Application of a cell-based protease assay for testing inhibitors of picornavirus 3C proteases.

Proteolytical cleavage of the picornaviral polyprotein is essential for viral replication. Therefore, viral proteases are attractive targets for anti-viral therapy. Most assays available for testing proteolytical activity of proteases are performed in vitro, using heterologously expressed proteases and peptide substrates. To deal with the disadvantages associated with in vitro assays, we modified a cell-based protease assay for picornavirus proteases. The assay is based on the induction of expression of a firefly luciferase reporter by a chimeric transcription factor in which the viral protease and cleavage sites are inserted between the GAL4 binding domain and the VP16 activation domain. Firefly luciferase expression is dependent on cleavage of the transcription factor by the viral protease. This biosafe assay enables testing the effect of compounds on protease activity in cells while circumventing the need for infection. We designed the assay for 3C proteases (3C(pro)) of various enteroviruses as well as of viruses of several other picornavirus genera, and show that the assay is amenable for use in a high-throughput setting. Furthermore, we show that the spectrum of activity of 3C(pro) inhibitor AG7088 (rupintrivir) not only encompasses enterovirus 3C(pro) but also 3C(pro) of foot-and-mouth disease virus (FMDV), an aphthovirus. In contrary, AG7404 (compound 1), an analogue of AG7088, had no effect on FMDV 3C(pro) activity, for which we provide a structural explanation.

A randomized two arm phase III study in patients post radical resection of liver metastases of colorectal cancer to investigate bevacizumab in combination with capecitabine plus oxaliplatin (CAPOX) vs CAPOX alone as adjuvant treatment.

BACKGROUND: About 50% of patients with colorectal cancer are destined to develop hepatic metastases. Radical resection is the most effective treatment for patients with colorectal liver metastases offering five year survival rates between 36-60%. Unfortunately only 20% of patients are resectable at time of presentation. Radiofrequency ablation is an alternative treatment option for irresectable colorectal liver metastases with reported 5 year survival rates of 18-30%. Most patients will develop local or distant recurrences after surgery, possibly due to the outgrowth of micrometastases present at the time of liver surgery. This study aims to achieve an improved disease free survival for patients after resection or resection combined with RFA of colorectal liver metastases by adding the angiogenesis inhibitor bevacizumab to an adjuvant regimen of CAPOX. METHODS/DESIGN: The Hepatica study is a two-arm, multicenter, randomized, comparative efficacy and safety study. Patients are assessed no more than 8 weeks before surgery with CEA measurement and CT scanning of the chest and abdomen. Patients will be randomized after resection or resection combined with RFA to receive CAPOX and Bevacizumab or CAPOX alone. Adjuvant treatment will be initiated between 4 and 8 weeks after metastasectomy or resection in combination with RFA. In both arms patients will be assessed for recurrence/new occurrence of colorectal cancer by chest CT, abdominal CT and CEA measurement. Patients will be assessed after surgery but before randomization, thereafter every three months after surgery in the first two years and every 6 months until 5 years after surgery. In case of a confirmed recurrence/appearance of new colorectal cancer, patients can be treated with surgery or any subsequent line of chemotherapy and will be followed for survival until the end of study follow up period as well. The primary endpoint is disease free survival. Secondary endpoints are overall survival, safety and quality of life. CONCLUSION: The HEPATICA study is designed to demonstrate a disease free survival benefit by adding bevacizumab to an adjuvant regime of CAPOX in patients with colorectal liver metastases undergoing a radical resection or resection in combination with RFA. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT00394992.

Solution structure of the second PDZ domain of the neuronal adaptor X11alpha and its interaction with the C-terminal peptide of the human copper chaperone for superoxide dismutase.

Protection against reactive oxygen species is provided by the copper containing enzyme superoxide dismutase 1 (SOD1). The copper chaperone CCS is responsible for copper insertion into apo-SOD1. This role is impaired by an interaction between the second PDZ domain (PDZ2alpha) of the neuronal adaptor protein X11alpha and the third domain of CCS (McLoughlin et al. (2001) J. Biol. Chem., 276, 9303-9307). The solution structure of the PDZ2alpha domain has been determined and the interaction with peptides derived from CCS has been explored. PDZ2alpha binds to the last four amino acids of the CCS protein (PAHL) with a dissociation constant of 91 +/- 2 microM. Peptide variants have been used to map the interaction areas on PDZ2alpha for each amino acid, showing an important role for the C-terminal leucine, in line with canonical PDZ-peptide interactions.

