Three-dimensional ultrashort echo time (3D UTE) MRI of Achilles tendon at 4.7T MRI with comparison to conventional sequences in an experimental murine model of spondyloarthropathy.

BACKGROUND: Due to the very short T2 of its components, the normal anatomy of Achilles enthesis is impossible to define with "conventional" long echo time (TE) T2 sequences. However, this is a common site affected by rheumatologic disease. Early abnormalities related to inflammatory processes are impossible to detect in this location. PURPOSE: To assess the feasibility of a 3D-UTE (ultrashort echo time) sequence to evaluate normal and pathological Achilles entheses, determining both anterior fibrocartilaginous and posterior collagenic portions at 4.7T, in a rat model of spondyloarthropathy (SpA) with histological correlation. To assess whether this sequence detects SpA enthesopathy prior to long TE T2 sequences, enabling disease monitoring. STUDY TYPE: Prospective case-control study. ANIMAL MODEL: Twelve immunocompetent Wistar male rats imaged before (controls); the model was induced in eight rats (16 tendons) imaged at day 6, day 13, and day 21 with regular sacrifice for ex vivo imaging and histological correlation. FIELD STRENGTH: 4.7T Bruker Biospec Systems. 3D balanced steady-state free precession (bSSFP) and 3D-UTE sequences, performed at baseline (day 0, n = 12 animals / 24 tendons), day 6 (n = 8/16), 13 (n = 4/8), and day 21 (n = 2/4). ASSESSMENT: Visual analysis and signal intensity measurements (signal to noise ratio, SNR) of both bSSFP and UTE images were performed by two independent musculoskeletal radiologists at different locations of the Achilles enthesis and preinsertional area. STATISTICAL TESTS: Normal and pathological rat values were compared by Wilcoxon signed-rank tests, as well as interobserver differences. MRI findings were compared against histological data. RESULTS: The 3D-UTE sequence identified the anterior fibrocartilage and posterior collagenic areas of Achilles entheses in all cases. Visual analysis and signal intensity measurements distinguished SpA-affected entheses from healthy ones at days 6 and 13 (P = 0.002 and P = 0.006, respectively). Neither the normal anatomy of the enthesis nor its pathological pattern could be identified on T2 bSSFP sequences. DATA CONCLUSION: Unlike bSSFP T2 sequences, 3D-UTE sequences enable visualization of normal enthesis anatomy and early detection of abnormalities in pathological conditions. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2019;50:127-135.

In vivo MRI discrimination between live and lysed iron-labelled cells using balanced steady state free precession.

OBJECTIVES: The goal of this study was to evaluate the ability of balanced steady state free precession (b-SSFP) magnetic resonance imaging sequence to distinguish between live and lysed iron-labelled cells. METHODS: Human breast cancer cells were labelled with iron oxide nanoparticles. Cells were lysed using sonication. Imaging was performed at 3 T. The timing parameters for b-SSFP and the number of iron-labelled cells in samples were varied to optimise the b-SSFP signal difference between live and lysed iron-labelled cell samples. For in vivo experiments, cells were mixed with Matrigel and implanted into nude mice. Three mice implanted with live labelled cancer cells were irradiated to validate this method. RESULTS: Lysed iron-labelled cells have a significantly higher signal compared with live, intact iron-labelled cells in bSSFP images. The contrast between live and dead cells can be maximised by careful optimisation of timing parameters. A change in the b-SSFP signal was measured 6 days after irradiation, reflecting cell death in vivo. Histology confirmed the presence of dead cells in the implant. CONCLUSIONS: Our results show that the b-SSFP sequence can be optimised to allow for the discrimination of live iron-labelled cells and lysed iron-labelled cells in vitro and in vivo.

Optimizing 4D abdominal MRI: image denoising using an iterative back-projection approach.

4D-MRI is a promising tool for organ exploration, target delineation and treatment planning. Intra-scan motion artifacts may be greatly reduced by increasing the imaging frame rate. However, poor signal-to-noise ratios (SNR) are observed when increasing spatial and/or frame number per physiological cycle, in particular in the abdomen. In the current work, the proposed 4D-MRI method favored spatial resolution, frame number, isotropic voxels and large field-of-view (FOV) during MR-acquisition. The consequential SNR penalty in the reconstructed data is addressed retrospectively using an iterative back-projection (IBP) algorithm. Practically, after computing individual spatial 3D deformations present in the images using a deformable image registration (DIR) algorithm, each 3D image is individually enhanced by fusing several successive frames in its local temporal neighborood, these latter being likely to cover common independent informations. A tuning parameter allows one to freely readjust the balance between temporal resolution and precision of the 4D-MRI. The benefit of the method was quantitatively evaluated on the thorax of 6 mice under free breathing using a clinically acceptable duration. Improved 4D cardiac imaging was also shown in the heart of 1 mice. Obtained results are compared to theoretical expectations and discussed. The proposed implementation is easily parallelizable and optimized 4D-MRI could thereby be obtained with a clinically acceptable duration.

