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C Baum

Publications and source records attributed to C Baum.

At least 55 records · Page 3Linked to original sources
PubMed2000

Human multidrug resistance-1 gene transfer to long-term repopulating human mobilized peripheral blood progenitor cells.

Mobilized peripheral blood progenitor cells (PBPC) are an attractive target for the retrovirus-mediated transfer of cytostatic drug resistance genes. We analyzed NOD/SCID mouse repopulating CD34+ PBPC from cancer patients following retroviral Transwell transduction in various cytokine combinations with the FMEV-based (Friend-mink cell focus forming/murine embryonic stem cell virus) hybrid vector SF-MDR carrying the human multidrug resistance-1 (MDR1) gene. Five to 10 weeks following transplantation of 2.0 x 10(6) CD34+ PBPC into NOD/SCID mice we observed medium to high levels of human cell engraftment with up to 33%. The extent of vector-marked human cells was assessed by a quantitative real-time polymerase chain reaction (PCR). SF-MDR gene transfer into long-term in vivo repopulating human hematopoietic cells was optimal in the presence of either IL-3/IL-6/SCF/FL or FL/TPO/SCF resulting in three-fold (12.4% +/- 1.7%) or four-fold (16.5% +/- 6.8%) higher average proportions of gene-marked human cells in NOD/SCID mice as compared to IL-3 alone (P < 0.01). In conclusion, we could optimize the engraftment capacity and the retroviral gene transfer to CD34+ PBPC using cocktails of early acting cytokines in combination with the recombinant fibronectin fragment CH-296. Our data suggest that the NOD/SCID model provides a valid assay to estimate the gene transfer efficiency to repopulating human PBPC that may be achievable in clinical autologous transplantation settings.

Animals↗
PubMed2000

Variegation of retroviral vector gene expression in myeloid cells.

We have comparatively evaluated the efficiency of a series of retroviral vectors transducing the gp91-phox gene, whose defects are responsible for impaired production of superoxide anion (O2-) by phagocytic cells and lead to the X-linked form of chronic granulomatous disease (X-CGD). These vectors included four constructs based on the MoMuLV backbone and expressing gp91-phox from the viral long terminal repeat (LTR) or from internal promoters, and one construct based on the myelotropic FMEV vector. Expression of the therapeutic gene from the MoMuLV LTR was unsatisfactory after transduction of the PLB985 X-CGD knockout cell line and of primary CD34+ hematopoietic progenitors from X-CGD patients. The presence of either constitutive or inducible internal promoters did not result in important improvements in the efficiency of O2- production and lowered the titers of the viral preparations. In contrast, sustained levels of superoxide generation were obtained upon transduction with the FMEV vector. To analyze the efficiency of transgene expression at the single cell level, over 150 cellular clones were generated from bulk cultures of PLB985 X-CGD cells transduced with this vector, each one representative of an individual transduction event. These clones revealed a markedly heterogeneous pattern of gp91-phox expression, ranging from complete silencing to full restoration of superoxide production. Within each clone, expression of the therapeutic gene correlated with the number of expressing cells rather than with the average levels of expression from each cell, indicating that at the single cell level, the proviral promoter is regulated by a binary, on/off mechanism. Moreover, both transduced bulk and clonal cell populations displayed a tendency to a progressive extinction of expression over time, with a mechanism involving LTR methylation. The design of novel retroviral vectors escaping silencing is highly desirable for efficient gene therapy.

Gene Expression↗
PubMed2000

De novo lipogenesis in adipose tissue of lean and obese women: application of deuterated water and isotope ratio mass spectrometry.

