M. C. Dal Canto

A low expressor line of transgenic mice carrying a mutant human Cu,Zn superoxide dismutase (SOD1) gene develops pathological changes that most closely resemble those in human amyotrophic lateral sclerosis

Abstract About 15–20% of patients with familial amyotrophic lateral sclerosis (ALS) carry one of several missense mutations in the gene for Cu,Zn superoxide dismutase (SOD1). We have previously reported on an animal model of this disease produced by the transgenic expression of a mutant form of human SOD1 containing a Gly93→Ala amino acid substitution. Several lines of transgenic mice were produced, characterized by a differing tempo and severity of disease that generally correlated with the number of mutant gene copies that these lines expressed. We reported that mice expressing high copy numbers (18–25) developed a disease with a relatively short course and with a pathology mainly characterized by severe vacuolar degeneration of motor neurons and their processes. Lewy-like bodies and swollen axons were also present. The exquisite localization to motor neurons was the feature that made the pathology in these overexpressors germane to the human disease. Severe vacuolar degeneration, however, was considered to be at variance with human ALS, in which similar changes have not been described. In the present study, we have made a temporal characterization of microscopic and immunohistochemical changes in a line of transgenic mice expressing lower copy numbers of the mutant gene. These mice, designated G5/G5, survive more than 400 days and present pathological changes which are virtually identical to those in the human disease. In fact, in these animals, anterior horn cell depletion, atrophy, astrocytosis, and the presence of numerous ubiquitinated Lewy-like bodies and axonal swellings are the main pathological features, while vacuolar pathology is minimal. This study underscores the importance of the level of expression of the mutant enzyme in the resulting clinical and pathological disease, and supports the value of this transgenic model as an excellent tool for investigating both pathogenesis of human ALS and possible therapeutic avenues.

Striking Similarity of Murine Nectin-1α to Human Nectin-1α (HveC) in Sequence and Activity as a Glycoprotein D Receptor for Alphaherpesvirus Entry

ABSTRACT A cDNA encoding the murine homolog of human nectin-1α (also known as poliovirus receptor-related protein 1 [Prr1] and herpesvirus entry protein C [HveC]) was isolated. The protein encoded by this cDNA proved to be 95% identical in sequence to the human protein and to have similar herpesvirus entry activity. Upon expression of the murine cDNA in hamster cells resistant to alphaherpesvirus entry, the cells became susceptible to the entry of herpes simplex virus types 1 and 2 (HSV-1 and -2), pseudorabies virus, and bovine herpesvirus 1. HSV envelope glycoprotein D (gD), a viral ligand for human nectin-1α, is also a ligand for the murine homolog based on evidence that (i) a soluble hybrid protein composed in part of the murine nectin-1 ectodomain bound specifically to purified soluble forms of HSV-1 and HSV-2 gD as demonstrated by enzyme-linked immunosorbent assay, (ii) a soluble hybrid of HSV-1 gD bound to hamster cells expressing murine nectin-1α but not to control cells, and (iii) cells expressing both murine nectin-1α and one of the alphaherpesvirus gDs were resistant to entry of HSV-1, indicative of interference with entry resulting from interactions of cell-associated gD with the entry receptor. Northern blot analysis revealed that nectin-1 is expressed in most of the mouse tissues examined and at high levels in the brain, skin, and kidneys. Immunocytochemical localization demonstrated the presence of nectin-1 in epithelial cells of the mouse vagina and also in neuronal cells of the central nervous system, suggesting an expression pattern relevant to both infection at a portal of entry and spread of infection to the brain.

Effect of Theiler's murine encephalomyelitis virus and cytokines on cultured oligodendrocytes and astrocytes

