Drabek, D.D.

Heavy chain-only antibodies and tetravalent bispecific antibody neutralizing Staphylococcus aureus leukotoxins (Article)

2011-09-27T00:00:00Z

Panton-Valentine leukocidin (PVL) is a pore-forming toxin associated with current outbreaks of community-associated methicillin-resistant strains and implicated directly in the pathophysiology of Staphylococcus aureus-related diseases. Humanized heavy chain-only antibodies (HCAb) were generated against S. aureus PVL from immunized transgenicmice to neutralize toxin activity. The active form of PVL consists of the two components, LukS-PV and LukF-PV, which induce osmotic lysis following pore formation in host defense cells. One anti-LukS-PV HCAb, three anti-LukF-PV HCAbs with affinities in the nanomolar range, and one engineered tetravalent bispecific HCAb were tested in vitro and in vivo, and all prevented toxin binding and pore formation. Anti-LukS-PV HCAb also binds to γ-hemolysin C (HlgC) and inhibits HlgC/HlgB pore formation. Experiments in vivo in a toxin-induced rabbit endophthalmitis model showed that these HCAbs inhibit inflammatory reactions and tissue destruction, with the tetravalent bispecific HCAb performing best. Our findings show the therapeutic potential of HCAbs, and in particular, bispecific antibodies.

A role for PML in innate immunity (Article)

2011-07-12T00:00:00Z

The promyelocytic leukemia gene (PML) of acute promyelocytic leukemia is an established tumor suppressor gene with critical functions in growth suppression, induction of apoptosis, and cellular senescence. Interestingly, although less studied, PML seems to play a key role also in immune response to viral infection. Herein, we report that Pml-/-mice spontaneously develop an atypical invasive and lethal granulomatous lesion known as botryomycosis (BTM). In Pml-/-mice, BTM is the result of impaired function of macrophages, whereby they fail to become activated and are thus unable to clear pathogenic microorganisms. Accordingly, Pml-/-mice are resistant to lipopolysaccharide (LPS)-induced septic shock as a result of an ineffective production of cytokines and chemokines, suggesting a role for PML in the innate immune Toll-like receptor (TLR)/NF-κB prosurvival pathway. These results not only shed light on a new fundamental function of PML in innate immunity, but they also point to a proto-oncogenic role for PML in certain cellular and pathological contexts.

Tagged mutagenesis by efficient minos-based germ line transposition (Article)

2010-01-01T00:00:00Z

Germ line gene transposition technology has been used to generate "libraries" of flies and worms carrying genomewide mutations. Phenotypic screening and DNA sequencing of such libraries provide functional information resulting from insertional events in target genes. There is also a great need to have a fast and efficient way to generate mouse mutants in vivo to model developmental defects and human diseases. Here we describe an optimized mammalian germ line transposition system active during early mouse spermatogenesis using the Minos transposon. Transposon-positive progeny carry on average more than 2 new transpositions, and 45 to 100% of the progeny carry an insertion in a gene. The optimized Minos-based system was tested in a small rapid dominant functional screen to identify mutated genes likely to cause measurable cardiovascular "disease" phenotypes in progeny/embryos. Importantly this system allows rapid screening for modifier genes. Copyright

Stable expression of human growth hormone over 50 generations in transgenic insect larvae (Article)

2007-02-01T00:00:00Z

Developments in insect transgenesis using transposons combined with available mass rearing technology for insects such as the Medfly, Ceratitis capitata, provide opportunity for the production of protein for industrial, agricultural and healthcare purposes on a very large scale. In this study, we report the germ-line transformation and expression of a cDNA encoding human growth hormone (hGH) in transgenic Drosophila using the Minos transposon. Production and secretion of a bioactive hGH into the haemolymph of transgenic larvae was demonstrated by immunoblot analysis, ELISA and a proliferation bioassay. Stable expression of hGH was observed over 50 generations. The results indicate that mass reared transgenic diptera with a rapid period of larval growth could provide cost effective production systems for the manufacture of therapeutic and other high value proteins.

