Differential regulation of myofibrillar proteins in skeletal muscles of septic mice.

Sepsis elicits skeletal muscle atrophy as a result of decreased total protein synthesis and/or increased total protein degradation. It is unknown how and whether sepsis differentially affects the expression of specific myofibrillar proteins in respiratory and limb muscles. In this study, we measured the effects of sepsis myofibrillar mRNAs and their corresponding protein levels in the diaphragm (DIA) and tibialis anterior (TA) muscles in a murine cecal ligation and perforation (CLP) model of sepsis. Male mice (C57/BL6j) underwent CLP-induced sepsis. Sham-operated mice were subjected to the same surgical procedures, except for CLP. Mice were euthanized 24, 48, or 96 h postsurgery. Transcript and protein levels of autophagy-related genes, ubiquitin E3 ligases, and several myofibrillar genes were quantified. Sepsis elicited transient fiber atrophy in the DIA and prolonged atrophy in the TA. Atrophy was coincident with increased autophagy and ubiquitin E3 ligase expression. Myosin heavy chain isoforms decreased at 24 h in the DIA and across the time-course in the TA, myosin light chain isoforms decreased across the time-course in both muscles, and troponins T and C as well as tropomyosin decreased after 24 and 48 h in both the DIA and TA. α-Actin and troponin I were unaffected by sepsis. Sepsis-induced decreases in myofibrillar protein levels coincided with decreased mRNA expressions of these proteins, suggesting that transcriptional inhibition is involved. We hypothesize that sepsis-induced muscle atrophy is mediated by decreased transcription and enhanced degradation of specific myofibrillar proteins, including myosin heavy and light chains, troponin C, troponin T, and tropomyosin.

Differential Regulation of the Autophagy and Proteasome Pathways in Skeletal Muscles in Sepsis.

OBJECTIVES: Skeletal muscle fiber atrophy develops in response to severe sepsis, but it is unclear as to how the proteolytic pathways that are involved in its development are differentially regulated. We investigated the link between sepsis-induced fiber atrophy and activation of the proteasome and autophagy pathways and whether the degree of activation is more severe and sustained in limb muscles than it is in the diaphragm. DESIGN: Randomized controlled experiment. SETTING: Animal research laboratory. SUBJECTS: Adult male C57/BL6 mice. INTERVENTIONS: Two groups of animals were studied. The sepsis group was subjected to a cecal ligation and perforation technique, whereas the control (sham) group was subjected to abdominal surgery without cecal ligation and perforation. Measurements for both groups were performed 24, 48, and 96 hours after the surgical procedure. MEASUREMENTS AND MAIN RESULTS: Atrophy was quantified in the diaphragm and tibialis anterior by measuring fiber diameter. Autophagy was evaluated using electron microscopic detection of autophagosomes and by measuring LC3B protein lipidation and autophagy-related protein expressions. Proteasomal degradation was quantified by measuring chymotrypsin-like activity of the 26S proteasome and messenger RNA expressions of muscle-specific E3 ligases. Sepsis triggered transient fiber atrophy in the diaphragm that lasted for 24 hours and prolonged atrophy in the tibialis anterior that persisted for 96 hours. The autophagy and proteasome pathways were activated in both muscles at varying intensities over the time course of sepsis. Activation was more pronounced in the tibialis anterior than in the diaphragm. Sepsis inhibited the V-Akt thymoma viral oncogene homolog 1 and complex 1 of the mammalian target of rapamycin pathways and stimulated the AMP-activated protein kinase pathway in both muscles. CONCLUSIONS: Sepsis triggers more severe and sustained muscle fiber atrophy in limb muscles when compared with respiratory muscle. This response is associated with enhanced proteasomal and autophagic proteolytic pathway activities and is triggered by inhibition of the AKT and complex 1 of the mammalian target of rapamycin pathways and activation of the AMPK pathway.

An immunogenic peptide in the A-box of HMGB1 protein reverses apoptosis-induced tolerance through RAGE receptor.

Apoptotic cells trigger immune tolerance in engulfing phagocytes. This poorly understood process is believed to contribute to the severe immunosuppression and increased susceptibility to nosocomial infections observed in critically ill sepsis patients. Extracellular high mobility group box 1 (HMGB1) is an important mediator of both sepsis lethality and the induction of immune tolerance by apoptotic cells. We have found that HMGB1 is sensitive to processing by caspase-1, resulting in the production of a fragment within its N-terminal DNA-binding domain (the A-box) that signals through the receptor for advanced glycation end products (RAGE) to reverse apoptosis-induced tolerance. In a two-hit mouse model of sepsis, we show that tolerance to a secondary infection and its associated mortality were effectively reversed by active immunization with dendritic cells treated with HMGB1 or the A-box fragment, but not a noncleavable form of HMGB1. These findings represent a novel link between caspase-1 and HMGB1, with potential therapeutic implications in infectious and inflammatory diseases.

