M.E. Truckenmiller

N‐Acetylation of L‐Aspartate in the Nervous System: Differential Distribution of a Specific Enzyme

Abstract: L‐Aspartate N‐acetyltransferase, a nervous system enzyme that mediates the synthesis of N‐acetyl‐L‐aspartic acid, has been characterized. In the presence of acetyl‐CoA, L‐aspartate was acetylated 10‐fold more efficiently than L‐glutamate, and the acetylation of aspartylglutamate was not detectable. Within the nervous system, a 10‐fold variation in the enzyme activity was observed, with the brainstem and spinal cord exhibiting the highest activity (10–15 pmol/min/mg tissue) and retina the lowest detectable activity (1–1.5 pmol/min/mg). No enzyme activity was detected in pituitary, heart, liver, or kidney. The enzyme activity was found to be membrane‐associated and was solubilized by treatment with Triton X‐100.

Corticosterone impairs dendritic cell maturation and function

Dendritic cells (DC) play a critical role in initiating and directing adaptive immune responses against pathogens and tumours. Immature DC are thought to act as sentinels in peripheral tissues where their main function is to capture antigen at sites of infection, whereas mature DC are highly efficient at priming T‐cell‐mediated immune responses against infectious pathogens. The DC maturation process is thought to be an important step in the efficient generation of cytotoxic T lymphocytes (CTL). It is well established that many aspects of immune function, including CTL‐mediated antiviral immunity, are modulated by neuroendocrine‐derived products. Corticosterone (CORT), an adrenal hormone produced at increased concentrations during a stress response, has been shown to play a role in impaired CTL responses in stressed animals, leading to high mortality in mice normally resistant to viral infection. While direct effects of neuroendocrine mediators on CTL have been studied, little is known about their effects on DC that are critical for CTL priming. Here, we found that physiologically relevant concentrations of CORT, acting via the glucocorticoid receptor, functionally compromise DC maturation. DC exposed to CORT remained phenotypically and functionally immature after stimulation with lipopolysaccharide and were impaired for the production of interleukin (IL)‐6, IL‐12, and tumour necrosis factor‐α. These effects were biologically significant, as CORT treatment resulted in a marked reduction in the ability of DC to prime naive CD8+ T cells in vivo. These findings offer a potential mechanism underlying stress‐associated immunosuppression.

The dual role of CD8+ T lymphocytes in the development of stress-induced herpes simplex encephalitis

Despite the generally restrictive nature of the blood-brain barrier (BBB), circulating lymphocytes can infiltrate into the central nervous system (CNS) during a variety of disease states. Although the contributions of these lymphocytes to CNS-associated disease have been identified in some viral models, the factors which govern this infiltration following herpes simplex virus (HSV) infection remain to be elucidated. We have developed a murine model of HSV encephalitis (HSE) to define the relationship among psychological stress, the recruitment of HSV-specific T cells into the CNS, and the development of HSE. Naive mice, as well as mice that had been vaccinated with a recombinant vaccinia virus (rVVESgB498-505) that elicits the generation of HSV-1 gB498-505-specific CD8(+) T cells, were infected intranasally (i.n.) with HSV-1 McIntyre. Beginning one day prior to HSV-1 infection and continuing for a total of 9 days, naive and vaccinated mice were exposed to a well-established stressor, restraint stress. Naive, stressed mice exhibited increased symptoms of HSE and HSE-associated mortality as compared to non-stressed controls. A concomitant increase in CD4(+) and CD8(+) T cells in the brain was observed throughout the infection, with CD8(+) T cells outnumbering CD4(+) T cells. The development of HSE in these naive, stressed mice was accompanied by a delayed infiltration of gB498-505-specific CD8(+) T cells after HSV spread into the brain. In contrast, both stressed and non-stressed rVVESgB498-505-vaccinated mice possessed gB498-505-specific CD8(+) T cells prior to HSV challenge and were protected against HSE despite having detectable HSV-1 DNA in the brain. Together, these findings suggest that a delayed infiltration of CD8(+) T cells into the brain may promote HSE in naive mice, while the presence of HSV-specific CD8(+) T cells in the brain prior to HSV challenge is protective, possibly by limiting HSV replication and spread within the CNS.

