Facilitated detection of adventitious agents using genetically engineered cell lines.

Proposals to expand the number and types of cellular substrates used in the production of live attenuated vaccines, especially to include those of tumour origin, have raised concerns about the current capacity to detect adventitious agents that may be present in vaccine stocks. Detection of unknown agents is especially difficult because the culture systems used may not be optimal. We hypothesize that failure to grow certain viruses in culture may be the result of the cellular suicide mechanism, known as apoptosis, killing virus-infected cells before the virus can effectively replicate and spread. Our earlier work with an HIV-1 culture system which overexpresses the cellular anti-apoptotic gene, bcl-2, demonstrated that interfering with the apoptotic programme could facilitate HIV-1 expression and accelerate the kinetics of an acute spreading HIV-1 infection. These findings may have implications for improving cell culture detection systems to screen for potentially harmful infectious agents in current and developmental vaccine substrates. In this paper, we briefly review earlier work and discuss future studies aimed at manipulating the cellular apoptotic programme to facilitate the replication of adventitious and transforming viral agents in vitro.

Simian foamy virus infection among zoo keepers.

We investigated 322 North American zoo workers in an anonymous serosurvey for antibodies to simian foamy viruses to establish the potential risk of zoonotic transmission by these retroviruses. 4 of 133 (3%) individuals who worked specifically with mammals including primates were seropositive, primarily with chimp-like viruses, indicating the importance of work practices to reduce exposure to these agents.

Survey of veterinary conference attendees for evidence of zoonotic infection by feline retroviruses.

OBJECTIVE: To examine exposure risks, possibility of zoonosis, and potential disease associations for feline retroviruses among a group of occupationally exposed individuals. DESIGN: Unlinked voluntary cross-sectional epidemiologic survey. SAMPLE POPULATION: 204 veterinarians, laboratory scientists, and other occupationally exposed individuals who attended a veterinary conference on feline geriatric medicine. PROCEDURE: Blood was collected from participants who also completed a 13-question survey requesting demographic, occupational, exposure, and health information. Blood specimens were fractionated into plasma and mononuclear cell components. Plasma was tested for antibodies against feline immunodeficiency virus (FIV) and feline foamy virus (FeFV), as well as p27 antigen of FeLV. Mononuclear cell lysates were tested for FeLV provirus. RESULTS: Subjects reported extensive duration of work with cats (mean, 17.3 years) and multiple high-risk exposures (eg, cat bites, scratches, and injuries with sharp instruments) per year. However, neither serologic nor molecular evidence of zoonosis with any of the 3 feline retroviruses was detected. CONCLUSIONS AND CLINICAL RELEVANCE: Veterinarians encounter occupational exposures to animal material that place them at high risk for zoonoses. For feline retroviruses, the risk of zoonosis among healthy adult humans appears to be extremely small. However, potential for retroviral zoonosis, especially for viruses such as FeLV and FeFV that can replicate in human cells, cannot be eliminated, and universal precautions to reduce potential exposures should be used when handling sick cats.

Persistent zoonotic infection of a human with simian foamy virus in the absence of an intact orf-2 accessory gene.

Although foamy viruses (FVs) are endemic among nonhuman primates, FV infection among humans is rare. Recently, simian foamy virus (SFV) infection was reported in 4 of 231 individuals occupationally exposed to primates (1.8%). Secondary transmission to spouses has not been seen, suggesting that while FV is readily zoonotic, humans may represent dead-end hosts. Among different simian species, SFV demonstrates significant sequence diversity within the U3 region of the long terminal repeat (LTR) and 3' accessory open reading frames (ORFs). To examine if persistent human SFV infection and apparent lack of secondary transmission are associated with genetic adaptations in FV regulatory regions, we conducted sequence analysis of the LTR, internal promoter, ORF-1, and ORF-2 on a tissue culture isolate and peripheral blood mononuclear cell samples from a human infected with SFV of African green monkey origin (SFV-3). Compared to the prototype SFV-3 sequence, the LTR, internal promoter, and FV transactivator (ORF-1) showed sequence conservation, suggesting that FV zoonosis is not dependent on host-specific adaptation to these transcriptionally important regions. However, ORF-2 contains a number of deleterious mutations predicted to result in premature termination of protein synthesis. ORF-2 codes in part for the 60-kDa Bet fusion protein, proposed to be involved in the establishment of persistent cellular SFV infections. These results suggest that persistent human infection by SFV and reduced transmissibility may be influenced by the absence of a functional ORF-2.

