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PubMedDate not supplied

Ultrasonic diagnostic system for interactive interrogation of adult brain through intact skull.

An integrated ultrasonic system has been developed employing static gray scale imaging, digital image processing, and analyses of quantitative ultrasonic backscatter for interactive interrogation of brain through the adult human skull. Operating at 750 kHz, to avoid severe spatial and temporal pulse distortion which accompany ultrasonic transkull transmission at higher frequencies, storage and image processing of successive images taken at selected gain settings in the same image plane allow the reconstruction of a composite cross-section of the brain and skull and enable visualization of internal brain structure. Analyses of digitized backscatter data obtained with the visualization transducer over selectable interrogation paths referenced to the cross-sectional image permit quantitative classification of tissue type independent of qualitative visual image analysis, such that enhanced diagnostic potential is provided. A previous paper described in detail the modifications of the commercial ultrasound diagnostic unit which serves as the core of this system. The present paper presents the rationale for the integrated system design approach, as well as a description of graphics display and tissue classification features which have been incorporated into the instrumentation configuration and are deemed necessary for successful transkull ultrasonic imaging and diagnosis.

Brain Diseases↗
PubMed1994

The bilayer lipid membrane as a generic electrochemical transducer of hydrolytic enzyme reactions.

Bilayer lipid membranes (BLMs) can be used as generic transducers to monitor hydrolytic enzyme reactions occurring at the membrane surface. The representative enzymatic reactions presented herein were between membrane associated urease and penicillinase with urea and penicillin, respectively. Transient electrochemical signals from BLMs which contained enzyme were obtained by proper selection of the lipid composition of membranes. Negatively charged lipid membranes composed of egg phosphatidylcholine (PC) and 35% dipalmitoylphosphatidic acid were used for this purpose. The results were consistent with an electrostatic mechanism of perturbation of the surface structure of the BLMs, where changes of local hydronium ion activity associated with the enzymatic reaction altered the extent of ionization of the headgroups of the acidic constituent of the membranes, thereby providing a transient charging current which lasted for a period on the order of seconds. The delay time for observation of the transient was directly and reproducibly related to the concentration of the substrate which could be determined over a range of microM to mM levels. The results indicate that BLMs can be used as generic selective electrochemical transducers and as switchable biosensors to monitor rapid enzymatic reactions which alter pH.

Biosensing Techniques↗
PubMed2003

Rational design of cavitand receptors for mass sensors.

This Communication describes a rational approach to the fabrication of selective mass sensors, using multiple hydrogen-bonding type interactions encoded in a single cavitand receptor. The introduction of two inward facing hydrogen bond acceptor PO groups at the upper rim of a cavitand renders the corresponding receptor layer deposited on a mass transducer highly selective toward alcohol vapors. The molecular origin of the increased selectivity has been identified in the presence of two energetically and geometrically equivalent interaction modes available to the guest, giving to the corresponding complexes an entropic advantage over their mono PO analogues.

Journal Article↗
PubMed1989

Optimized ultrasound imaging catheters for use in the vascular system.

Clinical experience with 6 and 9 Fr ultrasound imaging catheters (UICs) reveals that several transducer and catheter tip varieties are needed for optimum imaging of diseased intravascular sites. Our UIC design has combined established catheter design and very high frequency ultrasound imaging technology to create a versatile, user configured system for intravascular ultrasound imaging. Optimum use requires proper strategic selection of transducer and catheter sizes, frequencies of operation, and interventional accessories.

Angioplasty, Balloon↗
PubMed2004

Hematopoietic stem cell gene therapy of murine protoporphyria by methylguanine-DNA-methyltransferase-mediated in vivo drug selection.

Erythropoietic protoporphyria (EPP) is an inherited defect of the ferrochelatase (FECH) gene characterized by the accumulation of toxic protoporphyrin in the liver and bone marrow resulting in severe skin photosensitivity. We previously described successful gene therapy of an animal model of the disease with erythroid-specific lentiviral vectors in the absence of preselection of corrected cells. However, the high-level of gene transfer obtained in mice is not translatable to large animal models and humans if there is no selective advantage for genetically modified hematopoietic stem cells (HSCs) in vivo. We used bicistronic SIN-lentiviral vectors coexpressing EGFP or FECH and the G156A-mutated O6-methylguanine-DNA-methyltransferase (MGMT) gene, which allowed efficient in vivo selection of transduced HSCs after O6-benzylguanine and BCNU treatment. We demonstrate for the first time that the correction and in vivo expansion of deficient transduced HSC population can be obtained by this dual gene therapy, resulting in a progressive increase of normal RBCs in EPP mice and a complete correction of skin photosensitivity. Finally, we developed a novel bipromoter SIN-lentiviral vector with a constitutive expression of MGMT gene to allow the selection of HSCs and with an erythroid-specific expression of the FECH therapeutic gene.

