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Biomedical subjects

J C Olsen

Publications and source records attributed to J C Olsen.

At least 19 recordsLinked to original sources
PubMed2006

Influenza M2 envelope protein augments avian influenza hemagglutinin pseudotyping of lentiviral vectors.

Lentivirus-based gene transfer has the potential to efficiently deliver DNA-based therapies into non-dividing epithelial cells of the airway for the treatment of lung diseases such as cystic fibrosis. However, significant barriers both to lung-specific gene transfer and to production of lentivirus vectors must be overcome before these vectors can be routinely used for applications to the lung. In this study, we investigated whether the ability to produce lentiviral vectors pseudotyped with fowl plague virus hemagglutinin (HA) could be improved by co-expression of influenza virus M2 in vector-producing cells. We found that M2 expression led to a 10-30-fold increase in production of HA-pseudotyped lentivirus vectors based upon equine infectious anemia virus (EIAV) or human immunodeficiency virus type 1 (HIV-1). Experiments using the M2 inhibitor amantadine and a drug-resistant mutant of M2 established that the ion channel activity of M2 was important for M2-dependent augmentation of vector production. Furthermore, the neuraminidase activity necessary for particle release from producer cells could also be incorporated into producer cells by co-expression of influenza NA cDNA. Lentiviral vectors pseudotyped with influenza envelope proteins were able to efficiently transduce via the apical membrane of polarized mouse tracheal cultures in vitro as well as mouse tracheal epithelia in vivo.

Animals↗
PubMed2005

Optimization of equine infectious anemia derived vectors for hematopoietic cell lineage gene transfer.

Gene transfer into hematopoietic cells may allow correction of a variety of hematopoietic and metabolic disorders. Optimized HIV-1 based lentiviral vectors have been developed for improved gene transfer and transgene expression into hematopoietic cells. However, the use of HIV-1 based vectors for human gene therapy may be limited due to ethical and biosafety issues. We report that vectors based on the non-primate equine infectious anemia virus (EIAV) transduce a variety of human hematopoietic cell lines and primary blood cells. To investigate optimization of gene expression in hematopoietic cells, we compared a variety of post-transcriptional elements and promoters in the context of EIAV vectors. We observed cell specific increase in the number of transgene expressing cells with the different post-transcriptional elements, whereas the use of elongation factor alpha 1 (EFalpha1) promoter resulted in significant increases in both the number of transgene expressing cells and the level of transgene protein in all cell types tested. We then demonstrate increased transduction of hematopoietic cells using a second-generation EIAV vector containing a self-inactivating EIAV LTR and the EIAV central polypurine tract (cppt). These data suggest that optimized EIAV vectors may be a suitable alternative to HIV-1 vectors for use in hematopoietic gene therapy.

Animals↗
PubMed2003

Analysis of gene transfer and expression in skeletal muscle using enhanced EIAV lentivirus vectors.

Skeletal muscle is an attractive target tissue for gene therapy involving both muscle and nonmuscle disorders. HIV-1-based vectors transduce mature skeletal muscle; however, the use of these vectors for human gene therapy may be limited by biosafety concerns. In this study, we investigated gene transfer using lentivirus vectors based on the equine infectious anemia virus (EIAV) in skeletal muscle in vitro and in vivo. EIAV vectors transduce proliferating and differentiating C2C12 mouse muscle cells; furthermore, the addition of the woodchuck hepatitis posttranscriptional element to EIAV vectors markedly increases gene expression in these cells. A single injection of EIAV vectors into skeletal muscle of adult mice led to detectable gene marking and gene expression for the duration of the 3-month study. Use of a second-generation EIAV self-inactivating vector (E-SIN) increased transduction in muscle cells in vitro, and injection of E-SIN vectors into skeletal muscle resulted in increased gene marking and gene expression compared to first-generation EIAV vectors.

Animals↗
PubMed2002

Comparison of gene transfer efficiencies and gene expression levels achieved with equine infectious anemia virus- and human immunodeficiency virus type 1-derived lentivirus vectors.

