic murine leukemia virus PVC-211.

PVC-211 murine leukemia virus (MuLV) is a neuropathogenic variant of Friend MuLV (F-MuLV). Previous studies from our laboratory demonstrated that unlike the parental F-MuLV, PVC-211 MuLV can infect rat brain capillary endothelial cells efficiently and that it has acquired genetic changes responsible for its expanded cellular tropism. To determine if PVC-211 MuLV also has expanded its host range, we tested its infectivity on Chinese hamster ovary-derived CHO-K1 cells, which are generally resistant to ecotropic MuLV. The results indicated that PVC-211 MuLV, but not F-MuLV, was highly infectious for CHO-K1 cells. Studies using glycosylation inhibitors and glycosylation mutants of CHO-K1 cells, as well as interference studies, suggested that PVC-211 MuLV has acquired the ability to interact with the ecotropic MuLV receptor on CHO-K1 cells that has undergone glycosylation-dependent modification. Using chimeric viruses between PVC-211 MuLV and F-MuLV, we were able to localize the viral genetic element crucial for CHO-K1 cell tropism within the env gene of PVC-211 MuLV and show that glycine at position 116 and lysine at position 129 of the envelope glycoprotein SU were important. These viral determinants also appear to confer tropism for other hamster cells resistant to ordinary ecotropic MuLVs. Further studies on the interaction between PVC-211 MuLV and the receptor on hamster cells may provide novel insights into the molecular mechanisms for receptor recognition and binding by viral envelope glycoproteins.

In vitro infection of primary and retrovirus-infected human leukocytes by human foamy virus.

The infectivity of human foamy virus (HFV) was examined in primary and cultured human leukocytes. Cell-free infectious viral stocks of HFV were prepared from the human kidney cell line 293 transfected with an infectious molecular clone of HFV. HFV productively infects a variety of human myeloid and lymphoid cell lines. In addition, primary cell cultures enriched for human CD4+, monocytes and brain-derived microglial cells, were readily infected by HFV. Interestingly, while infected primary CD4+ lymphocytes and microglial cells showed marked cytopathology characteristic of foamy virus, HFV-infected monocyte-derived macrophages failed to show any cytopathology. In addition, marked cytotoxicity due to HFV infection was seen in both human T-cell leukemia virus type 1- and human immunodeficiency virus type 1-infected T-cell lines and in human immunodeficiency virus type 1-infected monocytoid cell lines. Thus, HFV infection produces differential cytopathology in a wide host range of primary human leukocytes and hematopoietic cell lines.

Induction of protective CTL responses in newborn mice by a murine retrovirus.

The susceptibility of neonates to virus-induced disease is thought to reflect, in part, the immaturity of their immune systems. However, inoculation of newborn mice with low doses of Cas-Br-M murine leukemia virus induced a protective cytotoxic T lymphocyte (CTL) response. The inability of neonates to develop a CTL response to high doses of virus was not the result of immunological immaturity but correlated with the induction of a nonprotective type 2 cytokine response. Thus, the initial viral dose is critical in the development of protective immunity in newborns.

Effects of subtle changes in the SU protein of ecotropic murine leukemia virus on its brain capillary endothelial cell tropism and interference properties.

PVC-211 murine leukemia virus (MuLV) is a neuropathogenic variant of Friend MuLV (F-MuLV) that causes a rapidly progressive neurodegenerative disease in susceptible rodents. PVC-211 MuLV, but not the parental F-MuLV, can infect rat brain capillary endothelial cells (BCEC) efficiently, and the major determinant for BCEC tropism of PVC-211 MuLV is localized within the XbaI-BamHI fragment of the viral genome containing the 5' half of the env gene. To further dissect the XbaI-BamHI region for its effects on BCEC tropism, we constructed recombinant viruses between PVC-211 MuLV and F-MuLV and tested their infectivity on a cell line established from rat BCEC. Our results indicated that Glu116-to-Gly and Glu129-to-Lys substitutions in the background of the F-MuLV envelope SU protein were sufficient for conferring BCEC tropism on the virus. Interference studies of these viruses on Rat-1 fibroblastic cells showed that the structure of the SU protein encoded by the XbaI-BamHI region also has significant effects on their affinity for the rat ecotropic MuLV receptor. These results support the possibility that structural elements I and II of the SU protein are important determinants for virus-receptor interaction.

