Quantitative ion determination in eccrine sweat gland cells correlates to sweat reduction of antiperspirant actives.

OBJECTIVE: Axillary wetness represents an unwanted effect of the physiologically vital sweating mechanism, especially when it becomes excessive. Cosmetic products reducing sweat secretion rely on aluminium salts as the active ingredient acting by physically blocking the sweat gland. Driven by the interest to better understand the sweat mechanism and to develop alternative technologies against excessive sweating a search for an effective testing approach started as up to now, cost- and time-consuming in vivo studies represent the standard procedure for testing and identifying these alternatives. MATERIAL AND METHODS: The herein described in vitro test system is based on the measurement of intracellular changes of the ion equilibrium in cultured eccrine sweat gland cells. Subsequently, in vivo studies on the back of volunteers were conducted to verify the sweat-reducing effect of in vitro newly discovered substance. RESULTS: In this study, we describe an effective cell-based in vitro method as a potent tool for a more targeted screening of alternatives to aluminium salts. Testing the commonly used aluminium chlorohydrate as one example of an aluminium-based active in this screening procedure, we discovered a distinct influence on the ion equilibrium: Intracellular levels of sodium ions were decreased while those of chloride increased. Screening of various substances revealed a polyethyleneimine, adjusted to pH 3.5 with hydrochloric acid, to evoke the same alterations in the ion equilibrium as aluminium chlorohydrate. Subsequent in vivo studies showed its substantial antiperspirant action and confirmed the high efficiency of the polyethyleneimine solution in vivo. Further, specific investigations connecting the chloride content of the tested substances with the resulting sweat reduction pointed towards a substantial impact of the chloride ions on sweating. CONCLUSION: The newly described in vitro cell-based screening method represents an effective means for identifying new antiperspirant actives and suggests an additional biological mechanism of action of sweat-reducing ingredients which is directed towards unbalancing of the ion equilibrium inside eccrine sweat gland cells.

OBJECTIF: l'humidité axillaire représente un effet indésirable du mécanisme physiologiquement vital de la sudation, en particulier lorsqu'elle devient excessive. Les produits cosmétiques réduisant la sécrétion de sueur reposent sur les sels d'aluminium comme principe actif agissant en bloquant physiquement la glande sudoripare. Motivée par l'intérêt de mieux comprendre le mécanisme de la sudation et de développer des technologies alternatives contre l'hypersudation, une recherche pour une approche de test efficace a commencé car, jusqu'à présent, les études in vivo coûteuses et chronophages représentent la procédure standard pour tester et identifier ces alternatives. MATÉRIELS ET MÉTHODES: le système de test in vitro décrit ici est basé sur la mesure des changements intracellulaires de l'équilibre ionique dans les cellules des glandes sudoripares exocrines cultivées. Par la suite, des études in vivo sur le dos de volontaires ont été menées pour vérifier l'effet réducteur de la sudation d'une substance nouvellement découverte in vitro. RÉSULTATS: dans cette étude, nous décrivons une méthode cellulaire efficace in vitro en tant qu'outil puissant pour un dépistage plus ciblé des alternatives aux sels d'aluminium. En testant le chlorohydrate d'aluminium couramment utilisé comme exemple d'un principe actif à base d'aluminium dans cette procédure de dépistage, nous avons découvert une influence distincte sur l'équilibre ionique : les taux intracellulaires d'ions sodium ont diminué tandis que ceux du chlorure ont augmenté. La recherche de diverses substances a révélé une polyéthylèneimine, ajustée au pH 3,5 avec de l'acide chlorhydrique, pour évoquer les mêmes altérations de l'équilibre ionique que le chlorohydrate d'aluminium. Des études in vivo ultérieures ont montré son action anti-transpirante substantielle et ont confirmé la haute efficacité de la solution de polyéthylèneimine in vivo. De plus, des études spécifiques établissant un lien entre la teneur en chlorure des substances testées et la réduction de la sudation qui en résulte ont indiqué que les ions chlorure ont un impact substantiel sur l'hypersudation. CONCLUSION: la nouvelle méthode de dépistage cellulaire in vitro décrite représente un moyen efficace d'identifier de nouveaux agents anti-transpirants actifs et suggère un mécanisme d'action biologique supplémentaire des ingrédients réducteurs de la sudation, dirigé vers le déséquilibre de l'équilibre ionique à l'intérieur des cellules des glandes sudoripares exocrines.