Mega-dose vitamin C in treatment of the common cold: a randomised controlled trial.

OBJECTIVE: To determine the effect of large doses of vitamin C in the treatment of the common cold. STUDY DESIGN: Double-blind, randomised clinical trial with four intervention arms: vitamin C at daily doses of 0.03g ("placebo"), 1 g, 3g, or 3g with additives ("Bio-C") taken at onset of a cold and for the following two days. PARTICIPANTS AND SETTING: 400 healthy volunteers were recruited from staff and students of the Australian National University, Canberra, ACT, between May 1998 and November 1999. The trial continued for 18 months. INTERVENTIONS: Participants were instructed to commence medication when they had experienced early symptoms of a cold for four hours, and to record daily their symptoms, severity, doctor visits and use of other medications. MAIN OUTCOME MEASURES: Duration of symptoms and cold episodes; cumulative symptom severity scores after 7, 14 and 28 days; doctor visits; and whether participants guessed which medication they were taking. RESULTS: 149 participants returned records for 184 cold episodes. No significant differences were observed in any measure of cold duration or severity between the four medication groups. Although differences were not significant, the placebo group had the shortest duration of nasal, systemic and overall symptoms, and the lowest mean severity score at 14 days, and the second lowest at 7 and 28 days. CONCLUSIONS: Doses of vitamin C in excess of 1 g daily taken shortly after onset of a cold did not reduce the duration or severity of cold symptoms in healthy adult volunteers when compared with a vitamin C dose less than the minimum recommended daily intake.

Non-invasive MR thermometry by 2D spectroscopic imaging of the Pr[MOE-DO3A] complex.

Future progress in regional hyperthermia requires a practical method for non-invasive thermometry. In magnetic resonance tomography, spin density, T1 relaxation time, diffusion coefficient and proton resonance frequency are candidates to measure temperature distributions. When used clinically in the pelvic region, all these methods are compromized by artifacts arising from different tissues, tissue alterations under hyperthermia, physiological and random movements, inhomogeneities, drift phenomena, and field instabilities. In this study a paramagnetic complex was evaluated, Pr[MOE-DO3A], with praseodymium as central atom, similar to common gadolinium containing MRI contrast media. The temperature dependence of its methoxy side group approximately -24 ppm downfield from the water resonance at 25 degrees C was employed to determine 2-D temperature distributions in a cylindrical agar phantom containing 9.5 mM of Pr[MOE-DO3A]. The phantom was heated externally through a water jacket creating a stationary temperature distribution throughout the phantom. At first, the correlation between temperature and the chemical shift of the methyl group of the lanthanide complex Pr[MOE-DO3A] was determined. Calibration curves obtained exhibited a linear relationship of 0.12 +/- 0.01 ppm/degree C, nearly independent from the surrounding medium. Local temperature distributions were determined employing the spatially resolved method of spectroscopic imaging (SI). 2-D spectroscopic images for three orthogonal slices were obtained by narrow-band excitation and 16 phase encoding steps in two dimensions. The FOV was 180 mm and the slice thickness in all cases was 20 mm for maximal spatial temperature resolution (11.2 x 11.2 mm2). The results indicate a measurement time of about 5s per acquisition under the following conditions: An estimated concentration of 1 mmol/l, a reduced matrix size of 8 x 8, and a reduced repetition time of 3 x T1 (TR approximately 85 ms). Those SI measurements produced a SNR of approximately 4 per acquisition, a measurements duration of 10-20 s, equivalent to two to four acquisitions per spectrum, seem sufficient for online temperature monitoring during hyperthermia. The in vitro data suggest the spectroscopic temperature measurement utilizing a temperature-sensitive Pr[MOE-DO3A] complex with a therapeutically realistic concentration of 1 mmol/l to be suitable for clinical use. Compared to the methods tested so far (rho, T1, diffusion, proton resonance), the method presented has the unique advantage of being less susceptible to artifacts. The competing methods of non-invasive thermometry employing magnetic resonance imaging are currently being investigated using the same experimental setup.

MR of the eye with retrobulbar anesthesia.