This report compares gene transfer efficiencies as well as durations and levels of gene expression for human immunodeficiency virus (HIV) and equine infectious anemia virus (EIAV) lentiviral vectors in a variety of human cell types in vitro. EIAV and HIV vectors transduced equivalent numbers of proliferating and G1/S- and G2/M-arrested cells, and both had very low efficiencies of transduction into G0-arrested cells. Analysis of the levels of both the enhanced green fluorescent protein (EGFP) and mRNA demonstrated that the HIV-transduced cells expressed greater levels of EGFP protein and RNA than the EIAV-transduced cells. Measurements of vector-derived EGFP RNA half-lives were fourfold higher with the HIV vector than with the EIAV vector. Long-term culture of EIAV-transduced human cells showed a significant decrease in the number of cells expressing the transgene; however, no corresponding loss was found in EIAV-transduced equine cells. In contrast, only a moderate decrease in the number of transgene-expressing cells was seen with the HIV vectors. Taken together, these results demonstrate that the EIAV vectors transduced human cells with efficiencies similar to those of the HIV vectors. However, our data indicate that transgene expression from EIAV vectors is limited by the instability of vector-derived RNA transcripts and silencing of the EIAV vectors over time.

Animals↗
PubMed2001

Functional correction of fanconi anemia group C hematopoietic cells by the use of a novel lentiviral vector.

Lentiviral vectors transduce nondividing hematopoietic cells more efficiently than other currently available vector systems. Here we report the results of human hematopoietic cell gene transfer using lentiviral vectors based upon human immunodeficiency virus (HIV-1) and equine infectious anemia virus (EIAV). EIAV is a nonprimate lentivirus and is severely restricted in its host range to horses and closely related equines. We employed the EIAV vector system to test for gene transfer to human Fanconi anemia (FA) hematopoietic cells by comparison with HIV-1- and Moloney murine leukemia virus-based systems. Fanconi anemia is characterized by bone marrow failure secondary to stem cell dysfunction. Fanconi anemia group C EBV-transformed lymphoblasts were transduced with all three viral vectors. Phenotypic correction of FA cells, as measured by mitomycin C drug resistance, was observed with a similar efficiency in all vector systems. This is the first description of lentiviral correction of FA cells and suggests that lentiviral vectors may be useful for FA gene transfer.

Cell Cycle↗
PubMed2001

EIAV, CAEV and other lentivirus vector systems.

Lentiviruses that infect non-primates make up a diverse collection of viruses. Although these viruses have some features in common with HIV and other primate viruses, differences in genome organization and viral gene function have made the successful derivation of vectors from non-primate lentiviruses unpredictable. This Chapter discusses the construction and application of gene transfer systems derived from four non-primate lentiviruses including equine infectious anemia virus (EIAV), caprine arthritis encephalitis virus (CAEV), visna virus, and Jembrana disease virus (JDV).

Animals↗
PubMed2001

Patient movement in the Pacific theater: changing the strategic focus from airplane to patient.

The U.S. military health care system forms a vast network across thousands of miles to serve patients in the Pacific theater. The medical treatment facilities in the Pacific, however, act independently and do not effectively track patients in the air evacuation system. The patients and the tracking systems are so disconnected that patients' whereabouts are unknown to both the command structure and the medical treatment facilities as soon as the plane leaves the ground. Furthermore, the databases cannot analyze treatment trends, and the cost of transport--a major part of the cost of health care in the Pacific--is hidden inside the air evacuation system. To ensure that managed care works effectively in the Pacific, Tricare Pacific has created an Internet-based database that will support a new network of case managers and effectively track patients. The Lead Agency's analysis of aeromedical evacuation also concluded that the wartime method of routine patient transport is not efficient in peacetime and, in fact, delays treatment. The recommendations from this financial analysis will reduce patient delays, enhance access, and save millions of health care dollars.

