[Essential levels of care and mode of admission: a trial of cost-volume contracts]. / LEA e riconversione dei ricoveri: sperimentazione di contratti per costo e volume.

Within a research project funded by the Ministry of Health, the purpose of this work was to define the procedures of a contract for the provision of health services (hospital admissions), between the regional health administration (the "buyer") and the University teaching hospital (the "provider"), with the aim of improving efficiency. The contract concerned a few DRGs, among those identified as being "at risk of inappropriateness", in the national decree on "essential levels of care" (LEA). The contract defined production levels (number and percentage of admissions in day hospital), financing rules and methods for evaluation of results and improvement of performance within the hospital. This trial, even if run for a limited time and for demonstration purposes, showed that some results can be attained, provided some organizational adjustments are made.

Identification and characterization of two classes of receptors for oxytocin and vasopressin in porcine tunica albuginea, epididymis, and vas deferens.

The neurohypophysial hormones oxytocin (OT) and vasopressin (VP) are involved in the regulation of the contractility of the male genital tract in several animal species. We investigated the presence of specific binding sites for [3H]OT and [3H]arginine VP (AVP) in membranes prepared from tunica albuginea, epididymis, and vas deferens from prepubertal pigs 2-16 weeks of age. Membranes were incubated with [3H]OT and [3H]AVP in the presence or absence of the corresponding unlabeled peptides. Binding equilibrium was reached in 60 min at 22 C. Millimolar concentrations of Mg2+ increased the specific binding of both ligands. Analysis of families of self- and cross-displacement curves using the computer program LIGAND clearly demonstrated that two classes of binding sites were present in all tissues investigated. The first class of sites, designated the OT site, shows high affinity for OT, AVP, lysine vasopressin, arginine vasotocin, the selective OT agonists [Thr4,Gly7]OT and [Asu1,6]OT, and the OT antagonists derived from ornithine vasotocin (OVT), namely d(CH2)5Tyr(Et)OVT and dEt2OVT. The second class of sites, designated the VP site, shows high affinity for AVP, lysine vasopressin, arginine vasotocin, and the selective V1 antagonist d(CH2)5Tyr(Me)AVP. The V2 agonist [1-deamino,4-valine]8-D-AVP shows low affinity for both sites. Isotocin, desglycinamide [Arg-8]AVP and tocinoic acid were ineffective in displacing [3H]AVP or [3H]OT. The highest density of OT receptors was found in tunica albuginea and epididymis, whereas the highest density of AVP receptors was found in vas deferens. Adenylate cyclase was not activated in any of the tissues studied by concentrations of AVP or OT up to 100-fold greater than their Kd values. This is the first demonstration and pharmacological characterization of specific OT and V1 VP receptors in the tunica albuginea, epididymis, and vas deferens. The recent demonstration of high local concentration of neurohypophysial hormones in the gonads of several mammals support a physiological role of these OT and VP receptors in regulation of the motility of the male genital tract.

Immunoglobulins from Graves' patients stimulate phospholipase-A2 in FRTL5 thyroid cells.

The well documented ability of immunoglobulins G (IgGs) from Graves' patients to stimulate cAMP production is believed to be involved in the pathophysiology of this disease. It is still under discussion whether other intracellular messengers known to regulate thyroid function might play a similar role. This study shows that phospholipase-A2, a signal pathway unrelated to cAMP, is activated by Graves' IgGs. The IgGs from 67 patients with active Graves' disease, 8 patients with Graves' disease in remission, 5 patients with idiopathic myxedema, 2 patients with Hashimoto's thyroiditis, 57 patients with nonautoimmune thyroid disease, and 65 normal subjects were tested for their ability to stimulate phospholipase-A2 activity, as measured by arachidonic acid release from FRTL5 thyroid cells. The IgGs from patients with active Graves' disease caused a significant increase in arachidonic acid release compared to those from normal subjects, patients with nonautoimmune thyroid diseases, and patients with Graves' disease in remission (P less than 0.0001). The IgGs from active Graves' patients were also able to increase cAMP accumulation in FRTL5 cells. This effect did not correlate with the ability of the same IgGs to induce arachidonic acid release, suggesting that Graves' IgGs stimulate these two pathways by separate mechanisms. Moreover, a subgroup of IgGs that stimulated phospholipase-A2 did not increase the cAMP levels in FRTL5 cells. Our data suggest a novel mechanism of action of Graves' IgGs, the activation of phospholipase-A2, well distinguishable from the known effect on cAMP accumulation. The assay we describe could be helpful in improving the diagnosis and therapy of Graves' disease and in distinguishing it from nonautoimmune thyroid diseases. It also supplies the basis for a prospective subclassification of the Graves' patients, which might become useful to clarify the pathophysiology of this disease.

