MR-guided focused ultrasound (MRgFUS) ablation for the treatment of nonspinal osteoid osteoma: a prospective multicenter evaluation.

BACKGROUND: Magnetic resonance-guided focused ultrasound (MRgFUS) is a novel imaging-guided surgical technique that allows the performance of noninvasive and radiation-free ablation. Presently, computed tomography (CT)-guided radiofrequency ablation, a minimally invasive percutaneous technique, is the standard for treating symptomatic osteoid osteomas. The purpose of this study was to evaluate the use of MRgFUS ablation for the treatment of nonspinal osteoid osteomas in terms of technical success, complications, and clinical success through one year of follow-up. METHODS: In this prospective multicenter study, thirty consecutive patients with a nonspinal osteoid osteoma were enrolled between May 2010 and April 2012 at three different university centers; twenty-nine of the patients were treated with use of MRgFUS. Lesions had been previously diagnosed on the basis of imaging, including dynamic contrast-enhanced MR. The mean number of sonications and energy deposition were determined. Technical success was evaluated through an assessment of complications immediately after treatment. Clinical success was determined on the basis of pain reduction as measured with a visual analog scale (VAS), recurrence, and long-term complications through twelve months. RESULTS: Technical success of MRgFUS was observed for all twenty-nine patients. The mean number of sonications (and standard deviation) was 7 ± 3, and the mean delivered acoustic energy was 1180 ± 736 J. At the twelve-month follow-up, complete clinical success was observed in twenty-six (90%) of the twenty-nine patients (95% confidence interval [CI] = 84 to 95; mean VAS, 0 ± 0 points). Partial success was observed in three (10%) of the twenty-nine patients (95% CI = 5 to 16; mean VAS score, 5 ± 0 points); two of these patients subsequently underwent CT-guided radiofrequency ablation, and one underwent open surgery. Pain score values showed a significant reduction (p < 0.001) between baseline (mean VAS score, 8 ± 1 points) and post treatment (mean VAS score, 1 ± 2 points). No complications were observed. CONCLUSIONS: MRgFUS may be an effective and safe alternative approach in the treatment of nonspinal osteoid osteoma. A complete clinical success rate of 90% was demonstrated without adverse events. MRgFUS is totally noninvasive and eliminates radiation exposure.

Squirrel monkeys (Saimiri sciureus) infected with the agent of bovine spongiform encephalopathy develop tau pathology.

Squirrel monkeys (Saimiri sciureus) were infected experimentally with the agent of classical bovine spongiform encephalopathy (BSE). Two to four years later, six of the monkeys developed alterations in interactive behaviour and cognition and other neurological signs typical of transmissible spongiform encephalopathy (TSE). At necropsy examination, the brains from all of the monkeys showed pathological changes similar to those described in variant Creutzfeldt-Jakob disease (vCJD) of man, except that the squirrel monkey brains contained no PrP-amyloid plaques typical of that disease. Constant neuropathological features included spongiform degeneration, gliosis, deposition of abnormal prion protein (PrP(TSE)) and many deposits of abnormally phosphorylated tau protein (p-Tau) in several areas of the cerebrum and cerebellum. Western blots showed large amounts of proteinase K-resistant prion protein in the central nervous system. The striking absence of PrP plaques (prominent in brains of cynomolgus macaques [Macaca fascicularis] with experimentally-induced BSE and vCJD and in human patients with vCJD) reinforces the conclusion that the host plays a major role in determining the neuropathology of TSEs. Results of this study suggest that p-Tau, found in the brains of all BSE-infected monkeys, might play a role in the pathogenesis of TSEs. Whether p-Tau contributes to development of disease or appears as a secondary change late in the course of illness remains to be determined.

Characterization of a US sheep scrapie isolate with short incubation time.

