Development and Validation of a Risk-Adapted Scoring Model for Metachronous Upper Tract Urothelial Carcinoma following Radical Cystectomy.

PURPOSE: The incidence and risk factors for metachronous upper tract urothelial carcinoma (UTUC) following radical cystectomy (RC) remain incompletely defined, which has limited the ability to individualize postoperative surveillance. MATERIALS AND METHODS: A retrospective review of 2 institutional registries was performed to identify patients undergoing RC for urothelial carcinoma. Multivariable Cox proportional hazard models for metachronous post-RC UTUC were developed in one institutional data set and validated in the second institutional data set. A post-RC UTUC risk score was then developed from these models. RESULTS: A total of 3,170 RC patients were included from the training cohort and 959 RC patients from the validation cohort. At a median followup after RC of 4.6 years (IQR 2.1-8.7), 167 patients were diagnosed with UTUC. On multivariable analysis in the training cohort, risk factors for metachronous UTUC were the presence of positive urothelial margin (HR 2.60, p <0.01), history of bacillus Calmette-Guérin treatment prior to RC (HR 2.20, p <0.01), carcinoma in situ at RC (HR 2.01, p <0.01) and pre-RC hydronephrosis (HR 1.48, p=0.04). These factors had similar discriminative capacity in the training and validation cohorts (C-statistic 0.71 and 0.73, respectively). A UTUC risk score was developed with these variables which stratified patients into low (0 points), intermediate (1-3 points), and high risk (4+ points) for post-RC UTUC, with respective 5-year UTUC-free survivals of 99%, 96%, 89% in the training cohort and 98%, 96%, and 91% in the validation cohort. CONCLUSIONS: We developed and validated a risk score for post-RC UTUC that may optimize UTUC surveillance protocols after RC.

Crystal structure of the extracellular domain from P0, the major structural protein of peripheral nerve myelin.

P0, the major protein of peripheral nerve myelin, mediates membrane adhesion in the spiral wraps of the myelin sheath. We have determined the crystal structure of the extracellular domain from P0 (P0ex) at 1.9 A resolution. P0ex is folded like a typical immunoglobulin variable-like domain; five residues at the C-terminus are disordered, suggesting a flexible linkage to the membrane. The requirements for crystallization of P0ex are similar to those for maintaining the native extracellular spacing of adjacent myelin lamellae; thus, given the self-adhesive character of P0ex, the crystal itself may reveal some of the natural interactions that occur between P0 molecules in myelin. The structure leads to the suggestion that P0 extracellular domains may emanate from the membrane surface as tetramers that link to tetramers on the opposing membrane surface, to result in the formation of networks of molecules. We report analytical ultracentrifugation data for P0ex that support this idea.

Open-label sildenafil treatment of partial and non-responders to double-blind treatment in men with antidepressant-associated sexual dysfunction.

Fifty partial and non-responders (Clinical Global Impression-Sexual Function (CGI-SF) score>2), out of 76 men who completed a 6-week, double-blind, placebo-controlled trial of sildenafil treatment for serotonergic antidepressant-associated sexual dysfunction, were eligible for an additional 6-week trial of open-label sildenafil (50 mg adjustable to 100 mg) under the same protocol, with blind maintained to initial assignment. Participation (double-blind and open-label) required major depressive disorder in remission (MDD-R) and continuing antidepressant medication. Forty-three entered open-label study: 16/17 initially randomized to sildenafil (sildenafil/sildenafil) and 27/33 initially randomized to placebo (placebo/sildenafil). Thirty-five of 43 (81%) achieved full response (CGI-SF

Solution structure of an isolated antibody VL domain.