Cellular inhibitors of apoptosis proteins cIAP1 and cIAP2 are required for efficient caspase-1 activation by the inflammasome.

Pathogen and danger recognition by the inflammasome activates inflammatory caspases that mediate inflammation and cell death. The cellular inhibitor of apoptosis proteins (cIAPs) function in apoptosis and innate immunity, but their role in modulating the inflammasome and the inflammatory caspases is unknown. Here we report that the cIAPs are critical effectors of the inflammasome and are required for efficient caspase-1 activation. cIAP1, cIAP2, and the adaptor protein TRAF2 interacted with caspase-1-containing complexes and mediated the activating nondegradative K63-linked polyubiquitination of caspase-1. Deficiency in cIAP1 (encoded by Birc2) or cIAP2 (Birc3) impaired caspase-1 activation after spontaneous or agonist-induced inflammasome assembly, and Birc2(-/-) or Birc3(-/-) mice or mice administered with an IAP antagonist had a dampened response to inflammasome agonists and were resistant to peritonitis. Our results describe a role for the cIAPs in innate immunity and further demonstrate the evolutionary conservation between cell death and inflammation mechanisms.

Caspase-12 controls West Nile virus infection via the viral RNA receptor RIG-I.

Caspase-12 has been shown to negatively modulate inflammasome signaling during bacterial infection. Its function in viral immunity, however, has not been characterized. We now report an important role for caspase-12 in controlling viral infection via the pattern-recognition receptor RIG-I. After challenge with West Nile virus (WNV), caspase-12-deficient mice had greater mortality, higher viral burden and defective type I interferon response compared with those of challenged wild-type mice. In vitro studies of primary neurons and mouse embryonic fibroblasts showed that caspase-12 positively modulated the production of type I interferon by regulating E3 ubiquitin ligase TRIM25-mediated ubiquitination of RIG-I, a critical signaling event for the type I interferon response to WNV and other important viral pathogens.

Control of intestinal homeostasis, colitis, and colitis-associated colorectal cancer by the inflammatory caspases.

Inflammatory caspases are essential effectors of inflammation and cell death. Here, we investigated their roles in colitis and colorectal cancer and report a bimodal regulation of intestinal homeostasis, inflammation and tumorigenesis by caspases-1 and -12. Casp1(-/-) mice exhibited defects in mucosal tissue repair and succumbed rapidly after dextran sulfate sodium administration. This phenotype was rescued by administration of exogenous interleukin-18 and was partially reproduced in mice deficient in the inflammasome adaptor ASC. Casp12(-/-) mice, in which the inflammasome is derepressed, were resistant to acute colitis and showed signs of enhanced repair. Together with their increased inflammatory response, the enhanced repair response of Casp12(-/-) mice rendered them more susceptible to colorectal cancer induced by azoxymethane (AOM)+DSS. Taken together, our results indicate that the inflammatory caspases are critical in the induction of inflammation in the gut after injury, which is necessary for tissue repair and maintenance of immune tolerance.

Turbellarian black spot disease in bluespine unicornfish Naso unicornis in New Caledonia, caused by the parasitic turbellarian Piscinquilinus sp.

Turbellarian black spot disease is described in a bluespine unicornfish Naso unicornis (Perciformes, Acanthuridae) collected from the wild off Nouméa, New Caledonia, South Pacific. Each cyst contained a whitish worm, 2 to 4 mm in length, which was able to swim freely in seawater. Cyst walls consisted of fibrous tissue with a scattering of melanocytes. Worms had 2 eyespots at the anterior end, a pharynx at the posterior end and a largely undeveloped reproductive system. They were identified as Piscinquilinus sp. (= Ichthyophaga sp.), a genostomatid turbellarian. The disease spread within 2 wk from a single infected fish to 3 other unicornfish in the same tank, in spite of copper sulphate and diluted seawater treatment; however, other fish from several families kept in the same tank were not infected, suggesting specificity of these parasites. Praziquantel injections into the infected fish eliminated the infection. Other cases of turbellarian infections are reported from 7 fish species off New Caledonia; prevalence is very low (0.3%).

Caspase-12 modulates NOD signaling and regulates antimicrobial peptide production and mucosal immunity.