Scrapie is a naturally occurring fatal neurodegenerative disease of sheep and goats. Susceptibility to the disease is partly dependent upon the genetic makeup of the host. In a previous study it was shown that sheep intracerebrally inoculated with US scrapie inoculum (No. 13-7) developed terminal disease within an average of 19 months. We have since produced an inoculum, No. x124 from pooled brains of US-origin sheep scrapie, that results in incubations nearly threefold shorter. The present study documents clinicopathologic findings and the distribution of abnormal prion proteins (PrP(Sc)) by immunohistochemical (IHC) and Western blot (WB) techniques, in tissues of sheep inoculated with No. x124. All inoculated sheep developed clinical disease and were euthanatized within an average of 7.7 months postinoculation (MPI). Sheep that had valine/valine or alamine/valine at codon 136 of prion protein (PRNP) gene developed the disease faster and were euthanatized at an average of 4.3 and 5.6 MPI, respectively. Also, the inoculum was able to induce disease in a short time (7 MPI) in a sheep that was relatively resistant (QR at codon 171) to scrapie. This indicates that inoculum No. x124 appears to induce scrapie in shorter time than inoculum No. 13-7, especially in sheep homozygous or heterozygous for valine at codon 136.

Prion removal effect of a specific affinity ligand introduced into the manufacturing process of the pharmaceutical quality solvent/detergent (S/D)-treated plasma OctaplasLG.

BACKGROUND AND OBJECTIVES: A new chromatographic step for the selective binding of abnormal prion protein (PrP(Sc)) was developed, and optimization for PrP(Sc) capture was achieved by binding to an affinity ligand attached to synthetic resin particles. This step was implemented into the manufacturing process of the solvent/detergent (S/D)-treated biopharmaceutical quality plasma Octaplas to further improve the safety margin in terms of risk for variant Creutzfeldt-Jakob disease (vCJD) transmission. MATERIALS AND METHODS: Intermediates and Octaplas final container material, spiked with hamster brain-derived PrP(Sc)-containing fractions, were used for experiments to establish the feasibility of introducing this novel chromatography step. The binding capacity per millilitre of ligand gel was determined under the selected manufacturing conditions. In addition, the specificity of the ligand gel to bind PrP(Sc) from human sources was investigated. A validated Western blot test was used for the identification and quantification of PrP(Sc). RESULTS: A reduction factor of > or = 3.0 log(10) could be demonstrated by Western blotting, utilizing the relevant Octaplas matrix from manufacturing. In this particular cell-free plasma solution, the PrP(Sc) binding capacity of the selected gel was very high (> or = 6 log(10) ID(50)/ml, equivalent to roughly 10 log(10) ID(50)/column at manufacturing scale). The gel binds specifically PrP(Sc) from both animal (hamster and mouse) and human (sporadic and variant CJD) sources. CONCLUSION: This new single-use, disposable PrP(Sc)-harvesting gel ensures a very high capacity in terms of removing the pathogenic agent causing vCJD from the new generation OctaplasLG, in the event that prions can be found in plasma from donors incubating the disease and thereby contaminating the raw material plasma used for manufacturing.

Characterization of scrapie-infected and normal hamster blood as an experimental model for TSE-infected human blood.

Ideally, the distribution and separation properties of blood-associated infectivity in humans infected with transmissible spongiform encephalopathies (TSEs) would be investigated using endogenously infected human blood. However, technical limitations prevent these studies. We report here an extensive comparison of component separations of TSE-infected and normal hamster blood with normal human blood to evaluate its suitability as a model for TSE-infected human blood. Two leukotrap filtration systems were used to assess the similarities and differences in cell separation and distribution between human and hamster blood. Human-sized units of scrapie-infected hamster whole blood pools were produced, leukoreduced and processed following current standard blood bank procedures. Three centrifugation steps were performed, and all but one centrifugation resulted in cell separations both similar to those obtained with human blood and in compliance with the American Association of Blood Banks standards. Analysis of white blood cell removal by leukoreduction indicated that all filters performed within specification. The only deviation from human blood was for hamster platelets, which were not retained by the whole blood leukoreduction filter and partitioned together with red blood cells when blood was centrifuged to produce platelet rich plasma. This study indicates that hamster blood is a realistic substitute for human blood for investigations of the component distribution of TSE infectivity in blood.

Standards for the assay of Creutzfeldt-Jakob disease specimens.