Naloxone administration does not affect gonadotropin secretion in agonadal men either basally or during testosterone treatment.

Naloxone administration has no effect on plasma gonadotropin levels of agonadal men. The present study was designed to evaluate whether testosterone replacement therapy could restore LH responsiveness to naloxone in such men. We measured plasma LH and FSH levels at 15-min intervals during naloxone infusion (8 mg in 1 min followed by 12 mg in 3 h) and for the following 3 h in a group of agonadal men both before and after at least 2 months of three different schedules of testosterone replacement therapy: 1) testosterone undecanoate, 40 mg three times a day by mouth; 2) testosterone enanthate 200 mg im every 2 weeks; and 3) testosterone enanthate 100 mg im once a week. Mean plasma gonadotropin levels as well as LH pulse frequency did not vary during naloxone infusion vs. placebo either basally or during each testosterone regimen. These results suggest that long term testosterone therapy does not affect the altered opioid modulation of gonadotropin secretion which is present in agonadal men.

Platelet adhesion to subendothelium--effect of shear rate, hematocrit and platelet count on the dynamic equilibrium between platelets adhering to and detaching from the surface.

The adherence of human 3H-adenine-labeled platelets to rat subendothelium was quantitated using a rotating probe device. Platelet adhesion increased in relation to the rotation time, reaching a plateau value in about 4-6 min without any further increase. A non-linear fitting analysis of experimental data allowed calculations of initial rate and plateau value of platelet adhesion. Increasing the shear rates (from 35 to 150 sec-1) or the hematocrit (from 10% to 40%), both the adhesion rate and the plateau value were increased. When different platelet concentrations were used the adhesion rate and the plateau calculated increased with platelet concentration. Different plateau values were obtained in the experimental conditions considered. This suggests that the plateau was not reached for the complete occupation of the subendothelial surface by the adherent platelets. Experiments using two different vessels rotated in the same platelet suspension or, viceversa, the same vessel rotated successively in two fresh platelet suspensions, showed that the plateau was not determined by reduced platelet reactivity. Rotating the same vessel first in radiolabeled platelets, until the plateau was reached, and secondly in non labeled platelets, or viceversa, showed that the plateau was indeed a dynamic condition where the number of platelets adhering and detaching reached equilibrium. These observations suggest that the platelet adhesion to subendothelium is the final equilibrium of two platelet fluxes, one adhering to the surface and another detaching from the surface.

Some tools for the diffusion of biomedical information using research networks.

Research and academic computer networks provide e-mail and other services to all members of participating institutions. Their usage by biomedical researchers and clinicians is still limited because of several reasons, including limited awareness of the available network resources. An increased use of these networks within the biomedical community would allow fast, effective communications and convenient remote access to information sources. As an example and pilot study, we prepared two network tools to make some information services maintained by our institution also accessible through e-mail. Both tools were implemented using PMDF e-mail software on a DEC MicroVAX connected to the Italian academic and research network (GARR), which is linked to the U.S. Internet. A network server takes care of automatic distribution of documents (files) reporting results of an oncology research/education project. An information server provides for semiautomated support of a consulting service on use of drugs. The feasibility of implementing these tools, based on existing software, further illustrates the potential usefulness of research computer networks for the dissemination of biomedical information.

Similarities and differences between D-ALA2 MET5 enkephalin amide and morphine in the induction of tolerance to their effects on catalepsy and on dopamine metabolism in the rat brain.

Rats were made tolerant to morphine or to DALA, a synthetic analogue of met-enkephalin, by prolonged exposure to these compounds. Tolerance was assessed by evaluating the resistance of the treated rats to present catalepsy after an acute dose of the opiates. Both morphine and DALA induced tolerance and cross-tolerance to the cataleptic effect. Acute administration of morphine and DALA increased the concentration of DOPAC in striatum, limbic area and s.nigra of control rats. This increase was not present when morphine was given acutely to chronically morphine-treated rats, indicating that these animals were tolerant to this effect. Chronically morphine-treated rats given DALA presented partial tolerance to the biochemical effect of the peptide in limbic area and in s.nigra but not in striatum, indicating that only in certain areas was cross-tolerance produced by chronic morphine. When DALA was administered at different doses to chronically DALA treated rats, the peptide induced rise in DA catabolite was similar to that produced in control animals, so clearly there was no tolerance to this biochemical effect. In these animals cross tolerance to morphine's effect on DA metabolism was present in s.nigra but not in the other two areas, indicating that s.nigra is particularly sensitive to opiate-induced tolerance on DA metabolism.

