RESUMEN

Natalizumab and fingolimod are effective multiple sclerosis (MS) therapies that disrupt lymphocyte migration but have differential effects on B cell maturation and trafficking. We investigated their effects on peripheral blood (PB) and cerebrospinal fluid (CSF) B cell repertoires using next-generation deep sequencing. Paired CSF and PB B cell subsets (naïve, CD27+ memory, and CD27-IgD- double-negative B cells and plasmablasts) were collected by applying flow cytometry at baseline and after 6 months of treatment and their respective heavy-chain variable region repertoires assessed by Illumina MiSeq. Treatment with fingolimod contracted, whereas natalizumab expanded circulating PB B cells. CSF B cell numbers remained stable following fingolimod treatment but decreased with natalizumab therapy. Clonal overlap between CSF and PB B cells was reduced with natalizumab treatment but remained stable with fingolimod therapy. Lineage analyses of pre- and posttreatment CSF B cell repertoires revealed large, clonally expanded B cell clusters in natalizumab-treated MS patients but no intrathecal clonal expansion following fingolimod therapy. Our findings suggest that natalizumab diminishes the exchange of peripheral and intrathecal B cells without impacting intrathecal clonal expansion. In contrast, fingolimod treatment fails to alter blood-brain barrier B cell exchange but diminishes intrathecal clonal expansion. Sphingosine-1 phosphate receptor inhibition may alter intrathecal B cell biology in MS.

Asunto(s)

Linfocitos B/efectos de los fármacos , Clorhidrato de Fingolimod/uso terapéutico , Esclerosis Múltiple/tratamiento farmacológico , Natalizumab/uso terapéutico , Adolescente , Adulto , Anciano , Linaje de la Célula/efectos de los fármacos , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células B de Memoria/efectos de los fármacos , Persona de Mediana Edad , Esclerosis Múltiple/sangre , Esclerosis Múltiple/líquido cefalorraquídeo , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral , Adulto Joven

RESUMEN

OBJECTIVE: Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that becomes latent in B-lymphocytes and has been implicated in the pathogenesis of multiple sclerosis (MS). We searched for latent and active EBV infection in MS brain and CSF. METHODS: Nested and non-nested real-time PCR were used to detect cell-specific and EBV-specific transcripts in 15 fresh-frozen and 5 formalin-fixed paraffin-embedded MS plaques and in single MS CSF B-lymphocytes and plasma cells. Intrathecal anti-EBV antibody synthesis was measured by ELISA. Immunocytochemistry was used to detect binding of MS CSF and recombinant antibodies (rAbs) generated from clonally expanded plasma cells in MS CSF to EBV-infected cells. RESULTS: No EBV RNA was found in MS CSF B-lymphocytes or plasma cells. In active MS plaques, EBV-encoded RNA (EBER)-1 was the only and rarely detected transcript. The frequency of detected intrathecal anti-EBV antibody synthesis in patients with MS did not differ from that in non-MS inflammatory CNS disease control patients. Anti-EBV antibodies were detected in the CSF of patients with MS, but MS rAbs did not react with EBV. CONCLUSIONS: Application of real-time PCR to multiple sclerosis brain and single B-lymphocytes in CSF did not reveal any evidence of active Epstein-Barr virus infection.

Asunto(s)

Encéfalo/patología , Encéfalo/virología , Infecciones por Virus de Epstein-Barr/complicaciones , Herpesvirus Humano 4/metabolismo , Esclerosis Múltiple/patología , Esclerosis Múltiple/virología , Linfocitos B/virología , Biomarcadores/análisis , Encéfalo/fisiopatología , Líquido Cefalorraquídeo/citología , Humanos , Esclerosis Múltiple/líquido cefalorraquídeo , Valor Predictivo de las Pruebas , ARN Viral/análisis , ARN Viral/genética , ARN Viral/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Virales/biosíntesis , Proteínas Virales/genética