Assays for the agent of Creutzfeldt-Jakob disease (CJD) include measurement of infectivity in different animal systems, such as wild-type or transgenic mice, and detection of PrP(Sc) by different methods and formats. The various assays could be best calibrated against each other by use of uniform readily available materials, and samples of four human brains, two from sporadic CJD patients, one from a variant CJD patient and one from a non-CJD patient, have been prepared as 10% homogenates dispensed in 2000 vials each for this purpose. Results of in vitro methods, particularly immunoblot assays, were compared in the first collaborative study described here. While dilution end-points varied, the minimum detectable volume was surprisingly uniform for most assays and differences in technical procedure, other than the sample volume tested, had no detectable systematic effect. The two specimens from sporadic CJD cases contained both type 1 and type 2 prion proteins in approximately equal proportions. The materials have been given the status of reference reagents by the World Health Organization and are available for further study and assessment of other in vitro or in vivo assay procedures.

Partitioning of TSE infectivity during ethanol fractionation of human plasma.

The practice of validating processes for their capacity to inactivate a range of non-enveloped and enveloped viruses also provides confidence that plasma products will be safe from emerging viral pathogens with known aetiology. Of greater concern are diseases of unknown or poorly defined aetiology such as the group of neurological diseases collectively called the transmissible spongiform encephalopathies (TSEs), or prion diseases, for which the best known human disease is Creutzfeldt-Jakob Disease (CJD) and its variant form (vCJD). The goal of the current study was to investigate the potential for manufacturing steps used in the production of albumin and immunoglobulin products by Kistler-Nitschmann fractionation, and the utility of nanofiltration of immunoglobulin to remove TSE agents. Two different scrapie model systems were used. In the first system infectious material used for spiking was scrapie sheep brain homogenate with infectivity titres being measured in hamsters. In the second system purified scrapie agent was used (PrP fibrils) with Western blot analysis measuring reduction in the proteinase K resistant form being used as a measure of removal. The data demonstrated substantial removal of the infectious agent by the manufacturing process in both model systems although some differences were observed in partitioning of the two different infectious materials. The hamster infectivity studies were shown to be approximately 1000 fold more sensitive than the Western Blot assay. The data from both studies provide added confidence that these plasma products are safe with respect to their potential to transmit TSE.

Mutant ubiquitin expressed in Alzheimer's disease causes neuronal death.

Ubiquitin-B+1 (UBB+1) is a mutant ubiquitin that accumulates in the neurones of patients with Alzheimer's disease (AD). Here we report on the biochemical and functional differences between ubiquitin and UBB+1 and the effect of the mutant protein on neuronal cells. UBB+1 lacks the capacity to ubiquitinate, and although it is ubiquitinated itself, UBB+1 is not degraded by the ubiquitin-proteasomal system and is quite stable in neuronal cells. Overexpression of UBB+1 in neuroblastoma cells significantly induces nuclear fragmentation and cell death. Our results demonstrate that accumulation of UBB+1 in neurones is detrimental and may contribute to neuronal dysfunction in AD patients.

Cdc2 phosphorylation of nucleolin demarcates mitotic stages and Alzheimer's disease pathology.

Nucleolin is a major multifunctional nuclear phosphoprotein that is phosphorylated by Cdc2 kinase in mitosis and that participates in a number of cellular processes. The monoclonal antibody TG-3 generated against neurofibrillary tangles (NFT) found in Alzheimer's disease (AD) is highly specific for mitotic cells in culture. We here demonstrate that phosphorylation of nucleolin by Cdc2 kinase generates the TG-3 epitope. The unique pool of TG-3 immunoreactive nucleolin appears abruptly during the prophase. It is associated with chromosomes through the metaphase and it gradually disappears during separation of chromosomes and exit from mitosis. In the brain, nucleolin was localized not only to nuclei but also to neuronal cytoplasm, and it is a marker for early NFT. In patients with AD, Cdc2 phosphorylated nucleolin was present in NFT. These findings suggest that phosphorylation of nucleolin by Cdc2 kinase is a critical event and the point of convergence of two distinct pathways, mitosis and neurodegeneration.

Ubiquitination and degradation of the zebrafish paired-like homeobox protein VSX-1.

