Yoshiro Ohara

Targeting of adoptively transferred experimental allergic encephalitis lesion at the sites of wallerian degeneration.

SummaryTo clarify the implication of the major histocompatibility complex class II (Ia) antigen induction in microglia following Wallerian degeneration in the central nervous system (CNS), experimental allergic encephalitis (EAE) was adoptively transferred to Lewis rats in which Ia antigens had been induced in microglia at the sites of Wallerian degeneration. In addition to randomly distributed typical EAE lesions, the recipient rats developed distinct inflammatory lesions in accord with the distribution of Ia-positive microglia; i.e., in the ipsilateral thalamus after cortical cryoinjury, and in the ipsilateral optic nerve, the contralateral optic tract and superior colliculus after unilateral eye ball enucleation. Thus, the EAE locus may be targeted by this approach. The inflammatory response was inducible by transfer of myelin basic protein-stimulated lymphocytes but not by transfer of phytohemagglutinin-stimulated or non-stimulated lymphocytes. When examined using monoclonal antibody surface markers; OX-6 for Ia antigen, W3/13 for pan T lymphocyte and OX-8 for cytotoxic/suppresser T lymphocyte, the types of lymphocytes in these lesions did not differ from those in ordinary EAE lesions in the spinal cord. The potential role of non-immunologically induced Ia-positive cell clusters that serve as a target for autoimmune CNS diseases was discussed.

A comparative study of acute and chronic diseases induced by two subgroups of Theiler’s murine encephalomyelitis virus

Abstract Theiler’s murine encephalomyelitis viruses (TMEV) are divided into two subgroups on the basis of their different biological activities. The GDVII strain produces acute polioencephalomyelitis in mice, whereas the DA strain produces demyelination with virus persistence in the spinal cord. A comparative study of GDVII and DA strains suggested that low host immune responses are responsible for the development of acute GDVII infection and that the persistence of infected macrophages plays a crucial role in the development of chronic white matter lesions in DA infection. All 78 mice infected with GDVII died or became moribund by day 13, while none of 54 mice infected with DA died. In the acute stage, the distribution of viral antigens in the central nervous system (CNS) tissue was similar in both GDVII and DA infections, although the virus titer was higher in GDVII infection. In DA infection, a substantial number of T cells were recruited to the CNS on day 6 when they were virtually absent in GDVII infection. The titer of neutralizing antibody was already high on day 6 in DA infection but was negligible in GDVII infection. Development of chronic paralytic disease from day 35 of the DA infection was accompanied by focal accumulation of viral antigen-positive macrophages in the spinal white matter. In addition, white matter lesions comparable to those in chronic DA infection were induced in the spinal cord within 7 days after intracerebral injection of DA-infected murine macrophages.

Isolation and characterization of subacute sclerosing panencephalitis virus (Yamagata-1 strain) from a brain autopsy.

Subacute sclerosing panencephalitis (SSPE) is a rare, progressive and usually fatal disease of the central nervous system which affects children and young adults. Structures like nucleocapsids of paramyxoviruses (2) and the viral antigens of measles virus (5) have been found in the brains of patients. High titers of antibody against measles virus in patients serum and cerebrospinal fluid (5) have also been demonstrated. Since Chen et al (3) and Horta-Barbosa et al (8, 9) succeeded in recovering measles-like viruses by co-cultivation of brain cells of the patients with susceptible tissue culture cells, a large number of SSPE virus-carrying cell lines have been established (1, 7). There is considerable variation, however, in their properties, e.g., virion production, synthesis of M protein as well as other virion constituents, and neurovirulence in experimental animals (7), that makes it difficult to relate a particular property of the virus to the etiology of SSPE. All of the four isolates in Japan have been reported to be defective in production of infectious virus (6, 11, 16, 19). We studied three of them and succeeded in recovering a small number of cell-free infectious virus particles from each of the virus-carrying cell lines by treatment of the cells with either EDTA or freezing and thawing though spontaneous release of the infectious virus was still not demonstrable (12, 13). Nevertheless, neurovirulence of these virus-carrying cells for mice was evident (14); thus these viruses differed from the so-called productive SSPE virus such as the Mantooth, Halle and LEC-S strains (18). These results seem to indicate that the property of less production of SSPE virus, like the isolates in Japan, is a requisite for neurovirulence. Accordingly, comparative study of these less-productive strains should provide a better understanding of the neurovirulent factors of this virus. In this paper, we describe preliminarily an additional new isolate, from the brain of an SSPE patient, of an SSPE virus which has characteristics similar to those of the other Japanese strains in terms of growth of the virus but distinct from them in neurovirulence and histopathology of the brain in mice.

