Platelet-derived growth factor-alpha receptor-positive oligodendroglia are frequent in multiple sclerosis lesions.

Platelet-derived growth factor (PDGF) ligand is a potent glial cell mitogen. When its cognate receptor (PDGF-alphaR) is expressed on oligodendroglial lineage cells, such cells are considered capable of division, and the receptor thus serves as a phenotypic marker for oligodendrocyte precursor cells. Here we identify using immunohistochemistry a considerably enlarged, PDGF-alphaR-expressing oligodendrocyte cell population within multiple sclerosis (MS) white matter lesions compared to control brains. Numerous PDGF-alphaR-positive oligodendroglia also colabel heavily with the nuclear cell proliferation marker antibody Ki-67. Our finding of large numbers of proliferating oligodendroglia in MS brains expressing up-regulated PDGF-alphaR suggests that these progenitor-like cells represent an important source of regenerating cells for the healing MS lesion.

Cell death and birth in multiple sclerosis brain.

The hallmark of the brain pathology in multiple sclerosis is the white matter plaque, characterized by myelin destruction and oligodendrocyte loss. To examine the role that cell death plays in the development of MS lesions, we used the in situ TUNEL technique, a method that sensitively detects DNA fragmentation associated with death at the single cell level. We found that patchy areas within acute MS lesions have massive numbers of inflammatory and glial cells undergoing cell death. The punched out areas of some long-standing chronic lesions also had labeled glial cells showing that the attack was not a single event. Immunocytochemical identification of the dying cells with glial specific marker co-labeling showed that 14-40% were the myelin-sustaining oligodendroglial cell. Confocal microscopic evaluation of fluorescein-labeled TUNEL positive cells revealed nuclei with morphologic characteristics of apoptosis, and electrophoresed MS brain DNA produced a ladder characteristic of apoptotic DNA cleavage confirming that substantial numbers of labeled cells, but not necessarily all, were dying by apoptotic mechanisms rather than cell necrosis. Companion studies using a marker for cell proliferation on MS lesions revealed that unexpectedly large populations of perivascular inflammatory cells and parenchymal glial cells had entered the cell proliferation cycle. These findings establish that two opposing glial cell responses - relentless cell death and coincident brisk cellular proliferation - are important features of MS pathology. In the end, however, glial cell loss prevails, and we suspect apoptosis may be the critical death mechanism responsible for the depletion of myelin observed in this condition.

Involvement of the CD95 (APO-1/Fas) receptor/ligand system in multiple sclerosis brain.

Immunohistochemical methods were used to search for Fas receptor/Fas ligand system involvement in multiple sclerosis (MS) white matter brain lesions. We found large numbers of Fas ligand (Fas-L)-bearing cells present in two acute lesions and 12 of 16 chronic MS lesions, and very few positive cells in non-inflammatory controls. Four of six brains from non-MS neuropathologic conditions associated with inflammation and white matter disease were, however, also positive for Fas-L. Double staining with cell-specific markers revealed that the pattern of ligand-positive cells in chronic MS lesions was complex and composed of several different cell types which were primarily resident glial cells with a small overlay of macrophages. Fas/APO 1 (CD95) receptor expression in MS tissue was also evaluated and marked upregulation of the receptor was found. In addition, Fas receptor was induced, but to a lesser extent, in numerous control brains. The observations that TUNEL-positive dying cells were present in MS lesions and showed excellent co-localization with Fas-L, indicate that the Fas death system may contribute to plaque pathogenesis and could lead to the development of a new category of therapeutic agents for MS.

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 of 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.

Canine distemper virus L gene: sequence and comparison with related viruses.

We cloned the genomic RNA of canine distemper virus (CDV) and determined the nucleotide sequence of the large (L) protein-coding gene. The L gene is 6573 nucleotides long and contains a single open reading frame coding for a polypeptide of 2161 amino acids (MW 246,354). The precise 5' end of the viral genome consists of a 38-nucleotide leader region. The CDV L protein shows over 77% amino acid similarity with its morbilliform relative measles virus (MV) with nearly 67% of their amino acids conserved. The sequence homology of 11 negative strand viruses L proteins is compared and relatedness was found in the following decreasing order: CDV, MV, Sendai virus, parainfluenza virus type 3, simian virus 5, parainfluenza virus type 2, mumps virus, Newcastle disease virus, respiratory syncytial virus, vesicular stomatitis virus, rabies virus. The consensus sequence of proposed functional domains involved in L gene catalytic activities was well conserved in the CDV L protein.

Spinal cord disease in children with HIV-1 infection: a combined molecular biological and neuropathological study.

