Two weeks of repetitive gut-challenge reduce exercise-associated gastrointestinal symptoms and malabsorption.

Debilitating gastrointestinal symptoms is a common feature of endurance running and may be exacerbated by and/or limit the ability to tolerate carbohydrate intake during exercise. The study aimed to determine whether two weeks of repetitive gut-challenge during running can reduce exercise-associated gastrointestinal symptoms and carbohydrate malabsorption. Endurance runners (n=18) performed an initial gut-challenge trial (GC1) comprising 2-hour running exercise at 60% VO2max (steady state) while consuming a formulated gel-disk containing 30 g carbohydrates (2:1 glucose-fructose, 10% w/v) every 20 minutes, followed by a 1-hour running effort bout. Gastrointestinal symptoms, feeding tolerance, and breath hydrogen (H2 ) were determined along the gut-challenge trial. After GC1, participants were randomly assigned to a blinded carbohydrate (CHO, 90 gCHO hour-1 ) or placebo (PLA, 0 gCHO hour-1 ) gut-training group. This comprised of consuming the group-specific feeding intervention during 1-hour running exercise at 60% VO2max equivalent, daily over a period of two weeks. Participants then repeated the gut-challenge trial (GC2). In GC2, a reduced gut discomfort (P=.012), total (P=.009), upper- (P=.015), and lower-gastrointestinal (P=.008) symptoms, and nausea (P=.05) were observed on CHO, but not PLA. Feeding tolerance did not differ between GC1 and GC2 on CHO and PLA. H2 peak was attenuated in GC2 (6±3 ppm) compared to GC1 (13±6 ppm) on CHO (P=.004), but not on PLA (GC1 11±7 ppm, and GC2 10±10 ppm). The effort bout distance was greater in GC2 (12.3±1.3 km) compared with GC1 (11.7±1.5 km) on CHO (P=.035) only. Two weeks of repetitive gut-challenge improve gastrointestinal symptoms and reduce carbohydrate malabsorption during endurance running, which may have performance implications.

Intermittent energy restriction and weight loss: a systematic review.

BACKGROUND/OBJECTIVES: Intermittent energy restriction (IER) is an eating pattern of regular daily periods of restricted energy intake followed by periods of unrestricted energy intake. This is gaining prominence as an alternative weight-loss strategy to daily energy restriction (DER). The aim of this systematic review was to determine the effectiveness of IER on weight loss in overweight and obese adults and compare this with DER. SUBJECTS/METHODS: A systematic literature search was conducted using the CINAHL, Embase, Medline, PsycINFO, Cochrane and Scopus databases. Eight studies that assigned overweight or obese adults to IER or to a DER 'control' were deemed eligible for inclusion. RESULTS: All studies reported significant weight loss for IER groups. Average weight loss was approximately 0.2-0.8 kg per week. IER resulted in comparable weight loss to DER when overall energy restriction remained similar between diets. The majority of studies that reported body composition outcomes have shown equal efficacy for fat mass, fat-free mass and waist circumference. CONCLUSIONS: Weight loss was achieved in overweight and obese adults following IER and this loss was comparable to a DER diet. IER may be an effective alternative strategy for health practitioners to promote weight loss for selected overweight and obese people.

Glutathione transferases P1/P2 regulate the timing of signaling pathway activations and cell cycle progression during mouse liver regeneration.

Glutathione transferases (GST) are phase II enzymes catalyzing the detoxification of endogenous noxious compounds and xenobiotics. They also regulate phosphorylation activities of MAPKinases in a catalytic-independent manner. Previous studies have demonstrated the regulation of JNK-dependent pathway by GSTP1/2. Considering the crucial role of JNK in the early steps of the hepatocyte cell cycle, we sought to determine whether GSTP1/2 were essential for hepatocyte proliferation following partial hepatectomy (PH). Using a conventional double knockout mouse model for the Gstp1 and Gstp2 genes, we found that the lack of GSTP1/P2 reduced the rate of DNA replication and mitotic index during the first wave of hepatocyte proliferation. The lowered proliferation was associated with the decrease in TNFalpha and IL-6 plasma concentrations, reduced hepatic HGF expression and delayed and/or altered activation of STAT3, JNK and ERK1/2 signaling pathways. In addition, the expression and/or activation of cell cycle regulators such as Cyclin D1, CDK4, E2F1 and MCM7 was postponed demonstrating that the absence of GSTP1/2 delayed the entry into and progression through the G1 phase of the cell cycle and impaired the synchrony of proliferation in hepatocytes following PH. Furthermore, while JNK and its downstream targets c-Jun and ATF2 were activated during the early steps of the liver regeneration in wild-type animals, the constitutively active JNK found in the quiescent liver of Gstp1/2 knockout mice underwent a decrease in its activity after PH. Transient induction of antioxidant enzymes and nitric oxide synthase were also delayed or repressed during the regenerative response. Altogether our results demonstrate that GSTP1/2 are a critical regulators of hepatocyte proliferation in the initial phases of liver regeneration.

