Subacute Sclerosing Panencephalitis Brain Research Articles

Screening Random Peptide Libraries with Subacute Sclerosing PanencephalitisBrain-Derived Recombinant Antibodies Identifies Multiple Epitopes in the C-Terminal Region of the Measles Virus Nucleocapsid Protein

Infectious and inflammatory diseases of the CNS are often characterized by a robust B-cell response that manifests as increased intrathecal immunoglobulin G (IgG) synthesis and the presence of oligoclonal bands. We previously used laser capture microdissection and single-cell PCR to analyze the IgG variable regions of plasma cells from the brain of a patient with subacute sclerosing panencephalitis (SSPE). Five of eight human IgG1 recombinant antibodies (rAbs) derived from SSPE brain plasma cell clones recognized the measles virus (MV) nucleocapsid protein, confirming that the antibody response in SSPE targets primarily the agent causing disease. In this study, as part of our work on antigen identification, we used four rAbs to probe a random phage-displayed peptide library to determine if epitopes within the MV nucleocapsid protein could be identified with SSPE brain rAbs. All four of the SSPE rAbs enriched phage-displayed peptide sequences that reacted specifically to their panning rAb by enzyme-linked immunosorbent assay. BLASTP searches of the NCBI protein database revealed clear homologies in three peptides and different amino acid stretches within the 65 C-terminal amino acids of the MV nucleocapsid protein. The specificities of SSPE rAbs to these regions of the MV nucleocapsid protein were confirmed by binding to synthetic peptides or to short cDNA expression products. These results indicate the feasibility of using peptide screening for antigen discovery in central nervous system inflammatory diseases of unknown etiology, such as multiple sclerosis, neurosarcoidosis, or Behcet's syndrome.

A comparison of complete untranslated regions of measles virus genomes derived from wild-type viruses and SSPE brain tissues

We compared complete untranslated regions (UTRs) of two subacute sclerosing panencephalitis (SSPE) measles virus (MV) strains and two wild-type (wt) MV strains, all belonging to the same genotype (D6). In comparison to wt MVs of the same genotype, base changes were identified in the two SSPE measles virus strains at 27 and 33 noncoding positions, respectively. Majority of these residues are unique for each of the SSPE virus sequences in comparison to all other reported measles virus strain sequences. The location of some of these changes indicates that they may modify cis-acting regulatory sequences including gene-end signal of the P gene, H/L gene junction and Kozak consensus element of the L gene. Further, within the long UTR between M and F genes, deletions and insertions were identified. Thus, our study could be significant for additional investigation using reverse genetics and recombinant viruses, of possible influence of mutations in UTRs on establishment and maintenance of chronic progressive CNS disease caused by MV persistence.

Recombinant antibodies generated from both clonal and less abundant plasma cell immunoglobulin G sequences in subacute sclerosing panencephalitis brain are directed against measles virus

Increased immunoglobulin G (IgG) and intrathecally produced oligoclonal bands (OGBs) are characteristic of a limited number of inflammatory central nervous system (CNS) diseases and are often directed against the cause of disease. In subacute sclerosing panencephalitis (SSPE), the cause of disease and the target of the oligoclonal response is measles virus (MV). The authors previously showed that clonally expanded populations of CD38+ plasma cells in SSPE brain, the likely source of OGBs, are directed against MV. In characterizing the breadth of the plasma cell reactivities, the authors found that a large proportion of the less abundant plasma cells are also directed against MV. The intrathecal response may be useful in determining the causes of other inflammatory CNS diseases, such as multiple sclerosis, Behcet's disease, and neurosarcoidosis.