MR imaging with retrobulbar anesthesia was performed in eight patients with uveal melanoma. Injection of 2 mL prilocain hydrochloride in 2% epinephrin into the eye muscle cone resulted in improved image quality in seven patients, without side effects. Ocular MR imaging can be indicated to clarify indeterminate sonographic findings in cases of extrascleral growth or to exclude optic nerve invasion in patients with tumors located at the posterior pole of the globe.

[Thermometry by measuring the chemical shift of lanthanide complex]. / Thermometrie durch Messung der chemischen Verschiebung eines Lanthanidenkomplexes.

BACKGROUND: In the long-term, non-invasive thermometry is vital for the continued clinical and technological development of regional hyperthermia. In magnetic resonance tomography. T1 relaxation time, diffusion and proton resonance frequency are used to measure temperature distributions. When used clinically in the pelvic region, all of these methods are plagued with errors and artefacts on account of the tissue relationships, tissue changes under hyperthermia, physiological and stochastic movements, inhomogeneities, drift phenomena and instabilities. MATERIAL AND METHOD: We tested the relationship between the temperature and the chemical shift of a methyl group of a lanthanide complex with central atom praseodymium (Pr-MOE-DO3A. Schering AG). To do this we used cylindrical phantoms containing a 5-mmol-solution of this temperature-sensitive substance. High resolution spectra and relaxation times were determined in a Bruker AMX at 11.5 T. A calibration curve was then recorded by a Siemens Magnetom SP63 at 1.5 T. Local temperature distributions were determined using the chemical shift imaging method, with a matrix size of 16 x 8 and a narrow-band excitation pulse. The temperature distribution was created using a Nd:YAG laser applicator. RESULTS: At a distance of -25.7 ppm from the water line, we found a singlet line with a temperature-dependent chemical shift of 0.13 ppm/C. In the phantom experiment we found that the chemical shift had a linear relationship with a gradient independent of the surroundings, and a temperature resolution of +/-0.6 degree C. With a concentration of 1 mmol/l, a matrix size of 8 x 8 and a measurement period of 5 s per acquisition, phantom measurements using the CSI method produced a signal to noise ratio of 3.5 per acquisition, i.e a measurement period of 10 to 20 s per spectrum. CONCLUSIONS: Our in vitro data show that spectroscopic temperature measurement using a temperature-sensitive praseodymium complex with a therapeutically practical concentration of 1 mmol/l already appears to be suitable for clinical use Compared with the methods tested so far (T1, diffusion, proton resonance), this method has the special advantage of not being very susceptible to artefacts. The competing methods of non-invasive thermometry using magnetic resonance tomography/spectroscopy will be investigated next.

Effects of magnetic fluid hyperthermia (MFH) on C3H mammary carcinoma in vivo.

Magnetic fluids (MF) have a potential for hyperthermia due to their good power absorption capabilities. Recent in vitro experiments with the so-called 'Magnetic Fluid Hyperthermia (MFH)' have shown that human tumours cells are homogeneously inactivated after AC magnetic field excitation of extracellular MF. The aim of the present study was the evaluation of a high dose MFH on intramuscularly implanted mammary carcinoma of the mouse. The tumours originated from initial in vivo passages of a spontaneous parent tumour. Because of larger variations of tumour growth in this rather primary model, logistic regression of non-averaged volumes was performed for each treatment modality. All growing tumours were randomized 30 days after transplantation (day of treatment) with an overall size distribution between 120-400 mm3. An intratumoural steady state temperature of 47 +/- 1.0 degrees C was maintained for 30 minutes with whole-body AC magnetic fields of 6-12.5 kA/m at 520 kHz. The magnetic fluid was #P6, which is a high biocompatible dextran magnetite. #P6 was given intratumourally (1.5 x 10(-2) mg ferrite/mm3) 20-30 minutes before excitation and was combined with magnetic targeting (50 mT), which yielded a 2.5-fold enhancement of the intratumoural iron concentration. Histological examinations of tumour tissue after intralesional ferrofluid administration alone indicated deep infiltration of the fluid into the carcinoma tissue, but no evidence of tissue damage as compared with untreated controls. In contrast, widespread tumour necrosis was observed after MFH. After application of either dextran or ferrofluid alone (no difference, p = 0.665), tumour growth was slightly delayed in comparison with untreated controls (p < 0.001). In contrast to the good fit of the controls (R = 0.92-0.87), tumour growth after MFH was much more heterogeneous; some tumours showed no evidence for regrowth at 50 days whereas others had grown quite readily. This most probably reflected the critical problem of homogeneity of the intratumoural MF distribution, which was also confirmed qualitatively by Magnetic Resonance Imaging (MRI), heterogeneous pigmentation of MFH treated tumours, and up to 1 degree C differences between temperature probes in the same tumour during AC magnetic field application. However, a quantitative comparison between intratumoural MF-heterogeneity and tumour response could not be performed in this study. Despite these current limitations, the regression analysis of the MFH data yielded a smaller tumour volume of about 1000 mm3 at 50 days growth time in contrast to all three controls. In conclusion, encouraging results have been obtained, which show, that one single high dose MFH is already able to induce local tumour control in many cases within 30 days after treatment. To overcome the uncertainties of intratumoural MF heterogeneity, advanced intralesional application methods are currently under development.

