Quantitative analysis of globin gene induction in single human erythroleukemic cells.

The mechanisms involved in the normal developmental regulation of globin gene expression, and the response to pharmacological agents that elevate fetal hemoglobin, may be expected to involve either changes in each cell or a selection process affecting subsets of differentiating erythroid cells. To study these mechanisms we have developed assays to measure mRNA levels in single erythroid cells. The assay involved the use of globin-specific probes, with no detectable cross-reactivity, in real-time, fluorescence-based quantitative PCR (Q-PCR). We had previously used this Q-PCR method to measure globin mRNA levels in cultures of primary erythroid cells demonstrating that drugs like hydroxyurea, 5-azacytidine and butyric acid each yielded increases in gamma/( gamma + ss) mRNA ratios, with differential effects on ss-globin levels. We have now extended this approach to measure globin mRNA levels in single K562 cells, a human erythroleukemic cell line, with and without 30 microM hemin treatment. Hemin exposure increases total hemoglobin levels by approximately 9-fold and total alpha-, epsilon- and gamma-globin mRNA levels by 1.5-2.3-fold. Single cell analyses showed initial wide distributions of each of the three individual globin mRNA levels with most cells having detectable but very low levels of each globin transcript. Hemin induction shifted the distributions to higher levels, with a tendency to residual left skewing as some cells remained with very low expression levels despite the effect of hemin in increasing expression in most of these low expressing cells. Thus transcriptional heterogeneity remains a crucial variable, even in this extensively used model of human erythroid biology, and clearly influences strongly the response to inducing agents. These methods may enable us to define better possible molecular and/or cellular models of globin gene modulation.

A kinetic analysis of calcium-triggered exocytosis.

Although the relationship between exocytosis and calcium is fundamental both to synaptic and nonneuronal secretory function, analysis is problematic because of the temporal and spatial properties of calcium, and the fact that vesicle transport, priming, retrieval, and recycling are coupled. By analyzing the kinetics of sea urchin egg secretory vesicle exocytosis in vitro, the final steps of exocytosis are resolved. These steps are modeled as a three-state system: activated, committed, and fused, where interstate transitions are given by the probabilities that an active fusion complex commits (alpha) and that a committed fusion complex results in fusion, p. The number of committed complexes per vesicle docking site is Poisson distributed with mean n. Experimentally, p and n increase with increasing calcium, whereas alpha and the pn ratio remain constant, reducing the kinetic description to only one calcium-dependent, controlling variable, n. On average, the calcium dependence of the maximum rate (R(max)) and the time to reach R(max) (T(peak)) are described by the calcium dependence of n. Thus, the nonlinear relationship between the free calcium concentration and the rate of exocytosis can be explained solely by the calcium dependence of the distribution of fusion complexes at vesicle docking sites.

Evolution and structure of the fibrinogen genes. Random insertion of introns or selective loss?

Chromosomal linkage as well as sequence homologies provide unequivocal evidence that the genes for the alpha, beta and gamma chains of fibrinogen arose by successive duplication of a single ancestral gene. Yet, when the three fibrinogen chains are aligned by amino acid homology, the positions of intervening sequences coincide at only two positions for all three chains. While one additional intron occurs at a homologous site in the beta and gamma chains, none of the positions of the remaining 11 introns in the three genes is shared. This arrangement of introns in the three fibrinogen genes suggests that either introns were selectively lost, implying that there is essential information in the retained introns, or the common introns were present in the ancestral fibrinogen gene and introns have been randomly inserted since the triplication of the original gene. The more likely possibility of selective loss of introns implies that the ancestral gene, as it existed about one billion years ago, must have been composed of numerous small exons.

Combining insulin-like growth factor-I and mean spontaneous nighttime growth hormone levels for the diagnosis of growth hormone deficiency.