Air Ambulances↗
PubMed2000

Megakaryocyte-targeted synthesis of the integrin beta(3)-subunit results in the phenotypic correction of Glanzmann thrombasthenia.

Glanzmann thrombasthenia is an inherited bleeding disorder characterized by qualitative or quantitative defects of the platelet-specific integrin, alphaIIbbeta(3). As a result, alphaIIbbeta(3) cannot be activated and cannot bind to fibrinogen, leading to a loss of platelet aggregation. Thrombasthenia is clinically characterized by mucocutaneous hemorrhage with episodes of intracranial and gastrointestinal bleeding. To develop methods for gene therapy of Glanzmann thrombasthenia, a murine leukemia virus (MuLV)-derived vector, -889Pl(A2)beta(3), was transduced into peripheral blood CD34(+) cells from 2 patients with thrombasthenia with defects in the beta(3) gene. The human alphaIIb promoter was used in this vector to drive megakaryocyte-targeted expression of the wild-type beta(3) subunit. Proviral DNA and alphaIIbbeta(3) biosynthesis were detected after in vitro differentiation of transduced thrombasthenic CD34(+) cells with megakaryocyte growth and development factor. Flow cytometric analysis of transduced patient samples indicated that 19% of megakaryocyte progeny expressed alphaIIbbeta(3) on the surface at 34% of normal receptor levels. Treatment of transduced megakaryocytes with a combination of agonists including epinephrine and the thrombin receptor-activating peptide induced the alphaIIbbeta(3) complex to form an activated conformation capable of binding fibrinogen as measured by PAC-1 antibody binding. Transduced cells retracted a fibrin clot in vitro similar to megakaryocytes derived from a normal nonthrombasthenic individual. These results demonstrate ex vivo phenotypic correction of Glanzmann thrombasthenia and support the potential use of hematopoietic CD34(+) cells as targets for alphaIIb promoter-driven MuLV vectors for gene therapy of platelet disorders. (Blood. 2000;95:3645-3651)

Antigens, CD↗
PubMed2000

Patient perceptions of the specialty of emergency medicine.

The study objective was to determine emergency department (ED) patients' perceptions of the specialty of emergency medicine. We surveyed a convenience sample of adult ED patients regarding their knowledge of the specialty of emergency medicine. Responses included: 22% believing that ED physicians have their own practice outside the ED; 26% of patients with primary care physicians expected to be seen by their primary care physician in the ED; 19% thought ED physicians care for patients after admission; 26% thought that ED physicians perform surgery, 62% perceived emergency medicine to be a specialty; 15% have heard of the American College of Emergency Physicians; 71% thought that ED physicians are board certified and 15% thought paramedics were ED physicians. Patients estimated ED physicians' mean annual mean salary to be $100,000 and 61% believe that ED physicians are hospital employees. In conclusion, the specialty of emergency medicine is not well understood by our patients.

Adolescent↗
PubMed2000

Video analysis of emergency medicine residents performing rapid-sequence intubations.

The purpose of this study was to evaluate Emergency Medicine resident physicians' compliance with our institution's rapid sequence intubation (RSI) protocol by the use of videotape analysis. We conducted a prospective, observational study of Emergency Medicine resident physicians (EM 1,2,3) as they were videotaped performing RSI on medical and trauma patients. The videotapes were reviewed by the study investigators to assess the rates of deviation from our standard RSI protocol. Forty-four RSIs performed by 33 residents were studied. The most common deviations from our standard RSI protocol concerned proper use of the Sellick maneuver (45%) and use of the end-tidal CO(2) detector (34%). Videotape analysis provides an objective measure of Emergency Medicine resident performance of RSI.

Clinical Competence↗
PubMed2000

Pseudotyped human lentiviral vector-mediated gene transfer to airway epithelia in vivo.