Development and validation of a Western immunoblot assay for detection of antibodies to porcine endogenous retrovirus.

BACKGROUND: Reports that pig endogenous retrovirus (PERV) infects human cells in vitro have heightened the importance of molecular and serologic monitoring of xenograft recipients for evidence of infection with PERV. We report the development and validation of a PERV-specific Western immunoblot assay for the diagnostic testing of porcine xenografts recipients. This assay is based upon the serological cross-reactivity observed between PERV variants capable of infecting human cells in vitro and other mammalian C type retroviruses. METHODS AND RESULTS: Strong reactivity between PERV expressing embryonic pig kidney PK-15 cells and antisera raised against whole virus preparations of murine leukemia virus, gibbon ape leukemia virus (GALV), and simian sarcoma-associated virus was demonstrated by an immunofluorescence assay, suggesting specific antigenic cross-reactivity between this group of viruses and PERV. Western immunoblot analysis demonstrated that anti-GALV antisera reacted with three proteins in PK-15 cells having molecular masses of 30, 55, and 66 kDa. Antisera specific for the Gag proteins of either GALV or simian sarcoma-associated virus reacted with the 30-kDa (major) and 55-kDa (minor) proteins present in PK-15 cells and in PERV-infected 293 human kidney cells, likely representing reactivity to the processed and precursor forms of the PERV Gag protein, respectively. No reactivity was seen in uninfected 293 cells. Analysis of plasma samples from 200 United States blood donors and from 58 human immunodeficiency virus-1, 18 human immunodeficiency virus-2, 13 human T-cell lymphotrophic virus-I, 21 human T-cell lymphotrophic virus-II, and 15 cytomegalovirus infected controls were negative. CONCLUSIONS: As this assay is based on PERV antigen derived from infected human cells, it clearly has the capacity to detect a serologic response towards PERV variants that have zoonotic potential and will allow for the accurate determination of PERV-specific seroreactivity in porcine xenograft recipients.

Xenotransplantation and the risk of retroviral zoonosis.

It is hypothesized that xenotransplantation could facilitate the emergence of new human pathogens. Retroviruses might pose the greatest public health risk because of the possibility of undetected transmission within a population. Evidence from naturally occurring retroviral zoonoses and cross-species infections by animal retroviruses provides a basis for reasoned speculation on the risks posed by xenotransplantation.

Antioxidant defenses influence HIV-1 replication and associated cytopathic effects.

HIV-infected cells often exhibit reduced levels of antioxidant enzymes and thiols. To investigate the role of cellular antioxidant defenses in the progression of an acutely spreading HIV-1 infection, human Sup-T1 T cells were engineered to overexpress the selenium-dependent glutathione peroxidase, GSHPx-1. This enzyme represents a major cellular defense mechanism against toxicity associated with reactive oxygen species (ROS). T cells engineered to produce elevated GSHPx-1 activity displayed accelerated viral replication and associated cytopathic effects compared to control cells. Conversely, the inhibition of the synthesis of glutathione with buthione sulfoximine (BSO) resulted in the attenuation of viral replication in Sup-T1 cells. Similarly, exposure of human peripheral blood lymphocytes (PBLs) to low, nontoxic levels of BSO resulted in an approximately 80% decline in HIV-1 replication as indicated by Western blot analysis of viral proteins.

A T-cell model for the biological role of selenium-dependent glutathione peroxidase.

The cytosolic form of selenium-dependent glutathione peroxidase detoxifies both hydrogen and lipid peroxides and therefore represents a major component of the cellular anti-oxidant defenses. In order to study the biological role of this enzyme, we generated an expression construct in a retroviral vector, which when introduced into immortalized human T-cells, resulted in significant increases in the activity of this important enzyme. This effect is stable over extended maintenance in culture. The anti-oxidant defenses in these same cells are also shown to be attenuated by chemically reducing cellular glutathione levels. Collectively, the ability to both increase and decrease the anti-oxidant defenses in human T cells results in a useful model system for the study of oxidative stress and signaling in this cell type.

bc1-2 expression facilitates human immunodeficiency virus type-1 mediated cytopathic effects during acute spreading infections.

