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Aims of the study Based on large sets of routine hospital data from inpatient cases, we aimed to explore multimorbidity and intervention clusters showing high risks for in-hospital mortality and unplanned readmissions using data-driven analytical methods. Methods We performed an explorative, historical cohort study of consecutive inpatient cases at a tertiary care centre with an integrated platform for routine healthcare data in Switzerland. From January 2012 through to December 2017, all inpatients aged g18 years at hospital admission were eligible for study inclusion. We predefined all-cause in-hospital death and unplanned hospital readmission as co-primary outcomes. In a first step, we explored and visualised multimorbidity and intervention clusters using mutual information analysis. In a subsequent step, we trained multi-layer Bayesian networks to identify clusters associated with in-hospital death and/or unplanned hospital readmission. Results Among 190,837 inpatient cases, 7994 unique diagnoses and 6639 interventions were routinely recorded during the six-year study period. Based on the mutual information analysis, we identified 32 multimorbidity clusters and 24 intervention clusters n of which several were directly related to in-hospital mortality and/or unplanned readmission in the subsequent Bayesian network analysis. Conclusions Bayesian network analysis may be used as a tool to mine large healthcare databases in order to explore intervention targets for quality improvement programmes. However, the resulting associations should be substantiated in consecutive investigations using specific causal models. (Trial registration no EKNZ 2016-02128.).

We describe a scalable platform for research-oriented analyses of routine data in hospitals, which evolved from a state-of-the-art business intelligence architecture for enterprise resource planning. This platform involves an in-memory database management system for data modeling and analytics and a high-performance cluster for more computing-intensive analytical tasks. Setting up platforms for research-oriented analyses is a highly dynamic, time-consuming, and costly process. In some health care institutions, effective research platforms may be derived from existing business intelligence systems.

ZusammenfassungDie erfolgreiche Haltungskontrolle setzt eine effektive und effiziente Interaktion aller posturalen Systeme voraus, die sowohl aktiv als auch reaktiv mittels muskulärer Kräfte den Körper im Gleichgewicht halten. Eine Störung dieses komplexen Posturalsystems auf sensorischer, zentraler oder motorischer Ebene führt in der Folge zu einer zunehmenden Instabilität und zu einer erhöhten Sturzneigung. Insofern scheinen die zuverlässige Erhebung der posturalen Kontrolle und die damit verbundene Sturzprävalenz ein wichtiges Element der ambulanten und stationären Versorgung zu sein. Die motorisch-funktionellen Assessments, die sich aus verschiedenen Gleichgewichts- und Stabilitätsaufgaben ergeben, stellen noch immer den Goldstandard zur Quantifizierung der posturalen Kontrolle dar. Diese Verfahren sind jedoch oftmals nicht in der Lage, die Gleichgewichtsfähigkeit eines Patienten vollumfänglich abzubilden, oder sind aufgrund der subjektiven Bewertung oder unzureichender Standardisierung nur bedingt vergleichbar. Das Ziel der vorliegenden Studie war daher die Entwicklung eines quantitativen Messverfahrens zur Überprüfung der posturalen Kontrolle, basierend auf dem neuroorthopädischen Therapiegerät Posturomed®. Das Messsystem wurde in Form eines reaktiven Screeningverfahrens ausgeführt. Bei diesem werden mechanische Perturbationen erzeugt mittels Elektromagneten, die an der Unterstützungsfläche des Probanden appliziert werden. Der Eignungsnachweis dieses Systems für eine Quantifizierung der posturalen Kontrolle erfolgte durch eine zweiarmige Querschnittsstudie mit 115 gesunden Probanden (Referenzgruppe) und 149 neurologischen Patienten.Die Gruppe der neurologischen Patienten setzte sich zusammen aus– 69 Patienten mit zerebraler Mikroangiopathie (ZMA) mit in den letzten 12 Monaten klinisch eher leichter, aber chronisch progredienter Symptomatik,– 31 Patienten mit Morbus Parkinson Stadium I bis III nach Hoehn & Yahr als chronisch progrediente, multilokuläre Netzwerkerkrankung und – 49 Patienten mit klinisch vordergründig restierender Hemiparese bei Zustand nach zerebraler Embolie (Kraftgrad des betroffenen Beins 4/5 oder latente Parese) außerhalb der Subakutphase. Die Ergebnisse der Studie belegen, dass Perturbationsversuche zur Quantifizierung der posturalen Kontrolle für ein breites Spektrum neurologischer Patienten geeignet sind. Die Validität des Verfahrens konnte durch eine hohe inhaltliche Übereinstimmung zwischen der dynamischen Posturografie und den konvergenten Maßen der Motorik (Berg Balance Scale; Dynamic Gait Index) nachgewiesen werden. Das vorgestellte System erscheint folglich geeignet, das reaktive Gleichgewicht als eine Eigenschaft der motorischen Standkontrolle zu bestimmen. Die dynamische Posturografie auf Basis eines Perturbationsversuchs kann im klinischen und therapeutischen Umfeld zur Quantifizierung der posturalen Kontrolle eingesetzt werden. Zielgruppenlimitationen und damit verbundene Einschränkungen der Generalisierbarkeit von motorisch-funktionellen Testverfahren werden mit ihm überwunden. Darüber hinaus können auf der Basis der reaktiven posturalen Kompetenz individuelle Rückschlüsse auf das Sturzrisiko gezogen werden. Die Prognosegüte dieser Sturzrisikobewertung entspricht denen der motorisch-funktionellen Testverfahren und ist je nach Zielgruppe teilweise sogar besser. Für Patienten mit Morbus Parkinson konnte für den Dämpfungskoeffizienten bei einem Grenzwert von Δτ = − 0,45 s die höchste Güte ermittelt werden. Eine Sensitivität von 79 % und Spezifität von 78 % lassen eine gute Verwendung als sturzbezogenes Assessment erkennen. Der routinemäßige klinische Einsatz zur Quantifizierung der Sturzgefährdung, z. B. im Rahmen einer Krankenhausaufnahme, erscheint auf Grundlage der Ergebnisse empfehlenswert. Eine Grundvoraussetzung dafür, dass sich ein Assessment für eine Verlaufsbestimmung im Rahmen von Behandlungen einer Erkrankung eignet, ist seine ausreichende Reliabilität. Die laterale Perturbation und dann die Wertung von mittlerer Schwingungsgeschwindigkeit und Schwingungsdistanz sind geeignet für die Beurteilung der posturalen Stabilität im Verlauf neurologischer Erkrankungen und damit auch für eine quantitative Erfassung auftretender Therapieeffekte.