MAIT cell activation in adolescents is impacted by bile acid concentrations and body weight.

Bile acids (BAs) are produced by liver hepatocytes and were recently shown to exert functions additional to their well-known role in lipid digestion. As yet it is not known whether the mucosal-associated invariant T (MAIT) cells, which represent 10-15% of the hepatic T cell population, are affected by BAs. The focus of the present investigation was on the association of BA serum concentration with MAIT cell function and inflammatory parameters as well as on the relationship of these parameters to body weight. Blood samples from 41 normal weight and 41 overweight children of the Lifestyle Immune System Allergy (LISA) study were analyzed with respect to MAIT cell surface and activation markers [CD107a, CD137, CD69, interferon (IFN)-γ, tumor necrosis factor (TNF)-α] after Escherichia coli stimulation, mRNA expression of promyelocytic leukemia zinc finger protein (PLZF) and major histocompatibility complex class I-related gene protein (MR1), the inflammatory markers C-reactive protein (CRP), interleukin (IL)-8 and macrophage inflammatory protein (MIP)-1α as well as the concentrations of 13 conjugated and unconjugated BAs. Higher body weight was associated with reduced MAIT cell activation and expression of natural killer cell marker (NKp80) and chemokine receptor (CXCR3). BA concentrations were positively associated with the inflammatory parameters CRP, IL-8 and MIP-1α, but were negatively associated with the number of activated MAIT cells and the MAIT cell transcription factor PLZF. These relationships were exclusively found with conjugated BAs. BA-mediated inhibition of MAIT cell activation was confirmed in vitro. Thus, conjugated BAs have the capacity to modulate the balance between pro- and anti-inflammatory immune responses.

Triphenylsulfonium topophotochemistry.

The products from the 193 nm irradiation of triphenylsulfonium nonaflate (TPS) embedded in a poly(methyl methacrylate) (PMMA) film have been characterized. The analysis of the photoproduct formation was performed using chromatographic techniques including HPLC, GPC and GC-MS as well as UV-vis and NMR spectroscopic methods. Two previously unreported TPS photoproducts, triphenylene and dibenzothiophene, were detected; additionally, GPC and DOSY-NMR spectroscopic analyses after irradiation suggested that TPS fragments had been incorporated into the polymer film. The irradiation of acetonitrile solutions containing 10% w/v PMMA and 1% w/v TPS in a 1 cm-path-length cuvette showed only a trace amount of triphenylene or dibenzothiophene, indicating that topochemical factors were important for the formation of these molecules. The accumulated evidence indicates that both products were formed by in-cage, secondary photochemical reactions: 2-(phenylthio)biphenyl to triphenylene, and diphenylsulfide to dibenzothiophene.

Impact of pharmacist interventions in older patients: a prospective study in a tertiary hospital in Germany.

