No abstract

Partial injury to the spinal cord can propagate itself, sometimes leading to paralysis attributable to degeneration of initially undamaged neurons. We demonstrated recently that autoimmune T cells directed against the CNS antigen myelin basic protein (MBP) reduce degeneration after optic nerve crush injury in rats. Here we show that not only transfer of T cells but also active immunization with MBP promotes recovery from spinal cord injury. Anesthetized adult Lewis rats subjected to spinal cord contusion at T7 or T9, using the New York University impactor, were injected systemically with anti-MBP T cells at the time of contusion or 1 week later. Another group of rats was immunized, 1 week before contusion, with MBP emulsified in incomplete Freund's adjuvant (IFA). Functional recovery was assessed in a randomized, double-blinded manner, using the open-field behavioral test of Basso, Beattie, and Bresnahan. The functional outcome of contusion at T7 differed from that at T9 (2.9 Ϯ 0.4, n ϭ 25, compared with 8.3 Ϯ 0.4, n ϭ 12; p Ͻ 0.003). In both cases, a single T cell treatment resulted in significantly better recovery than that observed in control rats treated with T cells directed against the nonself antigen ovalbumin. Delayed treatment with T cells (1 week after contusion) resulted in significantly better recovery (7.0 Ϯ 1; n ϭ 6) than that observed in control rats treated with PBS (2.0 Ϯ 0.8; n ϭ 6; p Ͻ 0.01; nonparametric ANOVA). Rats immunized with MBP obtained a recovery score of 6.1 Ϯ 0.8 (n ϭ 6) compared with a score of 3.0 Ϯ 0.8 (n ϭ 5; p Ͻ 0.05) in control rats injected with PBS in IFA. Morphometric analysis, immunohistochemical staining, and diffusion anisotropy magnetic resonance imaging showed that the behavioral outcome was correlated with tissue preservation. The results suggest that T cell-mediated immune activity, achieved by either adoptive transfer or active immunization, enhances recovery from spinal cord injury by conferring effective neuroprotection. The autoimmune T cells, once reactivated at the lesion site through recognition of their specific antigen, are a potential source of various protective factors whose production is locally regulated.

The ability of rats or mice to withstand the consequences of injury to myelinated axons in the CNS was previously shown to depend on the ability to manifest a T cell-mediated protective immune response, which is amenable to boosting by myelin-specific T cells. Here we show that this ability, assessed by retinal ganglion cell survival after optic nerve injury or locomotor activity after spinal cord contusion, is decreased if the animals were immunized as neonates with myelin proteins (resulting in their nonresponsiveness as adults to myelin proteins) or injected with naturally occurring regulatory CD4 ؉ CD25 ؉ T cells immediately after the injury, and is improved by elimination of these regulatory T cells. In nude BALB͞c mice replenished with a splenocyte population lacking CD4 ؉ CD25 ؉ regulatory T cells, significantly more neurons survived after optic nerve injury than in nude mice replenished with a complete splenocyte population or in matched wild-type controls. In contrast, neuronal survival in wildtype BALB͞c mice injected with CD4 ؉ CD25 ؉ regulatory T cells immediately after injury was significantly worse than in noninjected controls. These findings suggest that the ability to cope with the sequelae of a CNS insult is affected unfavorably by nonresponsiveness to myelin self-antigens and favorably by conditions allowing rapid expression of an autoimmune response. The regulatory T cells might represent an evolutionary compromise between the need to avoid autoimmune diseases and the need for autoimmunity on alert for the purpose of tissue maintenance.

Primary damage caused by injury to the CNS is often followed by delayed degeneration of initially spared neurons. Studies in our laboratory have shown that active or passive immunization with CNS myelin-associated self-antigens can reduce this secondary loss. Here we show, using four experimental paradigms in rodents, that CNS trauma spontaneously evokes a beneficial T cell-dependent immune response, which reduces neuronal loss.(1) Survival of retinal ganglion cells in rats was significantly higher when optic nerve injury was preceded by an unrelated CNS (spinal cord) injury. (2) Locomotor activity of rat hindlimbs (measured in an open field using a locomotor rating scale) after contusive injury of the spinal cord (T8) was significantly better (by three to four score grades) after passive transfer of myelin basic protein (MBP)-activated splenocytes derived from spinally injured rats than in untreated injured control rats or rats similarly treated with splenocytes from naive animals or with splenocytes from spinally injured rats activated ex vivo with ovalbumin or without any ex vivo activation. (3) Neuronal survival after optic nerve injury was 40% lower in adult rats devoid of mature T cells (caused by thymectomy at birth) than in normal rats. (4) Retinal ganglion cell survival after optic nerve injury was higher (119 Ϯ 3.7%) in transgenic mice overexpressing a T cell receptor (TcR) for MBP and lower (85 Ϯ 1.3%) in mice overexpressing a T cell receptor for the non-self antigen ovalbumin than in matched wild types. Taken together, the results imply that CNS injury evokes a T cell-dependent neuroprotective response.

Homologues of signal peptide peptidase (SPPLs) are putative aspartic proteases that may catalyse regulated intramembrane proteolysis of type II membrane-anchored signalling factors. Here, we show that four human SPPLs are each sorted to a different compartment of the secretory pathway. We demonstrate that SPPL2a and SPPL2b, which are sorted to endosomes and the plasma membrane, respectively, are functional proteases that catalyse intramembrane cleavage of tumour necrosis factor alpha (TNFalpha). The two proteases promoted the release of the TNFalpha intracellular domain, which in turn triggers expression of the pro-inflammatory cytokine interleukin-12 by activated human dendritic cells. Our study reveals a critical function for SPPL2a and SPPL2b in the regulation of innate and adaptive immunity.