Immunogenic determinants of a neuropathogenic murine leukemia virus.

Previous studies of Cas-Br-M murine leukemia virus (MuLV) (Cas-MuLV) infection demonstrated that cytotoxic T cells (CTL) of the CD8+ phenotype play a role in resistance to the neuropathogenic effects of the virus in NFS/N mice. In the current study, we sought to identify the Cas-MuLV epitopes that are immunogenic for the CTL response. Infection of adult NFS/N mice with a well-characterized neuropathogenic variant of Friend MuLV, PVC-211 MuLV (PVC-MuLV), was not immunogenic for MuLV-specific CTL. Therefore, we constructed chimeric viruses between Cas-MuLV and PVC-MuLV. Infectious chimeras contained the Cas-MuLV env gene on a PVC-MuLV background (PVC-CasenvMuLV) and the PVC-MuLV env gene on a Cas-MuLV background (Cas-PVCenvMuLV). Cas-MuLV-specific CTL were found following inoculation of both the chimeric viruses and the parental Cas-MuLV but not the parental PVC-MuLV, despite evidence of antibody responses to both parental and chimeric MuLV. CTL generated in response to infection with PVC-CasenvMuLV and Cas-PVCenvMuLV were exclusively of the CD8+ phenotype. These results indicate that both the env and gag-pol regions of Cas-MuLV express epitopes that are immunogenic for CTL.

Ultraviolet-light-inactivated Cas-Br-M murine leukemia virus induces a protective CD8+ cytotoxic T lymphocyte response in newborn mice.

Newborn NFS/N mice are susceptible to the neurological disease induced by infection with Cas-Br-M murine leukemia virus (Cas), and do not develop a protective cytotoxic T cell (CTL)-mediated response to Cas infection. Here we demonstrate that whole UV light-inactivated Cas (UV-Cas), inoculated in newborn NFS/N mice, induced a strong, Cas-specific CTL response detectable 2 weeks postinoculation and persisting in vivo for > or = 36 weeks. The magnitude of the UV-Cas-induced splenic CTL response, mediated by CD8+ T cells, inversely correlated with the level of proviral cas env sequences detectable in the spleen of the UV-Cas-inoculated mice, as revealed by PCR amplification of tissue DNA. The transfer of UV-Cas-primed splenocytes, with Cas-specific CTL activity, protected 100% of recipient newborn mice from the development of neurological disease induced by infection with live Cas, for more than 28 weeks, and reduced the level of viral replication in the recipients.

Extracellular human T-cell lymphotropic virus type I Tax protein induces cytokine production in adult human microglial cells.

Tropical spastic paraparesis (HAM/TSP) is caused by human T-cell lymphotropic virus type I (HTLV-I) infection. Although the virus infects T cells in vivo and is capable of infecting microglia in vitro, the inflammatory demyelination has not been linked to virus in central nervous system tissue. Thus, indirect mechanisms (e.g., cytokines) could be involved in demyelination and inflammation. The ability of HTLV-I Tax protein to induce tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and IL-6 in primary adult human microglia and peripheral blood macrophages (PBMs) was examined by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction (RT-PCR). Tax (20 ng/ml) induced TNF-alpha in microglia (from undetectable or low basal levels to 215-1,075 pg/ml, mean 576 +/- 375 pg/ml, n = 4) and in PBMs (70-1,900, mean 646 +/- 844 pg/ml, n = 4). This induction was dose dependent, Tax specific, and maximal at 8 hours after stimulation. IL-6 levels in microglia increased from a basal level of 368 +/- 194 to 664 +/- 270 pg/ml 24 hours after Tax stimulation. In contrast, IL-1 beta levels were modestly induced (< or = 26 pg/ml). An increase in mRNA levels of the three cytokines was observed by semiquantitative RT-PCR (TNF-alpha = 28-fold; IL-6 = 5.6-fold; IL-1 beta = 3.6-fold). Thus, in HAM/TSP, extracellular Tax released from infiltrating T cells could induce cytokine release by microglia and contribute to demyelination and inflammation in the absence of detectable virus.