RESUMEN

OBJECTIVE: To demonstrate the specificity of expanded CD138(+) plasma cell clones recovered from the CSF of a patient with subacute sclerosing panencephalitis (SSPE) for measles virus (MV). METHODS: IgG variable region sequences of single-antibody-secreting CD138(+) cells sorted from SSPE CSF were amplified by single-cell PCR and analyzed. Human IgG1 recombinant antibodies (rAbs) were produced from four expanded CD138(+) clones and assayed for immunoreactivity against MV proteins. RESULTS: Clonal expansion was a prominent feature of the SSPE plasma cell repertoire, and each of the four rAbs assayed was specific for either the MV fusion or the MV nucleocapsid protein. CONCLUSIONS: Expanded plasma cell clones in the CSF of patients with subacute sclerosing panencephalitis produce disease-relevant antibodies. Recombinant antibodies derived from CSF B cells could provide a tool to identify target antigens in idiopathic inflammatory disorders.

Asunto(s)

Anticuerpos Antivirales/líquido cefalorraquídeo , Virus del Sarampión/inmunología , Células Plasmáticas/virología , Panencefalitis Esclerosante Subaguda/líquido cefalorraquídeo , Panencefalitis Esclerosante Subaguda/virología , Proteínas Virales/inmunología , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos/inmunología , Separación Celular , Células Cultivadas , Niño , Células Clonales/inmunología , Humanos , Inmunoglobulina G/líquido cefalorraquídeo , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Masculino , Proteínas de la Nucleocápside/inmunología , Células Plasmáticas/inmunología , Proteínas Recombinantes de Fusión/líquido cefalorraquídeo , Proteínas Recombinantes de Fusión/inmunología , Panencefalitis Esclerosante Subaguda/inmunología , Proteínas Virales de Fusión/inmunología

RESUMEN

Two-dimensional gel electrophoresis and peptide mass fingerprinting were used to identify proteins in cerebrospinal fluid (CSF) pooled from three patients with multiple sclerosis (MS) and in CSF pooled from three patients with non-MS inflammatory central nervous system (CNS) disorders. Resolution of CSF proteins on three pH gradients (3-10, 4-7 and 6-11) enabled identification of a total of 430 spots in the MS CSF proteome that represented 61 distinct proteins. The gels containing MS CSF revealed 103 protein spots that were not seen on control gels. All but four of these 103 spots were proteins known to be present in normal human CSF. The four exceptions were: CRTAC-IB (cartilage acidic protein), tetranectin (a plasminogen-binding protein), SPARC-like protein (a calcium binding cell signalling glycoprotein), and autotaxin t (a phosphodiesterase). It remains unknown whether these four proteins are related to the cause and pathogenesis of MS.

Asunto(s)

Líquido Cefalorraquídeo/química , Esclerosis Múltiple Recurrente-Remitente/líquido cefalorraquídeo , Proteómica , Adulto , Electroforesis en Gel Bidimensional , Femenino , Humanos , Persona de Mediana Edad , Mapeo Peptídico , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

Proteomics combines two-dimensional gel electrophoresis and peptide mass fingerprinting and can potentially identify a protein(s) unique to disease. Such proteins can be used either for diagnosis or may be relevant to the pathogenesis of disease. Because patients with multiple sclerosis (MS) have increased amounts of immunoglobulin (Ig) G in their cerebrospinal fluid (CSF) that is directed against an as yet unidentified protein, we are applying proteomics to MS CSF, studies that require optimal separation of proteins in human CSF. We found that recovery of proteins from CSF of MS patients was improved using ultrafiltration, rather than dialysis, for desalting. Resolution of these proteins was enhanced by acetone precipitation of desalted CSF before electrophoresis and by fractionation of CSF using Cibacron Blue sepharose affinity chromatography. Improved protein recovery and resolution will facilitate excision from gels for analysis by peptide mass fingerprinting.