There is no gold standard for the diagnosis of GH deficiency. Recent data show that spontaneous GH levels may lack sensitivity, and that GH stimulation tests lack specificity as currently performed. Serum insulin-like growth factor-I (IGF-I) measurements lack both sensitivity and specificity. Some of these problems may be explained by nutritional effects. In children, overnutrition decreases GH and increases IGF-I, while undernutrition decreases IGF-I and increases GH. To overcome these difficulties and improve diagnostic accuracy, we combined mean spontaneous nighttime GH levels with IGF-I levels in a statistically based bivariate model. On a two-dimensional plot of mean spontaneous nighttime GH level (in SD units) vs. IGF-I level (in SD units), we defined a new variable, S (sum) score, where S = (1/square root of 2) x (nighttime mean GH SD+IGF-I SD). While IGF-I (SD) and the mean spontaneous nighttime GH (SD) showed a significant correlation with body mass index, the S score was independent of body mass. We, therefore, used the S score to define a new test for GH deficiency. A child failed this bivariate test if his S score was less than -2 SD. We applied this model to 47 normal children and 48 short or slowly growing children (all prepubertal). We measured spontaneous nighttime GH levels and IGF-I levels in all children. In addition, the short children underwent 3 GH stimulation tests. Forty-six of the 47 normal children passed the bivariate test for GH sufficiency. Twenty-three of the 48 short or slowly growing children failed the bivariate test, whereas only 11 children had an abnormally low mean spontaneous nighttime GH measurement alone. Sixteen of 23 children who were GH deficient by the bivariate test were also GH deficient by the stimulation tests. In summary, the bivariate test for GH deficiency appears 1) to be independent of body mass, unlike either IGF-I or GH individually; 2) to identify more children than the mean spontaneous nighttime GH level alone; and 3) to be highly specific in the normal population, unlike stimulation tests.

A comparison of the standard high dose dexamethasone suppression test and the overnight 8-mg dexamethasone suppression test for the differential diagnosis of adrenocorticotropin-dependent Cushing's syndrome.

To improve the overnight 8-mg dexamethasone (DEX) suppression test (DST) for differential diagnosis of Cushing's syndrome and to develop optimal criteria for its interpretation, we increased the number of blood samples and measured the suppression of both plasma ACTH and cortisol. Forty-one patients who were subsequently proven at surgery to have Cushing's syndrome were studied (34 Cushing's disease and 7 ectopic ACTH secretion). DEX (8 mg, orally) was administered at 2300 h. Blood samples for ACTH and cortisol measurements were obtained at 0800, 0830, and 0900 h on the day before and at 0700, 0800, 0900, and 1000 h on the morning after DEX treatment. The conventional 6-day DST was also performed, with measurement of both urinary free cortisol and urinary 17-hydroxysteroids as indices of suppression. Optimal criteria for the diagnosis of Cushing's disease were developed for both the overnight 8-mg and the 6-day tests using receiver operating characteristic curves. The results were compared with those using the previously published criteria for diagnosis of Cushing's disease by the overnight 8-mg test (> 50% suppression of plasma cortisol at 0700-0800 h). In our patients, the previously published criterion for the overnight 8-mg test yielded high sensitivity (88%), but low specificity (57%), in making the diagnosis of Cushing's disease. When the time of cortisol measurement and the diagnostic criteria for Cushing's disease were revised to achieve 100% specificity, the sensitivity of the overnight 8-mg test was 71%, which was not significantly different from that of the 6-day test (79%; P = NS). Addition of plasma ACTH levels to the test did not improve diagnostic accuracy compared to that with measurement of plasma cortisol levels alone. When the revised 8-mg overnight dexamethasone suppression test was combined with the 6-day dexamethasone suppression test, sensitivity increased to 91%, with a specificity of 100%, which was significantly better than that of the overnight 8-mg test alone (P < 0.002). We conclude that the overnight 8-mg DST has low specificity for the diagnosis of Cushing's disease when performed as originally described. However, with revised sampling times and diagnostic criteria, the overnight test has sensitivity and specificity similar to those of the conventional 6-day DST. The diagnostic performance of a criterion that combines the results of both tests is better than the diagnostic performance of either test alone.

Disease progression in sporadic inclusion body myositis: observations in 78 patients.