Endotoxin transiently inhibits protein synthesis through Akt and MAPK mediating pathways in C2C12 myotubes.

In this study, the effect of lipopolysaccharide (LPS) on protein synthesis (PS) and intracellular signaling factors that regulate it have been investigated in C2C12 murine-derived myotubes. In particular, the role of Akt/mammalian target of rapamycin (mTOR) and the mitogen-activated protein kinases (MAPKs) [p38 and extracelluar regulated protein kinase (ERK1/2)] have been examined. The direct effect of LPS on PS was measured at 3 and 18 h. LPS significantly decreased PS at 3 h but not at the 18-h time point. This effect was preceded by decreased Akt phosphorylation at 5 and 30 min after LPS administration. The mTOR phosphorylation exhibited a long time dose-dependent increase at all the time points. Similarly, the activity-related phosphorylation of p38 and ERK1/2 significantly increased in a time- and dose-dependent manner at all the time points. Polymyxin B abolished the LPS-induced decrease in PS rate. The phosphatidylinositol 3-kinase inhibitor LY-0294002 in combination with LPS significantly decreased the rate of PS by 81% and alone by 66%, respectively, for the 3- and 18-h time points, whereas p38 and ERK inhibitors in combination with LPS significantly decreased the rate PS rate at the 18-h time point by 41% and 59%, respectively, compared with control cells. In conclusion, LPS alone transiently decreased the rate of PS by 50% at 3 h; this effect is most likely mediated via the Toll-like receptor 4 (TLR4)-Akt/mTOR pathway, and both p38 and ERK when inhibited in the presence of LPS at 3 h have a similar effect in preventing the LPS-induced reduction in PS.

Detection of epileptic spikes by magnetoencephalography and electroencephalography after sleep deprivation.

INTRODUCTION: In diagnosis of epilepsies electrophysiological findings play a key role. While spontaneous electroencephalography (EEG) and EEG with sleep deprivation (EEGsd) are widely evaluated and used, application of magnetoencephalography (MEG) in this field is primarily limited to presurgical assessment of focal epilepsies. METHODS: In this study we retrospectively compared MEG (M/EEG) and EEGsd in 63 (55) patients with focal and generalized epilepsy with regard to occurrence of epileptic spikes. RESULTS: MEG could record epileptic spikes in 38 patients (60%), while EEGsd recorded spikes in only 32 patients (51%). In a group of 55 patients simultaneous MEG/EEG (M/EEG) was able to record spikes in 38 patients (71%) compared to epileptic spikes in 28 patients (51%) recorded by EEGsd. In a subgroup of 17 MR-negative patients simultaneous M/EEG could record epileptic spikes in all patients, while EEGsd was successful in only 11 (64%) of them. CONCLUSION: In this study, MEG showed a tendency to record epileptic spikes in more patients than EEGsd. Furthermore, simultaneous M/EEG has been shown to be especially successful in detection of epileptic spikes in patients with MR-negative epilepsy. This might at least in parts be explained by neocortical predominance of MR-negative epilepsy. Thus, this study motivates prospective studies to evaluate the substitutability of EEGsd by MEG more extensively.

[Quality improvement of multiple choice examinations: in psychiatry, psychosomatic medicine, psychotherapy, and neurology]. / Qualitätsverbesserung von Multiple-Choice-Prüfungen: In Psychiatrie, Psychosomatik, Psychotherapie und Neurologie.

