Application of an in vitro drug screening assay based on the release of phosphoglucose isomerase to determine the structure-activity relationship of thiazolides against Echinococcus multilocularis metacestodes

OBJECTIVES: The disease alveolar echinococcosis (AE), caused by the larval stage of the cestode Echinococcus multilocularis, is fatal if treatment is unsuccessful. Current treatment options are, at best, parasitostatic, and involve taking benzimidazoles (albendazole, mebendazole) for the whole of a patient's life. In conjunction with the recent development of optimized procedures for E. multilocularis metacestode cultivation, we aimed to develop a rapid and reliable drug screening test, which enables efficient screening of a large number of compounds in a relatively short time frame. METHODS: Metacestodes were treated in vitro with albendazole, the nitro-thiazole nitazoxanide and 29 nitazoxanide derivatives. The resulting leakage of phosphoglucose isomerase (PGI) activity into the medium supernatant was measured and provided an indication of compound efficacy. RESULTS: We show that upon in vitro culture of E. multilocularis metacestodes in the presence of active drugs such as albendazole, the nitro-thiazole nitazoxanide and 30 different nitazoxanide derivatives, the activity of PGI in culture supernatants increased. The increase in PGI activity correlated with the progressive degeneration and destruction of metacestode tissue in a time- and concentration-dependent manner, which allowed us to perform a structure-activity relationship analysis on the thiazolide compounds used in this study. CONCLUSIONS: The assay presented here is inexpensive, rapid, can be used in 24- and 96-well formats and will serve as an ideal tool for first-round in vitro tests on the efficacy of large numbers of antiparasitic compounds.

SiRNA inhibits replication of Langat virus, a member of the tick-borne encephalitis virus complex in organotypic rat brain slices

Tick-borne encephalitis virus is the causative agent of tick-borne encephalitis, a potentially fatal neurological infection. Tick-borne encephalitis virus belongs to the family of flaviviruses and is transmitted by infected ticks. Despite the availability of vaccines, approximately 2000-3000 cases of tick-borne encephalitis occur annually in Europe for which no curative therapy is available. The antiviral effects of RNA mediated interference by small interfering RNA (siRNA) was evaluated in cell culture and organotypic hippocampal cultures. Langat virus, a flavivirus highly related to Tick-borne encephalitis virus exhibits low pathogenicity for humans but retains neurovirulence for rodents. Langat virus was used for the establishment of an in vitro model of tick-borne encephalitis. We analyzed the efficacy of 19 siRNA sequences targeting different regions of the Langat genome to inhibit virus replication in the two in vitro systems. The most efficient suppression of virus replication was achieved by siRNA sequences targeting structural genes and the 3' untranslated region. When siRNA was administered to HeLa cells before the infection with Langat virus, a 96.5% reduction of viral RNA and more than 98% reduction of infectious virus particles was observed on day 6 post infection, while treatment after infection decreased the viral replication by more than 98%. In organotypic hippocampal cultures the replication of Langat virus was reduced by 99.7% by siRNA sequence D3. Organotypic hippocampal cultures represent a suitable in vitro model to investigate neuronal infection mechanisms and treatment strategies in a preserved three-dimensional tissue architecture. Our results demonstrate that siRNA is an efficient approach to limit Langat virus replication in vitro.

Ruminant organotypic brain-slice cultures as a model for the investigation of CNS listeriosis

Central nervous system (CNS) infections in ruminant livestock, such as listeriosis, are of major concern for veterinary and public health. To date, no host-specific in vitro models for ruminant CNS infections are available. Here, we established and evaluated the suitability of organotypic brain-slices of ruminant origin as in vitro model to study mechanisms of Listeria monocytogenes CNS infection. Ruminants are frequently affected by fatal listeric rhombencephalitis that closely resembles the same condition occurring in humans. Better insight into host-pathogen interactions in ruminants is therefore of interest, not only from a veterinary but also from a public health perspective. Brains were obtained at the slaughterhouse, and hippocampal and cerebellar brain-slices were cultured up to 49 days. Viability as well as the composition of cell populations was assessed weekly. Viable neurons, astrocytes, microglia and oligodendrocytes were observed up to 49 days in vitro. Slice cultures were infected with L. monocytogenes, and infection kinetics were monitored. Infected brain cells were identified by double immunofluorescence, and results were compared to natural cases of listeric rhombencephalitis. Similar to the natural infection, infected brain-slices showed focal replication of L. monocytogenes and bacteria were predominantly observed in microglia, but also in astrocytes, and associated with axons. These results demonstrate that organotypic brain-slice cultures of bovine origin survive for extended periods and can be infected easily with L. monocytogenes. Therefore, they are a suitable model to study aspects of host-pathogen interaction in listeric encephalitis and potentially in other neuroinfectious diseases.