Expression of the interferon-α/β-inducible MxA protein in brain lesions of subacute sclerosing panencephalitis

The type-I interferon (IFN) inducible human MxA protein exhibits antiviral activity against a variety of RNA viruses including the measles virus (MV). In this study, we investigated the association between the expression of MV antigens and MxA in subacute sclerosing panencephalitis (SSPE) brains. We analyzed the MxA expression in and around lesions in brains of three SSPE patients and compared it with normal brains. Double staining with antibodies against MxA and the MV nucleocapsid revealed that MxA was highly expressed in a belt surrounding MV-antigen-positive lesions in SSPE brains. In normal appearing regions distant from a lesion in SSPE brains and in normal brains, MxA was not detected. Furthermore, MxA was often less or not expressed in the center of lesions expressing high amounts of MV antigens. Such a pattern of MxA expression in SSPE brains clearly indicates that newly infected cells release type I IFN and will become demarcated by a protecting barrier of MxA expressing cells. Double staining with antibodies against MxA and glial fibrillary acidic protein (GFAP) showed that the MxA protein was expressed mainly in the cytoplasm of astrocytes. MxA expression did not correlate with the presence of cellular infiltrates of inflammatory cells, although some lymphoid cells were also positive for MxA. Since MxA inhibits the replication of MV, these findings suggest that the IFN-induced MxA protein plays an important role in slowing down the viral spread in SSPE brains and by doing so may contribute to the persistence of the MV-infection.

An Immunohistochemical Study of the Distribution of the Measles Virus Receptors, CD46 and SLAM, in Normal Human Tissues and Subacute Sclerosing Panencephalitis

We have compared the expression of the known measles virus (MV) receptors, membrane cofactor protein (CD46) and the signaling lymphocyte-activation molecule (SLAM), using immunohistochemistry, in a range of normal peripheral tissues (known to be infected by MV) as well as in normal and subacute sclerosing panencephalitis (SSPE) brain. To increase our understanding of how these receptors could be utilized by wild-type or vaccine strains in vivo, the results have been considered with regard to the known route of infection and systemic spread of MV. Strong staining for CD46 was observed in endothelial cells lining blood vessels and in epithelial cells and tissue macrophages in a wide range of peripheral tissues, as well as in Langerhans' and squamous cells in the skin. In lymphoid tissues and blood, subsets of cells were positive for SLAM, in comparison to CD46, which stained all nucleated cell types. Strong CD46 staining was observed on cerebral endothelium throughout the brain and also on ependymal cells lining the ventricles and choroid plexus. Comparatively weaker CD46 staining was observed on subsets of neurons and oligodendrocytes. In SSPE brain sections, the areas distant from lesion sites and negative for MV by immunocytochemistry showed the same distribution for CD46 as in normal brain. However, cells in lesions, positive for MV, were negative for CD46. Normal brain showed no staining for SLAM, and in SSPE brain only subsets of leukocytes in inflammatory infiltrates were positive. None of the cell types most commonly infected by MV show detectable expression of SLAM, whereas CD46 is much more widely expressed and could fulfill a receptor function for some wild-type strains. In the case of wild-type stains, which are unable to use CD46, a further as yet unknown receptor(s) would be necessary to fully explain the pathology of MV infection.

Approaches in the understanding of morbillivirus neurovirulence.

Certain members of the morbillivirus genus, canine distemper virus, phocine distemper virus, and the cetacean viruses of dolphins and porpoises exhibit high levels of central nervous system (CNS) infection in their natural hosts. CNS complications are rare for measles virus (MV) and are not associated with rinderpest virus (RPV) and peste des petits ruminants virus (PPRV) infection. However, both RPV and PPRV are neurovirulent in permissive murine strains. Human postmortem tissue, neural cell cultures, and animal models have been used to answer major questions concerning morbillivirus neurovirulence. Studies of the MV CNS complication subacute sclerosing panencephalitis (SSPE) indicate that virus could enter the CNS either by direct infection of endothelial cells or in infected leucocytes, followed by infection of predominately neurones and oligodendrocytes. It has been established that MV neurovirulence in mice is partially determined by the virus-receptor specificity. The two known MV receptors, CD46 and SLAM, have been examined in normal and SSPE brain tissue and the findings suggest that further receptors may be necessary to explain infection of the CNS with wild-type strains of MV. In both humans and mice (and in vitro), once infection of neurones has been established, virus spreads transneuronally. It is possible that all morbilliviruses transiently infect the CNS in their natural hosts, but development of disease is dependent on the efficiency of the immune response. Alternatively, for RPV and PPRV, virus entry may be restricted due either to absence of viral receptors or failure of virus to replicate or spread in the CNS.

