14-3-3- and II/3-10-gene expression as molecular markers to address viability and growth activity of Echinococcus multilocularis metacestodes

The present study aimed to search for and characterize parasite molecules, whose expression levels correlate with the viability and growth activity of Echinococcus multilocularis metacestodes. We focused on the expression profiles of 2 parasite-derived genes, 14-3-3 and II/3-10, as putative molecular markers for viability and growth activity of the larval parasite. In experiments in vivo, gene expression levels of 14-3-3 and II/3-10 were relatively quantified by real-time reverse transcription-PCR using a housekeeping gene, beta-actin, as a reference reaction. All three reactions were compared with growth activity of the parasite developing in permissive nu/nu and in non-permissive wild type BALB/c mice. At 2 months p.i., the transcription level of 14-3-3 was significantly higher in parasites actively proliferating in nu/nu mice compared to parasites moderately growing in wild type mice. Immunoblotting experiments confirmed at the protein level that 14-3-3 was over-expressed in parasites derived from nu/nu mice at 2 months p.i. In vitro treatment of E. multilocularis with an anti-echinococcal drug nitazoxanide resulted in a significant decrease of both 14-3-3 and II/3-10 transcription levels found after 8 days of treatment, which correlated with the kinetics of a housekeeping gene, beta-actin. The conclusion is that 14-3-3, combined with II/3-10, exhibits good potential as a molecular marker to assess viability and growth activity of the parasite.

Structure-activity relationships from in vitro efficacies of the thiazolide series against the intracellular apicomplexan protozoan Neospora caninum

Nitazoxanide (2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide; NTZ) represents the parent compound of a novel class of broad-spectrum anti-parasitic compounds named thiazolides. NTZ is active against a wide variety of intestinal and tissue-dwelling helminths, protozoa, enteric bacteria and a number of viruses infecting animals and humans. While potent, this poses a problem in practice, since this obvious non-selectivity can lead to undesired side effects in both humans and animals. In this study, we used real time PCR to determine the in vitro activities of 29 different thiazolides (NTZ-derivatives), which carry distinct modifications on both the thiazole- and the benzene moieties, against the tachyzoite stage of the intracellular protozoan Neospora caninum. The goal was to identify a highly active compound lacking the undesirable nitro group, which would have a more specific applicability, such as in food animals. By applying self-organizing molecular field analysis (SOMFA), these data were used to develop a predictive model for future drug design. SOMFA performs self-alignment of the molecules, and takes into account the steric and electrostatic properties, in order to determine 3D-quantitative structure activity relationship models. The best model was obtained by overlay of the thiazole moieties. Plotting of predicted versus experimentally determined activity produced an r2 value of 0.8052 and cross-validation using the "leave one out" methodology resulted in a q2 value of 0.7987. A master grid map showed that large steric groups at the R2 position, the nitrogen of the amide bond and position Y could greatly reduce activity, and the presence of large steric groups placed at positions X, R4 and surrounding the oxygen atom of the amide bond, may increase the activity of thiazolides against Neospora caninum tachyzoites. The model obtained here will be an important predictive tool for future development of this important class of drugs.

The multicenter trial SAKK 37/95 of cladribine, cyclophosphamide and prednisone in the treatment of chronic lymphocytic leukemias and low-grade non-Hodgkin's lymphomas

A multicenter trial was performed to confirm the therapeutic efficacy and the toxicity profile of the combination of cladribine, cyclophosphamide and prednisone in low-grade non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Twenty-three adults with previously treated (61%) or untreated (39%) NHL International Working Formulation A or Binet B and C CLL were administered cladribine 0.1 mg/kg/day as a subcutaneous bolus for 5 days, intravenous cyclophosphamide 500 mg/m2 on day 1, and oral prednisone 40 mg/m2 on days 1-5, every 4 weeks. Unexpected early hematological toxicities led to dose modifications for pretreated patients who received cladribine for 3 days only up to a maximum of five courses. Responses were observed in 75%, with 7 patients obtaining a complete clinical and hematological response. Median duration of complete response was 9 months. Median time to progression or relapse was 31 months. Myelosuppression and infections were dose limiting whereas posttreatment complications, including fatalities, resulted from infections. Median overall survival time from trial entry was 60 months. Activity of the combination of cladribine, cyclophosphamide and prednisone was confirmed. However, in the specific setting of a multicenter trial, unexpected fatal infectious episodes occurred in pretreated patients. Great caution is thus required in these susceptible patients and the routine use of corticosteroids should probably be abandoned.