Bacterial sensing by intracellular Nod proteins and other Nod-like receptors (NLRs) activates signaling pathways that mediate inflammation and pathogen clearance. Nod1 and Nod2 associate with the kinase Rip2 to stimulate NF-kappaB signaling. Other cytosolic NLRs assemble caspase-1-activating multiprotein complexes termed inflammasomes. Caspase-12 modulates the caspase-1 inflammasome, but unlike other NLRs, Nod1 and Nod2 have not been linked to caspases, and mechanisms regulating the Nod-Rip2 complex are less clear. We report that caspase-12 dampens mucosal immunity to bacterial infection independent of its effects on caspase-1. Caspase-12 deficiency enhances production of antimicrobial peptides, cytokines, and chemokines to entric pathogens, an effect dependent on bacterial type III secretion and the Nod pathway. Mechanistically, caspase-12 binds to Rip2, displacing Traf6 from the signaling complex, inhibiting its ubiquitin ligase activity, and blunting NF-kappaB activation. Nod activation and resulting antimicrobial peptide production constitute an early innate defense mechanism, and caspase-12 inhibits this mucosal antimicrobial response.

Applications of direct detection device in transmission electron microscopy.

A prototype direct detection device (DDD) camera system has shown great promise in improving both the spatial resolution and the signal to noise ratio for electron microscopy at 120-400 keV beam energies (Xuong et al., 2007. Methods in Cell Biology, 79, 721-739). Without the need for a resolution-limiting scintillation screen as in the charge coupled device (CCD), the DDD camera can outperform CCD based systems in terms of spatial resolution, due to its small pixel size (5 microm). In this paper, the modulation transfer function (MTF) of the DDD prototype is measured and compared with the specifications of commercial scientific CCD camera systems. Combining the fast speed of the DDD with image mosaic techniques, fast wide-area imaging is now possible. In this paper, the first large area mosaic image and the first tomography dataset from the DDD camera are presented, along with an image processing algorithm to correct the specimen drift utilizing the fast readout of the DDD system.

Alteration of soil rhizosphere communities following genetic transformation of white spruce.

The application of plant genetic manipulations to agriculture and forestry with the aim of alleviating insect damage through Bacillus thuringiensis transformation could lead to a significant reduction in the release of pesticides into the environment. However, many groups have come forward with very valid and important questions related to potentially adverse effects, and it is crucial to assess and better understand the impact that this technology might have on ecosystems. In this study, we analyzed rhizosphere soil samples collected from the first B. thuringiensis-transformed trees [with insertion of the CryIA(b) toxin-encoding gene] grown in Canada (Val-Cartier, QC, Canada) as part of an ecological impact assessment project. Using a robust amplified rRNA gene restriction analysis approach coupled with 16S rRNA gene sequencing, the rhizosphere-inhabiting microbial communities of white spruce (Picea glauca) genetically modified by biolistic insertion of the cryIA(b), uidA (beta-glucuronidase), and nptII genes were compared with the microbial communities associated with non-genetically modified counterparts and with trees in which only the genetic marker genes uidA and nptII have been inserted. Analysis of 1,728 rhizosphere bacterial clones (576 clones per treatment) using a Cramér-von Mises statistic analysis combined with a Monte Carlo comparison clearly indicated that there was a statistically significant difference (P < 0.05) between the microbial communities inhabiting the rhizospheres of trees carrying the cryIA(b), uidA, and nptII transgenes, trees carrying only the uidA and nptII transgenes, and control trees. Clear rhizosphere microbial community alterations due to B. thuringiensis tree genetic modification have to our knowledge never been described previously and open the door to interesting questions related to B. thuringiensis genetic transformation and also to the impact of commonly used uidA and nptII genetic marker genes.

Safety and effectiveness of radioactive coil embolization of aneurysms: effects of radiation on recanalization, clot organization, neointima formation, and surrounding nerves in experimental models.