The solution structure of the isolated VL domain of the anti-digoxin antibody 26-10 has been determined using data derived from heteronuclear multi-dimensional nuclear magnetic resonance (n.m.r.) experiments. Analytical ultracentrifugation and n.m.r. data demonstrate that the VL domain is only weakly associating (Kd = 2.5 (+/- 0.7) mM) and that it experiences a rapid monomer/dimer equilibrium under the n.m.r. experimental conditions. Therefore, the results reported here represent the first structure determination of an antibody VL domain in the absence of fixed quaternary interactions. The structure determination is based on 930 proton-proton distance constraints, 113 dihedral angle constraints, and 46 hydrogen bond constraints. Eighty initial structures were calculated with the variable target function program DIANA; of these, 31 were accepted on the basis of satisfaction of constraints (no distance constraint violations > 0.5 A; target function < 3.0 A2). Accepted DIANA structures were refined by restrained energy minimization using the X-PLOR program. The 15 best energy-minimized DIANA structures were chosen as a representative ensemble of solution conformations. The average root-mean-square differences (r.m.s.d.) between the individual structures of this ensemble and the mean coordinates is 0.85 (+/- 0.10) A for all backbone atoms and 1.29 (+/- 0.10) A for all heavy atoms. For beta-strands A, B, C, D, E and F, the average backbone atom r.m.s.d. to the mean structure is 0.46 (+/- 0.06) A. A higher-resolution ensemble, with all backbone atom and all heavy atom r.m.s.d.s. to the mean coordinates of 0.54 (+/- 0.08) A and 0.98 (+/- 0.12) A, respectively, was obtained by X-PLOR simulated annealing refinement of the 15 energy-minimized DIANA structures. A detailed analysis of the original ensemble of 15 energy-minimized DIANA structures is presented, as this ensemble retains a broader, and possibly more realistic, sampling of conformation space. The backbone atom and all heavy atom r.m.s.d.s between the mean energy-minimized DIANA structure and the X-ray derived coordinates of the VL domain within the Fab/digoxin complex are 1.05 A and 1.56 A, respectively. Subtle differences between the solution and X-ray structures occur primarily in CDR2, CDR3, beta-strands A, F and G, and localized regions of hydrophobic packing. Overall, these results demonstrate that the 26-10 VL domain conformation is determined primarily by intradomain interactions, and that quaternary VL-VH association induces relatively minor conformational adjustments.

Amino acid sequence of the N-terminal domain of calf thymus histone H2A.Z.

The minor histone H2A subtype, H2A.Z, has been purified to homogeneity from calf thymus and subjected to automated Edman degradation. The sequence of the first 30 amino acids possesses only 60% homology with major H2A subtypes of the same tissue. This sequence difference is more extreme than that exhibited between evolutionarily distant major H2A subtypes. However, an analysis of secondary structure reveals that H2A.Z and major H2A subtypes exhibit the same general topographical features within their N-terminal domains.

Tight ligand binding affinities determined from thermodynamic linkage to temperature by titration calorimetry.

A general isothermal titration calorimetry method is described that can be used to determine equilibrium binding constants for high-affinity interactions of ligands with biological macromolecules. The method exploits the thermodynamic linkage between the ligand binding equilibrium constant and temperature. By measuring the binding enthalpy change for an interaction as a function of temperature directly, the change in affinity can be calculated with an integrated form of the van't Hoff equation that is applicable to ligand binding to biological macromolecules. When the temperature dependence of the affinity is combined with the absolute affinity determined independently at a convenient temperature (where the affinity can most accurately or most easily be measured), the absolute binding affinity over the entire temperature range is determined.

Determining confidence intervals for parameters derived from analysis of equilibrium analytical ultracentrifugation data.

In the above discussion, we have introduced the profiling approach of Bates and Watts. It is an easy to implement, empirical approach to the determination of confidence intervals for parameters in nonlinear models. We have applied the approach to the analysis of equilibrium sedimentation data and have demonstrated that, although models for analyzing such data are formally nonlinear they are functionally linear. As such, linear approximation confidence intervals for the parameters are adequate for these models and data sets. Further, we have been able to examine the effect of implementing a multiple independent variable approach (in this case, using multiple rotor speeds) on the precision of the analysis. We found that the standard errors of the parameters were reduced and that this is accounted for by either the increase in the number of data points or the decreases in parameter correlation. In this case, profiling helped to visualize the effect on the sum of squares surface of reducing parameter correlation, making the effect of the small decreases in the correlation of some parameters more evident. Using profiling, it should be easy to explore other methods for the improvement of the analysis of ultracentrifugation data and to be able to quantitate the improvement. With the above discussion as an example, it is likely that the profiling approach should be quite useful and broadly applicable in the analysis of data in terms of nonlinear models.

Measurement of protein interaction bioenergetics: application to structural variants of anti-sCD4 antibody.

This chapter has described a bioenergetic analysis of the interaction of sCD4 with an IgG1 and two IgG4 derivatives of an anti-sCD4 MAb. The MAbs have identical VH and VL domains but differ markedly in their CH and CL domains, raising the question of whether their antigen-binding chemistries are altered. We find the sCD4-binding kinetics and thermodynamics of the MAbs are indistinguishable, which indicates rigorously that the molecular details of the binding interactions are the same. We also showed the importance of using multiple biophysical methods to define the binding model before the bioenergetics can be appropriately interpreted. Analysis of the binding thermodynamics and kinetics suggests conformational changes that might be coupled to sCD4 binding by these MAbs are small or absent.

