Viral persistence in surface and drinking water: Suitability of PCR pre-treatment with intercalating dyes.

After many outbreaks of enteric virus associated with consumption of drinking water, the study of enteric viruses in water has increased significantly in recent years. In order to better understand the dynamics of enteric viruses in environmental water and the associated viral risk, it is necessary to estimate viral persistence in different conditions. In this study, two representative models of human enteric viruses, adenovirus 41 (AdV 41) and coxsackievirus B2 (CV-B2), were used to evaluate the persistence of enteric viruses in environmental water. The persistence of infectious particles, encapsidated genomes and free nucleic acids of AdV 41 and CV-B2 was evaluated in drinking water and surface water at different temperatures (4 °C, 20 °C and 37 °C). The infectivity of AdV 41 and CV-B2 persisted for at least 25 days, whatever the water temperature, and for more than 70 days at 4 °C and 20 °C, in both drinking and surface water. Encapsidated genomes persisted beyond 70 days, whatever the water temperature. Free nucleic acids (i.e. without capsid) also were able to persist for at least 16 days in drinking and surface water. The usefulness of a detection method based on an intercalating dye pre-treatment, which specifically targets preserved particles, was investigated for the discrimination of free and encapsidated genomes and it was compared to virus infectivity. Further, the resistance of AdV 41 and CV-B2 against two major disinfection treatments applied in drinking water plants (UV and chlorination) was evaluated. Even after the application of UV rays and chlorine at high doses (400 mJ/cm(2) and 10 mg.min/L, respectively), viral genomes were still detected with molecular biology methods. Although the intercalating dye pre-treatment had little use for the detection of the effects of UV treatment, it was useful in the case of treatment by chlorination and less than 1 log10 difference in the results was found as compared to the infectivity measurements. Finally, for the first time, the suitability of intercalating dye pre-treatment for the estimation of the quality of the water produced by treatment plants was demonstrated using samples from four drinking-water plants and two rivers. Although 55% (27/49) of drinking water samples were positive for enteric viruses using molecular detection, none of the samples were positive when the intercalating dye pre-treatment method was used. This could indicate that the viruses that were detected are not infectious.

WE-G-BRB-06: Real-Time Radiation Field Tracking Using Long Scintillating Fibers.

PURPOSE: To ensure the quality assurance of small field, dynamic radiotherapy, we present and validate a radiation tracking system based on long scintillating fibers that allows for the real-time measurement of the position and energetic fluence of a small incident radiation field. METHOD: We aligned 60 parallel scintillating fibers on a thin grooved acrylic slab with a 100-cm source-to-fibers distance. Both ends of each scintillating fiber were coupled to clear optical fibers to enable light collection by a single CCD camera using an f/0.95, 50 mm focal length lens. Using a small, static photon radiation field of 2×2 cm2 of a Varian Clinac iX, we changed the interaction position on the prototype using the linac treatment couch. The interaction position parallel and perpendicular to the scintillating fiber array were deduced using the optical attenuation of the scintillating fibers. The energetic fluence of the incident field was calculated from the fibers light fluxes, corrected for the position dependent optical attenuation and scintillation efficiency. RESULTS: Considering a treatment couch positioning error of ±0.5 mm, the system was able to measure the field position with a mean error of 0.1 mm perpendicular and 0.8 mm parallel to the scintillating fiber array. The maximum error measured using this setup was of 0.13 mm perpendicular and 3.2 mm parallel to the scintillating fiber array. The energetic fluence was determined with a mean error of 0.5% and a maximum error of 2.2%. CONCLUSIONS: This work demonstrates the capacity of a long scintillating fibers array to detect in real-time both the position and the energetic fluence of an incident small radiation field. Such methodology would allow for the real-time tracking of small field in both photon and particle radiation therapy.

Sci-Thur PM: YIS - 05: Tomographic dosimetry using scintillating fibers and its application to 2D and 3D dosimetry.

