Ingrid V. Allen

A histological, histochemical and biochemical study of the macroscopically normal white matter in multiple sclerosis

In a combined histological, biochemical and histochemical study of the macroscopically normal white matter in multipe sclerosis 72% of samples were histologically abnormal. The significance of this fact in the interpretation of previous biochemical studies and in the design of future studies is discussed. The present study showed a significant elevation of the lysosomal enzyme beta-glucosaminidase in the microscopically normal white matter in MS as compared with controls. Studies on lysosomes separated from microscopically normal or mild to moderately gliosed white matter in multiple sclerosis showed an increase in lysosomal fragility. Histochemical study of the microscopically normal white matter in multiple sclerosis revealed an increase in the number of acid phosphate-containing cells as compared with normal and neurological control material. The significance of these findings is discussed and it is suggested that irrespective of the primary or secondary nature of these abnormalities, the white matter may be rendered more susceptible to the pathogenetic process in this disease.

Ocular pathology in multiple sclerosis: retinal atrophy and inflammation irrespective of disease duration

There has been growing interest in the use of retinal imaging for tracking disease progression in multiple sclerosis. However, systematic and detailed pathological descriptions of retinal tissue in multiple sclerosis are lacking. Graded, histological evaluations on eyes from 82 patients with multiple sclerosis and 10 subjects with other neurological diseases, with immunohistochemistry on a subset, were performed and correlated with clinical and pathological findings. Multiple sclerosis cases demonstrated evidence of retinal atrophy and inflammation even in late-stage disease. Retinal ganglion cell loss was significant and remaining neurons appeared shrunken and were partially engulfed by human leukocyte antigen-DR positive cells with the phenotype of microglia in samples subjected to immunohistochemistry. Neurofilament staining revealed variable but prominent degrees of axonal loss and injury. Neuronal loss was noted in the inner nuclear layer with focal reduction in cell density. Foamy-appearing human leukocyte antigen-DR positive cells were evident near vessels and periphlebitis was found in a small but significant number of multiple sclerosis cases. Glial fibrillary acidic protein staining showed extensive astrocyte hypertrophy and proliferation with prominent gliosis in multiple sclerosis cases. Frequent but previously unreported abnormalities in the iris were documented in the majority of chronic multiple sclerosis cases. The injury to both iris and retina could be seen at all stages of disease. Severity of retinal atrophy was correlated with overall brain weight at time of autopsy (P = 0.04) and a trend for increased atrophy was seen with longer disease duration (P = 0.13). This study provides the first large-scale pathological description of retinas in multiple sclerosis, including patients with different subtypes of disease at all stages, and with variable clinical severity. Changes were seen not only in the retinal nerve fibre layer and ganglion cell layer, but also in the inner nuclear layer, suggesting that retinal injury is more widespread than previously appreciated. Furthermore, the human retina is devoid of myelin, but inflammation was demonstrated to be prominent in multiple sclerosis and to persist in the retina at late stages of disease. The prominent gliosis and inflammation surrounding vessels of the inner retina could potentially impact optical coherence tomography evaluations in multiple sclerosis-as standard techniques exploit presumed differences in tissue reflectivity and utilize automated edge detection algorithms to judge axon loss in the nerve fibre layer. Deciphering the relationships between the different types of retinal pathology may aid us in understanding the factors that drive both inflammation and tissue atrophy in multiple sclerosis.

Pathological abnormalities in the normal-appearing white matter in multiple sclerosis

Abstract. In established cases of multiple sclerosis (MS), the normal-appearing white matter (NAWM), as defined for magnetic resonance imaging (MRI), is abnormal in the majority of cases. The clinical significance of these NAWM abnormalities is the subject of debate, but there is strong correlation with degree and progression of disability. New lesions form in NAWM before blood-brain barrier breakdown, as evidenced by gadolinium enhancement. The pathological basis of these neuroimaging abnormalities is largely unknown. Definitive pathological studies on the NAWM are few and are often based on small numbers of samples and of cases. Despite a variety of MS NAWM pathological studies, major research questions, of importance to our understanding of basic pathogenetic mechanisms and consequent rational therapies, remain unanswered. These relate to the frequency and extent of oligodendrocyte/myelin and axonal abnormalities in MS NAWM, and to the cellular basis of very early MS lesions detected by neuroimaging. In a pilot study of MS NAWM, microglial activation was demonstrated in 9 of 10 MS cases. We are currently testing the hypothesis that microglial activation, as defined by altered phenotype and HLA-DR positivity, will act as a marker for oligodendrocyte/myelin and axonal pathology in MS NAWM.