Cloning the Antibody Response in Humans with Chronic Inflammatory Disease: Immunopanning of Subacute Sclerosing Panencephalitis (SSPE) Brain Sections with Antibody Phage Libraries Prepared from SSPE Brain Enriches for Antibody Recognizing Measles Virus Antigens In Situ

In central nervous system (CNS) infectious and inflammatory diseases of known cause, oligoclonal bands represent antibody directed against the causative agent. To determine whether disease-relevant antibodies can be cloned from diseased brain, we prepared an antibody phage display library from the brain of a human with subacute sclerosing panencephalitis (SSPE), a chronic encephalitis caused by measles virus, and selected the library against SSPE brain sections. Antibodies that were retrieved reacted strongly with measles virus cell extracts by enzyme-linked immunosorbent assay and were specific for the measles virus nucleocapsid protein. These antibodies immunostained cells in different SSPE brains but not in control brain. Our data provide the first demonstration that diseased brain can be used to select in situ for antibodies directed against the causative agent of disease and point to the potential usefulness of this approach in identifying relevant antibodies in chronic CNS or systemic inflammatory diseases of unknown cause.

Cloning the Antibody Response in Humans with Inflammatory Central Nervous System Disease: Analysis of the Expressed IgG Repertoire in Subacute Sclerosing Panencephalitis Brain Reveals Disease-Relevant Antibodies That Recognize Specific Measles Virus Antigens

The presence of increased IgG in the brains of humans with infectious and inflammatory CNS diseases of unknown etiology such as multiple sclerosis may be a clue to the cause of disease. For example, the intrathecally synthesized oligoclonal bands (OGBs) in diseases such as subacute sclerosing panencephalitis (SSPE) or cryptococcal meningitis have been shown to represent Ab directed against the causative agents, measles virus (MV) or Cryptococcus neoformans, respectively. Using SSPE as a model system, we have developed a PCR-based strategy to analyze the repertoire of IgG V region sequences expressed in SSPE brain. We observed abnormal expression of germline V segments, overrepresentation of particular sequences that correspond to the oligoclonal bands, and substantial somatic mutation of most clones from the germline, which, taken together, constitute features of Ag-driven selection in the IgG response. Using the most abundant or most highly mutated gamma H chain and kappa or lambda L chain sequences in various combinations, we constructed functional Abs in IgG mammalian expression vectors. Three Abs specifically stained MV-infected cells. One Ab also stained cells transfected with the MV nucleoprotein, and a second Ab stained cells transfected with the MV-fusion protein. This technique demonstrates that functional Abs produced from putative disease-relevant IgG sequences can be used to recognize their corresponding Ags.

Cloning the antibody response in humans with inflammatory CNS disease: isolation of measles virus-specific antibodies from phage display libraries of a subacute sclerosing panencephalitis brain

We have developed a strategy to identify the disease-relevant antigens in a chronic inflammatory CNS disease exhibiting intrathecally expressed oligoclonal IgG. Using subacute sclerosing panencephalitis (SSPE), a chronic inflammatory measles virus infection of the brain as a model system, we constructed a phage display antibody Fab library from the amplified products of IgG expressed in the brain. Selection of the library against measles virus-infected cell lysates yielded four distinct Fabs which, by ELISA and by immunostaining, reacted specifically with measles virus-infected cells. Three Fabs immunoprecipitated a 72 kDa protein from infected cell cultures corresponding to the measles virus phosphoprotein. The fourth Fab immunoprecipitated and recognized by immunoblotting a 60 kDa protein corresponding to the measles virus nucleoprotein. The results demonstrate that functional antibodies from an inflammatory CNS disease can be expressed in bacteria and used to identify disease-relevant antigens. This approach could be applied to chronic inflammatory CNS diseases of unknown cause such as multiple sclerosis.

Absence of measles virus receptor (CD46) in lesions of subacute sclerosing panencephalitis brains.

