Systemic injection of AAV9-GDNF provides modest functional improvements in the SOD1G93A ALS rat but has adverse side effects.

Injecting proteins into the central nervous system that stimulate neuronal growth can lead to beneficial effects in animal models of disease. In particular, glial cell line-derived neurotrophic factor (GDNF) has shown promise in animal and cell models of Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis (ALS). Here, systemic AAV9-GDNF was delivered via tail vein injections to young rats to determine whether this could be a safe and functional strategy to treat the SOD1G93A rat model of ALS and, therefore, translated to a therapy for ALS patients. We found that GDNF administration in this manner resulted in modest functional improvement, whereby grip strength was maintained for longer and the onset of forelimb paralysis was delayed compared to non-treated rats. This did not, however, translate into an extension in survival. In addition, ALS rats receiving GDNF exhibited slower weight gain, reduced activity levels and decreased working memory. Collectively, these results confirm that caution should be applied when applying growth factors such as GDNF systemically to multiple tissues.

Drosophila modifier screens to identify novel neuropsychiatric drugs including aminergic agents for the possible treatment of Parkinson's disease and depression.

Small molecules that increase the presynaptic function of aminergic cells may provide neuroprotection in Parkinson's disease (PD) as well as treatments for attention deficit hyperactivity disorder (ADHD) and depression. Model genetic organisms such as Drosophila melanogaster may enhance the detection of new drugs via modifier or 'enhancer/suppressor' screens, but this technique has not been applied to processes relevant to psychiatry. To identify new aminergic drugs in vivo, we used a mutation in the Drosophila vesicular monoamine transporter (dVMAT) as a sensitized genetic background and performed a suppressor screen. We fed dVMAT mutant larvae ∼ 1000 known drugs and quantitated rescue (suppression) of an amine-dependent locomotor deficit in the larva. To determine which drugs might specifically potentiate neurotransmitter release, we performed an additional secondary screen for drugs that require presynaptic amine storage to rescue larval locomotion. Using additional larval locomotion and adult fertility assays, we validated that at least one compound previously used clinically as an antineoplastic agent potentiates the presynaptic function of aminergic circuits. We suggest that structurally similar agents might be used to development treatments for PD, depression and ADHD, and that modifier screens in Drosophila provide a new strategy to screen for neuropsychiatric drugs. More generally, our findings demonstrate the power of physiologically based screens for identifying bioactive agents for select neurotransmitter systems.

Role of extracellular calcium influx in EGF-induced osteoblastic cell proliferation.

This study investigated the effects of epidermal growth factor (EGF) on cytosolic calcium ([Ca++]i) levels in rat calvarial osteoblasts, the nature of the regulation of this event, and the role these EGF-induced [Ca++]i changes have in osteoblastic cell proliferation. EGF significantly increased [Ca++]i measured in fura-2-loaded, individual cells. This increase was related to extracellular calcium influx. Activation of protein kinase C(PKC) by pretreating the cells with phorbol esters blocked the EGF-induced increase in [Ca++]i. EGF failed to increase inositol trisphosphate levels measured by high performance liquid chromatographic analysis. However, it did increase inositol bisphosphate and inositol tetrakisphosphate production. The EGF-dependent increase in DNA synthesis was partially blocked by the addition of calcium channel blockers. Therefore, it appears that the mechanism of action of EGF-induced osteoblastic cell proliferation is mediated by changes in [Ca++]i primarily due to extracellular calcium influx.

A rapid desktop theophylline assay. Evaluation of use in clinical management.

STUDY OBJECTIVES: To assess the practicality and precision of a simple desktop theophylline assay (Biotrack 516TM) in comparison to a standard laboratory assay system (Abbott TDxTM). DESIGN: A prospective blinded paired sample study. SETTING: The respiratory ward and outpatient clinics, the pulmonary function and biochemistry laboratories of a university teaching hospital. PATIENTS: Sixty patients with asthma or COPD attending the respiratory service. MEASUREMENTS AND RESULTS: Paired specimens for theophylline assay were collected simultaneously for analysis. There was a highly significant (p < 0.001) correlation between the two assay systems for both the total range studied (1.2-39.1 micrograms/mL; r = 0.98), and the clinically important range of 5-15 micrograms/mL (r = 0.95). The limits of agreement for the data by the Bland and Altman method indicated a +/-2 micrograms/mL limit for the 5-15 micrograms/mL range and a +/-2.7 micrograms/mL limit for the total range studied. CONCLUSIONS: The Biotrack 516 is an easy-to-use system, which provides rapid and reasonably precise measurements of serum theophylline levels. The device should be of particular value in smaller centers without an on-site laboratory assay system.

