Upregulation of antithrombotic ectonucleotidases by aspirin in human endothelial cells in-vitro

Abstract— Ecto ATP-diphosphohydrolase (apyrase) activity of human endothelial cells following aspirin treatment has been studied in-vitro. It was shown by HPLC analysis of supernatant samples that pre-incubation of the cultures with aspirin resulted in a significantly increased turnover of supplemented ATP into its degradation products (ADP and AMP). Enhanced expression of ectoenzyme after aspirin treatment could be observed as demonstrated by immunofluorescence-staining with monoclonal anti-apyrase antibodies. This suggests enhancement of endothelial ATP-diphosphohydrolase activity induced by aspirin. The present data obtained in human vascular cells in-vitro are in line with results from previous animal studies in-vivo, suggesting a novel cyclo-oxygenase-independent antithrombotic activity of aspirin.     

High efficiency gene transfer to the central nervous system of rodents and primates using herpes virus vectors lacking functional ICP27 and ICP34.5.

The safe and efficient use of herpes simplex virus (HSV)-based vectors to deliver genes of potentially therapeutic benefit to the central nervous system will require their effective disablement by the inactivation of viral genes required for lytic growth. Here we report that viruses lacking functional genes for ICP27 (which is required for growth in all cell types) and ICP34.5 (which is required for growth in nondividing cell types) can deliver a marker gene to both the rodent and primate CNS with high efficiency whilst producing relatively minimal damage and having no effect on sodium currents in dorsal root ganglion neurons. Such viruses paradoxically deliver genes at much higher efficiency than the less disabled single mutant lacking ICP34.5 alone and also, as expected, produce less damage in vivo. Moreover, unlike the single mutant lacking ICP27 the double mutant viruses cannot revert to wild-type by acquistion of complimenting gene sequences during growth of virus stocks in vitro on dividing cells expressing ICP27 since artificial expression of ICP34.5 in these cells is not required. Such ICP27-; ICP34.5- viruses thus offer a platform for the development of vectors which are sufficiently safe for ultimate use in human gene therapy.     

Pharmacological profile of SCH39166: a dopamine D1 selective benzonaphthazepine with potential antipsychotic activity

SCH39166 [(-)-trans-6,7,7a,8,9,13b-hexahydro-3-chloro-2-hydroxy-N-methyl- 5H-benzo[d]naptho-(2,1-b)azepine] is a benzonaphthazepine that has been evaluated as a selective D1 dopamine receptor antagonist. In vitro, SCH39166 (Ki = 3.6 nM) inhibited the binding of [3H]SCH23390 (a D1 specific compound) and blocked dopamine-stimulated adenylate cyclase (Ki = 9.1 nM); in contrast the Ki for SCH39166 to displace [3H]spiperone (D2) was greater than 1 microM and its Ki vs. [3H]-ketanserin (5-hydroxytryptamine2) binding was greater than 300 nM. In vivo, SCH39166 inhibited both rat and squirrel monkey conditioned avoidance responding (minimal effective dose = 10 and 1.78 mg/kg p.o., respectively) and had a duration of at least 6 hr in both species. In addition, SCH39166 antagonized apomorphine-induced stereotypy in rats (minimal effective dose = 10 mg/kg p.o.). These in vivo actions of SCH39166 are similar to the activity of typical dopamine antagonists. However, in contrast to D2-selective antagonists, SCH39166 failed to increase plasma prolactin levels, did not block apomorphine-induced emesis in the dog and had minimal effects on the striatal levels of homovanillic acid or dihydroxyphenylacetic acid. Furthermore, although immobility was seen after p.o. administration of SCH39166 using the inclined screen test, the drug did not cause catalepsy at doses up to 10 times its minimal effective dose in the rat conditioned avoidance response test. Additionally, SCH39166 inhibited apomorphine-induced climbing at lower doses than it inhibited apomorphine-induced sniffing in mice. The results from these latter two tests suggest that SCH39166 may have a reduced liability to produce extrapyramidal side effects. Therefore, based on this profile of activity, SCH39166 is a selective D1 dopamine receptor antagonist both in vitro and in vivo. Additionally, because this compound is longer acting in the primate than previously available D1 antagonists, it has potential utility as a clinically useful drug.     