Injury to the CNS is often followed by a spread of damage (secondary degeneration), resulting in neuronal losses that are substantially greater than might have been predicted from the severity of the primary insult. Studies in our laboratory have shown that injured CNS neurons can benefit from active or passive immunization with CNS myelin-associated antigens. The fact that autoimmune T-cells can be both beneficial and destructive, taken together with the established phenomenon of genetic predisposition to autoimmune diseases, raises the question: will genetic predisposition to autoimmune diseases affect the outcome of traumatic insult to the CNS? Here we show that the survival rate of retinal ganglion cells in adult mice or rats after crush injury of the optic nerve or intravitreal injection of a toxic dosage of glutamate is up to twofold higher in strains that are resistant to the CNS autoimmune disease experimental autoimmune encephalomyelitis (EAE) than in susceptible strains. The difference was found to be attributed, at least in part, to a beneficial T-cell response that was spontaneously evoked after CNS insult in the resistant but not in the susceptible strains. In animals of EAE-resistant but not of EAEsusceptible strains devoid of mature T-cells (as a result of having undergone thymectomy at birth), the numbers of surviving neurons after optic nerve injury were significantly lower (by 60%) than in the corresponding normal animals. Moreover, the rate of retinal ganglion cell survival was higher when the optic nerve injury was preceded by an unrelated CNS (spinal cord) injury in the resistant strains but not in the susceptible strains. It thus seems that, in normal animals of EAE-resistant strains (but not of susceptible strains), the injury evokes an endogenous protective response that is T-cell dependent. These findings imply that a protective T-cell-dependent response and resistance to autoimmune disease are regulated by a common mechanism. The results of this study compel us to modify our understanding of autoimmunity and autoimmune diseases, as well as the role of autoimmunity in non-autoimmune CNS disorders. They also obviously have far-reaching clinical implications in terms of prognosis and individual therapy.

Liver gene transfer is a highly sought goal for the treatment of inherited and infectious diseases. Lentiviral vectors (LVs) have many desirable properties for hepatocyte-directed gene delivery, including the ability to integrate into nondividing cells. Unfortunately, upon systemic administration, LV transduces hepatocytes relatively inefficiently compared with nonparenchymal cells, and the duration of transgene expression is often limited by immune responses. Here, we investigated the role of innate antiviral responses in these events. We show that administration of LVs to mice triggers a rapid and transient IFN␣␤ response. This effect was dependent on functional vector particles, and in vitro challenge of antigen-presenting cells suggested that plasmacytoid dendritic cells initiated the response. Remarkably, when LVs were administered to animals that lack the capacity to respond to IFN␣␤, there was a dramatic increase in hepatocyte transduction, and stable transgene expression was achieved. These findings indicate that, even in the setting of acute delivery of replication-defective vectors, IFNs effectively interfere with transduction in a celltype-specific manner. Moreover, because disabling a single component of the innate/immune network was sufficient to establish persistent xenoantigen expression, our results raise the hope that the immunologic barriers to gene therapy are less insurmountable than expected. IntroductionLentiviral vectors (LVs) are a promising candidate system for therapeutic gene transfer. Because of their capacity to transduce nondividing cells and stably integrate a gene expression cassette of relatively large size and complexity, LVs have significant potential for achieving long-term expression of a therapeutic molecule. Several groups, including our own, have carried out studies using LV for in vivo gene delivery in rodents. [1][2][3][4][5] Efficient gene transfer to the liver could be achieved; however, hepatocytes, which were the main target of the therapy, were transduced at a relatively low efficiency compared with nonparenchymal cells. At a low vector dose, this effect was particularly pronounced. While a high frequency of Kupffer cells (KCs) were found to be vector positive, only a small fraction of hepatocytes were transduced.Interestingly, by increasing the concentration of injected vector a threshold is reached in which hepatocyte transduction becomes dose responsive, and improved hepatocyte gene transfer is achieved. This may be due to the requirement for saturating the particleclearance systems of the sinusoid-lining cells in blood-filtering organs. 6 Nonetheless, a better understanding of the rate-limiting factors in transduction would help to improve both the dose-effect relationship and risk-benefit ratio of systemic LV administration.A high incidence of transgene-specific immunity has also been observed in studies using LVs for in vivo gene delivery. [1][2][3][4][5] This response resulted in elimination of transduced cells and/or neutralization of the transgene product,...

Myosin VI is the only myosin that moves toward the minus end of actin filaments, suggesting a unique biological function. Here, we show that myosin VI is present in the nucleus of mammalian cells where it colocalizes with newly transcribed mRNA and with RNA polymerase II (RNAPII) and is detected in the RNAPII complex. The colocalization and interaction of myosin VI with RNAPII require transcriptional activity. Chromatin immunoprecipitation (ChIP) demonstrates that myosin VI is recruited to the promoter and intragenic regions of active genes, encoding urokinase plasminogen activator (uPA), eukaryotic initiation factor 6 (p27/eIF6), and low-density lipoprotein receptor (LDLR), but not to noncoding, nonregulatory intergenic regions. Downregulation of myosin VI reduces steady-state mRNA levels of these genes in vivo, and antibodies to myosin VI reduce transcription in vitro. We suggest that myosin VI modulates RNAPII-dependent transcription of active genes, implicating the possibility of an actin-myosin based mechanism of transcription.