OBJECTIVE: To evaluate the feasibility of using deuterated water and isotope ratio mass spectrometry to measure de novo fatty acid synthesis in adipose tissue, and to compare this parameter in obese and lean women. SUBJECTS: Six lean and six obese premenopausal Caucasian women in the main study and three obese Pima Indians in a pilot study. MEASUREMENTS: Deuterated water was administered orally twice daily for 14 days to create stable deuterium enrichment in body water, during which series of blood samples were collected to measure body water deuterium enrichment and deuterium incorporation into plasma total Triacylglycerol (TG) fatty acids and total cholesterol. Subcutaneous fat at different sites were sampled at the beginning and the end of deuterium administration to measure deuterium incorporation into TG fatty acids. RESULTS: Fractional de novo synthesis rate of TG fatty acids in adipose tissue was 0. 014+/-0.005 and 0.014+/-0.007% in lean and obese Caucasian women, corresponding to 2+/-0.7 and 5.6+/-3.2 g (P=0.3) of fatty acids synthesized daily, respectively. Plasma TG fatty acids and cholesterol synthesis rates were comparable to those reported previously. A pilot study showed that de novo lipid synthesis in adipose tissue of obese Pima Indians was also quantitatively minor. CONCLUSION: Human adipose tissue, like the liver, does not make a major contribution to whole body lipogenesis under eucaloric conditions. A combination of deuterated water and isotope ratio mass spectrometry is a useful research tool for studying accumulation of de novo synthesized lipids in human adipose tissue.

Adipocytes↗
PubMed2000

The effects of dose, route of administration, drug scheduling and MDR-1 gene transfer on the genotoxicity of etoposide in bone marrow.

We have used the bone marrow micronucleus assay (BMMN) as a measure of clastogenicity, in response to etoposide exposure in murine bone marrow. Oral delivery of etoposide resulted in a reduced number of micronucleated polychromatic erythrocytes (MPE) relative to the same dose delivered intraperitoneally (P < 0.001). Daily fractionation of the oral schedule of etoposide led to a more than six-fold increase in cumulative MPE frequency over that observed with the same total, unfractionated dose, with the potency of the response increasing with serial exposure (r = 0.79). Retrovirally-mediated expression of MDR1 in murine bone marrow resulted in partial protection against the clastogenic activity of etoposide relative to mock transduced control mice. The model system developed has indicated a variety of factors able to influence the genotoxicity of etoposide. It should now be possible to further exploit this model in order to define other factors governing haemopoietic sensitivity to etoposide.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

New perspectives for cancer chemotherapy by genetic protection of haematopoietic cells.

The effectiveness of anti-cancer chemotherapy can be limited by acute suppression of the bone marrow (myelosuppression). There is also a risk of therapy-related secondary haematopoietic malignancy as well as acute and longer term effects in other tissues. Clinical strategies have been established to address some of these problems, particularly toxic effects on the bone marrow (acute myelotoxicity); however, there is still substantial scope for improving the management of chronic toxicity and mutagenicity to haematopoietic cells and collateral damage to non-haematopoietic cells during chemotherapy. In this review, we have discussed a novel strategy that involves the transfer and expression of drug-resistance functions into haematopoietic stem cells and more-mature blood progenitor cells, to overcome both the acute and long-term deleterious effects of anti-tumour treatment in bone marrow. The potential advantages of this approach include: (1) the in vivo selection of protected cell populations, which offers the possibility of intensification or escalation of chemotherapeutic drug doses; (2) a reduction in the frequency of therapy-related leukaemia and (3) tumour sensitisation to chemotherapy at the same time as haematopoietic protection.

News↗
PubMed1999

Efficient gene transfer by hybrid retroviral vectors to murine spermatogenic cells.

Using murine spermatogenic cell lines GC-1 spg and GC-2 spd(ts) as target cells, an attempt was made to design a retroviral vector that would transduce genes efficiently. Promoter activities of various retroviral long terminal repeats (LTRs) were examined by using chloramphenicol acetyltransferase (CAT) as a reporter. The U3 region of spleen focus-forming virus (SFFVp) showed higher enhancer activity than that of Moloney murine leukemia virus (Mo-MuLV) in both cell lines. The U3 region of myeloproliferative sarcoma virus (MPSV) showed higher activity only in GC-1 spg cells. Expression was suppressed by the repressor element of the primer-binding site (PBS) of the Moloney-related virus. The efficiency of transduction of the multidrug-resistance gene (mdr-1) by an Mo-MuLV-based vector was compared with hybrid vectors consisting of the murine embryonic stem cell virus (MESV) PBS and the LTR of either SFFVp or MPSV. Rhodamine efflux assays and colchicine-resistant colony-forming assays demonstrated higher gene expression by the hybrid vectors. Amphotropic and ecotropic receptors were found to be expressed and functional in both cell lines. Thus, these hybrid vectors represent a powerful tool by which to transfer genes into spermatogenic cells.

