Impact of a negative prior stress test on emergency physician disposition decision in ED patients with chest pain syndromes

OBJECTIVE: Many emergency department (ED) patients with potential acute coronary syndromes (ACS) have prior visits and prior cardiac testing; however, the effect of knowledge of prior testing on the emergency physician disposition decision making is not known. We studied the impact of prior noninvasive testing (ie, stress testing) for myocardial ischemia on disposition decision making in ED patients with potential ACS. METHODS: We performed a prospective cohort study of ED patients with chest pain who received an electrocardiogram for potential ACS. Data included demographics, medical history, stress test history, and TIMI risk score. Patients were followed in-house; 30-day telephone interviews were performed for follow-up. Main outcomes were ED disposition (admit/discharge) and a composite of 30-day death, acute myocardial infarction, and revascularization stratified on the basis of prior stress testing known at the time of presentation. Standard statistical techniques were used with 95% confidence intervals (CI). RESULTS: There were 1853 patients enrolled and 97% had follow-up. Patients had a mean age of 53 +/- 14 years; 60% were women, 67% were black. There were 1491 (79%) patients without a prior stress test, 291 (16%) had a normal prior stress test result, and 89 (5%) had an abnormal prior stress test result. Admission rates were 92% (95% CI, 87%-98%) for patients with a prior abnormal stress test, 73% (95% CI, 67%-78%) for patients with a normal prior stress test, and 70% (95% CI, 67%-72%) for patients without a prior stress test. Adverse outcomes were the highest among patients with prior abnormal stress test but did not differ significantly between patients with no prior stress test and patients with prior normal stress test (10.1% [95% CI, 3.6-16.7%] vs 5.2% [95% CI, 4.1-6.4%] vs 4.8% [95% CI, 2.4-7.3%]). CONCLUSION: Patients without prior stress tests and patients with prior normal stress tests were admitted for potential ACS at the same rate and had the same 30-day cardiovascular event rates. This suggests that prior stress testing does not affect subsequent disposition decisions. Perhaps cardiac catheterization or computed tomography coronary angiograms would have more of an impact on subsequent visits, making them potentially more cost-effective in the low-risk patient.     

An empirical evaluation of the performance of antibody identification tasks

Four empirical studies were conducted for better understanding of the nature of problem-solving activities by medical technologists and medical technology students when performing antibody identification tasks. The results indicated the importance of strategies that ensure the collection of converging evidence, as these strategies protect against the fallibility of commonly used heuristics and against errors due to simple slips. The results also indicate that not only do students make significant numbers of errors, but so do practicing technologists. In one of the studies covering a 1-year period, for instance, a group of 16 technologists made a total of 41 errors in 1057 cases. On the basis of these findings, several alternatives are proposed to reduce errors.     

Thermolabile protein kinase molecules in a temperature-sensitive murine sarcoma virus pseudotype.

Murine sarcoma virus-associated protein kinases that bind to actin have been purified by affinity chromatography on actin coupled to Sepharose. Heat inactivation studies showed the presence of thermolabile enzyme activity in pseudotypes containing a temperature-sensitivity mutant of murine sarcoma virus (MSV) but not in two independent wild-type MSV pseudotypes. Studies with Sephadex G-75 column fractions showed that a low molecular weight form, approximately 15,000, is the major thermolabile kinase in the temperature-sensitive MSV virions. Antibodies raised against the MSV-coded p60 protein, when added to the in vitro reaction mixtures, showed specific phosphorylation of the IgG heavy chain and a simultaneous reduction in the extent of phosvitin phosphorylation catalyzed by the various MSV pseudotype kinases. Thus a transforming retrovirus-coded enzyme activity that interacts directly with a major cytoskeletal protein and whose activity parallels the transforming ability of a conditional MSV mutant has now been identified.     

Analysis of p53 mutations in a large series of lymphoid hematologic malignancies of childhood

p53 mutations are found in a wide variety of cancers, including hematologic malignancies. These alterations apparently contribute to development of the malignant phenotype. We analyzed a large series of lymphoid (330 cases) and a smaller series of myeloid (29 cases) malignancies of childhood for p53 mutations by single-strand conformational polymorphism (SSCP) following polymerase chain reaction. Samples with abnormal SSCP were reamplified and analyzed by direct sequencing method. p53 mutations were detected within the known mutational hotspots (exons 5 to 8) in 8 of 330 lymphoid malignancies, and in none of 29 myeloid malignancies, showing that the frequency of p53 mutations in childhood lymphoid malignancies was very low (8 of 330 cases [2%]). Four of these patients had very aggressive, fatal acute lymphocytic leukemia (ALL). None of 13 infants and none of 48 patients with T-lineage leukemia had detectable p53 mutations in their ALL cells. Exceptionally, p53 mutations were comparatively frequent in a small sample of B-cell non-Hodgkin's lymphomas (2 of 8 cases). Mutations were detected in samples from two patients with ALL at relapse; these were not detected in samples at initial diagnosis from the same patients, suggesting that p53 mutations may be associated with progression to a more malignant phenotype. Seven of eight alterations of p53 were missense mutations, and seven of eight samples may be heterozygous for the mutant p53, indicating that p53 protein may act in a dominant negative fashion.     