Induction of haem oxygenase-1 causes cortical non-haem iron increase in experimental pneumococcal meningitis: evidence that concomitant ferritin up-regulation prevents iron-induced oxidative damage

Desferrioxamine inhibits cortical necrosis in neonatal rats with experimental pneumococcal meningitis, suggesting that iron-induced oxidative damage might be responsible for neuronal damage. We therefore examined the spatial and temporal profile of changes in cortical iron and iron homeostatic proteins during pneumococcal meningitis. Infection was associated with a steady and global increase of non-haem iron in the cortex, particularly in neuronal cell bodies of layer II and V, and in capillary endothelial cells. The non-haem iron increase was associated with induction of haem oxygenase (HO)-1 in neurones, microglia and capillary endothelial cells, whereas HO-2 levels remained unchanged, suggesting that the non-haem iron increase might be the result of HO-1-mediated haem degradation. Indeed, treatment with the haem oxygenase inhibitor tin protoporphyrin (which completely blocked the accumulation of bilirubin detected in HO-1-positive cells) completely prevented the infection-associated non-haem iron increase. The same cells also displayed markedly increased ferritin staining, the increase of which occurred independently of HO activity. At the same time, no increase in DNA/RNA oxidation was observed in infected animals (as assessed by in situ detection of 8-hydroxy[deoxy]guanosine), strongly suggesting that ferritin up-regulation protected the brain from iron-induced oxidative damage. Thus, although pneumococcal meningitis leads to an increase of cortical non-haem iron, protective mechanisms up-regulated in parallel prevent iron-induced oxidative damage. Cortical damage does not appear to be a direct consequence of increased iron, therefore.

Evidence for low-grade systemic proinflammatory activity in patients with posttraumatic stress disorder

Posttraumatic stress disorder (PTSD) may increase cardiovascular risk but the psychophysiological mechanisms involved are elusive. We hypothesized that proinflammatory activity is elevated in patients with PTSD as diagnosed by the Clinician Administered PTSD Scale (CAPS) interview. Plasma levels of proinflammatory C-reactive protein (CRP), interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha, and of anti-inflammatory IL-4 and IL-10 were measured in 14 otherwise healthy PTSD patients and in 14 age- and gender-matched healthy non-PTSD controls. Levels of TNF-alpha (p=0.038; effect size Cohen's d=0.58) and of IL-1beta (p=0.075, d=0.68) were higher in patients than in controls. CRP (d=0.10), IL-6 (d=0.18), IL-4 (d=0.42), and IL-10 (d=0.37) were not significantly different between groups. Controlling for traditional cardiovascular risk factors, mood, and time since trauma revealed lower IL-4 in patients than in controls (p=0.029) and rendered group differences in TNF-alpha and IL-1beta insignificant. In all subjects, TNF-alpha correlated with total (frequency and intensity) PTSD symptom cluster of re-experiencing (r=0.49, p=0.008), avoidance (r=0.37, p=0.050), and hyperarousal (r=0.42, p=0.026), and with PTSD total symptom score (r=0.37, p=0.054). Controlling for time since trauma attenuated these associations. The correlation between IL-1beta and total avoidance symptoms (r=0.42, p=0.028) became insignificant when controlling for anxiety and depression. IL-4 correlated with total hyperarousal symptoms (r=-0.38, p=0.047), and after controlling for systolic blood pressure and smoking status, with PTSD total symptom score (r=-0.41, p=0.035). PTSD patients showed a low-grade systemic proinflammatory state, which, moreover, was related to PTSD symptom levels suggesting one mechanism by which PTSD could contribute to atherosclerotic disease.

The efficacy of non-pharmacological interventions in the management of procedural pain in preterm and term neonates. A systematic literature review

BACKGROUND: Neonates in a neonatal intensive care unit are exposed to a high number of painful procedures. Since repeated and sustained pain can have consequences for the neurological and behaviour-oriented development of the newborn, the greatest attention needs to be paid to systematic pain management in neonatology. Non-pharmacological treatment methods are being increasingly discussed with regard to pain prevention and relief either alone or in combination with pharmacological treatment. AIMS: To identify effective non-pharmacological interventions with regard to procedural pain in neonates. METHODS: A literature search was conducted via the MedLine, CINAHL, Cochrane Library databases and complemented by a handsearch. The literature search covered the period from 1984 to 2004. Data were extracted according to pre-defined criteria by two independent reviewers and methodological quality was assessed. RESULTS: 13 randomised controlled studies and two meta-analyses were taken into consideration with regard to the question of current nursing practice of non-pharmacological pain management methods. The selected interventions were "non-nutritive sucking", "music", "swaddling", "positioning", "olfactory and multisensorial stimulation", "kangaroo care" and "maternal touch". There is evidence that the methods of "non-nutritive sucking", "swaddling" and "facilitated tucking" do have a pain-alleviating effect on neonates. CONCLUSIONS: Some of the non-pharmacological interventions have an evident favourable effect on pulse rate, respiration and oxygen saturation, on the reduction of motor activity, and on the excitation states after invasive measures. However, unambiguous evidence of this still remains to be presented. Further research should emphasise the use of validated pain assessment instruments for the evaluation of the pain-alleviating effect of non-pharmacological interventions.