The ABRF-MIRG'02 study: assembly state, thermodynamic, and kinetic analysis of an enzyme/inhibitor interaction.

Fully characterizing the interactions involving biomolecules requires information on the assembly state, affinity, kinetics, and thermodynamics associated with complex formation. The analytical technologies often used to measure biomolecular interactions include analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). In order to evaluate the capabilities of core facilities to implement these technologies, the Association of Biomolecular Resource Facilities (ABRF) Molecular Interactions Research Group (MIRG) developed a standardized model system and distributed it to a panel of AUC, ITC, and SPR operators. The model system was composed of a well-characterized enzyme-inhibitor pair, namely bovine carbonic anhydrase II (CA II) and 4-carboxybenzenesulfonamide (CBS). Study participants were asked to measure one or more of the following: (1) the molecular mass, homogeneity, and assembly state of CA II by AUC; (2) the affinity and thermodynamics for complex formation by ITC; and (3) the affinity and kinetics of complex formation by SPR. The results from this study provide a benchmark for comparing the capabilities of individual laboratories and for defining the utility of the different instrumentation.

Serum albumin and the metabolism of disulfiram.

The effectiveness of tetraethylthiuram disulfide (DSF) as a drug used in the treatment of alcohol abuse has been limited by the fact that it is degraded rapidly in the tissues and in the serum. Hence, a useful dose-response curve for this drug cannot be determined easily. The degradation in the tissues has been well characterized; however, its fate in the serum is less well understood. Here we kinetically describe the first steps in the degradation of DSF in the serum which results from a covalent interaction of this drug with the free sulfhydryl of serum albumin. DSF and its cleavage product diethyldithiocarbamate (DDC) both absorb significantly in the ultraviolet region. The reduction of DSF with mercaptoethanol to two molecules of DDC resulted in a large change in absorption in this region. The reaction of serum albumin with DSF produced a similar but much slower change in the ultraviolet absorption. As a result of the existence of this slow spectral change, we have been able to directly and continuously monitor the interaction of serum albumin and DSF and have determined that it is an overall first-order process. A model is proposed wherein DSF and serum albumin rapidly form a noncovalent adduct and, subsequently, in a slow unimolecular process, DSF is reduced to one mole of free DDC and one mole of the serum albumin-DDC mixed disulfide. At pH 9 the half-time for this process was 30 to 40 sec, and at pH 7.4 the half-time for this process was 1 to 1.5 min. These results suggest that degradation of DSF by serum albumin is physiologically and clinically important since the drug is maximally active only many hours after administration.

Antidepressant medication change in a clinical treatment setting: a comparison of the effectiveness of selective serotonin reuptake inhibitors.

BACKGROUND: This investigation focuses on the 3 most frequently used selective serotonin reuptake inhibitors (SSRIs) (paroxetine, fluoxetine, sertraline) and examines the rate of medication switches as a measure of effectiveness. We answer 2 questions: (1) What is the likelihood that a patient starting treatment with an SSRI will complete treatment with the same agent? and (2) Depending on the initial SSRI agent used, do patients switch at different frequencies? METHOD: A retrospective chart review was performed on 2779 patients treated in a university outpatient clinic from March 1995 to January 1997. Of these, 263 patients given antidepressants were randomly selected: 214 were prescribed SSRIs; 24, novel antidepressants; and 25, tricyclic antidepressants. RESULTS: There was no significant difference in rate of switching between the different classes of antidepressant (p = .1) nor between drugs within the SSRI class (p = .513). When medication change was the independent factor, significant differences between the groups were total time in treatment and number of visits (p < .001 and p = .011, respectively). Age, education, and Clinical Global Impressions-Severity of Illness scale scores (admission, discharge, and change) were not significantly different between the groups. CONCLUSION: Approximately 25% of patients started with an SSRI will switch to another antidepressant in the course of their treatment. The SSRIs appear to be equivalent in effectiveness. They are not interchangeable, because patients who discontinue one SSRI for lack of tolerability or response can generally be treated effectively with another.

Sildenafil for iatrogenic serotonergic antidepressant medication-induced sexual dysfunction in 4 patients.

