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Background: As a consequence of the discovery of an extracellular component responsible for the progression of tau pathology, tau immunotherapy is being extensively explored in both preclinical and clinical studies as a disease modifying strategy for the treatment of Alzheimer’s disease. Objective: Describe the characteristics of the anti-phospho (T212/T217) tau selective antibody PT3 and its humanized variant hPT3. Methods: By performing different immunization campaigns, a large collection of antibodies has been generated and prioritized. In depth, in vitro characterization using surface plasmon resonance, phospho-epitope mapping, and X-ray crystallography experiments were performed. Further characterization involved immunohistochemical staining on mouse- and human postmortem tissue and neutralization of tau seeding by immunodepletion assays. Results and Conclusion: Various in vitro experiments demonstrated a high intrinsic affinity for PT3 and hPT3 for AD brain-derived paired helical filaments but also to non-aggregated phospho (T212/T217) tau. Further functional analyses in cellular and in vivo models of tau seeding demonstrated almost complete depletion of tau seeds in an AD brain homogenate. Ongoing trials will provide the clinical evaluation of the tau spreading hypothesis in Alzheimer’s disease.

Multiple animal models have been created to gain insight into Alzheimer’s disease (AD) pathology. Among the most commonly used models are transgenic mice overexpressing human amyloid precursor protein (APP) with mutations linked to familial AD, resulting in the formation of amyloid β plaques, one of the pathological hallmarks observed in AD patients. However, recent evidence suggests that the overexpression of APP by itself can confound some of the reported observations. Therefore, we investigated in the present study the AppNL-G-Fmodel, an App knock-in (App-KI) mouse model that develops amyloidosis in the absence of APP-overexpression. Our findings at the behavioral, electrophysiological, and histopathological level confirmed an age-dependent increase in Aβ1–42 levels and plaque deposition in these mice in accordance with previous reports. This had apparently no consequences on cognitive performance in a visual discrimination (VD) task, which was largely unaffected in AppNL-G-F mice at the ages tested. Additionally, we investigated neurophysiological functioning of several brain areas by phase-amplitude coupling (PAC) analysis, a measure associated with adequate cognitive functioning, during the VD task (starting at 4.5 months) and the exploration of home environment (at 5 and 8 months of age). While we did not detect age-dependent changes in PAC during home environment exploration for both the wild-type and the AppNL-G-F mice, we did observe subtle changes in PAC in the wild-type mice that were not present in the AppNL-G-F mice.

The tau spreading hypothesis provides rationale for passive immunization with an anti-tau monoclonal antibody to block seeding by extracellular tau aggregates as a disease-modifying strategy for the treatment of Alzheimer's disease (AD) and potentially other tauopathies. As the biochemical and biophysical properties of the tau species responsible for the spatio-temporal sequences of seeding events are poorly defined, it is not yet clear which epitope is preferred for obtaining optimal therapeutic efficacy. Our internal tau antibody collection has been generated by immunizations with different tau species: aggregated-and non-aggregated tau and human postmortem AD brain-derived tau fibrils. In this communication, we describe and characterize a set of these anti-tau antibodies for their biochemical and biophysical properties, including binding, tissue staining by immunohistochemistry, and epitope. The antibodies bound to different domains of the tau protein and some were demonstrated to be isoform-selective (PT18 and hTau56) or phospho-selective (PT84). Evaluation of the antibodies in cellular-and in vivo seeding assays revealed clear differences in maximal efficacy. Limited proteolysis experiments support the hypothesis that some epitopes are more exposed than others in the tau seeds. Moreover, antibody efficacy seems to depend on the structural properties of fibrils purified from tau Tg mice-and postmortem human AD brain.

A nanophotonic sensor for sensitive detection of methane in water solution is presented. Cryptophane-A doped waveguide cladding provides for methane pre-concentration on a chip, resulting in a detection limit of 60 ppm (86 nM).

Summary The ultrastructural dianges in Candida albicans after exposure in vitro to different doses of clotrimazole and miconazole have been investigated. The changes obtained with low dose levels of both drugs concerned preferentially the cell periphery with eventual hyperactivity of the central vacuolar system. With high dose levels, on the other hand, the morphologic changes differed markedly between the two drugs with respect to the number of cells involved, the intensity of the lesions and their subcellular location. This discrepancy in response of C. albicans towards high doses of the imidazoles, shed some doubts on the previously propagated mode of action of these drugs. Additionally to the morphological description of the involutionary processes in C. albicans, the behavior of some oxidative and peroxidative enzymes has been investigated after miconazole treatment. In the light of these findings, an alternate hypothesis is formulated proposing hydrogen peroxide intoxication as the cause of cell death. Zusammenfassung Die ultrastrukturellen Veranderungen bei Candida albicans nach Behandlung in vitro mit verschiedenen Dosen von Clotrimazol und Miconazol wurden untersucht. Die bei niedrigen Dosen beider Arzneimittel entstandenen Veranderungen beziehen sich vorzugsweise auf die Zellperipherie, mit moglicher Hyperalttivitat des zentralen Vakuolensystems. Andererseits zeigten beide Arzneimittel bei hohen Dosen cine stark verschiedene morphologische Wirkung in Hinsicht auf die beteiligte Zellzahl, den Lasionsgrad und ihre subzellulare Lokalisierung. Infolge dieser Reaktionsverschiedenheit von C. albicans gegenuber hohen Dosen der Imidazole kommt die fruher verbreitete Wirkungsweise dieser Arzneimittel wieder in Frage. Auser der morphologischen Beschreibung der Zurudrbildungsprozesse bei C. albicans wurde das Verhalten einiger oxydativer und peroxydativer Enzyme nach Mico-nazol-Behandlung geprufk. In Anbetracht dieser Befunde wird eine neue Hypothese formuliert, nach der der Zelltod auf Wasserstoffperoxydvergiftung zuruckgehen wurde.