Animals↗
PubMed1999

A first-generation packaging cell line for Epstein-Barr virus-derived vectors.

On the basis of the B lymphotropic Epstein-Barr virus (EBV), we have constructed a virus-free packaging cell line that allows encapsidation of plasmids into herpesvirus particles. This cell line harbors an EBV mutant whose packaging signals have been deleted. The gene vectors, which can encompass very large, contiguous pieces of foreign DNA, carry all cis-acting elements involved in amplification and encapsidation into virus-like particles as well as those essential for extrachromosomal maintenance in the recipient cell. Although this first-generation packaging cell line suffers from unwanted recombination between the helper virus genome and gene vector DNAs, this approach opens the way to delivery and stable maintenance of any transgene in human B cells.

B-Lymphocytes↗
PubMed1999

Bicistronic retroviral vectors for combining myeloprotection with cell-surface marking.

We have developed a retroviral vector coexpressing the multidrug-resistance 1 (MDR1) cDNA for inducing cancer drug resistance and the truncated version of the low-affinity nerve growth factor receptor (DeltaLNGFR) for cell-surface marking of transduced cells. The vector is based on the FMEV backbone which mediates high levels of gene expression in hematopoietic cells. To achieve optimal expression levels of both cDNAs, untranslated regions from MDR1 and DeltaLNGFR were removed and three different connections were tested: retroviral splice signals, an internal ribosomal entry site (IRES) from encephalomyocarditis virus, and an internal promoter from the chicken beta-actin gene. As determined by two-color flow cytometry, the best correlation of the expression of both cDNAs was obtained using the vector SF1mSdelta which utilized retroviral splice signals for co-expression. Simultaneous expression of both cDNAs at the single cell level was also shown by confocal laser microscopy. Lymphoid and hematopoietic progenitor cells, including primary human CD34+ cells, transduced with SF1mSdelta acquired dominant multidrug resistance. Transduced primary CD34+ cells could be enriched in vitro based on expression of DeltaLNGFR, avoiding exposure to cytostatic agents. Thus, monitoring the selection of chemotherapy-resistant cells and analyzing their biological properties may be alleviated, both in vitro and in vivo.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

A novel dual function retrovirus expressing multidrug resistance 1 and O6-alkylguanine-DNA-alkyltransferase for engineering resistance of haemopoietic progenitor cells to multiple chemotherapeutic agents.

Following transduction with a retrovirus (SF1MIH) expressing both the multidrug resistance 1 (MDR1) and O6-alkylguanine-DNA-alkyltransferase (ATase) proteins, human erythroleukaemic progenitor (K562) cells were isolated which were resistant to killing by the MDR1 substrate, colchicine. In colony-forming survival assays, K562-SF1MIH cells exhibited resistance to colchicine and doxorubicin, as well as to the O6-alkylating agents N-Methyl-N-nitrosourea (MNU) and temozolomide. Furthermore, the resistance to doxorubicin was abolished by preincubation with the MDR1 inhibitor verapamil while resistance to MNU was ablated by the specific ATase inactivator, O6-benzylguanine (O6-beG) confirming that resistance to doxorubicin and MNU was conferred by MDR1 and ATase, respectively. When K562-SF1MIH were exposed to combinations of colchicine and MNU or doxorubicin and temozolomide, simultaneous resistance to these agents was observed. Thus, transduction of K562 with SF1MIH conferred dual resistance to these cells. These data offer the prospect of designing vectors that will confer resistance to entire regimens of chemotherapy rather than just to individual components of such drug cocktails, thereby substantially increasing the efficacy of therapy. Furthermore, the use of such dual expression constructs is likely to be highly informative for the design of effective in vivo selection protocols, an issue likely to make a major impact in a clinical context in gene therapy in the near future.

Antineoplastic Agents↗
PubMed1999

Design of 5' untranslated sequences in retroviral vectors developed for medical use.