The relationship between rate of venous sampling and visible frequency of hormone pulses.

In this paper, a stochastic model of episodic hormone secretion is used to quantify the effect of the sampling rate on the frequency of pulses that can be detected by objective computer methods in time series of plasma hormone concentrations. Occurrence times of secretion pulses are modeled as recurrent events, with interpulse intervals described by Erlang distributions. In this way, a variety of secretion patterns, ranging from Poisson events to periodic pulses, can be studied. The notion of visible and invisible pulses is introduced and the relationship between true pulses frequency and mean visible pulse frequency is analytically derived. It is shown that a given visible pulse frequency can correspond to two distinct true frequencies. In order to compensate for the 'invisibility error', an algorithm based on the analysis of the original series and its undersampled subsets is proposed and the derived computer program is tested on simulated and clinical data.

EXPFIT: a program for simultaneous analysis of families of exponential decay curves.

We have developed a program to facilitate the simultaneous analysis and weighted least-squares curve fitting of families of exponential decay curves, subject to appropriate constraints. The simultaneous analysis of all curves allows one to pool information from several subjects or experiments, and avoids the need for approximations inherent in normalizing or transforming data. Selected parameters of the model can be constrained, i.e. shared among several curves, or set to a constant value. Hypotheses about the system under study can be tested in an objective, statistically valid manner. A BASIC computer program for routine data analysis is presented, with an example of its application to illustrative data.

A simplified method to determine binding parameters in a two-site case using linear regression.

A program using a simplified method to compute estimates of binding parameters in a system with two independent classes of binding sites is presented. An iterative method is used to approximate step-by-step the two lines describing the binding activities separately, starting from a curvilinear Scatchard or Hofstee plot. This method is designed for use on microcomputers with graphic facilities and on programmable hand-held calculators.

A versatile method for simultaneous analysis of families of curves.

We have developed a versatile new approach to the simultaneous analysis of families of curves, which combines the simplicity of empirical methods with several of the advantages of mathematical modeling, including objective comparison of curves and statistical hypothesis testing. The method uses weighted smoothing cubic splines; the degree of smoothing is adjusted automatically to satisfy constraints on curve chape (monotonicity, number of inflection points). By simultaneous analysis of a family of curves, one can extract the shape common to all the curves. Up to four linear scaling parameters are used to match the shape to each curve, and to provide optimal superimposition of the several curves. By applying constraints to these scaling factors, one can test a variety of hypotheses concerning comparisons of curves (e.g., identity, parallelism, or similarity of shape of two or more curves), and thus evaluate the effects of experimental manipulation. By optimal pooling of data one can avoid the need for arbitrary selection of a typical experiment, and can detect subtle but reproducible effects that might otherwise be overlooked. This approach can facilitate the development of an appropriate model. The method has been implemented in a Turbo-Pascal program for IBM-PC compatible microcomputers, and in FORTRAN-77 for the DEC-10 mainframe, and has been utilized successfully in a wide variety of applications.

Evaluation of pulse-detection algorithms by computer simulation of hormone secretion.

A versatile method is presented for generating synthetic hormonal time series, containing peaks at known locations, to be used to objectively evaluate both the false-negative (F-) and false-positive (F+) statistical error rates of computerized pulse-detection algorithms. Synthetic data are generated by assuming hormone secretion to occur as a succession of instantaneous release pulses, distributed as Poisson events, separated by quiescent intervals. The pulses are convolved to simulate cumulation of consecutive events and clearance of the hormone. Randomly generated errors, corresponding in magnitude to typical experimental measurement error, are then added to the convolved series. The choice of different values for simulation parameters (e.g., frequency and amplitude of pulses) allows one to emulate some typical physiological patterns of hormone secretion for luteinizing hormone, growth hormone, and thyrotropin or other hormones. Various subsets can be extracted from a simulated time series to study the effect of sampling frequency on the detection of pulses. We show that in sampled series the "observable frequency" of pulses is less than the true nominal frequency. Methods for evaluating pulse-detection algorithms and expressing the results are presented. Simulations of LH secretion were analyzed with the program DETECT. We show that minimizing F+ error rates only might lead to excessively high F- rates. A proper choice of sampling frequency and program probability levels can be made to provide acceptable F+ and F- error rates for various patterns of hormone secretion.

NL-FIT: a microcomputer program for non-linear fitting.