Clinical experiences with magnetic drug targeting: a phase I study with 4'-epidoxorubicin in 14 patients with advanced solid tumors.

Anticancer drugs reversibly bound to magnetic fluids (ferrofluids) could be concentrated in locally advanced tumors by magnetic fields that are arranged at the tumor surface outside of the organism. If certain requirements are met, systemic toxicity might be minimized, and local tumor efficacy might be increased. We have conducted a Phase I clinical trial using this approach in patients with advanced and unsuccessfully pretreated cancers or sarcomas. Nine such patients received two treatment courses, 3 patients received one course, and 2 patients received three courses of magnetic drug targeting consisting of the infusion of epirubicin in increasing doses (from 5 to 100 mg/m2) that had been chemically bound to a magnetic fluid and the application of magnetic fields to the tumors for 60-120 min. In 2 of 14 patients, the same dose of epirubicin not bound to a magnetic fluid was administered systemically 3 weeks after drug targeting for intraindividual comparisons. Magnetic drug targeting with epirubicin was well tolerated. In one case, a planned second treatment was withdrawn, because of an episode of chills 130 min after infusion of the magnetic drug. Two patients received a third treatment because of good responses after the first two therapies. Based on magnetic resonance tomographic techniques, pharmacokinetics, and the histological detection of magnetites, it was shown that the ferrofluid could be successfully directed to the tumors in about one-half of the patients. Organ toxicity did not increase with the treatment, but epirubicin-associated toxicity appeared at doses greater than 50 mg/m2. Although treatment with magnetic drug targeting seems safe, improvements are necessary to make it more effective and independent of patient- or disease-related problems. A study design to compare conventional treatments with the new treatment form within one patient seems crucial to eliminate interindividual differences.

Interleukin 2, 4 and 5 are sequentially produced in mitogen-stimulated murine spleen cell cultures.

Lymphokine production was analyzed in murine spleen lymphocytes stimulated with different T cell mitogens. Using in situ hybridization, frequencies of cells and the kinetics of production of interleukin (IL) 2, 4 and 5 were analyzed. The different mitogens varied in their ability to induce the three interleukins. IL2 was most successfully induced with a high dose of the calcium ionophore A23187 combined with phorbol 12-myristate 13-acetate (PMA). Significant frequencies of cells containing IL4 or IL5 mRNA were found among cells stimulated with an anti-CD3 antibody together with PMA, or pokeweed mitogen. The combination of anti-CD3 and PMA induced relatively high frequencies of all three cytokines. The production was sequential with the highest levels of IL2 mRNA present during the first 24 h, IL4 mRNA reaching a peak on day 2 and finally IL5 peaking on day 3. When cells that had been stimulated with mitogens in vitro were restimulated, the lymphokines were produced more rapidly. The order of production was maintained with IL2 mRNA reaching a maximum already at 3 h of culture, IL4 mRNA at 8 h and IL5 mRNA at 24 h.

[The effect of x-ray contrast media on magnetic resonance tomography of the spinal canal]. / Der Einfluss von Röntgenkontrastmitteln bei der Magnetresonanztomographie des Spinalkanals.

The effects of x-ray contrast media on signal intensities were studied. Relaxation times of myelographic contrast media were measured. Whereas relaxation times of Amipaque were longer, compared to CSF, the relaxation times of Duroliopaque, Omnipaque und Solutrast were significantly shorter than that of CSF. Therefore, the qualitative and quantitative effects of different contrast media on signal intensities varied. Most notable was the increase of signal intensity on T1-weighted images due to oily contrast media.