title: Immune and Epstein-Barr virus gene expression in cerebrospinal fluid and peripheral blood mononuclear cells from patients with relapsing-remitting multiple sclerosis.
abstract: BACKGROUND
  Gene expression analyses in paired cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMC) from patients with multiple sclerosis (MS) are restrained by the low RNA amounts from CSF cells and low expression levels of certain genes. Here, we applied a Taqman-based pre-amplification real-time reverse-transcription polymerase chain reaction (RT-PCR) (PreAmp RT-PCR) to cDNA from CSF cells and PBMC of MS patients and analyzed multiple genes related to immune system function and genes expressed by Epstein-Barr virus (EBV), a herpesvirus showing strong association with MS. Using this enhanced RT-PCR method, we aimed at the following: (1) identifying gene signatures potentially useful for patient stratification, (2) understanding whether EBV infection is perturbed in CSF and/or blood, and (3) finding a link between immune and EBV infection status.
  
  
  METHODS
  Thirty-one therapy-free patients with relapsing-remitting MS were included in the study. Paired CSF cells and PBMC were collected and expression of 41 immune-related cellular genes and 7 EBV genes associated with latent or lytic viral infection were determined by PreAmp RT-PCR. Clinical, radiological, CSF, and gene expression data were analyzed using univariate and multivariate (cluster analysis, factor analysis) statistical approaches.
  
  
  RESULTS
  Several immune-related genes were differentially expressed between CSF cells and PBMC from the whole MS cohort. By univariate analysis, no or only minor differences in gene expression were found associated with sex, clinical, or radiological condition. Cluster analysis on CSF gene expression data grouped patients into three clusters; clusters 1 and 2 differed by expression of genes that are related mainly to innate immunity, irrespective of sex and disease characteristics. By factor analysis, two factors grouping genes involved in antiviral immunity and immune regulation, respectively, accurately discriminated cluster 1 and cluster 2 patients. Despite the use of an enhanced RT-PCR method, EBV transcripts were detected in a minority of patients (5 of 31), with evidence of viral latency activation in CSF cells or PBMC and of lytic infection in one patient with active disease only.
  
  
  CONCLUSIONS
  Analysis of multiple cellular and EBV genes in paired CSF cell and PBMC samples using PreAmp RT-PCR may yield new information on the complex interplay between biological processes underlying MS and help in biomarker identification.
authors.affiliation: Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy. caterina.veroni@iss.it.
authors.forename: Caterina
authors.initials: C
authors.lastname: Veroni
authors.affiliation: Neurology 2-CRESM (Multiple Sclerosis Regional Reference Center), AOU San Luigi Gonzaga, Regione Gonzole 10, 10043, Orbassano, Italy. fabiana.marnetto@gmail.com.
authors.forename: Fabiana
authors.initials: F
authors.lastname: Marnetto
authors.affiliation: Neurology 2-CRESM (Multiple Sclerosis Regional Reference Center), AOU San Luigi Gonzaga, Regione Gonzole 10, 10043, Orbassano, Italy. letiziagranieri@libero.it.
authors.forename: Letizia
authors.initials: L
authors.lastname: Granieri
authors.affiliation: Neurology 2-CRESM (Multiple Sclerosis Regional Reference Center), AOU San Luigi Gonzaga, Regione Gonzole 10, 10043, Orbassano, Italy. antonio.bertolotto@gmail.com.
authors.forename: Antonio
authors.initials: A
authors.lastname: Bertolotto
authors.affiliation: Department of Neuroscience, Drug and Child Health (NEUROFARBA), University of Florence, Viale Pieraccini 6, 50137, Florence, Italy. clara.ballerini@unifi.it.
authors.forename: Clara
authors.initials: C
authors.lastname: Ballerini
authors.affiliation: Multiple Sclerosis Center, Neurology 2 Division, Careggi University Hospital, University of Florence, Viale Morgagni 85, 50134, Florence, Italy. repicea@gmail.com.
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Repice
authors.affiliation: Department of Public Health, Clinical and Molecular Medicine, Multiple Sclerosis Center, University of Cagliari, Via Is Guadazzonis, 2, 09126, Cagliari, Italy. lucia_schirru@tiscali.it.
authors.forename: Lucia
authors.initials: L
authors.lastname: Schirru
authors.affiliation: Department of Public Health, Clinical and Molecular Medicine, Multiple Sclerosis Center, University of Cagliari, Via Is Guadazzonis, 2, 09126, Cagliari, Italy. gccoghe@gmail.com.
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Coghe
authors.affiliation: Department of Public Health, Clinical and Molecular Medicine, Multiple Sclerosis Center, University of Cagliari, Via Is Guadazzonis, 2, 09126, Cagliari, Italy. ecocco@unica.it.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Cocco
authors.affiliation: Department of Experimental Medicine, La Sapienza University, Viale Regina Elena 324, 00161, Rome, Italy. elenasta@gmail.com.|Department of Pathology, Beth Israel Deaconess Medical Center/Harvard Medical School, 330 Brookline Avenue, Boston, MA, 02215, USA. elenasta@gmail.com.
authors.forename: Eleni
authors.initials: E
authors.lastname: Anastasiadou
authors.affiliation: Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy. maria.puopolo@iss.it.
authors.forename: Maria
authors.initials: M
authors.lastname: Puopolo
authors.affiliation: Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy. francesca.aloisi@iss.it.
authors.forename: Francesca
authors.initials: F
authors.lastname: Aloisi
mesh_terms: D000328:Adult; D000368:Aged; D002555:Cerebrospinal Fluid / Q000166:cytology* / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D005814:Genes, Viral / Q000235:genetics; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000378:metabolism; D000949:Histocompatibility Antigens Class II / Q000097:blood / Q000134:cerebrospinal fluid / Q000235:genetics*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D020529:Multiple Sclerosis, Relapsing-Remitting / Q000097:blood / Q000134:cerebrospinal fluid / Q000235:genetics*; D015999:Multivariate Analysis; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results
chemical_list: D000949:Histocompatibility Antigens Class II