Application and comparison of classification algorithms for recognition of Alzheimer's disease in electrical brain activity (EEG)

The early detection of subjects with probable Alzheimer's disease (AD) is crucial for effective appliance of treatment strategies. Here we explored the ability of a multitude of linear and non-linear classification algorithms to discriminate between the electroencephalograms (EEGs) of patients with varying degree of AD and their age-matched control subjects. Absolute and relative spectral power, distribution of spectral power, and measures of spatial synchronization were calculated from recordings of resting eyes-closed continuous EEGs of 45 healthy controls, 116 patients with mild AD and 81 patients with moderate AD, recruited in two different centers (Stockholm, New York). The applied classification algorithms were: principal component linear discriminant analysis (PC LDA), partial least squares LDA (PLS LDA), principal component logistic regression (PC LR), partial least squares logistic regression (PLS LR), bagging, random forest, support vector machines (SVM) and feed-forward neural network. Based on 10-fold cross-validation runs it could be demonstrated that even tough modern computer-intensive classification algorithms such as random forests, SVM and neural networks show a slight superiority, more classical classification algorithms performed nearly equally well. Using random forests classification a considerable sensitivity of up to 85% and a specificity of 78%, respectively for the test of even only mild AD patients has been reached, whereas for the comparison of moderate AD vs. controls, using SVM and neural networks, values of 89% and 88% for sensitivity and specificity were achieved. Such a remarkable performance proves the value of these classification algorithms for clinical diagnostics.

Prognostic factors affecting long-term outcomes in patients with resected stage IIIA pN2 non-small-cell lung cancer: 5-year follow-up of a phase II study

The aim was to investigate the efficacy of neoadjuvant docetaxel-cisplatin and identify prognostic factors for outcome in locally advanced stage IIIA (pN2 by mediastinoscopy) non-small-cell lung cancer (NSCLC) patients. In all, 75 patients (from 90 enrolled) underwent tumour resection after three 3-week cycles of docetaxel 85 mg m-2 (day 1) plus cisplatin 40 or 50 mg m-2 (days 1 and 2). Therapy was well tolerated (overall grade 3 toxicity occurred in 48% patients; no grade 4 nonhaematological toxicity was reported), with no observed late toxicities. Median overall survival (OS) and event-free survival (EFS) times were 35 and 15 months, respectively, in the 75 patients who underwent surgery; corresponding figures for all 90 patients enrolled were 28 and 12 months. At 3 years after initiating trial therapy, 27 out of 75 patients (36%) were alive and tumour free. At 5-year follow-up, 60 and 65% of patients had local relapse and distant metastases, respectively. The most common sites of distant metastases were the lung (24%) and brain (17%). Factors associated with OS, EFS and risk of local relapse and distant metastases were complete tumour resection and chemotherapy activity (clinical response, pathologic response, mediastinal downstaging). Neoadjuvant docetaxel-cisplatin was effective and tolerable in stage IIIA pN2 NSCLC, with chemotherapy contributing significantly to outcomes.

Multicenter phase II trial of gefitinib first-line therapy followed by chemotherapy in advanced non-small-cell lung cancer (NSCLC): SAKK protocol 19/03

BACKGROUND: Gefitinib is active in patients with pretreated non-small-cell lung cancer (NSCLC). We evaluated the activity and toxicity of gefitinib first-line treatment in advanced NSCLC followed by chemotherapy at disease progression. PATIENTS AND METHODS: In all, 63 patients with chemotherapy-naive stage IIIB/IV NSCLC received gefitinib 250 mg/day. At disease progression, gefitinib was replaced by cisplatin 80 mg/m(2) on day 1 and gemcitabine 1250 mg/m(2) on days 1, 8 for up to six 3-week cycles. Primary end point was the disease stabilization rate (DSR) after 12 weeks of gefitinib. RESULTS: After 12 weeks of gefitinib, the DSR was 24% and the response rate (RR) was 8%. Median time to progression (TtP) was 2.5 months and median overall survival (OS) 11.5 months. Never smokers (n = 9) had a DSR of 56% and a median OS of 20.2 months; patients with epidermal growth factor receptor (EGFR) mutation (n = 4) had a DSR of 75% and the median OS was not reached after the follow-up of 21.6 months. In all, 41 patients received chemotherapy with an overall RR of 34%, DSR of 71% and median TtP of 6.7 months. CONCLUSIONS: First-line gefitinib monotherapy led to a DSR of 24% at 12 weeks in an unselected patients population. Never smokers and patients with EGFR mutations tend to have a better outcome; hence, further trials in selected patients are warranted.