We describe a continuous improvement process in planning, performance, and evaluation of multiple choice examination questions in psychiatry, neurology, psychosomatic medicine, and psychotherapy. We analyzed 640 multiple choice questions of 1,419 students during a period of 4 years. Crucial changes concerned the abolishment of problematic question types, implementation of validated new question formats, extension of case-based questions, elongation of question stems, quantitative evaluation of item difficulty, discriminatory value, and the introduction of a peer review system. Consequences of these improvements were greater item difficulty (average 18%) and discriminatory value (average 67%) and reduced post hoc analysis times. Introduction of peer reviews resulted in longer preparation time, which was however appreciated by the peers due to a clear improvement in item quality.

Effects of peer-assisted training during the neurology clerkship: a randomized controlled study.

OBJECTIVE: To determine the efficacy of peer-assisted clinical skills training for students during their neurology clerkship. METHODS: Students (n = 122) were randomized to get clinical skills training from either student (peer) instructors (experimental group) or from experienced clinical staff (control group). The remaining schedule during the clerkship did not differ between both groups. Primary endpoint was students' practical skills and knowledge tested at the end of the course by a written test and objective structured clinical examination (OSCE). Secondary endpoints were evaluation of the practical training and self-estimated gain in theoretical and practical competence. RESULTS: In the written test, the peer-trained group (n = 66) scored 69.5 +/- 10.2 (95% CI 67-72) points of 100 and the postgraduates-trained group (n = 56) 66.7 +/- 11.4 (95% CI 63.6-69.8) (P = 0.15). In the OSCE the peer-trained group scored 93.7 +/- 6.3 (95% CI 92.1 to 95.2) points of 100 and the postgraduates-trained group 92 +/- 5.1 (95% CI 90.6 to 93.4) (P = 0.11). In the evaluation and self-assessment items, there was no significant difference between the two groups except for the postgraduates' higher competence (P = 0.004). CONCLUSION: Peer-trained students pass written exam and OSCE as efficient as postgraduates-trained students. Self-assessed learning success is equally rated in both groups.

[The Erlangen examination. An alternative to multiple choice testing for German neurology students]. / ERlanger Neurologisches Examen-STrukturiert (ERNEST). Alternative zur Multiple-Choice-Prüfung zum Abschluss des Blockpraktikums Neurologie.

BACKGROUND: With the latest revision of the German Medical Licensing Regulation in 2002, educating faculties gained more freedom in the organisation and assessment of trainees. The Erlangen Neurological Exam Structured (ERNEST) is an alternative for assessing competency in clinical knowledge at the end of the neurological general education. The answers must be given in written, short essay format. METHOD: The students (mostly 5th year of medical education) underwent the ERNEST including eight sections with ten to 15 questions each and the MC examination as had been applied earlier. The results were given in percentage scores. The examination was evaluated by a questionnaire using Likert scales. RESULTS: A total of 128 students (81 women, 47 men) with a mean age of 25.3 years (range 22-33) completed their initial training by the exam. The mean score was 69.6% in the ERNEST and 73.4% in the MC part (P<0.001). Of the students 12.5% in ERNEST and 11.7% in the MC (nonsignificant) failed to reach the projected score of 60% to pass the exam. Correlation between the ERNEST and MC results was significant, with r=0.784 (Pearson's coefficient, P<0.001). The students evaluated the aspects innovation, length, format, clarity of the tasks, closeness to reality, and compatibility with general physician's practice as predominantly positive. CONCLUSION: The ERNEST is a viable alternative form of assessment as compared to the conventional MC exam. The basic quantitative parameters of the assessment comply with the requirements of medical assessments. The students evaluated ERNEST as mainly positive.

Cyclosporin-A inhibits stretch-induced changes in myosin heavy chain expression in C2C12 skeletal muscle cells.

Studies in vivo, have shown that passive stretch of skeletal muscle induces changes in contractile protein expression. In the present study the effects of passive stretch upon myosin heavy chain (MyHC) expression were examined in C2C12 cell myotubes. Passive stretch induced an upregulation of adult fast and slow MyHCs, which was prevented by cyclosporin A (CsA), an inhibitor of calcineurin. Calcineurin has been shown to act via the dephosphorylation of NFAT and MEF2 transcriptional factors. In this study no significant change in the phosphorylation state of these factors was observed. In contrast stretch induced an alteration in the levels of the myogenic regulatory factors (MRFs) MyoD, myogenin and myf5. The modulation in the level of these MRFs was also inhibited by CsA. These data indicate that changes in muscle phenotype in C2C12 can be modulated by passive stretch and some of these changes are calcineurin dependent.