In this study we investigated pathological changes of the expression of the measles virus (MV) receptor, CD46, in subacute sclerosing panencephalitis (SSPE) brains. We analyzed CD46 expression in lesions of brain specimens from five SSPE patients in comparison to uninfected regions of the same brains and to normal human brains. The correlation between CD46 and MV infection, in individual cells in SSPE brains, was analyzed by double-staining procedures using monoclonal antibodies (mAbs) and in situ hybridization to detect MV-specific mRNAs. We found that CD46 was expressed at relatively low levels by neurons and astrocytes in normal brains in comparison to neuroblastoma and astrocytoma cell lines. Within heavily infected (MV-positive) brain lesions of all five SSPE cases, CD46 was either not detected or was expressed to a lesser degree by neural cells, irrespective of whether MV antigens were detectable or not. In contrast, normal levels of CD46 were found in SSPE brain tissue distant from the lesion. Using in situ hybridization, mRNAs of both MV nucleocapsid and MV hemagglutinin (MV-H) were detected in all SSPE lesions, while no or only small amounts of MV-H protein were detected. MV-infected neurons were never found to express CD46. Although a strict correlation between levels of the MV-H protein and the absence CD46 could not be seen, these findings suggest that the CD46 expression is reduced by the MV infection in lesions of SSPE brains.

Extraction and purification of active IgG from SSPE and MS brain

Immunoglobulin (Ig) G was purified from soluble and membrane fractions of postmortem subacute sclerosing panencephalitis (SSPE) brain, multiple sclerosis (MS) brain plaque-periplaque white matter, and normal human brain (NHB) white matter. After homogenization in 0.32 M sucrose and removal of cell debris and nuclei by low-speed centrifugation, soluble and crude membrane fractions were separated by ultracentrifugation. After removal of sucrose by dialysis, IgG was isolated from the soluble fraction by protein A affinity chromatography. IgG was obtained from the membrane fraction by elution at low pH and purification from the eluate by protein A chromatography. Whereas very little IgG was in NHB white matter, significant levels of IgG were recovered from both SSPE and MS brain. Both immunocytochemical staining of measles virus-infected cells in tissue culture and protein immunoblotting of virus-infected cell lysates showed that the IgG from SSPE brain contained activity specific for measles virus protein. The abundance, purity and functional activity of IgG extracted from SSPE and MS brain indicate that IgG extracted from the brain of humans with an inflammatory disease of unknown etiology can be used to identify its corresponding antigen.

Leukaemia inhibitory factor mRNA is expressed in the brains of patients with subacute sclerosing panencephalitis

We have examined the in situ transcription of leukaemia inhibitory factor (LIF) in brain tissue from 3 cases of subacute sclerosing panencephalitis (SSPE) and in 2 non-neurological control brains. This has been compared with expression of interleukin 2 (IL-2), interleukin 6 (IL-6) and tumour necrosis factor beta (TNF beta) in the same tissues. All of the cytokines in the study were expressed in cells in the inflammatory infiltrate as well as in glial cells. LIF mRNA was also found to be expressed in neurons, in foci where these cells were also virally infected. No hybridization was found with any of the probes in areas of SSPE brain, which were negative for measles virus RNA or in the non-neurological control cases, although expression was demonstrated in the latter by use of reverse transcription-polymerase chain reaction (RT-PCR). Differentiated, cultured human neuronal cells were also positive, by RT-PCR, for LIF. This is the first demonstration of LIF expression in human brain and the results suggest that this cytokine is up-regulated, in several cell types, including neurons, following virus infection.

The use of microwave irradiation as a pretreatment to in situ hybridization for the detection of measles virus and chicken anaemia virus in formalin-fixed paraffin-embedded tissue.