PURPOSE: To present the proof of concept and the experimental validation of tomographic dosimetry (tomodosimetry), where a tomographic acquisition of the incident deposited dose is performed using long scintillating fibers. METHOD: 2D tomodosimetry: 50 long scintillating fibers were aligned on a 20cm diameter disk inside a 30cm diameter masonite phantom. 3D tomodosimetry: 128 long scintillating fibers of various orientation were simulated on the surface of two cylindrical regions of radius 7.5 and 3.75cm inside a 20cm diameter, 20cm long cylindrical phantom. In both case, the dose projections were acquire each 5 degrees over a 180 degrees (2D) or 360 degrees (3D) rotation of the device, and the dose in each scintillating fiber plane was reconstructed using a total variation minimization reconstruction iterative algorithm at a resolution of 1×1mm2 . The 3D dose was obtained by interpolating between in each cylindrical plane in the 3D prototype. RESULTS: 3%/3mm gamma tests conducted in the isocentre plane for both configurations achieved a success rate of more than 99% of the dose pixels in the region over 50% of the maximum dose. Absolute dose differences in the high dose low gradient region of each scintillating fiber plane were on average below 1% for the 2D configuration and below 1.3% for the 3D configuration. CONCLUSIONS: This work illustrates the potential and capacity of scintillating fiber based 2D and 3D tomodosimeters. The presented methodology allows for millimeter resolution dosimetry in a whole 2D plane or 3D volumes in real-time using only a limited number of detectors.

Enhancing VSV oncolytic activity with an improved cytosine deaminase suicide gene strategy.

Oncolytic viruses (OVs) are promising therapeutic agents for cancer treatment, with recent studies emphasizing the combined use of chemotherapeutic compounds and prodrug suicide gene strategies to improve OV efficacy. In the present study, the synergistic activity of recombinant vesicular stomatitis virus (VSV)-MΔ51 virus expressing the cytosine deaminase/uracil phosphoribosyltransferase (CD::UPRT) suicide gene and 5-fluorocytosine (5FC) prodrug was investigated in triggering tumor cell oncolysis. In a panel of VSV-sensitive and -resistant cells-prostate PC3, breast MCF7 and TSA, B-lymphoma Karpas and melanoma B16-F10-the combination treatment increased killing of non-infected bystander cells in vitro via the release of 5FC toxic derivatives. In addition, we showed a synergistic effect on cancer cell killing with VSV-MΔ51 and the active form of the drug 5-fluorouracil. Furthermore, by monitoring VSV replication at the tumor site and maximizing 5FC bioavailability, we optimized the treatment regimen and improved survival of animals bearing TSA mammary adenocarcinoma. Altogether, this study emphasizes the potency of the VSV-CD::UPRT and 5FC combination, and demonstrates the necessity of optimizing each step of a multicomponent therapy to design efficient treatment.

Delayed onset of severe neonatal toxoplasmosis.

We report a case of severe neonatal infection on day 6 of life due to Toxoplasma gondii mimicking septic shock syndrome associated with multiple organ failure such as acute respiratory distress syndrome with persistent pulmonary hypertension, neurological distress, thrombocytopenia with disseminated intravascular coagulopathy and transaminitis. Clinicians facing an unexplained life-threatening condition in the first week of life should take into consideration the possibility of neonatal toxoplasmosis.

[Neonatal hypocalcaemic dilated myocardiopathy due to a 22q11 microdeletion]. / Myocardiopathie dilatée hypocalcémique néonatale révélatrice d'une microdélétion 22q11.

Here we report 2 cases of hypocalcemic cardiomyopathy revealing a 22q11 microdeletion syndrome. This presentation at diagnosis is rare as the cardiac phenotype is mainly made of conotruncal congenital heart defects in this condition. Cardiac failure was diagnosed during the neonatal period in the 2 cases and was associated with profound hypocalcemia. As usual, treatment with calcium and vitamin D led to the regression of the hypocalcemia and the left ventricular function was fully restored. While this circumstances are unusual, we recommend that screening for 22q11 deletion should be performed when confronted to hypocalcemic cardiomyopathy or left ventricular systolic dysfunction in conotruncal defects in neonates.

Complete elucidation of a minimal class I MHC natural killer cell receptor haplotype.

The BALB/c inbred mouse is widely used in models of infectious disease, transplantation, and cancer. The differences in the immune responses of BALB/c compared to C57BL/6 mice are especially valuable for the identification of immune regulation genes. One striking immune variance between these mice is in the function of natural killer (NK) cells, and there is strong evidence implicating differential expression of Ly49 genes. In this study, the complete BALB/c Ly49 gene cluster has been sequenced and found to contain six functional genes and two pseudogenes. Compared to C57BL/6 mice, there is a 200 kb region absent in the BALB/c cluster including a complete lack of Ly49h-related genes, which explains the increased susceptibility of BALB/c to cytomegalovirus infection. In addition, there is no BALB/c Ly49d allele, explaining the inability of BALB/c NK cells to kill certain tumor cells. The Ly49 region has now been sequenced in three different inbred mouse strains, and comparisons indicate that the evolution of each haplotype is not straightforward and has involved large-scale deletions/insertions, gene recombination, and unequal crossing over between divergent haplotypes. This study confirms that relatively small murine class I MHC receptor haplotypes exist, analogous to observations made of human killer cell Ig-like receptor gene haplotypes.