Summary: The antifungal activity of ketoconazole was tested in a medium that promotes mycelial growth (enriched EMEM medium) for 45 clinical isolates of Candida albicans isolated from pathological conditions in man. The mean antifungal activity of ketoconazole for all tested strains at a concentration of 10-7 M (0.05 μg/ml) was 97.3 %. At this concentration ketoconazole was fungicidal for 6 strains and in all other strains prevented the formation of mycelium and pseudomycelium, the invasive forms in pathology. Zusammenfassung: Auf dem fur Myzelwachstum gunstigen angereicherten EMEM Nahrboden wurde die antimykotische Wirksamkeit von Ketoconazol auf 45 klinische Isolate von Candida albicans, alle aus pathologischem Material beim Menschen isoliert, gepruft. Bei einer Konzentration von 10-7 M betrug die mittlere antimykotische Wirksamkeit des Ketoconazols 97,3% fur alle untersuchten Stamme. Bei dieser Konzentration wirkte Ketoconazol auf 6 Stamme fun-gizid, wahrend bei alien anderen Stammen die Myzel- bzw. Pseudomyzelbildung, das heist die invasiven pathologischen Formen unterdruckt wurden.

Summary. Guinea pigs, infected with either Trichophyton mentagrophytes or Microsporum canis, were treated orally or topically with azole antifungals daily for two weeks. Fungi located in the stratum corneum were affected similarly by both treatment schedules, showing typical cell wall changes after azole exposure and necrosis of internal organelles. Fungi located in the hair sheaths were affected only by the oral treatment, which not only prevented invasion of the inner hair structures and inflammatory responses but also led to a complete clearance of the infection within 7 days. Topically applied azole treatment was not able to injure fungi in the hair sheaths and did not suppress invasion into the hair shafts. These observations are in favour of oral antifungal medication with azoles for the treatment of der-matophyte infections involving hairy skin. Zusammenfassung. Mit Trichophyton mentagrophytes oder Microsporum canis infizierte Meer-schweinchen wurden zwei Wochen lang taglich oral bzw. lokal mit antimykotischen Azolen behandelt. Beide Behandlungsweisen zeigten ahnliche Auswirkungen auf im Stratum corneum angesiedelte Pilze nach Azol-Exposition, namlich typische Zellwandveranderungen und Nekrose interner Organellen. Bei in den Haarscheiden angesiedelten Pilzen zeigte nur die orale Behandlung Wirkung. Es wurden nicht nur eine Invasion der inneren Haarstruktur und entzundliche Reaktionen vermieden, sondern die Infektion wurde innerhalb von 7 Tagen vollstandig zum Abklingen gebracht. Die lokale Azolbehandlung konnte die Pilze in den Haarscheiden nicht schadigen und verhinderte nicht das Eindringen in die Haarschafte. Diese Beobachtungen sprechen fur eine orale antimykotische Medikation mit Azolen zur Behandlung von Dermatophyten-Infektionen behaarter Hautpartien.

Dermatomycoses are among the most widespread and common superficial and cutaneous fungal infections in humans. These typically nonfatal conditions are difficult to treat, especially infections of the nail. Dermatomycoses are caused by filamentous fungi such as Trichophyton, Microsporum or Epidermophyton species. These filamentous fungi have a high affinity for keratin, an important component of hair, skin and nails, which are the primary areas of infection by dermatophytes. The antifungal agents currently marketed for dermatomycoses are mainly inhibitors of ergosterol biosynthesis, except for griseofulvin, which interferes with the cytoplasmic and nuclear microtubular system. Three different types of inhibitors of the ergosterol biosynthetic pathway have been proven to be effective in clinic: the azoles (e.g. topical miconazole and topical/oral ketoconazole, itraconazole and fluconazole), the allylamines (e.g. terbinafine) and morpholines (amorolfine). Even today more effective antifungal azoles with less adverse effects and short-term therapy are deemed necessary to treat dermatophytosis. A promising novel triazole compound in this respect is R126638, which showed potent in vitro and in vivo activity.

Background— Dysfunctional and normally perfused remote regions show equal myolysis and glycogen accumulation in pig hibernating myocardium. We tested the hypothesis that these arose secondary to elevations in preload rather than ischemia. Methods and Results— Expression of structural protein (desmin, desmoplakin, titin, cardiotin, α-smooth muscle actin, lamin-A/C, and lamin-B2) in viable dysfunctional myocardium was analyzed by immunohistochemistry. We performed blinded analysis of paired dysfunctional left anterior descending coronary artery and normal remote subendocardial samples from stunned (24 hours; n=6), and hibernating (2 weeks; n=6) myocardium versus sham controls pigs (n=7). Within 24 hours, cardiac myocytes globally reexpressed α-smooth muscle actin. In stunned myocardium, cardiotin was globally reduced, whereas reductions in desmin were restricted to the dysfunctional region. Alterations progressed with the transition to hibernating myocardium, in which desmin, cardiotin, and titin were globally reduced. A qualitatively similar reorganization of cytoskeletal proteins occurred 3 hours after transient elevation of left ventricular end-diastolic pressure to 33±3 mm Hg. Conclusions— Qualitative cardiomyocyte remodeling similar to that in humans with chronic hibernation occurs rapidly after a critical coronary stenosis is applied, as well as after transient elevations in left ventricular end-diastolic pressure in the absence of ischemia. Thus, reorganization of cytoskeletal proteins in patients with viable dysfunctional myocardium appears to reflect chronic and/or cyclical elevations in preload associated with episodes of spontaneous regional ischemia.

