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Biomedical subjects

D Pan

Publications and source records attributed to D Pan.

At least 37 records · Page 2Linked to original sources
PubMed2000

Retroviral vector design studies toward hematopoietic stem cell gene therapy for mucopolysaccharidosis type I.

To optimize a gene transfer system for hematopoietic stem cell gene therapy of patients with mucopolysaccharidosis (MPS) type I, 10 retroviral vectors were constructed to express the human alpha-L-iduronidase (IDUA) cDNA. These vectors were designed to evaluate the potential effects of specific promoters, the addition of selectable markers, and the use of multiple promoters versus an internal ribosome entry site for expression of IDUA and selectable maker genes. The effect of vector design was investigated in primary patient fibroblasts (F(MPS)) or murine fibroblast cell lines; while overall comparison of transgene expression was determined in patients' peripheral blood lymphocytes (PBL(MPS)) and CD34+ progenitors (PBPC(MPS)). We observed that the human PGK promoter introduced the highest IDUA activity per 1% relative transgene frequency in F(MPS). Use of the same promoter to separately regulate both the therapeutic gene and a drug-resistance gene resulted in decreased expression of the unselected gene. Co-selection using bicistronic vectors not only increased the number of transductants, but also elevated transgene expression under selective pressure in transgene-positive progenitors. Bicistronic vector LP1CD overcame down-regulation and practically introduced the highest IDUA level in unselected PBL(MPS) and an intermediate level in PBPC(MPS). These studies provide a better understanding of factors contributing to efficient gene expression in hematopoietic cells.

Animals↗
PubMed1999

Combined ultrafiltration-transduction in a hollow-fiber bioreactor facilitates retrovirus-mediated gene transfer into peripheral blood lymphocytes from patients with mucopolysaccharidosis type II.

The process of growing and transducing large quantities of human primary peripheral blood lymphocytes (PBLs) with high gene transfer efficiency continues to be one of the major challenges for clinical and experimental gene therapy. Toward developing a clinical trial of lymphocyte gene therapy for mucopolysaccharidosis type II (i.e., Hunter syndrome), we investigated a novel method that exploited the innate capability of a hollow-fiber bioreactor system to filter large quantities of vector supernatant and facilitate transduction. An aliquot (5 x 10(7)) of PBL apheresis product was precultured in a gas-permeable culture bag or a bioreactor, and then transduced with a retroviral vector L2SN containing the iduronate-2-sulfatase (IDS) and neomycin resistance genes. We observed that the total number of PBLs could be expanded up to 187-fold, yielding up to 10(10) cells at the end of a 7-day culture period. The multiplicity of infection could be increased (up to 20-fold) by ultrafiltrating a large volume of vector supernatant through the semipermeable membrane of this system. A high level of transduction efficiency (up to 57%) was achieved, resulting in IDS enzyme activity as high as 1250 U/mg/hr in transduced PBL(MPS) 15 days after transduction. This level was markedly increased from that of nontransduced cells (<3 U/mg/hr) and was even greater than that of normal PBLs (mean, 809; n = 10). After 12 days of G418 selection, PBL(MPS) transductants exhibited a proviral IDS enzyme level approximately threefold higher than that in normal PBLs. These results indicated that the hollow-fiber bioreactor could be used to culture and transduce human primary PBLs in clinically useful quantities with relatively high gene transfer efficiency and transgene expression.

Bioreactors↗
PubMed1999

Identification of an oxygen responsive enhancer element in the glyceraldehyde-3-phosphate dehydrogenase gene.

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is induced by hypoxia in endothelial cells (EC). Upregulation occurs primarily at the level of transcription and occurs to a much greater extent in EC than in other cell types. To characterize EC specific hypoxia response elements within the GAPDH gene, we performed transient transfection studies in EC, fibroblasts and smooth muscle cells using portions of the GAPDH promoter linked to a CAT reporter gene. These initial studies identified an EC specific hypoxia responsive region that was further characterized (using SV40-promoter-CAT reporter constructs) as a 19-nucleotide sequence (-130 to -112) containing both an hypoxia inducible factor-1 (HIF-1)-binding site and a novel flanking sequence. Electrophoretic mobility shift assays confirmed inducible EC protein binding to this fragment. Mutation of either the HIF-1-binding site or the flanking sequence resulted in complete loss of function and loss of inducible protein binding. Thus, a single HIF-1-binding site is necessary, but not sufficient, for hypoxic regulation of GAPDH in EC. Furthermore, the novel HIF-1 flanking sequence required for GAPDH upregulation and the protein(s) that bind to it may be EC specific.