Animals↗
PubMed1993

Evaluation of expression of transferred genes in differentiating myeloid cells: expression of human glucocerebrosidase in murine macrophages.

The retroviral vector LGSN, in which the human glucocerebrosidase (GC) cDNA is driven by the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR), was tested for expression in the murine myelomonocytic leukemia cell line M1 before and after induction of differentiation with interleukin-6 (IL-6). Southern analysis of the seven transduced clones selected for neomycin resistance in Geneticin (G-418 sulfate) demonstrated one to eight copies of intact provirus with rearrangements in only two clones. Absolute levels of human GC RNA and protein increased with increased copy numbers of provirus in the clones. Upon induction with IL-6 of the seven transduced clones to the macrophage phenotype, there was no significant change, overall, in RNA levels but some increase in human GC protein levels could be detected. Although this was the average trend, considerable clonal variation in RNA and protein levels was observed upon induction. Transduction of the M1 cells did not interfere with the ability of the cells to differentiate from blasts to macrophages as seen by the appearance of membrane receptors for the constant region of immunoglobulins (Fc gamma RI) and lysozyme production in the differentiated M1 cells. Thus, the M1 cell line can be used for testing retroviral vector expression in myeloid lineages at early and late stages of differentiation. This rapid in vitro testing of potential retroviral vectors will be beneficial for gene therapy of disorders that affect differentiated macrophages such as Gaucher's disease.

Animals↗
PubMed1998

Efficient protection of cells from the genotoxicity of nitrosoureas by the retrovirus-mediated transfer of human O6-methylguanine-DNA methyltransferase using bicistronic vectors with human multidrug resistance gene 1.

Retrovirus-mediated transfer of O6-methylguanine-DNA methyltransferase (MGMT; E.C. 2.1.1.63) and a human multidrug-resistance gene (MDR1) confers resistance to nitrosoureas and natural product antitumor agents, respectively. In a previous study, we constructed two bicistronic retroviral vectors, Ha-MDR-IRES-MGMT and Ha-MGMT-IRES-MDR, that allow co-expression of the MGMT gene and the MDR1 gene to protect cells from the toxicity of combination chemotherapy. Each cell transduced with Ha-MDR-IRES-MGMT or Ha-MGMT-IRES-MDR showed high-level resistance to vincristine and 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosou rea (ACNU), indicating that the two drug-resistance genes can be functionally co-expressed from these vectors. In the present study, we examined whether the expression of MGMT from these MDR1-MGMT bicistronic retroviral vectors could protect cells from the genotoxicity of nitrosoureas. Three independent Ha-MDR-IRES-MGMT-transduced clones and three independent Ha-MGMT-IRES-MDR-transduced clones of HeLa MR cells showed 12-23-fold and 27-30-fold higher MGMT activity than the parental cells. These clones are more resistant to ACNU mutagenicity measured by the frequency of the emergence of 6-thioguanine-resistant colonies after ACNU treatment over the frequency seen in the parental cells. The ACNU-induced sister chromatid exchange (SCE) was markedly suppressed in these clones. Murine bone marrow cells were transduced with either Ha-MDR-IRES-MGMT or Ha-MGMT-IRES-MDR. Non-selected populations of the transduced cells showed only marginal increases in drug resistance and MGMT activity. Remarkable increase in drug resistance and MGMT activity were observed after a short exposure of the transduced cells to vincristine. The Ha-MDR-IRES-MGMT-transduced, vincristine-selected bone marrow cells showed 27-fold resistance to vincristine, 7-fold resistance to ACNU, and 10-fold higher MGMT activity than the non-transduced, non-selected cells. The Ha-MGMT-IRES-MDR-transduced, vincristine-selected cells showed 8-fold resistance to vincristine, 16-fold resistance to ACNU and 19-fold higher MGMT activity than the non-transduced, non-selected cells. The rates of ACNU-induced SCE in the vincristine-selected cells were as follows: non-transduced cells (non-selected) and HaMDR-transduced cells>Ha-MDR-IRES-MGMT-transduced cells>Ha-MGMT-IRES-MDR-transduced cells. Again, the only marginal levels of increases in the rates of ACNU-induced SCE were observed in non-selected population of the transduced cells. These results indicate that the MDR1-MGMT bicistronic retrovirus vectors would be useful to protect normal hematopoietic cells from nitrosourea-induced mutagenesis, and drug-selectable bicistronic constructs would have great advantage over non-selectable vectors.