Activation of non-ischemic, hypoxia-inducible signalling pathways up-regulate cytoprotective genes in the murine liver

BACKGROUND/AIMS: We investigated the molecular response of a non-ischemic hypoxic stress in the liver, in particular, to distinguish its hepatoprotective potential. METHODS: The livers of mice were subjected to non-ischemic hypoxia by clamping the hepatic-artery (HA) for 2h while maintaining portal circulation. Hypoxia was defined by a decrease in oxygen saturation, the activation of hypoxia-inducible factor (HIF)-1 and the mRNA up-regulation of responsive genes. To demonstrate that the molecular response to hypoxia may in part be hepatoprotective, pre-conditioned animals were injected with an antibody against Fas (Jo2) to induce acute liver failure. Hepatocyte apoptosis was monitored by caspase-3 activity, cleavage of lamin A and animal survival. RESULTS: Clamping the HA induced a hypoxic stress in the liver in the absence of severe metabolic distress or tissue damage. The hypoxic stimulus was sufficient to activate the HIF-1 signalling pathway and up-regulate hepatoprotective genes. Pre-conditioning the liver with hypoxia was able to delay the onset of Fas-mediated apoptosis and prolong animal survival. CONCLUSIONS: Our data reveal that hepatic cells can sense and respond to a decrease in tissue oxygenation, and furthermore, that activation of hypoxia-inducible signalling pathways function in part to promote liver cell survival.

Induction of EROD activity by 1-phenylimidazole and beta-naphthoflavone in rainbow trout cultured hepatocytes: a comparative study

The classical pathway for induction of cytochrome P4501A (CYP1A) by xenobiotics is ligand binding to the aryl hydrocarbon receptor (AhR). However, several studies with mammalian cell systems point out a range of xenobiotics including imidazole derivatives, which are able to activate CYP1A through non-classical mechanisms. The objective of the present work is to compare induction of CYP1A (determined at the catalytic level as 7-ethoxyresorufin-O-deethylase, EROD) in rainbow trout (Oncorhynchus mykiss) hepatocytes by the prototypic AhR ligand, beta-naphthoflavone (betaNF), and by the imidazole derivative, 1-phenylimidazole (PIM). PIM was able to induce EROD activity although its potency was clearly lower than that of betaNF. In order to assess the relative importance of classical AhR ligand binding and alternative signaling pathways in CYP1A induction by PIM, co-exposure experiments with the partial AhR antagonist alpha-naphthoflavone (alphaNF) or with inhibitors of protein kinase C (staurosporine) and tyrosine kinases (genistein, herbimicine) were performed. alphaNF and herbimicin provoked a decrease of EROD induction both by betaNF and PIM, whereas staurosporine and genistein remained without effect. The overall similarities in the response of betaNF and PIM to the various inhibitors suggest that both compounds, in apparent contrast to the behaviour of some other imidazole derivatives, induce CYP1A following similar mechanisms.

Tissue-specific induction of EROD activity and CYP1A protein in Sparus aurata exposed to B(a)P and TCDD

The purpose of this study was to compare xenobiotic CYP1A induction in liver, gills, and excretory kidney of gilthead seabream, Sparus aurata. Fishes were exposed via water for 20 days to different concentrations of benzo(a)pyrene (B(a)P) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). CYP1A was measured at the enzyme activity level as 7-ethoxyresorufin-O-deethylase (EROD) activity, and at the protein level by means of ELISA. The liver displayed the highest absolute levels of EROD activity, both under non-exposed and exposed conditions. Organ- or inducer-related differences in the time course of CYP1A induction were moderate; however, the magnitude of the induction response varied between the organs and between B(a)P and TCDD. In the case of TCDD, liver, and kidney yielded a comparable induction response, whereas in the case of B(a)P, the kidney showed a substantially higher maximum induction factor than the liver. In the gills, the two xenobiotics resulted in similar maximum induction factors. In B(a)P-exposed seabream, EROD activities and CYP1A protein levels showed a good correlation in all three organs, whereas with TCDD as inducer the correlation was poor, what was mainly due to a decrease of EROD activities at the higher concentrations of TCDD, while CYP1A protein levels showed no concomitant decline. Overall, the study revealed both similarities and differences in the time-, concentration-, and inducer-dependent CYP1A responses of the three target organs, liver, kidney, and gills. Although, the findings of this study principally confirm the notion of the liver as the major metabolic organ in fish, they also provide evidence for substantial metabolic potential in gills and particularly in the kidney.

