Diversity of Raft-Like Domains in Late Endosomes

BACKGROUND: Late endosomes, the last sorting station in the endocytic pathway before lysosomes, are pleiomorphic organelles composed of tubular elements as well as vesicular regions with a characteristic multivesicular appearance, which play a crucial role in intracellular trafficking. Here, we have investigated whether, in addition to these morphologically distinguishable regions, late endosomal membranes are additionally sub-compartmentalized into membrane microdomains. METHODOLOGY/PRINCIPAL FINDINGS: Using sub-organellar fractionation techniques, both with and without detergents, combined with electron microscopy, we found that both the limiting membrane of the organel and the intraluminal vesicles contain raft-type membrane domains. Interestingly, these differentially localized domains vary in protein composition and physico-chemical properties. CONCLUSIONS/SIGNIFICANCE: In addition to the multivesicular organization, we find that late endosomes contain cholesterol rich microdomains both on their limiting membrane and their intraluminal vesicles that differ in composition and properties. Implications of these findings for late endosomal functions are discussed

Corneal Alterations during Combined Therapy with Cyclodextrin/Allopregnanolone and Miglustat in a Knock-Out Mouse Model of NPC1 Disease

BACKGROUND: Niemann Pick disease type C1 is a neurodegenerative disease caused by mutations in the NPC1 gene, which result in accumulation of unesterified cholesterol and glycosphingolipids in the endosomal-lysosomal system as well as limiting membranes. We have previously shown the corneal involvement in NPC1 pathology in form of intracellular inclusions in epithelial cells and keratocytes. The purpose of the present study was to clarify if these inclusions regress during combined substrate reduction- and by-product therapy (SRT and BPT). METHODOLOGY/PRINCIPAL FINDINGS: Starting at postnatal day 7 (P7) and thereafter, NPC1 knock-out mice (NPC1(-/-)) and wild type controls (NPC1(+/+)) were injected with cyclodextrin/allopregnanolone weekly. Additionally, a daily miglustat injection started at P10 until P23. Starting at P23 the mice were fed powdered chow with daily addition of miglustat. The sham group was injected with 0.9% NaCl at P7, thereafter daily starting at P10 until P23, and fed powdered chow starting at P23. For corneal examination, in vivo confocal laser-scanning microscopy (CLSM) was performed one day before experiment was terminated. Excised corneas were harvested for lipid analysis (HPLC/MS) and electron microscopy. In vivo CLSM demonstrated a regression of hyperreflective inclusions in all treated NPC1(-/-)mice. The findings varied between individual mice, demonstrating a regression, ranging from complete absence to pronounced depositions. The reflectivity of inclusions, however, was significantly lower when compared to untreated and sham-injected NPC1(-/-) mice. These confocal findings were confirmed by lipid analysis and electron microscopy. Another important CLSM finding revealed a distinct increase of mature dendritic cell number in corneas of all treated mice (NPC1(-/-) and NPC1(+/+)), including sham-treated ones. CONCLUSIONS/SIGNIFICANCE: The combined substrate reduction- and by-product therapy revealed beneficial effects on the cornea. In vivo CLSM is a non-invasive tool to monitor disease progression and treatment effects in NPC1 disorder

What is the role of routine follow-up for localised limb soft tissue sarcomas? A retrospective analysis of 174 patients

BACKGROUND: There are neither prospective data nor agreement on the optimal routine follow-up procedures in patients treated for soft tissue sarcoma of the limb. METHODS: Data on 174 consecutive patients with a soft tissue sarcoma of the limb undergoing follow-up by oncologists at a single centre from 2003 to 2009 were included in this analysis. The rate and site of recurrence and mode of detection were analysed. Outcome of the patients was assessed. RESULTS: Eighty-two patients (47%) experienced relapse of any type. Isolated local recurrence occurred in 26 patients and local relapse with synchronous pulmonary metastases in five patients. Local recurrences were detected clinically in 30 of these 31 patients; magnetic resonance imaging identified only one local recurrence. Twenty-eight patients developed isolated lung metastases; in nine patients these were amenable to resections, seven of whom are currently free of disease after treatment. Lung metastases were detected by chest x-ray (CXR) in 19 patients, computed tomography scanning in 3 patients, and clinically in 11 patients. Twenty-three patients developed non-pulmonary metastases. More than 80% of relapses occurred in the first 2 years of follow-up; however, later recurrences were also observed. CONCLUSIONS: Routine follow-up CXR can detect lung metastases suitable for surgical resection, although the optimal interval of imaging has yet to be defined. Local relapse is almost always detected by patients or physicians, and routine scanning of the primary site is of doubtful benefit. Patient and physician education to detect local relapse may be helpful. Prospective evaluation of follow-up is recommended