R126638 is a new triazole agent with potent antifungal activity in vitro against various dermatophytes, Candida spp., and Malassezia spp. Its activity against Malassezia spp. in vitro was superior to that of ketoconazole, the agent currently used for the treatment of Malassezia -related infections. R126638 showed activity comparable to or lower than that of itraconazole against dermatophytes in vitro; however, in guinea pig models of dermatophyte infections, R126638 given orally consistently showed antifungal activity superior to that of itraconazole, with 50% effective doses (ED 50 s) three- to more than eightfold lower than those of itraconazole, depending on the time of initiation and the duration of treatment. The ED 50 of R126638 in a mouse dermatophytosis model was more than fivefold lower than that of itraconazole. These data indicate that if the effects of R126638 seen when it is used to treat animals can be extrapolated to humans, the novel compound would be expected to show effects at doses lower than those of existing drugs and, hence, present a lower risk for side effects.

There is increasing evidence that apoptosis in postmitotic neurons is associated with a frustrated attempt to reenter the mitotic cycle. Okadaic acid, a specific protein phosphatase inhibitor, is currently used in models of Alzheimer's research to increase the degree of phosphorylation of various proteins, such as the microtubule-associated protein tau. Okadaic acid induces programmed cell death in the human neuroblastoma cell lines TR14 and NT2-N, as evidenced by fragmentation of DNA and attenuation of this process by protein synthesis inhibitors. In differentiated TR14 cells, okadaic acid increases the fraction of cells in the S phase, induces the appearance of cyclin B1 and cyclin D1 markers of the cell cycle, and triggers a time-dependent increase in DNA fragmentation after release of a thymidine block. Fully differentiated NT2-N cells are forced to enter the mitotic cycle as shown by DNA staining. Chromatin condensation and chromosome formation are initiated, but the cells fail to complete their mitotic cycle. These data suggest that okadaic acid forces differentiated neuronal cells into the mitotic cycle. This pattern of cyclin up-regulation and cell cycle shift is compared with apoptosis induced by neurotrophic factor deprivation in differentiated rat pheochromocytoma PC12 cells.

Aims Cardiomyocyte apoptosis is known to occur in infarct border zones, where cardiomyocyte dedifferentiation, as seen in hibernating myocardium, can also be observed. The aim of the study is to determine whether dedifferentiated cardiomyocytes represent a population of cells stably surviving or undergoing apoptosis. Methods and Results Microinfarctions were induced in sheep (n = 8) by intracoronary injection of polymer macrobeads. The sheep were killed when cardiac function was gradually decreased (ejection fraction 37 +/- 6%, mean +/- SEM), but not earlier than 6 weeks after embolization. Transmural biopsies were taken from embolized and remote areas, based on flow measurements with positron emission tomography. Cells were classified as dedifferentiated when sarcomere content was depleted by >10%, and glycogen content increased. Apoptosis was detected using the Tdt-mediated nick-end labelling (TUNEL) method and activated caspase-3 immunolabelling. Dedifferentiated cardiomyocytes were identified by morphology and by immunohistochemical evaluation of dedifferentiation related expression patterns of desmin, titin. cardiotin and a-smooth muscle actin. Cardiomyocyte apoptosis was detected in both the infarction border zones and remote areas. Dedifferentiated cardiomyocytes accounted for up to 30%. of the cells in embolized areas and were almost exclusively non-apoptotic. Conclusion In embolization induced microinfarcted tissue, dedifferentiated cardiomyocytes are preferentially spared to undergo apoptosis. It is hypothesized that dedifferentiated cardiomyocytes and apoptotic cardiomyocytes represent two different cell populations. The dedifferentiated cells can be considered as stable surviving cells.

Experimental stroke can be induced by permanent or temporal reduction of oxygen supply in focal brain regions of a variety of animal species in order to produce a reliable functional deficit with or without the formation of reproducible infarcts. The resulting lesion depends on the size of the occluded vessels and the territory that they supply. This can include a complete hemisphere, a major perfusion territory, specific functional regions or unpredictable lacunar fields. The outcome depends on the residual flow to the affected area and the timely restoration of blood supply. Areas of low flow, either in the occluded region or in the apposing penumbra, deserve attention for therapeutic rescue. This is also true for areas remote from the compromised structure. Since the progression of injury and the response of the different brain constituents are multifactorial and behave in a rather unpredictable and variable way, treatment is a complex matter. The evolution of acute or delayed neuronal injury and secondary phenomena such as edema formation, inflammatory reaction and retrograde degeneration may require well-timed approaches based on various mechanisms of action. In this paper current experimental models are reviewed and pharmacotherapeutic measures will be discussed in relation to the pathogenesis of stroke.