Generation of heavy-chain-only antibodies in mice. (Article)

2006-10-10T00:00:00Z

We have generated transgenic mice containing hybrid llama/human antibody loci that contain two llama variable regions and the human D, J, and Cmu and/or Cgamma constant regions. Such loci rearrange productively and rescue B cell development efficiently without LC rearrangement. Heavy-chain-only antibodies (HCAb) are expressed at high levels, provided that the CH1 domain is deleted from the constant regions. HCAb production does not require an IgM stage for effective pre-B cell signaling. Antigen-specific heavy-chain-only IgM or IgGs are produced upon immunization. The IgG is dimeric, whereas IgM is multimeric. The chimeric HCAb loci are subject to allelic exclusion, but several copies of the transgenic locus can be rearranged and expressed successfully on the same allele in the same cell. Such cells are not subject to negative selection. The mice produce a full antibody repertoire and provide a previously undescribed avenue to produce specific human HCAb in the future.

Transposition of the Drosophila hydei Minos transposon in the mouse germ line. (Article)

2003-02-01T00:00:00Z

We tested the suitability of the fly transposon Minos, a member of the Tc1/mariner superfamily, for insertional mutagenesis in the mouse germ line. We generated a transgenic mouse line expressing Minos transposase in growing oocytes and another carrying a tandem array of nonautonomous transposons. The frequency of transposition in the progeny derived from oocytes carrying both transgenes is 8.2%. Analysis of the new integration sites shows a high frequency of transpositions to a different chromosome. Thus Minos transposition could be an effective system for insertional mutagenesis and functional genomic analysis in the mouse.

Intracellularly expressed single-domain antibody against p15 matrix protein prevents the production of porcine retroviruses (Article)

2003-01-01T00:00:00Z

The presence of porcine endogenous retroviruses presents a potential risk of transmission of infectious diseases (xenozoonosis) if tissues and organs from genetically modified pigs are to be used in xenotransplantation. Here, we report that intracellular expression of a llama single-domain antibody against p15, the matrix domain protein of the porcine endogenous retrovirus Gag polyprotein, blocks retrovirus production, providing the possibility of eliminating the risk of infection in xenotransplantation.

Functional and comparative analysis of globin loci in pufferfish and humans (Article)

2003-01-01T00:00:00Z

To further our understanding of the regulation of vertebrate globin loci, we have isolated cosmids containing alpha- and beta-globin genes from the pufferfish Fugu rubripes. By DNA fluorescence in situ hybridization (FISH) analysis, we show that Fugu contains 2 distinct hemoglobin loci situated on separate chromosomes. One locus contains only alpha-globin genes (alpha-locus), whereas the other also contains a beta-globin gene (alpha beta-locus). This is the first poikilothermic species analyzed in which the physical linkage of the alpha- and beta-globin genes has been uncoupled, supporting a model in which the separation of the alpha- and beta-globin loci has occurred through duplication of a locus containing both types of genes. Surveys for transcription factor binding sites and DNaseI hypersensitive site mapping of the Fugu alpha beta-locus suggest that a strong distal locus control region regulating the activity of the globin genes, as found in mammalian beta-globin clusters, may not be present in the Fugu alpha beta-locus. Searching the human and mouse genome databases with the genes surrounding the pufferfish hemoglobin loci reveals that homologues of some of these genes are proximal to cytoglobin, a recently described novel member of the globin family. This provides evidence that duplication of the globin loci has occurred several times during evolution, resulting in the 5 human globin loci known to date, each encoding proteins with specific functions in specific cell types.

The role of the -50 region of the human gamma-globin gene in switching. (Article)

2001-09-17T00:00:00Z

During the switch from human gamma- (fetal) to beta- (adult) globin gene expression, the gamma and beta genes are expressed competitively by an alternating transcription mechanism. The -50 region of the gamma gene promoter has been proposed to be responsible for the early competitive advantage of the gamma genes and to act as a stage selector element (SSE) in hemoglobin switching. We analyzed the effect of mutating the -50 region of the gamma gene in the presence of a competing beta gene in transgenic mice. This shows that the -50 region does not affect silencing of the beta gene in early development and does not act as a stage selector. However, it affects the ratio of gamma versus beta gene expression in the early, but not later, stages of fetal development. Interestingly, both the wild-type and mutant minilocus constructs show a higher frequency of alternate transcription than observed in the complete locus, suggesting that sequences normally present between the gamma and beta genes facilitate the interaction of the locus control region (LCR) and beta-globin gene in the complete locus.