Functional decline for each decade at symptom onset and need for cane, walker, or wheelchair were assessed in 78 biopsy-proved patients with sporadic inclusion body myositis. Patients with disease onset between 40 and 59 years used a walker after 10.2 +/- 5.8 years, whereas those with disease onset between 60 and 79 years used a walker after 5.7 +/- 5.0 years (p = 0.05). Because patients progress faster to disability when symptoms begin after the age of 60, age at disease onset may define patient subsets for stratification in clinical trials.

Telephone triage by primary care physicians.

To determine if experienced primary care physicians are more likely to reach correct decisions on the telephone than their less experienced colleagues, we asked 31 first-year and 29 third-year residents, 21 faculty, and 36 private practitioners in pediatrics and family practice to evaluate three pediatric patients via a telephone interview with a simulated mother and to decide whether each patient needed to be seen that evening. Compared with first-year residents, the third-year residents, faculty and private practitioners decided less frequently to see children who were not severely ill (P less than .05) or injured (P less than .01); however, less than half obtained histories considered adequate to rule out potential serious illnesses. Faculty did better than either residents or private practitioners in managing a severely dehydrated child; 100% of the faculty, but less than 60% of the residents or private practitioners, chose to see the patient promptly (P less than .001). More than one third of all residents and private practitioners reached inappropriate management decisions despite obtaining information that should have altered their decisions. In these simulations, experience in private practice was not associated with improved telephone management of very sick children. Faculty physicians appeared to be better able to identify severely ill children without inappropriately evaluating those who were less ill. In all three simulations, attainment of the correct decision appeared to be determined not by the number or type of questions asked, but rather by the physician's interpretation of the information collected.

Central venous pressure or pulmonary capillary wedge pressure as the determinant of fluid replacement in aortic surgery.

Both central venous pressure (CVP) and pulmonary capillary wedge pressure (PCWP) have been used as guides to fluid replacement in patients undergoing abdominal aortic surgery. To test the hypothesis that changes in PCWP (deltaPCWP) may be reliably estimated from changes in CVP (deltaCVP), 55 patients had simultaneous measurements (302 paired observations) of CVP and PCWP, as well as of cardiac output, before and during operation. The correlation between deltaCVP and deltaPCWP was high (r = 0.716, P less than 0.001). The estimated slope for the linear regression was 0.88. The standard error of the estimate was +/- 3.75 torr, making the prediction of deltaPCWP from deltaCVP accurate only to within 7.5 torr. There were five patients who each had a low and failing CVP accompanied by a stable cardiac output and a rising PCWP; for them, a decision to administer fluid based on the CVP might have resulted in pulmonary edema. Although deltaCVP accurately predicted direction and magnitude of deltaPCWP in the majority of patients (90%), there was a small group in whom there was no substitute for direct measurement of PCWP.

Variation of hepatic fibrosis and granuloma size among mouse strains infected with Schistosoma mansoni.

To investigate the relation between the size of circumoval granulomas and hepatic fibrosis, a variety of mouse strains infected with Schistosoma mansoni were examined and the number of eggs in the tissues, the fibrotic responses to the eggs, and the volume of the granulomas were determined. Marked differences in granuloma volume and in hepatic fibrosis were found between mouse strains, and those strains with the largest granulomas also showed the most hepatic fibrosis. On the other hand no significant correlation between granuloma size and hepatic fibrosis was found in the progeny of the F2 generation and backcrosses between F1 mice and the parental strains when crosses were made between Nmri mice (high granuloma volume and high fibrosis) and C57BL/6 mice (low granuloma volume and low fibrosis). Hepatic fibrosis per egg decreased with increasing infection intensity while granuloma volume was unaffected, indicating that fibrosis and granuloma size are at least in part modulated by different factors. The number of eggs found in the tissues per worm pair and the proportion of eggs in the liver also decreased as infection intensity increased. Some influence of the major histocompatibility complex on both granuloma size and fibrosis was found. Congenic mice on the C57BL/10 and C3H/HeSn backgrounds showed larger granulomas in H-2b than in H-2k mice, but no such correlation was found in comparing C57BL/6 mice with B6.H-2k mice. Less hepatic fibrosis was found in B10.M (H-2f), B10.SM (H-2v), and B10.RIII (H-2r) animals than in C57BL/10 mice. The regulation of granuloma size and of hepatic fibrosis is clearly complex and involves genes both outside of and within the major histocompatibility complex.

