Reversal of phosphate induced decreases in force by the benzimidazole pyridazinone, UD-CG 212 CL, in myofilaments from human ventricle.

UD-CG 212 Cl, (Fig. 1: 4,5-dihydro-6-[2-(4-hydroxyphenyl)-1 H-benzimidazole-5-yl]-5-methyl-3(2H)-pyridazinone), is the primary metabolite of the positive inotropic agent pimobendan (UDCG 115 BS, Acardi). Our previous studies showed in detergent extracted preparations of canine ventricular muscle that sub-nanomolar concentrations of UD-CG 212 Cl increased submaximal myofilament force, but only when the activation state had been altered by relatively high (5-10 mM) concentrations of inorganic phosphate (Pi) or relatively low (20 microM) concentrations of MgATP. In the present study, we investigated the effects of UD-CG 212 Cl on the pCa-force relationship of detergent extracted bundles of human cardiac fibers before and after addition of Pi. As expected, treatment with 5 mM Pi depressed maximal force at pCa 4.5 by 27.0 +/- 0.4% (mean +/- SEM). Force generated at the half-maximally activating Ca2+ concentration (pCa50) of control fibers (5.98 +/- 0.2) was significantly (p < .05) reduced following the addition of 5 mM Pi (pCa50 = 5.69 +/- 0.3). The addition of UD-CG 212 Cl over a range of concentrations (10(-11)-10(-6) M) had no effect on Ca(2+)-sensitivity under control conditions, but in the presence of 5 mM Pi, there was a 23.1 +/- 0.1% increase in the percent maximal force at pCa5.9. Ca(2+)-sensitivity was also significantly increased in the presence of Pi and 10(-8) M UD-CG 212 Cl (pCa50 = 5.74 +/- 0.3, p < .05). We conclude that UD-CG 212 Cl potentially increases sub-maximal force of human ventricular myofilaments with an inotropic action depending on a state of myofilament activation associated with ischemic conditions.

Denial of emergency department authorization of potentially high-risk patients by managed care.

This study was designed to evaluate patients presenting to a large urban university emergency department (ED) who were subsequently denied authorization for reimbursed care by their managed care provider and to characterize the denial as potentially safe or unsafe based on published triage criteria. A consecutive case surveillance was performed from October 1, 1994 to September 30, 1995 at a university-based ED (30,000 visits per year) for adult patients in inner-city Chicago. Cases were comprised of adult managed care participants whose providers refused by telephone to authorize payment for ED services and who then left the ED without treatment. Chief complaints and vital signs were used to categorize patients as high-risk or nonemergent based on previously published criteria. A total of 2,965 adult managed care patients presented to the ED during the study period, representing 11.1% of the total ED census. Of these patients, 244 (8.2%) were denied authorization for payment of their care. By previously established criteria, 115 (47.1%) were identified as potentially unstable, 61 (53%) due to abnormal vital signs and 54 (47%) with other high-risk indications such as severe pain, chest pain, or abdominal pain. These potentially high-risk patients may subsequently suffer adverse outcomes. Current guidelines used for telephone triage by managed care to divert patients from our ED do not meet previously published safe triage criteria.

An evaluation of a chest pain diagnostic protocol to exclude acute cardiac ischemia in the emergency department.