BACKGROUND: Inappropriate pharmacotherapy among older adults remains a critical issue in our health care systems. Besides polypharmacy and multiple comorbidities, the age-related pharmacokinetic and pharmacodynamic changes may increase the risk of adverse drug reactions and medication errors. OBJECTIVE: The main target of this study was to describe the characteristics of pharmaceutical interventions in two geriatric wards (orthogeriatric ward and geriatric day unit) of a general teaching hospital and to evaluate the clinical significance of the detected medication errors. MATERIALS AND METHODS: The study was conducted between August 2014 and October 2015 and was based on a triple approach that included validation of medical orders, medication reconciliation at patients' admission, and a predischarge planning appointment with the patient. The validation of medical orders was based on analyzing the suitability of the drugs prescribed, the drug dose depending on the patient's characteristics, the presence of contraindications and interactions between drugs, and the proposal of alternative drugs included in the hospital formulary. RESULTS: A total of 2,307 interventions associated to a medication error in 15,282 medical orders for 1,859 older patients were recorded. The greater part of the interventions carried out on the orthogeriatric ward at admission and at discharge were due to omission of a drug in the medical order (20.0%) and clinically significant interactions requiring monitoring (30.4%), respectively. The main factor triggering pharmacist's recommendations on the geriatric day unit was clinically significant interactions (21.1%). With regard to the clinical severity of the detected errors, 68.1% were considered significant, 24.8% were of minor significance, and 7.2% were clinically serious. CONCLUSION: Our findings show the importance of clinical pharmacist involvement in the optimization of pharmacotherapy in older adults, ensuring that they receive effective, safe, and efficient drug therapy.

Comparing Humoral and Cellular Immune Response Against HBV Vaccine in Kidney Transplant Patients.

Host protection upon vaccination usually results from the complex interplay of humoral and cellular components of the immune system. Exploring hepatitis B surface antigen (HBsAg)-specific T cell responses and their correlation with humoral responses under immunosuppression, we analyzed 51 renal transplant recipients, differing in HBV vaccine-specific antibody titers (non [NRs]-, low [LRs]-, and high responders [HRs]) and in 22 healthy controls (HCs) in a cross-sectional study. HBsAg-specific T cells were analyzed by flow cytometry according to expression of activation markers CD40L and/or CD69, and the cytokines IFNγ, IL-2, TNFα, and IL-17. No significant differences in responder rate and magnitude of HBsAg-specific T cell responses were found between HCs and HRs. Interestingly, HBsAg-specific Th-cells were also observed in 50% of humoral NRs. Frequencies of HBsAg-specific CD40L+ Th-cells were significantly higher in HRs compared to LRs (p = 0.009) and in LRs in comparison to NRs (p = 0.043). All but NRs showed a predominance of multi-potent HBsAg-specific TNFα+IL-2+ Th-cells. As expected, HBsAg-specific CD8(+) T cells were rarely found. In conclusion, mounting of hepatitis B vaccine-specific T cell responses is possible in kidney transplant recipients despite immunosuppression. Detection of HBV-specific Th-cells in a significant proportion of humoral NRs contributes to the current discussion on conferring immune protection by cellular memory in such patients.

The role of CD4(+) T cells in BKV-specific T cell immunity.

Reactivation of polyomavirus BK (BKV) infection represents a severe complication in kidney transplant (KTX) patients. We previously reported an association between a declining BK viral load and the reconstitution of CD4(+) T cell BKV-specific immunity in patients following kidney transplantation. However, the specific contribution of CD4(+) T cells in the regulation of BKV-replication is unknown. Nevertheless, in vitro enrichment of BKV-specific T cells and subsequent adoptive T cell transfer may improve the restoration of immune competence in KTX patients with BKV infection. To date, strategies to capture human BKV-specific T cells with the ensuing expansion to clinically useful numbers are lacking. Here, we demonstrated a comprehensive flow cytometric analysis of the BKV-specific T cell response that permits access to the majority of T cells specific for immunodominant BKV antigens. A full-spectrum evaluation of the BKV-specific T cell response was performed by stimulating peripheral blood mononuclear cells (PBMC) with a mixture of BKV immunodominant peptide pools at varying concentrations and measuring activation marker expression and cytokine secretion. We also examined the effects of co-stimulation and PBMC resting time prior to activation. We defined the narrow range of stimulation conditions that permit the capture and expansion of functional BKV-specific T cell lines. The generated BKV-specific T cell lines showed the highest specificity and functionality when the T cells were captured according to IFNγ-secretion. This study highlights the multifunctional and cytolytic BKV-specific CD4(+) T cells as a dominant population within the generated T cell product. This method offers a novel approach for the generation of BKV-specific T cell lines for adoptive immunotherapy and underscores the critical role of CD4(+) T cells in the clearance of BKV.