We used a replication defective human lentiviral (HIV) vector encoding the lacZ cDNA and pseudotyped with the vesicular stomatitis virus (VSV) glycoprotein (G) to evaluate the utility of this vector system in airway epithelia. In initial studies, apical application of vector to polarized well differentiated human airway epithelial cell cultures produced minimal levels of transgene expression whereas basolateral application of vector enhanced levels of transduction approximately 30-fold. Direct in vivo delivery of HIV vectors to the nasal epithelium and tracheas of mice failed to mediate gene transfer, but injury with sulfur dioxide (SO2) before vector delivery enhanced gene transfer efficiency to the nasal epithelium of both mice and rats. SO2 injury also enhanced HIV vector-mediated gene transfer to the tracheas of rodents. These data suggest that SO2 injury increases access of vector to basal cells and/or the basolateral membrane of airway surface epithelial cells. Quantification of gene transfer efficiency in murine tracheas demonstrated that transduction was more efficient when vector was delivered on the day of exposure (7.0%, n = 4) than when vector was delivered on the day after SO2 exposure (1.7%, n = 4).

Analysis of Variance↗
PubMed2000

Frequency of adverse reactions to prochlorperazine in the ED.

Prochlorperazine (Compazine, PCZ) is a frequently used medication in the emergency department (ED). Akathisia and dystonia are known adverse reactions to the use of this medication, but their incidence in the ED has not been well studied. We conducted a prospective, descriptive study to evaluate the frequency of akathisia and dystonia in the ED from the use of IV or IM PCZ in patients with nausea/vomiting or headache. Two hundred-twenty nine patients (> or =18 years old) were enrolled and contacted within 2 weeks of ED discharge to access the incidence of these adverse reactions. After the use of PCZ in the ED, 16% of patients developed akathisia and 4% developed dystonia. Emergency physicians and our patients need to be aware of these potential adverse reactions to the use of PCZ in the ED.

Adolescent↗
PubMed2000

Functional characterization of the dimer linkage structure RNA of Moloney murine sarcoma virus.

Several determinants that appear to promote the dimerization of murine retroviral genomic RNA have been identified. The interaction between these determinants has not been extensively examined. Previously, we proposed that dimerization of the Moloney murine sarcoma virus genomic RNAs relies upon the concentration-dependent interactions of a conserved palindrome that is initiated by separate G-rich stretches (H. Ly, D. P. Nierlich, J. C. Olsen, and A. H. Kaplan, J. Virol. 73:7255-7261, 1999). The cooperative action of these two elements was examined using a combination of genetic and antisense approaches. Dimerization of RNA molecules carrying both the palindrome and G-rich sequences was completely inhibited by an oligonucleotide complementary to the palindrome; molecules lacking the palindrome could not dimerize in the presence of oligomers that hybridize to two G-rich sequences. The results of spontaneous dimerization experiments also demonstrated that RNA molecules lacking either of the two stretches of guanines dimerized much more slowly than the full-length molecule which includes the dimer linkage structure (DLS). However, the addition of an oligonucleotide complementary to the remaining stretch of guanines restored the kinetics of dimerization to wild-type levels. The ability of this oligomer to rescue the kinetics of dimerization was dependent on the presence of the palindrome, suggesting that interactions within the G-rich regions produce changes in the palindrome that allow dimerization to proceed with maximum efficiency. Further, unsuccessful attempts to produce heterodimers between constructs lacking various combinations of these elements indicate that the G-rich regions and the palindrome do not interact directly. Finally, we demonstrate that both of these elements are important in maintaining efficient viral replication. Modified antisense oligonucleotides targeting the DLS were found to reduce the level of viral vector titer production. The reduction in viral titer is due to a decrease in the efficiency of viral genomic RNA encapsidation. Overall, our data support a dynamic model of retroviral RNA dimerization in which discrete dimerization elements act in a concerted fashion.

Animals↗
PubMed1999

Integrin alphaIIb promoter-targeted expression of gene products in megakaryocytes derived from retrovirus-transduced human hematopoietic cells.