Cytokine-gene expression in measles-infected adult human glial cells.

The expression of interleukin (IL)-1 beta, IL-6 and tumor necrosis factor (TNF) alpha transcripts in cultured human glial cells was examined using reverse transcription followed by polymerase chain reaction (PCR) amplification and Southern blot quantitation. Microglial cultures derived from brain biopsy specimens from three different individuals expressed transcripts for the three cytokines under basal culture conditions. This expression was enhanced in response to measles virus (MV) infection (IL-1 beta, 2.2-8.8-fold; IL-6, 2.5-8.4-fold; TNF alpha, 2.2-3.2-fold). Neither IL-1 beta nor TNF alpha transcripts were detectable in undissociated brain tissue from two individuals, suggesting that the basal expression of these cytokines in culture may have been induced by tissue dissociation or by the culture conditions. Oligodendrocytes did not express cytokine transcripts under basal culture conditions, and IL-1 beta and IL-6 but not TNF alpha transcripts could be induced by MV. Similarly, meningeal fibroblasts expressed IL-1 beta and IL-6 but not TNF alpha in response to MV-infection, suggesting that the production of TNF alpha is more cell type-restricted than either IL-1 beta or IL-6. The results indicate that adult human microglia can participate in the inflammatory response to MV infection in the CNS by producing cytokines that contribute to inflammation and demyelination. In addition, besides their role in myelination, oligodendrocytes can potentially influence immunoreactivity in the CNS by producing IL-1 beta and IL-6.

Viral determinants that control the neuropathogenicity of PVC-211 murine leukemia virus in vivo determine brain capillary endothelial cell tropism of the virus in vitro.

PVC-211 murine leukemia virus (MuLV) is a neuropathogenic, weakly leukemogenic variant of the nonneuropathogenic, highly leukemogenic Friend MuLV (F-MuLV). Chimeric viruses constructed from PVC-211 MuLV clone 3d and F-MuLV clone 57 indicate that the env gene of PVC-211 MuLV contains the determinant(s) responsible for pathological changes in the central nervous system. However, sequences within the 5' one-third (AatII-EcoRI region) of the PVC-211 MuLV genome, which include the 5' leader sequence, the gag gene, and the 5' quarter of the pol gene, are also needed in conjunction with the env gene determinant(s) to cause clinically evident neurological disease in the majority of virus-infected animals after a short latency. In the presence of the AatII-EcoRI region of the PVC-211 MuLV genome, the PVC-211 MuLV env gene sequences encoding the amino-terminal half of the SU protein, which contains the receptor-binding region of the protein, were sufficient to cause rapidly progressive neurological disease. When PVC-211 MuLV, F-MuLV, and various chimeric viruses were tested for their ability to replicate in cultured brain capillary endothelial cells (BCEC), the primary site of PVC-211 MuLV replication within the central nervous system, there was a direct correlation between the replication efficiency of a virus in BCEC in vitro and its ability to cause neurological disease in vivo. This observation indicates that the sequences in PVC-211 MuLV that render it neuropathogenic affect its replication in BCEC and suggests that rapid and efficient replication of the virus in BCEC is crucial for the pathological changes in the central nervous system that result in development of neurological disease.

IFN-gamma production in response to neuropathogenic Cas-Br-M murine leukemia virus infection.