Corticosterone impairs MHC class I antigen presentation by dendritic cells via reduction of peptide generation

The presentation of viral peptide-MHC class I complexes by antigen presenting cells, such as dendritic cells (DCs), is obligatory for the generation of antiviral effector and memory CD8(+) cytotoxic T lymphocyte (CTL) responses. Prolonged psychological stress is immunosuppressive and undermines primary and memory CTL-mediated antiviral immunity; however, the mechanisms involved are unknown. Using a panel of novel reagents and techniques, we quantitatively measured the effect of the stress-induced hormone corticosterone (CORT) on the efficiency of DCs to process and present virally expressed antigen, characterized the conditions for this CORT-mediated effect, and delineated the components of the MHC class I pathway that were affected. We found that physiologically relevant levels of CORT, prior to infection and acting via the glucocorticoid receptor, suppressed the formation of peptide-MHC class I complexes on the surface of infected DCs. We further showed that this suppression of peptide-MHC class I complexes is via the action of CORT on elements of the class I pathway upstream from TAP that are involved in the generation of antigenic peptides. This CORT-mediated suppression of peptide-class I complexes on DCs also resulted in a marked reduction of their ability to activate a specific T cell hybridoma. These findings offer a mechanism contributing to the stress-induced suppression of host defenses against viral diseases and have implications for the efficacy of antiviral vaccines. At the most fundamental cellular level, this impairment of antigen processing has implications for the regulation of protein degradation in all cells, which is critical to many aspects of immune function.

Stress-Induced Glucocorticoids at the Earliest Stages of Herpes Simplex Virus-1 Infection Suppress Subsequent Antiviral Immunity, Implicating Impaired Dendritic Cell Function

The systemic elevation of psychological stress-induced glucocorticoids strongly suppresses CD8+ T cell immune responses resulting in diminished antiviral immunity. However, the specific cellular targets of stress/glucocorticoids, the timing of exposure, the chronology of immunological events, and the underlying mechanisms of this impairment are incompletely understood. In this study, we address each of these questions in the context of a murine cutaneous HSV infection. We show that exposure to stress or corticosterone in only the earliest stages of an HSV-1 infection is sufficient to suppress, in a glucocorticoid receptor-dependent manner, the subsequent antiviral immune response after stress/corticosterone has been terminated. This suppression resulted in early onset and delayed resolution of herpetic lesions, reduced viral clearance at the site of infection and draining popliteal lymph nodes (PLNs), and impaired functions of HSV-specific CD8+ T cells in PLNs, including granzyme B and IFN-γ production and the ability to degranulate. In knockout mice lacking glucocorticoid receptors only in T cells, we show that these impaired CD8+ T cell functions are not due to direct effects of stress/corticosterone on the T cells, but the ability of PLN-derived dendritic cells to prime HSV-1–specific CD8+ T cells is functionally impaired. These findings highlight the susceptibility of critical early events in the generation of an antiviral immune response to neuroendocrine modulation and implicate dendritic cells as targets of stress/glucocorticoids in vivo. These findings also provide insight into the mechanisms by which the clinical use of glucocorticoids contributes to altered immune responses in patients with viral infections or tumors.

A truncated SV40 large T antigen lacking the p53 binding domain overcomes p53-induced growth arrest and immortalizes primary mesencephalic cells.

Abstract As an alternative to primary fetal tissue, immortalized central nervous system (CNS)-derived cell lines are useful for in vitro CNS model systems and for gene manipulation with potential clinical use in neural transplantation. However, obtaining immortalized cells with a desired phenotype is unpredictable, because the molecular mechanisms of growth and differentiation of CNS cells are poorly understood. The SV40 large T antigen is commonly used to immortalize mammalian cells, but it interferes with multiple cell-cycle components, including p53, p300, and retinoblastoma protein, and usually produces cells with undifferentiated phenotypes. In order to increase the phenotypic repertoire of immortalized CNS cells and to address the molecular mechanisms underlying immortalization and differentiation, we constructed an expression vector containing a truncated SV40 large T gene that encodes only the amino-terminal 155 amino acids (T155), which lacks the p53-binding domain. Constructs were first transfected into a p53-temperature-sensitive cell line, T64-7B. Colonies expressing T155 proliferated at the growth-restrictive temperature. T155 was then transfected into primary cultures from embryonic day-14 rat mesencephalon. Two clonal cell lines were derived, AF-5 and AC-10, which co-expressed T155 and mature neuronal and astrocytic markers. Thus, the amino-terminal portion of SV40 large T is sufficient to: (1) overcome p53-mediated growth arrest despite the absence of a p53-binding region, and (2) immortalize primary CNS cells expressing mature markers while actively dividing. T155 and T155-transfectants may be useful for further studies of cell-cycle mechanisms and phenotyic expression in CNS cells or for further gene manipulation to produce cells with specific properties.

Viral vectors for inducing CD8 + T cell responses

CD8+ T cells (TCD8+) can mediate protective immunity to intracellular pathogens and tumours. Viruses generate strong TCD8+ responses and, therefore, represent attractive vectors for generating vaccines aimed at producing TCD8+-mediated protective immunity. This review will examine the immunological properties of viruses that make them good candidates as vaccine vectors, as well as the manipulations of both vector and antigen that may be required to produce an effective vaccine. The areas addressed include virus infection of dendritic cells in vivo, stimulation of the innate immune response via intracellular and extracellular pattern recognition receptors, the effect of antigenic form on the pathways of antigen presentation and the requirement for elimination of viral genes that target various aspects of the innate and adaptive immune response.