Animals↗
PubMed1990

Effect of cisapride on gastric motility.

UNLABELLED: The effect of cisapride on gastric motility was determined in vitro (gastroduodenal preparation of the guinea pig) and in vivo (conscious dog). In vitro, cisapride (10(-7)-10(-6) M) antagonized induced gastric relaxation. It enhanced the amplitude of gastric peristaltic waves and improved antroduodenal coordination. This effect was prevented by previous administration of atropine or tetrodotoxin indicating that cisapride exerted its effect via facilitation of cholinergic neurotransmission. In vivo, cisapride (0.08-5 mg/kg p.o.) enhanced the amplitude and the coordination of antral, pyloric and duodenal contractions, both in acute and chronic experiments in conscious dogs with force transducers. Cisapride selectively improved motility since it did not affect gastric secretion (basal or stimulated) in dogs with a gastric cannula. Cisapride (0.16-1.25 mg/kg p.o.) accelerated gastric emptying of a liquid and solid test meal in dogs equipped with a gastric or duodenal cannula, in which gastric emptying was delayed by intragastric or intraduodenal administration of oleic acid. IN CONCLUSION: Cisapride facilitates cholinergic neurotransmission in the gastric wall resulting in selective improvement of motor function and acceleration of delayed gastric emptying.

Animals↗
PubMed1999

Inhibition of HIV-1 by an anti-integrase single-chain variable fragment (SFv): delivery by SV40 provides durable protection against HIV-1 and does not require selection.

Human immunodeficiency virus type 1 (HIV-1) encodes several proteins that are packaged into virus particles. Integrase (IN) is an essential retroviral enzyme, which has been a target for developing agents to inhibit virus replication. In previous studies, we showed that intracellular expression of single-chain variable antibody fragments (SFvs) that bind IN, delivered via retroviral expression vectors, provided resistance to productive HIV-1 infection in T-lymphocytic cells. In the current studies, we evaluated simian-virus 40 (SV40) as a delivery vehicle for anti-IN therapy of HIV-1 infection. Prior work suggested that delivery using SV40 might provide a high enough level of transduction that selection of transduced cells might be unnecessary. In these studies, an SV40 expression vector was developed to deliver SFv-IN (SV(Aw)). Expression of the SFv-IN was confirmed by Western blotting and immunofluorescence staining, which showed that > 90% of SupT1 T-lymphocytic cells treated with SV(Aw) expressed the SFv-IN protein without selection. When challenged, HIV-1 replication, as measured by HIV-1 p24 antigen expression and syncytium formation, was potently inhibited in cells expressing SV40-delivered SFv-IN. Levels of inhibition of HIV-1 infection achieved using this approach were comparable to those achieved using murine leukemia virus (MLV) as a transduction vector, the major difference being that transduction using SV40 did not require selection in culture whereas transduction with MLV did require selection. Therefore, the SV40 vector as gene delivery system represents a novel therapeutic strategy for gene therapy to target HIV-1 proteins and interfere with HIV-1 replication.

Blotting, Western↗
PubMed2003

In vivo expansion of transduced murine hematopoietic cells with a selective amplifier gene.

