Cytokine production by primary bone marrow megakaryocytes

Primary human bone marrow megakaryocytes were studied for their ability to express and release cytokines potentially relevant to their proliferation and/or differentiation. The purity of the bone marrow megakaryocytes was assessed by morphologic and immunocytochemical criteria. Unstimulated marrow megakaryocytes constitutively expressed genes for interleukin-1 beta (IL-1 beta), IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha), by the polymerase chain reaction (PCR) and Northern blot analysis. At the protein level, megakaryocytes secreted significant amounts of IL-1 beta (53.6 +/- 3.6 pg/mL), IL-6 (57.6 +/- 15.6 pg/mL), and GM-CSF (24 +/- 4 pg/mL) but not TNF-alpha. Exposure of human marrow megakaryocytes to IL-1 beta increased the levels of IL-6 (87.3 +/- 2.3 pg/mL) detected in the culture supernatants. Transforming growth factor- beta was also able to stimulate IL-6, IL-1 beta, and GM-CSF secretion, but was less potent than stimulation with phorbol-12-myristate-13- acetate (PMA). The secreted cytokines acted additively to maintain and increase the number of colony-forming unit-megakaryocytes colonies (approximately 35%). These studies demonstrate the production of multiple cytokines by isolated human bone marrow megakaryocytes constitutively or stimulated in vitro. The capacity of human megakaryocytes to synthesize several cytokines known to modulate hematopoietic cells supports the concept that there may be an autocrine mechanism operative in the regulation of megakaryocytopoiesis.     

Predictors and Outcomes of Frequent Emergency Department Users

OBJECTIVE: To identify predictors and outcomes associated with frequent emergency department (ED) users. METHODS: Cross-sectional intake surveys, medical chart reviews, and telephone follow-up interviews of patients presenting with selected chief complaints were performed at five urban EDs during a one-month study period in 1995. Frequent use was defined by four or more self-reported, prior ED visits. Multivariate logistic regression identified predictors of frequent ED visitors from five domains (demographics, health status, health access, health care preference, and severity of acute illness). Associations between high use and selected outcomes were assessed with logistic regression models. RESULTS: All study components were completed by 2,333 of 3,455 eligible patients (67.5%). Demographics predicting frequent use included being a single parent, single or divorced marital status, high school education or less, and income of less than $10,000 (1995). Health status predictors included hospitalization in the preceding three months, high ratings of psychological distress, and asthma. Health access predictors included identifying an ED or a hospital clinic as the primary care site, having a primary care physician (PCP), and visiting a PCP in the past month. Choosing the ED for free care was the only health preference predictive of heavy use. Illness severity measures were higher in frequent visitors, although these were not independently predictive in the multivariate model. Outcomes correlated with heavy use include increased hospital admissions, higher rates of ED return visits, and lower patient satisfaction, but not willingness to return to the ED or follow-up with a doctor. CONCLUSIONS: Frequent ED visits are associated with socioeconomic distress, chronic illness, and high use of other health resources. Efforts to reduce ED visits require addressing the unique needs of these patients in the emergency and primary care settings.     

Amino-terminal protein-protein interaction motif (POZ-domain) is responsible for activities of the promyelocytic leukemia zinc finger-retinoic acid receptor-alpha fusion protein.

Promyelocytic leukemia zinc finger-retinoic acid receptor a (PLZF-RARalpha), a fusion receptor generated as a result of a variant t(11;17) chromosomal translocation that occurs in a small subset of acute promyelocytic leukemia (APL) patients, has been shown to display a dominant-negative effect against the wild-type RARalpha/retinoid X receptor alpha (RXRalpha). We now show that its N-terminal region (called the POZ-domain), which mediates protein-protein interaction as well as specific nuclear localization of the wild-type PLZF and chimeric PLZF-RARalpha proteins, is primarily responsible for this activity. To further investigate the mechanisms of PLZF-RARalpha action, we have also studied its ligand-receptor, protein-protein, and protein-DNA interaction properties and compared them with those of the promyelocytic leukemia gene (PML)-RARalpha, which is expressed in the majority of APLs as a result of t(15;17) translocation. PLZF-RARalpha and PML-RARalpha have essentially the same ligand-binding affinities and can bind in vitro to retinoic acid response elements (RAREs) as homodimers or heterodimers with RXRalpha. PLZF-RARalpha homodimerization and heterodimerization with RXRalpha were primarily mediated by the POZ-domain and RARalpha sequence, respectively. Despite having identical RARalpha sequences, PLZF-RARalpha and PML-RARalpha homodimers recognized with different affinities distinct RAREs. Furthermore, PLZF-RARalpha could heterodimerize in vitro with the wild-type PLZF, suggesting that it may play a role in leukemogenesis by antagonizing actions of not only the retinoid receptors but also the wild-type PLZF and possibly other POZ-domain-containing regulators. These different protein-protein interactions and the target gene specificities of PLZF-RARalpha and PML-RARalpha may underlie, at least in part, the apparent resistance of APL with t(11;17) to differentiation effects of all-trans-retinoic acid.     