Direct sequence comparison of two divergent class I MHC natural killer cell receptor haplotypes.

The murine Ly49 gene family encoding natural killer cell receptors for class I MHC is an example of a rapidly evolving cluster of immune response genes. Determining the genomic sequence of the 129S6/SvEvTac (129S6) Ly49 cluster and comparing it to the known sequence of the C57BL/6 (B6) region provided insight into the mechanisms of Ly49 gene evolution. 129S6 contains 20 Ly49, many of which are pseudogenes and 40% of the genes have no counterpart in the B6 genome. The difference in gene content between these two strains is primarily the result of distinct patterns of gene duplication. Phylogenetic analyses of individual exons showed that Ly49 genes form distinct sub-families and an ancestral haplotype can be surmised. Dotplot analysis supports limited allelism in the two haplotypes; however, large regions of variation punctuate these islands of co-linearity. These variable regions contain a high concentration of repetitive elements that are predicted to contribute to the dynamic evolution of this cluster. The extreme variation in Ly49 haplotype content between mouse strains provides a genetic explanation for the documented differences in natural killer cell phenotype, and also indicates that differences in natural killer cell function observed between B6 and 129-derived gene-targeted mice should be interpreted with caution.

Peripheral secretin-induced Fos expression in the rat brain is largely vagal dependent.

I.v. injection of secretin activates neurons in brain areas controlling autonomic function and emotion. Peripheral administration of secretin inhibits gastric functions through a central mechanism that is mediated by vagal dependent pathways. We investigated whether the vagus nerve is involved in i.p. injection of secretin-induced brain neuronal activation in conscious rats as monitored by Fos immunohistochemistry. Secretin (40 or 100 microg/kg, i.p., 90 min) induced a dose-related increase in the number of Fos positive neurons in the central nucleus of the amygdala (CeA), and a plateau Fos response in the area postrema (AP), nucleus tractus solitarii (NTS), locus coeruleus (LC), Barrington's nucleus (Bar), external lateral subnucleus of parabrachial nucleus (PBel) and arcuate nucleus, and at 100 microg/kg, in the dorsal motor nucleus of the vagus (DMV) compared with i.p. injection of vehicle. Double immunohistochemistry showed that secretin (40 microg/kg, i.p.) activates tyrosine hydroxylase neurons in the NTS. Subdiaphragmatic vagotomy (7 days) abolished Fos expression-induced by i.p. secretin (40 microg/kg) in the NTS, DMV, LC, Bar, PBel and CeA, while a significant rise in the AP was maintained. In contrast, s.c. capsaicin (10 days) did not influence the Fos induction in the above nuclei. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR showed that secretin receptor mRNA is expressed in the nodose ganglia and levels were higher in the right compared with the left ganglion. These results indicate that peripheral secretin activates catecholaminergic NTS neurons as well as neurons in medullary, pontine and limbic nuclei regulating autonomic functions and emotion through vagal-dependent capsaicin-resistant pathways. Secretin injected i.p. may signal to the brain by interacting with secretin receptors on vagal afferent as well as on AP neurons outside the blood-brain barrier.

A secretin i.v. infusion activates gene expression in the central amygdala of rats.

For the last 100 years secretin has been extensively studied for its hormonal effects on digestion. Recent observations that the deficits in social reciprocity skills seen in young (3-4-year-old) autistic children are improved after secretin infusions suggest an additional influence on neuronal activity. We show here that i.v. administration of secretin in rats induces Fos protein expression in the neurons of the central amygdala as well as the area postrema, bed nucleus of the stria terminalis, external lateral parabrachial nucleus and supraoptic nucleus. However, secretin infusion did not induce Fos expression in the solitary tract nucleus or paraventricular nucleus, regions normally activated by related peptides such as cholecystokinin. The peak blood levels of secretin that induce Fos protein expression in rat brain are similar to the peak blood levels observed during i.v. treatment with secretin in humans. The amygdala is known to be critical for developing reciprocal social interaction skills and abnormalities in this brain region have been demonstrated in autistic children.

Dysferlin expression after normal myoblast transplantation in SCID and in SJL mice.