Microwave irradiation was investigated as a pretreatment to in situ hybridization on formalin-fixed, paraffin-embedded tissue. Two probe/tissue systems were used: a single-stranded RNA probe for the detection of measles virus nucleocapsid genome in subacute sclerosing panencephalitis brain tissue, and a double stranded DNA probe for chicken anaemia virus in thymus of chicken infected with the virus. Microwaving, when used as sole pretreatment, was not as effective as the more traditional enzyme pretreatments for in situ hybridization. However, when used in combination with existing pretreatments, a significant increase was found in hybridization signal in both brain and thymus tissue. This was emphasized when combination enzyme/microwave pretreatments were used prior to detection of measles virus by in situ hybridization in a series of five archival subacute sclerosing panencephalitis cases. The use of microwave irradiation would be recommended as a means of supplementing in situ hybridization methods, especially when using long-term formalin fixed paraffin-embedded tissue.

Defective Measles Virus in Human Subacute Sclerosing Panencephalitis Brain

Evidence is presented showing that the brain cells of patients with subacute sclerosing panencephalitis (SSPE) contain mutant measles (MV) genomes having the characteristics of 5′ copy-back defective interfering (DI) RNAs. Using a polymerase chain reaction-based amplification specific for copy-back DIs, abundant, discrete cDNAs representing different-sized MV defective RNA species were generated from each SSPE brain. The defective genomes were cloned in two portions. The most common of these defective species were sequenced, confirming their MV genome origin and 5′ copy-back nature. We deduced that the minimum DI stem length in these species was 95 nucleotides, further delimiting the prerequisite 5′ regulatory region sequences specifying MV genomic replication/encapsidation functions. This calculation assumes a precise copy-back mechanism and complete complementarity of the panhandle structure. Since the SSPE-derived viral genome encodes dysfunctional viral envelope proteins, we hypothesize that SSPE brains may lack the high degree of selective pressure encountered in tissue culture MV infections. This allows for the coexistence of numerous replication-competent defective particles in each SSPE brain. A role for viral defective particles as modulators of this persistent measles virus infection of humans is proposed.

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.

As 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.

Detection of measles virus genomic sequences in sspe brain tissue by the polymerase chain reaction

The polymerase chain reaction (PCR) was modified to detect RNA genomic sequences by generating cDNA copies of these sequences as a preliminary step. Oligonucleotide primer pairs complementary to sequences in each of the five major structural protein genes of the measles virus (nucleocapsid protein, phosphoprotein, matrix protein, fusion protein, and hemagglutinin protein) were synthesized. PCR products were tentatively identified by visualization of bands of the appropriate size by ethidium bromide staining after gel electrophoresis, and identity was confirmed by subsequent restriction enzyme cleavage of the products at predetermined sites to yield fragments of predicted size. This method successfully amplified 400-500 base regions from each of these five genes in RNA extracts of wild measles virus cultured in Vero cells and in RNA extracted from most of the SSPE brain tissues tested, but not in RNA from any control brain tissues. Measles virus genome was detected in SSPE brain tissues stored frozen for as long as 27 years and formalin-fixed paraffin-embedded subacute sclerosing panencephalitis (SSPE) brain tissues as old as 9 years. This method provides a simple, rapid and highly sensitive means of detecting and identifying sequences of RNA genomes by PCR. The success of this method in detecting measles virus in SSPE brain tissue suggests that PCR is appropriate to investigate the possible presence of RNA viruses in other neurological disorders of unknown etiology.

Accumulated measles virus mutations in a case of subacute sclerosing panencephalitis: Interrupted matrix protein reading frame and transcription alteration

Subacute sclerosing panencephalitis (SSPE) is a fatal disease affecting the human central nervous system several years after acute measles infection. Measles virus (MV) genomes replicating in SSPE brains do not give rise to budding particles and present various defects in gene expression, mostly concerning the matrix (M) protein. For one SSPE case (K), shown previously to be devoid of M protein expression, we examined here in detail the features involved in this defect. In the brain of patient K the normal, monocistronic MV M mRNA was completely substituted by a bicistronic RNA containing the coding sequence of the preceding phosphoprotein (P) gene in addition to the M coding sequence. Analysis of the P-M intercistronic region by direct cDNA sequencing showed that the consensus sequence at this RNA processing site was unaltered but revealed several distant point mutations. cDNA cloning and sequencing of the entire M coding region established that one of the point mutations leads to a stop codon at triplet 12 of the M reading frame. It is unknown whether this defect, explaining by itself the lack of M protein, is related also to the block of M mRNA formation. In addition we note that as much as 1% of the nucleotides different between two overlapping clones from the same brain. This high sequence variability could possibly account for the diversity of defects observed in MV gene expression in SSPE brains and may be a general phenomenon associated with RNA virus persistence.