Effects of light on T-cells in HIV-infected subjects are not dependent on history of seasonal affective disorder.

In order to evaluate the effect of light on helper- and suppressor-T-cell counts in HIV-infected individuals, with and without a history of seasonal affective disorder (SAD), we treated 35 subjects with 45 min of light therapy in the morning, in a crossover design involving two 2 week treatment conditions: visible white light (half-peak band width, 530-620 nm; 10,000 lux) and visible red light (half-peak band width, 615-685 nm; 175 lux). We found small but significant differences between the two treatment conditions, with higher CD4 and CD8 levels during the white, as compared with the red, condition. There were no differences between baseline and treatment conditions. Both light treatment conditions were associated with significant mood improvements in the SAD, but not the non-SAD, subjects. There was no evidence that the higher cell counts seen under the bright light conditions were mediated by the effects of light on mood or on plasma cortisol levels. While the size of the light effect on T-cells renders questionable the potential therapeutic value of this treatment modality for HIV, the finding is of theoretical interest and is reassuring for those HIV-infected individuals who require light treatment for other reasons.

Recovery of reproductive function in rats treated with the aromatase inhibitor fadrozole.

The aromatase inhibitor, fadrozole hydrochloride (CGS 16949A), was developed for the treatment of breast cancer, and has not been available for pediatric use because of the lack of information about potential reproductive toxicology. To determine the effect of fadrozole on subsequent fertility and reproductive performance in rats, peripubertal male and female Sprague-Dawley rats (10/group) were given fadrozole by oral gavage once a day for 60 consecutive days (age 21 through 80 d) at a dose of 0, 1200, or 6000 micrograms/kg/d (dose range in women with breast cancer: 60 to 240 micrograms/kg/d). Following a 30-d recovery period (days 81 through 111 of age), cohabitation with untreated rats of the opposite sex was accomplished for 30 d or until positive evidence of mating was obtained (daily vaginal smears). The nonfadrozole-treated males used for cohabitation were proven fertile breeders; the females were virgin with proven 4-d estrous cycles. The duration of pregnancy, number, sex, condition, and body weight of pups were determined. Pregnant rats were weighed on gestational days 7, 14, and 20. There was a profound decrease in the number of estrous cycles at both dose levels of fadrozole compared to the control (P < 0.001). During the 30-d recovery period, estrous cycles were reestablished within a few days in the treated rats and the number and length of estrous cycles were not statistically different between fadrozole-treated and control rats. The gestational body weights of fadrozole-treated and untreated females did not differ significantly. There were no statistically significant differences in the number of matings/number of pairings, gestational length, mean live pups/litter, % pups born alive/litter, and % male pups/litter in the three groups (vehicle-, low-, and high-dose fadrozole-treated females, cohabited with untreated males and fadrozole-treated males, cohabited with untreated females). Thus, young male and female rats treated for 60 d with large doses of fadrozole had no detectable adverse effect on subsequent reproductive function.

Probability machines: consistent probability estimation using nonparametric learning machines.

BACKGROUND: Most machine learning approaches only provide a classification for binary responses. However, probabilities are required for risk estimation using individual patient characteristics. It has been shown recently that every statistical learning machine known to be consistent for a nonparametric regression problem is a probability machine that is provably consistent for this estimation problem. OBJECTIVES: The aim of this paper is to show how random forests and nearest neighbors can be used for consistent estimation of individual probabilities. METHODS: Two random forest algorithms and two nearest neighbor algorithms are described in detail for estimation of individual probabilities. We discuss the consistency of random forests, nearest neighbors and other learning machines in detail. We conduct a simulation study to illustrate the validity of the methods. We exemplify the algorithms by analyzing two well-known data sets on the diagnosis of appendicitis and the diagnosis of diabetes in Pima Indians. RESULTS: Simulations demonstrate the validity of the method. With the real data application, we show the accuracy and practicality of this approach. We provide sample code from R packages in which the probability estimation is already available. This means that all calculations can be performed using existing software. CONCLUSIONS: Random forest algorithms as well as nearest neighbor approaches are valid machine learning methods for estimating individual probabilities for binary responses. Freely available implementations are available in R and may be used for applications.