TCR repertoire analysis by next generation sequencing allows complex differential diagnosis of T cell-related pathology.

Clonotype analysis is essential for complete characterization of antigen-specific T cells. Moreover, knowledge on clonal identity allows tracking of antigen-specific T cells in whole blood and tissue infiltrates and can provide information on antigenic specificity. Here, we developed a next generation sequencing (NGS)-based platform for the highly quantitative clonotype characterization of T cells and determined requirements for the unbiased characterization of the input material (DNA, RNA, ex vivo derived or cell culture expanded T cells). Thereafter we performed T cell receptor (TCR) repertoire analysis of various specimens in clinical settings including cytomegalovirus (CMV), polyomavirus BK (BKV) reactivation and acute cellular allograft rejection. Our results revealed dynamic nature of virus-specific T cell clonotypes; CMV reactivation was linked to appearance of new highly abundant antigen-specific clonalities. Moreover, analysis of clonotype overlap between BKV-, alloantigen-specific T cell-, kidney allograft- and urine-derived lymphocytes provided hints for the differential diagnosis of allograft dysfunction and enabled appropriate therapy adjustment. We believe that the established approach will provide insights into the regulation of virus-specific/anti-tumor immunity and has high diagnostic potential in the clinical routine.

Elastin expression in a newly developed full-thickness skin equivalent.

The resilience of the human skin is mediated by elastic fibres mainly consisting of fibrillins and elastin. In order to establish a model system to study the impact of cosmetic and pharmaceutical compounds on the elastic system in vitro, we analyzed the expression of elastin in a newly developed full-thickness skin model. After a 5-week cultivation period the skin model developed a fully differentiated epidermis including a stratum corneum. The dermis contains fibroblasts embedded in extracellular matrix proteins. The models were viable until at least 51 days at the air-liquid interface (ALI) culture. Using immunohistochemistry we detected elastin first on day 7 of ALI. With proceeding culture time, elastin-positive fibres of different lengths and distribution patterns accumulated in the dermal compartment. Elastin mRNA expression started on day 7 of ALI, increased until day 10 and then dropped to a level comparable to that of day 7. Our results demonstrate that in our full-thickness skin model an in vivo-like elastic system, which clearly mimics at least two subsets of dermal elastic fibres, is generated. This physiological property favours the model as a promising animal-free approach to study those processes leading to an environment- and age-dependent decrease in skin elasticity.

Inhibition of carotis venous bypass graft disease by intraoperative nucleic acid-based therapy in rabbits.

BACKGROUND: Bypass graft stenosis after venous revascularisation procedures is characterised by massive neointimal and vascular smooth muscle cell proliferation triggered via endothelin-1 synthesis in the vessel wall. Decoy oligodesoxynucleotides (ODN) against the transcription factor activator protein-1 (AP-1) inhibits pre-pro-endothelin-1 expression. METHODS: In 20 rabbits, an end-to-side jugular vein bypass to the carotid artery was performed: (group A) 8 grafts were treated with consensus AP-1 decoy ODN, (group B) 8 with mutated control ODN and (group C) 4 received no treatment. Explantation, histomorphometric and immunohistochemical evaluation was performed after 28 days. RESULTS: Median intimal thickness of groups: (A) 28.3 microm, (B) 48.4 microm, (C) 71.1 microm. The decoy ODN-treated group showed a significant reduction of neointima formation ( P = 0.029) and a downregulation of the endothelin receptor. CONCLUSIONS: In this model, neointima formation was reduced by local transfection with consensus decoy ODN against AP-1. Endothelin A and B receptor expression is downregulated. Molecular target nucleic acid-based therapies seem to be a future means of overcoming neointima proliferation in pressure-induced venous graft failure. Intraoperative local application makes it easy to use in routine revascularisation procedures.

Novel aspects on RANK ligand and osteoprotegerin in osteoporosis and vascular disease.