The cytopathic effects (CPE) resulting from the infection of CD4+ T cells by human immunodeficiency virus (HIV) have generally been characterized as single-cell killing associated with apoptosis and/or the generation of syncytia resulting from the direct cell-to-cell transmission of the virus. Little is known, however, about the cellular factors influencing host cell susceptibility to HIV-mediated CPE. Because expression of the antiapoptosis gene, bcl-2, enhances cell viability after exposure to cytotoxic agents or stimuli, the effect of bcl-2 expression on HIV infection of stably transfected T-cell clones was investigated. Unexpectedly, bcl-2 expression by these cells accelerated the kinetics of an acute spreading HIV infection, as evidenced by a rapid loss of culture viability associated with the appearance of CPE and reverse transcriptase activity in the culture supernatant. This unexpected effect of bcl-2 expression results from the arrest of syncytial apoptosis, directly facilitating the cell-to-cell transmission of HIV. In addition, bcl-2 expression is associated with enhanced HIV replication as determined by HIV type 1-specific Western blot (immunoblot) analysis. These results suggest that the inhibition of apoptosis is essential for this mode of viral transmission.

Deep cerebral venous thrombosis presenting as an encephalitic illness.

Three cases of deep cerebral vein thrombosis presenting as encephalitic illnesses are described. Thyrotoxicosis was present in one case, ulcerative colitis in one case and an anticardiolipin antibody was detected in two cases. All three patients were on oestrogen and progesterone. Magnetic resonance imaging and angiography allowed rapid confirmation of the diagnosis and permitted non-invasive follow up of this condition. The first two patients made complete clinical recoveries despite having thalamic infarction, in one case bilaterally, demonstrable radiologically.

New strategies for treating AIDS.

To date, the effective management of HIV-1 infection by anti-retroviral drugs has proved remarkably difficult to achieve. This is primarily due to the ease with which HIV-1 becomes resistant to drugs which initially may be very effective at blocking viral replication. In a recent issue of Science, two promising new AIDS treatments were reported. The first described the use of retroviral-type zinc finger structures found in the HIV-1 nucleocapsid protein as targets for antiretroviral drugs. THe second demonstrated the feasibility of the reverse transcriptase inhibitor (R)-9-(2-phosphonylmethoxypropyl) adenine as a postexposure prophylaxis in blocking HIV-1 infection.

Lipid hydroperoxide-induced apoptosis: lack of inhibition by Bcl-2 over-expression.

Increased membrane lipid peroxidation has recently been implicated as being associated with apoptosis. In the present study the addition of 15-hydroperoxyeicosatetraenoic acid (15-HPETE) or 13-hydroperoxydodecadienoic acid (13-HPODE) to A3.01 T cells is shown to induce marked chromatin condensation coincident with DNA fragmentation, indicative of apoptosis. 15-HPETE also evoked an immediate and sustained rise in cytoplasmic calcium which was required for the induction of apoptosis. A3.01 cells transfected with the bcl-2 proto-oncogene were 6- to 8-fold more resistant to apoptotic killing by tumor necrosis factor-alpha, but only 0.4-fold more resistant to 15-HPETE. Thus, Bcl-2 is not capable of protecting cells from undergoing apoptosis following the direct addition of lipid hydroperoxides.

Redox regulation of programmed cell death in lymphocytes.

A redox imbalance caused by an over-production of prooxidants or a decrease in antioxidants seems to play a role in the programmed cell death that occurs in various developmental programs. Such a physiological function for oxidative stress is particularly applicable to the immune system, wherein individual lymphocytes undergo continuous scrutiny to determine if they should be preserved or programmed to die. Following activation, lymphocytes produced increased levels of reactive oxygen species (ROS) which may serve as intracellular signaling molecules. The ultimate outcome of this increased ROS formation, i.e., lymphocyte proliferation versus programmed cell death, may be dictated by macrophage-derived costimulatory molecules that bolster or diminish lymphocyte antioxidant defenses. HIV-1-infected individuals display multiple symptoms of redox imbalance consistent with their being in oxidative stress, and lymphocytes from such individuals are more prone to undergo apoptosis in vitro. It is suggested that oxidative stress, and lymphocytes from such individuals are more prone to undergo apoptosis in vitro. It is suggested that oxidative stress is a physiological mediator of programmed cell death in lymphoid cells, and that HIV disease represents an extreme case of what can happen when regulatory safeguards are compromised.