Animals↗
PubMed1999

Real-time quantitative polymerase chain reaction to assess gene transfer.

Accurate quantification of gene transfer (or gene correction) is a universal challenge in the field of gene therapy. In developing a clinical trial of lymphocyte gene therapy for Hunter syndrome (mucopolysaccharidosis type II), methods using Southern blot or automated DNA sequencing technology were employed, but found to be laborious and subject to considerable variation. As an alternative approach, we explored a real-time kinetic PCR assay appropriate to new instrumentation (PE Biosystems model 7700). A TaqMan probe was designed to hybridize directly across the exon 2-exon 3 junction of the iduronate-2-sulfatase transgene cDNA. In this assay system, cDNA from the retroviral vector L2SN generates a PCR product that is 84 nucleotides long and readily quantified by TaqMan probe binding and subsequent cleavage. Evaluation of this method demonstrated sensitivity over at least 5 logs with respect to the standard (vector plasmid pL2SN). There was no detectable signal from genomic DNA from nontransduced cells, thus indicating the specificity of this assay. The sample preparation method used to prepare specimens was a relatively simple cell lysis procedure, without DNA extraction, and represents a significant advancement over the more complex methods of DNA extraction that are typically used for such assays. This specific assay, and comparison to previous methods, illustrates the utility of a new method that is readily generalized to many gene therapy studies, and that has the potential to be extended to measure gene expression by means of quantitative RT-PCR.

Base Sequence↗
PubMed1999

Retroviral transduction and expansion of peripheral blood lymphocytes for the treatment of mucopolysaccharidosis type II, Hunter's syndrome.

BACKGROUND: Gene therapy using autologous peripheral blood lymphocytes (PBLs) has been used to produce adenosine deaminase with which to treat patients with severe combined immunodeficiency. Patients with mucopolysaccharidosis type II (MPS II) lack iduronate-2-sulfatase (IDS), and serial PBL gene therapy may benefit these patients. STUDY DESIGN AND METHODS: The purpose of these studies was to develop a method to transduce PBLs from a patient with MPS II by using a retroviral vector, LS2N, containing the IDS gene. PBLs were collected by apheresis and cryopreserved in aliquots for the performance of multiple transductions and expansions. The PBLs were expanded in number and then transduced in a hollow-fiber bioreactor (HFBR). Additional culture allowed for further expansion. RESULTS: Fresh PBLs (6.2 x 10(7)) from a patient with MPS II were transduced with L2SN and expanded in an HFBR with an extracapillary space of 11 mL. After 10 days of culture, 4.1 x 10(9) cells were harvested. Cryopreserved MPS II PBLs could not be reliably expanded if they were placed in the HFBR immediately after being thawed; however, cells were successfully transduced and expanded in the HFBR if they were first cultured in a bag. To increase the cell yield, PBLs were expanded in a 60-mL HFBR after transduction and expansion in an 11-mL HFBR. In four separate experiments, 2 x 10(8) cryopreserved PBL were cultured for 3 days in a bag and transferred to an 11-mL HFBR, where they were transduced daily with L2SN for 3 days and then expanded for 4 additional days. Cells were then transferred into a 60-mL HFBR and expanded for an additional 7 days. In the four experiments, 5.5 x 10(9), 7.4 x 10(9), 1.12 x 10(9), and 19.4 x 1(9) cells were produced. The vector was detected in the harvested cells, but the proportion of cells transduced was less than 2.5 percent, the lowest standard used in the assay. In two of the experiments, cells harvested from the HFBR were used in a gene therapy clinical trial. CONCLUSION: Autologous cryopreserved PBLs can be transduced and expanded to produce >1 x 10(10) cells. This procedure is being used for a Phase I/II clinical trial of lymphocyte gene therapy.

Cells, Cultured↗
PubMed1998

Targeted expression of teashirt induces ectopic eyes in Drosophila.

teashirt was initially identified as a gene required for the specification of the trunk segments in Drosophila embryogenesis and encodes a transcription factor with zinc finger motifs. We report here that targeted expression of teashirt in imaginal discs is sufficient to induce ectopic eye formation in non-eye tissues, a phenotype similar to that produced from targeted expression of eyeless, dachshund, and eyes absent. Furthermore, teashirt and eyeless induce the expression of each other, suggesting that teashirt is part of the gene network that functions to specify eye identity.

Animals↗
PubMed1998

Rapid synthesis of a 5'-fluorinated oligodeoxy-nucleotide: a model antisense probe for use in imaging with positron emission tomography (PET).