Search for virus nucleic acid sequences in postmortem human brain tissue using in situ hybridization technology with cloned probes: some solutions and results on progressive multifocal leukoencephalopathy and subacute sclerosing panencephalitis tissue.

In order to obtain a useful and readily applicable in situ hybridization (ISH) protocol for progressive central nervous system (CNS) diseases of unknown etiology that are possibly due to persistent viral infection, known and well described diseases were studied, namely, progressive multifocal leukoencephalopathy (PML) and subacute sclerosing panencephalitis (SSPE). The procedures described were validated by confirming results obtained by other investigators using histology, immunocytochemistry, electron microscopy, and ISH. A number of frequently encountered problems of tissue preparation are addressed as well as techniques to reduce autoradiography exposure times. A multi-staged specific, sensitive, reliable, and valid procedure for detection of viral genomes, mRNA and proteins is approached. Formalin-fixed and paraffin-embedded (FFPE) brain material from six patients who died with PML and one patient who died from SSPE were studied using ISH with a tritium-labeled cloned JC virus DNA probe and a measles-cloned nucleocapsid (NC) gene cDNA probe, respectively. This report constitutes a methodological framework as well as a detailed neuropathological analysis of identified brain cell populations within which in situ hybridization was detected. In early PML lesions, swollen nuclei or oligodendrocytes were the predominant cells labeled, whereas older lesions revealed increased numbers of reactive and bizarre hypertrophic astrocytes hybridized at the outer periphery of the demyelinated lesions. The hybridization varied greatly in intensity in different cells. Intense hybridization was noted very rarely in microglial cells, including rod cells and rarely in venular pericytes, intravascular mononuclear cells, or in vascular endothelial cells. These results, considered together with previous findings, indicate that in PML the viral infection runs different courses in the various cells: in astrocytes the viral genome persists for a long time inducing pathological changes in some cells. In oligodendrocytes the infection rapidly lyses the cells. There was a good correlation between chromatic changes observable in routinely stained sections and virus presence. In addition, in situ hybridization using a measles-NP-cloned probe in white matter from FFPE SSPE brain is presented confirming earlier results in SSPE cryopreserved brain.

Subacute sclerosing panencephalitis: measles virus matrix protein nucleic acid sequences detected by in situ hybridization.

Subacute sclerosing panencephalitis (SSPE) is characterized by a hyperimmune state toward the polypeptides of measles virus except the matrix (M) protein. Using cloned (3H)-labeled complementary DNA probes for in situ hybridization, we found the M protein and nucleocapsid (NP) protein nucleotide sequences in glial cells and neurons of cryostat sections from two SSPE brains. In one SSPE brain, M protein was lacking, but the other measles polypeptides were present. IgG and IgM antibodies eluted from that brain lacked antibodies to M protein, but antibodies to other measles polypeptides were present. In SSPE brain, the viral M-protein defect is not a deletion of the M gene, but rather a block in gene expression.

Enrichment of measles virus-like RNA in the nucleocapsid fraction isolated from subacute sclerosing panencephalitis brains

A procedure has been developed which facilitates the detection of measles virus RNA sequences in human brains. The procedure involves isolating subviral components (nucleocapsids) from brain tissues prior to RNA purification, followed by hybridization of these RNAs to cDNA synthesized from measles virus 50 S RNA template. Using these techniques we were able to obtain an RNA fraction which was manyfold enriched in measles virus-specific RNA, relative to unfractionated subacute sclerosing panencephalitis (SSPE) brain RNAs. 70–100% of the measles virus-specific RNA present in these SSPE brain samples were recovered in this enriched fraction.