Limb girdle muscular dystrophy type 2B form and Miyoshi myopathy are both caused by mutations in the recently cloned gene dysferlin. In the present study, we have investigated whether cell transplantation could permit dysferlin expression in vivo. Two transplantation models were used: SCID mice transplanted with normal human myoblasts, and SJL mice, the mouse model for limb girdle muscular dystrophy type 2B and Miyoshi myopathy, transplanted with allogeneic primary mouse muscle cell cultures expressing the beta-galactosidase gene under control of a muscle promoter of Troponin I. FK506 immunosuppression was used in the non-compatible allogeneic model. One month after transplantation, human and mouse dysferlin proteins were detected in all transplanted SCID and SJL muscles, respectively. Co-localization of dysferlin and human dystrophin or beta-galactosidase-positive fibers was observed following the transplantation of myoblasts. Dysferlin proteins were monitored by immunocytochemistry and Western blot. The number of dysferlin-positive fibers was 40-50% and 20-30% in SCID and SJL muscle sections, respectively. Detection of dysferlin in both SCID mice and dysferlin-deficient SJL mouse shows that myoblast transplantation permits the expression of the donor dysferlin protein.

Non-amines, drugs without an amine nitrogen, potently block serotonin transport: novel antidepressant candidates?

The serotonin transporter (SERT) is a principal site of action of therapeutic antidepressants in the brain. Without exception, these inhibitors of serotonin transport contain an amine nitrogen in their structure. We previously demonstrated that novel compounds without an amine nitrogen in their structure (non-amines), blocked dopamine transport in cells transfected with the human dopamine transporter. The present study investigated whether, in the absence of an amine nitrogen, certain non-amines bind selectively to the SERT and block the transport of serotonin. At 10 microM concentration, select non-amines displayed no, or little, affinity for 9 serotonin, 5 dopamine, 7 adrenergic, 5 muscarinic cholinergic, 3 opiate and histamine receptors. The affinities of non-amines for [(3)H]citalopram binding sites on the SERT and their potencies for blocking [(3)H]serotonin transport were measured in cloned human SERT stably or transiently expressed in HEK-293. Whether oxa- or carba-based, non-amines bound to [(3)H]citalopram-labeled sites and blocked [(3)H]serotonin transport in the low nanomolar range, at values equal to or higher than those of some conventional antidepressants. A non-amine, O-1809, was 99-fold more selective for the serotonin over the dopamine transporter. As substituents on the aromatic ring of non-amines confer high affinity for the SERT, we investigated the hypothesis that aromatic-aromatic interactions may contribute significantly to non-amine/transporter association. A SERT mutant was produced in which a highly conserved aromatic amino acid, phenylalanine 548, was replaced by an alanine (F548A). Although the affinities of several non-amines were unchanged in the mutant SERT, the affinity of imipramine was decreased, revealing possible differences in amine and non-amine binding domains on the SERT. The similar affinities of non-amines and conventional antidepressant drugs for the SERT support the view that an amine nitrogen is not essential for drugs to block serotonin transport with high affinity. Non-amines open avenues for developing a new generation of antidepressants.

Orally bioavailable, indole-based nonpeptide GnRH receptor antagonists with high potency and functional activity.

Stereospecific introduction of a methyl group to the indole-3-side chain enhanced activity in our tryptamine-derived series of GnRH receptor antagonists. Further improvements were achieved by variation of the bicyclic amino moiety of the tertiary amide and by adjustment of the tether length to a pyridine or pyridone terminus. These modifications culminated in analogue 24, which had oral activity in a rat model and acceptable oral bioavailability and half-life in dogs and monkeys.

Substituted indole-5-carboxamides and -acetamides as potent nonpeptide GnRH receptor antagonists.

The 2-aryltryptamine class of GnRH receptor antagonists has been modified to incorporate carboxamide and acetamide substituents at the indole 5-position. With either a phenol or methanesulfonamide terminus on the N-aralkyl side chain, potent binding affinity to the GnRH receptor was achieved. A functional assay for GnRH antagonism was even more sensitive to structural modification and revealed a strong preference for branched tertiary amides.

Potent nonpeptide GnRH receptor antagonists derived from substituted indole-5-carboxamides and -acetamides bearing a pyridine side-chain terminus.

A pyridine side-chain terminus has been incorporated into the indole-5-carboxamide and indole-5-acetamide series of GnRH antagonists. Potent activity was observed in binding and functional assays. Certain branched or cyclic tertiary amides were identified as preferred in each series. Alkylation of the side-chain secondary amine had generally unfavorable effects. Variations of the gem-dialkyl substituents in the indole-5-acetamide series were also investigated.

2-(3,5-Dimethylphenyl)tryptamine derivatives that bind to the GnRH receptor.