5'-Deoxy-5'-fluoro-O4-methylthymidine was synthesized by the reaction of the corresponding 5'-O-tosylate with KF in the presence of Kryptofix [222] and coupled to a 5'-phosphoramidite-activated CPG-bound oligodeoxynucleotide. The sequence of reactions and purifications were accomplished within 4 h, a necessary condition of the development of radiofluorinated antisense oligodeoxynucleotide probe for use with PET.

Bridged Bicyclo Compounds, Heterocyclic↗
PubMed1998

Mobilization and transduction of peripheral blood progenitor cells in patients with mucopolysaccharidosis I.

Mucopolysaccharidosis type I (MPS I) results from a deficiency of alpha-L-iduronidase enzyme (IDUA), an enzyme responsible for the catabolism of glycosaminoglycans. Genetically modified progenitor cells may permit a therapeutic effect similar to that obtained from allogeneic BMT without the associated risks. To that end, CD34+ peripheral blood hematopoietic progenitor cells from patients with MPS I were mobilized using G-CSF, collected by apheresis, and enriched using avidin-biotin separation techniques. These cells were cultured in a hollow fiber bioreactor and transduced with a retroviral vector (LP1CD) containing the cDNA for human IDUA and a murine dihydrofolate reductase (DHFR) enzyme. Approximately 4%-16% of the colonies expressed methotrexate drug resistance. Expression of the IDUA enzyme in the progenitor cells was initially high and declined after approximately 10 days of culture. These results indicate that PBPC from patients with MPS I can be mobilized, isolated, enriched, and transduced with a therapeutic gene.

Adult↗
PubMed1998

Primary and secondary control over age-related changes in physical appearance.

Beliefs about appearance-related changes due to aging were used to test the effects of perceived control and secondary control (acceptance) in a sample of 412 young, early-middle-age, and late-middle-age college-educated adults. Mean difference in aging-related appearance control and hypotheses regarding the adaptiveness of primary and secondary control were examined. Primary control over aging-related appearance was lower in older adults and secondary control was higher. In addition, the results indicated support for the Primacy/Back-Up Model that primary perceived control is important at all levels of actual control. Those with stronger beliefs in their primary control were less distressed. Secondary control served a back-up function in that it was related to less distress only for those who had medium or lower beliefs in primary control. The implications of these findings, that primary control may be advantageous even in low-control circumstances, are discussed.

Adult↗
PubMed1998

Gross anatomy of the pancreatic lobes and ducts in six breeds of domestic ducks and six species of wild ducks in China.

A previously unreported pancreatic duct was found by Liu (1989) in Pekin ducks. This duct has now been consistently found in six breeds of domestic ducks and six species of wild ducks in China. For purposes of Nomina Anatomica Avium it is hereby called the 'first pancreatic duct' (Ductus pancreaticus primus) since it enters the duodenum at or near the flexure where the descending duodenum becomes the ascending duodenum. All other pancreatic ducts enter the duodenum later, closer to where it joins the jejunum. This first pancreatic duct drains the caudal extremity of the dorsal lobe of the pancreas and can be easily exteriorized for experimental purposes. Within the parenchyma of the dorsal lobe of the pancreas this duct communicates with the dorsal pancreatic duct. In the present study of the gross anatomy of the pancreatic lobes of domestic and wild Chinese ducks we describe and illustrate variations in position and number of all biliary and pancreatic ducts.

Animals↗
PubMed1998

Arachidonic acid inhibits lipogenic gene expression in 3T3-L1 adipocytes through a prostanoid pathway.

This report examines the effect of polyunsaturated fatty acids (PUFA) on lipogenic gene expression in cultured 3T3-L1 adipocytes. Arachidonic acid (20:4, n-6) and eicosapentaenoic acid (20:5, n-3) suppressed mRNAs encoding fatty acid synthase (FAS) and S14, but had no effect on beta-actin. Using a clonal adipocyte cell line containing a stably integrated S14CAT fusion gene, oleic acid (18:1, n-9), arachidonic acid (20:4, n-6) and eicosapentaenoic acid (20:5, n-3) inhibited chloramphenicol acetyltransferase (CAT) activity with an ED50 of 800, 50, and 400 microM, respectively. Given the high potency of 20:4, n-6, its effect on adipocyte gene expression was characterized. Arachidonic acid suppressed basal CAT activity, but did not affect glucocorticoid-mediated induction of S14CAT expression. The effect of 20:4, n-6 on S14CAT expression was blocked by an inhibitor of cyclooxygenase implicating involvement of prostanoids. Prostaglandins (PGE2 and PGF2alpha at 10 microM) inhibited CAT activity through a pertussis toxin-sensitive Gi/Go-coupled signalling cascade. Our results suggest that 20:4, n-6 inhibits lipogenic gene expression in 3T3-L1 adipocytes through a prostanoid pathway. This mechanism of control differs from the polyunsaturated fatty acid-mediated suppression of hepatic lipogenic gene expression.