BACKGROUND: Hematopoietic stem-cell-directed gene transfer has achieved limited success in transducing clinically relevant levels of target cells. The expansion of gene-modified cells is one way to circumvent the problem of inefficient transduction with current vectors. To this end, we have developed 'selective amplifier genes' (SAGs) that encode chimeric proteins that are a fusion of granulocyte colony-stimulating factor receptor and the steroid-binding domain. Prototype SAGs conferred estrogen-responsive growth on murine hematopoietic progenitors. METHODS: We constructed a retroviral vector coexpressing an SAG for 4-hydroxytamoxifen (Tm)-specific proliferation and the enhanced green fluorescent protein (EGFP). Murine bone marrow cells were transduced with this vector and transplanted into myeloablated mice. Subsequently, recipients were challenged with Tm, and EGFP(+) cells were enumerated. RESULTS: The challenge induced a significant increase in EGFP(+) leukocytes (21 +/- 4% to 27 +/- 5%), while EGFP(+) cells decreased in untreated animals (21 +/- 5% to 10 +/- 3%). Three months later, bone marrow cells were transplanted from the unchallenged mice to secondary hosts. Again the administration of Tm resulted in an increase of EGFP(+) cells (16 +/- 4% to 35 +/- 3%), contrasting to a decrease in controls (22 +/- 4% to 12 +/- 4%), and the difference was significant for more than 3 months. A detailed study of lineage showed a preferential expansion of EGFP(+) cells in granulocytes and monocytes following Tm administration. CONCLUSIONS: Long-term repopulating cells were transduced with the SAG, and the transduced granulocyte/monocyte precursors were most likely to be expandable in vivo upon Tm stimulation.

Animals↗
PubMed2003

Improved herpes simplex virus type 1 amplicon vectors for proportional coexpression of positron emission tomography marker and therapeutic genes.

For the development of efficient and safe gene therapy protocols for clinical application it is desirable to determine the tissue dose of vector-mediated therapeutic gene expression noninvasively in vivo. The herpes simplex virus type 1 thymidine kinase gene (HSV-1-tk) has been shown to function as a marker gene for the direct noninvasive in vivo localization of thymidine kinase (TK) expression by positron emission tomography (PET). Using bicistronic or multicistronic gene-expressing cassettes with tk as the PET marker gene, the quantitative analysis of tk gene expression may indirectly indicate the distribution and the level of expression of linked and proportionally coexpressed genes. Here, we describe the construction and functional evaluation of HSV-1 amplicon vectors mediating proportional coexpression of HSV-1-tk as PET marker gene and the enhanced green fluorescent protein gene (gfp) as proof of principle and cell culture marker gene and the Escherichia coli cytosine deaminase (cd) as therapeutic gene. Several double-/triple-gene constructs expressing HSV-1-tk, gfp, and E. coli cd were engineered based on gene fusion or the use of an internal ribosome entry site (IRES). Functional analysis in cell culture (green fluorescent protein [GFP] fluorescence and sensitivity to the prodrugs ganciclovir [GCV] and 5-fluorocytosine [5-FC]) and Western blots were carried out after infection of proliferating rat 9L gliosarcoma and human Gli36 glioma cells with helper virus-free packaged HSV-1 amplicon vectors. To study the ability of PET to differentiate various levels of tk expression noninvasively in vivo, retrovirally transduced and selected populations of rat F98 and human Gli36dEGFR glioma cells with defined levels of proportionally coexpressed tk and gfp genes were grown as subcutaneous tumors in nude rats and nude mice, and tk imaging by PET was performed. To study HSV-1 amplicon vector-mediated gene coexpression in vivo, HSV-1 amplicon vectors bearing coexpression constructs were injected (4 x 10(7) to 1 x 10(8) transducing units) into subcutaneously growing Gli36dEGFR gliomas in nude animals, and tk imaging was performed 24 hr later. All vector constructs mediated GFP expression and sensitized 9L and Gli36 cells toward GCV- and 5-FC-mediated cell killing in a drug dose-dependent manner, respectively. The levels of gene expression varied depending on the location of the genes within the constructs indicating the influence of the IRES on the level of expression of the second gene. Moreover, functional proportional coexpression of the PET marker gene HSV-1-tk and the linked therapeutic E. coli cd gene was observed. In selected tumor cell populations, subtle IRES-dependent differences of tk gene expression could be noninvasively distinguished by PET with good correlation between quantitative assays for IRES-dependent attenuated GFP and TK expression in culture and in vivo. After infection of subcutaneously growing gliomas with HSV-1 amplicon vectors, various levels of TK expression were found ranging from 0.011-0.062 percentage injected dose per gram (%ID/g). These values were 4.0- to 5.7-fold lower than positive control tumor cells. TK expression could be imaged by PET in vivo even with the tk gene located at the weak position downstream from the IRES. In conclusion, these HSV-1 amplicon vectors carrying HSV-1-tk as PET marker gene and any linked therapeutic gene will serve an indirect noninvasive assessment of the distribution of therapeutic gene expression by PET. Monitoring the correlation between primary transduction and therapeutic efficiency of a given vector is highly desirable for the development of safe and efficient gene therapy and vector application protocols in clinical applications.