Utilizing genetic modules of simple retroviruses, we have developed a novel generation of gene transfer vectors with improved therapeutic potential. In the 5' untranslated "leader" sequences, all AUG codons which may aberrantly initiate translation and all viral coding sequences were removed. Thus, the probability of expressing unwanted peptides and the potential for homologous recombination with retroviral genes were largely reduced, and the cloning capacity was increased. The transgene was inserted to replace the viral gag sequences, and a new minimal splice acceptor was introduced, resulting in increased expression with all genes tested (those coding for human multidrug resistance 1 and enhanced green fluorescent protein, as well as the lacZ gene). These vectors may represent attractive tools for human gene therapy, because they increase the efficiency of transgene expression and may also increase safety in medical applications.

5' Untranslated Regions↗
PubMed1999

Retroviral vectors pseudotyped with lymphocytic choriomeningitis virus.

Pseudotyping can improve retroviral vector stability and transduction efficiency. Here, we describe a novel pseudotype of murine leukemia virus packaged with lymphocytic choriomeningitis virus (LCMV). This pseudotype was stable during ultracentrifugation and infected several cell lines from different species. Moreover, LCMV glycoproteins were not cell toxic.

Animals↗
PubMed1999

Retroviral vector-mediated gene expression in hematopoietic cells.

Gene transfer vectors based on simple retroviruses and more complex lentiviruses are currently the most reliable tools for stable establishment of transgenes in hematopoietic cells. While important hurdles in basic gene transfer technologies have been overcome in recent years, there is still some uncertainty in the choice of the cis-regulatory elements of the vector. These elements dictate the overall level, clonal variability, response to differentiation and persistence of transgene expression in vivo and thus have a significant influence on the outcome of therapeutic applications of somatic gene transfer. The rationale underlying the further improvement of such cis-elements is reviewed here.

Animals↗
PubMed1998

Dominant selection of hematopoietic progenitor cells with retroviral MDR1 co-expression vectors.

When transferring the human multidrug resistance 1 (MDR1) cDNA, FMEV retroviral vectors mediate high-dose multidrug resistance and, thus, background-free selection in primary human hematopoietic progenitor cells. Here, we analyzed strategies for co-expression of a second gene from an FMEV:MDR1 vector. When linking the cDNAs with the internal ribosomal entry site (IRES) of poliovirus or retroviral splice signals, almost all multidrug-resistant hematopoietic colonies simultaneously coexpressed the 3' positioned second gene, neomycin-phosphotransferase (neoR). The IRES strategy allowed functional co-transfer of a 4.2-kb lacZ-neoR fusion gene, resulting in a total proviral genome size of 11 kb, corresponding to the packaging limit of retroviral vectors. Preselection based on multidrug resistance elevated the expression of the second gene in IRES constructs, but not in splice vectors. Moreover, three intriguing observations were made. First, up to 30% of cells preselected for functional transfer of the 3' positioned cDNA (neoR) showed infunctional MDR1; this occurred irrespective of the linking principle and was associated with instability of the MDR1 transcription unit. Second, the levels of multidrug resistance achieved with the co-expression vectors were moderately lower (15-30% reduced) than those mediated by the monocistronic counterpart. Third, transduction with FMEV:MDR1 co-expression vectors still resulted in high-dose cancer drug resistance and background-free selection of hematopoietic progenitor cells (including primary human CD34+ colony-forming units). Thus, for the first time, we describe MDR1 co-expression vectors that maintain their desired function in early and primary human hematopoietic cells. However, careful interpretation of the data reveals that further vector improvements are required to obtain clinically useful MDR1 co-expression vectors.

Antigens, CD34↗
PubMed1998

Long-term results of Salter and Chiari hip osteotomies in developmental hip dysplasia. A survey of over 10 years follow-up with a new hip evaluation score.