3T3 Cells↗
PubMed1997

Kuzbanian controls proteolytic processing of Notch and mediates lateral inhibition during Drosophila and vertebrate neurogenesis.

Notch and the disintegrin metalloprotease encoded by the kuzbanian (kuz) gene are both required for a lateral inhibition process during Drosophila neurogenesis. We show that a mutant KUZ protein lacking protease activity acts as a dominant-negative form in Drosophila. Expression of such a dominant-negative KUZ protein can perturb lateral inhibition in Xenopus, leading to the overproduction of primary neurons. This suggests an evolutionarily conserved role for KUZ. The Notch family of receptors are known to be processed into smaller forms under normal physiological conditions. We provide genetic and biochemical evidence that Notch is an in vivo substrate for the KUZ protease, and that this cleavage may be part of the normal biosynthesis of functional Notch proteins.

Animals↗
PubMed1997

Retroviral transduction of peripheral blood leukocytes in a hollow-fiber bioreactor.

BACKGROUND: Peripheral blood white cells (leukocytes) (PBLs) have been used as effective targets for genetic manipulation by transduction with retroviruses in open systems. A semi-closed hollow-fiber bioreactor was tested for culturing and transducing lymphocytes. STUDY DESIGN AND METHODS: PBLs were isolated from five healthy donors, and 5 x 10(7) cells were cultured in hollow-fiber bioreactors for 4 days after stimulation with anti-CD3 in medium containing 200 units per mL of recombinant interleukin 2. Transduction with retroviral vector containing the gene for iduronate-2-sulfatase and G 418 resistance, L2SN, was performed daily on Days 4, 5, 6, and 7, and the cells were expanded for an additional 3 days. RESULTS: PBLs from three donors were harvested from the bioreactor after transduction and expansion, and 4.5 x 10(9), 7.0 x 10(9), and 2.9 x 10(9) cells were recovered, representing 90-, 136-, and 58-fold expansions. The transduction frequency of L2SN was 10, 5, and 1 percent, respectively. For additional expansion of PBLs, in two cases the bioreactor was reinoculated with 5 x 10(7) cells, which were expanded again for 16 and 8 days, respectively, yielding 1.4 x 10(9) and 3.1 x 10(9) cells, which reflected an additional 28- and 62-fold expansion of cells. PBLs from two other donors were transduced and expanded in the bioreactor, and then 0.8 mg per mL of G 418 was added to the medium in an attempt to enrich the transduced population. Although 2.5 and 10 percent of the cells were transduced, cell death and absence of expansion in the presence of G 418 resulted in final cell lots with viabilities of only 4 and 8 percent. In all cases, the harvested cells tested negative in bacterial and fungal cultures. CONCLUSION: Hollow-fiber bioreactors are an efficient and effective system for the retroviral transduction and expansion of PBLs for clinical gene therapy.

Bioreactors↗
PubMed1997

Nuances of aesthetic blepharoplasty.

The orbits and surrounding tissues form the emotional and expressive center of the human face. Eye contact forms a large part of human interaction and, unfortunately, it is often this region that first surrenders to the aging process. A successful blepharoplasty depends on the careful evaluation of a patient's eyes, correct assessment of the cosmetic problems, and the surgical dexterity to repair such defects adequately. Consistent success may be achieved only with mastery of aesthetic consideration of the eyes, which aid in the diagnosis of the deformity of asymmetry. From that, the appropriate surgical intervention may ensue. This discussion will review the anatomy related to different orbital expressions, delineate the effects of aging, and briefly touch on techniques in our surgical armamentarium to correct and improve eyes through blepharoplasty.

Adult↗
PubMed1996

The cell surface metalloprotease/disintegrin Kuzbanian is required for axonal extension in Drosophila.

It has long been suspected that proteolytic activity associated with advancing growth cones may be required for axon extension. We have isolated mutations in the kuzbanian (kuz) gene, which is expressed in the nervous system and encodes a putative zinc metalloprotease with a disintegrin domain. Drosophila embryos with loss-of-function mutations in kuz have dramatic defects in the development of central nervous system axon pathways, with many axons stalling and failing to extend through the nerve cord. This phenotype is rescued by panneural expression of kuz mRNA in the embryo. These results show that the Kuz metalloprotease is required for axon extension, suggesting a requirement for proteolytic activity at the growth cone surface.

Animals↗