The ocular penetration of systemically administered antiretroviral drugs (ARVs), which is important in the clinical setting of HIV uveitis, is unknown. This study aimed to assess the ocular penetration of different antiretroviral drugs in an animal model.Twenty-five male New Zealand white rabbits were assigned to one of five treatment groups. Each group received a single oral dose of an antiretroviral drug (lamivudine, tenofovir, efavirenz, lopinavir, and raltegravir). These 5 drugs represent 4 different ARV drug classes [nucleoside reverse transcriptase inhibitors (NRTI), non-NRTI, protease inhibitors, and integrase inhibitors]. Serum, cerebrospinal fluid (CSF), and aqueous and vitreous humor samples were collected at the time of theoretical maximum serum concentration of each respective drug, for example, lamivudine 1 h, tenofovir 1 h, efavirenz 5 h, lopinavir 4 h, and raltegravir 3 h. The drug concentration in each sample was determined by means of high-performance liquid chromatography and mass spectrometry.After a single oral dose, measurable levels of all five ARVs administered could be detected in all 4 body compartments. The limit of detection based on a signal-to-noise ratio of 1:3 for the antiretroviral agents was as follows: 1 part per billion (ppb) for lamivudine, tenofovir, and efavirenz and 0.1 ppb for lopinavir and raltegravir. The IC50 (inhibitory concentration where 50% of viral replication is inhibited by a drug) was reached for all drugs in the serum and CSF. In the aqueous humor, lopinavir failed to reach IC50 and in the vitreous humor, only efavirenz and lopinavir attained IC50 levels.After a single oral dose, measurable levels of all 4 classes of ARVs could be detected in all 4 body compartments sampled. In the eye, IC50 levels were lower in the vitreous humor than in aqueous humor. IC50 levels in the serum were higher than in the CSF.

Objectives We aimed to quantify the shared information between medical diagnoses of an adult inpatient population to explore both multimorbidity patterns and vice versa the unrelatedness of medical diagnoses. Study Design and Setting This was a cross-sectional study, performed at a tertiary care center in Switzerland. Diagnoses were routinely coded using the International Classification of Diseases, 10th revision. Results Among 190,837 inpatient cases, 7,994 unique diagnoses were coded. There were 31.9 million possible diagnosis pairs; the respective mutual information scores in diagnosis pairs were low (range, 10−7 to 0.237). There were 148 pairs of diagnoses with a mutual information score higher than 0.01, which formed several clinically plausible disease clusters; 27.2% of cases did not have a diagnosis that belonged to one of the morbidity clusters. Conclusion In an explorative analysis, we observed a high unrelatedness of diagnoses in a tertiary-care inpatient population. This finding indicates that although multimorbidity patterns can be observed, inpatient cases frequently have further, unrelated diagnoses, which share little information with specific other diagnoses. Therefore, management of multimorbid patients should be individualized and may not be generalized based on a few multimorbidity patterns or clusters.

This paper summarizes the panel discussion at the IEEE Engineering in Medicine and Biology Point-of-Care Healthcare Technology Conference (POCHT 2013) held in Bangalore India from Jan 16–18, 2013. Modern medicine has witnessed interdisciplinary technology innovations in healthcare with a continuous growth in life expectancy across the globe. However, there is also a growing global concern on the affordability of rapidly rising healthcare costs. To provide quality healthcare at reasonable costs, there has to be a convergence of preventive, personalized, and precision medicine with the help of technology innovations across the entire spectrum of point-of-care (POC) to critical care at hospitals. The first IEEE EMBS Special Topic POCHT conference held in Bangalore, India provided an international forum with clinicians, healthcare providers, industry experts, innovators, researchers, and students to define clinical needs and technology solutions toward commercialization and translation to clinical applications across different environments and infrastructures. This paper presents a summary of discussions that took place during the keynote presentations, panel discussions, and breakout sessions on needs, challenges, and technology innovations in POC technologies toward improving global healthcare. Also presented is an overview of challenges and trends in developing and developed economies with respect to priority clinical needs, technology innovations in medical devices, translational engineering, information and communication technologies, infrastructure support, and patient and clinician acceptance of POC healthcare technologies., This paper summarizes the panel discussion at the IEEE Engineering in Medicine and Biology Point of Care Healthcare Technology Conference (POCHT 2013) held in Bangalore India from Jan 16-18, 2013.