An autopsy study was performed on spinal cords from 18 children who died with HIV-1 infection, using standard histopathologic techniques as well as in situ hybridization and immunocytochemistry for HIV-1. Of 16 spinal cords examined by histology, nine had inflammatory cell infiltrates and six had multinucleated cells; both types of lesion are associated with the presence of HIV-1 in central nervous system tissue. HIV-1 type lesions were often present in the spinal cord and brain from the same patient. Pallor of myelin in corticospinal tracts in the cord was present in half of the cases; this change correlated with diffuse myelin pallor in the corresponding brains, but not with the HIV-1 associated changes in the cords. In situ hybridization for HIV-1 nucleic acid sequences gave positive results in seven of 18 spinal cords, with hybridizing signal usually localized to inflammatory cell infiltrates and multinucleated cells. Positive in situ hybridization, on frozen sections, correlated with the presence of HIV-1 associated changes on paraffin sections from the same cases. Immunocytochemistry for p25 core protein of HIV-1, using a monoclonal antibody on frozen sections, was positive in multinucleated cells, macrophages, and microglia. In this series there were two cases of vacuolar myelopathy, one a 30-month-old boy who had concomitant measles virus in the spinal cord grey matter, and the other nine-year-old girl who had severe HIV-1 infection of the cord. Other than the single case of measles virus, there were no opportunistic infections in the cords in this series. HIV-1 frequently involves the spinal cord in children with AIDS, while opportunistic infections are rare. Vacuolar myelopathy occurs in children with HIV-1 infection, although its occurrence is much less frequent than in adults with AIDS.

Sequence variability and function of measles virus 3' and 5' ends and intercistronic regions.

Sequences critical for the activity of the measles virus (MV) RNA polymerase in transcription and replication were analyzed using a MV genomic cDNA library containing overlapping clones encompassing the entire MV genome. Clones corresponding to the 3' and 5' ends of the MV genome were identified and sequenced, and these sequences were confirmed by primer extension experiments. Neither (+) nor (-) strand leader RNAs were detected in MV-infected cell extracts, using high specific activity riboprobes made form these clones. Clones representing each of the MV gene boundaries were also sequenced, and variations including point mutations, insertions, and deletions were noted. Together with the sequence of the MV L gene region, this report completes the sequence determination of the MV genome.

Measles virus L protein evidences elements of ancestral RNA polymerase.

We have determined the nucleotide sequence of the measles virus (MV) L gene using a cDNA library encompassing the entire MV genome (J. Crowley et al. (1987) Intervirology, 28, 65-77). The L gene is 6639 nucleotides in length, and contains a single long open reading frame that could code for a protein of 247,611 kDa. Both the L gene and in particular the predicted L protein of MV bear substantial homology to their counterparts in Sendai virus and Newcastle disease virus, suggesting that the multifunctional nature of paramyxovirus L proteins imposes strong evolutionary constraints. The predicted MV L protein also contains distinct elements of a postulated ancestral RNA polymerase.

Molecular cloning of 99% of measles virus genome, positive identification of 5' end clones, and mapping of the L gene region.

Molecular probes specific for paramyxovirus 3' and 5' end sequences are essential for studying viral transcription and replication and the role of defective interfering particles in these processes. We have constructed from measles virus genomic RNA an ordered series of overlapping clones containing over 99% of measles virus genetic information. Clones corresponding to the 3' and 5' ends of the genome were identified by means of a probe made from 'capped' genomic RNA, and their location was confirmed by primer extension experiments. Clones representing the N, P/C, M, F, and H genes were identified by reference to previously characterized clones, and their positions in the genome were defined by restriction analysis and S1 nuclease mapping of intercistronic clones. The L gene region was characterized by site-to-site restriction mapping and measured as 6,550 nucleotides in length.

Transcriptional map of the measles virus genome.

We have constructed a recombinant DNA library of the measles virus genome and identified gene-specific clones containing sequences coding for portions of each of the six viral structural proteins, as well as clones coding for intercistronic sequences. By Northern blotting, we could order the clones according to the pattern of individual gene-specific and readthrough mRNAs. Clones corresponding to the N, P/C and M genes were identified by correlation of the mRNAs with their in vitro translation products; clones corresponding to the H, F and L genes were identified by indirect evidence. The results indicated that the gene order in measles virus is that of a typical paramyxovirus (3'-N,P/C,M,F,H,L-5'), but that the M and F transcripts each contain 1.5 times the coding capacity needed for synthesis of these proteins in vivo.

Mycoplasma antibody in Guillain-Barré syndrome and other neurological disorders.

Counterimmunoelectrophoresis (CIEP) was used to determine precipitating antibodies to Mycoplasma pneumoniae retrospectively in sera from 100 patients with Guillain-Barré syndrome (GBS), 125 medical and neurological controls, and 40 normal individuals. Sera from 7 patients produced precipitin lines. These positive cases included 5 patients with GBS, 1 with acute cerebellar ataxia, and 1 with acute disseminated encephalomyelitis. A complement-fixation test performed with the same antigen showed titers to M. pneumoniae of 1:512 or greater in these 7 sera. In contrast, sera from other patient controls and normal individuals were negative by CIEP and had only low mycoplasma complement-fixation antibody titers. No distinguishing clinical features separated the 5 seropositive GBS patients from the whole group except for their young age, which parallels that for human mycoplasma infection in general. Additional laboratory findings consistent with acute mycoplasma infection were demonstrated in 6 of the 7 seropositive patients.