Asunto(s)

Proteínas del Líquido Cefalorraquídeo/análisis , Electroforesis en Gel Bidimensional/métodos , Esclerosis Múltiple/líquido cefalorraquídeo , Albúminas , Humanos , Focalización Isoeléctrica/métodos , Sales (Química)

RESUMEN

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 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 developed a strategy to identify the antigenic targets of the intrathecal disease-relevant IgG in chronic human inflammatory and demyelinating diseases of the CNS. Libraries of cDNA Ags were displayed on the surface of T7Select bacteriophage and biopanned on IgG extracted from the brain of an SSPE patient, or on a monospecific recombinant Fab identified from SSPE brain. After three or six rounds of biopanning on either Ab, positive phage-displayed Ags reacting with IgG were enriched to 35-77% of all panned clones. Sequence analysis of the positive clones identified fragments of the nucleocapsid protein of MV, the cause of SSPE. The sensitivity of the system was determined by diluting the positive clones from this SSPE phage-displayed library at a ratio of 10(-6) into another phage-displayed library that did not contain any detectable MV Ags; after six rounds of panning, the positive clones comprised 34% of all phage and were also shown to be MV nucleocapsid specific. This strategy will be useful to identify potentially rare Ags in diseases of unknown cause.

Asunto(s)

Antígenos Virales/inmunología , Encéfalo/inmunología , Inmunoglobulina G/inmunología , Virus del Sarampión/inmunología , Biblioteca de Péptidos , Panencefalitis Esclerosante Subaguda/inmunología , Adulto , Antígenos Virales/genética , Humanos , Masculino , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/inmunología , Sensibilidad y Especificidad

RESUMEN

Identification of the causative agent of multiple sclerosis (MS) has long eluded investigators and has become the "Holy Grail" of researchers in the field. The immune response in cerebrospinal fluid of patients with MS, indicated by an increased IgG level and the presence of specific oligoclonal bands after electrophoresis, strongly parallels that found in various infectious diseases of the central nervous system. To understand the nature of B-lymphocyte activation in MS, 4 laboratories studied the antigen-binding regions of antibodies found in MS brain demyelinative plaques and cerebrospinal fluid. Each analysis revealed (1) limited germline expression, results not expected for a random bystander response; (2) features consistent with a specific antigen-targeted process; and (3) the clonal expansion of populations of B lymphocytes in MS. The screening of libraries expressing protein products derived from chronic MS plaque messenger RNA with antibodies purified from plaques, cerebrospinal fluid, or serum of patients with MS has thus far not revealed the antigenic target(s) of the MS antibody response. Because putative MS antigens could be in low abundance, the screening of large libraries of random peptides expressed on phage surfaces might offer an alternative approach to identify peptide sequences recognized by MS antibodies. New sophisticated molecular immunologic techniques described herein should enhance our ability to identify putative antigen(s) targets in MS.

Asunto(s)

Antígenos CD/genética , Antígenos CD/inmunología , Linfocitos B/inmunología , Encéfalo/inmunología , Inmunoglobulinas/inmunología , Esclerosis Múltiple/genética , Esclerosis Múltiple/inmunología , Anticuerpos Monoclonales/genética , Anticuerpos Antivirales/inmunología , Afinidad de Anticuerpos/inmunología , Antígenos CD/líquido cefalorraquídeo , Enfermedad Crónica , Clonación Molecular/métodos , ADN Complementario/genética , ADN Complementario/inmunología , ADN Recombinante , Progresión de la Enfermedad , Epítopos , Estudios de Factibilidad , Biblioteca de Genes , Humanos , Inmunoglobulinas/líquido cefalorraquídeo , Inmunoglobulinas/genética , Esclerosis Múltiple/líquido cefalorraquídeo , Biblioteca de Péptidos , Polimorfismo Genético/genética , Polimorfismo Genético/inmunología , ARN Mensajero/genética , ARN Mensajero/inmunología

RESUMEN

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of unknown cause that afflicts the central nervous system. MS is typified by a highly clonally restricted antigen-driven antibody response that is confined largely to the central nervous system. The major antigenic targets of this response and the role of antibody in disease pathogenesis remain unclear. To help resolve these issues, we cloned the IgG repertoire directly from active plaque and periplaque regions in MS brain and from B cells recovered from the cerebrospinal fluid of a patient with MS with subacute disease. We found that high-affinity anti-DNA antibodies are a major component of the intrathecal IgG response in the patients with MS that we studied. Furthermore, we show DNA-specific monoclonal antibodies rescued from two subjects with MS as well as a DNA-specific antibody rescued from an individual suffering from systemic lupus erythematosus bound efficiently to the surface of neuronal cells and oligodendrocytes. For two of these antibodies, cell-surface recognition was DNA dependent. Our findings indicate that anti-DNA antibodies may promote important neuropathologic mechanisms in chronic inflammatory disorders, such as MS and systemic lupus erythematosus.