Animals↗
PubMed2004

Nuclear-localizing O6-benzylguanine-resistant GFP-MGMT fusion protein as a novel in vivo selection marker.

OBJECTIVE: We characterized a novel in vivo selectable fusion protein, green fluorescence protein-O6-benzylguanine (BG)-resistant O6-methylguanine-methyltransferase (GFP-MGMT* [*refers to mutant MGMT]) used to delineate optimum selection regimens for transduced hematopoietic stem cells (HSC) ex vivo and in vivo. MATERIALS AND METHODS: We transduced human or mouse HSC with retrovirus vector encoding GFP-MGMT* where BG-resistant forms of human P140K-hMGMT* and mouse P144K-mMGMT* were studied. We evaluated selection of transduced HSC ex vivo and in vivo using either BG/1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or BG/temozolomide (TMZ) combinations, evaluating transduction marking by flow cytometry and real-time TaqMan PCR. RESULTS: GFP-MGMT* transduction confers nuclear-localized GFP fluorescence and BG resistance. Optimum selection ex vivo of GFP-MGMT*-transduced HSC occurred with BG (2.5-10 microM)/BCNU (5-10 microM) or TMZ (100-200 microM), which increases marking while preserving maximum viable transduced cells. Starting at low levels (0.1%) or high levels (>30%) of in vivo bone marrow gene making in mice, in vivo selection with BG/BCNU (20/6 mg/kg) (weeks 4 and 5) or BG/TMZ (20/60 mg/kg) (daily x 5 at week 4) increased bone marrow marking to 8.58% +/- 3.52% or 82.0% +/- 3.4% GFP+ cells, respectively, in the low- or high-level initial marking mice. CONCLUSIONS: GFP-MGMT* is an informative tool to explore optimization of in vivo selection regimens using BG/BCNU or BG/TMZ to increase gene marking of HSC. Both timing and dosing of selection regimens and the starting level of marking may all be important to the level of selective increase of in vivo marking achieved.

Animals↗
PubMed1998

Retroviral gene transfer: effects on endothelial cell phenotype.

BACKGROUND: Endothelial cells (EC) are an attractive target for somatic cell gene therapy, both for the treatment of cardiovascular disease and for the systemic delivery of recombinant gene products directly into the circulation. Recent evidence, however, suggests that viral transduction may induce unfavorable changes in EC phenotype. We examined the proliferative capacity and cell adhesion molecule (CAM) profile of EC after retroviral gene transfer (GT), employing a clinically relevant ex vivo GT protocol. METHODS: Human umbilical vein EC (HUVEC, N = 14 isolates) were exposed to supernatants containing the MFG.nlsLACZ vector, which codes for a nuclear localized beta-galactosidase. Control HUVEC were exposed to empty virus (CRIP) or no virus (NT). Efficiency of GT was quantitated by direct counting of beta-galactosidase-stained cells on a grid. Proliferation was quantitated by a 1-week assay of viable cell counts. Expression of EC activation molecules (Class II major histocompatibility antigen [MHC II], E-selectin, intercellular adhesion molecule-1 [ICAM-1], and vascular cell adhesion molecule-1 [VCAM-1]) was examined using fluorescent cytometry (FACS) at rest and after cytokine stimulation. RESULTS: GT was reproducibly efficient (mean 57%, range 40-77%) using sequential viral exposures without selection. NT, CRIP, and LACZ-transduced HUVEC exhibited identical FACS profiles for E-selectin, ICAM-1, VCAM-1, and MHC II at rest, consistent with a nonactivated state. Upregulation of expression by cytokine was quantitatively similar for all groups. Growth rates were likewise not different between groups. CONCLUSIONS: Retroviral vectors may be employed to achieve high percentages of transduced EC for ex vivo GT without the use of selection. Transduced EC generated in this fashion are not activated, demonstrate an unaltered pattern of inducible CAM expression, and exhibit normal cell growth. The effects of GT on target cell phenotype are likely to be both vector and protocol specific and should be carefully assessed in each case prior to in vivo applications.

