Subject(s)
Hamartoma Syndrome, Multiple/complications , Hamartoma Syndrome, Multiple/genetics , PTEN Phosphohydrolase/genetics , Psychotic Disorders/genetics , Adult , Brain Neoplasms/complications , Brain Neoplasms/pathology , Brain Neoplasms/surgery , Female , Ganglioneuroma/complications , Ganglioneuroma/pathology , Ganglioneuroma/surgery , Germ-Line Mutation , Hamartoma Syndrome, Multiple/psychology , Humans , Magnetic Resonance ImagingABSTRACT
Peripheral nerve sheath tumours are hallmarks of neurofibromatosis type 1 (NF1). Development of plexiform neurofibromas to malignant peripheral nerve sheath tumours (MPNST) is common. The NF1 gene promoter harbours a hypomethylated CpG island. Thus, methylation changes may be involved in the development of different types of neurofibromas and malignant transformation. We investigated NF1-associated dermal (n=9) and plexiform neurofibromas (n=7), MPNST (n=5) and non-NF1 leucocyte samples (n=20) for their methylation pattern by bisulphite genomic sequencing. We could not find global hypermethylation in the NF1 promoter in our series. Nevertheless, site-specific methylation, involving transcription factor binding sites for SP1, CRE (-10), and AP-2, was observed. One region of the 5'-UTR (untranslated region) overlapping with a putative AP-2 binding site was methylated at 30-100% in 4/20 control samples. In conclusion, we did not find hypermethylation in NF1-associated tumours. Instead, low level methylation could parallel a global genomic hypomethylation in malignancy.
Subject(s)
Nerve Sheath Neoplasms/metabolism , Neurofibromatosis 1/metabolism , Promoter Regions, Genetic/genetics , Antioxidants/metabolism , Clone Cells , DNA Methylation , DNA, Neoplasm/metabolism , Female , Humans , Leukocytes/metabolism , Loss of Heterozygosity , Male , Molecular Sequence Data , Nerve Sheath Neoplasms/genetics , Neurofibromatosis 1/genetics , Polymerase Chain Reaction/methods , Sulfites/metabolism , Transcription, GeneticABSTRACT
Recently, combinations of antiretroviral drugs (highly active antiretroviral therapy [HAART]) have led to a dramatic reduction of human immunodeficiency virus type 1 (HIV-1)-related clinical symptoms. Success of treatment is defined as almost complete suppression of plasma viremia, although in a sizable fraction of patients this goal is not achieved. We characterized primary HIV-1 isolates from 2 cohorts of patients in which HAART failed in terms of viral suppression. One cohort showed clinical benefit and stable or increasing CD4+ T cell numbers despite high viral load. The second viremic cohort had no CD4+ T cell recovery and exhibited typical AIDS-related symptoms. Primary isolates from HAART patients with minor clinical symptoms used CXCR4 as the most relevant receptor on primary cells. Thus, for the first time, it is shown that patients improving clinically under HAART harbor relatively high viral loads with viruses preferring CXCR4 as coreceptor.
Subject(s)
Anti-HIV Agents/therapeutic use , HIV Infections/drug therapy , HIV-1/physiology , Receptors, HIV/metabolism , Amino Acid Sequence , Antibodies/immunology , CD4 Lymphocyte Count , CD4-Positive T-Lymphocytes/immunology , Chemokine CXCL12 , Chemokines, CXC/therapeutic use , Cohort Studies , Drug Therapy, Combination , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/genetics , HIV Infections/virology , HIV-1/genetics , HIV-1/isolation & purification , Humans , Molecular Sequence Data , Neutralization Tests , Peptide Fragments/chemistry , Peptide Fragments/genetics , Receptors, CXCR4/genetics , Receptors, CXCR4/metabolism , Receptors, Chemokine/genetics , Receptors, Chemokine/metabolism , Receptors, HIV/genetics , Sequence Analysis, DNA , Viral Load , Viremia/drug therapy , Viremia/virologyABSTRACT
To generate T cell-specific retroviral vectors an scFv phage display library derived from immunized mice was selected for binding to the human T cell line Molt-4/8. The scFv cDNAs recovered from the selected phages were transiently expressed as an N-terminal fusion of the spleen necrosis virus (SNV) transmembrane protein (TM) subunit of the viral envelope protein (Env) in the cell line DSH-cxl, which packages the beta-galactosidase gene into SNV particles. Screening of supernatants from about 150 transfections resulted in the identification of 5 scFvs that mediated efficient transduction of Molt-4/8 cells. Using stable packaging cell lines vector preparations with titers greater than 10(4) EFU/ml on human T cells were obtained. The scFv 7A5 in particular was able to mediate selective transduction of human T cells with high efficiency. Titers of up to 106 EFU/ml were reached on Molt-4/8, Jurkat, and A301 cells, while titers on HeLa cells, TE671 cells, 293T cells, and HT1080 cells were below 102 EFU/ml. Transduction of stimulated primary human peripheral blood cells, which consisted mainly of T cells, was about fivefold more efficient than transduction of B cells. Western blot analysis of supernatant from the 7A5 packaging cells demonstrated incorporation of 7A5-TM into vector particles and indicated proteolytic processing of the coexpressed unmodified TM during particle formation. Binding of bacterially expressed 7A5-scFv to a panel of cell lines correlated well with the transduction results. These data provide the first proof of concept that a general approach can be taken to obtain scFvs able to mediate selective gene transfer into target cells.