Phorbol ester tumor promoter regulation of natural antitumor antibody binding depends on protein kinase C and an intact microfilament system.

Phorbol ester tumor promoter treatment produced a biphasic effect on the binding of polyclonal whole-serum natural antibody (NAb) by L5178Y-F9 murine lymphoma cells. In vitro tumor growth in 100 ng/ml 12-O-tetradecanoylphorbol-13-acetate (TPA) produced a rapid decrease followed by a reversible and unstable increase in NAb binding detected at 4 degrees C. The latter was associated with a functional decrease in NAb binding at 37 degrees C and increases in the tumorigenic and metastatic potentials in vivo. Colchicine, cytochalasin B and sodium azide inhibited the NAb binding of TPA-treated cells, while only colchicine reduced the binding of controls, suggesting the dependence of the TPA-induced increase in NAb binding on microfilament organization and active energy production. The non-tumor-promoting, non-PKC-activating TPA analogue 4-O-Me-TPA failed to alter NAb binding, arguing against nonspecific effects of TPA. The non-tumor-promoting, PKC-activating diacylglycerol, OAG, reproduced the initial decrease in NAb binding but was unable to mimic the subsequent TPA-induced increase. The PKC inhibitor H-7, but not HA1004, could block the TPA-induced increase in NAb binding. Together the data argue that PKC activation is required for both TPA-induced changes in NAb binding but that it is not sufficient to generate the energy- and microfilament-system-dependent, unstable high-NAb-binding phenotype associated with increased tumor progression.

Inhibition of activation-induced death in T cell hybridomas by thiol antioxidants: oxidative stress as a mediator of apoptosis.

N-Acetylcysteine (NAC) is a well established thiol antioxidant which, after uptake, deacylation and conversion to glutathione functions as both a redox buffer and a reactive oxygen intermediate scavenger. We report here that NAC completely blocks activation induced death and associated DNA fragmentation of myelin basic protein (MBP) specific T cell hybridomas. Conversely, NAC had very little effect on the antigen driven proliferation of a MBP specific T cell line similar to that from which the hybridomas were derived. NAC displayed an analogous absolute inhibition of mitogen mediated activation induced death, even if added up to 3 h post activation. Although glutathione was as efficient as NAC at blocking activation induced death, dithiothreitol displayed minimal inhibition while L-cysteine had no effect at all. The observation that certain thiol antioxidants such as NAC and glutathione can completely block the activation induced death of T cell hybridomas implicates redox regulation in this process.

Oxidative stress as a mediator of apoptosis.

Many agents which induce apoptosis are either oxidants or stimulators of cellular oxidative metabolism. Conversely, many inhibitors of apoptosis have antioxidant activities or enhance cellular antioxidant defenses. Mammalian cells exist in a state of oxidative siege in which survival requires an appropriate balance of oxidants and antioxidants. Thomas Buttke and Paul Sandstrom suggest that eukaryotic cells may benefit from this perilous existence by invoking oxidative stress as a common mediator of apoptosis.

Lipid hydroperoxides induce apoptosis in T cells displaying a HIV-associated glutathione peroxidase deficiency.

8E5 is a chronically human immunodeficiency virus (HIV)-infected human T cell line, which we have previously shown to be extremely susceptible to hydrogen peroxide (H2O2)-induced apoptosis due to a HIV-associated catalase deficiency. Here we report that HIV gene expression additionally renders 8E5 cells 10-fold more sensitive than either uninfected A3.01 cells or HIV-infected but nonexpressing 8E5L cells to killing by 15-hydroperoxyeicosatetraenoic acid (15-HPETE), as well as several other hydroperoxy fatty acids. Whereas the viability of A3.01 and 8E5L cells was relatively unaffected by exposure to 10 microM 15-HPETE, similarly treated 8E5 cells underwent apoptosis, as demonstrated by morphological changes and the presence of fragmented DNA. The unique susceptibility of 8E5 cells was attributable to their inability to convert 15-HPETE to 15-hydroxy-eicosatetraenoic acid (15-HETE) owing to a marked reduction in glutathione peroxidase activity. Since oxidized lipids have been reported to accumulate in oxidatively stressed, HIV-infected individuals, a HIV-associated glutathione peroxidase deficiency may contribute to the depletion of CD4 T cells that occurs in the acquired immune deficiency syndrome (AIDS).