BACKGROUND: Although accelerated diagnostic protocols are being increasingly used in emergency departments to diagnose acute cardiac ischemia, there have been no prospective evaluations of a chest pain diagnostic protocol with serial determinations of creatine kinase MB isoenzyme and mandatory exercise electrocardiography (ExECG). METHODS: Prospective cross-sectional study in which chest pain protocol results were compared with final (reference) diagnoses of acute cardiac ischemia (including acute myocardial infarction and unstable angina). Patients in need of hospital admission but at low probability (by a validated algorithm) for acute myocardial infarction were examined for exclusions: known coronary artery disease, cardiac complications, severe comorbidities, or inability to perform exercise testing. A 12-hour diagnostic protocol included serial measurements of creatine kinase MB, ECG, and clinical assessments followed by ExECG for those with negative initial serial testing. Reference diagnoses were established during hospitalization and diagnostic accuracy was assessed. RESULTS: The study group of 317 patients was 54% male and 65% black, and had a mean age of 46.6 years; 9.5% had a final diagnosis of acute cardiac ischemia. For this diagnosis, the protocol had a sensitivity of 90.0% (95% confidence interval, 72.3%-97.4%); specificity, 50.5% (95% confidence interval, 44.6%-56.4%); positive predictive value, 16.0%; and negative predictive value, 98.0%. Creatine kinase MB, serial ECGs, and ExECG each made a contribution to improved sensitivity and accuracy, whereas clinical reassessments were less discriminating, as indicated by protocol's receiver operating characteristic curve. CONCLUSIONS: A chest pain diagnostic protocol achieved high sensitivity and improved specificity over the standard emergency department workup. There were no adverse advents associated with early ExECG.

A novel curriculum for teaching research methodology.

We sought to develop a unique educational course for emergency medicine residents that provides the essential knowledge, the practical skills, and the motivation to complete a research project during the residency. A 5-day mandatory course was designed for first-year emergency medicine residents. Unique features of the curriculum include 1) didactic lectures paired with workshops teaching practical skills and 2) the use of a hypothetical research question ("mock project") that is utilized for practical experience at each step and results in an oral presentation of the completed research project. The course was evaluated with pre- and post-tests of general research knowledge, quality of the final presentations, and a post-course questionnaire. Following completion of the course, the 16 emergency medicine resident participants showed significant improvement in research knowledge, as evidenced by higher median post-test scores and an absolute increase in the pass rate of 50%. Residents' final course presentations were given ratings of "above average" to "superior" by all observing faculty members. Finally, 87.5% of the residents felt that developing a mock project during the course ("hands-on" experience) increased their confidence and interest in conducting future academic research. Thus, an introductory course in research methodology that utilizes didactic lectures paired with appropriate practical workshops and incorporates completion of a mock research project may provide an effective method for teaching emergency medicine residents to conduct research.

Forced air speeds rewarming in accidental hypothermia.

STUDY OBJECTIVE: To compare the rates of rewarming of forced-air and passive insulation as a treatment for accidental hypothermia. METHODS: We carried out a prospective, randomized clinical trial in two urban, university-affiliated emergency departments. Our subjects were 16 adult hypothermia victims with core temperatures less than 32 degrees C. A convective cover inflated with air at about 43 degrees C (forced-air group) or cotton blankets (control group) were applied until the patient's core temperature reached 35 degrees C. Members of both groups were given IV fluids warmed to 38 degrees C and warmed, humidified oxygen at 40 degrees C by inhalation. RESULTS: The mean +/- SD initial temperature was 28.8 degrees +/- 2.5 degrees C (range, 25.5 degrees C to 31.9 degrees C) in the patients who underwent forced-air rewarming and 29.8 degrees +/- 1.5 degrees C (range, 28.2 degrees C to 31.9 degrees C) in those given blankets. Core temperature increased about 1 degree C/hour faster in patients treated with forced-air rewarming (about 2.4 degrees C/hour) than in patients given only cotton blankets (about 1.4 degrees C/hour, P = .01). Core-temperature afterdrop was detected in neither group. CONCLUSION: Forced air accelerated the rate of rewarming without producing apparent complications in hypothermic patients.

Differential regulation of slow-skeletal and cardiac troponin I mRNA during development and by thyroid hormone in rat heart.