Influence of sodium fluoride on the stability of human blood samples and blood gas measurements

OBJECTIVE: This study aimed to reduce the analytical error associated with measuring oxygen and carbon dioxide partial pressures as well as the pH in arterial blood samples an hour after sample collection. The standard blood sample preparation procedure involving sample cooling down to 0 degrees C is known to have several drawbacks. Therefore, another approach using NaF at room temperature as an inhibitor of metabolic reactions was introduced. DESIGN AND METHODS: Arterial heparin blood samples from six volunteers were distributed over 104 single capillaries prepared with different concentrations of NaF. The capillaries were filled simultaneously and under the same conditions with blood samples, and the blood gas parameters of each sample were measured. Changes in pO2, pCO2, and pH during storage were evaluated with the aid of t test statistics. RESULTS: During the storage period under investigation, fluctuations of the carbon dioxide partial pressure and the pH were low, whereas there was a significant (P < 0.01) decrease of the oxygen partial pressure. This was observed at all NaF concentrations. Depending on the addition of NaF, a significant baseline shift for the time-resolved pH and pCO2 values could be observed. Whereas the partial pressure of carbon dioxide and the pH could be kept stable by adding a defined amount of NaF, the partial pressure of oxygen decreased significantly over 70 min. CONCLUSIONS: The proposed new method can be practically applied to a comparative blood gas study, significantly reducing the blood sample volume required. The application of analytical grade NaF is an improvement compared to previous work because a pH decrease could not be observed.     

Arg-Gly-Asp-dependent occupancy of GPIIb/IIIa by applaggin: evidence for internalization and cycling of a platelet integrin

Using indirect immunofluorescence microscopy we examined the distribution and cycling of GPIIb/IIIa after binding to applaggin, a high-affinity Arg-Gly-Asp (RGD)--containing ligand. Resting, unfixed platelets were incubated with applaggin for 30 minutes at 37 degrees C, and bound applaggin was detected by an affinity-purified rabbit anti- applaggin antibody. Examination of intact cells showed a rim pattern for applaggin, consistent with its binding to the platelet surface. Staining of Triton X-100--permeabilized cells showed an intracellular pool of applaggin. Competition of applaggin binding by either AP-2, an anti-GPIIb/IIIa monoclonal antibody (MoAb) that blocks fibrinogen binding, or the synthetic peptide RGDW eliminated both surface and intracellular staining, indicating that applaggin is binding to GPIIb/IIIa in an RGD-dependent manner. Inhibition of platelet activation by PGE1 and theophylline had no effect on the observed staining patterns, indicating that cellular activation is not required for surface binding and subsequent internalization. To evaluate whether occupancy of functional binding sites on GPIIb/IIIa is required for internalization, we used mAb15, an anti-GPIIIa antibody that neither blocks fibrinogen binding nor induces the expression of ligand-induced binding sites on GPIIb/IIIa. In these studies mAb15 was internalized in a manner analogous to both AP-2 and applaggin, showing that occupancy of the RGD binding site is not required to initiate receptor internalization. To estimate the size of the newly internalized pool of applaggin, 125I-applaggin--binding studies were performed. Displacement of bound 125I-applaggin by excess unlabeled applaggin or EDTA showed that at least 17% of bound applaggin was nondisplaceable when binding was performed under conditions permitting membrane flow and internalization. These data indicate that GPIIb/IIIa is internalized in unstimulated platelets independent of cellular activation or occupancy of the functional binding site(s) of GPIIb/IIIa by RGD-containing ligands. Thus, internalization of GPIIb/IIIa may represent a mechanism by which the surface expression of this adhesion receptor is regulated.     

Effect of an educational program on medical students’ conversations with patients about advance directives

Advance directives, such as the durable power of attorney for health care (DPAHC), help patients and physicians make end-of-life health care decisions. Medical education should prepare student physicians to be knowledgeable about and comfortable with discussing advance directives. The authors developed an educational module for the third-year medical school curriculum and conducted a randomized trial to evaluate in students its effect on various outcome measures regarding the DPAHC. Over a six-week period, students who received written material about the DPAHC and a two-hour seminar significantly increased knowledge about and reported increased skill, comfort, and experience with the DPAHC.     

Induction of sperm release in frogs by mammalian gonadotropin-releasing hormone

A single subcutaneous injection of a synthetic mammalian gonadotropin-releasing hormone (LH/FSH-RH) stimulated a rapid release of sperm (spermiation) from the testes of the intact treefrog Hyla regilla; the median effective dose was between 35 and 75 ng. The rate of response was dose-dependent, ranging from about 5 to 30 min. Even large injections of RH failed to stimulate spermiation in hypophysectomized frogs, but 100 ng RH was effective after transplantation of a pituitary into these animals. Thus, the action of RH is mediated by the frogs pituitary. These data confirm the lack of species-specificity in the hypothalamic gonadotropin-releasing factor.