Microglia used as vehicles for both inducible thymidine kinase gene therapy and MRI contrast agents for glioma therapy.

Microglia are phagocytic cells that are chemoattracted by brain tumors and can represent up to 70% of the tumor cell population. To get insight into gene therapy against glioma, we decided to take advantage of those microglia properties and to use those cells as vehicles to transport simultaneously a suicide gene (under the control of a heat-sensitive promoter) and contrast agents to localize them by magnetic resonance imaging before applying any therapeutic treatment. Thymidine kinase (TK) expression and its functionality after gancyclovir administration were investigated. After the heat shock (44 degrees C and 20 min), TK was expressed in 50% of the cells. However, after gancyclovir treatment, 90% of the cells died by apoptosis, showing an important bystander effect. Then, the cells were incubated with new lanthanide contrast agents to check both their potential toxicity and their MR properties. Results indicate that the nanoparticles did not induce any cell toxicity and yield a hypersignal on MR images at 4.7 T. These in vitro experiments indicate that microglia are good candidates as vectors in gene therapy against brain tumors. Finally, microglia containing gadolinium-grafted nanoparticles were injected in the close vicinity of C6 tumor, in a mouse. The hyperintensive signal obtained on in vivo images as well as its retention time show the potential of the novel contrast agents for cellular imaging.

[Small cell lung cancer and cancer-associated retinopathy]. / Carcinoma broncogénico microcítico y retinopatía asociada a cáncer.

We report the case of a patient who presented with cancer-associated retinopathy and small cell carcinoma of the lung, which was treated surgically because the initial diagnostic biopsy finding was squamous cell carcinoma. The patient then underwent chemotherapy and radiation therapy. We discuss the characteristics and pathogenesis of this paraneoplastic syndrome as well as its association with the lung tumor's aberrant production of a protein that competes with retinal recoverin at the photoreceptors of the retinal cone.

Prostaglandins mediate lipopolysaccharide effects upon cholecystokinin and neurotensin phenotypes in neuroendocrine corticotropin-releasing hormone neurons: in situ hybridization evidence in the rat.

Intraperitoneal injection of the endotoxin lipopolysaccharide produces an inflammation accompanied by immune system activation and secretion of cytokines that stimulate the hypothalamo-pituitary-adrenal (HPA) axis to release the anti-inflammatory corticosterone. Upstream in HPA axis are neuroendocrine corticotropin-releasing hormone neurons in the paraventricular nucleus whose multipeptidergic phenotype changes during inflammation: coexisting corticotropin-releasing hormone and cholecystokinin mRNAs are up-regulated whereas neurotensin mRNA expression is induced de novo. These changes may be mediated by prostaglandins released from perivascular and microglial cells in response to circulating cytokines. We examined by quantitative in situ hybridization histochemistry whether blockade of prostaglandin synthesis by indomethacin alters phenotypic expression in paraventricular nucleus neurons after lipopolysaccharide. Because indomethacin also elevated circulating corticosterone, animals were adrenalectomized and corticosterone replaced. Results showed that i.p. indomethacin administration suppressed lipopolysaccharide effects in a phenotype non-specific manner: one injection was sufficient to prevent both the increase in corticotropin-releasing hormone and cholecystokinin mRNAs expression and the induction of neurotensin mRNA expression. Therefore, neuroendocrine corticotropin-releasing hormone neurons with different peptidergic phenotypes appear to respond as a whole in the acute phase response to systemic infection.

Is the mineralocorticoid receptor in Brown Norway rats constitutively active?

