Stanley G. Rabinowitz

Experimental models of virus‐induced demyelination of the central nervous system

One of the arguments in favor of a viral pathogenesis for multiple sclerosis is the existence of several experimental and natural animal models of virus‐induced primary demyelination. This review deals comprehensively with such models. Well‐known examples of demyelinating viral infections in their natural host are JHM, Theiler, visna, and canine distemper encephalomyelitides. Recent reports of experimental murine infections with pathogens such as vesicular stomatitis, Chandipura, herpes simplex, Venezuelan equine encephalomyelitis, and Semliki Forest viruses are also discussed. The thrust of the review is to include viral models suspected of producing primary demyelination on an immunopathological basis.

Measurement and comparison of the proliferative and antibody responses of neonatal, immature and adult murine spleen cells to T-dependent and T-independent antigens

Abstract Mouse spleen cell antigenic responses to the thymic-dependent antigen sheep red blood cells (SRBC), and the thymic-independent antigens, E. Coli lipopolysaccharide (LPS) and pneumococcal polysaccharides Type I and II (SI, SII) were studied as as a function of age, employing both in vitro spleen cell stimulation and plaque-forming cell (PFC) assay systems. Primary spleen cell proliferative and PFC responses to SRBC, were either absent or meager in comparison to adult (8–12 weeks) values for the first 3 weeks of life. Thereafter responses rose achieving adult values between 4 and 8 weeks of age. The inability of young mice to respond to SRBC was not because of a different immunizing dose requirement for SRBC, since immunization with SRBC over a 200-fold range did not enhance their capability to respond. Also, addition of adherent cells or macrophages from adult mice did not enhance the immune responses of young mice. Furthermore, immunization of 2–4 week old mice with SRBC inhibited the secondary response to SRBC. In contrast, young murine spleen cell proliferative and PFC responses to SI, SII, and LPS were approximately the same as the adult by 7–14 days of life. These data suggest that B-cell immunologic activity, as measured by immunologic assays utilized in this study, develops much earlier than does T-cell responsiveness.

A simplified plaque assay for influenza viruses in Madin-Darby kidney (MDCK) cells

A variety of influenza A and B viruses plaque in MDCK cell in trypsin is added only at the time of viral adsorption to the monolayer. Therefore, a conventional soft-agar overlay can be employed without addition of proteolytic enzymes. Plaquing efficiency was comparable to that when embryonated eggs were used to determine infectivity. Finally the method is simple and economical.

Virus-induced demyelination. Production by a viral temperature-sensitive mutant.

Infection of mice with a temperature-sensitive (ts) mutant of Chandipura virus (CV) ts472 CV, induced a slower disease than the respective parental virus and white matter lesions characterized by perivascular mononuclear infiltrates accompanied by primary demyelination. The pattern of these lesions was very similar to that in EAE, a prototypic autoimmune disease and in Theilers virus infection in which an immunopathologic mechanism of myelin injury is strongly suggested. Results obtained in nude mice supported the possible immunopathological nature of myelin injury in ts472 CV infection. No inflammatory response was elicited in either grey or white matter. However, whereas grey matter presented extensive necrosis, no alterations were present in white matter. Such data suggest that whereas grey matter lesions are produced by direct viral cytolytic activity, white matter pathology is probably dependent on the host immune response for its development. The finding of additional models of virus-induced demyelination with a possible immunopathologic mechanism of myelin injury is significant as it suggests that this type of virus-induced myelin degeneration is not restricted to a single virus like Theilers, but it may represent a more general mechanism of virus-induced demyelination.

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.