[Hypertension in the elderly]

Many aspects of hypertension diagnosis and treatment are similar in young and old patients. However, some differences exist. Due to the increasing vascular stiffness most elderly patients have isolated systolic hypertension and its prevalence in the population is high. Blood pressure should be measured in the sitting position and also with the patient standing to exclude orthostatic hypotension, a frequent problem in elderly patients. Pseudohypertension, a source of inadequate measurements in elderly patients, should be recognized. In comparison to other health problems there is good scientific evidence for antihypertensive treatment in elderly patients. As treatment does not only improve survival, but also reduces cardiovascular events such as non-fatal stroke or myocardial infarction, antihypertensive therapy is an important measure to prevent functional decline and disability.

Registry for the Evaluation of the Prognostik value of a novel integrated imaging approach combining Single Photon Emission Computed Tomography with coronary calcification imaging (REPROSPECT)

AIMS: Although an added diagnostic and prognostic value of the global coronary artery calcification (CAC) score as an adjunct to single-photon emission computed tomography (SPECT)-myocardial perfusion image (MPI) has been repeatedly documented, none of the previous studies took advantage of the anatomic information provided by the unenhanced cardiac CT. Therefore, no co-registration has so far been used to match a myocardial perfusion defect with calcifications in the subtending coronary artery. To evaluate the prognostic value of integrating SPECT-MPI with CAC images were obtained from non-enhanced cardiac computed tomography (CT) for attenuation correction to predict major adverse cardiac events (MACE). METHODS AND RESULTS: Follow-up was obtained in 462 patients undergoing a 1-day stress/rest (99m)Tc-teterofosmin SPECT and non-enhanced cardiac CT for attenuation correction. Survival free of MACE was determined using the Kaplan-Meier method. After integrating MPI and CT findings, patients were divided into three groups (i) MPI defect matched by calcification (CAC ≥ 1) in the subtending coronary artery (ii) unmatched MPI and CT finding (iii) normal finding by MPI and CT. At a mean follow-up of 34.5 ± 13 months, a MACE was observed in 80 patients (33 death, 6 non-fatal myocardial infarction, 9 hospitalizations due to unstable angina, and 32 revascularizations). Survival analysis revealed the most unfavourable outcome (P < 0.001 log-rank test) for patients with a matched finding. CONCLUSION: In the present study, a novel approach using a combined integration of cardiac SPECT-CAC imaging allows for refined risk stratification, as a matched defect emerged as an independent predictor of MACE.

Equine botulism and acute pasture myodystrophy: new soil-borne emerging diseases in Switzerland?

In Switzerland, the incidence of equine botulism and acute pasture myodystrophy have remarkably increased in the last five years. Equine fodder-borne botulism in Europe is most likely caused by Clostridium botulinum types C and D that produce the toxins BoNT/C and BoNT/D. Horses showing signs suggestive of botulism (muscle weakness and tremors, reduced tongue tone, slow chewing, salivation and difficulties swallowing, drooping eyelids, mydriasis), especially patients that have fed on suspect fodder (mostly haylage), must be treated with anti-serum as soon as possible.They also need intensive care, which is often difficult to provide and always expensive in the face of a guarded to poor prognosis. Therefore, prevention (high standards of forage quality and vaccination) is all the more important. Pasture myodystrophy is an acute disease with signs of rhabdomyolysis and lethality rate over 90%. It affects grazing horses under frosty, windy and rainy conditions. Preliminary results indicate that Clostridium sordellii and Clostridium bifermentans producing lethal toxin may play a role in pasture myodystrophy. Our efforts concentrate on developing a new subunit vaccine for equine botulism and understanding the ethiology and pathogenesis of pasture myodystrophy with the goal of improving prevention against these highly fatal diseases that present a significant risk to our horse population.