The clinical use of all-trans-retinoic acid (ATRA) in the treatment of cancer is significantly hampered by the prompt emergence of resistance, believed to be caused by increased ATRA catabolism. Inhibitors of ATRA catabolism may therefore prove valuable for cancer therapy. Liarozole-fumarate is an anti-tumour drug that inhibits the cytochrome P450-dependent catabolism of ATRA. ATRA, but also its naturally occurring catabolites, 4-oxo-ATRA and 5,6-epoxy-ATRA, as well as its stereoisomers, 9-cis-RA and 13-cis-RA, show significant antiproliferative activity in MCF-7 human breast cancer cells. To further elucidate its mechanism of action, we investigated whether liarozole-fumarate was able to enhance the antiproliferative activity of ATRA catabolites and isomers. Liarozole-fumarate alone up to a concentration of 10(-6) M had no effect on MCF-7 cell proliferation. However, in combination with ATRA or the ATRA catabolites, liarozole-fumarate (10(-6) M) significantly enhanced their antiproliferative activity. On the contrary, liarozole-fumarate (10(-6) M) was not able to potentiate the antiproliferative activity of the ATRA stereoisomers, most probably because of the absence of cytochrome P450-dependent catabolism. Together, these findings show that liarozole-fumarate acts as a versatile inhibitor of retinoid catabolism in that it not only blocks the breakdown of ATRA, but also inhibits the catabolic pathway of 4-oxo-ATRA and 5,6-epoxy-ATRA, thereby enhancing their antiproliferative activity.

All-trans-retinoic acid (ATRA) is well known to inhibit the proliferation of human breast cancer cells. Much less is known about the antiproliferative activity of the naturally occurring metabolites and isomers of ATRA. In the present study, we investigated the antiproliferative activity of ATRA, its physiological catabolites 4-oxo-ATRA and 5,6-epoxy-ATRA and isomers 9-cis-RA and 13-cis-RA in MCF-7 human breast cancer cells by bromodeoxyuridine incorporation. MCF-7 cells were grown in steroid- and retinoid-free medium supplemented with growth factors. Under these culture conditions, ATRA and its naturally occurring catabolites and isomers showed significant antiproliferative activity in MCF-7 cells in a concentration-dependent manner (10[-11] M to 10[-6] M). The antiproliferative activity of ATRA catabolites and isomers was equal to that of the parent compound ATRA at concentrations of 10(-8) M and 10(-7) M. Only at 10(-6) M were the catabolites and the stereoisomer 13-cis-RA less potent. The stereoisomer 9-cis-RA was as potent as ATRA at all concentrations tested (10[-11] M to 10[-6] M). In addition, we show that the catabolites and isomers were formed from ATRA to only a limited extent. Together, our findings suggest that in spite of their high antiproliferative activity the catabolites and isomers of ATRA cannot be responsible for the observed growth inhibition induced by ATRA.

It has been previously demonstrated that lysophosphatides accumulate rapidly in ischaemic tissue, and may play a key role in the genesis of ischaemia-reperfusion injury. The present study investigated the effects of exogenously added lysophosphatidylcholine (1-20 microM) on single isolated cardiomyocytes from adult rabbit hearts. Quiescent cells exposed toor = 8 microM lysophosphatidylcholine dose-dependently displayed irreversible hypercontraction, whereas after 60 min at 3 microM lysophosphatidylcholine, most cells remained rod-shaped (87.2 +/- 2.0%, mean +/- S.E.M.). However, when combined with electrical field stimulation (1 Hz), exposure to 3 microM lysophosphatidylcholine resulted in irreversible hypercontracture of most cells after 60 min: only 27.5 +/- 7.5% of the cells remained rod-shaped. Contracture depended upon the presence of extracellular Ca2+, and coincided with a significant rise in the median intracellular free Ca2+ level from 72.2 to 352.1 nM (P = 0.0001), suggesting intracellular Ca(2+)-overload. Pretreatment with 10(-6) M flunarizine or R 56865 significantly reduced the fraction of damaged cells when exposed to 3 microM lysophosphatidylcholine and electrical stimulation: 78.3 +/- 12.2% and 56.3 +/- 13.1% respectively of the cells remained rod-shaped. No protection was observed when quiescent cells were exposed to 10 microM lysophosphatidylcholine. Cytochemical localization of Ca2+ showed that lysophosphatidylcholine induced a loss of sarcolemma-bound Ca2+ precipitate and an accumulation of Ca2+ clusters in mitochondria of damaged cells in a dose and time dependent way. These results suggest that lysophosphatidylcholine induces functional and structural damage (Ca(2+)-overload) in isolated cardiomyocytes and that this can be prevented by cytoprotective drugs.

Experimental induction of subarachnoid haemorrhage in rats resulted in acute haemodynamic changes. Heart rate decreased concomitantly with a rise in arterial blood pressure. Intracranial pressure increased and consequently cerebral perfusion pressure dropped. These changes as well as the observed electrocardiographic (ECG) changes were comparable to those reported in patients. Apart from blood also saline, when introduced into the cisterna magna, was able to elicit such abnormalities. The haemodynamic and electrocardiographic changes, which result from subarachnoid haemorrhage, may even become aggravated, when repetitive injections of blood or saline are given into the cisterna magna and when cerebral angiography is performed prior to induction of the subarachnoid haemorrhage. Chronic intracranial pressure monitoring during the 48 hours following subarachnoid haemorrhage revealed no significant rise in pressure. A thorough control of the experimental conditions is thus of utmost importance in order to give a valid interpretation of the observed anomalies.

5'-Nucleotidase and purine nucleoside phosphorylase, two key enzymes in nucleoside metabolism, have been localized electronmicroscopically in left ventricular myocardium of the human, dog, pig, rabbit, guinea pig and rat. Ectonucleotidase activity was present in all species at the plasma membrane of pericytes. Reactive endothelial cells in the microcirculatory bed were restricted to those covering resistance arterioles. Cardiomyocytes were reactive only in the rat. Purine nucleoside phosphorylase was localized uniformly in the vascular endothelium of all species. The strongest activity was seen in the pericytes of guinea pig, rat and dog. Pericytes of rabbit and pig were virtually unreactive, whereas a minority of cells in human samples were positive. Cardiomyocytes were unreactive in all species. These variations in the distribution pattern of adenosine metabolic sites may have definite consequences for disposal and recovery of adenylates and their breakdown products in ischaemia and for the effects to be expected from interference with nucleoside transport inhibition.