Protection against lethal Venezuelan equine encephalitis (VEE) virus infection by cell-free supernatant obtained from immune spleen cells

Considerable effort has been directed at unraveling the mechanisms underlying protection and recovery in acute viral infections. Various factors, including antibody, interferon, lymphokines and certain lymphocyte and macrophage cell constituents, have been shown to play a role in host recovery. We have previously studied an adoptive transfer system in experimental VEE virus CNS infection in a mouse model, and shown that both T cells and B cells exert an influence in protecting the host from lethal CNS infection with this virus. Because of the complexity involved in the adoptive transfer of protection, we sought a simpler and more easily defined experimental approach to dissect the various factors responsible for protection in our model. We here report that immune cell-free supernatant, derived from mice immunized with vaccine against VEE virus, exerts as much protection as the whole cell transfer system against lethal VEE virus infection. Such cell-free supernatant allowed us to look more closely at individual host immune factors in their relationship to protection against infection. Studies herein reported demonstrate that the protection conferred by immune cell-free supernatants is not dependent on neutralizing antibody or interferon, but rather appears to be related to the presence of both interleukin-1 and -2 and the activation and potentiation of NK cell activity against VEE virus.

Endogenous myelin basic protein-serum factors (MBP-SFS) and anti-MBP antibodies in a patient with post-herpes simplex virus acute disseminated encephalomyelitis☆

The measurement of myelin basic protein serum factors (MBP-SFs) and anti-MBP antibodies in specimens from a patient with post-herpes simplex acute disseminated encephalomyelitis (ADE) is described. Transitory appearance of high affinity anti-MBP antibodies in the absence of detectable MBP-SFs was observed. This pattern was similar to that found previously in acute multiple sclerosis and experimental allergic encephalomyelitis. These findings are consistent with the hypothesis that a possible normal role of MBP-SFs is as neuroautotolerogens, preventing autoreactive lymphocyte clones from damaging myelin.

Comparative Mucigenic Responses of T-Cells and B-Cells in Spleens of Mice of Varying Age

Mouse spleen cell mitogenic responses to Phytohemagglutinin (PHA), conconavalin A (con A), staphylococcal enterotoxin B (SEB) and lipopolysaccharide (LPS) were studied as a function of age. In CD-1 mice, spleen cell DNA synthesis in response to stimulation by PHA, con A and SEB varied from 18% - 33% of adult values for the first 3 weeks of life. By 6 weeks, the PHA response was 63% of the adult response and achieved adult values by 8 weeks of life. In contrast to these T-cell responses, DNA spleen cell synthesis in response to LPS was approximately that of adults as early as 7 days after birth. When spleen cell mitogenic responses were studied in C57B1/6J mice a similar pattern emerged. C57 spleen cell responses to PHA, con A and SEB varied from 24% - 54% of adult values for the first 3 weeks of life. By 4 weeks, spleen cell responses to PHA and con A were approximately those of adults. Again, C57 spleen cell responses to LPS were equivalent to adult values as early as 7 days after birth.

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.

Altered neurovirulence of temperature-sensitive vesicular stomatitis virus mutants in a murine model by inoculation of bombesin: A neuropeptide: I. Clinical, virological and pathological observations

Previous work in our laboratory has demonstrated that only certain temperature-sensitive (ts) mutants of vesicular stomatitis virus (VSV) appear capable of producing central nervous system (CNS) infection in a mouse model system. Considerable effort has been devoted to studies directed at unraveling the mechanisms underlying host virulence with these tsVSV mutants. With the previous demonstration that certain neuropeptides, capable of lowering body temperature, alter avirulent into virulent infection, we explored the role of one of these neuropeptides, bombesin, in CNS infection induced by normally avirulent tsG11 VSV, as well as certain tsVSV mutants derived from persistently infected (pi) carrier cultures. Our observations indicate that bombesin dramatically alters CNS infection with either tsG11 VSV as well as tsVSV mutants derived from persistent carrier cultures. When virus alone was inoculated intracerebrally, no sign of illness was observed and no animal died. When bombesin was injected along with normally avirulent tsG11 VSV, or glioma derived tsG31 VSV, 50% of mice died within 6-8 days after inoculation. Moreover, mice infected with virus and neuropeptide demonstrated striking pathological alterations in the CNS. These studies are in agreement with previously published results from others as well as our own laboratory and strongly suggest a direct correlation between CNS temperature and the capacity of certain tsVSV mutants to induce clinical and pathological disease.