Dysphagia in elderly women: consider tetanus

BACKGROUND: Dysphagia is seldom caused by tetanus; however, it is a common symptom of tetanus. Treating patients with tetanus is a rare event in industrialized countries and awareness is needed to recognize early signs of this serious disease. In Switzerland, the most recently reported tetanus cases occurred in elderly women with insufficient seroprotection. PATIENTS: We report on three elderly women presenting with dysphagia as an initial symptom of tetanus. RESULTS: Generalized tetanus was diagnosed in two patients upon admission, the third presented with cephalic tetanus with secondary generalization. All three patients had undetectable levels of tetanus antibodies and had no documented prior tetanus immunizations. Cultures of wound swabs grew Clostridium tetani in all cases. Electromyography was highly suggestive for tetanus in two patients. Treatment involved mechanical ventilation, intravenous benzodiazepine and metronidazole therapy, and active and passive tetanus immunization. The disease had a favorable outcome in two cases and was fatal in one. CONCLUSION: Tetanus remains a threat in patients with insufficient seroprotection and efforts are needed to improve tetanus immunization in these individuals. Tetanus should be considered in the differential diagnosis of dysphagia.

Alteration of the pulmonary surfactant system in full-term infants with hereditary ABCA3 deficiency

RATIONALE: ABCA3 mutations are known to cause fatal surfactant deficiency. OBJECTIVE: We studied ABCA3 protein expression in full-term newborns with unexplained respiratory distress syndrome (URDS) as well as the relevance of ABCA3 mutations for surfactant homeostasis. METHODS: Lung tissue of infants with URDS was analyzed for the expression of ABCA3 in type II pneumocytes. Coding exons of the ABCA3 gene were sequenced. Surfactant protein expression was studied by immunohistochemistry, immunoelectron microscopy, and Western blotting. RESULTS: ABCA3 protein expression was found to be greatly reduced or absent in 10 of 14 infants with URDS. Direct sequencing revealed distinct ABCA3 mutations clustering within vulnerable domains of the ABCA3 protein. A strong expression of precursors of surfactant protein B (pro-SP-B) but only low levels and aggregates of mature surfactant protein B (SP-B) within electron-dense bodies in type II pneumocytes were found. Within the matrix of electron-dense bodies, we detected precursors of SP-C (pro-SP-C) and cathepsin D. SP-A was localized in small intracellular vesicles, but not in electron-dense bodies. SP-A and pro-SP-B were shown to accumulate in the intraalveolar space, whereas mature SP-B and SP-C were reduced or absent, respectively. CONCLUSION: Our data provide evidence that ABCA3 mutations are associated not only with a deficiency of ABCA3 but also with an abnormal processing and routing of SP-B and SP-C, leading to severe alterations of surfactant homeostasis and respiratory distress syndrome. To identify infants with hereditary ABCA3 deficiency, we suggest a combined diagnostic approach including immunohistochemical, ultrastructural, and mutation analysis.

Multicycle dose-intensive chemotherapy for women with high-risk primary breast cancer: results of International Breast Cancer Study Group Trial 15-95