The impact of age on delivered dose intensity and hospitalizations for febrile neutropenia in patients with intermediate-grade non-Hodgkin's lymphoma receiving initial CHOP chemotherapy: a risk factor analysis.

The purpose of this historical case series study was to evaluate the association of age on delivered dose intensity of initial CHOP (cyclophosphamide/doxorubicin/ vincristine/prednisone) chemotherapy and the occurrence of hospitalizations for febrile neutropenia for patients with intermediate-grade non-Hodgkin's lymphoma (NHL). Findings are reported for 12 managed community and academic practices. Medical records of 930 NHL patients not enrolled on clinical trial protocols were reviewed. We reported on 577 of the study patients (62%) who received initial CHOP chemotherapy. Median age of the patients was 65.1 years. Older patients (age > or = 65 years) had more hospitalizations for febrile neutropenia (28% vs. 16%; P < 0.05) than younger patients (age, 18-64 years). In patients with advanced-stage NHL (stage III/IV), older patients received fewer cycles of CHOP (< 6 cycles, 35% vs. 22%; P < 0.05) than younger patients. Older patients were planned for lower average relative dose intensity (ARDI < or = 80%; P < 0.05) and had more heart disease and comorbid conditions (P < 0.05) than younger patients. Multiple logistic regression models showed that older patients were more likely to receive a lower dose intensity (ARDI < or = 80%; odds ratio = 2.46, 95% confidence interval [CI]: 1.62-3.72) during their first 3 cycles of therapy and to experience more hospitalizations for febrile neutropenia (odds ratio = 2.17, 95% CI: 1.43-3.30). We found the dose intensity of delivered CHOP chemotherapy for elderly patients to be less than standard CHOP therapy and the risk of hospitalizations for febrile neutropenia to be greater than in younger patients. Prospective clinical trials examining supportive care measures, such as colony-stimulating factor, for elderly NHL patients are recommended.

Health resource utilization in ABMT with and without G-CSF in stage III/IV breast cancer patients.

G-CSF has been available since 1991 for use in patients receiving high-dose chemotherapy/ABMT, and while it has been shown to effectively reduce the risk of febrile neutropenia, its cost effectiveness has been open to question. In this small retrospective study, five indicators of the consumption of health care resources were examined in stage III/IV breast cancer patients who received high-dose chemotherapy with ABMT or peripheral stem cell support. The study covered the time periods before and after the availability of G-CSF. The results showed that patients who received G-CSF had reductions in length of hospital stay of 20% (the purged marrow group) and 17% (nonpurged group), compared with similar groups that did not receive the growth factor; the shortest lengths of stay were seen in the peripheral stem cell group, all of whom received G-CSF. Other findings, including number of days the ANC fell below 500, total days of G-CSF use, and total days of antibiotic use, are presented.

Patterns of chemotherapy administration in patients with intermediate-grade non-Hodgkin's lymphoma.

Records from 653 patients treated between 1991 and 1998 in the Oncology Practice Patterns Study (OPPS) were analyzed to determine contemporary chemotherapy delivery patterns in patients with intermediate-grade non-Hodgkin's lymphoma (NHL). Of the 653 patient records reviewed, 90 (14%) omitted an anthracycline or mitoxantrone (Novantrone) from primary therapy. Among patients receiving CHOP (cyclophosphamide [Cytoxan, Neosar], doxorubicin HCl, vincristine [Oncovin], prednisone) or CNOP (cyclophosphamide, mitoxantrone, vincristine, prednisone), 134 (27%) of 492 received an average relative dose intensity of less than 80% of the literature-referenced dose, due either to an inadequate planned or delivered dose. Of 181 advanced-stage patients with responsive disease, 28 (15%) failed to receive at least six treatment cycles. Overall, 283 (43%) of 653 patients potentially received suboptimal chemotherapy due either to choice of regimen or chemotherapy delivered. Patient age > or = 65 years and cardiac comorbidity appeared to have the greatest influence on a physician's decision regarding chemotherapy administration. Among the 492 patients who received CHOP or CNOP, 235 (48%) experienced a delay or reduction in chemotherapy dose (usually neutropenia-related), 100 (20%) developed mucositis, and 116 (24%) were hospitalized for febrile neutropenia. Growth factor was administered to 261 patients (53%), and its primary prophylactic use was associated with a significant reduction in the incidence of hospitalizations for febrile neutropenia in all patient subgroups receiving appropriate chemotherapeutic dose intensity (P = .02). This assessment of chemotherapy delivery to patients with intermediate-grade NHL showed significant variation from current standards. Further analysis of factors influencing chemotherapy delivery might improve therapeutic outcomes.