BACKGROUND AND PURPOSE: Recanalization after coil embolization can be prevented by radiation emitted from 32P coils. We wanted to determine the upper limits of 32P activities that could be implanted onto coils with respect to the potential injury to nearby nerves, delay in organization of the clot, and effects on neointima formation and recanalization. METHODS: We studied the effects of various 32P activities on recanalization and organization of thrombus after coil occlusion of canine arteries and on neointima formation at the neck of canine carotid bifurcation aneurysms. We also tested potential injury to nerves in the vicinity of radioactive or nonradioactive coils in 3 models: the brachial plexus (near proximal vertebral arteries) and the lingual nerve in a lingual artery bifurcation aneurysm model, both models being treated by radioactive or standard coil occlusion. Finally, we wrapped lingual nerves with nonradioactive or high-activity coils and studied their effects on lingual nerves and tongues. Results were assessed with a pathological scoring system and compared with Mann-Whitney and Kruskal-Wallis tests. RESULTS: No deleterious effect of radiation on nerves could be detected. Neointima formation was not hampered, scores of aneurysms treated with 32P-coils being significantly better when compared with treatments with standard coils (P=0.002). Arteries treated with high-activity coils (>3.39 microCi) showed absent recanalization but delayed organization of the clot at 3 months compared with low-activity or nonradioactive coils (P<0.05). CONCLUSIONS: beta-Radiation can prevent recanalization after coil occlusion. We could not demonstrate any deleterious effects of radioactivity on nervous structure or on neointima formation. Delayed organization of thrombus provides a rational basis to establish an upper limit for 32P activities to be implanted onto coils.

Production of radioactive particles for endovascular therapeutic interventions.

Recanalization is a common phenomenon that decreases the efficacy of embolization procedures. It can be inhibited by beta-radiation. Two novel ways of producing radioactive particles are described, by neutron beam irradiation of gold-containing microspheres, or by using the 32P binding capacity of zirconium-containing microspheres. Particles were tested in vivo, to assess their ability to deliver radioactivity locally, using canine renal artery, porcine rete mirabile, and rabbit ear embolization models. Both radioactive microspheres (198Au and 32P) showed no detectable activity outside the target territory. 32P microspheres demonstrated typical radiation changes in a porcine rete mirabile arteriovenous malformation model.

Active pixel sensor array as a detector for electron microscopy.

A new high-resolution recording device for transmission electron microscopy (TEM) is urgently needed. Neither film nor CCD cameras are systems that allow for efficient 3-D high-resolution particle reconstruction. We tested an active pixel sensor (APS) array as a replacement device at 200, 300, and 400 keV using a JEOL JEM-2000 FX II and a JEM-4000 EX electron microscope. For this experiment, we used an APS prototype with an area of 64 x 64 pixels of 20 microm x 20 microm pixel pitch. Single-electron events were measured by using very low beam intensity. The histogram of the incident electron energy deposited in the sensor shows a Landau distribution at low energies, as well as unexpected events at higher absorbed energies. After careful study, we concluded that backscattering in the silicon substrate and re-entering the sensitive epitaxial layer a second time with much lower speed caused the unexpected events. Exhaustive simulation experiments confirmed the existence of these back-scattered electrons. For the APS to be usable, the back-scattered electron events must be eliminated, perhaps by thinning the substrate to less than 30 microm. By using experimental data taken with an APS chip with a standard silicon substrate (300 microm) and adjusting the results to take into account the effect of a thinned silicon substrate (30 microm), we found an estimate of the signal-to-noise ratio for a back-thinned detector in the energy range of 200-400 keV was about 10:1 and an estimate for the spatial resolution was about 10 microm.

Endovascular treatment of intracranial aneurysms with radioactive coils: initial clinical experience.

BACKGROUND AND PURPOSE: Endovascular treatment of intracranial aneurysms is safe and effective but is associated with angiographic recurrences. Beta radiation prevents recanalization after coil embolization in experimental models. We wanted to assess the feasibility of using radioactive coil embolization to improve long-term results of endovascular treatment. METHODS: Platinum coils were ion-implanted with 0.13 to 0.26 microCi/cm of 32P. Forty-one patients aged 34 to 84 years with 44 aneurysms with a high propensity for recurrences were included. Radioactive coils were introduced into aneurysms to reach a target volumetric activity of 0.018 microCi/mm3. Nonradioactive coils were also used to ensure the same safety and the same angiographic results as the standard procedure. Angiographic results, procedure-related complications, and neurological events during follow-up were recorded. Angiographic follow-up data are available in 36 lesions 6 months after treatment. RESULTS: Forty of 44 aneurysms (91%) could be treated with radioactive coils. Target activities could be reached in 88% of lesions that could actually be coiled (35/40). Total activities ranged from 1.72 to 80.9 microCi, for a mean of 20.13+/-20.80 microCi. Procedure-related complications occurred in 7% of patients. Initial angiographic results were satisfactory (complete occlusions or residual necks) in 75% of lesions. Angiographic recurrences occurred in 11 (31%) of patients followed, within the expected range for standard coils. There was no complication from beta radiation during a mean follow-up period of 10 months. CONCLUSIONS: Radioactive coil embolization is feasible; target volumetric activities can be reached in most aneurysms considered for endovascular treatment.

