Advantages of using recombinant measles viruses expressing a fluorescent reporter gene with vibratome slice technology in experimental measles neuropathogenesis.

AIMS: In this study of experimental measles neuropathogenesis, the utility of enhanced green fluorescent protein (EGFP) as a sensitive indicator of measles virus (MV) cell-to-cell spread in the central nervous system (CNS) has been assessed in vibratome-cut brain slices to demonstrate the degree and mechanism of viral spread in the rodent CNS. METHODS: Recombinant MVs expressing EGFP were visualized at different levels in 200-microm vibratome-cut brain sections from infected animals by confocal scanning laser microscopy (CSLM). Comparison was made with 7-microm microtome sections, stained for the N protein of measles by immunocytochemistry (ICC). RESULTS: The recombinant viruses were readily visualized in infected brain tissue, with no loss of neuropathogenicity. No difference was found in the sites of infection when MV infection was detected through EGFP fluorescence or by ICC. MV-infected cells were detected in the cerebral cortex, olfactory bulb and tract, hippocampus, thalamus, hypothalamus, ependyma and subventricular zone. However, the 200-microm vibratome-cut sections and confocal microscopy proved excellent for demonstrating virus distribution in neurites and for in-depth analysis of the extent of tract infection in the white matter of the cerebral hemispheres such as selective infection of the internal capsule and anterior commissure. CONCLUSIONS: The use of self-tracing recombinant MVs, viewed in thick vibratome-cut sections by CSLM, demonstrated that in experimental MV neuropathogenesis the infection is selective and spreads predominately by neurites using defined anatomical pathways.

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 (<6 months). However, in two of these cases viral RNA but not antigen was detected in the spinal cord. In long duration cases (>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.

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.

Use of immunocytochemistry and biotinylated in situ hybridisation for detecting measles virus in central nervous system tissue.

Optimised immunocytochemical (ICC) and in situ hybridisation (ISH) protocols for long term, formalin fixed, central nervous system tissue infected with measles virus were developed. The effectiveness of 10 proteases for the enzymatic unmasking of formalin fixed antigen and nucleic acid was investigated. Protease VIII gave maximal signal generation with optimal tissue preservation and no background staining for both techniques. The use of a microwave oven as an additional pre-hybridisation step for RNA-RNA in situ hybridisation produced a significant increase in the number of cells labelled for genomic RNA. The ability to show the presence of antigen and nucleic acid in long term, formalin fixed tissue facilitates the use of stored necropsy material available in pathology departments for ICC and ISH investigations.

Comparison of reporter molecules for viral in situ hybridization.

A number of streptavidin-linked reporter molecules at the endpoint of a five-step detection protocol for viral in situ hybridization using biotinylated probes were examined. DNA-DNA and RNA-RNA model systems were used. Streptavidin linked to either peroxidase or fluorescein was found to be optimal in terms of sensitivity and resolution within individual cells. All other reporter molecules labelled similar numbers of cells with low background reaction. However, streptavidin-5 nm gold followed by silver enhancement gave very high background staining making interpretation of positive signals very difficult.

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.

Screening of multiple sclerosis cerebrospinal fluid for autoantibodies.

An enzyme-linked immunoadsorbent assay, using nitrocellulose discs as solid phase and small sample volumes (50 microliter), was developed for the measurement of antibodies. This was used to screen CSF samples for autoantibodies against tissue components. Extracts from a selection of tissues from both "normal" and MS patients and from 3 glial cell lines were made in phosphate-buffered saline; in the case of neural and lymphoid samples the remaining particulate materials were subsequently solubilised with octylglucoside. The saline-soluble components were screened against CSF samples from MS patients (18), patients with other neurological disorders (10), and matched orthopaedic patients but no differences were found among the 3 groups. However, when the detergent-soluble components were screened a significant (at the P less than or equal to 0.01 level) elevation of reactivity towards brain was found in 6/16 MS patients and 2/12 patients with other neurological diseases when compared to their controls.

Antibodies to simian virus 5 in patients with multiple sclerosis and other neurological disorders.

Antibodies to the paramyxovirus simian virus 5 were measured in cerebrospinal fluid samples using an enzyme-linked immunosorbent assay. Six of the 13 clinically definite MS patients had elevated levels of antibodies compared with other neurological disease and orthopaedic controls. None of the samples from MS patients classed as probable or possible had increased amounts of SV5 antibodies. Simian virus 5 antibodies and measles antibodies showed a weak correlation and it is suggested that the elevated levels of the former are a manifestation of the increased antiviral response found in some MS patients.

An immunohistochemical study of neurofibrillary tangle formation in post-encephalitic Parkinsonism.

Neurofibrillary tangle (NFT) formation is a feature of postencephalitic Parkinsonism (PEP) and Alzheimer's disease (AD). Tangle formation has been compared immunohistochemically in these 2 conditions. Staining patterns for tau protein, ubiquitin and beta/A4 amyloid protein were studied in frontal lobe, hippocampus, and midbrain in 2 classical cases of PEP, 2 cases of AD and 2 controls matched for age and sex. NFTs were present in all cases, but with varying frequency: all tangles were tau-positive and many were ubiquitinated. In the frontal cortex and hippocampus, irrespective of the case category, tangle formation was associated with beta/A4 amyloid deposition. A similar association was present in the 2 AD cases in the midbrain. However, in PEP tangle formation in the midbrain was not associated with adjacent beta/A4 amyloid deposition. This finding raises the possibility that the pathogenetic mechanism of tangle formation in PEP is different from that of AD, although the final cellular morphological expression of abnormality in both conditions is similar.

Primary malignant melanoma of the meninges.

A case of primary malignant melanoma of the meninges is described in which the clinical presentation is that of visual loss and limb weakness. Clinically a diagnosis of carcinomatous meningitis was made and subsequently malignant cells were found in the cerebrospinal fluid (CSF). At autopsy the diagnosis of primary malignant melanoma of the meninges was made.

Neurotoxicity of 1-methoxycycloheptatriene--a Purkinje cell toxicant.

The toxicity of 1-methoxycycloheptatriene (1-MCHT), a sensory irritant, has been investigated in beagles. It was found to produce gross inco-ordination of the limbs at intravenous doses greater than 10 mg kg-1. The main histological abnormalities were in the cerebellum and consisted of Purkinje cell death and subsequent reactive gliosis. A few necrotic neurons were seen in the diencephalon, pons and medulla. Haematological abnormalities, e.g. leucocytosis with relative lymphopenia, were seen, while biochemical changes included hyperglycaemia and a rise in plasma aminotransferases. The no-effect dose for the histological and biochemical changes was the same. These abnormalities are compared with cerebellar changes observed in acrylamide and other toxic states.