Megakaryocyte-specific expression of the platelet-adhesion receptor, integrin alphaIIbbeta3, is caused by the presence of regulatory elements of the alphaIIb promoter that direct high-level, selective gene transcription early in megakaryocytopoiesis. To develop methods for targeted expression of transgenes, we transduced human CD34+ peripheral blood cells with a murine leukemia virus (MuLV) vector controlled by the human integrin alphaIIb promoter (nucleotides -889 to +35). A naturally occurring cDNA encoding the Pl(A2) alloantigen form (Pro(33)) of the integrin beta3 subunit was subcloned into this construct (-889Pl(A2)beta3) and transduced into cells that endogenously synthesized Pl(A1)beta3 (Leu(33)) as a marker for detection of provirus-derived beta3. The ability of this vector to target expression of Pl(A2)beta3 to megakaryocytes was first examined in cell lines. Immunoblot analysis with human anti-Pl(A2) alloserum detected synthesis of Pl(A2)beta3 in transduced promegakaryocytic cells; however, Pl(A2)beta3 protein was not detected in transduced epithelial cells. Human hematopoietic CD34+ cells were transduced with -889Pl(A2)beta3 virions and induced to differentiate with megakaryocyte growth and development factor. A hybrid alphaIIbbeta3 complex was formed in progeny megakaryocytes where provirus-derived Pl(A2)beta3 was detected associated with endogenous alphaIIb subunit. Another alphaIIb promoter-driven MuLV vector (-889nlacZ) encoding Escherichia coli beta-galactosidase was used to demonstrate that transgene expression was selectively targeted to the megakaryocyte progeny of transduced CD34+ cells. These studies demonstrate the feasibility of using alphaIIb promoter-driven MuLV vectors for gene transfer of hematopoietic CD34+ cells to target transgene expression in developing megakaryocytes and platelets and indicate potential applications toward human gene therapy for platelet disorders.

Antigens, CD34↗
PubMed1999

Moloney murine sarcoma virus genomic RNAs dimerize via a two-step process: a concentration-dependent kissing-loop interaction is driven by initial contact between consecutive guanines.

Retroviruses contain two plus-strand genomic RNAs, which are stably but noncovalently joined in their 5' regions by a dimer linkage structure (DLS). Two models have been put forward to explain the mechanisms by which the RNAs dimerize; each model emphasizes the role of specific molecular determinants. The kissing-loop model implicates interactions between palindromic sequences in the DLS region. The second model proposes that purine-rich stretches in the region form purine quartet structures. Here, we present an examination of the in vitro dimerization of Moloney murine sarcoma virus (MuSV) RNA in the context of these two models. Dimers were found to form spontaneously in a temperature-, time-, concentration-, and salt-dependent manner. In contrast to earlier reports, we found that deletion of neither the palindrome nor the consensus purine motifs (PuGGAPuA) affected the level of dimer formation at low concentrations of RNA. Rather, different purine-rich sequences, i.e., consecutive stretches of guanines, were found to enhance both in vitro RNA dimerization and in vivo viral replication. Biochemical evidence further suggests that these guanine-rich (G-rich) stretches form guanine quartet structures. We also found that the palindromic sequences could support dimerization at significantly higher RNA concentrations. In addition, the G-rich stretches were as important as the palindromic sequence for maintaining efficient viral replication. Overall, our data support a model that entails contributions from both of the previously proposed mechanisms of retroviral RNA dimerization.

Animals↗
PubMed1998

Death in the emergency department.

Issues regarding the deaths of patients in the ED arise on a regular basis for emergency physicians. These issues include physician discomfort with death notification, the approach to families after ED deaths, autopsies, donation of organs and tissues, and procedures on the newly dead. If physicians were more comfortable with death notification, not only would families be better served but benefits to society could be realized through the increased use of autopsy and organ/tissue donation. The controversial topic of physician education through practice of medical procedures on the newly dead weighs the benefits to society against the rights of the individual. Improved physician education, including the need for a death notification plan and enlistment of the support of nursing personnel, social workers, and clergy, may improve the experience of events surrounding ED deaths for physicians, families, and society. We review the literature and give recommendations on approaches to deal with these issues.

Attitude to Death↗