Asunto(s)

Anticuerpos Antinucleares/inmunología , Linfocitos B/inmunología , Encéfalo/inmunología , Esclerosis Múltiple/inmunología , Adulto , Anticuerpos Antinucleares/sangre , Anticuerpos Antinucleares/líquido cefalorraquídeo , Femenino , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/inmunología , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Cadenas kappa de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/inmunología , Esclerosis Múltiple/sangre , Esclerosis Múltiple/líquido cefalorraquídeo

RESUMEN

Analyses have shown that the repertoire of Ig heavy chain sequences (VH) expressed in multiple sclerosis (MS) plaques or cerebrospinal fluid is consistent with B cell clonal expansion and affinity maturation. PCR amplification of VH sequences from MS lesions obtained from an acute MS patient at autopsy revealed oligoclonal and extensively mutated VH sequences from plaque-periplaque regions with discrete intraclonal differences indicative of B cell clonal expansion in the groups of overrepresented major sequences. None of the VH sequences expressed in plaque regions were detected in peripheral blood lymphocytes from this patient. These data indicate the presence of a CNS-targeted antigen-driven response in MS plaques.

Asunto(s)

Autoanticuerpos/genética , Enfermedades Autoinmunes/inmunología , Subgrupos de Linfocitos B/inmunología , Encéfalo/inmunología , Reordenamiento Génico de Cadena Pesada de Linfocito B , Genes de Inmunoglobulinas , Inmunoglobulina G/análisis , Cadenas Pesadas de Inmunoglobulina/genética , Esclerosis Múltiple/inmunología , Secuencia de Aminoácidos , Enfermedades Autoinmunes/sangre , Enfermedades Autoinmunes/patología , Subgrupos de Linfocitos B/patología , Células Sanguíneas/inmunología , Encéfalo/patología , Células Clonales/inmunología , Células Clonales/patología , Análisis Mutacional de ADN , Humanos , Región Variable de Inmunoglobulina/genética , Datos de Secuencia Molecular , Esclerosis Múltiple/sangre , Esclerosis Múltiple/patología , Mutación Puntual , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico

RESUMEN

OBJECTIVE: To better understand B-cell activation in MS by analyzing the immunoglobulin (Ig)G heavy chain variable region (VH) repertoire found in MS brains and comparing it with brain VH sequences in individuals with subacute sclerosing panencephalitis (SSPE)--a chronic encephalitis produced by measles virus (MV)-and characterized by an antigen-driven oligoclonal IgG response to MV antigens. BACKGROUND: The specificity of oligoclonal IgG in MS CSF and plaques, and their relevance to the pathogenesis of MS is unknown. METHODS: Nested PCR was used to amplify and sequence the rearranged IgG heavy-chain VH repertoire in plaques of three acute MS brains and in three SSPE brains. A representative population of VH sequences from each tissue was aligned to the known 51 functional VH germline segments. From this the authors determined the closest VH family germline segment, and the degree and location of somatic mutations for each unique IgG. RESULTS: As expected for an antigen-driven response against MV antigens, most VH sequences from the SSPE brains were mutated extensively compared with their closest germline segments. Furthermore, SSPE VH sequences accumulated replacement mutations preferentially in the complementary-determining regions (CDRs) relative to framework regions-features normally observed during antigen-driven selection. A comparison of VH family and germline usage also demonstrated that each SSPE brain had its own unique IgG response. When the authors compared the VH response in MS plaques with SSPE, MS VH sequences were also mutated extensively, displayed a preferential accumulation of replacement mutations in CDRs, and were unique in each MS brain. CONCLUSION: The presence of an antigen-driven response in MS, rather than a nonconventional mechanism of B-cell activation, warrants additional analysis of the specificity of IgG in MS brain and CSF.