The clinical coincidence of osteoporosis and vascular disease has long indicated that common mediators may adversely affect bone metabolism and vascular integrity alike. Receptor activator of NF-kappaB ligand (RANKL) is an important cytokine for bone resorption that acts through its osteoclastic receptor, receptor activator of NF-kappaB (RANK), while osteoprotegerin serves as a decoy receptor that binds RANKL and prevents activation of RANK. Skeletal and vascular cells are sources and targets of RANKL and OPG both in vitro and in vivo. Modulation of the RANKL/RANK/OPG system in animals results in a skeletal and vascular phenotype, and administration of OPG may prevent osteoporosis and vascular calcification. Recent studies on OPG serum levels and gene polymorphisms also suggest an important role of this cytokine system in skeletal and vascular diseases. In summary, there is increasing evidence that RANKL and OPG may link the skeletal with the vascular system.

[LDL-Apheresis for the treatment of hyperchylomicronemia-induced pancreatitis]. / LDL-Apherese bei der hyperchylomikronämieinduzierten Pankreatitis.

Severe hyperchylomicronemia due to defects of lipoprotein lipase or apoC-II is a rare cause for acute pancreatitis. Food with a high content of fat, as well as alcoholic or hormonal influences, can lead to excessive hypertriglyceridemia. Especially hyperchylomicronemia due to hormonal influences during pregnancy are troublesome. Here, we are confronted with both the risk to the mother as well as the vital risk to the unborn. Conventional plasma apheresis has been used to successfully eliminate chylomicrons and, thus, the primary cause of chylomicron-induced pancreatitis. Most recently, we reported the use of selective LDL-apheresis in a 24-year-old pregnant woman (thirteenth week of pregnancy), who was admitted with the signs of acute pancreatitis to our hospital. The patient was known to have a history of severe hyperchylomicronemia and she had also been treated several years before for acute pancreatitis by LDL-apheresis. Her triglycerides were severely elevated (11500 mg/dl) and, in order to achieve a rapid decrease of chylomicrons, we decided to treat her by selective LDL-apheresis utilizing HELP-apheresis. The treatment was well tolerated and within half an hour the patient was free of any abdominal pain. However, due to the enormous triglyceride load, we needed to change the precipitate filters several times and at the end of the treatment triglyceride levels were 6600 mg/dl. Under a low-fat diet (<30 gram fat per day), the follow-up was uneventful and the patient delivered a healthy baby at the end of week 39. We conclude that LDL-apheresis is a safe and rapid procedure to eliminate chylomicrons in chylomicron-induced pancreatitis.

[Comparison of extracorporeal shockwave therapy (ESWT) with roentgen irradiation in supraspinatus tendon syndrome--a prospective randomized single-blind parallel group comparison]. / Vergleich der extrakorporalen Stosswellentherapie (ESWT) mit der Röntgenreizbestrahlung beim Supraspinatussehnensyndrom--Ein prospektiver randomisierter einfachblinder Parallelgruppenvergleich.

AIM: Extracorporal shock wave therapy (FSWT) is applied in the case of supraspinatus tendinitis if conservative therapies have failed. So far there has been no controlled study comparing the effectiveness of ESWT with an established conservative method of therapy such as X-ray stimulation radiotherapy. METHOD: Thirty patients with chronic supraspinatus tendinitis were admitted into the prospective randomised study. After randomisation, the patients were treated either three times with 2000 pulses (energy flux density ED+ 0.33 mJ/mm2) with a Storz Minilith SL1 after one week, or with X-ray stimulation radiotherapy with 6 x 0.5 Gy on the ICRU reference point (1 neutral fraction/day) with cobalt 60 gamma rays. Primary endpoint was the age-corrected constant score. RESULTS: In the ESWT group the average age-corrected constant score rose from 50.1 points before ESWT to 91.5 points after 12 weeks and to 97.8 after 52 weeks. In the radiotherapy group it improved from 47.6 through 79.5 points to 87.4 points. CONCLUSION: No statistically significant differences were proven between ESWT and radiotherapy. ESWT appears to be at least equivalent to radiotherapy in treating chronic supraspinatus tendinitis syndrome and can avoid a dose of radiation for patients and staff. A comprehensive randomised study is, however necessary to ensure the equivalence of ESWT.