T cell-mediated production of IFN-gamma followed infection of adult, but not neonatal NFS/N mice with Cas-Br-M murine leukemia virus (Cas). The IFN-gamma response was associated with the appearance of CTL specific for Cas and with age-dependent resistance to neurologic disease. While both immune responses were mediated by a CD8-enriched population of T cells, IFN-gamma did not play a role in the activation of the Cas-specific CTL response. However, when given exogenously, IFN-gamma delayed the onset and reduced the incidence of Cas-induced neurologic disease. These data suggest that the IFN-gamma response to Cas infection may be an important host defense mechanism whose effects on virus replication and neurologic disease expression are independent of its effect on Cas-specific CTL.

Human T-cell leukemia virus type I infection of monocytes and microglial cells in primary human cultures.

The pathogenesis of progressive spastic paraparesis [HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP)], a serious consequence of human T-cell leukemia virus type I (HTLV-I) infection, is unclear. T and B lymphocytes can be naturally infected by HTLV-I, but the susceptibility to HTLV-I infection of other cell types that could contribute to the pathogenesis of HAM/TSP has not been determined. We found that a human monocyte cell line (THP-1), primary human peripheral blood monocytes, and isolated microglial cells but not astrocytes or oligodendroglial cells derived from adult human brain were infected by HTLV-I in vitro. Infection with HTLV-I enhanced the secretion of interleukin 6 in human microglial cell-enriched cultures but did not stimulate the release of interleukin 1 from monocytes or microglial cells. Tumor necrosis factor alpha production was stimulated by HTLV-I infection of monocytes and microglial cells and could be enhanced by suboptimal amounts of lipopolysaccharide. Since both tumor necrosis factor alpha and interleukin 6 have been implicated in inflammatory demyelination and gliosis, our findings suggest that human microglial cells and monocytes infected with and activated by HTLV-I could play a role in the pathogenesis of HAM/TSP.

Cellular tropism and localization in the rodent nervous system of a neuropathogenic variant of Friend murine leukemia virus.

BACKGROUND: We studied PVC-211 murine leukemia virus (MuLV) (1), a neuropathogenic variant of Friend MuLV, to determine its cellular tropism and distribution in the nervous system of infected rats and the factors that affected disease expression. EXPERIMENTAL DESIGN: Rats from five different strains and mice from 3 strains were inoculated intracerebrally or intraperitoneally from birth to 10 days of age and observed for signs of neurologic disease and tumors for 24 weeks. Nervous system pathology, MuLV gp70 expression, and virus production were evaluated weekly for 4 weeks after perinatal infection of Fisher (F344) rats. Blood-brain-barrier integrity and ultrastructure were evaluated in 21-day-old symptomatic infected rats. Microvessel and mixed glial cell cultures were prepared from brains of infected and uninfected 21-day-old F344 rats and evaluated for virus production, MuLV gp70 expression, and the presence of PVC-211 MuLV DNA. RESULTS: Tremor, ataxia, spasticity, and hindlimb weakness occurred in rats and mice as early as 3 weeks after neonatal infection. Severity, latency, and progression varied among mouse and rat strains but exposure to PVC-211 MuLV before 6 days of age was required for disease expression. Rapid PVC-211 MuLV replication in brain capillary endothelial cells (BCEC) early in the perinatal period was followed by widespread astrogliosis, neuropil vacuolation, and finally, neuronal degeneration in the spinal cord, brainstem, cerebellum, and subcortex. MuLV gp70 expression in vivo increased during infection, was restricted to BCEC, but was not associated with perivascular inflammatory infiltrates. BCEC cultured from microvessel preparations but not astrocytes or microglia in mixed glial cell cultures isolated from infected rats contained PVC-211 MuLV DNA, expressed MuLV gp70, and produced infectious virus. CONCLUSIONS: The rapid replication of PVC-211 MuLV that occurs in the nervous system of infected rodents is restricted to BCEC. These infected BCEC appear to play a critical role in initiating the astroglial response in this neurodegenerative process through mechanisms that remain to be defined.