A simple and rapid method for light and electron microscopic visualization of degenerating neuronal processes and axon terminals is described. Hundred-micrometer vibratome sections of perfusion-fixed rat brain were incubated briefly in a 5% silver nitrate solution in a conventional microwave oven. After a rinse in 1% acetic acid, the sections were silver enhanced. Differentiation and counterstaining was done respectively in ethanol 100% and cresyl violet. In the light microscope, degenerating neuronal processes appeared as black dots against a clear background. Areas of calcification were also positively stained. The presence of silver deposits in degenerating presynaptic terminals and dendrites was confirmed ultrastructurally.

Intracellular Ca2+ overload is considered to be the final pathway leading to cell death under pathological conditions. However, both the route of Ca2+ entry and the site of action of cardioprotective drugs remain obscure. This was investigated using isolated adult rat and rabbit cardiomyocytes exposed to the experimental pathological stimuli veratridine, singlet O2, lysophosphatidylcholine, and ouabain. Under these conditions, the majority of cells displayed irreversible hypercontraction as a consequence of intracellular Ca2+ overload. Nifedipine did not prevent Ca2+ overload, but tetrodotoxin (TTX) and reduction of the extracellular Na+ concentration protected against the above pathological stimuli. This strongly suggests that intracellular Ca2+ overload after exposure to these pathological stimuli may be mediated via fast Na+ channel dysfunction, causing excessive entry of Na+, followed by Ca2+ overload via Na(+)-Ca2+ exchange. The new cardioprotective drug R 56865 dose dependently prevented hypercontracture induced by each of these stimuli, suggesting that R 56865 may interfere with this modified Na+ channel that is in a way different from class I antiarrhythmic drugs. This is regarded as a new cardiac cytoprotective principle.

We examined the role of calcium in the pathogcnesis of ischemie cardiac cell death in the isolated working rabbit heart subjected to normothermic global ischemia followed by rcperfusion. Apart from measurements of cardiodynamic function and ultrastructural examination, we also used a cytochemical procedure to localize exchangeable calcium pools at the ultrastructural level. The effects of verapamil (1.5 × 10 −8 M, 3 × 10 −8 M) (high affinity for L-type calcium channels) were compared with those of R 56 865 (4 × 10 −7 M) (Ca 2+ overload blocker with low affinity for the L-type calcium channels). A severe depression of cardiac function was observed after solvent or verapamil pretreatment and 25 min of ischemia followed by reperfusion. R 56 865 treatment resulted in a significantly improved postischemic recovery when compared to solvent and verapamil treatment groups. The ultrastructural and cytochemical results corroborated the hcmodynamic findings. In solvent and verapamil-treated hearts, irreversible damage was observed mainly in mid- and cndocardial areas. Ultrastructural changes were accompanied by shifts in calcium localization: i.e. loss of sarcolcmmal calcium binding capacity, accumulation of calcium precipitate in the mitochondria. In the R 56 865 treatment group, damage was limited to some cells scattered in the midcardial areas. In conclusion, R 56 865, which has little affinity for the slow channels was highly effective in protecting against ischemie damage, indicating that, in this experimental set-up, the calcium responsible for cellular Ca 2+ overload is not entering via L-typc calcium channels.

The effect of nebivolol treatment on the survival of cardiomyopathic hamsters (BIO82.62) who had developed congestive heart failure was investigated. Five percent of the hamsters included in the study had died between 200 and 220 days of age, indicating development of congestive heart failure. At age 220 days, a treatment protocol was initiated and the remaining animals received either nonmedicated food (n = 34) or food supplemented with nebivolol (1 mg/kg/day, n = 33). The death rate was similar in both groups during the first 27 days of treatment, after which nebivolol significantly delayed mortality (p = 0.011). The median survival time was 293 days in controls vs 321 days in the nebivolol group. It is concluded that nebivolol prolongs survival in this animal model of congestive heart failure, although further investigation is needed to clarify the mechanism of action.

The effect of pretreatment with selective beta 1-adrenoceptor blockers (dl-nebivolol or atenolol) on myocardial mechanical activity, mitochondrial function, morphology, and calcium cytochemistry was studied during normothermic ischemic arrest and reperfusion of isolated working rabbit hearts. The hearts subjected to 25 min of ischemia followed by 30 min of post-ischemic reperfusion showed typical signs of severe myocardial ischemic damage. The ultrastructural changes showed a good relation with the changes in mechanical activity and mitochondrial function. To determine whether these changes could be prevented or reduced by beta 1-adrenoceptor blockade, dl-nebivolol or atenolol (0.62 mg/liter) was added to the perfusate 30 min before the induction of ischemia. The results showed that dl-nebivolol exerted a protective effect on recovery of mechanical activity, on mitochondrial function during reperfusion as well as on the ultrastructure as examined at the end of the reperfusion period. On the other hand, atenolol failed to protect the myocardium against ischemia-reperfusion damage in the isolated working rabbit heart.

Fungal cultures were incubated for various periods of time with saperconazole at concentrations ranging from 10(-10) to 10(-5) M: Candida albicans (4 and 24 h), Pityrosporum ovale (2 and 7 days), and Trichophyton rubrum (1, 2, 3, 7 and 14 days). At the end of the incubation period, fungal morphology was compared with that of control cultures by transmission and scanning electron microscopy. In all three species, major ultrastructural changes were seen from concentrations of 10(-8) to 10(-7) M saperconazole onwards, depending on the species and time of incubation. The main changes were inhibition of hyphal outgrowth (C. albicans), abortive hyphal outgrowth (T. rubrum) and deposition of electron-dense vesicles in a thickened cell wall (C. albicans, T. rubrum). In P. ovale, a direct, necrotizing action was seen without concomitant wall changes.

