OBJECTIVE: To compare the long-term clinical, quality-of-life, and economic outcomes after an initial prescription for fluoxetine, imipramine hydrochloride, or desipramine hydrochloride. DESIGN: Randomized, controlled trial. SETTING: Primary care clinics of a staff-model health maintenance organization in the Seattle, Wash, area. PATIENTS: Four hundred seventy-one adults beginning antidepressant drug treatment for depression. INTERVENTION: Random assignment of initial medication (desipramine, fluoxetine, or imipramine), with treatment (dosing, medication changes or discontinuation, and follow-up visits) managed by a primary care physician. MEASUREMENTS: Interviews at baseline and at 6, 9, 12, 18, and 24 months examined medication use, clinical outcomes (Hamilton Depression Rating Scale and depression subscale of the Hopkins Symptom Checklist), and quality of life (Medical Outcomes Study SF-36 Health Survey). Medical costs were assessed using the health maintenance organization's accounting data. RESULTS: Patients assigned to fluoxetine therapy were significantly more likely to continue taking the initial antidepressant but no more likely to continue any antidepressant therapy. The fluoxetine group did not differ significantly from either tricyclic drug group on any measure of depression severity or quality of life. For 24 months, antidepressant drug costs were approximately $250 higher for patients assigned to fluoxetine therapy, but total medical costs were essentially identical. CONCLUSIONS: Initial selection of fluoxetine or a tricyclic antidepressant drug should lead to similar clinical outcomes, functional outcomes, and overall costs. Differences in antidepressant prescription costs are blunted by the large minority of tricyclic-treated patients who switch to use of more expensive medications. Restrictions on first-line use of fluoxetine in primary care will probably not reduce overall treatment costs.

Adrenergic Uptake Inhibitors↗

PubMedDate not supplied

Polycation-based DNA complexes for tumor-targeted gene delivery in vivo.

BACKGROUND: Efficient and target-specific in vivo gene delivery is a major challenge in gene therapy. Compared to cell culture application, in vivo gene delivery faces a variety of additional obstacles such as anatomical size constraints, interactions with biological fluids and extracellular matrix, and binding to a broad variety of non-target cell types. METHODS: Polycation-based vectors, including adenovirus-enhanced transferrinfection (AVET) and transferrin-polyethylenimine (Tf-PEI), were tested for gene delivery into subcutaneously growing tumors after local and systemic application. DNA biodistribution and reporter gene expression was measured in the major organs and in the tumor. RESULTS: Gene transfer after intratumoral application was 10-100 fold more efficient with Tf-PEI/DNA or AVET complexes in comparison to naked DNA. Targeted gene delivery into subcutaneously growing tumors after systemic application was achieved using electroneutral AVET complexes and sterically stabilized PEGylated Tf-PEI/DNA complexes, whereas application of positively charged polycation/DNA complexes resulted in predominant gene expression in the lungs and was associated by considerable toxicity. CONCLUSION: For systemic application, the physical and colloidal parameters of the transfection complexes, such as particle size, stability, and surface charge, determine DNA biodistribution, toxicity, and transfection efficacy. By controlling these parameters, DNA biodistribution and gene expression can be targeted to different organs.

Adenoviridae↗

PubMedDate not supplied

Transfection of epithelial cells is enhanced by combined treatment with mannitol and polyethyleneglycol.

BACKGROUND: Gene transfer efficiency drops significantly when polarized mammary epithelial cells are transfected instead of actively growing cells. However, fully differentiated cells are the targets for gene transfer in many in vivo applications. Therefore, a simple and effective method for the transfection of polarized mammary epithelial cells in confluent monolayers was developed. METHODS: Reporter gene plasmids were complexed with polyethylenimine with an average molecular weight of 25 kDa (PEI 25), or other agents, to transfect confluent monolayers of ovine mammary epithelial cells (OMEC II) or human carcinoma cells (CaCo-2) in vitro. The improved technique included pretreatment of the cells with a hyperosmotic mannitol solution (7%) which caused a loosening of the tight contacts between the cells. Alternatively, the mannitol shock could be replaced by a short treatment with trypsin or EDTA. In addition to the pretreatment, 12.5% polyethyleneglycol with an average molecular weight of 8000 kDa (PEG 8000) was included in the transfection mixture containing the DNA complexes. RESULTS: The combined application of mannitol and PEG resulted in a very reliable 5- to 30-fold increase in reporter gene expression in OMEC II and CaCo-2 cells, but not K562 cells (an example of another cell type). The improved technique can also be combined with other polymer-based transfection agents. The transfection rate was enhanced for confluent monolayer cells with fully developed epithelial polarity but also for subconfluent, growing epithelial cell cultures. CONCLUSIONS: A novel transfection protocol for epithelial cells is presented. The combined treatment of cells with mannitol and polyethyleneglycol results in substantial enhancement of in vitro transfection of epithelial cell lines.