Zusammenfassung : Hintergrund: : Über demographische und medizinische Charakteristika von chronischen Schmerzpatienten mit depressiven Symptomen in der Praxis ist bisher wenig bekannt. Methodik: : Die Charakteristika hinsichtlich regionaler Herkunft und Schmerz intensität zu Beginn einer antidepressiven Behandlung werden von 585 chronischen Schmerzpatienten mit depressiver Symptomatik aus 122 Arztpraxen der ganzen Schweiz (Innere Medizin, Allgemeinmedizin, Psychiatrie) beschrieben. Aufgrund der klinischen Erfahrungen im Schweizer Gesundheitssystem wurde vermutet, dass Internisten und Allgemeinpraktiker im Vergleich zu Psychiatern eher ältere und geringergradig depressive Patienten mit weniger intensiven chronischen Schmerzen behandeln, die vermehrt aus mitteleuropäischen Ländern stammen. Ergebnisse: : Tatsächlich behandelten Internisten (und Allgemeinpraktiker) gegenüber Psychiatern häufiger ältere Patienten aus Mitteleuropa mit leichteren depressiven Symptomen und geringerer Schmerzintensität sowie seltener Schmerzen im Kopfbereich. Weiter litten Patienten aus Ost- und Südeuropa gegenüber Mitteleuropäern unter intensiveren Gesamtschmerzen, und dies vor allem im Kopf-, Extremitäten-, Rücken- und Brustbereich, während Südeuropäer gegenüber Mitteleuropäern zusätzlich auch vermehrt unter Brustschmerzen litten. Schlussfolgerung: : Es handelt sich um eine vergleichsweise große Stichprobe, die praxisnah und aus allen Regionen der Schweiz niedergelassene, in Schmerzbehandlungen involvierte Internisten, Allgemeinpraktiker und Psychiater einbezieht und damit die Verhältnisse von Patienten mit körperlichen Schmerzen und einer depressiven Begleitsymptomatik hinsichtlich regionaler Herkunft und Schmerzintensität gut repräsentiert

Background Recent publications suggest a benefit from surgical removal of urothelial carcinoma metastases (UCM) for a subgroup of patients. Objective We report the combined experience and outcome of patients undergoing resection of UCM gained at 15 uro-oncologic centers in Germany. Design, setting, and participants Retrospective survey of 44 patients with distant UCM of the bladder or upper urinary tract who underwent complete resection of all detectable metastases in 15 different German uro-oncological centers between 1991 and 2008. Intervention Resected metastatic sites were the following: retroperitoneal lymph nodes (56.8%), distant lymph nodes (11.3%), lung (18.2%), bone (4.5%), adrenal gland (2.3%), brain (2.3%), small intestine (2.3%), and skin (2.3%). Systemic chemotherapy was administered in 35 of 44 patients (79.5%) before and/or after UCM surgery. Measurements Overall, cancer-specific and progression-free survival from time of diagnosis and metastasectomy of UCM. Results and limitations Median survival from initial diagnosis of UCM and subsequent resection was as follows: overall survival, 35 mo and 27 mo; cancer-specific survival, 38 mo and 34 mo; and progression-free survival, 19 mo and 15 mo. Overall 5-yr survival from metastasectomy for the entire cohort was 28%. Seventeen patients were still alive without progression at a median follow-up of 8 mo. Seven patients without disease progression survived for >2 yr and remained free from tumor progression at a median follow-up of 63 mo. No significant prognostic factors could be determined due to the limited patient number. Conclusions Long-term cancer control and possible cure can be achieved in a subgroup of patients following surgical removal of UCM. Metastasectomy in patients with disseminated UCM remains investigational and should only be offered to those with limited disease as a combined-modality approach with systemic chemotherapy.

Background Intramedullary nailing of clavicular midshaft fractures using the titanium elastic nail has been described as a technically easy, minimally invasive operation with few complications and an early return to competitive sports. Hypothesis The results reported thus far have been positive. The titanium elastic nail is associated with multiple intraoperative and postoperative problems. Study Design Case series; Level of evidence, 4. Methods From April 2004 to March 2007, 34 patients at our institution were treated with intramedullary nailing. A standard titanium elastic nail was used in 19 cases and a titanium elastic nail with an end cap in 15 cases. The titanium elastic nail was inserted and advanced under fluoroscopic control. A short incision at the fracture site was made for open reduction if needed. Postoperatively, free range of motion was allowed. Results In 62% of patients, open reduction was necessary independent of fracture type, flattening of the titanium elastic nail, or transverse fragments. Operating time was 44 minutes (range, 10—105 minutes) and fluoroscopy time 9 minutes (range, 2—25 minutes). In 70% of patients, problems or complications occurred (7 medial perforations, 7 lateral penetrations, 1 titanium elastic nail breakage, 1 titanium elastic nail dislocation, 7 hardware irritations). The reoperation rate was 36%. Medial migration and pain were significantly reduced by using an end cap. Conclusion Intramedullary nailing of clavicular midshaft fractures using the titanium elastic nail is a technically demanding operation with various complications in the postoperative phase. This study might explain why the implant has not yet achieved widespread application, despite the fact that other authors have reported good results. Amending the operative technique and postoperative treatment might improve the outcome in the future.