Summary Background An alternative approach to retinoid therapy is to inhibit the cytochrome P450 (CYP)-mediated catabolism of endogenous all-trans retinoic acid in the skin by applying retinoic acid metabolism blocking agents such as talarozole (R115866). Objectives To study the effects of topical talarozole on retinoid biomarkers in normal skin in a randomized phase I trial. Methods Gels containing talarozole (0·35% or 0·07%) and vehicle were applied once daily for 9 days on either buttock of 16 healthy volunteers. Epidermal shave biopsies (for mRNA analysis) and punch biopsies (for histology and immunofluorescence analysis) were collected from the treatment areas. Genes encoding the following were studied by quantitative real-time polymerase chain reaction: cellular retinoic acid binding protein 2 (CRABP2), cytokeratins (KRT2 and KRT4), CYP26A1, CYP26B1, CYP26C1 and CYP2S1, two enzymes in the retinol metabolism (retinal dehydrogenase-2 and retinol acyltransferase) and two proinflammatory cytokines [interleukin (IL)-1α and tumour necrosis factor-α]. Results Talarozole treatment increased the mRNA expression of CRABP2, KRT4, CYP26A1 and CYP26B1 dose dependently, and decreased the expression of KRT2 and IL-1α compared with vehicle-treated skin. No mRNA change in retinol-metabolizing enzymes was obtained. There was no induction of epidermal thickness or overt skin inflammation in talarozole-treated skin. Immunofluorescence analysis confirmed an upregulation of KRT4 protein, but no upregulation of CYP26A1 and CYP26B1 expression was detected. Conclusions Talarozole influences the biomarker pattern consistently with increased retinoic acid stimulation. The low irritancy of talarozole at the two examined dosages is a possible advantage over topical retinoids.

The Jackson methodology for systems development is seen as a formal methodology. Although it is formal, the analyst has a freedom to specify models of problems in alternative ways without giving any indication which one is the better. Proposals to extend the methodology with the concepts ‘(near) static entity’ and ‘static roles’ give the analyst even more freedom than before. From the maintenance point of view this can lead to problems of understandability of the models. Also some enhancements cannot be done without completely redesigning entities. In this paper we propose to separate the existence aspect of an entity in one process. A second proposal is to model relationships (i.e. aspects comon to two or more entities) between entities explicitly using entity-relationship notation. In this way, more maintainable specifications will be obtained.

The cytochemical localization of NADH-oxidase, a possible source of oxygen derived toxic species was studied in the isolated working rabbit heart subjected to normothermic global ischaemia. The activity of this oxidase could be important for the damage observed during ischaemia, when cellular defence mechanisms against free radicals are depleted. In non-ischaemic myocardium only small amounts of the NADH-oxidase reaction product were present in the mitochondria. Although the reaction product could already be observed after 45 min of incubation, prolonged incubation times up to 2h were necessary to clearly define these reactive sites. The reaction product is substrate dependent and is not affected by cyanide. Exposure of the hearts to ischaemia resulted in an alteration of the enzyme activity depending on the degree of ischaemic damage. In ultrastructurally slightly altered areas a high degree of activity was observed in the mitochondria. In infarcted tissue, mitochondria contained little or no reaction product. This cytochemical study supports the hypothesis that hydrogen peroxide and oxygen radicals produced in the mitochondria by NADH-oxidase activity may contribute to the mitochondrial damage observed during ischaemia when NADH is no longer oxidized by the respiratory chain and cellular defence mechanisms are impaired.

Summary Background R115866 (RambazoleTM; Barrier Therapeutics NV, Geel, Belgium), a new-generation retinoic acid metabolism-blocking agent, is a nonretinoid compound enhancing intracellularly the endogenous levels of all-trans-retinoic acid by blocking its catabolism. By virtue of this property, and the proven positive effects of retinoids in the treatment of acne, R115866 could potentially be a useful drug for acne. Objectives To explore the efficacy, safety and tolerability of systemic R115866 in male patients with moderate to severe facial acne vulgaris (at least 15 papules and/or pustules and at least two nodulocystic lesions). Methods In this exploratory trial, 17 patients were treated with oral R115866 1 mg once daily for 12 weeks, followed by a 4-week treatment-free period. Results At the end of treatment (week 12, n = 16) a mean reduction in inflammatory lesion count of 77·4% (P

Functional outcome after revascularization of chronic hibernating left ventricular segments is less predictable than initially thought. The earlier proposed criterium of the hibernating state — that functional recovery occurs immediately after revascularization — has been called into question. The time-course and the degree of functional recovery after revascularization are two of the unresolved issues. The morphologic changes in the myocardium of hypocontractile segments, especially the possible structural counterparts underlying chronic ischemia, are not well documented. In recent studies (1, 4, 6, 10) light and electron microscopy was performed on myocardial tissue removed from the anterior wall of the left ventricle during coronary artery bypass grafting.