We have examined mRNA levels for the cardiac troponin I (cTnI) and slow-skeletal (ssTnI) in perinatal rat hearts. Northern blots showed that hypothyroidism was associated with a delay in the expected isoform switching. RNA slot blots showed a six-fold increase in cTnI mRNA from day 3 to day 21 in hearts from postnatal euthyroid rats compared to a three-fold increase in cTnI for the same period in the hypothyroid hearts. On the other hand, ssTnI mRNA levels were higher after 3 days in hearts from the hypothyroid animals and fell to undetectable levels after 21 days. In euthyroid hearts ssTnI was not detectable after 14 days and was not re-expressed in adult hearts. In the ventricles from 28-day-old animals the most significant differences in cTnI mRNA levels were between the euthyroid and T3-treated hypothyroid preparations and in the 87 to 125-day-old group between euthyroid and hypothyroid ventricles. T3 treatment of the 87 to 125-day-old hypothyroid animals did not increase the cTnI mRNA above euthyroid levels despite elevated serum T3. These results show that thyroid hormone influences expression of the cTnI isoform in postnatal and young adult rats, but not to the same extent in animals greater than 28 days of age.

Selective isolation of human breast carcinoma cells resistant to the growth-inhibitory effects of retinol.

The anchorage-dependent and anchorage-independent growth of the human mammary carcinoma cell line MDA-MB-231 is inhibited by vitamin A (retinol). Clones resistant to growth inhibition by retinol were isolated from this cell line in soft agar without the use of mutagens. This paper describes the isolation and characterization of the resistant lines. The clones were selectively resistant to retinol. There was significant growth inhibition after treatment with retinoic acid and 13-cis-retinoic acid. The resistant clones maintain their resistance to retinol through multiple passages. Resistance is specific for inhibition of growth, because treatment of the resistant clones results in stimulation of plasminogen activator activity without alteration of proliferation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows no significant qualitative or quantitative difference in the clones when compared with the MDA-MB-231 parent line. Although the clones do not regrow in soft agar, they are tumorigenic in athymic mice. Tumors are produced at a rate similar to the parent line. The advantage of this isolation method is that sensitive and resistant malignant cells derived from the same parent cell line are now available to study the molecular events involved in the inhibition of cellular proliferation after treatment with retinol.

Effect of retinoids on xenotransplanted human mammary carcinoma cells in athymic mice.

Previous studies have shown dose-dependent growth inhibition of the human mammary carcinoma cell line MDA-MB-231 xenotransplanted in athymic mice using retinol. In this study, the growth inhibitory effect of retinoic acid (RA) and 13-cis-retinoic acid (13-cis-RA) was examined in vitro and in vivo. With both agents there was dose-related growth inhibition in monolayer culture. The MDA-MB-231 cell line was more sensitive in monolayer culture to 13-cis-RA than to RA. Anchorage-independent growth of the MDA-MB-231 cell line was also inhibited by both of these agents but only in a dose-dependent manner with 13-cis-RA. Athymic mice inoculated with MDA-MB-231 human mammary carcinoma cells were treated with various doses of RA and 13-cis-RA for 30 days. RA doses greater than 90 micrograms were clinically toxic to the animals. There was a decrease in tumor size with all doses of RA tested but not in a dose-related fashion. Response at the higher doses of RA may be related to subclinical toxicity. Doses of 13-cis-RA above 300 micrograms were clinically toxic. Unlike RA, there was no statistically significant decrease in tumor size with treatment with 13-cis-RA. These findings show that there is significant reduction in growth in vivo of the human mammary carcinoma cell line MDA-MB-231 after treatment with RA. However, in vivo response to the retinoids is not always predicted by in vitro methods.

Effects of orally administered retinol on natural killer cell activity in wild type BALB/c and congenitally athymic BALB/c mice.