Antibody Inhibition of a Viral Type 1 Interferon Decoy Receptor Cures a Viral Disease by Restoring Interferon Signaling in the Liver

Type 1 interferons (T1-IFNs) play a major role in antiviral defense, but when or how they protect during infections that spread through the lympho-hematogenous route is not known. Orthopoxviruses, including those that produce smallpox and mousepox, spread lympho-hematogenously. They also encode a decoy receptor for T1-IFN, the T1-IFN binding protein (T1-IFNbp), which is essential for virulence. We demonstrate that during mousepox, T1-IFNs protect the liver locally rather than systemically, and that the T1-IFNbp attaches to uninfected cells surrounding infected foci in the liver and the spleen to impair their ability to receive T1-IFN signaling, thus facilitating virus spread. Remarkably, this process can be reversed and mousepox cured late in infection by treating with antibodies that block the biological function of the T1-IFNbp. Thus, our findings provide insights on how T1-IFNs function and are evaded during a viral infection in vivo, and unveil a novel mechanism for antibody-mediated antiviral therapy.

A Marked Reduction in Priming of Cytotoxic CD8+ T Cells Mediated by Stress-Induced Glucocorticoids Involves Multiple Deficiencies in Cross-Presentation by Dendritic Cells

Protracted psychological stress elevates circulating glucocorticoids, which can suppress CD8+ T cell-mediated immunity, but the mechanisms are incompletely understood. Dendritic cells (DCs), required for initiating CTL responses, are vulnerable to stress/corticosterone, which can contribute to diminished CTL responses. Cross-priming of CD8+ T cells by DCs is required for initiating CTL responses against many intracellular pathogens that do not infect DCs. We examined the effects of stress/corticosterone on MHC class I (MHC I) cross-presentation and priming and show that stress/corticosterone-exposed DCs have a reduced ability to cross-present OVA and activate MHC I-OVA257–264-specific T cells. Using a murine model of psychological stress and OVA-loaded β2-microglobulin knockout “donor” cells that cannot present Ag, DCs from stressed mice induced markedly less Ag-specific CTL proliferation in a glucocorticoid receptor-dependent manner, and endogenous in vivo T cell cytolytic activity generated by cross-presented Ag was greatly diminished. These deficits in cross-presentation/priming were not due to altered Ag donation, Ag uptake (phagocytosis, receptor-mediated endocytosis, or fluid-phase uptake), or costimulatory molecule expression by DCs. However, proteasome activity in corticosterone-treated DCs or splenic DCs from stressed mice was partially suppressed, which limits formation of antigenic peptide–MHC I complexes. In addition, the lymphoid tissue-resident CD11b−CD24+CD8α+ DC subset, which carries out cross-presentation/priming, was preferentially depleted in stressed mice. At the same time, CD11b−CD24+CD8α− DC precursors were increased, suggesting a block in development of CD8α+ DCs. Therefore, glucocorticoid-induced changes in both the cellular composition of the immune system and intracellular protein degradation contribute to impaired CTL priming in stressed mice.

T155g-immortalized kidney cells produce growth factors and reduce sequelae of cerebral ischemia.

Fetal rat kidney cells produce high levels of glial-derived neurotrophic factor (GDNF) and exert neuroprotective effects when transplanted into the brain in animal models of Parkinsons disease and stroke. The purpose of the present experiment was to produce kidney cell lines that secrete GDNF. Genes encoding two truncated N-terminal fragments of SV40 large T antigen, T155g and T155c, which does not code for small t antigen, were used. T155g was transduced into E17 cultured fetal Sprague-Dawley rat kidney cortex cells using a plasmid vector, and T155c was transduced with a plasmid and a retroviral vector. Sixteen clones were isolated from cultures transfected with the T155g-expressing plasmid. No cell lines were obtained with T155c. Four clones produced GDNF at physiological concentrations ranging from 55 to 93 pg/ml of medium. These four clones were transplanted into the ischemic core or penumbra of rats that had undergone middle cerebral artery occlusion (MCAO). Three of the four clones reduced the volume of infarction and the behavioral abnormalities normally resulting from MCAO. Blocking experiments with antibodies to GDNF and platelet-derived growth factor (PDGF) suggested that these growth factors contributed only minimally to the reduction in infarct volume and behavioral abnormality. These cell lines may be useful for intracerebral transplantation in animal models of brain injury, stroke, or Parkinsons disease.