Expression and function of CCAAT/enhancer binding proteinbeta (C/EBPbeta) LAP and LIP isoforms in mouse mammary gland, tumors and cultured mammary epithelial cells.

CCAAT/Enhancer binding proteins (C/EBPs) play important roles in the regulation of cell growth and differentiation. This study investigated the expression and function of C/EBPbeta isoforms in the mouse mammary gland, mammary tumors, and a nontransformed mouse mammary epithelial cell line (HC11). C/EBPbeta mRNA levels are 2-5-fold higher in mouse mammary tumors derived from MMTV/c-neu transgenic mice compared with lactating and involuting mouse mammary gland. The "full-length" 38 kd C/EBPbeta LAP ("Liver-enriched Activator Protein") isoform is the predominant C/EBPbeta protein isoform in mammary tumor whole cell lysates, however, the truncated 20 kd C/EBPbeta LIP ("Liver-enriched Inhibitory Protein") isoform is also present at detectable levels (mean LAP:LIP ratio 5.3:1). The mammary tumor C/EBPbeta LAP:LIP ratio decreases 70% (from 5.3:1 to 1.6:1) when lysate preparation is switched from a rapid whole cell lysis protocol to a multistep nuclear/cytoplasmic fractionation protocol. In contrast to mammary tumors, only the C/EBPbeta LAP isoform is detectable in the mammary gland whole cell and nuclear lysates; the truncated "LIP" isoform is undetectable regardless of isolation protocol. Ectopic over expression of C/EBPbeta LIP or C/EBPbeta LAP did not alter HC11 growth rates. However, C/EBPbeta LIP over expressing HC11 cells (LAP:LIP ratio of approximately 1:1) exhibited a consistent 2-4 h delay in G(0)/S phase transition. C/EBPbeta LIP overexpressing HC11 cells did not express beta-casein mRNA (mammary epithelial cell differentiation marker) in response to lactogenic hormones. This defect in beta-casein expression was not corrected by carrying out the differentiation protocol in the presence of an artificial extracellular matrix. These results demonstrate that the "full-length" C/EBPbeta LAP isoform is the predominant C/EBPbeta protein isoform expressed in mouse mammary gland in vivo and mouse mammary epithelial cell cultures in vitro. C/EBPbeta LIP detected in mammary tumor lysates may result from in vivo production or ex vivo isolation-induced proteolysis of C/EBPbeta LAP. Ectopic overexpression of C/EBPbeta LIP (LAP:LIP ratio of approximately 1:1) inhibits mammary epithelial cell differentiation (beta-casein expression).

Strategies to optimize CAD prevention in modern cardiology. The "Marburg CAD Prevention Project".

In recent years we identified numerous cardiovascular risk factors and had been able to reduce cardiovascular events by a variety of different interventions. There is no question that we can improve the course of coronary artery disease (CAD) in patients with a high-risk profile by modifying these factors. Despite this knowledge, many patients with known CAD or myocardial infarction are not treated for secondary prevention as recommended by well established guidelines (http:¿www.chd-taskforce.com). In order to improve secondary prevention in our patients we started a project, the so called "Marburg CAD Prevention Project". By this we combine our computer data base of the CAD preventional routine laboratory with the computer program CARDDAS. Individual risk factors and the angiographic findings were analyzed. Patients as well as local physicians were informed on the need to treat the important risk factors. This approach ensures that at least all of our patients with documented CAD receive the appropriate preventional recommendations and treatment.

Effect of Apis mellifera propolis from two Brazilian regions on caries development in desalivated rats.