Retinoids have been shown to inhibit the growth and development of neoplastic cells in many systems. One mechanism of action may be through activation of the immune system, specifically natural killer (NK) cell activity. The effect of retinol on NK cell cytotoxicity was examined in three groups of mice: BALB/c (wild-type), BALB/c nu/nu (athymic), and BALB/c nu/nu previously injected with human tumor cells. In untreated mice, NK activity was highest in athymic mice without tumors and lowest in wild-type mice, although serum and liver retinol concentrations were identical in all three groups. In mice fed graded, nontoxic doses of retinol daily for 3 weeks, serum retinol levels in all three groups exhibited a sharp peak and decline following daily bolus retinol administration. Retinol stores in the livers showed a dose-dependent increase in all treated animals. However, NK cell activity, differed for each group. Athymic mice without tumors exhibited no change in NK activity as a result of retinol treatment. Athymic mice with tumors had NK levels that tended to increase with increasing retinol doses, but these changes were not statistically significant. Wild-type mice, on the other hand, demonstrated significantly higher NK levels after treatment with retinol doses of 300 and 600 micrograms/day. In subsequent time course experiments, there was a peak in NK activity 1 h following bolus retinol administration similar to the peak seen in serum retinol concentrations, suggesting either an acute activation or recruitment of cytotoxic cells. Retinol thus appears to increase NK activity in wild-type BALB/c mice, and this activity may be an important component of its antineoplastic activity.

Growth inhibition by retinol of a human breast carcinoma cell line in vitro and in athymic mice.

Although the anticarcinogenic and antiproliferative effects of vitamin A (retinol) have been extensively studied in vitro, there are few data regarding the response of human tumor cells to this agent in vivo. We have studied the effects of retinol on the human breast carcinoma cell line, MDA-MB-231. Initial in vitro studies on monolayer cultures demonstrated a retinol-induced growth inhibition that was reversible as well as time and dose dependent. A similar dose-dependent decrease in tumor cell growth was shown in vivo when BALB/c-nu/nu (athymic) mice were inoculated s.c. with MDA-MB-231 cells and given graded nontoxic doses of retinol intragastrically for 3 weeks. Tumor cells were also inhibited from lung colonization as "artificial" metastatic lesions when injected i.v. into athymic mice following retinol treatment. Spleen cells from these mice were assayed for natural killer cells as determined by their cytotoxic activity on 51Cr-labeled target cells. There was no change in natural killer activity with any dose of retinol. We conclude that retinol has a dose-dependent antiproliferative effect on human breast carcinoma in vivo as well as in vitro. Further, the retinol-induced tumor inhibition seen in T-cell-deficient mice does not appear to be due to enhancement of host immunity and thus may be solely a direct effect of retinol on the tumor.

Carcinoembryonic antigen expression and patient survival in carcinoma of the breast.

Immunoperoxidase staining was used to examine carcinoembryonic antigen (CEA) expression in 167 breast cancer cases. Patients with histological evidence of regional or localized breast cancer who lived less than 3 years or greater than 10 years were assessed. Overall expression of CEA was 65%. There was no significant correlation in CEA expression and survival in either regional or localized breast cancer cases. There was no association between CEA expression and number of lymph nodes involved, size of tumor, parity, gravidity, blood type, or menopausal status of the patients in either group. When the lymph nodes of cases with regional breast cancer were examined, there was a statistically significant number of short survivors whose primary tumor was negative for CEA, but whose metastatic tumor expressed the marker when compared to long survivors with regional lymph node involvement.

Localization of cellular retinol-binding protein in several rat tissues.

The distribution of cellular retinol-binding protein (CRBP) in rat liver, ileum, and epididymis was examined by the peroxidase-antiperoxidase immunolocalization technique. Positive cytoplasmic staining was seen in the liver when antiserum prepared against purified CRBP was used but not when antiserum absorbed with purified CRBP was used. Ileal mucosa, a tissue that contains no detectable CRBP, showed no positive staining. The epididymis showed strong positive staining in the caput but not in the cauda. Staining was present in principal and basal cells but not in peritubular or interstitial cells. Radioimmunoassay revealed that the CRBP within the caput epididymidis was localized in the initial segment and proximal region, areas known to be involved in the synthesis and secretion of factors necessary for sperm maturation. The results demonstrate that the expression of CRBP may vary within the same cell type, as well as between different cell types within the same tissue.