ResumenObjetivoVenlafaxina ha demostrado ser eficaz en el tratamiento de la depresión y del dolor. Carecemos de datos sobre el resultado de los pacientes con dolor crónico y síntomas depresivos tratados con venlafaxina en el marco de la atención primaria. Este estudio de observación intentó definir la eficacia de venlafaxina y su prescripción por los médicos de atención primaria y psiquiatras suizos en pacientes con dolor crónico y sintomatología depresiva.Sujetos y métodosEstudiamos a 505 pacientes con síntomas depresivos que sufrían dolor crónico en un estudio prospectivo naturalista de observación con venlafaxina en la atención primaria suiza. Estos pacientes fueron tratados con venlafaxina por 122 médicos (psiquiatras, médicos generalistas e internistas).ResultadosPor término medio, los pacientes recibieron con 143 ± 75 mg (0-450 mg) de venlafaxina al día durante tres meses. Venlafaxina demostró ser eficaz tanto en el tratamiento de los síntomas depresivos como del dolor crónico.DiscusiónAunque no se observaron efectos secundarios en la mayor parte de los pacientes, posiblemente no se consiguió una respuesta satisfactoria de la depresión por administrar dosis bajas de venlafaxina. Nuestros resultados reflejan la complejidad del tratamiento de dolor crónico en pacientes con síntomas depresivos en la atención primaria.ConclusiónSon necesarios nuevos estudios aleatorizados que determinen la dosis apropiada de venlafaxina para el tratamiento de la depresión y del dolor coexistente.

Purpose.Venlafaxine has shown benefit in the treatment of depression and pain. Worldwide data are extensively lacking investigating the outcome of chronic pain patients with depressive symptoms treated by venlafaxine in the primary care setting. This observational study aimed to elucidate the efficacy of venlafaxine and its prescription by Swiss primary care physicians and psychiatrists in patients with chronic pain and depressive symptomatology.Subjects and methods.We studied 505 patients with depressive symptoms suffering from chronic pain in a prospective naturalistic Swiss community based observational trial with venlafaxine in primary care. These patients have been treated with venlafaxine by 122 physicians, namely psychiatrists, general practitioners, and internists.Results.On average, patients were treated with 143 ± 75 mg (0–450 mg) venlafaxine daily for a follow-up of three months. Venlafaxine proved to be beneficial in the treatment of both depressive symptoms and chronic pain.Discussion.Although side effects were absent in most patients, physicians might have frequently omitted satisfactory response rate of depression by underdosing venlafaxine. Our results reflect the complexity in the treatment of chronic pain in patients with depressive symptoms in primary care.Conclusion.Further randomized dose-finding studies are needed to learn more about the appropriate dosage in treating depression and comorbid pain with venlafaxine.

PURPOSE To report two cases of life-threatening aortic infection after percutaneous endovascular coil embolization prior to endovascular abdominal aortic aneurysm (AAA) repair (EVAR). CASE REPORT Two 76-year-old patients were readmitted 5 days and 3 weeks, respectively, after technically successful percutaneous coil embolization of aortic side branches in advance of scheduled EVAR. In the first patient, the right hypogastric artery, the inferior mesenteric artery (IMA), and a lumbar artery had been embolized, whereas in the second patient only the right hypogastric artery and the IMA had been occluded. On admission, both patients presented with severe abdominal pain. Investigations revealed acute aortic infection in both patients, combined with substantial AAA enlargement in one. Open surgical infrarenal aortic replacement was performed using homografts, and antibiotic therapy was initiated. After uneventful recovery, both patients were asymptomatic, had intact aortic homografts, and showed no evidence of infection after 12 and 18 months of follow-up, respectively. CONCLUSION Endovascular infections are a potentially serious complication following percutaneous coil embolization of major aortic branches. Early diagnosis and dedicated therapy are mandatory. Immediate resection of the infected aorta and replacement with homografts in association with prolonged antibiotic treatment showed good midterm results.

Surface recombination and diffusion screen measurements of the bulk lifetime in photoluminescence decay experiments. In this work, we introduce a linearization method, derived from our analytical solution to the three-dimensional diffusion problem in cylindrical coordinates, demonstrating that it allows an accurate measurement of the bulk lifetime in GaAs from 77K to 700K. This robust linearization method enables temperature-dependent bulk lifetime measurements on thick, unpassivated, strongly absorbing materials, which allow for characterization and identification of the defects that limit lifetime and photovoltaic efficiency. We apply this linearization technique to perform a temperature-dependent lifetime analysis, and find evidence that the EL2 defect is the dominant nonradiative recombination center.

In this work, we show that extraction of the true bulk lifetime from the biexponential decay that follows from low initial carrier density photoluminescence decay experiments is not generally possible, and introduce new models to enable extraction of the bulk lifetime in the case where the initial carrier density exceeds the doping level. From measurements with high initial carrier density, we establish quasi-equilibrium between localized and free carrier states and accurately measure the bulk lifetime. Using our new models, we measure the time constants associated with localization processes as well as nonradiative and radiative bulk recombination in our GaAs double heterostructures grown with molecular beam epitaxy from experiments with varied excitation strength providing initial carrier densities that range from around 1014 to 1017 cm–3. We demonstrate that this approach can be applied to lightly doped (1016 cm–3) materials where the strongest excitation yields initial carrier densities that exceed th...

In this work, we have decoupled the radiative and nonradiative contributions to recombination by developing analytical models to fit observed non-monoexponential photoluminescence decay and bulk-lifetime temperature-dependence, which has enabled absolute measurement of the effective radiative efficiency and characterization of the dominant defect in lightly doped GaAs samples grown using molecular-beam-epitaxy. The measured effective radiative efficiency values under low level injection conditions range from (88.9 ± 0.1)% and (81.2 ± 0.1)% at 77 K to (0.028 ± 0.001)% and (0.034 ± 0.001)% at 700 K, with 300 K values of (17.3 ± 0.1)% and (10.5 ± 0.1)% in the p-type and n-type samples, respectively. A common defect-signature is observed in the temperature-dependence of the nonradiative lifetime in samples of both doping types, and our measured parameters are consistent with characteristics previously measured for the EL2 defect using deep-level transient spectroscopy.