The purpose of the present study was to evaluate the effect of Apis mellifera propolis collected from two regions of Brazil on caries development in desalivated rats. Ethanolic extracts of propolis (EEP) were prepared from crude propolis samples collected in Minas Gerais state (MG), southeastern Brazil, and Rio Grande do Sul state (RS), southern Brazil. The flavonoid composition of EEP was analyzed by high-performance thin-layer chromatography (HPTLC) and reversed-phase high-performance liquid chromatography (HPLC). For the animal study, 30 specific pathogen-free Wistar rats were infected with Streptococcus sobrinus 6715 and surgically desalivated. The rats were randomly divided into three groups which were treated with 80% ethanol (control), EEP from MG and EEP from RS. The animals were placed in a König-Höfer programmed feeder and received 17 meals of diet 2000 daily at hourly intervals. The solutions were applied on the rat molars (25 microl on molars of each quadrant) twice a day, by using graduate syringes. After 3 weeks, the animals were killed by CO(2) asphyxiation. For microbial assessment, the left jaw was removed and sonicated in 154 mM NaCl solution. Dental caries was evaluated according to Larson's modification of Keyes' system. The HPTLC patterns and HPLC profiles demonstrated that both quality and quantity of flavonoid aglycones of EEP from MG were different compared to EEP from RS. In general, it is apparent that EEP from RS contained the highest concentrations of pinocembrin, chrysin, acacetin and galangin. The group of animals treated with EEP from RS showed the lowest smooth-surface and sulcal caries scores as well as less caries severity in smooth-surface and sulcal lesions, and these data were statistically different when compared with the control group. The group treated with EEP from MG only demonstrated a significant difference in the severity of sulcal lesions when compared to the control group. The percentage of S. sobrinus was lower in the groups treated with EEP, but did not differ statistically from the control group. The results showed that the cariostatic effect of propolis depends on its composition, and consequently the region of collection of propolis samples.

Lifibrol enhances the low density lipoprotein apolipoprotein B-100 turnover in patients with hypercholesterolemia and mixed hyperlipidemia.

Lifibrol (4-(4'-tert-butylphenyl)-1-(4'carboxyphenoxy)-2-butanol), a new hypocholesterolemic drug, effectively reduces total cholesterol (CH), low density lipoprotein (LDL)-CH, and apolipoprotein (apo) B in experimental animals and in humans. The impact of Lifibrol on the metabolism of apoB-100 containing lipoproteins in patients with hyperlipoproteinemia using endogenous labeling with stable isotopes is examined. Kinetic studies were performed in four male hypercholesterolemic individuals (type IIa) before and on treatment with 450 mg of Lifibrol daily for 4 weeks, and in five male individuals suffering from mixed hyperlipidemia (type IIb) before and on therapy for 12 weeks. Kinetic parameters were estimated by multicompartmental modeling. Lifibrol therapy reduced total CH by 16% (P = 0.012) in all patients, increased triglycerides (TG) by 11% (not significant) in type IIa patients and decreased TG by 34% (P = 0.059) in type IIb patients. During Lifibrol therapy, LDL apoB-100 concentrations decreased by 19% (P = 0.011) in all patients. The decrease in LDL apoB concentrations with Lifibrol therapy was due to an overall increase (75%, P = 0.006) of the fractional catabolic rates (FCR) of LDL apoB. This increase was partially attenuated by a 33% increase in LDL apoB production rate (PR) (P = 0.041). The overall production of apoB increased only slightly. Our data suggest that the major mechanism by which Lifibrol lowers LDL-CH is an increase in receptor-mediated catabolism of LDL rather than a decrease in hepatic apoB production.

Physico-chemical stability of infusion solutions for epidural administration containing fentanyl and bupivacaine or lidocaine.