A program for a microcomputer (HP 85) has been written using BASIC language. Given the analytical form of a model y: R leads to R the program computes the optimal parameters for non-linear least-squares fitting and gives information for its statistical evaluation. The program uses the Gauss-Newton algorithm with Marquardt's modification and other tricks. During the run a message is displayed if there is a high correlation between one or more couples of parameters to establish an interactive dialogue with the user on critical problems. The authors' aim was to make the program sufficiently quick and easy to use, efficient for a wide class of models and robust enough to deal with bad starting parameters.

Detection and characterization of peaks and estimation of instantaneous secretory rate for episodic pulsatile hormone secretion.

We have developed a new computer program for detection of "peaks" in sequential hormone measurements in longitudinal studies of episodic hormone secretion. The program provides: (a) several statistically based approaches to the estimation of the random measurement error as a function of hormone level; (b) peak detection based on analysis of first derivatives with logic that has been optimized for asymmetrical peaks with exponential decays; (c) several approaches to the estimation of tolerances for the first and second derivatives; (d) a sensitive curve-fitting approach, to distinguish between upstrokes, exponential decays, and flat baselines; (e) ability to detect multiple overlapping peaks; (f) analysis of "robustness" by systematically varying the threshold around the most-likely value; (g) superimposition of detected peaks, to evaluate "average peak shape"; (h) analysis of the "decay rate," to obtain an estimate of the disappearance rate constant and half-life; (i) use of a "discrete deconvolution" approach, to solve for the apparent instantaneous rate of secretion, and provision of an error analysis to obtain estimates of the precision of these derived values; and (j) correlation with other relevant series as a means of cross validating. The program has been tested extensively on real and synthetic data, and appears to perform well. The frequency of "false positive" peaks can be held at any desired low level, and can be prevented from increasing as sampling frequency increases. The number of arbitrary assumptions, approximations, or thresholds is held to an absolute minimum. These methods are natural, logical, and follow from first principles of statistics.

A model-free approach to estimation of relative potency in dose-response curve analysis.

We have developed a new, general approach to analysis of dose-response curves from bioassay, immunoassay (including radioimmunoassay, immunoradiouretic assay, enzyme-linked immunosorbent assay), and other experimental procedures. It provides a test for parallelism, similarity of shape, and a measure of relative potency for any set of two or more curves. The method uses a constrained smoothing spline function to estimate the curve shape, together with a nonlinear least-squares fitting technique to estimate parameters for relative potency and slope. The use of "constrained splines" permits the analysis of nonlinear dose-response curves that cannot be described by a simple model or equation such as the symmetric four-parameter logistic. A microcomputer program is used for the analysis, providing relative potencies and their SE and evaluation of goodness of fit.

Objective assessment of concordance of secretory events in two endocrine time series.

A new objective method is presented for investigating the presence of a temporal relationship between episodic release of two hormones. The two time series of hormone concentrations are first analysed by an objective method for peak detection. Both data series are then transformed into "quantized" or discretized series by recording the occurrence of a hormone pulse as an "event", characterized by the onset, the maximum, or another unique feature. The two quantized series are then matched, and the number of concordant events and discordant events are counted. Each point in series A is compared with a "time-window" of a selected number of points in series B, to accommodate small degree of mismatch between events in the two series. An index of concordance is computed, compensating for any spurious random coincidence: the "Specific Concordance", to evaluate the frequency of concordant events in excess of those expected on the basis of chance alone. This calculation is systematically repeated, interposing a range of time-lags between the two series. A graph of Specific Concordance versus time-lag indicates the time-lag corresponding to a maximal concordance. Simulations of random series of events are performed, and their degree of concordance is evaluated in a similar fashion, thus generating frequency distributions of Specific Concordance values under the null hypothesis of no temporal relationship. This permits the selection of criteria for statistical significance at any desired p-level, for one or many lag times, and for one or multiple subjects. Various degrees of concurdance can also be stimulated to evaluate the performance (sensitivity, statistical power) of this approach. These methods have been implemented as a collection of short microcomputer programmes, and applied to the study of the temporal relationship between beta-endorphin and cortisol in normal subjects sampled every 10 min for 24 h. This analysis demonstrated concordance between events in the two series, with synchronous occurrence of beta-endorphin and cortisol release events significantly more frequently than expected on the basis of random association (p less than 0.01).

Oxytocin and V1 vasopressin receptors in rabbit endometrium during pregnancy.