Histopathological alterations, EROD activity, CYP1A protein and biliary metabolites in gilthead seabream Sparus aurata exposed to benzo(a)pyrene

This study compared for seabream, Sparus aurata exposed to benzo(a)pyrene-B(a)P-, the response of molecular cytochrome P450 1A (CYP1A) and cellular histopathology biomarkers. Male gilthead seabream, Sparus aurata specimens were exposed for 20 days via water to a series of high B(a)P concentrations. CYP1A was assessed by measuring enzymatic activity (EROD) and CYP1A protein content, and cellular responses were evaluated by routine histopathological methods. In addition, biliary metabolites were measured in order to verify that B(a)P was absorbed and metabolised. Histological lesions, both in liver and gills, increased in parallel to B(a)P concentrations, with the majority of changes representing rather non-specific alterations. Hepatic EROD and CYP1A proteins data showed a concentration-dependent induction, while in the gills, EROD activity but not CYP1A proteins showed a non-monotonous dose response, with a maximum induction level at 200 microg B(a)P.L-1 and decreasing levels thereafter. The findings provide evidence that short-term, high dose exposure of fish can result in significant uptake and metabolism of the lipophilic B(a)P, and in pronounced pathological damage of absorptive epithelia and internal organs.

Increased expression of the auxiliary beta2-subunit of ventricular L-type Ca2+ channels leads to single-channel activity characteristic of heart failure

BACKGROUND: Increased activity of single ventricular L-type Ca(2+)-channels (L-VDCC) is a hallmark in human heart failure. Recent findings suggest differential modulation by several auxiliary beta-subunits as a possible explanation. METHODS AND RESULTS: By molecular and functional analyses of human and murine ventricles, we find that enhanced L-VDCC activity is accompanied by altered expression pattern of auxiliary L-VDCC beta-subunit gene products. In HEK293-cells we show differential modulation of single L-VDCC activity by coexpression of several human cardiac beta-subunits: Unlike beta(1) or beta(3) isoforms, beta(2a) and beta(2b) induce a high-activity channel behavior typical of failing myocytes. In accordance, beta(2)-subunit mRNA and protein are up-regulated in failing human myocardium. In a model of heart failure we find that mice overexpressing the human cardiac Ca(V)1.2 also reveal increased single-channel activity and sarcolemmal beta(2) expression when entering into the maladaptive stage of heart failure. Interestingly, these animals, when still young and non-failing ("Adaptive Phase"), reveal the opposite phenotype, viz: reduced single-channel activity accompanied by lowered beta(2) expression. Additional evidence for the cause-effect relationship between beta(2)-subunit expression and single L-VDCC activity is provided by newly engineered, double-transgenic mice bearing both constitutive Ca(V)1.2 and inducible beta(2) cardiac overexpression. Here in non-failing hearts induction of beta(2)-subunit overexpression mimicked the increase of single L-VDCC activity observed in murine and human chronic heart failure. CONCLUSIONS: Our study presents evidence of the pathobiochemical relevance of beta(2)-subunits for the electrophysiological phenotype of cardiac L-VDCC and thus provides an explanation for the single L-VDCC gating observed in human and murine heart failure.

EEG reactivity and EEG activity in never-treated acute schizophrenics, measured with spectral parameters and dimensional complexity

Our approaches to the use of EEG studies for the understanding of the pathogenesis of schizophrenic symptoms are presented. The basic assumptions of a heuristic and multifactorial model of the psychobiological brain mechanisms underlying the organization of normal behavior is described and used in order to formulate and test hypotheses about the pathogenesis of schizophrenic behavior using EEG measures. Results from our studies on EEG activity and EEG reactivity (= EEG components of a memory-driven, adaptive, non-unitary orienting response) as analyzed with spectral parameters and "chaotic" dimensionality (correlation dimension) are summarized. Both analysis procedures showed a deviant brain functional organization in never-treated first-episode schizophrenia which, within the framework of the model, suggests as common denominator for the pathogenesis of the symptoms a deviation of working memory, the nature of which is functional and not structural.