Specific cellular immune response and neutralizing antibodies in goats immunized with native or recombinant envelope proteins derived from human T-lymphotropic virus type IIIB and in human immunodeficiency virus-infected men.

Animals immunized with native or recombinant envelope proteins from the human immunodeficiency virus (HIV, formerly referred to as human T-lymphotropic virus type III) human T-lymphotropic virus type IIIB and naturally HIV-infected men were assessed for neutralizing antibodies and cell-mediated immunity toward the virus. Immunization of rabbits or goats with the native external envelope glycoprotein gp120 or with corresponding recombinant proteins elicited strictly type-specific neutralizing antibodies. A broad, group-specific cellular immune response to gp120 and to three different HIV isolates was seen in goats immunized with the native gp120 but not in animals immunized with the nonglycosylated recombinant envelope proteins. In HIV-infected people, no T-cell response was seen, even though their T-cell response toward other foreign antigens was intact. The results show type- and group-specific epitopes on gp120, some of which may be of importance for the development of a vaccine against HIV infection.     

Microtubule-targeting agents augment the toxicity of DNA-damaging agents by disrupting intracellular trafficking of DNA repair proteins

The paradigm that microtubule-targeting agents (MTAs) cause cell death via mitotic arrest applies to rapidly dividing cells but cannot explain MTA activity in slowly growing human cancers. Many preferred cancer regimens combine a MTA with a DNA-damaging agent (DDA). We hypothesized that MTAs synergize with DDAs by interfering with trafficking of DNA repair proteins on interphase microtubules. We investigated nine proteins involved in DNA repair: ATM, ATR, DNA-PK, Rad50, Mre11, p95/NBS1, p53, 53BP1, and p63. The proteins were sequestered in the cytoplasm by vincristine and paclitaxel but not by an aurora kinase inhibitor, colocalized with tubulin by confocal microscopy and coimmunoprecipitated with the microtubule motor dynein. Furthermore, adding MTAs to radiation, doxorubicin, or etoposide led to more sustained γ-H2AX levels. We conclude DNA damage-repair proteins traffic on microtubules and addition of MTAs sequesters them in the cytoplasm, explaining why MTA/DDA combinations are common anticancer regimens.First developed as anticancer agents in the 1950s, microtubule targeting agents (MTAs) are used in the treatment of a wide variety of malignancies and until now have been thought to kill cells by arresting them in mitosis (1, 2). Although this explanation applies to rapidly dividing cells in preclinical models, it cannot explain the activity of these agents in tumors in humans because these cells divide much more slowly. For the latter situtation, a different paradigm must explain the activity of MTAs, and we have proposed that interfering with microtubule (MT) trafficking in interphase cells is the principal mechanism of MTA action (3–5). In breast, ovarian, lung, and head and neck cancers, as well as in most lymphomas, combination regimens that include a MTA and a DNA-damaging agent (DDA) are preferred (Table S1). Although the frequency with which these combinations are used might be fortuitous, it is likely there is a mechanistic basis for this outcome. We hypothesized that by hampering the trafficking of essential DNA repair proteins, MTAs synergize with DDAs, augmenting their toxicity. To explore this theory further we studied the effects of combining a MTA and a DDA in a number of cell models and examined the distribution and biology of nine different proteins involved in DNA repair. We have confirmed the hypothesis and report our findings.     

Detection of minimal residual disease in acute lymphoblastic leukemia by in vitro amplification of rearranged T-cell receptor delta chain sequences

Human T-cell receptor (TCR) delta-chain diversity mainly originates from high junctional variability, since only a limited number of germline elements is available. This extraordinary diversity at the V.J junction, due to the use of two D delta elements and extensive incorporation of N nucleotides, constitutes a specific clonal marker for cell populations exhibiting rearranged TCR delta genes. To this end we amplified in vitro by polymerase chain reaction (PCR) the TCR delta junctional region of five acute lymphoblastic leukemias (ALL), isolated respective DNA fragments, and used them directly as clonospecific probes. The combination of PCR technology and hybridization to clonospecific probes permitted the detection of leukemia DNA at dilution of 1:100,000 in all five cases. Moreover, we were able to investigate one of the ALL patients 11 months after achieving continuous complete remission. Conventional Southern blot analysis failed to detect rearranged TCR genes at this stage. However, residual leukemic cells could readily be detected by PCR technique. We conclude that the strategy proposed here is a very sensitive tool to detect minimal residual disease in a significant proportion of human lymphoid neoplasias.     