Cost-effectiveness of autologous bone marrow transplantation.

The impact of filgrastim on the use of health care resources during recovery from autologous bone marrow transplantation (ABMT) was studied. The charts for patients with metastatic breast cancer treated with ABMT at a general hospital between November 1989 and July 1993 were reviewed by Blue Cross of Western Pennsylvania. The 58 patients were divided into five groups: group 1-bone marrow purged, no filgrastim therapy; group 2-bone marrow not purged, no filgrastim therapy; group 3-bone marrow purged, filgrastim therapy after ABMT; group 4-bone marrow not purged, filgrastim therapy after ABMT; and group 5-peripheral blood stem cells (PBSCs) given, followed by filgrastim therapy. The groups were compared for total length of stay (LOS), number of days the absolute neutrophil count (ANC) was < 500/cu mm, total number of days of filgrastim therapy, and total number of cumulative unit days of antimicrobial use. Total LOS was shorter for patients who received filgrastim (groups 3-5) than for patients who did not. Filgrastim was associated with fewer days of ANC < 500/cu mm in groups 4 and 5. The total number of cumulative unit days of antimicrobial use was lower in filgrastim recipients. Patients who received PBSCs needed fewer days of filgrastim therapy than the other filgrastim recipients. The health insurance company determined that, as a result of filgrastim therapy and PBSC transplantation, ABMT costs to the company have dropped by more than 50% since 1990. Patients now have available an alternative to conventional therapy for metastatic breast cancer without prejudice or penalty from their payer.

Developing an in-house physician advisor program.

The rapid rise in health care costs during the 1980s has led to a growing demand for utilization management companies, supported by teams of physician advisors. The increasing involvement of physician advisors in day-to-day case review has also led to a growing necessity for their being hired on an in-house basis. This article attempts to show a basic process for developing a functional and efficient in-house physician advisor program.

A managed care approach to outpatient review.

The recent growth in outpatient services has been the result of both increased use of new and improved technology, along with a shift to an ambulatory setting for many previously performed inpatient tests and procedures. Employers have seen an almost unchecked rapid growth in this portion of health care costs, with no signs of slowing down. This article attempts to show a basic step-by-step approach to identifying and reviewing some of the major cost generators through a managed care program.

Development of an outpatient utilization review program.

Usage of outpatient services has reached an all time high. Insurers and employers seek means to monitor the escalating costs for providing ambulatory care. This article offers insight about the objectives of managed care groups and what they are looking for in their outpatient utilization review endeavors.

The impact of lifestyle health risk on the bottom line: a case study.

As the Lifestyle Advantage study continues, HPS expects to follow the utilization trends for a consistent group of participants and nonparticipants, maintaining full confidentiality. Several key issues will be addressed in the process: As the participation rate increases, will the claims payment gap between participants and nonparticipants be reduced? Can a company hope to see a return on an investment in a health promotion program? If so, over what period of time? Finding answers to these questions will contribute to the larger issues of long-term health care cost containment and health care reform.

The mouse Thy-1.2 glycoprotein gene: complete sequence and identification of an unusual promoter.

Recombinant bacteriophage and cosmid clones containing the gene for the mouse Thy-1.2 glycoprotein were isolated and characterized. The complete sequence of the gene was determined, including a previously unidentified exon located 2.1 kb upstream of the portion of the gene encoding the Thy-1.2 glycoprotein. The transcriptional initiation site was located by S1 nuclease protection mapping in both T lymphocytes and neural cells and was found to be located immediately upstream of this exon. S1 nuclease protection mapping was also used to define the 3' end of the Thy-1.2 transcription unit, and no evidence for alternate mRNA processing was found. Thus, the mouse Thy-1.2 gene is 5447 base pairs in length, including promoter sequences, rather than 2094 as previously described. The mouse and rat Thy-1 genes are highly homologous in both introns and exons. However, the mouse Thy-1 cDNA and rat Thy-1 cDNA differ significantly in sequence in the 5' untranslated region. This suggests that the transcriptional initiation site of the mouse and rat genes may be located at different positions within the genomic sequence and may be related to the differing tissue distribution of Thy-1 in the two species.

Molecular organization of the human CD3 gene family on chromosome 11q23.