When conservative treatment of developmental hip dysplasia (DDH) does not render satisfactory results, surgical methods are indicated to gain a maximum of joint posture and stability. To assess the efficiency of these surgical procedures, the long-term results of the most often used pelvic osteotomies were examined. As common evaluation scores do not apply for DDH, we developed a specific score system of 100 points to integrate subjective factors such as patient's complaints with objective functional and radiological findings after surgical intervention. Our survey with an average postoperative follow-up examination of 10 years indicated that Salter's innominate osteotomy can render long-term pain relief and enable normal hip development, whereas Chiari's capsular arthroplasty does not. Here, subjective patient complaints do not correlate with clinical and radiological findings, which leads to unsatisfactory results. Ideally, patients aged 2-3 years can undergo Salter's innominate osteotomy whereas the Chiari osteotomy should be considered for patients older than 15 years. Even under optimal surgical conditions, Chiari's osteotomy cannot significantly prevent the development of joint arthritis. Undoubtedly, late results of both surgical procedures are dependent upon the initial stage of DDH. Additional upper femoral correction osteotomies have no further influence on hip development.

Adolescent↗
PubMed1998

FMEV vectors: both retroviral long terminal repeat and leader are important for high expression in transduced hematopoietic cells.

FMEV retroviral vectors combine the long terminal repeat of Friend mink cell focus-forming viruses with the 5' untranslated leader region of the murine embryonic stem cells virus. These modules were connected to achieve high transgene expression in hematopoietic progenitor and stem cells. Here, we report the cloning of safety-improved and versatile FMEV vectors allowing module-wise exchange of crucial elements for comparative studies. By transfer and expression of four different marker genes (neomycin phosphotransferase, lacZ, enhanced green fluorescent protein and truncated low affinity nerve growth factor receptor), we formally demonstrate that both the long terminal repeat and the leader contribute to the high expression of FMEV in transduced hematopoietic cells. Most prominent are the data recorded in the absence of selection in myelo-erythroid progenitor cells. Here, FMEV vectors mediate up to two orders of magnitude increased transgene expression levels when compared with vectors based on the Moloney murine leukemia virus.

5' Untranslated Regions↗
PubMed1998

Foamy virus particle formation.

Subgenomic expression plasmids for the so-called human foamy virus (HFV) structural gag, gag/pol, and env genes were constructed and used to analyze foamy virus particle formation by electron microscopy. Expression of an R-U5-gag-pol construct under control of the human cytomegalovirus immediate-early enhancer-promoter resulted in the formation of viral cores with a homogeneous size of approximately 50 nm located in the cytoplasm. Upon coexpression of an envelope construct, particles were observed budding into cytoplasmic vesicles and from the plasma membrane. Expression of the Gag protein precursor pr74 alone led to aberrantly formed viral particles of heterogeneous size and with open cores. Normal-shaped cores were seen after transfection of a construct expressing the p70gag cleavage product, indicating that p70gag is able to assemble into capsids. Coexpression of p70gag and Env resulted in budding virions, ruling out a requirement of the reverse transcriptase for capsid or virion formation. In sharp contrast to other retroviruses, the HFV cores did not spontaneously bud from cellular membranes. Radiochemical labeling followed by protein gel electrophoresis also revealed the intracellular retention of Env-deprived HFV capsids.

Humans↗
PubMed1998

cis-Active elements of Friend spleen focus-forming virus: from disease induction to disease prevention.

The polycythemic strain of the Friend spleen focus-forming virus (SFFVp) is a replication-defective, acutely transforming retrovirus inducing a bistage erythroleukemia in susceptible mice. The first stage of the disease is an acute polyclonal erythroblastosis induced by the proliferation-promoting effect of gp55. gp55 is expressed from a spliced subgenomic message of SFFVp and stimulates the cellular receptor for erythropoietin. Using a selectable SFFVp that otherwise mimics the specificity of the disease, we demonstrate that the kinetics of the polyclonal expansion depends on the transcriptional strength of the retroviral cis-active elements. By exchanging gp55 for apathogenic genes, we show that SFFVp enhancer and splice signals can be successfully utilized for the development of retroviral vectors mediating very efficient transgene expression in hematopoietic cells. Apathogenic selectable SFFVp-based vectors carrying distinct enhancer alterations are a valuable tool to analyze transcriptional control of leukemia viruses in the absence of oncogenic proteins. Moreover they might have therapeutic potential.

Animals↗