In vivo binding of SCH 39166: a D-1 selective antagonist

SCH 39166 [(-)-trans-6,7,7a,8,9,13b-hexahydro-3-chloro-2-hydroxy-N-methyl- 5H-benzo[d]naphtho-[2,1b]-azepine] has been identified previously as a potent and selective D-1 antagonist. These studies demonstrated that SCH 39166 binds to the D-1 receptor in vitro and inhibits the rat conditioned avoidance response, a test predictive of antipsychotic activity. The current study demonstrates that SCH 39166 inhibits the in vivo binding of [125I]SCH 38840 to D-1 receptors in rat striatal tissue with an ED50 of 11.67 nmol/animal or 0.016 mg/kg s.c. SCH 39166 did not inhibit the in vivo binding of [125I]SCH 38840 to rat frontal cortex, suggesting that, unlike other D-1 antagonists, SCH 39166 was not binding to 5-hydroxytryptamine (5-HT)2 receptors in vivo. The in vivo binding of SCH 39166 to D-2 receptors was studied using [3H]raclopride and demonstrated that SCH 39166 did not bind to D-2 receptors up to doses of 100 mumol/animal or approximately 150 mg/kg s.c. Further studies to determine the in vivo selectivity of SCH 39166 utilized N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) to inactivate selected neurotransmitter receptors. Preadministration of SCH 39166, at doses as low as 0.01 mg/kg s.c., produced a statistically significant protection of D-1 receptors from EEDQ inactivation. SCH 39166 produced a similar protection of 5-HT2 receptors only at the highest dose tested, 10 mg/kg s.c., whereas there was no protection of D-2 sites even at this high dose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Smallpox Vaccination: A National Survey of Emergency Health Care Providers

Concerns about bioterrorism have prompted a national voluntary smallpox (SP) vaccination program in the United States. Although emergency health care providers are among the first targeted for vaccination, little is known about how these providers view the risks and benefits of SP vaccination. OBJECTIVES: To assess the willingness of emergency health care personnel to receive pre-event SP vaccination prior to the start of the national program. METHODS: The authors conducted a national cross-sectional, anonymous survey of 1,701 emergency physicians, nurses, and mid-level practitioners working full time in 13 adult and pediatric academic emergency departments in large U.S. cities in November and December 2002. The main outcome measure was willingness to be vaccinated against SP. Secondary outcomes included the prevalence of self-reported contraindications, and reasons for and against vaccination. RESULTS: 732 emergency health care providers returned questionnaires (response rate 43%). Overall, 73% (95% CI = 66% to 80%) were willing to receive pre-event SP vaccination. 18% (95% CI = 14% to 23%) reported contraindications to vaccination, and 50% (95% CI = 39% to 61%) of these providers were willing to receive pre-event SP vaccination. Self-protection (72%) was the most common reason cited for desiring vaccination against SP; concern about vaccine-related adverse events (54%) was the most common reason cited for not wanting immunization. CONCLUSIONS: Most emergency health care providers express a willingness to receive pre-event SP immunization; self-protection is a principal motivating reason. A subset of health care providers, however, may place themselves at increased risk by desiring vaccination despite contraindications.     

In vitro proliferation of hematopoietic stem cells in the absence of an adherent monolayer

Experiments on long-term murine bone marrow cultures indicate that the production and maintenance of the hematopoietic stem cell is dependent on the establishment of an adherent monolayer and a secondary repopulation of the culture with fresh marrow. In contrast, we have found that bone marrow cultures derived from the Syrian hamster do not require a repopulation step and produce stem cells that proliferate and differentiate for more than 12 wk in the absence of an adherent layer. Stem cells were grown in Fisher's medium (pH 7.0-7.2) containing 20% horse serum in a fully humidified atmosphere of 5% CO2 in air at 37 degrees C. Cultures were fed twice weekly by removal of half of the medium and supernatant cells and replacement with an equal volume of fresh medium. No hormones or exogenous growth factors were required for the maintenance of myeloid cells, monocytes, and megakaryocytes in either the adherent or suspension cells cultures.     

Presence of mixed colony-forming cells in long-term hamster bone marrow suspension cultures: response to pokeweed spleen conditioned medium

In contrast to the murine system, long-term hamster bone marrow suspension cultures maintain proliferation of both pluripotent and committed stem cells in the absence of an adherent layer and without addition of exogenous factors, such as hydrocortisone. Addition of pokeweed-mitogen-stimulated hamster spleen conditioned medium (SCM) to these long-term suspension cultures produces an increase in the number of mixed colonies assayed in soft-agar, These mixed colonies, which contained four cell lineages--granulocytic, erythroid, megakaryocytic, and macrophage--could be generated from cells grown in suspension for over 6 mo. Addition of SCM also induces an initial rapid expansion of the myeloid compartment, and this expansion results in 70% of the cells being terminally differentiated granulocytes. In contrast, addition of SCM to hamster bone marrow cultures containing both adherent cells and hematopoietic stem cells produced no change in the number of mixed colonies generated in the culture. This system allows the in vitro study of the process of stem cell proliferation and differentiation and also provides a means to examine the relationship of adherent and supernatant bone marrow populations.