Neurohypophysial hormone receptors were identified and characterized in rabbit endometrium and decidua by radioligand binding methods. The results strongly support the presence of a heterogeneity of sites in the decidua of parturient rabbits. The oxytocin site (R1) binds oxytocin and oxytocin analogues ([Thr4, Gly7]oxytocin and OTA) with high affinity, whereas the AVP site (R2) was selective for the V1 AVP analogues, [Phe2, Orn8]VT and d(CH2)5TyrMeAVP. The concentration of oxytocin receptors was low (50-100 fmol/mg protein) at oestrus (Day 0) and on Day 29 of pregnancy, but increased significantly (about 8-fold, P less than 0.05) during parturition. Conversely, V1 AVP receptors were more concentrated than the oxytocin sites at the end of pregnancy (150 fmol/mg protein) but did not change during parturition. These results indicate that neurohypophysial hormones have specific receptors not only in the myometrium but also in the uterine mucosa and we suggest that these receptors may participate in the regulation of uterine activity during pregnancy.

Identification and characterization of a vasopressin isoreceptor in porcine seminal vesicles.

Neurohypophysial hormones stimulate the motility of tunica albuginea, epididymis, and vas deferens acting through oxytocin (OT) and V1 vasopressin receptors. To test the hypothesis that these hormones are involved also in the regulation of seminal vesicle physiology, we studied binding of [3H]OT and [3H] arginine vasopressin ([3H]AVP) to porcine seminal vesicle membranes. Neurohypophysial hormones bind to two different classes of sites. The first class shows low capacity (35 fmol per mg of protein) and a very high affinity (Kd less than 1 nM) for both the labeled ligands. The second class is characterized by a high capacity (2000 fmol per mg of protein) and a high affinity for AVP (Kd approximately equal to 2.5 nM), whereas OT has 160 times lower affinity. Lysine vasopressin and the V1 antagonist [1-deaminopenicillamine, 2-(O-methyl)tyrosine]Arg8-vasopressin compete with high affinity with [3H]AVP binding, whereas the V2 agonist [1-deamino,4-valine]D-Arg8-vasopressin (dVDAVP) is 110 times less potent than AVP. The OT agonist [Thr4,Gly7]OT and the OT antagonist [1(beta-mercapto-beta, beta-cyclopentamethylene propionic acid), 2-(O-ethyl)tyrosine, 8-ornithine]vasotocin failed to affect [3H]AVP binding. These findings seem to suggest that AVP interacts with the V1 vasopressin isoreceptor in porcine seminal vesicle membranes. However, AVP stimulates adenylate cyclase activity in a dose-dependent fashion with an EC50 of 14 nM, whereas OT or dVDAVP has no effect at 100 nM. Moreover, a well-characterized V1 vasopressin antagonist, [1-(beta-mercapto-beta, beta-cyclopentamethylene propionic acid),2-(O-methyl)tyrosine]Arg8-vasopressin [d(CH2)5Tyr(Me)AVP], competes with [3H]AVP binding with an IC50 of 0.17 microM. These pharmacological properties are distinct from the previously described V1 and V2 vasopressin receptors and indicate the presence of a new class of AVP receptors. Although this vasopressin isoreceptor shares some pharmacological characteristics with the V1 (pressor) isoreceptor, it has low affinity for the V1 antagonist d(CH2)5-Tyr(Me)AVP and is linked to the adenylate cyclase system. The extremely high density of AVP receptors in porcine seminal vesicles (2 pmol per mg of protein) is comparable to the density of V2 vasopressin receptors in porcine renal medulla, suggesting a physiological role for vasopressin in the seminal vesicle.

Specific concordance index defines the physiological lag between LH and progesterone in women during the midluteal phase of the menstrual cycle.

Using a recently developed statistically based method for assessment of the degree of concordance, we evaluated the degree of specific concordance (SC) between luteinizing hormone (LH) and progesterone secretory patterns. Eight healthy women volunteered for this study, undergoing a 12-h pulsatility study, sampling every 10 min. LH and progesterone pulse frequencies were estimated with the program DETECT (9.75 +/- 1 and 11.5 +/- 0.9 pulses/12 h, respectively; mean +/- SEM). The temporal relationship between LH and progesterone secretions was evaluated with cross-correlation analysis and with the computation of the SC index. Cross-correlation showed concordance between LH and progesterone (p less than 0.05) at a range of lag between 0 and 40 min, while the SC index indicated that LH and progesterone pulses were significantly (p less than 0.05) and maximally correlated at 10-min lag. In conclusion, our data demonstrated that the specific concordance confirms the statistically significant concordance of LH and progesterone secretory events in women during the midluteal phase. In addition, the use of this new, objective, statistically based approach permits, compared to traditional cross-correlation analysis, a more precise definition of the physiological time lag for temporal coupling of secretory events between the two hormones.