Vsx-1 is a paired-like : CVC homeobox protein dynamically expressed during zebrafish development. Previous results indicate that Vsx-1 influences bipolar cell differentiation and maintenance of these cells in the adult retina. To understand the developmental regulation of this transcription factor, we investigated ubiquitination as a possible posttranslational mechanism. In vitro, Vsx-1 was conjugated with multiple ubiquitin moieties. Proteasome inhibitors and added ubiquitin increased the accumulation of Vsx-1-ubiquitin(n) complexes and stabilized unmodified Vsx-1. Also, in transiently transfected COS-7 cells, Vsx-1 is ubiquitinated, and pulse-chase experiments show that Vsx-1 proteolysis occurs. Vsx-1 proteins with C-terminal deletions retained the capacity for initial modification by ubiquitin but lost the capacity for efficient chain elongation. These results show that Vsx-1 is a substrate of the ubiquitin/proteasome pathway and suggest that C-terminal sequences of Vsx-1 are critical for ubiquitin chain elongation. In addition, our findings suggest that ubiquitin-dependent proteolysis regulates Vsx-1 during zebrafish retinal development.

Endogenous presenilin 1 redistributes to the surface of lamellipodia upon adhesion of Jurkat cells to a collagen matrix.

Most familial early-onset Alzheimer's disease cases are caused by mutations in the presenilin 1 (PS1) gene. Subcellular localization of the endogenous PS1 is essential for understanding its function, interactions with proteins, and role in Alzheimer's disease. Although numerous studies revealed predominant localization of PS1 to endoplasmic reticulum and Golgi, there are conflicting reports on the localization of PS1 to the cell surface. We found that endogenous PS1 is highly expressed in T lymphocytes (Jurkat cells). Using a variety of methods, we present evidence that endogenous PS1 is localized to the cell surface in addition to intracellular membrane compartments. Moreover, PS1 appeared in high levels on the surface of lamellipodia upon adhesion of the cells to a collagen matrix. The redistribution of PS1 in adhered cells was strikingly similar to that of the well characterized adhesion protein CD44. Cell surface PS1 formed complexes in vivo with actin-binding protein filamin (ABP-280), which is known to form bridges between cell surface receptors and cytoskeleton and mediate cell adhesion and cell motility. Taken together, our results suggest a role of PS1 in cell adhesion and/or cell-matrix interaction.

Ubiquitin, cellular inclusions and their role in neurodegeneration.

Covalent binding of ubiquitin to proteins marks them for degradation by the ubiquitin/ATP-dependent pathway. This pathway plays a major role in the breakdown of abnormal proteins that result from oxidative stress, neurotoxicity and mutations. Failure to eliminate ubiquitinated proteins disrupts cellular homeostasis, causing degeneration. Inclusions containing ubiquitinated proteins are commonly detected in many neurological disorders. These aggregates are mostly cytosolic; nevertheless, ubiquitinated inclusions are found in endosomes/lysosomes in Alzheimer's disease and prion encephalopathies, and in nuclei in disorders associated with CAG/polyglutamine repeats, such as Huntington's disease and spinocerebellar ataxias. Ubiquitinated aggregates must result from a malfunction or overload of the ubiquitin/ATP-dependent pathway or from structural changes in the protein substrates, halting their degradation. Prevention of protein aggregation in these diseases might offer new therapeutic leads.

Binding of amyloid beta protein to the 20 S proteasome.

Neurodegenerative disorders of aging are characterized by the intraneuronal accumulation of ubiquitin conjugates into tangles and inclusions. Ubiquitin conjugates are degraded by cellular particles known as proteasomes. We have previously shown that amyloid beta protein (Abeta) inhibits proteasomal activity and thereby blocks ubiquitin conjugate degradation. In the present studies, we found that Abeta binds the 20 S proteasome and forms a proteasome-Abeta complex. The complex was detected by Western blot with anti-Abeta antibodies. Using a 1.4 nm Nanogold-labeled Abeta, we visualized proteasome-Abeta complexes by scanning transmission electron microscopy (STEM). Analysis of the side-on oriented proteasome-Abeta complexes revealed a single gold particle, corresponding to one gold-labeled Abeta, in the middle portion of the proteasome. On end-on views of proteasome-Abeta complexes, gold was detected within the area delimited by the proteasome circular projection. Both STEM views are consistent with Abeta localization inside the proteasome along the peptide channel. Direct interaction of Abeta with the inner catalytic compartment of the proteasome may explain the generation of ubiquitin-containing lesions in Alzheimer's disease and other neurodegenerative disorders. In addition, detection of Nanogold-labeled peptide inside the 20 S eukaryotic proteasome suggests that conformational constraints for protein degradation in eukaryotic proteasomes are different from those in archaebacteria proteasomes.