The pathogenesis of Theilers murine encephalomyelitis virus (TMEV)‐induced demyelinating disease is still controversial. Our hypothesis is that primary infection of oligodendrocytes (OLGs) is not a crucial event in the pathogenesis of demyelination in this model. In fact, it has been proposed that myelin may be destroyed, as an innocent bystander, following an antiviral delayed‐type hypersensitivity (DTH) response. This hypothesis would not need widespread oligodendroglial infection, because virus present in other cells would be sufficient to trigger a DTH response. The present study demonstrates that cultured OLGs and astrocytes from susceptible strains of mice (SJL and DBA) and immortalized OLGs can be infected with TMEV in vitro. Infection of OLGs, however, is at very low levels and does not result in overt cytolytic effect. In contrast, infection of immortalized OLGs is very efficient and results in clear cytolysis. Because an important characteristic of DTH responses is the liberation of potentially injurious cytokines into adjacent tissues, we also examined the effects of mouse recombinant tumor necrosis factor‐α (TNF‐α), interleukin‐1α (IL‐1α), and interferon‐γ (IFN‐γ) on cultured OLGs and immortalized OLGs. We found that TNF‐α caused immortalized. OLG cytotoxicity in a time‐ and dose‐dependent manner. In contrast, no cytotoxicity was observed on primary OLGs with any of the above cytokines. To determine whether functional effects could be demonstrated on primary OLGs by either virus or cytokines, we measured mRNA expression of different myelin proteins in primary and immortalized OLGs exposed to virus or TNF‐α. Neither the BeAn strain or the GDVII strain of TMEV interfered with myelin protein mRNA expression in primary OLGs, whereas GDVII virus dramatically reduced myelin OLG glycoprotein (MOG) mRNA in immortalized OLGs. Interestingly, although even high concentrations of TNF‐α (10,000 U/ml) did not produce primary OLG cytotoxicity, they resulted in a significant reduction in mRNA for both myelin basic protein (MBP) and MOG in these cells. TNF‐α (at 500 U/ml) also specifically reduced MOG mRNA in immortalized OLGs. Because immortalized OLGs are considered to be arrested at an early stage of maturation, our results suggest that immature OLGs are susceptible to both virus‐ and cytokine‐dependent cytotoxicity whereas mature OLGs are resistant to cytolysis by either TMEV or cytokines. TNF‐α however, is capable of reducing mRNA expression of myelin proteins in primary OLGs; therefore, it may participate in the induction of demyelination, as suggested by the DTH‐mediated hyphotesis.

The Uncoupled Relationship between the Temperature-sensitivity and Neurovirulence in Mice of Mutants of Vesicular Stomatitis Virus

Inoculation of wild-type (wt) VSV intracerebrally (i.c.) in Swiss weanling mice results in a rapidly fatal illness with death in two to three days. In contrast, i.c. inoculation of temperature-sensitive (ts) VSV mutants G3I and G22, but not ts GII or ts G4I, results in a more slowly progressive central nervous system (CNS) disease with distinct neurological signs. Studies undertaken to evaluate the neurovirulence of ts VSV mutants indicated that the ability of ts mutants to produce pathological changes in the CNS of mice appeared related to their ability to replicate to high titre in brain and spinal cord. However, replication of ts VSV mutants in brain alone was not sufficient to produce clinical illness. More importantly, the ability of ts VSV mutants to replicate at non-permissive temperatures in vitro did not appear to correlate with neurovirulence. VSV harvests from brains and spinal cords of mice infected with each of the ts mutants were temperature-insensitive. In spite of their temperature-insensitivity, the biological behaviour of viruses recovered from CNS tissues was, surprisingly, not that which was characteristic of revertant clones. Virus isolates recovered from infected CNS tissues, despite their temperature-insensitivity, behaved biologically like the orignal stocks of ts mutant virus. These data suggest that temperature-sensitivity is not directly correlated with the unique pathogenesis elicited by infection with ts VSV mutants.

Experimental allergic neuritis in Lewis rats: altered pattern of disease induced by pertussis vaccine.

Abstract Experimental allergic neuritis (EAN) was induced in Lewis rats by a single injection of sciatic nerve-complete Freunds adjuvant either with or without a simultaneous injection of pertussis vaccine as a supplemental immunopotentiating agent. Fibrin deposition, documented by immunofluorescence and electron microscopy, was found to be an important immunohistopathologic concomitant of this autoimmune peripheral nervous system disorder irrespective of whether pertussis vaccine was included in the sensitization regimen. In contrast to the well-known capacity of pertussis vaccine to accelerate and intensify other analogous experimental autoallergies, Lewis rats receiving pertussis vaccine in addition to peripheral nerve sensitization clearly developed a less intense form and altered pattern of EAN as manifested by: (a) decreased thoracic cage muscular weakness and associated respiratory impairment and (b) decreased sciatic nerve inflammation. Diminished inflammation of sciatic nerves was accompanied by increased fibrin deposition and focal cellular infiltration within the brain. Pertussis vaccine would appear to exert an immunosuppressive effect on EAN while simultaneously retaining its known capacity to augment immunologic injury of the central nervous system.