Asunto(s)

Encéfalo/inmunología , Encéfalo/patología , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Esclerosis Múltiple/genética , Panencefalitis Esclerosante Subaguda/genética , Adolescente , Adulto , Northern Blotting , Femenino , Biblioteca de Genes , Humanos , Masculino , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/patología , Sondas ARN , Panencefalitis Esclerosante Subaguda/inmunología , Panencefalitis Esclerosante Subaguda/patología

RESUMEN

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.

Asunto(s)

Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/aislamiento & purificación , Encéfalo/inmunología , Virus del Sarampión/inmunología , Panencefalitis Esclerosante Subaguda/inmunología , Especificidad de Anticuerpos , Bacteriófagos , Encéfalo/virología , Cápside/inmunología , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Biblioteca de Péptidos

RESUMEN

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.

Asunto(s)

Anticuerpos Antivirales/biosíntesis , Antígenos Virales/inmunología , Química Encefálica/genética , Química Encefálica/inmunología , Inmunoglobulina G/biosíntesis , Virus del Sarampión/inmunología , Panencefalitis Esclerosante Subaguda/genética , Panencefalitis Esclerosante Subaguda/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/química , Anticuerpos Antivirales/genética , Antígenos Virales/química , Chlorocebus aethiops , Clonación Molecular , Epítopos/química , Epítopos/inmunología , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/genética , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/biosíntesis , Cadenas kappa de Inmunoglobulina/química , Cadenas kappa de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/biosíntesis , Cadenas lambda de Inmunoglobulina/química , Cadenas lambda de Inmunoglobulina/genética , Datos de Secuencia Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Panencefalitis Esclerosante Subaguda/metabolismo , Transfección , Células Vero

RESUMEN

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.

Asunto(s)

Anticuerpos Antivirales/aislamiento & purificación , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/inmunología , Virus del Sarampión/inmunología , Panencefalitis Esclerosante Subaguda/inmunología , Adolescente , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Antígenos Virales/análisis , Antígenos Virales/aislamiento & purificación , Bacteriófagos , Encéfalo/inmunología , Encéfalo/virología , Clonación Molecular , ADN Complementario , Ensayo de Inmunoadsorción Enzimática , Epítopos , Biblioteca de Genes , Humanos , Immunoblotting , Fragmentos Fab de Inmunoglobulinas/análisis , Inmunoglobulina G/análisis , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Masculino , Pruebas de Precipitina

RESUMEN

Multiple sclerosis (MS) cerebrospinal fluid and brain contain increased IgG and oligoclonal bands. Whether this oligoclonal and polyclonal IgG is directed against a disease-relevant antigen remains unknown. To distinguish between random activation versus a targeted B-cell response, we analyzed the IgG heavy chain variable region (VH) repertoire expressed in different lesions of an acute MS brain. To obtain a representative sample of the VH repertoire, we constructed directional complementary DNA libraries from plaque-periplaque messenger RNA and amplified VH regions from the library by nested polymerase chain reaction. When MS VH sequences were aligned to germline segments, about 60% of different VH sequences in the acute MS brain were VH4 germline segments, significantly greater than the known approximately 20% VH4 germline prevalence. Specific VH sequences were overrepresented and expressed at multiple plaque sites. Within some overexpressed populations, there were distinct sequence differences (clonal variants) indicative of clonal expansion. Alignment of VH sequences to their closest germline counterparts revealed extensive somatic mutation and the preferential accumulation of amino acid replacement mutations in complementarity determining regions. These observations suggest the limited B-cell response found in this acute MS brain was antigen driven.