To study pharmacotherapy of traumatic brain injury in rats, a modified closed head injury model was used that expresses clinically relevant features including intracranial hypertension and morphological alterations. Long-term survival under ethically acceptable conditions would greatly improve its clinical relevance. To ensure this goal with great reproducibility, the experimental protocol was adapted, in particular the impact-acceleration kinetics. Variations in impact-acceleration conditions were obtained by modifying the stiffness of the impact site and changing the height of a 400 g weight dropped from 51.5 to 31.5 cm (51.5/400; 31.5/400). Impact and acceleration were measured with a force sensor incorporated in a rigid dummy-rat and an accelerometer mounted on the platform onto which the animals are positioned. Significant correlation was shown between impact and acceleration. Accelerations obtained in rats were significantly lower than those in the dummy. Unlike the 51.5/400 group, in the 31.5/400 group no mortality or cranial fractures were observed. In both groups intracranial pressure rose to pathological values immediately after trauma and remained elevated longer than 24 h. Diffuse axonal injury developed in all groups and remained present for at least 7 days. By reducing the impact-acceleration conditions, post-traumatic complications were diminished, while the clinically important features were maintained.

Bcl-2 is a gene with clear anti-apoptotic properties in neurodegenerative conditions. One of the earliest hallmarks of degeneration in neuronal cell cultures is the loss of neurite morphology. Therefore the effect of Bcl-2 on neuronal morphology and microtubule stability was studied in nerve growth factor differentiated PC12 cells. Microtubule dynamics were modulated using the microtubule stabilizer taxol and the microtubule destabilizer, okadaic acid, a protein phosphatase inhibitor. It was shown that Bcl-2 protects against both taxol- and okadaic acid induced neurite retraction. Bcl-2 overexpression also significantly reduced the increased ratio of acetylated tubulin over total tubulin induced by taxol treatment. Interestingly, Bcl-2 attenuates the decrease of the same ratio after exposure to okadaic acid, suggesting that Bcl-2 is able to normalize the level of acetylated tubulin. In addition, cell death and nuclear fragmentation, induced by okadaic acid, were reduced in Bcl-2 overexpressing cells. This protection is either downstream or independent of tau phosphorylation as quantitative immunocytochemistry with AT8 showed that Bcl-2 did not modify the level of tau phosphorylation. The data suggest that the protective effect of Bcl-2 on the neuronal cytoskeleton is probably linked to changes in the post-translational modification of tubulin.

Taxol-induced peripheral neuropathy is a commonly-occurring side-effect in the treatment of cancer patients with taxoteres or taxanes. Taxol is known to induce apoptosis in a number of tumor cells. This report documents that, similar to proliferating cells, taxol induces apoptosis in NGF-differentiated PC12 cells, as assessed by exogenous FITC-annexin-V binding and nuclear fragmentation. It is shown that PC12 cells that stably overexpress Bcl-2 are protected against the toxic effect of taxol, as evidenced by the XTT assay and by a decreased fraction of propididum iodide positive cells in a dye exclusion test. Also the number of annexin-V-positive cells and the number of fragmented nuclei are lower in the Bcl-2 transfected cells. The effect is similar to the protective effect of Bcl-2 against NGF deprivation in differentiated PC12 cells. Although taxol forced both wild-type and Bcl-2-overexpressing cells into a mitotic state, only in Bcl-2-overexpressing cells did this lead to the appearance of metabolically active, multi-nucleated cells. This suggests that Bcl-2 is able to induce an alternative escape pathway, downstream of the G2/M block, in taxol-treated differentiated PC12 cells.

All-trans-retinoic acid (RA) regulates epithelial differentiation and growth through activation of specific nuclear RA receptors (RARs). Because high-rate metabolism largely impairs the biological efficacy of RA, we have sought for compounds capable of inhibiting the metabolic breakdown of the retinoid. This study identifies R115866 as a novel inhibitor of the cytochrome P450 (CYP)-mediated metabolism of RA. In vitro, nanomolar concentrations of R115866 inhibited the conversion of RA by CYP26, a RA-inducible RA metabolizing enzyme. In vivo, oral administration of R115866 (2.5 mg/kg) to rats induced marked and transient increases of endogenous RA levels in plasma, skin, fat, kidney, and testis. Consistent with its ability to enhance endogenous RA content in tissues, R115866 was found to exert retinoidal activities. Like RA, the title compound: 1) inhibited vaginal keratinization in estrogen-stimulated rats; 2) induced epidermal hyperplasia in mouse ear skin; 3) transformed mouse tail epidermis from a para- to an orthokeratotic skin type; and 4) up-regulated the CYP26 mRNA expression in rat liver. Furthermore, we found that the keratinization-suppressive and CYP26-inducing activities of R115866 could be reversed by concomitant administration of the RAR antagonist, AGN193109. Our data characterize R115866 as a potent, orally active inhibitor of RA metabolism, capable of enhancing RA levels and displaying retinoidal actions. These activities are reversed by RAR antagonism, supporting the idea that the actions of R115866 result from increased availability of endogenous RA and improved RAR triggering.

The aim of this study was to modify the photochemical stroke model of Watson et al [23] so as to make possible microscopical investigation of the so-called penumbra, a tissue zone at risk that surrounds an infarction. The idea was to minimize photochemical challenge to endothelial membranes in such a way that thrombotic vascular obstruction is avoided but destabilization of the blood-brain barrier is still obtained. Morphological examination of the challenged area revealed open blood vessels, overt blood-brain barrier leakage over the entire area, severely swollen glial cells and structurally intact neurons. The lesion expanded over time due to progressive extravasation, formation of perivascular edema and consequent development of secondary ischemia through mechanical compression and microvascular congestion. In contrast to a photo-thrombotic infarct, in which the ischemic insult is more severe and blood vessels are completely congested by aggregated platelets, with this approach blood flow is partially preserved. In this way, an ischemic penumbra is created that mimics pathologic conditions secondary to stroke and trauma. The model may be useful in studying effects of drugs on pathologic phenomena that are characteristic of a penumbra, e.g. vasogenic and cellular edema, inflammation and infarction.

