Bone marrow from a normal male pig was transplanted into a related female pig with severe homozygous von Willebrand's disease (vWd). After engraftment the circulating leukocytes were of the male karyotype, and the platelets were strongly positive for von Willebrand factor (vWF) by indirect immunofluorescence. The average level of vWF was 1.96 U/dl and of ristocetin cofactor was 2.8 U/dl. The ear immersion bleeding time before transplantation was consistently more than 15 min and afterwards varied between 5 min and more than 15 min. Transfused vWF corrected the bleeding time at a level of 10 U/dl, which is lower than that required for a von Willebrand pig. We concluded that: (a) the plasmatic compartment is only minimally replenished by the vWF from platelets and megakaryocytes; and (b) the platelet vWF alone only partially corrects the abnormal tests of the hemostatic mechanism in severe vWd.
The precise factors involved in the development of a progressive motor dysfunction, a hallmark of immune-mediated demyelinating diseases such as multiple sclerosis, are not well defined. The ability to identify neurologic deficits that result in impaired motor performance early in disease may allow for the identification of therapeutic interventions that slow or eliminate the progression toward a permanent dysfunction. Here we describe the use of three objective, quantitative functional assays (spontaneous activity box, rotarod, and footprint analysis) to detect early neurologic deficits following the initiation of a demyelinating disease with Theiler's murine encephalomyelitis virus (TMEV). The results show that the assays are capable of revealing neurologic deficits at the early stages of the demyelinating disease process. These findings are the first to objectively characterize neurologic function in an animal model of progressive CNS demyelination.
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.
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