Asunto(s)

Química Encefálica , Mutación de Línea Germinal/genética , Inmunoglobulina G/genética , Esclerosis Múltiple/genética , Secuencia de Aminoácidos , ADN Complementario/análisis , Amplificación de Genes , Regulación de la Expresión Génica , Inmunoglobulina G/líquido cefalorraquídeo , Datos de Secuencia Molecular , Esclerosis Múltiple/líquido cefalorraquídeo , Esclerosis Múltiple/inmunología , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , ARN Mensajero/análisis , Valores de Referencia , Panencefalitis Esclerosante Subaguda/genética

RESUMEN

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.

Asunto(s)

Anticuerpos Antivirales/aislamiento & purificación , Química Encefálica/inmunología , Inmunoglobulina G/aislamiento & purificación , Esclerosis Múltiple/inmunología , Panencefalitis Esclerosante Subaguda/inmunología , Animales , Anticuerpos Antivirales/metabolismo , Línea Celular , Membrana Celular/inmunología , Chlorocebus aethiops , Colodión , Electroforesis en Gel de Poliacrilamida , Humanos , Immunoblotting , Inmunoglobulina G/metabolismo , Focalización Isoeléctrica , Riñón/citología , Virus del Sarampión/inmunología , Proteínas de la Nucleocápside/inmunología , Desnaturalización Proteica , Solubilidad

RESUMEN

Programmed cell death is an important process in many types of cell. In the central nervous system of vertebrates, up to 50% of neurons die during development. The fact that in many cases neuronal cell death depends on macromolecular synthesis suggests that there is a genetic program that must be activated for cell death to occur. In this review, general features of cell death are discussed; in addition, the role of putative death-associated genes is outlined. Finally, the influence of neurotrophic factors on cell death is described.

Asunto(s)

Apoptosis/fisiología , Factores de Crecimiento Nervioso/fisiología , Sistema Nervioso/citología , Vertebrados/anatomía & histología , Animales , Regulación de la Expresión Génica/fisiología , Neuronas/citología

RESUMEN

Chronic inflammatory and infectious diseases of the central nervous system (CNS) are characterized by increased IgG and oligoclonal bands (OGBs) in the brain and cerebrospinal fluid (CSF). The OGBs in CNS infectious diseases of known cause have been shown to be directed against the pathogenic agent. In multiple sclerosis (MS), the antigenic specificity of the OGBs is unknown, but could be directed against an infectious agent, an autoantigen, or both. In a molecular approach to identify antigens specific for MS, we constructed directional cDNA expression libraries with mRNA extracted from chronic and acute MS plaques and periplaque white matter. The libraries were: (1) screened to identify clones whose expression products react with MS CSF, but not with CSF from other infectious and inflammatory diseases of the CNS; (2) subtracted by hybridization to mRNA from normal human brain white matter and differentially screened to detect unique MS transcripts; and (3) used as template in polymerase chain reactions to amplify, clone, and sequence IgG heavy and light chain variable regions (VH and VL, respectively) expressed in MS plaques. Analysis of the VH and VL IgG repertoire in MS brain may identify disease-relevant IgG sequences that can be assembled into functional antibodies using recombinant phage technology. Such recombinant antibodies will be useful to probe brain tissue to identify antigens unique to MS.

Asunto(s)

Inmunoglobulina G/genética , Esclerosis Múltiple/inmunología , Análisis de Secuencia de ADN , ADN Complementario/aislamiento & purificación , Epítopos , Biblioteca de Genes , Humanos

RESUMEN

Plaque-periplaque areas from MS brain tissue were explanted and propagated in tissue culture. The same in vitro techniques that successfully rescued measles virus from SSPE brain, papovavirus from PML brain, and HSV from normal human trigeminal ganglia, were applied. MS brain cells were also inoculated into chimpanzees, multiple rodent species, and embryonated hens eggs. No neurologic disease developed in experimentally infected animals, and no cytopathic effect was observed in explanted cells, or after cocultivation or fusion of MS brain cells with indicator cells. Further analysis of explanted and cocultivated cells by indirect immunofluorescence with various antiviral antisera prepared against viruses associated with post-infectious encephalomyelitis, as well as antisera to other ubiquitous viruses, failed to detect viral antigen. Finally, attempts to detect a latent enveloped virus in MS brain cells by 'superinfecting' MS brain cells in culture with vesicular stomatitis virus (VSV) did not reveal a VSV non-neutralizable fraction. Nevertheless, since oligoclonal bands (OGBs) in the CSF of patients with chronic infectious diseases of the CNS are directed against the causative agent, it is likely that OGBs in MS CSF are antibody directed against the agent or antigen that triggered disease. Although the relevant antibody may be scarce relative to irrelevant antibody in MS CSF, and only small amounts of an MS-specific antigen may be present in brain, this report provides a rationale for strategies proposed in our companion report by Owens et al which will allow detection of an MS-specific antigen or its cognate RNA in brain.