Subacute panencephalitis associated with chronic graft-versus-host disease

SummaryA unique form of subacute panencephalitis developed in a child with aplastic anemia 8 months after an allogeneic bone marrow transplantation (BMT). It was characterized by parenchymal infiltration of CD3 lymphocytes, a marked increase in the number of microglia strongly expressing HLA-DR antigens in both the gray and white matter, and diffuse degeneration of the cerebral white matter. The onset of neurological symptoms coincided with the development of chronic systemic graft-versus-host disease (GVHD). Cellular infiltrates in the CNS lesions were exclusively CD3 lymphocytes intermingled with a small number of monocytes labeled with CD68. There was a preponderance of cells of the CD45RB phenotype. The pathological changes in visceral organs were consistent with those of chronic GVHD. In addition, scrutiny of immunohistochemistry disclosed sparse infiltration of CD3 lymphocytes and diffuse gliosis in the cerebral white matter of another child with chronic GVHD who died 9 months after allogeneic BMT. These cases are suggestive of a potential risk of CNS involvement in GVHD.

Sensitivity of the polymerase chain reaction for detecting human T-cell leukemia virus type I sequences in paraffin-embedded tissue. Effect of unbuffered formalin fixation

Recently, the application of the polymerase chain reaction (PCR) to formalin-fixed paraffin-embedded tissue has been reported. But formalin, especially unbuffered formalin, is known to break DNA into small fragments. DNA extracted from MT-2 cells fixed in unbuffered formalin for various periods of time were subjected to the PCR and the effect of unbuffered formalin fixation on the ability of the PCR to detect exogenous sequences; i.e., human T-cell leukemia virus type I (HTLV-I) proviral DNA, was examined. The sensitivity of the PCR decreased as a function of both the duration of fixation and the length of the expected DNA products. When the expected length of the PCR product was about 200 bp, a slight decrease in the sensitivity was observed after 4-day fixation. When it was about 300 bp, a similar decrease was observed following 4-h fixation. In the case of a 500 bp product, the sensitivity began to decrease after 30-min fixation and a 100-fold decrease was observed after 10-day fixation. A decrease was not observed, however, with a 100 bp product. The appropriate design of primers, especially with regard to the length of the amplified product, is essential to keep the sensitivity of the PCR, particularly when the target tissues have been fixed in unbuffered formalin.

Clinical manifestations of tularemia in Japan — Analysis of 1,355 cases observed between 1924 and 1987

SummaryA total of 1,355 cases of tularemia observed between 1924 and 1987 in Japan were viewed on the basis of clinical manifestations and the results were compared with those in the United States. The incubation period varied from one day to over one month. In 75.5% of cases, the symptoms of illness appeared within seven days with the peak on the third day. A sudden onset of flu-like symptoms was generally observed, and 92% of cases was followed by regional lymph node swelling which mostly appeared in axillary and cubital regions. They were observed predominantly at the left rather than the right side. In contrast with the cases in the United States, the number of cases of ulceroglandular type in Japan was only one third of those of glandular type. None of the pleuropulmonary cases or fatal tularemia have been reported in Japan. The number of oropharyngeal cases has remarkably increased after World War II, and is still on the rise, presumably because of the change of dietary habits in Japan. All these characteristics of Japanese tularemia are assumed to be caused by low virulence of Japanese strains ofFrancisella tularensis.Zusammenfassung1355 Fälle von Tularämie, die in Japan zwischen 1924 und 1987 beobachtet wurden, wurden nach klinischen Manifestationsformen aufgeschlüsselt und mit Fallbeobachtungen in den USA verglichen. Die Inkubationszeit variierte zwischen einem Tag und mehr als einem Monat. In 75,5% der Fälle traten die klinischen Krankheitserscheinungen innerhalb von sieben Tagen, am häufigsten um den dritten Tag auf. Im allgemeinen stellten sich anfangs grippeähnliche Symptome ein, denen in 92% der Fälle regionale Lymphknotenschwellungen, bevorzugt der Axillar- und Kubitalregionen, folgten, die links häufiger auftraten als rechts. Ulzeröse Formen des glandulären Typs machten in Japan nur ein Drittel der Fälle aus, während der Anteil in den USA erheblich höher war. In Japan wurde kein einziger Fall einer pleuropulmonalen Form der Tularämie oder einer Tularämie mit letalem Ausgang mitgeteilt. Oropharyngeale Fälle nahmen nach dem zweiten Weltkrieg erheblich zu und steigen weiter an. Dies könnte auf veränderte Essensgewohnheiten in Japan zurückzuführen sein. Es wird angenommen, daß die geringe Virulenz der japanischen Stämme vonFrancisella tularensis für die Besonderheiten der japanischen Tularämie verantwortlich sind.