[Hospitalization of non-stroke patients in a Stroke Unit]. / Einweisung von Nicht-Schlaganfallpatienten auf eine Stroke Unit.

BACKGROUND AND OBJECTIVE: Stroke care in Germany has substantially improved during the last decade. One column of modern stroke care is the institution of stroke unit which allows rapid diagnosis and treatment. The aspect of admission of nonstroke patients to a stroke unit is poorly evaluated. The aim of this study is to evaluate the number of patients who are admitted to a national stroke unit but do not suffer from stroke. Furthermore, we related the proportion of nonstroke referrals to the different referral modes. PATIENT AND METHODS: Observational study recording all suspected stroke referrals with regard to final diagnosis and type of referral during a 12-month period (1.8.2002-31.7.2003). RESULTS: 462 patients were admitted by 4 routes: 74 by paramedics or by self-presentation, 138 by emergency physicians, 144 by primary care doctors, and 106 were transferred from other hospitals. 88 patients (19 %) finally revealed no acute stroke. The most common nonstroke diagnoses were seizure (20 %), dissociative disorders (14 %), cranial nerve disorders (11 %), hypoglycaemia (8 %) and transient global amnesia (7 %). There was no significant difference among the proportion of nonstroke patients referred by ambulance paramedics and self-presentation (15 %), emergency physicians (21 %), primary care doctors (15 %) and interhospital transfer (24 %) [p = 0.222, X (2)-test according to Pearson]. CONCLUSION: Due to the fact that a number of clinical neurological conditions mimic acute stroke, misdiagnosis of stroke is common. We advocate that all stroke patients are seen early in the course of the disease by a neurologist. An alternative could be that in stroke units of internal medicine hospitals patients are seen by a consulting neurologist. Alternatively, telemedicine might be used and the neurologist on duty of a neurological stroke unit could be consulted.

Comparison of peak and trough level monitoring of cyclosporine treatment using two modern cyclosporine preparations.

AIMS: Determination of the peak cyclosporine blood level instead of the trough level promises to represent an improvement in cyclosporine therapy monitoring due to better correlation with the AUC. In kidney transplant recipients we investigated whether this conclusion applies also to a new dispersion formulation of cyclosporine (Cicloral). PATIENTS: 42 stable kidney transplant recipients were converted from Sandimmun Neoral (NEO) to Cicloral (CIC) in a 1:1 dose relation. METHODS: On the last day of NEO administration and 14 days after conversion to CIC a full 12 h cyclosporine AUC was performed using blood samples obtained prior to and at serial times after dosing. The correlations between cyclosporine levels at these time points and the AUC were determined for NEO and CIC. For each measurement, a predicted AUC was calculated by regression analysis. The prediction error for each sampling time was calculated separately for NEO and CIC. RESULTS: The cyclosporine trough levels showed the poorest correlation with AUC for both preparations (NEO: r = 0.187 vs CIC: r = 0.554). The best correlation was observed for samples obtained at three hours after intake of either CIC (r = 0.807) or NEO (r = 0.611). The number of 2 hours measurements that lead to an unacceptable estimate from the real AUC was somewhat lower for CIC (8/40 vs 11/41 with NEO). CONCLUSIONS: Two- or three-hour cyclosporine level monitoring with the newer cyclosporine preparation Cicloral has at least the same precision as that of the original Neoral(R). In this study, the newer preparation even showed a tendency towards superior monitoring properties.

Alfuzosin, an alpha1-adrenoceptor antagonist for the treatment of benign prostatic hyperplasia: once daily versus 3 times daily dosing in healthy subjects.