Using a monostable multivibrator, the signal-dependent afterpulsing background from a single photon avalanche diode was suppressed. This digital filter gate was characterized and optimized using photoluminescence decay lifetime measurements to show a reduction in systematic error that is 25% of the measured lifetime and ∼500X improvement in acquisition time when compared with the time taken to obtain a comparably reliable result by reduction of the repetition rate to suppress afterpulsing. At 10 MHz, there is an increase in the linear dynamic range from ∼2τ to ∼6τ, where the lifetime, t, was measured to be (5.0±0.1)ns. Lifetime measurements were performed with a pulsed 510 nm diode laser, a 500 nm GaAs layer (sandwiched between InGaP capping layers), a silicon single-photon avalanche photodiode and time-correlated single photon counting electronics.

Multiple ocular challenges or seasonal trials have demonstrated the efficacy of levocabastine and nedocromil sodium in the treatment of allergic conjunctivitis.This study was designed to compare the protective effect of levocabastine eye drops with that of nedocromil in a conjunctival provocation test with allergen.Twenty-four patients with allergic conjunctivitis to grass pollen were recruited. After a preliminary provocation to determine conjunctival reaction threshold (erythema of at least 50% of the conjunctiva with ocular itching), patients were randomized to receive either topical levocabastine (0.05%) or nedocromil (2%) 15 minutes before provocation. Erythema and pruritus intensity were recorded at each concentration of allergen up to the reaction threshold.The allergen concentration level necessary to reach reaction threshold was increased (p0.001) after treatment with both drugs. Comparison between screening and each treatment indicated that the shift in allergen concentration was significantly greater after levocabastine treatment than after nedocromil treatment (p = 0.019). Conjunctival itching (symptom score) and erythema (percent conjunctival surface) were also better controlled by levocabastine than by nedocromil during provocation (p0.05).In a provocation test with allergen, levocabastine and nedocromil were both effective in increasing the conjunctival tolerance to allergen, with better protection provided by levocabastine.

We derive phenomenological, C- and P-invariant lagrangians for the interaction of CP-exotic mesons (JPC = 0+−, 0−−, 1−+) with spin − 1 2 and spin − 3 2 baryons as well as with ordinary mesons. Our lagrangians are quite general and in principle independent of the subhadronic quark dynamics. Hence they can be used as a starting point for model-independent discussions of various aspects of exotic-meson phenomenology at LEAR. The difference between exotic-mesons interactions on the one hand and ordinary-mesons interactions on the other is explicitly displayed.

Background Constitutive expression and localization of antimicrobial human β-defensin-1 (HBD-1) in human kidneys as a potential mechanism of antimicrobial defense has been previously reported. Inducible expression of human β-defensin-2 (HBD-2) has been described in various epithelial organs but not for the urogenital tract. Methods We investigated the gene- and protein expression of HBD-1 and HBD-2 by reverse transcriptase-polymerase chain reaction, and immunohistochemistry in 15 normal human kidney samples and 15 renal tissues with chronic bacterial infection. Additionally, cell culture experiments were performed to study HBD gene expression by real-time RT-PCR in response to inflammatory cytokines TNFα and IL-1β as well as lipopolysaccharide from Gram-negative bacteria. Results Constitutive HBD-1 gene- and protein expression was detected in normal renal tissue and kidneys with chronic infection. As a novel finding, inducible HBD-2 gene- and protein expression was demonstrated in tubulus epithelia with chronic infection but not in normal renal tissue. In pyelonephritic kidneys HBD-1 and HBD-2 expression showed a similar pattern of localizaton in distal tubules, loops of Henle and in collecting ducts of the kidney. Furthermore, real-time RT-PCR of kidney derived cell lines stimulated with inflammatory agents TNF-α, IL-1β and LPS revealed a strong increase in relative HBD-2 transcription level and also a slight increase in relative HBD-1 transcription level. Conclusions Upregulated HBD-2 expression in renal tubulus epithelium indicates a role of a wider range of human defensins for antimicrobial host defense in the urogenital tract than previously recognized.

This open-label, prospective, multicentre, 4-week trial was undertaken to assess the efficacy and tolerability of twice daily levocabastine eye drops (0.5 mg/ml), with sodium cromoglycate nasal spray for the relief of concurrent nasal symptoms if required, in a total of 233 children with seasonal allergic conjunctivitis. No correlation between efficacy, tolerability and age was found. Investigator assessments revealed that the total severity of ocular symptoms decreased by 84 ± 34% in patients < 12 years and 85 ± 30% in those ≥ 12 years, with corresponding reductions in the total severity of ocular findings of 84% in both patient groups over the 4-week treatment period. Global assessments of therapeutic efficacy revealed the effect of therapy on ocular symptoms to be excellent or good in 81% of patients < 12 years and 82% of those ≥ 12 years after 2 weeks of treatment, with corresponding values at the end of the trial of 88% and 82% in the two groups, respectively. Treatment tolerability was considered to be excellent or good by 94% of patients overall. Application site reactions were the most common adverse event associated with ocular levocabastine, occurring in 13% of patients < 12 years and 9% of those ≥ 12 years. Twice daily levocabastine eye drops therefore appear to be effective and well tolerated for the treatment of seasonal allergic conjunctivitis in children.