This study examines the physico-chemical stability of infusion solutions for epidural administration containing bupivacaine hydrochloride 0.06% or 0.125% or lidocaine hydrochloride 0.25% in 0.9% sodium chloride, each with fentanyl 0.0002%. The solutions were prepared in polyvinyl chloride (PVC) infusion bags and stored without overwrap at room temperature (25-30 degrees C) or refrigerated (4-8 degrees C). Over a period of 32 days stability was determined by visual inspection, pH measurement, and HPLC assay of drug concentrations. Admixtures of bupivacaine/fentanyl and lidocaine/fentanyl proved to be chemically stable over a 32 day period, but physical incompatibility (sorption) with PVC-bags was discovered. The stability of the admixtures was influenced by pH and storage temperature. In none of the tested admixtures with an initial pH value lower than 6, did the concentrations of fentanyl or the local anesthetic decrease under 90% of the initial concentrations. A solution of fentanyl and lidocaine with an initial pH of 6.7 exhibited a rapid decrease of drug concentrations. Supposing fentanyl loss was due to sorption, buffered single drug fentanyl solutions of pH 5.5, 5.8, 6.3, and 6.7 were prepared in glass and PVC containers and stored under the same conditions. All solutions in PVC bags showed relevant fentanyl loss which was more evident at higher pH, whereas fentanyl concentration remained unchanged in glass containers at any of the tested pH values.

[Postoperative epidural analgesia with Bupivacaine-HCl 0.06%/Fentanyl 0.0002%. Experiences and physicochemical stability in infusion bags]. / Epidurale Analgesie mit Bupivacain-HCl 0.06% plus Fentanyl 0.0002%. Erfahrungen mit der rezepturmässigen Fertigstellung einer Mischlösung in Infusionsbeuteln.

Continuous epidural infusion of bupivacaine with the opioid fentanyl represents an effective analgesic method in the therapy of strong postoperative pain after major surgery. Preparation of the required infusion solution in syringes with a volume of 50 ml immediately prior to administration is routinely performed by nursing staff in Germany. The effort required for the preparation is associated with logistical and pharmaceutical difficulties. The preparation of a mixture of bupivacaine hydrochloride 0.06% and fentanyl 0.0002% in 250 ml infusion bags at the pharmacy of the University Hospital Mainz is described. To determine the physicochemical stability, the concentration of bupivacaine-HCl and fentanyl was assessed using HPLC over a period of 32 days; in addition the pH values were determined. After 32 days 95% of the bupivacaine hydrochloride and fentanyl baseline values were recorded. The pH baseline value had decreased from 5.48-5.52 to 0.5-0.7 units. The measured values confirm the physicochemical stability of the mixture of bupivacaine hydrochloride 0.06% and fentanyl 0.0002% over a period of 32 days. The infusion bag can be stored for 4 weeks at room temperature.

Thermostable variants of subtilisin selected by temperature-gradient gel electrophoresis.

Region-specific random mutagenesis in the weak calcium binding site of subtilisin Carlsberg and subsequent screening for variants with enhanced heat stability revealed two variants, which showed significantly enhanced residual activity at 68 degrees C, 0.1 mM CaCl2, pH 8.0. Preselected variants have been studied by temperature-gradient gel electrophoresis (TGGE) and were found to be stabilized due to different effects. Whereas the point mutation (Ser188Pro) mainly enhanced autoproteolytic stability of subtilisin, the double mutation (Ser188Pro; Ala194Glu) additionally increased the apparent Tm-value of the molecule for 2-3 degrees C under a variety of conditions. It was possible to differentiate between the effects of autoproteolysis and structural unfolding to a certain degree by TGGE and to show the complex influence of changed calcium affinity on thermal stability for the double variant.

Random mutagenesis of the weak calcium binding site in subtilisin Carlsberg and screening for thermostability by temperature-gradient gel electrophoresis.

A random mutagenesis approach was directed to the weak calcium binding site of subtilisin Carlsberg in order to enhance the thermal stability of the enzyme by changing its calcium affinity. The structural motif of the binding site was altered by two strategies, the ligand strategy, which was directed to the amino acid ligands of the calcium ion and the conformation strategy, by which a part of the calcium cave was redesigned. Subtilisin mutants were expressed in Bacillus subtilis and screened for enhanced thermostability by a filter assay and by temperature-gradient gel electrophoresis (TGGE). Characterization of selected mutants and application of TGGE to investigate the thermal stability of proteases and protease-inhibitor complexes in general is described.