Cell Division↗
PubMed2002

Modified blasticidin S resistance gene (bsrm) as a selectable marker for construction of retroviral vectors.

Retroviral vectors are commonly used in ex vivo gene therapy protocols. The structure of vectors basically consists of one gene of interest and a selectable marker gene. Fast selection without damaging cells is a critical step for ex vivo gene therapy protocols. Blasticidin S deaminase isolated from Bacillus cereus has a neutralizing action on the highly toxic antibiotic blasticidin S (BS). A commercially available gene coding for blasticidin S deaminase (bsr) when used to construct retroviral vectors, LBSN and LNSB, provided very low levels of BS deaminase activity, precluding their routine use in gene transfer experiments. However, with the introduction of specific mutations into the bsr gene based on the Kozak consensus sequences and deletion of a 5' untranslated sequence to generate bsrm, we were able to construct a retroviral vector encoding resistance to high doses of BS (at least 16-fold above the usual lethal dose in NIH3T3 cells), showing that bsrm/BS may provide a useful system for selection of transduced mammalian cells.

3T3 Cells↗
PubMed2000

Ex vivo T lymphocyte expansion for retroviral transduction: influence of serum-free media on variations in cell expansion rates and lymphocyte subset distribution.

OBJECTIVE: In the setting of allogeneic stem cell transplantation, suicide gene-manipulated donor T cells that can be selectively inactivated in vivo would potentially allow optimal control of the GVL (graft-vs-leukemia)/GVHD (graft-vs-host disease) balance. Retroviral T-cell transduction requires ex vivo cell expansion, which is often achieved by IL-2 and anti-CD3 stimulation. Traditionally, culture media for cell expansion are supplemented with fetal bovine serum (FBS) or human serum. While these sera promote cell growth and viability, they contain uncharacterized elements that may yield inconsistent results from batch to batch. Cell expansion in serum-free media would therefore be preferable. MATERIALS AND METHODS: We compared T-cell expansion rates in three commercially available serum-free culture media (X-VIVO 15, AIM-V, and Cellgro SCGM), with or without the addition of human serum (HS, 5%). We also aimed to evaluate how the in vitro expansion affected the composition of the various T-cell subsets. Buffy-coats from four healthy donors were expanded for 21 days. The media were compared to standard RPMI 1640 medium, supplemented with HS (5%) or FBS (10%). For retroviral transductions, the LN vector carrying the neomycin- resistance gene was used in four additional donors. RESULTS: In our hands, X-VIVO 15 gave the highest rate of serum-free expansion (a median of 79-fold expansion, range 20-117). For serum-free expansion, activation with OKT3 for 21 days gave slightly higher expansion rates than a 5-day course (however, without statistical significance). When serum was added, this discrepancy was not seen. Cytokine analysis (IFN-gamma, IL-10, and IL-4) showed a distinct type1 cytokine pattern with elevated IFN-gamma levels during the whole period of culture. Flow cytometric analyses showed substantial inter-media, but also some inter-donor, variability in T-cell subset compositions. Transduction of cells with the LN vector and G418 selection resulted in a 14-fold increase (range 3-18) for serum-free X-VIVO 15 based cultures. Cell phenotypes remained unchanged by the transduction procedure as compared to nontransduced cells. CONCLUSION: Among the tested serum-free media, X-VIVO 15 has shown to best support the in vitro expansion of T cells, resulting in equal percentages of CD4(+) and CD8(+) cells. These cells can easily be transduced and selected. There seem to be no significant benefits, regarding absolute cell numbers or T-cell subset compositions, with OKT3-stimulation for more than five days. The addition of low levels of HS increases the consistencies in the cell expansion rates for all media.

Animals↗
PubMed2001

Treatment of ischemic wounds using cultured dermal fibroblasts transduced retrovirally with PDGF-B and VEGF121 genes.