Patient-centered yes/no prognosis using learning machines.

In the last 15 years several machine learning approaches have been developed for classification and regression. In an intuitive manner we introduce the main ideas of classification and regression trees, support vector machines, bagging, boosting and random forests. We discuss differences in the use of machine learning in the biomedical community and the computer sciences. We propose methods for comparing machines on a sound statistical basis. Data from the German Stroke Study Collaboration is used for illustration. We compare the results from learning machines to those obtained by a published logistic regression and discuss similarities and differences.

Practical experiences on the necessity of external validation.

The validity of prognostic models is an important prerequisite for their applicability in practical clinical settings. Here, we report on a specific prognostic study on stroke patients and describe how we explored the prediction performance of our model. We considered two practically highly relevant generalization aspects, namely, the model's performance in patients recruited at a later time point (temporal transportability) and in medical centers different from those used for model building (geographic transportability). To estimate the accuracy of the model, we investigated classical internal validation techniques and leave-one-center-out cross validation (CV). Prognostic models predicting functional independence of stroke patients were developed in a training set using logistic regression, support vector machines, and random forests (RFs). Tenfold CV and leave-one-center-out CV were employed to estimate temporal and geographic transportability of the models. For temporal and external validation, the resulting models were used to classify patients from a later time point and from different clinics. When applying the regression model or the RFs, accuracy in the temporal validation data was well predicted from classical internal validation. However, when predicting geographic transportability all approaches had difficulties. We observed that the leave-one-center-out CV yielded better estimates than classical CV. On the basis of our results, we conclude that external validation in patients from different clinics is required before a prognostic model can be applied in practice. Even validating the model in patients recruited merely at a later time point does not suffice to predict how it may fare with regard to another clinic.

Job satisfaction of staff registered nurses in primary and team nursing delivery systems.

Job satisfaction for nurses in team and primary nursing delivery systems as measured by a human needs questionnaire was studied. Staff-level registered nurses from 27 units in a large research hospital completed a 22-item questionnaire. Each item measured present and desired job satisfaction and the importance of the item to the respondent. Data from 115 primary-system nurses and 65 team-system nurses were analyzed by Kruskal-Wallis tests of significance and chi-square contingency tables. Overall, primary-system nurses reported greater job satisfaction than team-system nurses. The primary system offered more opportunity for accountability and fulfilling higher level needs. Neither system provided sufficient opportunities for self fulfillment, decision-making, or independent judgment. The identified weakness of the primary system was the unmet need for supervision. Of all the nurses in this study, 75% were dissatisfied with the prestige accorded to them.

Giant papillary conjunctivitis and meibomian gland dysfunction blepharitis.

In this prospective pilot study we saw 42 consecutive giant papillary conjunctivitis (GPC) patients (80 eyes), all of whom were found to have some meibomian gland dysfunction blepharitis. Severity of GPC at presentation correlated with severity of meibomian gland dysfunction blepharitis (Kendall's tau b averaged 0.246 across the adjusted statistical analyses). Thirty-two patients (63 eyes) were refit after treating meibomian gland dysfunction blepharitis. Twenty-eight of these patients (55 eyes; 87%) continue to wear contact lenses, maintaining an improvement in GPC (mean follow-up 21 months; range 11-36 months). We hypothesize that meibomian gland dysfunction blepharitis may play a role in the pathogenesis of GPC and suggest that a large, controlled, multi-observer study be performed to further investigate this possible association.

Size and shape analysis of schistomsome egg-counts in Egyptian autopsy data.