OBJECTIVE: A new patented prolonged release formulation of the alpha1-adrenoceptor antagonist alfuzosin has been developed for once-daily (OD) administration in benign prostatic hyperplasia (BPH). This study was designed to compare 2 dose regimens: 10 mg OD alfuzosin and 2.5 mg TID alfuzosin at steady state. METHODS: In an open, randomized crossover study with a 9-day washout between treatments, 18 healthy male subjects (50 - 65 years) received OD or TID alfuzosin tablets orally over 5 days. Both formulations were administered according to the schedule recommended for therapeutic use: OD was administered 5 min after the evening meal, TID was administered in the evening, then in the morning and at noon (30 min before meals). On the fifth day, plasma concentrations were quantitated by HPLC with spectrofluorometric detection. RESULTS: The following pharmacokinetic parameters refer to the geometric mean values for both formulations. Mean Cmax value of 10 mg OD alfuzosin was 15.8 ng/ml at a median t(max) of 9.0 h; Cmax was higher and reached earlier from 2.5 mg alfuzosin TID: 19.3 ng/ml, 19.7 ng/ml and 20.3 at 1.0 hour after each dosing, respectively. Mean AUC(0-24) values after OD and TID were 228.3 and 226.0 ng x h/ml, respectively. Based on AUC(0-24) values corrected by the administered daily dose, the relative bioavailability of alfuzosin OD was 75.7% with a 90% confidence interval of 68.0 - 84.3%. Non-corrected AUC(0-24) values were bioequivalent with a ratio estimate of 101.0% and a 90% confidence interval of 90.7 - 112.5%. The higher daily dose compensated for the loss of bioavailability observed with the OD formulation. Mean t1/2z value was longer for the OD (8.9 h) than the TID formulation (6.9 h). Variability between individuals was similar for the 2 formulations. Both dose regimens were well tolerated. CONCLUSIONS: Alfuzosin 10 mg once-daily provides a suitable pharmacokinetic profile for a once-daily administration, equivalent bioavailability between the 2 dosage regimens and a good safety profile justify the use of alfuzosin 10 mg in patients with BPH.

Effect of polymorphism in the human glutathione S-transferase A1 promoter on hepatic GSTA1 and GSTA2 expression.

The patterns of expression of glutathione S-transferases A1 and A2 in human liver (hGSTA1 and hGSTA2, respectively) are highly variable, notably in the ratio of hGSTA1/hGSTA2. We investigated if this variation had a genetic basis by sequencing the proximal promoters (-721 to -1 nucleotides) of hGSTA1 and hGSTA2, using 55 samples of human liver that exemplified the variability of hGSTA1 and hGSTA2 expression. Variants were found in the hGSTA1 gene: -631T or G, -567T, -69C, -52G, designated as hGSTA1*A; and -631G, -567G, -69T, -52A, designated as hGSTA1*B. Genotyping for the substitution -69C > T by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), showed that the polymorphism was widespread in Caucasians, African-Americans and Hispanics, and that it appeared to conform to allelic variation. Constructs consisting of the proximal promoters of hGSTA1*A, hGSTA1*B or hGSTA2, with luciferase as a reporter gene, showed differential expression when transfected into HepG2 cells: hGSTA1*A approximately hGSTA2 > hGSTA1*B. Similarly, mean levels of hGSTA1 protein expression in liver cytosols decreased significantly according to genotype: hGSTA1*A > hGSTA1-heterozygous > hGSTA1*B. Conversely, mean hGSTA2 expression increased according to the same order of hGSTA1 genotype. Consequently, the ratio of GSTA1/GSTA2 was highly hGSTA1 allele-specific. Because the polymorphism in hGSTA1 correlates with hGSTA1 and hGSTA2 expression in liver, and hGSTA1-1 and hGSTA2-2 exhibit differential catalysis of the detoxification of carcinogen metabolites and chemotherapeutics, the polymorphism is expected to be of significance for individual risk of cancer or individual response to chemotherapeutic agents.

Clathrin-dependent and clathrin-independent endocytosis are differentially sensitive to insertion of poly (ethylene glycol)-derivatized cholesterol in the plasma membrane.

We examined the effect of a cholesterol derivative, poly (ethylene glycol) cholesteryl ether on the structure/function of clathrin-coated pits and caveolae. Addition of the compound to cultured cells induced progressive smoothening of the surface. Markedly, when the incorporated amount exceeded 10% equivalent of the surface area, fluid pinocytosis, but not endocytosis of transferrin, became inhibited in K562 cells. In A431 cells, both clathrin-independent fluid phase uptake and the internalization of fluorescent cholera-toxin B through caveolae were inhibited with concomitant flattening of caveolae. In contrast, clathrin-mediated internalization of transferrin was not affected until the incorporated poly (ethylene glycol) cholesteryl ether exceeded 20% equivalent of the plasma membrane surface area, at which point opened clathrin-coated pits accumulated. The cells were ruptured upon further addition of poly (ethylene glycol) cholesteryl ether. We propose that the primary reason for the differential effect of poly (ethylene glycol) cholesteryl ether is that the bulk membrane phase and caveolae are both more elastic than the rigid clathrin-coated pits. We analyzed the results with the current mechanical model (Rauch and Farge, Biophys J 2000;78:3036-3047) and suggest here that the functional clathrin-lattice is much stiffer than typical phospholipid bilayers.