A point mutation in the integrin beta 3 cytoplasmic domain (S752-->P) impairs bidirectional signaling through alpha IIb beta 3 (platelet glycoprotein IIb-IIIa)

Agonist-induced inside-out signaling results in an increased affinity of integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) for soluble ligands (fibrinogen [Fg] and PAC1). Ligand binding to integrins initiates outside-in signaling that leads to cellular responses such as cell spreading and focal adhesion formation. A point mutation in the beta 3 cytoplasmic domain (S752-->P) is associated with blocked inside- out alpha IIb beta 3 signaling in a variant Glanzmann's thrombasthenia. This mutation was introduced into beta 3 and cotransfected into Chinese hamster ovary cells with a chimeric alpha subunit consisting of the alpha IIb extracellular and transmembrane domains and the alpha 6B cytoplasmic domain. The substitution of the alpha IIb cytoplasmic domain with that of alpha 6 led to activation of alpha IIb beta 3 to bind PAC1, mimicking inside-out signaling. This effect was reversed by the S752-->P mutation, indicating a disruption of inside-out signaling by the mutation. In addition, transfectants expressing this beta 3 variant showed reduced alpha IIb beta 3-mediated cell spreading on immobilized Fg, focal adhesion, and fibrin clot retraction, suggesting an impairment in outside-in alpha IIb beta 3 signaling. Therefore, a single point mutation in the beta 3 cytoplasmic domain impaired bidirectional signaling through alpha IIb beta 3.     

Differential effect of phorbol esters and interleukin-3 on protein kinase C isoform content and kinase activity in the FDC-P1 cell line

FD/PMA is a subclone of the interleukin-3 (IL-3)-dependent, FDC-P1 cell line, which proliferates in response to either 12-O- tetradecanoylphorbol-13 acetate (PMA) or IL-3. While several endogenous substrates were phosphorylated in response to protein kinase C (PKC) activation in FDC-P1, phospholipid-dependent phosphorylation in the FD/PMA grown in PMA was not observed. Basal, phosphatidylserine- independent, and diolein-independent phosphorylation of cytosolic substrates with molecular weights of 17, 52, 57, and 105 Kd were enhanced in FD/PMA cells grown in PMA as compared with FDC-P1 cells cultured in IL-3. Phosphorylation of a 105-Kd substrate was enhanced in the particulate fraction of FD/PMA cells maintained in PMA. The 17-Kd substrate in FD/PMA cells comigrated with a substrate phosphorylated in a PKC-dependent manner in FDC-P1 cells. Phosphorylation of the 52- and 57-Kd substrates, but not of the 17-Kd substrate, was inhibited by H-7 and staurosporine. A portion of the PMA-induced cytosolic kinase activity coeluted with PKC on diethyl aminoethyl chromatography. While FD/PMA cells cultured in PMA contained negligible PKC-dependent phosphorylation of endogenous substrates or histone, alpha and epsilon PKC isoforms were detected by Western blot analysis. PKC phosphotransferase activity was observed in FD/PMA cells grown in PMA when peptides corresponding to residues 720 to 737 of PKC-epsilon or residues 4 to 14 of myelin basic protein were used as substrates. These data indicate that maintenance of FD/PMA cells in PMA stimulates proliferation and markedly alters PKC substrate specificity. Generation of at least two phospholipid-independent kinases occurs in PMA-treated cells.     

Purification of 120,000 dalton envelope glycoprotein from culture fluids of human immunodeficiency virus (HIV)-infected H9 cells

The outer envelope glycoprotein, gp120, has been purified from large volumes (greater than 100 liters) of HTLV-IIIB-infected H9 cell culture fluids using immunoaffinity chromatography resins prepared from immunoglobulins of AIDS patients plasma. By using a single-step immunoaffinity purification, between 7 and 28 micrograms of gp120 was recovered from each liter of culture fluid which represented between 60 and 95% of the total envelope glycoprotein present in the fluid. Envelope glycoprotein in culture media was concentrated more than 10,000 times over the starting material. The water-soluble gp120, containing trace contaminating proteins, was purified to apparent homogeneity by preparative polyacrylamide gel electrophoresis (PAGE). Envelope glycoprotein purified from culture fluids was immunogenic in laboratory animals in both native and PAGE-purified forms and was reactive with AIDS patient sera in immunoassays.