ABSTRACT
The identification of mutations in the transcriptional repressor methyl-CpG-binding protein 2 (MECP2) gene in Rett Syndrome (RTT) suggests that an inappropriate release of transcriptional silencing may give rise to RTT neuropathology. Despite this progress, the molecular basis of RTT neuropathogenesis remains unclear. Using multiple cDNA microarray technologies, subtractive hybridization, and conventional biochemistry, we generated comprehensive gene expression profiles of postmortem brain tissue from RTT patients and matched controls. Many glial transcripts involved in known neuropathological mechanisms were found to have increased expression in RTT brain, while decreases were observed in the expression of multiple neuron-specific mRNAs. Dramatic and consistent decreases in transcripts encoding presynaptic markers indicated a specific deficit in presynaptic development. Employing multiple clustering algorithms, it was possible to accurately segregate RTT from control brain tissue samples based solely on gene expression profile. Although previously achieved in cancers, our results constitute the first report of human disease classification using gene expression profiling in a complex tissue source such as brain.
Subject(s)
Brain/enzymology , Chromosomal Proteins, Non-Histone , DNA-Binding Proteins/biosynthesis , Gene Expression Profiling , Nerve Tissue Proteins/biosynthesis , Neurons/metabolism , Postmortem Changes , Repressor Proteins , Rett Syndrome/genetics , Adolescent , Adult , Algorithms , Amino Acid Sequence , Blotting, Southern , Cause of Death , Child , Child, Preschool , DNA-Binding Proteins/genetics , Dosage Compensation, Genetic , Female , Humans , Methyl-CpG-Binding Protein 2 , Molecular Sequence Data , Nerve Tissue Proteins/genetics , Neuroglia/metabolism , Oligonucleotide Array Sequence Analysis , RNA, Messenger/biosynthesis , Rett Syndrome/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Subtraction TechniqueABSTRACT
We have succeeded in stably maintaining the entire genome of SIVmac239 as a plasmid clone. Supercoiled proviral plasmid DNA was inoculated intramuscularly into two adult rhesus macaques and into a neonate. All three animals became viremic and seroconverted. Viral kinetics were followed prospectively by quantitative competitive reverse transcriptase polymerase chain reaction (QC-RT-PCR), measurement of proviral DNA load in peripheral blood mononuclear cells (PBMCs) by PCR, and virus isolation by cocultivation. The infant developed high virus loads and succumbed to AIDS and SIV-associated nephropathy at 10 weeks postinoculation. Both adults are still living but have progressed to AIDS; one adult has also developed severe thrombocytopenia. We conclude that infection through intramuscular inoculation of cloned plasmid DNA encoding the entire proviral genome is reproducible and will provide a useful tool for studying viral pathogenesis.
Subject(s)
Plasmids/genetics , Simian Acquired Immunodeficiency Syndrome/physiopathology , Simian Immunodeficiency Virus/pathogenicity , Viremia/virology , Animals , Animals, Newborn , DNA, Viral/blood , Macaca mulatta , Proviruses , Reverse Transcriptase Polymerase Chain Reaction , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/genetics , T-Lymphocyte Subsets/immunology , Viral LoadABSTRACT
A substantial risk in using live attenuated, multiply deleted viruses as vaccines against AIDS is their potential to induce AIDS. A mutant of the simian immunodeficiency virus (SIV) with large deletions in nef and vpr and in the negative regulatory element induced AIDS in six of eight infant macaques vaccinated orally or intravenously. Early signs of immune dysfunction were seen in the remaining two offspring. Prolonged follow-up of sixteen vaccinated adult macaques also showed resurgence of chronic viremia in four animals: two of these developed early signs of disease and one died of AIDS. We conclude that this multiply deleted SIV is pathogenic and that human AIDS vaccines built on similar prototypes may cause AIDS.
Subject(s)
Aging/immunology , SAIDS Vaccines , Simian Acquired Immunodeficiency Syndrome/prevention & control , Simian Acquired Immunodeficiency Syndrome/virology , Vaccines, Attenuated/immunology , Amniotic Fluid/virology , Animals , Disease Progression , Female , Gene Products, nef/genetics , Gene Products, vpr/genetics , Immunity, Mucosal , Macaca mulatta , Male , Molecular Sequence Data , Pregnancy , Pregnancy Complications, Infectious/immunology , Pregnancy Complications, Infectious/prevention & control , SAIDS Vaccines/immunology , Sequence Deletion , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Immunodeficiency Virus/immunologyABSTRACT
We report the complete nucleotide sequence of the genome of Rauscher murine leukemia virus (R-MuLV), the replication-competent helper virus present in the Rauscher virus complex, and its phylogenetic relationship with other murine leukemia virus genomes. An overall sequence identity of 97.6% was found between R-MuLV and the Friend helper virus (F-MuLV), and the two viruses were closely related on the phylogenetic trees constructed from either gag, pol, or env sequences. Moloney murine leukemia virus (Mo-MuLV) was the next closest relative to R-MuLV and F-MuLV on all trees, followed by Akv and radiation leukemia virus (RadLV). The most distantly related helper virus was Hortulanus murine leukemia virus (Ho-MuLV). Interestingly, Cas-Br-E branched with Mo-MuLV on the gag and pol trees, whereas on the env tree, it revealed the highest degree of relatedness to Ho-MuLV, possibly due to an ancient recombination with an Ho-MuLV ancestor. In summary, a phylogenetic analysis involving various MuLVs has been performed, in which the postulated close relationship between R-MuLV and F-MuLV has been confirmed, consistent with the pathobiology of the two viruses.