Cytopathic Effects in Mouse Neuroblastoma Cells during a Non-permissive Infection with a Mutant of Vesicular Stomatitis Virus

Morphological changes were extensive following infection of murine neuroblastoma N-18 cells with a temperature-sensitive (ts) mutant of vesicular stomatitis virus (VSV), G31 (complementation group III), and incubation at 39 degrees C, a non-permissive condition for virion maturation. Incubation for 24 h after infection resulted in extensive morphological degeneration of mitochondria with over 80% from that in uninfected cells. Janus green B supravital staining, was reduced by 81% from that in uninfected cells. Cellular ATP levels were reduced by 50% 12 h after infection. Mitochondrial degeneration still occurred in infected cells after the inactivation of lysosomes with chloroquine. Extensive cell fusion and cytoplasmic vacuole formation also occurred during the non-permissive infection with ts G31. Loss of plasma membrane integrity was not the cause of vacuole formation since 90% of the cells were able to exclude trypan blue 24 h after infection, nor were the vacuoles the result of inactivation of the mitochondria since cyanide-poisoned cells did not form vacuoles. The cytopathic alterations observed in N-18 cells during the non-permissive infection of N-18 cells with ts G31 did not occur during the non-permissive infection of N-18 cells with ts G11 (I), ts G41 (IV), or u.v.-inactivated ts G31. However, the non-permissive infection with ts O45 (V) led to mitochondrial degeneration and cytoplasmic vacuole formation, but no cell fusion occurred. These results are discussed in light of the ultrastructural features previously observed in the central nervous system of mice infected with ts G31 and cells in culture infected with wild-type VSV.

The Role of Astrocytes, Oligodendrocytes, Microglia and Endothelial Cells in TMEV Infection

Chronic TMEV infection in susceptible strains leads to an inflammatory demyelinating disease modeling MS. TMEV antigen can be found abundantly in microglia and infiltrating macrophage, but viral growth is restricted in these cells. Viral persistence has been seen in astrocytes, microglia, and oligodendrocytes. However, infiltrating macrophage and, to some extent, resident microglia play an important role in antigen presentation, inflammation and epitope spreading.

Theiler’s Virus Infection of the Central Nervous System of Mice: An Animal Model of Virus-Induced Demyelination

In the 1930s, Max Theiler recovered a virus from the central nervous systems (CNS) of spontaneously paralyzed mice [11]. Experimental transmission of the virus to other mice by intracerebral (IC) and intranasal routes of inoculation produced similar paralytic disease. It was subsequently shown that Theiler’s murine encephalomyelitis viruses are enteric pathogens which usually cause asymptomatic intestinal infections [8]. These viruses are now known to be small, nonenveloped viruses containing RNA, and are classified as picornaviruses. The clinical disease and the histopathology of the CNS lesions described by Theiler so closely resembled human poliomyelitis that for many years this infection served as an analog of human polio in a small laboratory animal.

Anti-MBP proteolytic activity from microglia and macrophages infected with theiler's murine encephalomyelitis virus

The susceptibility of oligodendrocytes and myelin to bystander inflammation in the CNS is a controversial issue. yet is of Importance in understanding the pathogenesis of demyelinating diseases such as MS and EAE. In order to induce a localised area of inflammation in CNS white matter, a suspension of male mouse mixed glial cells was grafted into normal rat spinal cord white matter. Survival of male mouse cells was monitored using a probe which hybridises specifically to the mouse Y chromosome. By 2 weeks following transplantation, xenogeneic cells could not be detected and a dense infIammatory infiltrate extended well beyond the area occupied by the graft. Demyelinated axons were seen at the centre of the inflammatory infiltrate. By 4 weeks, inflammation had subsided, and remyelinated axons were present in the localised area corresponding to the site previously occupied by the graft. Thus; although bystander demyelination occurred as a result of inflammation, the demyelinated area was small compared to the size of the inflammatory inliltrate. This suggests that bystander demyelination occurs only at the site immediately adjacent to the target of the immune response.