Animals↗

PubMedDate not supplied

Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo.

BACKGROUND: Efficient gene transfer is a major challenge for non-viral gene therapy. Understanding how non-viral vectors initiate gene expression could lead to the development of new future vectors with enhanced efficacy. METHODS: Linear or branched polyethylenimine (PEI)/DNA complexes were generated in varying salt conditions and their transfection efficiencies were compared in vitro and in vivo using reporter genes, luciferase and green fluorescent protein, and rhodamine labeled DNA (pGeneGrip). RESULTS: The transfection efficiency of linear PEI22/DNA in vitro was generally greater than that of branched PEI/DNA when complexes were generated in salt containing buffer. However, PEI complexes generated under salt-free conditions generally had low transfection activity in vitro. In contrast, PEI22/DNA salt-free complexes were highly active in vivo. Branched PEI/DNA and salt containing PEI22/DNA complexes were generally 10-100-fold less active than the salt-free PEI22/DNA complexes. Salt-free PEI22/DNA complexes were small, but subsequently grew into aggregates when salt was added. In contrast, PEI25/DNA complexes remained small even after salt was added under the same conditions. Furthermore, PEI22/pGeneGrips complexes formed large aggregates associated with the cell membrane, cytoplasm and nucleus, while branched PEI complexes remained as small distinct particles associated with the cell membrane or in the cytoplasm. CONCLUSIONS: Branched and linear PEI/DNA complexes differ in their ability to transfect cells. The greater efficiency of linear PEI might be due to an inherent kinetic instability under salt conditions. Understanding how to employ this kinetic instability of linear PEI could help in designing future vectors with greater flexibility and transfection efficiency in vivo.

Animals↗

PubMedDate not supplied

Frustrating patients: physician and patient perspectives among distressed high users of medical services.

OBJECTIVE: To identify differences between patients viewed as frustrating by their physicians and those considered typical and satisfying. DESIGN: This cross-sectional observational study focused on psychologically distressed high users of medical services. Frustrating patients were compared with typical and satisfying patients, using data from patient questionnaires, physician assessments, structured psychiatric interviews, and computerized utilization records. SETTING: Group Health Cooperative of Puget Sound, a large health maintenance organization. PATIENTS/PARTICIPANTS: Study patients were in the top decile for ambulatory visits, and bad elevated scores for anxiety, depression, and somatization. Among the 339 patients invited to participate in the study, 251 agreed, and 228 were rated by their physicians. MAIN RESULTS: A substantial proportion (37%) of the high users were viewed as frustrating by their physicians. Physicians' ratings of physical disease severity did not differ among the groups, but frustrating patients rated their own health status less favorably and reported more somatic symptoms and disabilities. The frustrating group utilized more medical services than did other distressed high utilizers. All three groups had a high prevalence of mental disorders. However, frustrating patients had higher rates of somatization and generalized anxiety disorder. CONCLUSIONS: Physicians and their frustrating patients had contrasting views of the patients' illnesses. The best predictors of physician frustration were somatization and increased medical service utilization. There is need for further research and clinical attention concerning optimal clinical management for patients with somatization.

Adult↗

PubMedDate not supplied

DNA-binding transferrin conjugates as functional gene-delivery agents: synthesis by linkage of polylysine or ethidium homodimer to the transferrin carbohydrate moiety.

We have previously demonstrated that transferrin-polycation conjugates are efficient carrier molecules for the introduction of genes into eukaryotic cells. We describe here a more specific method for conjugation of transferrin with DNA-binding compounds involving attachment at the transferrin carbohydrate moiety. We used the polycation poly(L-lysine) or the DNA intercalator, ethidium homodimer as DNA-binding domains. Successful transferrin-receptor-mediated delivery and expression of the Photinus pyralis luciferase gene in K562 cells has been shown with these new transferrin conjugates. The activity of the transferrin-ethidium homodimer (TfEtD) conjugates is low relative to transferrin-polylysine conjugates; probably because of incomplete condensation of the DNA. However, DNA delivery with TfEtD is drastically improved when ternary complexes of the DNA with TfEtD and the DNA condensing agent polylysine are prepared. The gene delivery with the carbohydrate-linked transferrin-polylysine conjugates is equal or superior to described conjugates containing disulfide linkage. The new ligation method facilitates the synthesis of large quantities (greater than 100 mg) of conjugates.

Carbohydrate Sequence↗

PubMedDate not supplied

Different strategies for formation of pegylated EGF-conjugated PEI/DNA complexes for targeted gene delivery.