The Entry and Intracellular Multiplication of Francisella tularensis in Cultured Cells: Its Correlation with Virulence in Experimental Mice

Five acriflavine agglutination test‐positive (acf+) colonies and five negative (acf–) colonies were isolated from each of the four strains (Ebina, CMB2, N9, and Schu) of Francisella tularensis, and the correlation between the virulence in experimental mice and the entry and intracellular multiplication in cultured mouse fibroblast cells (L‐929 cells) was examined. All of the acf– colonies derived from the Ebina and CMB2 strains were highly virulent in mice, readily entering and growing well in the cells, while all of the acf‐ colonies from N9 and Schu strains were of low virulence and neither entered nor grew in the cells effectively. On the other hand, regardless of their parent strains, the acf+ colonies were low virulent and most of those colonies did neither enter nor grow in L‐929 cells. In addition, two acf‐ colonies, one from the N9 and the other from the Schu strain, gained virulence through several passages in mice, and in parallel, their entry and multiplication also improved. However, two acf+ colonies from the Ebina strain and one acf+ colony from the N9 strain showed a moderate degree of the entry and multiplication although they were all low virulent. The overall results indicate that the entry and multiplication in cells are important factors regulating the virulence of F. tularensis. The results also showed, however, that they were not sole factors to elucidate the virulence of the bacterium in mice.

Search for human T-cell leukemia virus type I (HTLV-I) proviral sequences by polymerase chain reaction in the central nervous system tissue of HTLV-I-associated myelopathy

SummaryUsing the polymerase chain reaction (PCR), proviral DNA sequences of thepol andenv regions of human T-cell leukemia virus type I (HTLV-I) were directly amplified in paraffin-embedded and frozen tissue sections of active inflammatory central nervous system (CNS) lesions in three autopsy cases of HTLV-I-associated myelopathy (HAM) with serological confirmation. In parallel, the enumeration of UCHL-1 (monoclonal antibody reactive to T-cells) positive cells in the tissue sections subjected to PCR were carried out. Although the control DNA sequence of parathyroid hormone-like peptide gene was definitely amplified, no signals for HTLV-I proviral sequences were detected in these specimens. The number of UCHL-1 positive cell nuclei was almost on the border line of our PCR sensitivity in formalin-fixed tissue, which was estimated to be 20–200 copies. Therefore, it is unlikely that the central nervous system tissue damage in HAM/TSP is a consequence of productive infection of HTLV-I in the CNS tissue.

Cytotropism of Theiler's murine encephalomyelitis viruses in oligodendrocyte-enriched cultures

SummaryThe cytotropism of two strains, GDVII and DA, of Theilers murine encephalomyelitis viruses (TMEV) was studied in the oligodendrocyte-enriched murine neural cell cultures. Both GDVII and DA caused cytopathic effects in the neural cell cultures, and double immunostaining for galactocerebroside (Gal-Cer), a marker molecule for oligodendrocyte, and viral antigens disclosed a dual expression of Gal-Cer and viral antigens in over 80% of cells in both cultures 24h after infection with either GDVII or DA. The kinetics of cell-free and cell-associated infectivity were not significantly different between two cultures. These in vitro observations suggest that neither replication in oligodendrocyte nor cell-associated infectivity is a sole factor in discriminating those two subgroups of TMEV with regard to the demyelinating activity, and that virus cell binding may play an important role in virus persistence and TMEV-induced demyelination.

Phenotypes of mononuclear cell infiltrates in human central nervous system

SummaryUsing a panel of monoclonal antibodies applicable for identification of cell types in paraffin sections, the prevalence of mononuclear cell infiltrates with different phenotypes was estimated in large areas taken from 11 cases of acute and chronic inflammatory diseases in the human central nervous system. The present study clearly demonstrated a diversity of inflammatory mononuclear cell infiltrates, and the dominance of cell types in individual lesions appeared to be determined by both the nature of the diseases and the age of the lesions. The possible pathognomonic significance of a relatively high prevalence of CD4+CD45RO+ lymphocytes in acute rabies and in a convalescent stage of Japanese encephalitis and subacute sclerosing panencephalitis is discussed.