Kallikrein 6 (K6) is a member of the kallikrein gene family that comprises 15 structurally and functionally related serine proteases. In prior studies we showed that, while this trypsin-like enzyme is preferentially expressed in neurons and oligodendroglia of the adult central nervous system (CNS), it is up-regulated at sites of injury due to expression by infiltrating immune and resident CNS cells. Given this background we hypothesized that K6 is a key contributor to the pathophysiology of traumatic spinal cord injury (SCI), influencing neural repair and regeneration. Examination of K6 expression following contusion injury to the adult rat cord, and in cases of human traumatic SCI, indicated significant elevations at acute and chronic time points, not only at the injury site but also in cord segments above and below. Elevations in K6 were particularly prominent in macrophages, microglia and reactive astrocytes. To determine potential effects of elevated K6 on the regeneration environment, the ability of neurons to adhere to and extend processes on substrata which had been exposed to recombinant K6 was examined. Limited (1 h) or excess (24 h) K6-mediated proteolytic digestion of a growth-facilitatory substrate, laminin, significantly decreased neurite outgrowth. By contrast, similar hydrolysis of a growth-inhibitory substrate, aggrecan, significantly increased neurite extension and cell adherence. These data support the hypothesis that K6 enzymatic cascades mediate events secondary to spinal cord trauma, including dynamic modification of the capacity for axon outgrowth.

A major area of investigation in neurovirology is directed toward understanding the factors that participate in neuronal viral clearance versus viral persistence. Clearance of virus from the infected central nervous system (CNS) is unique because of the intact blood-brain barrier, the relative absence of major histocompatibility complex (MHC) molecules on neuronal cells, and the lack of well-established lymphatic drainage. Nevertheless, once viruses replicate in the CNS, there is a vigorous immune response directed toward the clearance of virus antigen from infected cells. Antibody plays a critical role in neutralizing extracellular viral particles and also has been proposed to participate in the clearance of intracellular virus (4, 21). However, the manner in which antibody enters cells or interacts with surface cellular receptors to prevent viral persistence in neurons is not well understood.The classical way in which intracellular virus is eliminated is by cytotoxic T cells that are restricted by class I MHC. The control of MHC expression on neurons is dependent upon electrical activity (36). Neurons with normal electrical activity suppress MHC expression, whereas silent or injured neurons up-regulate class I MHC expression, an activity that makes them susceptible to class I MHC-mediated injury. Disruption of electrical activity induces class II MHC expression on microglia and astrocytes (36). Following virus infection in the CNS, class I MHC is rapidly up-regulated (1, 26) in neuronal cells. In particular, soluble factors such as alpha/beta interferon (IFN-␣/␤) (39) are required for the up-regulation of MHC in most CNS cells, including neurons. Cytotoxic T-cell responses in brain infiltrating mononuclear inflammatory cells have been demonstrated (23,24,25,28) and have been shown to participate in viral clearance. However, the consequences to the CNS are a "double-edged sword." Virus is cleared at the expense of the destruction of neurons that are not renewable and whose death results in permanent functional deficits. For example, cytotoxic T cells have been shown to transect neurites expressing class I MHC (31). Therefore, this vigorous cytotoxic response may participate directly in immune-mediated pathology.However, there are examples where viruses are cleared from the CNS without significant destruction of brain parenchyma (2). In these situations, the hypothesis proposed is that factors secreted by cytotoxic lymphocytes participate in viral clearance without cytotoxicity. Of the factors that are secreted by immune cells and that are thought to play a critical role in viral clearance, IFN-␥ has received the most attention (33). IFN-␥ is a 50-kDa N-glycosylated noncovalent homodimer composed of two identical 17-kDa polypeptides. It is produced by activated NK cells and T cells. IFN-␥ induces many immunomodulatory effects on CNS cells, including activation of macrophages, promotion of leukocyte adhesion to allow trafficking of cells to the * Corresponding author. Mailing address:

For many emerging and re-emerging infectious diseases, definitive solutions via sterilizing adaptive immunity may require years or decades to develop, if they are even possible. The innate immune system offers alternative mechanisms that do not require antigen-specific recognition or a priori knowledge of the causative agent. However, it is unclear whether effective stable innate immune system activation can be achieved without triggering harmful autoimmunity or other chronic inflammatory sequelae. Here, we show that transgenic expression of a picornavirus RNA-dependent RNA polymerase (RdRP), in the absence of other viral proteins, can profoundly reconfigure mammalian innate antiviral immunity by exposing the normally membrane-sequestered RdRP activity to sustained innate immune detection. RdRP-transgenic mice have life-long, quantitatively dramatic upregulation of 80 interferon-stimulated genes (ISGs) and show profound resistance to normally lethal viral challenge. Multiple crosses with defined knockout mice (Rag1, Mda5, Mavs, Ifnar1, Ifngr1, and Tlr3) established that the mechanism operates via MDA5 and MAVS and is fully independent of the adaptive immune system. Human cell models recapitulated the key features with striking fidelity, with the RdRP inducing an analogous ISG network and a strict block to HIV-1 infection. This RdRP-mediated antiviral mechanism does not depend on secondary structure within the RdRP mRNA but operates at the protein level and requires RdRP catalysis. Importantly, despite lifelong massive ISG elevations, RdRP mice are entirely healthy, with normal longevity. Our data reveal that a powerfully augmented MDA5-mediated activation state can be a well-tolerated mammalian innate immune system configuration. These results provide a foundation for augmenting innate immunity to achieve broad-spectrum antiviral protection.

Axon injury is a major determinant of the loss of neurologic function in patients with multiple sclerosis (MS). It is unclear, however, whether damage to axons is an obligatory consequence of demyelination or whether it is an independent process that occurs in the permissive environment of demyelinated lesions. Previous investigations into the role of CD8+ T cells and perforin in the Theiler’s murine encephalomyelitis virus (TMEV) model of MS have used mouse strains resistant to TMEV infection. To test the role of CD8+ T cells in axon injury, we established a perforin-deficient mouse model on the H-2q MHC background thereby removing confounding factors related to viral biology in this TMEV-susceptible strain. This permitted direct comparison of clinical and pathological parameters between perforin-competent and perforin-deficient mice. The extent of demyelination was indistinguishable between perforin-competent and perforin-deficient H-2q mice but chronically infected perforin-deficient mice exhibited preservation of motor function and spinal axons despite the presence of spinal cord demyelination. Thus, demyelination is necessary but insufficient for axon injury in this model; the absence of perforin protects axons without impacting demyelination. These results suggest that perforin is a key mediator of axon injury and lend additional support to the hypothesis that CD8+ T cells are primarily responsible for axon damage in MS.

We compared CNS disease following intracere‐bral injection of SJL mice with Daniel's (DA) and BeAn 8386 (BeAn) strains of Theiler's murine encephalomyelitis virus (TMEV). In tissue culture, DA was more virulent then BeAn. There was a higher incidence of demyelination in the spinal cords of SJL/J mice infected with DA as compared to BeAn. However, the extent of demyelination was similar between virus strains when comparing those mice that developed demyelination. Even though BeAn infection resulted in lower incidence of demyelination in the spinal cord, these mice showed significant brain disease similar to that observed with DA. There was approximately 100 times more virus specific RNA in the CNS of DA infected mice as compared to BeAn infected mice. This was reflected by more virus antigen positive cells (macrophages/microglia and oligodendrocytes) in the spinal cord white matter of DA infected mice as compared to BeAn. There was no difference in the brain infiltrating immune cells of DA or BeAn infected mice. However, BeAn infected mice showed higher titers of TMEV specific antibody. Functional deficits as measured by Rotarod were more severe in DA infected versus BeAn infected mice. These findings indicate that the diseases induced by DA or BeAn are distinct.