The Z isomer of 4-trans-(N,N-dimethylamino)-cinnamaldoxime, (Z)-DMOX (lambda maxH2O 354 nm), forms a ternary complex with NAD+ and equine liver alcohol dehydrogenase. The 3-acetyl (3-acetyl-PdAD+), 3-thiocarboxamide (3-thio-NAD+), 3-iodo (io3PdAD+) and nicotinamide mononucleotide (NMN+) analogues of NAD+ also form ternary complexes with enzyme and (Z)-DMOX. These complexes are characterized by large red-shifts in the UV-visible spectrum of bound (Z)-DMOX (lambda max 428 nm for the NAD+ complex) and new spectral bands in the 280-340-nm region associated with the pyridine moieties of NAD+ and the NAD+ analogues. The ternary enzyme-NAD+-(Z)-DMOX complex is weakly fluorescent (lambda ex 430 nm; lambda em max 505 nm) and strongly quenches the residual tryptophan fluorescence of the enzyme-NAD+ binary complex. (Z)-DMOX binds with high affinity to the enzyme-NAD+ complex (Kd less than or equal to 4 X 10(-9) M at pH 8.75 and 25 degrees C), and similarly high affinities were found for the 3-acetyl-PdAD+, 3-thio-NAD+, and io3PdAD+ complexes. Binding is much weaker to the enzyme-NMN+ complex. The active site specifically substituted Co(II), Ni(II), Cu(II), and Cd(II) enzyme derivatives and the enzyme species lacking any metal ion at the active site (apoenzyme) also form ternary complexes with (Z)-DMOX in which the DMOX UV-visible spectrum is red-shifted (ranging from 43 to 83.5 nm). The complexes formed with the Zn(II) and Co(II) enzymes are characterized by relatively high affinities for (Z)-DMOX and by spectra that are independent of pH over the range 6-10. The affinity of the apoenzyme-NAD+ complex for (Z)-DMOX is much lower, and the spectrum of the complex is pH dependent with lambda max = 430 nm at pH 7 and lambda max = 397 nm at pH 10. The rate of (Z)-DMOX dissociation from the apoenzyme complex was found to be approximately 10(3)-fold greater than the rates observed for the metal ion substituted enzymes. The 280-340-nm spectral bands appear to result from the dihydropyridine moieties of covalent adducts formed between (Z)-DMOX and NAD+ and the NAD+ analogues. The large red-shifts of the (Z)-DMOX spectrum result from the bonding of the oxime nitrogen to a strong electrophilic center (either the active site zinc ion or the nicotinamide ring of NAD+.)(ABSTRACT TRUNCATED AT 400 WORDS)

A physicochemical characterization of the metal binding center of the active site of the dimeric enzyme horse liver alcohol dehydrogenase (HLADH) has been performed by replacing the zinc ion by various metal ions, e.g. Co, Ni, Cd, Cu. These metal ions have served as spectroscopic and kinetic probes to study the binding of coenzymes, substrates and inhibitors. The metal replacement is performed in two steps. First, the catalytic zinc ions are removed by treatment of crystal suspensions with chelating agents resulting in a species H4Zn(n)2-HLADH (where n denotes the non-catalytic zinc ions which remain in situ). Secondly, the H4Zn(n)2-HLADH can be reconstituted with different metal ions in a crystal suspension or in solution to yield Me(c)2Zn(n)2-HLADH (where c denotes the catalytic metal ion). X-ray crystallographic investigations have shown: (i) in H4Zn(n)2-HLADH only the catalytic zinc ion has been removed without any gross changes in the tertiary structure of HLADH or the ligand sphere of the catalytic metal ion; (ii) on binding NADH to H4Zn(n)2-HLADH, the transition from an ‘open’ to a ‘closed’ conformation occurs analogous to the native enzyme; and (iii) in Co(c)2Zn(n)2-HLADH the cobalt ion has been inserted into the active site accepting a coordination geometry very similar to the zinc ion in the native enzyme. The kinetics and the mechanism of the reconstitution of H4Zn(n)2-HLADH with Co(II), Zn(II) and Ni(II) has been shown to be a two-step process: The spectroscopic characterization of binary and ternary complexes of Me(c)2Zn(n)2-HLADH with coenzymes and/or inhibitors (substrates) has demonstrated that changes of the electronic structure of the catalytic metal ion during enzymatic turn-over are of fundamental importance. A novel kind of interaction of the catalytic metal ion with the coenzyme was noted: the rate of coenzyme dissociation and, therefore, the enzymatic turn-over depends on the kind of metal present in the active site. Concerning the interaction of the catalytic metal ion with non-protein ligands, we conclude: (a) previous conclusions drawn from measurements of the proton relaxation enhancement of the cobalt-bound ligand protons are invalid, since the cobalt ion is not a suitable relaxation probe in this protein; (b) the metal ion acts as a Lewis acid on inner-sphere bound aldehydes; and (c) the coordination number of the catalytic metal ion in binary and ternary complexes of LADH is a matter of controversy. Finally, we point out that previous postulated mechanisms for LADH are premature since the assignment of ionizing groups controlling catalysis is not firmly established. In particular, we have shown by 1H NMR spectroscopy of Co(c)2Zn(n)2-HLADH that the δ(NH) value of the metal ligand His-67 controls the acid-base equilibrium with pKa = 9.2 in the free enzyme. This possibility has never been considered in any previous mechanism.