Enforced expression of GATA-3 during T cell development inhibits maturation of CD8 single-positive cells and induces thymic lymphoma in transgenic mice (Article)

2001-01-01T00:00:00Z

The zinc finger transcription factor GATA-3 is of critical importance for early T cell development and commitment of Th2 cells. To study the role of GATA-3 in early T cell development, we analyzed and modified GATA-3 expression in vivo. In mice carrying a targeted insertion of a lacZ reporter on one allele, we found that GATA-3 transcription in CD4(+)CD8(+) double-positive thymocytes correlated with the onset of positive selection events, i.e., TCRalphabeta up-regulation and CD69 expression. LacZ expression remained high ( approximately 80% of cells) during maturation of CD4 single-positive (SP) cells in the thymus, but in developing CD8 SP cells the fraction of lacZ-expressing cells decreased to <20%. We modified this pattern by enforced GATA-3 expression driven by the CD2 locus control region, which provides transcription of GATA-3 throughout T cell development. In two independent CD2-GATA3-transgenic lines, approximately 50% of the mice developed thymic lymphoblastoid tumors that were CD4(+)CD8(+/low) and mostly CD3(+). In tumor-free CD2-GATA3-transgenic mice, the total numbers of CD8 SP cells in the thymus were within normal ranges, but their maturation was hampered, as indicated by increased apoptosis of CD8 SP cells and a selective deficiency of mature CD69(low)HSA(low) CD8 SP cells. In the spleen and lymph nodes, the numbers of CD8(+) T cells were significantly reduced. These findings indicate that GATA-3 supports development of the CD4 lineage and inhibits maturation of CD8 SP cells in the thymus.

Locus control regions and gene therapy (Doctoral Thesis)

1999-09-29T00:00:00Z

Gene therapy is a procedure in which exogenous genetic material is introduced into the cells of a patient in order to correct an genetic error or to provide the cells of the patient with a new functional property. Correction can be achieved by gene targeting via homologous recombination, at present achieved with very low efficiency (reviewed in Yanez and Porter 1998), or by addition of a therapeutic gene (augmentation). For gene therapy to succeed in clinical practice, the therapeutic gene must be delivered efficiently into the appropriate cells (tissue) and, once delivered, the gene must be expressed at a therapeutical level. In this chapter (section A) I will summarise some of the efforts currently underway for the development of practical, efficient and safe methods for gene transfer in man and the rationale underlying these approaches as well as the limitations and the problems involved (Tables IA and IB). I will also address the following issues: regulation of gene expression (section B), possible targets for gene therapy (section C), creation of animal models for human diseases and their relevance to somatic gene therapy (section D) and ethical and social implications of gene therapy (section E).

Microinjection of cre recombinase RNA induces site-specific recombination of a transgene in mouse oocytes (Article)

1998-01-01T00:00:00Z

We have developed a strategy for producing single copy transgenic mouse lines using Cre-loxP site specific recombination. The method is based on transient expression of the recombinase after injection of in vitro transcribed mRNA into the cytoplasm of fertilised eggs containing multiple copies of the transgene. The success rate of the recombination event is 100% (15 out of 15).