Microsporidia and Cyclospora: epidemiology and assessment of risk from the environment.

Two classes of parasites with an environmental stage in their lifestyle have recently emerged as significant gastrointestinal pathogens for humans. Microsporidia represent a group that contains a number of genera related to the genus Cryptosporidium. They are generally transmitted via direct human to human contact, but can survive in water and food, and recently have been found in surface water used as drinking source water. Their most common host range is in patients with clinical AIDS. Limited work to date suggests the group is susceptible to chlorine achievable CxT (concentration x time) values and is coagulated by filtration. Cyclospora cayetanensis is a species of parasite that has caused outbreaks from contaminated food. Its major risk is from the use of inadequately treated water used for irrigation. Cyclospora can infect normal and immunosuppressed hosts. Current information regarding the lifestyle, transmission, and control of both groups of parasites are discussed, with a health risk assessment analysis.

FADD/MORT1 and caspase-8 are recruited to TRAIL receptors 1 and 2 and are essential for apoptosis mediated by TRAIL receptor 2.

Apoptosis induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/APO-2L) has been shown to exert important functions during various immunological processes. The involvement of the death adaptor proteins FADD/MORT1, TRADD, and RIP and the apoptosis-initiating caspases-8 and -10 in death signaling by the two death-inducing TRAIL receptors 1 and 2 (TRAIL-R1 and TRAIL-R2) are controversial. Analysis of the native TRAIL death-inducing signaling complex (DISC) revealed ligand-dependent recruitment of FADD/MORT1 and caspase-8. Differential precipitation of ligand-stimulated TRAIL receptors demonstrated that FADD/MORT1 and caspase-8 were recruited to TRAIL-R1 and TRAIL-R2 independently of each other. FADD/MORT1- and caspase-8-deficient Jurkat cells expressing only TRAIL-R2 were resistant to TRAIL-induced apoptosis. Thus, FADD/MORT1 and caspase-8 are essential for apoptosis induction via TRAIL-R2.

Endocytosis switch controlled by transmembrane osmotic pressure and phospholipid number asymmetry.

The dynamics of endocytosis in living K562 cells was investigated after the osmotic pressure of the external medium was decreased and the transmembrane phospholipid number asymmetry was increased. When the external pressure was decreased by a factor of 0.54, a sudden inhibition of endocytosis was observed. Under these conditions, the endocytosis suddenly recovered after the phospholipid number asymmetry was increased. The phospholipid asymmetry was generated by the addition of exogenous phosphatidylserine, which is translocated by the endogenous flippase activity to the inner layer of the membrane. The recovery of endocytosis is thus consistent with the view that the phospholipid number asymmetry can act as a budding force for endocytosis. Moreover, we quantitatively predict both the inhibition and recovery of endocytosis as first-order phase transitions, using a general model that assumes the existence of a transmembrane surface tension asymmetry as the budding driving force. In this model, the tension asymmetry is considered to be elastically generated by the activity of phospholipid pumping. We finally propose that cells may trigger genetic transcription responses after the internalization of cytokine-receptor complexes, which could be controlled by variations in the cytosolic or external pressure.