In blunt head trauma, primary brain injury takes the form of surface contusions and fractures, diffuse axonal injury and intracranial hemorrhage. Secondary damage may result from delayed pathological events including cerebral ischemia, brain edema and increased intracranial pressure (ICP). These events reinforce one another and may lead to a fatal disturbance of the balance between oxygen delivery and oxygen demand. Improvement or maintenance of this balance is a main issue in the treatment of severe head-injured patients. In this respect reduction of ICP is a key tool as it may improve brain perfusion and attenuate the pathologic cascade. Therapeutic interventions that adequately reduce ICP do not necessarily improve and may even impair tissue oxygenation (Unterberg et al., 1997a). This addresses the need for adequate monitoring of brain oxygenation during and after treatment.

Left ventricular biopsies from 21 patients with clinically diagnosed chronic hibernating myocardium (CHM) were examined by light- and electron microscopy. A mean of 27% of cardiomyocytes were structurally altered and were characterized as hibernating, because of reduced amount of myofibrils and increased glycogen content. Electron microscopy of these cells showed reduction of T-tubules and numerous small mitochondria, but few changes associated with degeneration, acute ischemia or apoptosis. The structural changes found in CHM are reminiscent of dedifferentiation rather than degeneration. The expression patterns of some structural proteins show resemblance with those in embryonic cardiomyocytes. Histochemically, mitochondrial NADH-oxidase and proton translocating ATPase activities were absent, whereas cytochrome c activity was present. Intracellular calcium distribution indicated normally bound sarcolemmal calcium and absence of excess mitochondrial calcium accumulation. Nuclear chromatin ranged from normal to dispersed with only a few nuclei that were clumped. These results suggest that cardiomyocytes from human CHM hearts are structurally altered, but viable, and lack morphologic and cytochemical characteristics suggestive of apoptosis or acute ischemia.

The value of 99mTc-sestamibi (2-methoxy-isobutyl isonitrile [MIBI]) as a viability tracer was investigated in patients undergoing coronary artery bypass graft surgery.Initial studies claim that rest MIBI single-photon emission computed tomographic (SPECT) studies can be used to assess myocardial viability.Thirty patients with a severely stenosed left anterior descending coronary artery and wall motion abnormalities were prospectively included. The patients underwent a MIBI rest study, a positron emission tomographic (PET) flow (13NH3) and metabolism (18F-deoxyglucose) study and nuclear angiography before undergoing bypass surgery. A preoperative transmural biopsy specimen was taken from the left ventricular anterior wall. Morphometry was performed to assess percent fibrosis. After 3 months, radionuclide angiography was repeated.Statistically significant higher MIBI values were found in the group with myocardial viability as assessed by PET than in the group with PET-assessed nonviability (p0.01). Significantly higher MIBI values were found in the group with enhanced contractility at 3 months (76 +/- 13% vs. 53 +/- 22%, p0.01). A linear relation was found between MIBI uptake and percent fibrosis in the biopsy specimen (r = 0.78, p0.00001). When maximizing the threshold for assessment of viability with MIBI by using functional improvement as the reference standard, a cutoff value of 50% was found, with positive and negative predictive values of 82% and 78%, respectively.99mTc MIBI uptake was significantly higher in PET-assessed viable areas and in regions with enhanced contractility at 3 months. A linear relation was found between percent fibrosis and MIBI uptake. An optimal threshold of 50% was found for prediction of functional recovery.

Intracranial pressure (ICP) was studied in a modified experimental model of closed head injury, in which the dynamic process of impact versus impulse loading was separately controlled. In this model, mortality of Wistar rats was considerably higher as compared to Sprague-Dawley rats subjected to similar traumatic conditions. Therefore Sprague-Dawley rats were used for all further experiments. Twenty-four rats, divided into 4 groups, underwent either sham or gradually increasing impact-acceleration trauma. Four hours after closed head injury, ICP measurements showed a significant correlation between the severity of the traumatic challenge and the resultant pressure rise (r2 = 0.731; p0.001). At the moment of impact there was a momentary blood pressure peak immediately followed by a transient period of hypotension. ICP measurements following directly to an impact-acceleration trauma, revealed an abrupt rise in ICP reaching pathological levels within 5 minutes. In conclusion, this modified model of closed head injury produces a predictable and reproducible pathologic ICP in Sprague-Dawley rats.

Intracranial pressure (ICP) was studied in a modified experimental model of closed head injury, in which the dynamic process of impact versus impulse loading was separately controlled. In this model, mortality of Wistar rats was considerably higher as compared to Sprague-Dawley rats subjected to similar traumatic conditions. Therefore Sprague-Dawley rats were used for all further experiments. Twenty-four rats, divided into 4 groups, underwent either sham or gradually increasing impact-acceleration trauma. Four hours after closed head injury, ICP measurements showed a significant correlation between the severity of the traumatic challenge and the resultant pressure rise (r2 = 0.731; p < 0.001). At the moment of impact there was a momentary blood pressure peak immediately followed by a transient period of hypotension. ICP measurements following directly to an impact-acceleration trauma, revealed an abrupt rise in ICP reaching pathological levels within 5 minutes. In conclusion, this modified model of closed head injury produces a predictable and reproducible pathologic ICP in Sprague-Dawley rats.

Cramer et al . (Reports, 23 March 2012, p. 1503; published online 9 February 2012) tested bexarotene as a potential β-amyloid–lowering drug for Alzheimer’s disease (AD). We were not able to reproduce the described effects in several animal models. Drug formulation appears very critical. Our data call for extreme caution when considering this compound for use in AD patients.