HIV gene expression enhances T cell susceptibility to hydrogen peroxide-induced apoptosis.

A human T cell lineage was used to determine the possible effects of HIV infection on T cell antioxidant status. On inoculation into serum-free culture, 8E5, a constitutive HIV-expressing T cell line, underwent apoptosis whereas cell death was not observed with the uninfected A3.01 or latently HIV-infected 8E5L T cell lines. 8E5 survival was markedly prolonged by supplementing the serum-free medium with either A3.01-conditioned medium, catalase, vitamin E, or 2-mercaptoethanol, but supplementation with ascorbic acid, glutathione, or N-acetylcysteine had no effect. Consistent with their being in a state of oxidative stress, 8E5 cells displayed reduced levels of catalase activity, and were more susceptible to killing by exogenous hydrogen peroxide (H2O2) than A3.01 and 8E5L cells. These results demonstrate an inverse correlation between HIV gene expression and antioxidant status in human T cells. Enhanced cytotoxicity of HIV-infected, antioxidant-deficient CD4 T cells following exposure to H2O2 in lymphoid tissues responding to opportunistic pathogens may contribute to the depletion of CD4 T cells in AIDS.

Ethanol-induced insulin resistance suppresses the expression of embryonic ornithine decarboxylase activity.

In utero exposure to ethanol is associated with significant increases in fetal morbidity and mortality as well as with behavioral and learning problems that appear later in life. Growth suppression of the developing child is the most frequent physical effect of ethanol exposure and is correlated with specific molecular changes within the developing organism. The present report suggests that embryonic ethanol exposure suppresses the normal developmental increase in ornithine decarboxylase (ODC) activity. The loss of ODC activity during the early stages of development is dose-dependent and is correlated with the degree of growth suppression. Because ODC is the rate-limiting step for the synthesis of the polyamines and thus appears to be a focal enzyme for the regulation of growth, we have investigated the biochemical consequences of an ethanol-induced inhibition of ODC activity. Using intact chick embryos as well as cultured embryonic tissue, these studies indicate that ethanol-induced changes in tissue putrescine content result in growth suppression because a single dose of exogenous putrescine blocked the growth suppression. In cultured tissue, ethanol exposure inhibited the ability of a known trophic factor (insulin) to induce ODC activity. The loss of insulin-inducible decarboxylase activity as a result of ethanol exposure was specific to ODC, but ethanol per se had no effect on ODC activity in vitro. The data suggest that exposure to ethanol results in a resistance of the embryonic tissue to the action of insulin and thereby disrupts the molecular path by which this mitogenic compound induces the expression of ODC enzymatic activity.

Autocrine production of extracellular catalase prevents apoptosis of the human CEM T-cell line in serum-free medium.

CCRF-CEM is a human T-cell line originally isolated from a child with acute lymphoblastic leukemia. At cell densities > 2 x 10(5) cells per ml, CEM cells grow in serum-free medium, but at lower cell densities the cultures rapidly undergo apoptosis, or programmed cell death. The viability of low-density CEM cells could be preserved by supplementing the serum-free medium with "conditioned" medium from high-density CEM cultures, but a variety of known growth factors and lymphokines were ineffective. Fractionation of conditioned medium by sequential chromatography on DEAE-cellulose, propyl agarose, chromatofocusing, and hydrophobic-interaction HPLC resulted in the isolation of a 60-kDa protein capable of sustaining CEM growth in the absence of serum. The active protein was identified as human catalase based on its amino acid sequence and composition and was subsequently shown to exhibit catalase activity and to be replaceable by human erythrocyte catalase or bovine liver catalase. Comparison of the level of intracellular catalase activity with the amount released into the culture medium demonstrated that the latter accounted for < 3% of the total catalase activity present in the cell culture. These findings show that, despite its low amount, the catalase released by CEM cells, and perhaps by T cells in general, provides a critical first line of defense against hydrogen peroxide (H2O2) present in the extracellular milieu.