Asunto(s)

Encéfalo/virología , Esclerosis Múltiple/virología , Humanos , Inmunoglobulina G/líquido cefalorraquídeo , Infecciones/líquido cefalorraquídeo , Esclerosis Múltiple/epidemiología , Sistema Nervioso/virología , Prevalencia

RESUMEN

RP-8 is one of several mRNAs elevated during apoptosis in immature thymocytes. We used in situ hybridization to look for RP-8 mRNA in the cerebella of weaver mice. In the weaver mouse cerebellar granule cells fail to differentiate and instead die during the first two weeks of postnatal development [30]. We demonstrate that the dying cells exhibit DNA degradation, a characteristic feature of apoptosis. RP-8 is expressed in the weaver cerebellum during the period of granule cell death, and is limited to cells in the same region where apoptotic granule cells are located. Thus, RP-8 expression, first associated with PCD in thymocytes, correlates with granule cell death in the weaver cerebellum.

Asunto(s)

Apoptosis/fisiología , Cerebelo/fisiología , Expresión Génica , Neuronas/fisiología , Animales , Animales Recién Nacidos , Secuencia de Bases , Corteza Cerebelosa/citología , Corteza Cerebelosa/metabolismo , Cerebelo/citología , Genes , Hibridación in Situ , Ratones , Ratones Mutantes Neurológicos , Datos de Secuencia Molecular , Sondas de Oligonucleótidos/genética , ARN Mensajero/metabolismo

RESUMEN

Intracerebral transplants of ventral mesencephalic (VM) tissue have been well characterized. VM grafts contain numerous tyrosine hydroxylase immunoreactive neurons which send axons into the host brain. Transplanted neurons in VM grafts develop normally in that they contain tyrosine hydroxylase and GAP43. An overlooked aspect of graft development is cell death. It has been suggested that cell death in VM grafts was mostly necrotic. However, recent work in this laboratory suggested that developing grafts contain numerous apoptotic cells. In the present paper morphological, histochemical, and molecular correlates of apoptosis were used to assay cell death during VM graft development. At early times (5-15 days) after grafting VM grafts contained numerous apoptotic cells. In older grafts (21 and 28 days) few apoptotic cells were observed. In situ end labeling of fragmented DNA with biotinylated dUTP showed that early grafts contained numerous positive cells. The expression of RP8, a molecular correlate of apoptotic cell death, occurred in early grafts, but was not detectable in older grafts. These results indicate that apoptosis is a normal part of VM graft development. As in naturally developing neural systems, cell death in grafts may function to eliminate cells that fail to connect to appropriate targets.

Asunto(s)

Apoptosis , Trasplante de Tejido Encefálico/fisiología , Trasplante de Tejido Fetal/fisiología , Neuronas/fisiología , Sustancia Negra/fisiología , Sustancia Negra/trasplante , Animales , Elementos sin Sentido (Genética) , Secuencia de Bases , Corteza Cerebral/citología , Corteza Cerebral/fisiología , Femenino , Proteína GAP-43 , Sustancias de Crecimiento/análisis , Sustancias de Crecimiento/metabolismo , Hibridación in Situ , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/metabolismo , Mesencéfalo/embriología , Mesencéfalo/trasplante , Datos de Secuencia Molecular , Degeneración Nerviosa , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Ratas , Ratas Sprague-Dawley , Sustancia Negra/citología , Tirosina 3-Monooxigenasa/análisis , Tirosina 3-Monooxigenasa/metabolismo