We study egg-counts from a series of Egyptian autopsy cases with active schistosomiasis at death (Kamel, Cheever, Elwi, Mosimann and Danner 1977). The data are unique, and enable us to study the proportional distribution of eggs among various organs in relation to infection intensity for two species of schistosome worms. We develop a model for the distribution of eggs in three organs of the mesenteric circulation. Under a lognormal distribution assumption, several exact statistical procedures are presented and used to examine model consistency as well as isometry with respect to geometric means. Mesenteric egg counts for both Schistosoma mansoni and S. haematobium behave consistently with the model, and show little or no departure from isometry in cases without Symmers' fibrosis of the liver. We show that cases with Symmers' fibrosis have relatively more S. mansoni eggs in the small, as opposed to large, intestine. In the genito-urinary organs we show that the proportion of S. haematobium eggs in the bladder is high for low-intensity infections. This is consistent with the conclusion of Smith, Elwi, Kamel and Lichtenberg (1975), based on egg/gram data, that such infections may be initiated in the bladder. The correspondingly high proportion of eggs in the ureters with high-intensity infections indicates that increased intensity may have a disproportionately large pathologic effect on the infected individual.

Evaluating UMLS strings for natural language processing.

The National Library of Medicine's Unified Medical Language System (UMLS) is a rich source of knowledge in the biomedical domain. The UMLS is used for research and development in a range of different applications, including natural language processing (NLP). In this paper we investigate the nature of the strings found in the UMLS Metathesaurus and evaluate them for their usefulness in NLP. We begin by identifying a number of properties that might allow us to predict the likelihood of a given string being found or not found in a corpus. We use a statistical model to test these predictors against our corpus, which is derived from the MEDLINE database. For one set of properties the model correctly predicted 77% of the strings that do not belong to the corpus, and 85% of the strings that do belong to the corpus. For another set of properties the model correctly predicted 96% of the strings that do not belong to the corpus and 29% of the strings that do belong to the corpus.

In vivo microdialysis assessment of extracellular serotonin and dopamine levels in awake monkeys during sustained fluoxetine administration.

Fluoxetine (FLU) rapidly enhances extracellular (EC) serotonin (5-HT) in rodent brain, whereas the antidepressant effects of this drug in humans are typically not observed for 2-3 weeks. Thus, the effects of chronic oral FLU administration on neocortical and hippocampal EC 5-HT, and on caudate EC 5-HT and dopamine (DA), were examined in awake monkeys (Macaca fascicularis) using in vivo microdialysis (10.0 mg/kg; 3, 7, 14, and 21 days). On day 3, 5-HT was significantly increased above baseline levels in hippocampus (HC) and caudate. There was a trend for an increase in neocortex EC 5-HT levels. However, by day 7 5-HT remained significantly elevated only in HC, although 5-HT levels elsewhere had not completely returned to baseline. In contrast, levels of the 5-HT metabolite, 5-HIAA, were significantly reduced in all brain regions at all time points. Caudate DA levels tended to be decreased throughout FLU treatment. Local FLU and K(+) infusion were also used at various times during chronic systemic FLU administration to evaluate changes in functional synaptic regulation. In general, these results, along with the significant decrease in 5-HIAA levels and the tendency for basal EC 5-HT levels to remain modestly elevated only in HC during sustained FLU administration, suggest a reduction in releasable pools of 5-HT. Taken together with the trend for a decrease in caudate EC DA levels, these results do not appear to support the current hypothesis regarding the mechanism of action of SSRI antidepressants-that monoaminergic neurotransmission is progressively augmented during chronic treatment.

Comparison of breathing comfort during weaning with two ventilatory modes.

In twenty-one patients ventilated for > or = 3 days, we compared similar levels of partial support provided by synchronized intermittent mandatory ventilation (SIMV) and pressure support ventilation (PSV) in terms of breathing comfort. On a single day, eligible subjects experienced, in random order, both SIMV and PSV weaning protocols (sequential 20% reductions in support at timed intervals) separated by a 1 to 3 h rest. Breathing comfort was defined by subjective ratings of dyspnea and anxiety. Subjects reported significant levels of preweaning dyspnea and anxiety despite resting for at least 6 h. Dyspnea and anxiety were not significantly different between the two methods at any level of support. Our findings suggest that dyspnea and anxiety are higher than expected on "full" ventilator support, and that comfort may not differ between PSV and SIMV during active withdrawal of machine support.