OBJECTIVE: To evaluate the effect of sildenafil on iatrogenic serotonergic antidepressant-induced sexual dysfunction. METHOD: Four outpatients (2 men, 2 women) who developed sexual dysfunction (erectile impotence, anorgasmia) during treatment with a serotonin reuptake inhibitor antidepressant for psychiatric disorder were selected. Each subject was initially prescribed sildenafil 50 mg to be taken approximately 1 hour before sexual activity. The dose was increased to 100 mg for a partial or failed response. RESULTS: Four cases are detailed in case report fashion. All 4 had rapid reversal of their sexual dysfunction, usually with the first dose. Reversal equates to 1 successful use of sildenafil in each of 2 patients and 3 uses in 2 patients. CONCLUSION: Sildenafil may be an effective treatment for serotonergic antidepressant-induced sexual dysfunction and deserves further evaluation in randomized placebo-controlled studies.

Formulary restriction of selective serotonin reuptake inhibitors for depression: potential pitfalls.

The American healthcare market is currently estimated at more than 900 billion US dollars with double digit rising costs per year. Psychotropic agent costs have more than kept pace with market increases. Medication acquisition costs are an obvious focus for limiting costs in various care systems. Restrictive formularies are a common method of attempting to limit costs. To support our opinion that a single agent is ill advised, we explored the available evidence on the intended and unintended consequences of having a single or exclusive selective serotonin reuptake inhibitor (SSRI) on a formulary. Central to this position is an assumption of the interchangeability of SSRIs; we examined the evidence for and against this through a model to determine the probability of interchangeability. We conclude that the practice of having a single SSRI on the formulary for a healthcare plan seems ill founded. Patients who switch antidepressants remain in treatment 50% longer and cost approximately 50% more to treat in a more costly treatment setting. Giving the primary care physician several antidepressant choices can provide more options to continue treatment of his or her patient in the less expensive primary care setting. In terms of cost containment, formulary restrictions are far more likely to have the opposite effect.

Modeling the pharmacoeconomic cost of three selective serotonin reuptake inhibitors.

The authors present a method for modeling cost data on three selective serotonin reuptake inhibitors (SSRIs)-fluoxetine, paroxetine, and sertraline-from a large clinical outcomes study in a university-affiliated mental health center. Using data from 2,779 patients, average drug cost per day was calculated based on the percentage of patients on each daily dose of each medication. Given no overall significant difference between the SSRIs in effectiveness, the actual average cost per day determined by dose distribution was $1.79 for fluoxetine, $1.41 for paroxetine, and $1.21 for sertraline (using halved 100 mg tablets). The results suggest that cost can serve as one measure to help guide choice of medications.

Sildenafil for women patients with antidepressant-induced sexual dysfunction.

In an open study, sildenafil (Viagra) was prescribed for nine women outpatients who reported sexual dysfunction induced by antidepressant medication, primarily selective serotonin reuptake inhibitors. A 50 mg dose of sildenafil was prescribed, and patients were instructed to take it approximately one hour before sexual activity. They were told to increase the dose to 100 mg on the next occasion if they experienced a partial response or a lack of response to sildenafil. The nine patients, all of whom had experienced either anorgasmia or delayed orgasm with or without associated disturbances, reported significant reversal of sexual dysfunction, usually with the first dose of 50 mg of sildenafil.

Detection and consequences of recombinant protein isoforms: implications for biological potency.

Various types of structural variants have been observed in recombinant DNA - derived products. These isoforms include variations in post translational carbohydrate modifications where variations in site occupancy or unoccupied sites may occur. In addition, varying degrees of C-terminal processing and N-terminal substitutions have been observed. Isoforms may also be generated during processing and can include aggregated and/or chemically modified forms of the protein. Sophisticated analytical techniques exist for the identification and characterization of these structural variants. Several strategies have been used to isolate or enrich the isoform before molecular characterization. However, the effect these structural variations have on the biological activity of the product is less well understood. This may, in part, be due to the specificity and variability of the bioassay employed. This presentation describes the isolation and characterization of specific molecular isoforms for a monoclonal antibody product as well as an assessment of effects on biological activity.

Employing new grads: a plan for success.

The authors describe an elegant goal-driven program designed to prepare new nursing graduates to function optimally on a hospital unit by focusing first on their security and affiliative needs [belonging], and subsequently on their professional skill and knowledge acquisition. This developmental program has both a clear timeline and structure as well as well defined role expectations for the experienced RN guide/mentor, the orientee, and the unit's nurse manager and staff. The guide/mentors are carefully selected and prepared for their new roles as well. Shared goal-setting sessions are held weekly, along with three major evaluations during the first 90 days, with less frequent updates throughout the following year. This frequent feedback, reinforcement, and fine-tuning of goals allows the new graduate to gradually take on the full work load by the end of week 12 without feeling as overwhelmed or inadequate as those who are less carefully developed.