The healing of ischemic wounds is a particularly difficult clinical challenge. In this study, rabbit dermal fibroblasts transduced retrovirally with human platelet-derived growth factor B (PDGF-B) and human vascular endothelial growth factor 121 (VEGF121) genes were used to treat wounds in a rabbit ischemic ear model. The PDGF-B and VEGF121 genes were obtained from human umbilical vein endothelial cells (HUVECs) by reverse transcription-polymerase chain reaction, cloned into retroviral vectors under control of the beta-actin promoter, and introduced into primary rabbit dermal fibroblast cells. In vitro results demonstrated that rabbit dermal fibroblasts are transduced and selected readily using retroviral vectors, and are engineered to secrete PDGF-B and VEGF121 at steady-state levels of 150 ng per 10(6) cells per 24 hours and 230 ng per 10(6) cells per 24 hours respectively. These cells were then seeded onto polyglycolic acid (PGA) scaffold matrices and used to treat ischemic rabbit ear wounds. Immunohistochemistry showed intense staining for PDGF-B and VEGF121 in the wounds treated with these transduced cells compared with the control treatment groups. For the relatively more ischemic distal ear wounds, granulation tissue deposition was increased significantly in the wounds treated with PDGF-B- and VEGF121-transduced cells compared with wounds treated with PGA alone. These results demonstrate that gene augmentation of rabbit dermal fibroblasts with the PDGF-B and VEGF121 genes introduced into this ischemic wound model via PGA matrices modulates wound healing, and may have clinical potential in the treatment of ischemic wounds.

Animals↗
PubMed2005

Recognition of fresh human tumor by human peripheral blood lymphocytes transduced with a bicistronic retroviral vector encoding a murine anti-p53 TCR.

The p53 protein is markedly up-regulated in a high proportion of human malignancies. Using an HLA-A2 transgenic mouse model, it was possible to isolate high-avidity murine CTLs that recognize class I-restricted human p53 epitopes. We isolated the alpha- and beta-chain of a TCR from a highly avid murine CTL clone that recognized the human p53(264-272) epitope. These genes were cloned into a retroviral vector that mediated high efficiency gene transfer into primary human lymphocytes. Efficiencies of >90% for gene transfer into lymphocytes were obtained without selection for transduced cells. The p53 TCR-transduced lymphocytes were able to specifically recognize with high-avidity, peptide-pulsed APCs as well as HLA-A2.1+ cells transfected with either wild-type or mutant p53 protein. p53 TCR-transduced cells demonstrated recognition and killing of a broad spectrum of human tumor cell lines as well as recognition of fresh human tumor cells. Interestingly, both CD8+ and CD4+ subsets were capable of recognizing and killing target cells, stressing the potential application of such a CD8-independent TCR molecule that can mediate both helper and cytotoxic responses. These results suggest that lymphocytes genetically engineered to express anti-p53 TCR may be of value for the adoptive immunotherapy of patients with a variety of common malignancies.

Animals↗
PubMed1993

BQ123, an ETA receptor antagonist, inhibits endothelin-1-mediated proliferation of human pulmonary artery smooth muscle cells.

Endothelin (ET-1) has been shown to be co-mitogenic for vascular smooth muscle cells (SMC) from human systemic arteries. A more modest growth-promoting effect has also been described in SMC from the bovine and porcine pulmonary circulation. Whether ET-1 has mitogenic properties in the human pulmonary circulation, and which ET receptor subtype mediates this response, is unknown. We first examined the effects of ET-1, ET-3, and the selective ETB agonist, Sarafotoxin 6c, on human pulmonary artery SMC growth. Cells were harvested from normal lung transplant donors. Growth was assessed by change in cell number 3 days after stimulation of quiescent cells. ET-1 in the presence of 0.3% serum produced a dose-dependent increase (82 +/- 1.5%) in cell number (threshold, 10(-11) M; maximal, 10(-7) M). ET-3 also stimulated growth (36 +/- 3.8%) but was less potent than ET-1 (threshold, 10(-9) M; maximal, 10(-7) M). The ETB selective agonist Sarafotoxin 6c had no proliferative effect. The effects of BQ123, a selective ETA receptor antagonist, on ET-1-induced growth were then assessed. BQ123 inhibited (threshold, 1.5 x 10(-7) M; maximal, 1.5 x 10(-5) M) ET-1-induced growth but had no effect on proliferation stimulated by the non-ET receptor-mediated growth factors, platelet-derived growth factor BB and 5-hydroxytryptamine. These results suggest that ET-1 is a potent co-mitogen for human proximal pulmonary artery SMC and that this effect is transduced by selective activation of the ETA receptor.

Analysis of Variance↗