Substitution of certain divalent transition metal ions, i.e. Co(II), Ni(II), Cu(II) and Fe(II), for the active site ion of horse liver alcohol dehydrogenase yields catalytically active enzyme derivatives [1–4]. Since catalysis occurs via inner-sphere coordination of substrate [3,5,6], the ligand-to-metal charge-transfer (LMCT) and d-d electronic transitions of these metal ions are useful spectroscopic probes of the chemical events occurring during catalysis [1, 2, 7]. As will be shown in this report, when reaction is monitored via rapid-scanning, stopped-flow (RSSF) UV-visible spectroscopy, the resulting time-resolved spectra (3000 nm to 700 nm) provide detailed temporal information about transient intermediates formed during catalysis. We report here evidence from RSSF spectroscopy for the existence of ternary enzymeNAD+alkozide ion intermediates both in the Co(II)E-catalyzed reduction of aldehydes and in the Co(II)E-catalyzed oxidation of alcohols. Experimental Site-specifically-substituted Co(II)E was prepared by the method of Maret et al. [1]. The preparation used in this study was found to have 76 percent active site occupancy relative to total protein. The remaining active sites contained no metal ion. Substrates were purified prior to use by vacuum distillation. The highest purity grades of NADH and NAD+ were purchased from either Sigma Chemical Company or Boehringer Mannheim Corporation and used without further purification. For kinetic isotope studies, both (4R)-4-deuterio-nicotinamide adenine dinucleotide (NADD) and isotopically normal NADH were prepared and purified as previously described [5, 8, 9]. Pyrazole (Aldrich) was purified by vacuum sublimation, isobutyramide (Aldrich) was purified by recrystallization from hot water. The rapid kinetics systems for rapid-scanning, rapid-mixing and for single-wavelength studies have been previously described [10, 11]. Results and Discussion Reactions of horse liver alcohol dehydrogenase can be constrained to essentially a single turnover of sites when [coenzyme], [S] > [E] by the inclusion of fast-reacting, tight-binding inhibitors which bind selectively to either the E(NAD+) or the E(NADH) complex. Pyrazole (pyr) has proven to be an extremely useful ‘suicide’ inhibitor for trapping the E(NAD+) complex [3, 12, 13]. Isobutyramide (IBA) binds selectively to the E(NADH) complex and, at high concentraions, is effective in slowing the steady-state rate of alcohol turnover so that the first catalytic cycle is rapid relative to subsequent turnovers [14]. Using pyr and IBA, respectively, to trap the Co(II)E(NAD+) and Co(II)E(NADH) complexes, we have investigated the single-turnover time-courses for aldehyde reduction and for alcohol oxidation. Figures 1A and 1B show the families of time-resolved spectra during the reduction of benzaldehyde under single-turnover conditions obtained from RSSF experiments. The insets to these figures present single-wavelength time-courses at 325 nm (trace a), 350 nm (trace b), 398 nm (trace c), 575 nm (trace d) and 680 nm (trace e). These time courses establish that reaction occurs in two kinetically detectible relaxations. The spectral changes in the fast relaxation correspond to the oxidation of bound NADH(viz. Fig. 1A and traces a and b) and the appearance of a new d-d transition at 575 nm (Fig. 1B and trace d). Detailed analyses of the changes in Fig. 1A (not shown) indicate the fast process also is accompanied by perturbations of a LMCT band in the 350 nm region. As is evidenced by the apparent isoabsorptive points at 325 nm and 380 nm, the slow relaxation consists of the conversion of the species formed in the fast phase to the Co(II)E(NAD-pyr) adduct (characterized by spectrum 19 in each of Figs. 1A and 1B). Consequently, we conclude that the species formed in the fast relaxation is a ternary complex containing NAD+ and inner-sphere coordinated benzyl alcohol. Dissociation of benzyl alcohol from this complex is rate-limiting for the reaction of pyr with bound NAD+. Although there is a pronounced dependence of the fast and slow relaxation rates on substrate structure, the substrates investigated, acetaldehyde, benzaldehyde, anisaldehyde, cyclohexanone (this study) and p-nitrobenzaldehyde (Koerber et al., in press) give spectral changes similar to those shown in Fig. 1. Substitution of NADD for NADH did not alter the values of the measured rate constants for any of these substrates, compare traces 1 and 2 in inset (a) of Fig. 1. In contrast to these findings, the reduction of aromatic aldehydes catalyzed by the native Zn(II)E is characterized by a primary kinetic isotope effect when NADD is compared to NADH [12]. Consequently, these findings show that substitution of Co(II) for Zn(II) alters the rate limiting step for hydride transfer. Figures 2A and B present RSSF data for the Co(II)E-catalyzed oxidation of benzyl alcohol. The first two spectra (collected respectively at 8.6 msec and 17.2 msec) are shown in Fig. 2B offset above the full set of spectra. Single wavelength time-courses at 328 nm (trace a), 575 nm (trace b) and 680 nm (trace c) are shown as insets. The spectra in Fig. 2A and the time-course in trace (a) show a burst appearance of NADH followed by a slow, steady-state appearance of more NADH as the IBA-inhibited enzyme recycles. The spectra in Fig. 2B and the time-course in trace (b) show that the burst appearance of NADH seen in Fig. 2a is preceeded by the appearance of a new d-d transition with λmax ≅ 575 nm. The decay of this 575 nm species to a steady-state in which the spectrum of the Co(II)E(NADH, IBA) complex dominantes, coincides with the burst appearance of NADH. These data indicate that a ternary complex containing NAD+ and inner-sphere-coordinated benzyl alcohol is formed prior to hydride transfer. Similar spectral changes were obtained with ethanol, anisyl alcohol and cyclohexanol. Attempts to model the 575 nm spectral changes found in these reactions led to the investigation of the reaction of an inhibitor, 2,2,2-trifluoroethanol (TFE), with the Co(II)E in the presence of NAD+ (Fig. 3). We were surprised to find that the time-resolved spectra for the reaction of TFE with the Co(II)E-(NAD+) complex in two phases. During the fast phase, there appears a new 575 nm d-d transition. In the second, slower phase the new 575 nm band remains essentially unchanged while a new d-d transition appears at 680 nm. The spectrum of the first Co(II)E(NAD+, TFE) species (formed in the fast phase) appears to model the spectrum of the 575 nm intermediate formed prior to the oxidation of alcohol substrates (viz. Fig. 2), while the final (stable) Co(II)E(NAD+, TFE) complex models the species formed upon reduction of aldehydes (viz. Fig. 1). The 575 nm d-d transition which characterizes all these Co(II)E(NAD+, alc.) complexes also is present in the spectrum of the Co(II)E(NAD+, acetate ion) complex, and in the spectrum of the Co(II)E(NAD+) binary complex at pH 10.5. From the above observations, we infer that the 575 nm band arises from inner-sphere coordintation of oxy-anions to the active cobaltus ion forming Co(II)E(NAD+, RO−) ternary complexes, where RO− is the oxy-anion. If this interpretation is correct, then the transient 575 nm species detected in the single turnover reactions of aldehydes (Fig. 1) and of alcohols (Figs. 2 and 3) are Co(II)E(NAD+, RO−) ternary complexes. From similar reasoning, the spectrum of the Co(II)E(NAD+) at pH 10.5 arises from coordination of hydroxide ion to the active site cobalt Because no 575 nm band is present in the Co(II)E(NAD+) spectrum below pH 9, the apparent pKa for the ionization of a coordinated water molecule must be 9.0 < pKa < 10.5. Finally, we wish to draw attention to the apparent differences in the spectra of the Co(II)E(NAD+, RO−) species derived from aldehyde reduction (Fig. 1B) vis a vis the species formed during alcohol oxidation (Fig. 2B, spectrum 2). The transient formed in Fig. 1B is characterized by d-d transitions with λmax ≅ 575 nm, 640 nm, and 680 nm, whereas the transient formed in Fig. 2B is characterized by d-d transitions with λmax ≅ 575 nm and 650 nm. Both of these species are formed when TFE reacts the Co(II)E(NAD+) complex (Fig. 3); the species with λmax ≅ 575 nm and 650 nm is formed as a transient, the species with λmax ≅ 575 nm, 640 nm and 689 nm is the stable product. The Co(II)E d-d transitions in the 600–700 nm region are sensitive to the conformation state of the protein and/or to the coenzyme oxidation state [1, 2, 7]. Therefore, it is likely that the changes in this region of the spectrum which accompany the reaction of TFE signal changes in the Co(II) coordination sphere which result from conformational changes in the protein triggered by the coordination of TFE. Since it would appear that both types of alkoxide ion ternary complexes are kinetically competent [15], we propose that the protein conformation change for the interconversion of these complexes is an obligatory step in the catalytic mechanism of liver alcohol dehydrogenase.