The significance of measles virus antigen and genome distribution in the CNS in SSPE for mechanisms of viral spread and demyelination.

The distribution of measles virus (MV) antigen and genomic RNA in the central nervous system (CNS) in 10 cases of subacute sclerosing panencephalitis (SSPE) was determined using optimized immunocytochemistry and in situ hybridization techniques. As in previous reports neurons and oligodendrocytes were found to be the most frequently infected cells. It was confirmed that MV infection in neuronal processes was predominantly dendritic but there was also some evidence suggestive of occasional axonal involvement, a finding confirmed by electron microscopy. In addition MV genomic RNA was detected in neuronal processes, in some cases in the absence of demonstrable MV antigen. The relationship between myelin destruction and oligodendrocytic infection suggested that the demyelination may be solely the result of virus infection. A possible correlation between viral distribution and form and the clinical duration of disease was examined. Viral antigen and genome were equally abundant in the cerebral cortex in most short duration cases (lt; 6 months). However, in two of these cases viral RNA but not antigen was detected in the spinal cord. In long duration cases (gt;36 months) viral RNA was abundant in all areas of the CNS examined, frequently in the absence of demonstrable antigen. These findings may suggest viral spread in a cephalo-caudal direction, probably by transneuronal spread

THE PATHOLOGY OF MULTIPLE SCLEROSIS‐FACT, FICTION AND HYPOTHESIS

The present state of our pathological knowledge of multiple sclerosis is reviewed emphasizing the ‘hard facts’ while highlighting subjects of disputed interpretation and of ignorance.

Association of measles virus with neurofibrillary tangles in subacute sclerosing panencephalitis: a combined in situ hybridization and immunocytochemical investigation

Neurofibrillary tangle formation, a cardinal characteristic of Alzheimers disease, is also a feature of several other neurodegenerative disorders, including subacute sclerosing panencephalitis (SSPE). In the present study the association of measles virus genome with neurofibrillary tangle formation has been studied in five cases of SSPE, using in situ hybridization (measles genome) and immunocytochemistry (tau, ubiquitin and p7A4 amyloid). In two cases with duration of disease less than one year, neurofibrillary tangle formation was not observed. However, in those cases in which the disease was of several years duration, numerous tau– and ubiquitin positive neurofibrillary tangles were demonstrated. In the two cases of longest duration, double–labelling techniques demonstrated the frequent association of neurofibrillary tangle formation with neuronal measles virus genome positivity. Immunocytochemistry for p7A4 amyloid failed to demonstrate amyloid in any of the five cases. These findings support the hypothesis that neurofibrillary tangle formation can occur independently of amyloid formation and that this mechanism may operate in both Alzheimers disease and in virallyinduced disease.

Rational Attenuation of a Morbillivirus by Modulating the Activity of the RNA-Dependent RNA Polymerase

ABSTRACT Negative-strand RNA viruses encode a single RNA-dependent RNA polymerase (RdRp) which transcribes and replicates the genome. The open reading frame encoding the RdRp from a virulent wild-type strain of rinderpest virus (RPV) was inserted into an expression plasmid. Sequences encoding enhanced green fluorescent protein (EGFP) were inserted into a variable hinge of the RdRp. The resulting polymerase was autofluorescent, and its activity in the replication/transcription of a synthetic minigenome was reduced. We investigated the potential of using this approach to rationally attenuate a virus by inserting the DNA sequences encoding the modified RdRp into a full-length anti-genome plasmid from which a virulent virus (rRPVKO) can be rescued. A recombinant virus, rRPVKOL-RRegfpR, which grew at an indistinguishable rate and to an identical titer as rRPVKO in vitro, was rescued. Fluorescently tagged polymerase was visible in large cytoplasmic inclusions and beneath the cell membrane. Subcutaneous injection of 104 TCID50 of the rRPVKO parental recombinant virus into cattle leads to severe disease symptoms (leukopenia/diarrhea and pyrexia) and death by 9 days postinfection. Animals infected with rRPVKOL-RRegfpR exhibited transient leukopenia and mild pyrexia, and the only noticeable clinical signs were moderate reddening of one eye and a slight ocular-nasal discharge. Viruses that expressed the modified polymerase were isolated from peripheral blood lymphocytes and eye swabs. This demonstrates that a virulent morbillivirus can be attenuated in a single step solely by modulating RdRp activity and that there is not necessarily a correlation between virus growth in vitro and in vivo.