The expression of bacterial nitroreductase in eukaryotic cells results in their killing by the prodrug CB1954. (Article)

1997-01-01T00:00:00Z

The enzyme nitroreductase, isolated from Escherichia coli B, converts CB1954 ((5-aziridin-1-yl)-2,4-dinitro-benzamide) into a cytotoxic DNA interstrand cross-linking agent. The E. coli B gene (nfnB, NTR) encoding nitroreductase (NTR) was cloned into eukaryotic expression vectors. When driven by a CMV promoter, 5-10% of the stably transfected mouse fibroblasts expressed the NTR enzyme. These cells were killed at a concentration of 20 microM CB1954 in comparison to nonexpressing cells which were killed at a much higher concentration (500 microM). We subsequently generated transgenic mice to test the prodrug system in vivo. Nitroreductase was expressed specifically in T cells driven by the control elements of the human CD2 locus. Upon CB1954 treatment, transgenic mice show extensive cell depletion in thymus and spleen (14-16% of normal cell numbers), whereas all other tissues are unaffected by prodrug administration. These results raise the possibility of using the NTR gene in anticancer therapy.

Correction of the X-linked immunodeficiency phenotype by transgenic expression of human Bruton tyrosine kinase under the control of the class II major histocompatibility complex Ea locus control region. (Article)

1997-01-01T00:00:00Z

Bruton tyrosine kinase (Btk) is essential for the development of pre-B cells to mature B cell stages. Btk-deficient mice manifest an X-linked immunodeficiency (xid) defect characterized by a reduction of peripheral IgMlow IgDhigh B cells, a lack of peritoneal CD5+ B cells, low serum levels of IgM and IgG3, and impaired responses to T cell independent type II (TI-II) antigens. We have generated transgenic mice in which expression of the human Btk gene is driven by the murine class II major histocompatibility complex Ea gene locus control region, which provides gene expression from the pre-B cell stage onwards. When these transgenic mice were mated onto a Btk- background, correction of the xid B cell defects was observed: B cells differentiated to mature IgMlowIgDhigh stages, peritoneal CD5+ B cells were present, and serum Ig levels and in vivo responses to TI-II antigens were in the normal ranges. A comparable rescue by transgenic Btk expression was also observed in heterozygous Btk+/- female mice in those B-lineage cells that were Btk-deficient as a result of X chromosome inactivation. These findings indicate that the Btk- phenotype in the mouse can be corrected by expression of human Btk from the pre-B cell stage onwards.

The human β-globin locus control region confers an early embryonic erythroid-specific expression pattern to a basic promoter driving the bacterial β-galactosidase gene. (Article)

1996-01-01T00:00:00Z

The beta-globin locus control region (LCR) is contained on a 20 kb DNA fragment and is characterized by the presence of five DNaseI hypersensitive sites in erythroid cells, termed 5'HS1-5. A fully active 6.5 kb version of the LCR, called the muLCR, has been described. Expression of the beta-like globin genes is absolutely dependent on the presence of the LCR. The developmental expression pattern of the genes in the cluster is achieved through competition of the promoters for the activating function of the LCR. Transgenic mice experiments suggest that subtle changes in the transcription factor environment lead to the successive silencing of the embryonic epsilon-globin and fetal gamma-globin promoters, resulting in the almost exclusive transcription of the beta-globin gene in adult erythropoiesis. In this paper, we have asked the question whether the LCR and its individual hypersensitive sites 5'HS1-4 can activate a basic promoter in the absence of any other globin sequences. We have employed a minimal promoter derived from the mouse Hsp68 gene driving the bacterial beta-galactosidase (lacZ) gene. The results show that the muLCR and 5'HS3 direct erythroid-specific, embryonic expression of this construct, while 5'HS1, 5'HS2 and 5'HS4 are inactive at any stage of development. Expression of the muLCR and 5'HS3 transgenes is repressed during fetal stages of development. The transgenes are in an inactive chromatin conformation and the lacZ gene is not transcribed, as shown by in situ hybridization. These data are compatible with the hypothesis that the LCR requires the presence of an active promoter to adopt an open chromatin conformation and with models proposing progressive heterochromatization during embryogenesis. The results suggest that the presence of a beta-globin gene is required for LCR function as conditions become more stringent during development.

The human beta-globin locus control region confers an early embryonic erythroid-specific expression pattern to a basic promoter driving the bacterial lacZ gene (Article)

1996-01-01T00:00:00Z