Substitution of Co(II) for the catalytic site Zn(II) of horse liver alcohol dehydrogenase (LADH) yields an active enzyme derivative, CoIIE, with characteristic Co(II) charge-transfer and d-d electronic transitions that are sensitive to the events which take place during catalysis [Koerber, S. C., MacGibbon, A. K. H., Dietrich, H., Zeppezauer, M., & Dunn, M. F. (1983) Biochemistry 22, 3424-3431]. In this study, UV-visible spectroscopy and rapid-scanning stopped-flow (RSSF) kinetic methods are used to detect and identify intermediates in the LADH catalytic mechanism. In the presence of the inhibitor isobutyramide, the pre-steady-state phase of alcohol (RCH2OH) oxidation at pH above 7 is characterized by the formation and decay of an intermediate with lambda max = 570, 640, and 672 nm for both aromatic and aliphatic alcohols (benzyl alcohol, p-nitrobenzyl alcohol, anisyl alcohol, ethanol, and methanol). By comparison with the spectrum of the stable ternary complex formed with oxidized nicotinamide adenine dinucleotide (NAD+) and 2,2',2''-trifluoroethoxide ion (TFE-), CoIIE(NAD+, TFE-), the intermediate which forms is proposed to be the alkoxide ion (RCH2O-) complex, CoIIE(NAD+, RCH2O-). The timing of reduced nicotinamide adenine dinucleotide (NADH) formation indicates that intermediate decay is limited by the interconversion of ternary complexes, i.e., CoIIE(NAD+, RCH2O-) in equilibrium CoIIE(NADH, RCHO). From competition experiments, we infer that, at pH values below 5, NAD+ and alcohol form a CoIIE(NAD+, RCH2OH) ternary complex. RSSF studies carried out as a function of pH indicate that the apparent pKa values for the ionization of alcohol within the ternary complex, i.e., CoIIE(NAD+, RCH2OH) in equilibrium CoIIE(NAD+, RCH2O-) + H+, fall in the range 5-7.5. Using pyrazole as the dead-end inhibitor, we find that the single-turnover time courses for the reduction of benzaldehyde, p-nitrobenzaldehyde, anisaldehyde, and acetaldehyde at pH above 7 all show evidence for the formation and decay of an intermediate. Via spectral comparisons with CoIIE-(NAD+, TFE-) and with the intermediate formed during alcohol oxidation, we identify the intermediate as the same CoIIE(NAD+, RCH2O-) ternary complex detected during alcohol oxidation.

1. 1. Leuconostoc mesenteroides alcohol dehydrogenase is an acidic protein (pI =4.1) with a mol wt of 135,000 consisting of 4 identical subunits. 2. 2. It contains 2 Zn atoms and 5 cysteine residues per subunit. 3. 3. Thermal stability increases with increasing ionic strength. 4. 4. One NADPH molecule is bound per subunit. NADPH is bound about 10 times more strongly than NADH. Fluorescence spectra of bound coenzymes show characteristic blue shifts and intensity enhancements compared to those of free NAD(P)H. 5. 5. A comparison of molecular properties between horse liver-, yeast- and L. mesenteroides alcohol dehydrogenase shows the closer relationship of Leuconostoc ADH to yeast ADH.