Beta radiation and inhibition of recanalization after coil embolization of canine arteries and experimental aneurysms: how should radiation be delivered?

BACKGROUND AND PURPOSE: Beta radiation prevents recanalization after coil embolization. We sought to determine the effects of varying coil caliber, length, activity of 32P per centimeter of coil or per volume, and spatial distribution of coils on recanalization. METHODS: We studied the angiographic evolution of 81 canine maxillary, cervical, and vertebral arteries implanted with a variety of nonradioactive (n=29 arteries) or radioactive (n=52) devices. We compared 1- or 2-caliber 0.015 or 0.010 coils ion-implanted or not with 3 different activity levels (0.05 to 0.08, 0.06 to 0.12, 0.18 to 0.32 microCi/cm) of 32P and totaling 4, 8, and 16 cm in length for the same arterial volume. We also compared inhibition of recanalization by beta radiation delivered by stents, after coil occlusion proximal to or within the stent, with that delivered by coils placed within nonradioactive stents. We finally studied the angiographic evolution of canine lateral wall carotid aneurysms treated with 1 or 2 stents of various activity levels positioned inside the parent artery across the neck. Animals were killed at 4 and 12 weeks for macroscopic photography and pathological examination. RESULTS: All arteries (29 of 29) occluded with nonradioactive devices were recanalized, while 49 of 52 arteries (94%) implanted with 32P devices were occluded at 4 weeks. All aneurysms treated with stents, radioactive or not, demonstrated residual filling of the sac or of channels leading to the aneurysms at follow-up angiography at 4 weeks. CONCLUSIONS: The recanalization process found in the canine arterial occlusion model is minimally affected by coil caliber, number, and length or packing density. Beta radiation reliably inhibits this process, but thrombosis is an essential condition for the efficacy of a radioactive coil strategy.

Feasibility of radioactive embolization of intracranial aneurysms using 32P-implanted coils.

BACKGROUND AND PURPOSE: Beta radiation can prevent recanalization after embolization. Our goal was to assess the feasibility of endovascular treatment of intracranial aneurysms using coils of a predetermined activity of 32P per centimeter. METHODS: We studied the total length of coils deployed into 357 intracranial aneurysms. Aneurysmal volumes were estimated using 3 mathematical models. We simulated that coils were implanted with 0.26 microCi/cm of 32P, calculated resulting volumetric activities, and compared them with "effective" levels derived from experimental data and "safe" levels prescribed for the clinical use of 32P in cystic craniopharyngiomas. RESULTS: Effective activities would have been reached in 92% to 98% of lesions had the coils been radioactive at the time of treatment. CONCLUSIONS: Radioactive coil embolization of aneurysms is feasible in most patients.

In situ beta radiation to prevent recanalization after coil embolization of cerebral aneurysms.

BACKGROUND AND PURPOSE: Endovascular treatment of cerebral aneurysms, a minimally invasive alternative to surgery, is too often followed by recanalization and recurrences. The purpose of this work was to assess if in situ beta radiation can inhibit recanalization after coil embolization. METHODS: Radioactive platinum coils (32P-coils) were produced by ion implantation of 32P. A single-coil arterial occlusion model was used to compare angiographic and pathological results at 1 to 12 weeks after nonradioactive and 32P-coil embolization of maxillary, cervical, and vertebral arteries in 26 dogs. Coils of varying activities were used and results compared to define the minimal activity required to inhibit recanalization. Similar experiments were performed in 16 porcine maxillary and lingual and 8 rabbit axillary arteries. Results of 32P-coil embolization of bifurcation aneurysms were then compared with embolization with nonradioactive coils in 12 dogs at 3 months. RESULTS: Nonradioactive coil embolization of canine arteries led to occlusion at 1 week, followed by recanalization at 2 weeks, which persisted at 3 months in all cases. 32P-coils, ion-implanted with activities above 0.13 microCi/cm, led to persistent occlusion at 3 months in 80% of arteries. 32P-coils ion-implanted with the same activity inhibited recanalization in porcine and rabbit arteries. Bifurcation aneurysms treated with 32P-coils had better angiographic results at 3 months (P=0.006) than aneurysms treated with nonradioactive coils. Arteries occluded were filled with fibrous tissue at 3 months. Aneurysms embolized with 32P-coils showed more complete neointimal coverage of the neck, without recanalization, as compared with aneurysms treated with nonradioactive coils. CONCLUSION: In situ low-dose beta radiation inhibits recanalization after coil embolization and may improve long-term results of endovascular treatment of aneurysms.