In a previous study using corticosterone treatment of adrenalectomized rats, we hypothesized that mineralocorticoid receptor (MR)-related mechanisms are constitutively active and that glucocorticoid receptor (GR)-mediated mechanisms are more efficient in Brown Norway rats compared to Fischer 344 (F344) rats. In order to discriminate the mineralocorticoid from the glucocorticoid actions exerted by corticosterone, F344 and Brown Norway adrenalectomized rats were treated with increasing doses (1, 5 and 25 microg/ml of drinking water) of deoxycorticosterone (DOC, MR-specific ligand) or RU 28362 (GR-specific ligand). These rats were compared with long-term adrenalectomized (ADX) untreated rats and sham-ADX rats. This study confirms our previous results, notably the lack of effect of ADX on body weight and fluid intake in Brown Norway rats. Moreover, DOC treatment had no effect in Brown Norway rats whereas the higher dose restored fluid intake of the F344 ADX group to sham values. These results support the hypothesis of a constitutive activation of the MR and therefore the insensitivity of this receptor to its ligand in Brown Norway rats. Alternatively, RU 28362 treatment induced greater weight loss, decrease in food intake, anxiolysis, thymus involution, and decrease in plasma transcortin concentration and pituitary corticosteroid receptor densities in Brown Norway rats than in F344 rats, which is consistent with greater efficiency of GR mechanisms in Brown Norway rats than in F344 rats. Therefore, these strains are of great utility to disentangle MR and GR effects on complex phenotypes.

Evaluation of the chick embryo for the determination of relative virulence of Neisseria meningitidis.

The chick embryo model was evaluated as a method to compare virulence between selected strains of Neisseria meningitidis. Inoculation of 13-day-chick embryos via the egg yolk distinguished strains having an LD50 of 10(3) colony forming units (CFU) or greater (low virulence) from those having an LD50 of approximately 10(1) or less (high virulence). A strain of serogroup B and a spontaneous nonpiliated strain of group C were found to be of relatively high virulence while a strain of N. lactamica, a serogroup A carrier strain, and certain nongroupable strains were found to be of low virulence. Strains having an LD50 of 10(2) were not differentiated from either of these. Alternatively, inoculation of the chorioallantoic membrane (CAM) of 9-day-old chick embryos statistically differentiated most strains of N. meningitidis although inoculation via this route was less sensitive.

Utilization of high-performance liquid chromatography as an enrichment step for the determination of cyclic fatty acid monomers in heated fats and biological samples.

A method was developed to determine traces of cyclic fatty acid monomers (CFAM) in oils and animal tissues. This method is a combination of some techniques developed earlier but with the enrichment step being achieved by high-performance liquid chromatography (HPLC) instead of urea inclusion. After transformation of the lipids into methyl esters, the latter were hydrogenated after addition of an internal standard (methyl heptadecanoate or ethyl hexadecanoate). The mixture was enriched in CFAM by HPLC on a semi-preparative C18 reversed-phase column using acetonitrile-acetone (90:10, v/v) at 4 ml/min. The enriched fraction containing the CFAM and the internal standard was then analysed by gas chromatography on a polar column (cyanosilicone phase). This method was developed using known mixtures of CFAM isolated from both heated sunflower and linseed oils. Small amounts of CFAM (50 microg/g of sample) were determined with good reproducibility without any loss during the HPLC enrichment step and with no modification of the relative proportions of the CFAM in the mixture. This method can be applied to either heated fats and oils or biological samples (heart cell culture) that contain only traces of CFAM. Ethyl hexadecanoate (16:0 ethyl ester) can be used as an internal standard for samples containing small amounts of 17:0.

Incorporation of cyclic fatty acid monomers in lipids of rat heart cell cultures.

Primary cultures of newborn rat cardiomyocytes were grown in medium supplemented with cyclic fatty acid monomers (CFAM) which had been isolated from heated linseed oil. The cells were harvested, and lipids were extracted and fractionated using silica cartridges and high-performance liquid chromatography. The CFAM structures isolated from cellular lipids were determined and compared to those that had been supplemented to the medium, using gas-liquid chromatography coupled with mass spectrometry (GC/MS). We found that CFAM were incorporated into phospholipids and neutral lipids of cardiomyocytes. Furthermore, CFAM with a cyclopentyl ring structure were more abundant in cardiomyocytes than were the cyclohexyl ring isomers. Our data suggest that CFAM of the 5-carbon and 6-carbon ring series are metabolized differently in newborn rat cardiomyocytes.

Comparative effects of whole-body vibration on sensorimotor performance achieved with a mini-stick and a macro-stick in force and position control modes.

The aim of this investigation was to assess the performance of subjects in a target recentering task, performed under both normal and vibration conditions. A conventional helicopter stick and an arm-side controller were used in both position and force control modes. The task was designed to simulate instrument flying. The results showed that in the no-vibration situation, the highest performance was achieved in the force control mode and little difference was observed between the two sticks. They also showed that vibration impaired the velocity control of the performance. It is suggested that the subject might be switching over from a visual and arm afferent and efferent control in the no-vibration situation, to a visual control only under vibration condition. From this study, it appears that the more efficient stick to execute the designed task is the mini-stick operating in the force control mode.

PulseNet USA standardized pulsed-field gel electrophoresis protocol for subtyping of Vibrio parahaemolyticus.