Cytomegalovirus complement fixation antibody in Guillain-Barré syndrome.

Cytomegalovirus, measles, and adenovirus antibodies were measured in the sera of 92 Guillain-Barré patients and 120 controls. Thirty patients (33 percent) had markedly elevated levels of complement-fixing antibody to cytomegalovirus and in 21, a fourfold or more alteration in titer was demonstrated. Diagnostic falls in titer were seen in most instances and no significant elevation to the other viral agents was found. The serologic findings reported here suggest that cytomegalovirus may be a common agent involved in the pathogenesis of the Guillain-Barré syndrome.

Herpes simplex virus-IgM specific antibodies in Guillain-Barré syndrome and encephalitis.

Herpes simplex virus (HSV) has veen associated with a variety of inflammatory neurologic disorders. Recently we studied a patient with Guillain-Barré syndrome (GBS) following acute herpes vaginalis infection. Since IgM virus-specific antibody is thought to be a reliable indicator of acute viral infection, we employed a 2-h serologic assay for serum IgM antibodies to HSV using an indirect immunofluorescent technique. This patient demonstrated high serum IgM titers to HSV type 2 during the acute phase of her neurologic syndrome. The titer dropped substantially as convalescence progressed. A search for similar elevations in HSV-IgM specific antibody was made on sera from more than 70 other GBS patients. No other significant IgM antibody titers to either HSV type 1 or type 2 were found in this GBS series and a large number of neurologic controls. However, sera from two patients with a presumptive diagnosis of acute herpes encephalitis based on clinical and cerebrospinal fluid findings were positive, showing high titers in our test. The results of this study suggest that associated acute HSV infection is uncommon in GBS and an immunofluorescent seroassay of the type reported here may be a valuable noninvasive technique enabling the clinical laboratory to rapid confirm a diagnosis of herpes encephalitis.

Guillain-Barré syndrome in Greater New York-New Jersey.

Epidemiologic data on 176 patients with acute Guillain-Barré syndrome observed over a nine-year period in the New York-New Jersey metropolitan area are presented. A striking predilection for patients aged 16 to 25 years, with a second lesser peak between ages 45 to 60, was found. Seasonal clustering of patients occurred, with nearly half the patients afflicted during the four-month period of late summer and fall. The distribution between men and women was equal. In this series 23% of the patients required tracheostomy, and 5% had a fatal course.

Precipitating antibodies in mycoplasma infection.

The effectiveness of counterimmunoelectrophoresis (CIEP) for detecting human precipitating antibodies to mcyoplasma antigen was compared with the conventional complement fixation (CF) method in a double-blind experiment. Fifty-one sera from patients suspected of having acute mycoplasma infection were tested by both techniques. Dense precipitin lines to mycoplasma antigen developed in 28 sera with CIEP. Twenty-six of 28 had elevated CF titers to this antigen. No precipitin bands were observed in sera with low antibody titers to mycoplasma. These findings indicate that the CIEP test is a specific method for reliably detecting elevated serum CF antibody levels in patients with acute or recent mycoplasma infection.

Rapid fluorometric assay for cerebrospinal fluid immunoglobulin G.

Using a new quantitative immunofluorometric procedure, we measured cerebrospinal fluid immunoglobulin G levels in 59 patients and compared the results with values obtained by the most precise currently available method--the immunoprecipitin method of Kabat--and a radial immunodiffusion technique. The absolute immunoglobulin G values determined by the fluorometric assay correlated closely with the other methods. This technique offers several advantages over most conventional techniques for measuring low levels of immunoglobulin G: (1) Small, drop-sized (5 to 10 micronl) samp les of cerebrospinal fluid are used, (2) a large number of samples can be tested, (3) the range of sensitivity is sufficiently wide that cerebrospinal fluid and serum levels can be determined simultaneously, and (4) the technique is fast, requiring 3.5 hours to perform. The fluorometric method is rapid, reproducible, and easy. It suitability for laboratories engaged in the measurement of cerebrospinal fluid immunoglobulin G appears promising.

Rapid detection of antibodies to cytomegalovirus by counterimmunoelectrophoresis.

A new method for the detection of precipitating antibody to cytomegalovirus by counterimmunoelectrophoresis (CIEP) is described. Fourteen of 15 adult sera (95%) with IgM-specific antibodies (as detected by immunofluorescence) and complement-fixing antibodies to cytomegalovirus in high titers gave positive reactions by this method. Control sera from 156 patients and 40 normal subjects were negative by CIEP. One of 32 individuals acutely infected with other members of the herpesvirus group gave a positive reaction (3%). False-positive reactions were restricted to a group of sera containing rheumatoid factor (10 of 31), but this activity could be eliminated by preliminary absorption of the sera with aggregated gamma-globulin. The present findings demonstrate that CIEP is useful for the detection of precipitating antibodies to cytomegalovirus in sera from acutely infected patients and promises to be a rapid, inexpensive screening procedure of diagnostic value.