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1.
J Allergy Clin Immunol ; 133(4): 1116-23, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24332219

ABSTRACT

BACKGROUND: Recombination-activating gene 1 (RAG1) deficiency results in severe combined immunodeficiency (SCID) caused by a complete lack of T and B lymphocytes. If untreated, patients succumb to recurrent infections. OBJECTIVES: We sought to develop lentiviral gene therapy for RAG1-induced SCID and to test its safety. METHODS: Constructs containing the viral spleen-focus-forming virus (SF), ubiquitous promoters, or cell type-restricted promoters driving sequence-optimized RAG1 were compared for efficacy and safety in sublethally preconditioned Rag1(-/-) mice undergoing transplantation with transduced bone marrow progenitors. RESULTS: Peripheral blood CD3(+) T-cell reconstitution was achieved with SF, ubiquitous promoters, and cell type-restricted promoters but 3- to 18-fold lower than that seen in wild-type mice, and with a compromised CD4(+)/CD8(+) ratio. Mitogen-mediated T-cell responses and T cell-dependent and T cell-independent B-cell responses were not restored, and T-cell receptor patterns were skewed. Reconstitution of mature peripheral blood B cells was approximately 20-fold less for the SF vector than in wild-type mice and often not detectable with the other promoters, and plasma immunoglobulin levels were abnormal. Two months after transplantation, gene therapy-treated mice had rashes with cellular tissue infiltrates, activated peripheral blood CD44(+)CD69(+) T cells, high plasma IgE levels, antibodies against double-stranded DNA, and increased B cell-activating factor levels. Only rather high SF vector copy numbers could boost T- and B-cell reconstitution, but mRNA expression levels during T- and B-cell progenitor stages consistently remained less than wild-type levels. CONCLUSIONS: These results underline that further development is required for improved expression to successfully treat patients with RAG1-induced SCID while maintaining low vector copy numbers and minimizing potential risks, including autoimmune reactions resembling Omenn syndrome.


Subject(s)
Genetic Therapy , Genetic Vectors/genetics , Homeodomain Proteins/genetics , Lentivirus/genetics , Severe Combined Immunodeficiency/genetics , Severe Combined Immunodeficiency/therapy , Animals , Autoimmunity/genetics , Bone Marrow Cells/metabolism , Disease Models, Animal , Female , Gene Dosage , Gene Expression , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/metabolism , Humans , Immunophenotyping , Male , Mice , Mice, Knockout , Phenotype , Severe Combined Immunodeficiency/immunology , Spleen/immunology , T-Lymphocytes/metabolism , Thymus Gland/immunology , Transduction, Genetic , Transplantation Chimera
2.
J Gen Virol ; 93(Pt 12): 2652-2657, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22971823

ABSTRACT

Bats are the natural reservoir of a variety of viruses, including a polyomavirus (PyV) from a North American brown bat. We investigated 163 spleen samples from 22 bat species from French Guiana for the presence of PyVs. In total, we detected 25 PyV-positive animals belonging to nine different bat species. Phylogenetic analysis was performed on the genomes of eight representative PyVs, and showed that the bat PyVs form three distinct lineages within the genus Orthopolyomavirus and are genetically different from the previously described North American bat virus. Interestingly, two lineages cluster with PyVs found in chimpanzees, orangutans and gorillas. In addition, one group of bat PyVs is genetically related to the human Merkel cell polyomavirus.


Subject(s)
Chiroptera/virology , Polyomaviridae/genetics , Polyomaviridae/isolation & purification , Polyomavirus/genetics , Polyomavirus/isolation & purification , Animals , Disease Reservoirs/veterinary , Disease Reservoirs/virology , French Guiana , Genome, Viral , Gorilla gorilla/virology , Humans , Merkel cell polyomavirus/classification , Merkel cell polyomavirus/genetics , Molecular Sequence Data , Pan troglodytes/virology , Phylogeny , Polyomaviridae/classification , Polyomavirus/classification , Pongo/virology , South America , Species Specificity
3.
Breast Cancer Res Treat ; 114(1): 23-30, 2009 Mar.
Article in English | MEDLINE | ID: mdl-18351453

ABSTRACT

Endocrine treatment of breast cancer is widely applied and effective. However, in advanced disease cases, the tumors will eventually progress into an estrogen-independent and therapy-resistant phenotype. To elucidate the molecular mechanisms underlying this endocrine therapy failure, we applied retroviral insertion mutagenesis to identify the main genes conferring estrogen independence to human breast cancer cells. Estrogen-dependent ZR-75-1 cells were infected with replication-defective retroviruses followed by selection with the anti-estrogen 4-hydroxy-tamoxifen. In the resulting panel of 79 tamoxifen-resistant cell lines, the viral integrations were mapped within the human genome. Genes located in the immediate proximity of the retroviral integration sites were characterized for altered expression and their capacity to confer anti-estrogen resistance when transfected into breast cancer cells. Out of 15 candidate BCAR (breast cancer anti-estrogen resistance) genes, seven (AKT1, AKT2, BCAR1, BCAR3, EGFR, GRB7, and TRERF1/BCAR2) were shown to directly underlie estrogen independence. Our results show that insertion mutagenesis is a powerful tool to identify BCAR loci, which may provide insights into the molecular and cellular mechanisms of breast tumor progression and therapy resistance thereby offering novel targets for the development of tailor-made therapeutical and prevention strategies.


Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/physiopathology , Estrogens/physiology , Cell Line, Tumor , Estrogens/genetics , Female , Humans , Mutagenesis, Insertional , Retroviridae , Virus Integration
4.
Endocr Relat Cancer ; 17(1): 215-30, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19966015

ABSTRACT

Although endocrine treatment of breast cancer is effective and common practice, in advanced disease the development of resistance is nearly inevitable. To get more insight into individual genes that account for resistance against hormonal agents, we have executed functional genetic screens and subsequently evaluated the clinical relevance of several identified genes with respect to tumor aggressiveness and tamoxifen resistance in estrogen receptor-positive patients. Estrogen-dependent human breast cancer cells were transduced with different retroviral cDNA expression libraries and subjected to selective cultures with various anti-estrogens. From a total of 264 resistant cell clones, 132 different genes were recovered by PCR. By applying stringent selection criteria, we identified 15 breast cancer anti-estrogen resistance (BCAR) genes individually yielding resistance. BCAR genes were recovered with differential frequencies for the diverse culture conditions and anti-estrogen drugs. Analysis of the relation of BCAR genes (EIF1, FBXL10, HRAS, NRG1, PDGFRA, PDGFRB, RAD21, and RAF1) with tamoxifen treatment in patients with advanced disease showed significant association with clinical benefit and progression-free survival for EIF1 and PDGFRA mRNA levels. Furthermore, PDGFRA and HRAS mRNA levels were significantly associated with tumor aggressiveness in lymph node-negative patients who had not received adjuvant systemic therapy. In conclusion, our functional genetic screens showed that BCAR genes differ in their ability to confer resistance towards distinct anti-estrogens. Based on the clinical relevance of several BCAR genes, further studies are warranted to characterize the underlying mechanisms, which may ultimately lead to the development of novel treatments and more individualized management of breast cancer patients.


Subject(s)
Biomarkers, Pharmacological , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Drug Resistance, Neoplasm/genetics , Estrogen Antagonists/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Tamoxifen/therapeutic use , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adult , Aged , Aged, 80 and over , Animals , Antineoplastic Agents, Hormonal/pharmacology , Biomarkers, Pharmacological/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Crk-Associated Substrate Protein/genetics , Crk-Associated Substrate Protein/metabolism , Disease Progression , Drug Resistance, Neoplasm/drug effects , Estrogen Antagonists/metabolism , Female , Genetic Testing , Guanine Nucleotide Exchange Factors , HeLa Cells , Humans , Mice , Middle Aged , Neoplasm Invasiveness , Retrospective Studies , Substrate Specificity
5.
Infect Immun ; 73(11): 7252-8, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16239520

ABSTRACT

The NikR protein is a nickel-dependent regulatory protein which is a member of the ribbon-helix-helix family of transcriptional regulators. The gastric pathogen Helicobacter pylori expresses a NikR ortholog, which was previously shown to mediate regulation of metal metabolism and urease expression, but the mechanism governing the diverse regulatory effects had not been described until now. In this study it is demonstrated that NikR can regulate H. pylori nickel metabolism by directly controlling transcriptional repression of NixA-mediated nickel uptake and transcriptional induction of urease expression. Mutation of the nickel uptake gene nixA in an H. pylori 26695 nikR mutant restored the ability to grow in Brucella media supplemented with 200 microM NiCl2 but did not restore nickel-dependent induction of urease expression. Nickel-dependent binding of NikR to the promoter of the nixA gene resulted in nickel-repressed transcription, whereas nickel-dependent binding of NikR to the promoter of the ureA gene resulted in nickel-induced transcription. Subsequent analysis of NikR binding to the nixA and ureA promoters showed that the regulatory effect was dependent on the location of the NikR-recognized binding sequence. NikR recognized the region from -13 to +21 of the nixA promoter, encompassing the +1 and -10 region, and this binding resulted in repression of nixA transcription. In contrast, NikR bound to the region from -56 to -91 upstream of the ureA promoter, resulting in induction of urease transcription. In conclusion, the NikR protein is able to function both as a repressor and as an activator of gene transcription, depending on the position of the binding site.


Subject(s)
Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial/drug effects , Helicobacter pylori/genetics , Nickel/pharmacology , Repressor Proteins/metabolism , Transcription, Genetic/drug effects , Bacterial Proteins/genetics , Base Sequence , Cation Transport Proteins/genetics , Gene Expression Regulation, Bacterial/genetics , Helicobacter pylori/drug effects , Helicobacter pylori/enzymology , Molecular Sequence Data , Operator Regions, Genetic/genetics , Promoter Regions, Genetic/genetics , Protein Binding , Transcription, Genetic/genetics , Urease/genetics
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