Regulation of tumor necrosis factor-related apoptosis-inducing ligand sensitivity in primary and transformed human keratinocytes.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to exert potent cytotoxic activity against many tumor cell lines but not against normal cells. It has been hypothesized that this difference in TRAIL sensitivity between normal and transformed cells might be due to the expression of the non-death-inducing TRAIL receptors (TRAIL-R) TRAIL-R3 and TRAIL-R4, presumably by competition for limited amounts of TRAIL. To assess the regulation of resistance versus sensitivity to TRAIL in primary as well as transformed keratinocytes, we examined TRAIL sensitivity, TRAIL receptor expression, and intracellular signaling events induced by TRAIL. Although TRAIL induced apoptosis in primary as well as transformed keratinocytes, a marked difference in sensitivity could be observed with primary keratinocytes (PK) being 5-fold less sensitive to TRAIL than transformed keratinocytes (TK). Yet both cell types exhibited similar TRAIL receptor surface expression, suggesting that expression of TRAIL-R3 and TRAIL-R4 may not be the main regulator of sensitivity to TRAIL. Biochemical analysis of the signaling events induced by TRAIL revealed that PK could be sensitized for TRAIL and, similarly, for TRAIL-R1- and TRAIL-R2-specific apoptosis by pretreatment of the cells with cycloheximide (CHX). This sensitization concomitantly resulted in processing of caspase-8, which did not occur in TRAIL-resistant PK. These data indicate that an early block of TRAIL-induced apoptosis was present in PK compared with TK or PK treated with CHX. Interestingly, cellular FLICE inhibitory protein (cFLIP) levels, high in PK and low in TK and several other squamous cell carcinoma cell lines, decreased rapidly after treatment of PK with CHX, correlating with the increase in TRAIL sensitivity and caspase-8 processing. Furthermore, ectopic expression of cFLIP long (cFLIP(L)) in TK by transfection with a cFLIP(L) expression vector resulted in resistance to TRAIL-mediated apoptosis of these cells. Thus, our results demonstrate that TRAIL sensitivity in PK is primarily regulated at the intracellular level rather than at the receptor level.

Functional analysis of TRAIL receptors using monoclonal antibodies.

mAbs were generated against the extracellular domain of the four known TNF-related apoptosis-inducing ligand (TRAIL) receptors and tested on a panel of human melanoma cell lines. The specificity of the mAb permitted a precise evaluation of the TRAIL receptors that induce apoptosis (TRAIL-R1 and -R2) compared with the TRAIL receptors that potentially regulate TRAIL-mediated apoptosis (TRAIL-R3 and -R4). Immobilized anti-TRAIL-R1 or -R2 mAbs were cytotoxic to TRAIL-sensitive tumor cells, whereas tumor cells resistant to recombinant TRAIL were also resistant to these mAbs and only became sensitive when cultured with actinomycin D. The anti-TRAIL-R1 and -R2 mAb-induced death was characterized by the activation of intracellular caspases, which could be blocked by carbobenzyloxy-Val-Ala-Asp (OMe) fluoromethyl ketone (zVAD-fmk) and carbobenzyloxy-Ile-Glu(OMe)-Thr-Asp (OMe) fluoromethyl ketone (zIETD-fmk). When used in solution, one of the anti-TRAIL-R2 mAbs was capable of blocking leucine zipper-human TRAIL binding to TRAIL-R2-expressing cells and prevented TRAIL-induced death of these cells, whereas two of the anti-TRAIL-R1 mAbs could inhibit leucine zipper-human TRAIL binding to TRAIL-R1:Fc. Furthermore, use of the blocking anti-TRAIL-R2 mAb allowed us to demonstrate that the signals transduced through either TRAIL-R1 or TRAIL-R2 were necessary and sufficient to mediate cell death. In contrast, the expression of TRAIL-R3 or TRAIL-R4 did not appear to be a significant factor in determining the resistance or sensitivity of these tumor target cells to the effects of TRAIL.

Pharmacokinetics of new testosterone transdermal therapeutic systems in gonadotropin-releasing hormone antagonist-suppressed normal men.

In a phase I single-center, open, randomized pilot study with a three-way cross-over design the pharmacokinetics of three testosterone-containing transdermal therapeutic systems were evaluated in healthy male volunteers. Testosterone TTS HEXAL type 1 and 2 are nonscrotal membrane patches differing in the kind of adhesive used. 6 subjects were treated with low dose Testosterone TTS type 1, high dose Testosterone TTS type 1 and low dose Testosterone TTS type 2. To eliminate the influence of endogenous serum testosterone, the endogenous testosterone secretion was suppressed by the GnRH antagonist cetrorelix. In all subjects under GnRH antagonist treatment a marked suppression of LH, FSH, testosterone, DHT and estradiol was observed. Physiologic testosterone levels were achieved during the 24-hour-application period. Maximal serum levels were reached after 4 hours with both TTS systems. Both systems appear suited for further testing because both enable a physiological circadian profile to be achieved. GnRH-antagonist pretreatment is a useful model to evaluate the effect of exogenous testosterone in clinical studies, when, due to fluctuations in endogenous hormone levels, an estimation of the proportion of exogenous steroid is not possible.