Correlation of SfiI macrorestriction endonuclease fingerprint analysis of Candida parapsilosis isolates with source of isolation.

SfiI macrorestriction digests from whole chromosome DNA preparations of 46 isolates of Candida parapsilosis from vaginal (20 isolates), blood (23 isolates) and soil (three isolates) sources were examined by CHEF-MAPPER pulsed-field electrophoresis. The isolates were grouped into nine macrorestriction endonuclease fingerprint (MEF) classes according to the number or size of the macrorestriction fragments, or both. The electrophoretic karyotype (EK) was also examined and found to contain 18 karyotypic classes (named A-R). A comparison between SfiI MEF and EK demonstrated that the former correlated much better than the latter with the source of C. parapsilosis isolates. Five SfiI classes (I-V) contained only vaginal isolates (or vaginal and three soil isolates, class I), and the blood isolates were distributed between four classes (VI-IX). This relationship was less evident with the EK classes as several of these were composed of both vaginal and blood isolates (B, G, L and M). The three soil isolates were in class A which also included one vaginal isolate. We conclude that SfiI macrorestriction endonuclease patterns seem to be useful in discriminating among C. parapsilosis isolates, with apparent association with source of isolation.

Amyloid beta-protein inhibits ubiquitin-dependent protein degradation in vitro.

Intraneuronal accumulation of ubiquitin conjugates in inclusion bodies and neurofibrillary tangles is a pathological feature of neurodegenerative disorders such as Alzheimer's disease and Down's syndrome and of normal aging of the brain. Amyloid beta-protein (A beta) and its precursor are found in neurofibrillary tangle-containing neurons. A beta is the major component of extracellular plaques. We showed that A beta acts as an inhibitor of the ubiquitin-dependent protein degradation in vitro. We examined the effect of A beta on the steps of this proteolytic pathway that contribute to the level of ubiquitin conjugates in the cell. Neither conjugate formation nor conjugate deubiquitination was affected by the presence of A beta. However, A beta significantly reduced the rate of conjugate degradation. Our results indicate that A beta interacts with the proteolytic step of the ubiquitin degradative pathway. Since this step is performed by the 26 S proteasome, the effect of A beta on the catalytic core of this proteolytic complex, the 20 S proteasome, was determined. We found that A beta selectively inhibits the chymotrypsin-like activity of the 20 S proteasome. Under pathological conditions in the affected neuron, A beta could interfere with ubiquitin-dependent degradation by inhibiting the 26 S proteasome activity. This finding may explain the origin of the accumulation of ubiquitin conjugates.

Ubiquitin-mediated degradative pathway degrades the extracellular but not the intracellular form of amyloid beta-protein precursor.

The presence of ubiquitin and ubiquitin conjugates has been detected in patients affected by neurodegenerative diseases such as Alzheimer's disease. We investigated the role of ubiquitin in the degradation of amyloid beta-protein precursor (APP) and its participation in the process of amyloid beta-protein formation. APP was tested as a substrate for ubiquitin-mediated degradation, using both the extracellular and the intracellular forms of APP770, APP751 and APP695. The intracellular APP forms did not show appreciable ubiquitin-mediated degradation. In contrast, the three extracellular forms of APP were degraded in vitro by this proteolytic pathway, with similar degradation rates. Our results suggest a potential regulatory role for the ubiquitin-dependent degradation mechanism in the in vivo APP metabolic pathway.

Transthyretin sequesters amyloid beta protein and prevents amyloid formation.

The cardinal pathological features of Alzheimer disease are depositions of aggregated amyloid beta protein (A beta) in the brain and cerebrovasculature. However, the A beta is found in a soluble form in cerebrospinal fluid in healthy individuals and patients with Alzheimer disease. We postulate that sequestration of A beta precludes amyloid formation. Failure to sequester A beta in Alzheimer disease may result in amyloidosis. When we added A beta to cerebrospinal fluid of patients and controls it was rapidly sequestered into stable complexes with transthyretin. Complexes with apolipoprotein E, which has been shown to bind A beta in vitro, were not observed in cerebrospinal fluid. Additional in vitro studies showed that both purified transthyretin and apolipoprotein E prevent amyloid formation.