The genes encoding three invariant components of the human T-cell antigen receptor, the CD3 delta, gamma, and epsilon chains, are located on human chromosome 11 at band q23. We isolated cosmid clones containing the human CD3 delta and gamma chain genes in vectors designed for rapid and efficient chromosome "walking". The human CD3 epsilon gene was located in the region immediately downstream of the CD3 delta and gamma genes using synthetic oligonucleotide probes and the localization of this gene confirmed by DNA sequencing. Detailed restriction mapping of the CD3 locus demonstrated that all three CD3 subunits are encoded within 60 kb of DNA with the CD3 epsilon gene located 26 kb downstream of the CD3 delta and gamma genes. Analysis of genomic DNA on pulsed field gels using probes isolated from these cosmid clones defined a physical map of 750 kb spanning the CD3 locus on human chromosome 11q23. The CD3 genes thus comprise a multigene family encoding cell surface components important for transmembrane signaling on T lymphocytes. The arrangement of these genes suggest that they may share common regulatory elements for the control of gene expression during T-cell ontogeny.

Cyclic AMP decreases the expression of a neuronal marker (GAD67) and increases the expression of an astroglial marker (GFAP) in C6 cells.

C6 cells express proteins and mRNAs that are characteristic of both glia and neurons. Agents that increase intracellular levels of cyclic AMP (cAMP) decrease the enzymatic activity of glutamate decarboxylase (GAD), a neuronal marker, and the mRNA levels for one of the two GAD isoenzymes, GAD67. This reduction is accompanied by increased levels of glial fibrillary acidic protein (GFAP) mRNA, an astrocyte marker. Transient transfection assays, in which a 2-kb upstream regulatory region of the human GFAP gene was linked to a reporter gene, indicate that at least some of the cAMP-mediated increase of GFAP mRNA levels is due to increased transcription. Increases in intracellular cAMP appear to induce differentiation of C6 cells toward a more mature astrocyte phenotype.

Two invariant tryptophans on the alpha1 subunit define domains necessary for GABA(A) receptor assembly.

Two invariant tryptophan residues on the N-terminal extracellular region of the rat alpha1 subunit, Trp-69 and Trp-94, are critical for the assembly of the GABA(A) (gamma-aminobutyric acid, type A) receptor into a pentamer. These tryptophans are common not only to all GABA(A) receptor subunits, but also to all ligand-gated ion channel subunits. Converting each Trp residue to Phe and Gly by site-directed mutagenesis allowed us to study the role of these invariant tryptophan residues. Mutant alpha1 subunits, coexpressed with beta2 subunits in baculovirus-infected Sf9 cells, displayed high affinity binding to [(3)H]muscimol, a GABA site ligand, but no binding to [(35)S]t-butyl bicyclophosphorothionate, a ligand for the receptor-associated ion channel. Neither [(3)H]muscimol binding to intact cells nor immunostaining of nonpermeabilized cells gave evidence of surface expression of the receptor. When expressed with beta2 and gamma2 polypeptides, the mutant alpha1 polypeptides did not form [(3)H]flunitrazepam binding sites though wild-type alpha1 polypeptides did. The distribution of the mutant receptors on sucrose gradients suggests that the effects on ligand binding result from the inability of the mutant alpha1 subunits to form pentamers. We conclude that Trp-69 and Trp-94 participate in the formation of the interface between alpha and beta subunits, but not of the GABA binding site.

Recombinant adeno-associated virus (AAV) drives constitutive production of glutamate decarboxylase in neural cell lines.

Many neurological disorders result directly or indirectly from the loss of inhibitory function. Engineering the production of GABA, an inhibitory neurotransmitter, may therefore be able at least partly to restore the lost inhibition seen in epilepsy, Parkinson's disease, or Huntington's disease. In this article, we describe a set of recombinant adeno-associated viruses (AAVs) that can deliver cDNAs encoding the GABA-producing enzyme, glutamate decarboxylase (GAD), directly into neural cells. We have characterized these recombinant AAVs in several cell lines derived from the CNS. These recombinant AAVs effectively transduced all neural cell lines, although with different efficiencies. Transduction occurred in both proliferating and nonproliferating cells, but actively proliferating cell lines had approximately six times greater transduction efficiency than nonproliferating cells. Furthermore, these AAVs maintained long-term expression of GAD in an astrocytic cell line for at least seven passages. These recombinant AAVs are promising vehicles for investigating the potential therapeutic effects of GABA in animal models of epilepsy and neurodegenerative diseases.