ADHESION MOLECULE EXPRESSION AND LYMPHOCYTE ADHESION TO CEREBRAL ENDOTHELIUM : EFFECTS OF MEASLES VIRUS AND HERPES SIMPLEX 1 VIRUS

Expression of endothelial cell (EC) adhesion molecules is increased in inflammatory neurological disorders and this may regulate lymphocyte homing to the central nervous system (CNS). Viral encephalitis is characterised by lymphocytic infiltration of the CNS and one mechanism of this response may be EC adhesion molecule induction with consequent inflammatory cell/EC binding. This report characterises the effects of herpes simplex 1 (HSV1) or measles virus (MV) infection of BALB/c brain microvascular EC in vitro on adhesion of naive syngenic splenocytes and levels of ICAM-1. Adhesion was enhanced by 42% for MV-infected cells and by 73% for HSV-1-infected EC. At the multiplicities of infection employed, levels of ICAM-1 were upregulated on HSV-1-infected EC, but not on MV-infected EC. It is concluded that ICAM-1/ligand interactions do not play a role in mediation of MV enhancement of adherence, but represent one mechanism responsible for increased lymphocyte adherence to HSV-1-infected cerebral EC.

The F Gene of Rodent Brain-Adapted Mumps Virus Is a Major Determinant of Neurovirulence

ABSTRACT Prior to the introduction of live-attenuated vaccines, mumps virus (MuV) was the leading cause of virus-induced meningitis. Although vaccination has been effective at controlling the disease, the use of insufficiently attenuated strains has been associated with high rates of aseptic meningitis in vaccinees. The molecular basis of MuV attenuation is poorly understood, and no reliable molecular markers of virulence have been identified. In this study, reverse genetics has been used to identify molecular determinants of MuV neuropathogenesis. Recombinant viruses, containing the envelope-associated genes from the Kilham (MuVKH) rodent brain-adapted strain of MuV, were generated in the Jeryl Lynn 5 (MuVJL5) vaccine strain background. The syncytium phenotypes of the recombinant viruses on Vero cells differed depending on the source of the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins, with heterologous combinations showing either an increase or a decrease in the level of cell fusion compared to that of the homologous parental combinations. This was confirmed by transiently cotransfecting eukaryotic F and HN glycoprotein expression constructs. A Lewis rat model that discriminates between neurovirulent and nonneurovirulent MuV strains based on the extent of hydrocephalus induced in the rat brain after intracerebral inoculation was used to assess the phenotype of the recombinant viruses. Expression of the matrix (M), small hydrophobic (SH), or HN gene in isolation did not confer a neurovirulent phenotype. Expression of the F gene of the neurovirulent strain alone was sufficient to induce significant levels of hydrocephalus. Coexpression of the homologous HN gene led to a marginal increase in the level of hydrocephalus.

Measles virus infection of cells in perivascular infiltrates in the brain in subacute sclerosing panencephalitis: confirmation by non-radioactive in situ hybridization, immunocytochemistry and electron microscopy

SummaryAs part of continuing multidisciplinary studies on the neuropathogenesis of subacute sclerosing panencephalitis (SSPE), in situ hybridisation, immunocytochemistry and electron microscopy were used to detect measles virus nucleic acid, protein and nucleocapsids in brain perivascular infiltrates of three cases. Perivascular cuffing cells which contained measles virus nucleic acid and antigens were found in all cases. Infected cuffs occurred predominantly in areas of general parenchymal cell infection and in many of these a high proportion of the infiltrating cells were infected. Other cuffs in these areas were either uninfected or contained only a few infected cells. Occasional infected cells were also seen in cuffs in non-infected areas. In contrast, no specific immunocytochemical reactions or in situ hybridisation for measles virus was observed in brain tissue from a patient with herpes encephalitis. By electron microscopy viral nucleocapsid, consistent with measles virus, was found within the cytoplasm of plasma cells in the inflammatory cuffs in SSPE brain tissue. Possible explanations for our results are that infiltrates become infected on arrival in the CNS or alternatively, that the infected infiltrates reflect a generalised infection of the reticuloendothelial system. The frequent presence of uninfected cuffs favours the former explanation.