PURPOSE: To compare adjuvant dose-intensive epirubicin and cyclophosphamide chemotherapy administered with filgrastim and progenitor cell support (DI-EC) with standard-dose anthracycline-based chemotherapy (SD-CT) for patients with early-stage breast cancer and a high risk of relapse, defined as stage II disease with 10 or more positive axillary nodes; or an estrogen receptor-negative or stage III tumor with five or more positive axillary nodes. PATIENTS AND METHODS: Three hundred forty-four patients were randomized after surgery to receive seven cycles of SD-CT over 22 weeks, or three cycles of DI-EC (epirubicin 200 mg/m2 plus cyclophosphamide 4 gm/m2 with filgrastim and progenitor cell support) over 6 weeks. All patients were assigned tamoxifen at the completion of chemotherapy. The primary end point was disease-free survival (DFS). RESULTS: After a median follow-up of 5.8 years (range, 3 to 8.4 years), 188 DFS events had occurred (DI-EC, 86 events; SD-CT, 102 events). The 5-year DFS was 52% for DI-EC and 43% for SD-CT, with hazard ratio of DI-EC compared with SD-CT of 0.77 (95% CI, 0.58 to 1.02; P = .07). The 5-year overall survival was 70% for DI-EC and 61% for SD-CT, with a hazard ratio of 0.79 (95% CI, 0.56 to 1.11; P = .17). There were eight cases (5%) of anthracycline-induced cardiomyopathy (two fatal) among those who received DI-EC. Women with hormone receptor-positive tumors benefited significantly from DI-EC. CONCLUSION: There was a trend in favor of DI-EC with respect to disease-free survival. A larger trial or meta-analysis will be required to reveal the true effect of dose-intensive therapy.

Mutations of the myeloid transcription factor CEBPA are not associated with the blast crisis of chronic myeloid leukaemia

The transcription factor CEBPA is crucial for normal myeloid differentiation. CEBPA gene mutations have been reported in patients with acute myeloid leukaemia. The inevitable evolution of chronic myeloid leukaemia (CML) in chronic phase (CP) to a fatal blast crisis (BC) is assumed to result from the acquisition of additional genetic changes in the leukaemic clone. Gain of CEBPA mutations might represent a key event causing the differentiation block observed in myeloid CML-BC, but not in CML-CP. Here, no CEBPA mutation in 95 CML-BC patients was found, suggesting a limited role, if any, of CEBPA mutations in this disorder.

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.

Sleep-wake disturbances in sporadic Creutzfeldt-Jakob disease

BACKGROUND: The prevalence and characteristics of sleep-wake disturbances in sporadic Creutzfeldt-Jakob disease (sCJD) are poorly understood. METHODS: Seven consecutive patients with definite sCJD underwent a systematic assessment of sleep-wake disturbances, including clinical history, video-polysomnography, and actigraphy. Extent and distribution of neurodegeneration was estimated by brain autopsy in six patients. Western blot analyses enabling classification and quantification of the protease-resistant isoform of the prion protein, PrPSc, in thalamus and occipital cortex was available in four patients. RESULTS: Sleep-wake symptoms were observed in all patients, and were prominent in four of them. All patients had severe sleep EEG abnormalities with loss of sleep spindles, very low sleep efficiency, and virtual absence of REM sleep. The correlation between different methods to assess sleep-wake functions (history, polysomnography, actigraphy, videography) was generally poor. Brain autopsy revealed prominent changes in cortical areas, but only mild changes in the thalamus. No mutation of the PRNP gene was found. CONCLUSIONS: This study demonstrates in sporadic Creutzfeldt-Jakob disease, first, the existence of sleep-wake disturbances similar to those reported in fatal familial insomnia in the absence of prominent and isolated thalamic neuronal loss, and second, the need of a multimodal approach for the unambiguous assessment of sleep-wake functions in these patients.

Treatment of thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP), characterized by thrombocytopenia and microangiopathic haemolytic anaemia, was almost universally fatal until the introduction of plasma exchange (PE) therapy in the 1970s. Based on clinical studies, daily PE has become the first-choice therapy since 1991. Recent findings may explain its effectiveness, which may include, in particular, the removal of anti-ADAMTS13 autoantibodies and unusually large von Willebrand factor multimers and/or supply of ADAMTS13 in acquired idiopathic or congenital TTP. Based on currently available data, the favoured PE regimen is daily PE [involving replacement of 1-1.5 times the patient's plasma volume with fresh-frozen plasma (FFP)] until remission. Adverse events of treatment are mainly related to central venous catheters. The potential reduction of plasma related side-effects, such as transfusion-related acute lung injury (TRALI) or febrile transfusion reactions by use of solvent-detergent treated (S/D) plasma instead of FFP is not established by controlled clinical studies. Uncontrolled clinical observations and the hypothesis of an autoimmune process in a significant part of the patients with acquired idiopathic TTP suggest a beneficial effect of adjunctive therapy with corticosteroids. Other immunosuppressive treatments are not tested in controlled trials and should be reserved for refractory or relapsing disease. There is no convincing evidence for the use of antiplatelet agents. Supportive treatment with transfusion of red blood cells or platelets has to be evaluated on a clinical basis, but the transfusion trigger for platelets should be very restrictive. Further controlled, prospective studies should consider the different pathophysiological features of thrombotic microangiopathies, address the prognostic significance of ADAMTS13 and explore alternative exchange fluids to FFP, the role of immunosuppressive therapies and of new plasma saving approaches as recombinant ADAMTS13 and protein A immunoadsorption.