A series of 2-(3,5-dimethylphenyl)tryptamine derivatives was prepared and evaluated on a rat gonadotropin releasing hormone receptor assay. Some para-substituents on the 4-phenylbutyl side chain attached to the tryptamine nitrogen led to compounds with potent GnRH receptor binding. The study has helped define structural requirements for GnRH receptor binding for the 2-aryltryptamine GnRH antagonists.

Heterocyclic derivatives of 2-(3,5-dimethylphenyl)tryptamine as GnRH receptor antagonists.

A series of heterocyclic 2-(3,5-dimethylphenyl)tryptamine derivatives was prepared and evaluated on a rat gonadotropin releasing hormone receptor assay. The carbon tether length and heterocyclic ring attached to the amino group of 2-(3,5-dimethylphenyl)tryptamine were varied. Several of these derivatives were potent GnRH antagonists with the most potent compound having an IC50 of 16 nM.

Effect of MPTP-induced denervation on basal ganglia GABA(B) receptors: correlation with dopamine concentrations and dopamine transporter.

We investigated the effect of MPTP-induced lesion of the substantia nigra pars compacta (SNpc) dopaminergic neurons on GABA(B) receptors in the basal ganglia of mice and monkeys using receptor autoradiography and in situ hybridization. The extent of the lesion was measured with striatal catecholamine content, striatal binding of (125)I-RTI-121 to dopamine transporter (DAT), and DAT expression in the SNpc. GABA(B) receptors in mice brain were evaluated using (3)H-CGP54626 and its expression was measured with oligonucleotides probes targeting the mRNAs of GABA(B(1a+b)), GABA(B(1a)), GABA(B(1b)), GABA(B(2)) subunits. In monkeys, (125)I-CGP64213 and selective probes for GABA(B(1a+b)) and GABA(B(2)) mRNAs were used. In mice, dopamine content, (125)I-RTI-121 binding, and DAT expression were reduced by 44%, 40%, and 39% after a dose of 40 mg/kg of MPTP and 74%, 70%, and 34% after 120 mg/kg of MPTP, respectively. In monkeys, dopamine content and DAT expression were decreased by more than 90% and 80%, respectively. In the striatum and the subthalamic nucleus, GABA(B) receptors were unchanged following MPTP in both species. In the SNpc of mice, MPTP (120 mg/kg) induced a significant decrease of (3)H-CGP54626 binding (-10%) and of the expression of GABA(B(1a+b)) mRNA (-13%). The decrease of the expression of GABA(B(1a+b)) mRNA was correlated with dopamine content, (125)I-RTI-121 binding and DAT expression. In MPTP-treated monkeys, (125)I-CGP64213 binding (-40%), GABA(B(1a+b)) mRNA (-69%) and GABA(B(2)) mRNA (-66%) were also significantly decreased in the SNpc. Our results suggest that MPTP-induced denervation is associated with a decrease of GABA(B) receptors restricted to the SNpc. These observations may be relevant to the pathophysiology of motor disorders involving dysfunction of the basal ganglia such as Parkinson disease.

A potent, nonpeptidyl 1H-quinolone antagonist for the gonadotropin-releasing hormone receptor.

Extensive development of the structure-activity relationships of a screening lead determined three important pharmacophores for gonadotropin-releasing hormone (GnRH) receptor antagonist activity. Incorporation of the 3,4,5-trimethylphenyl group at the 3-position, 2-(2(S)-azetidinyl)ethoxy group at the 4-position, and N-4-pyrimidinylcarboxamide at the 6-position of the quinolone core resulted in the identification of 4-(2-(azetidin-2(S)-yl)ethoxy)-7-chloro-2-oxo-3-(3,4,5-trimethylphenyl)-1,2-dihydroquinoline-6-carboxylic acid pyrimidin-4-ylamide (1) as a potent antagonist of the GnRH receptor. A 10(4)-fold increase in in vitro binding affinity is observed for the GnRH receptor as compared to the initial screening lead. Compound 1 exhibits nanomolar binding activity and functional antagonism at the human receptor and is 7-fold less active at the rhesus receptor. Intravenous administration of compound 1 to rhesus monkeys results in a significant decrease of the serum levels of downstream hormones, luteinizing hormone (79% decrease in area under the curve) and testosterone (92% decrease in area under the curve), at a dose of 3 mg/kg. Quinolone 1 is a potent nonpeptidyl antagonist for the human GnRH receptor that is efficacious for the suppression of luteinizing hormone and testosterone in primates.