With the aim of generating gene delivery systems for tumor targeting, we have synthesized a conjugate consisting of polyethylenimine (PEI) covalently modified with epidermal growth factor (EGF) peptides. Transfection efficiency of the conjugate was evaluated and compared to native PEI in three tumor cell lines: KB epidermoid carcinoma cells, CMT-93 rectum carcinoma cells, and Renca-EGFR renal carcinoma cells. Depending on the tumor cell line, incorporation of EGF resulted in an up to 300-fold increased transfection efficiency. This ligand-mediated enhancement and competition with free EGF strongly suggested uptake of the complexes through the EGF receptor-mediated endocytosis pathway. Shielded particles being crucial for systemic gene delivery, we studied the effect of covalent surface modification of EGF-PEI/DNA complexes with a poly(ethylene glycol) (PEG) derivative. An alternative way for the formation of PEGylated EGF-containing complexes was also evaluated where EGF was projected away from PEI/DNA core complexes through a PEG linker. Both strategies led to shielded particles still able to efficiently transfect tumor cells in a receptor-dependent fashion. These PEGylated EGF-containing complexes were 10- to 100-fold more efficient than PEGylated complexes without EGF.

Animals↗

PubMedDate not supplied

Gene transfer into hepatocytes using asialoglycoprotein receptor mediated endocytosis of DNA complexed with an artificial tetra-antennary galactose ligand.

We have constructed an artificial ligand for the hepatocyte-specific asialoglycoprotein receptor for the purpose of generating a synthetic delivery system for DNA. This ligand has a tetra-antennary structure, containing four terminal galactose residues on a branched carrier peptide. The carbohydrate residues of this glycopeptide were introduced by reductive coupling of lactose to the alpha- and epsilon-amino groups of the two N-terminal lysines on the carrier peptide. The C-terminus of the peptide, containing a cysteine separated from the branched N-terminus by a 10 amino acid spacer sequence, was used for conjugation to 3-(2-pyridyldithio)propionate-modified polylysine via disulfide bond formation. Complexes containing plasmid DNA bound to these galactose-polylysine conjugates have been used for asialoglycoprotein receptor-mediated transfer of a luciferase gene into human (HepG2) and murine (BNL CL.2) hepatocyte cell lines. Gene transfer was strongly promoted when amphipathic peptides with pH-controlled membrane-disruption activity, derived from the N-terminal sequence of influenza virus hemagglutinin HA-2, were also present in these DNA complexes. Thus, we have essentially borrowed the small functional domains of two large proteins, asialoglycoprotein and hemagglutinin, and assembled them into a supramolecular complex to generate an efficient gene-transfer system.

Amino Acid Sequence↗

PubMedDate not supplied

Influence of membrane-active peptides on lipospermine/DNA complex mediated gene transfer.

To explore whether endosomal release presents a major barrier to lipospermine-mediated gene delivery, acidic membrane-active peptides derived from influenza virus or artificial sequences were incorporated into DNA/dioctadecylamidoglycylspermine (= Transfectam) complexes. Depending on the cell line used, gene expression levels are approximately 3-30-fold higher than those obtained by applying DNA complexed to optimal amounts of Transfectam alone. In addition, gene transfer efficiency of DNA complexes with lower amounts of Transfectam (1.5-2 charge equiv) is increased by a factor of up to 1000 by peptides INF6 (influenza virus derived sequence) and INF10 (artificial sequence). The helper lipids 1,2-dioleoylphosphatidylethanolamine, egg phosphatidylethanolamine, and 1,2-dioleoyl-racglycerol also can enhance the gene transfer. Thus, endosomal escape seems to be only a moderate barrier for optimized, positively charged DNA/Transfectam complexes, but a substantial bottleneck for less positively charged complexes.

Amino Acid Sequence↗

PubMedDate not supplied

Histopathologic findings in synthetic and biologic explanted grafts used in peripheral arterial reconstruction.

Surgically explanted vascular grafts, including 47 autografts, 14 allografts, 103 chemically processed human umbilical vein (HUV) grafts, and 406 polytetrafluoroethylene (PTFE) grafts, were studied histopathologically. Autografts, HUV, and PTFE grafts were implanted mostly in the femoropopliteal position, whereas allografts were used as a blood access for hemodialysis. The main cause for explantation in autografts (53.6%), HUV (74.8%), and PTFE (61.3%) was thrombosis, whereas allograft failure was usually related to aneurysmal degeneration (72.7%). The infection rates of HUV (20.9%) and PTFE (25%) grafts were higher than those of autografts (4.8%) and allografts (9.1%). Furthermore, bacterial colonization occurred more frequently in HUV (63.9%) and PTFE (64.7%) than in autografts (6.7%) and allografts (0%). Leukocyte infiltration was detected mostly in HUV (39.4%) and PTFE (27%) explants. Autografts and allografts both showed a 7.1% rate of lipid uptake, whereas lipid retention was observed in 84.9% of HUV and 79.8% of PTFE grafts. These results suggest that biologic vascular grafts are to be used preferentially in small diameter arterial reconstruction because of their resistance to bacterial colonization and lipid uptake.

Adult↗