[In Medea's gardens--two tales of colchicine]

Colchicine is a highly active alkaloid used in the treatment of acute inflammatory syndromes such as Mediterranean fever, M. Behçet or gouty arthritis. The two cases we present here illustrate exemplarily the pros and contras of colchicine therapy. In the first case, colchicine was successfully given for recurrent febrile attacks due to acute rheumatic fever. The second patient unfortunately had a fatal colchicine intoxication. The pharmacology of colchicine, the clinical features associated with overdose and the options for treatment are discussed. Colchicine should not be given in combination with macrolides, especially in patients with renal insufficiency.

Cyclic nucleotide specific phosphodiesterases of Leishmania major

Background Leishmania represent a complex of important human pathogens that belong to the systematic order of the kinetoplastida. They are transmitted between their human and mammalian hosts by different bloodsucking sandfly vectors. In their hosts, the Leishmania undergo several differentiation steps, and their coordination and optimization crucially depend on numerous interactions between the parasites and the physiological environment presented by the fly and human hosts. Little is still known about the signalling networks involved in these functions. In an attempt to better understand the role of cyclic nucleotide signalling in Leishmania differentiation and host-parasite interaction, we here present an initial study on the cyclic nucleotide-specific phosphodiesterases of Leishmania major. Results This paper presents the identification of three class I cyclic-nucleotide-specific phosphodiesterases (PDEs) from L. major, PDEs whose catalytic domains exhibit considerable sequence conservation with, among other, all eleven human PDE families. In contrast to other protozoa such as Dictyostelium, or fungi such as Saccharomyces cerevisiae, Candida ssp or Neurospora, no genes for class II PDEs were found in the Leishmania genomes. LmjPDEA contains a class I catalytic domain at the C-terminus of the polypeptide, with no other discernible functional domains elsewhere. LmjPDEB1 and LmjPDEB2 are coded for by closely related, tandemly linked genes on chromosome 15. Both PDEs contain two GAF domains in their N-terminal region, and their almost identical catalytic domains are located at the C-terminus of the polypeptide. LmjPDEA, LmjPDEB1 and LmjPDEB2 were further characterized by functional complementation in a PDE-deficient S. cerevisiae strain. All three enzymes conferred complementation, demonstrating that all three can hydrolyze cAMP. Recombinant LmjPDEB1 and LmjPDEB2 were shown to be cAMP-specific, with Km values in the low micromolar range. Several PDE inhibitors were found to be active against these PDEs in vitro, and to inhibit cell proliferation. Conclusion The genome of L. major contains only PDE genes that are predicted to code for class I PDEs, and none for class II PDEs. This is more similar to what is found in higher eukaryotes than it is to the situation in Dictyostelium or the fungi that concomitantly express class I and class II PDEs. Functional complementation demonstrated that LmjPDEA, LmjPDEB1 and LmjPDEB2 are capable of hydrolyzing cAMP. In vitro studies with recombinant LmjPDEB1 and LmjPDEB2 confirmed this, and they demonstrated that both are completely cAMP-specific. Both enzymes are inhibited by several commercially available PDE inhibitors. The observation that these inhibitors also interfere with cell growth in culture indicates that inhibition of the PDEs is fatal for the cell, suggesting an important role of cAMP signalling for the maintenance of cellular integrity and proliferation.