PulseNet is a national molecular subtyping network for foodborne disease surveillance composed of public health and food regulatory agencies. Participants employ molecular subtyping of foodborne pathogens using a standardized method of pulsed-field gel electrophoresis (PFGE) for conducting laboratory-based surveillance of foodborne pathogens. The PulseNet standardized PFGE protocols are developed through a comprehensive testing process. The reproducibility of the protocol undergoes an internal evaluation at the Centers for Disease Control and Prevention and an external evaluation in multiple PulseNet laboratories. Here we describe the development and evaluation of a rapid PFGE protocol for subtyping Vibrio parahaemolyticus for use in PulseNet activities. The protocol was derived from the existing standardized PulseNet protocols for Escherichia coli O157:H7 and Vibrio cholerae. An external evaluation of this protocol was undertaken in collaboration with three PulseNet USA participating public health laboratories. Comparative analysis of the PFGE fingerprints generated by each of these laboratories demonstrated that the protocol is both reliable and reproducible in the hands of multiple users.

PulseNet USA: a five-year update.

PulseNet USA is the molecular surveillance network for foodborne infections in the United States. Since its inception in 1996, it has been instrumental in detection, investigation and control of numerous outbreaks caused by Shiga toxin-producing Escherichia coli O157:[H7] (STEC O157), Salmonella enterica, Listeria monocytogenes, Shigella spp., and Campylobacter. This paper describes the current status of the network, including the methodologies used and its future possibilities. The currently preferred subtyping method in the network is pulsed-field gel electrophoresis (PFGE), a proven highly discriminatory molecular subtyping method. New simpler sequencebased subtyping methods are under development and validation to complement and eventually replace PFGE. PulseNet is essentially a cluster detection network, but the data in the system will now also be used in attribution analyses of sporadic infections. The PulseNet platform will also be used as a primary tool in preparedness and response to acts of food bioterrorism.

Increase of Fas-induced apoptosis by inhibition of extracellular phosphorylation of Fas receptor in Jurkat cell line.

Apoptosis signalling through the Fas pathway requires several steps of aggregation of the Fas receptor in the membrane, including aggregation that may occur in the absence of Fas ligand. Association of Fas domains is determinant to signal transmission following Fas ligand binding to a specific domain. The domains involved in Fas aggregation are located in its extracellular region and contain three potential protein kinase C-binding motifs. We therefore studied the possibility that phosphorylation of the extracellular region of Fas might be implicated in the regulation of Fas-mediated apoptosis. Inhibition experiments of extracellular phosphorylation were performed in human Jurkat T leukemia cells with K252b, an impermeant protein-kinase inhibitor. Extracellular phosphorylation of Fas receptor was related to ecto-kinase, as assessed by the [gamma-(32)P] ATP labelling of Fas-116 kDa aggregates, suppressed by K252b inhibitor which significantly increased the sensitivity to Fas-mediated apoptosis. Ecto-PKC involvement was demonstrated by bisindolylmaleimide VIII, a selective inhibitor of protein kinase C which significantly increased both Fas aggregation in the membrane and Fas-mediated apoptosis and by the addition of the PKC pseudo-substrate 19-36 which inhibited the phosphorylation of 116 kDa Fas aggregates. These data support a role for Fas phosphorylation in the decreased sensitivity to apoptosis in the Jurkat T leukemia cell line.

Development and validation of a PulseNet standardized pulsed-field gel electrophoresis protocol for subtyping of Vibrio cholerae.

PulseNet is a network that utilizes standardized pulsed-field gel electrophoresis (PFGE) protocols with the purpose of conducting laboratory-based surveillance of foodborne pathogens. PulseNet standardized PFGE protocols are subject to rigorous testing during the developmental phase and careful evaluation during a validation process assessing its robustness and reproducibility in different laboratories. Here we describe the development and validation of a rapid PFGE protocol for subtyping Vibrio cholerae for use in PulseNet International activities. While the protocol was derived from the existing PulseNet protocol for Escherichia coli O157, various aspects of this protocol were optimized for use with V. cholerae, most notably a change of the primary and secondary restriction enzyme to SfiI and NotI, respectively, and the use of a two-block electrophoresis program. External validation of this protocol was undertaken through a collaboration between three PulseNet Asia Pacific laboratories (Public Health Laboratory Centre, Hong Kong, National Institute of Infectious Diseases, Japan, and International Center for Diarrhoeal Diseases Research-Bangladesh) and PulseNet USA. Comparison of PFGE patterns generated by each of the participating laboratories demonstrated that the protocol is robust and reproducible.