Cells´ Flow and Immune Cell Priming under alternating g-forces in Parabolic Flight.

Gravitational stress in general and microgravity (µg) in particular are regarded as major stress factors responsible for immune system dysfunction in space. To assess the effects of alternating µg and hypergravity (hyper-g) on immune cells, the attachment of peripheral blood mononuclear cells (PBMCs) to adhesion molecules under flow conditions and the antigen-induced immune activation in whole blood were investigated in parabolic flight (PF). In contrast to hyper-g (1.8 g) and control conditions (1 g), flow and rolling speed of PBMCs were moderately accelerated during µg-periods which were accompanied by a clear reduction in rolling rate. Whole blood analyses revealed a "primed" state of monocytes after PF with potentiated antigen-induced pro-inflammatory cytokine responses. At the same time, concentrations of anti-inflammatory cytokines were increased and monocytes displayed a surface molecule pattern that indicated immunosuppression. The results suggest an immunologic counterbalance to avoid disproportionate immune responses. Understanding the interrelation of immune system impairing and enhancing effects under different gravitational conditions may support the design of countermeasures to mitigate immune deficiencies in space.

The Bonebridge: preclinical evaluation of a new transcutaneously-activated bone anchored hearing device.

OBJECTIVES: To assess the functional performance of the Bonebridge (BB, MED-EL), a newly-designed transcutaneous bone conduction implant that allows the skin to remain intact and to compare it with the current clinical model of choice, a percutaneous bone conduction implant (BAHA BP100, Cochlear Bone Anchored Solutions AG). MATERIALS AND METHODS: The devices were compared using two methods: (1) Measurements of cochlear promontory acceleration in five cadaver heads: Accelerations of the cochlear promontories on both ipsilateral and contralateral sides were measured using a Laser Doppler system, with free-field sound stimuli of 90 dB SPL in the frequency range of 0.3-10 kHz (2) Measurements of pure-tone sound field thresholds in 5 normally hearing human adult subjects under a condition of simulated hearing loss. For the latter measurements, the devices were applied to the head using a Softband, and measurements were performed in the frequency range of 0.25-8 kHz. Within investigation comparisons (i.e., in cadavers or listeners) and a cross-comparison analysis of the cadaver and human results were done. RESULTS: Results from the cadaver heads showed that the cochlear promontory acceleration with the BB was higher within 10 dB on the ipsilateral side and lower within 5 dB on the contralateral side than the acceleration with the BAHA, in the frequency range of 0.7-10 kHz. The transcranial attenuation of the acceleration for the BB was greater than for the BAHA within 20 dB. For the sound-field threshold assessments with human subjects, the BB and BAHA showed similar threshold improvements of more than 10 dB HL for the ipsilateral side. For the contralateral side, the threshold improvement with the BB was less than with the BAHA, indicating better separation between ipsilateral and contralateral sides. CONCLUSIONS: Preclinical results imply that the BB has functional performance similar to the BAHA and could be beneficial to patients suffering with conductive and mixed hearing losses as well as for those with unilateral impairment. Based on these preliminary results, a carefully designed clinical trial with conservative inclusion criteria can be recommended. This article is part of a special issue entitled "MEMRO 2012".

Accuracy of linear intraoral measurements using cone beam CT and multidetector CT: a tale of two CTs.

OBJECTIVES: The aim was to compare the accuracy of linear bone measurements of cone beam CT (CBCT) with multidetector CT (MDCT) and validate intraoral soft-tissue measurements in CBCT. METHODS: Comparable views of CBCT and MDCT were obtained from eight intact cadaveric heads. The anatomical positions of the gingival margin and the buccal alveolar bone ridge were determined. Image measurements (CBCT/MDCT) were performed upon multiplanar reformatted data sets and compared with the anatomical measurements; the number of non-assessable sites (NASs) was evaluated. RESULTS: Radiological measurements were accurate with a mean difference from anatomical measurements of 0.14 mm (CBCT) and 0.23 mm (MDCT). These differences were statistically not significant, but the limits of agreement for bone measurements were broader in MDCT (-1.35 mm; 1.82 mm) than in CBCT (-0.93 mm; 1.21 mm). The limits of agreement for soft-tissue measurements in CBCT were smaller (-0.77 mm; 1.07 mm), indicating a slightly higher accuracy. More NASs occurred in MDCT (14.5%) than in CBCT (8.3%). CONCLUSIONS: CBCT is slightly more reliable for linear measurements than MDCT and less affected by metal artefacts. CBCT accuracy of linear intraoral soft-tissue measurements is similar to the accuracy of bone measurements.

Murine adult neural progenitor cells alter their proliferative behavior and gene expression after the activation of Toll-like-receptor 3.

Viral infections during pregnancy significantly increase the risk for psychological pathologies like schizophrenia in the offspring. One of the main morphological hallmarks of schizophrenia is a reduced size of the hippocampus. Since new neurons are produced in this particular brain compartment throughout life, it might be possible that low neurogenesis levels triggered by a maternal viral infection contribute to developmental deficits of the hippocampus. We injected polyinosinic:polycytidylic acid (Poly I:C) in pregnant C57Bl/6 mice to stimulate an anti-viral response through TLR3 and examined gene expressions in the neuronal progenitor cells (NPCs) of the offspring at different ages. Additionally, we treated adult NPC lines with Poly I:C to investigate its direct effect. We could show for the first time that TLR3 and its downstream effector molecule IRF3 are expressed in adult NPCs. Poly I:C treatment in vitro and in vivo led to the regulation of proliferation and genes involved in antiviral response, migration, and survival. These findings indicate that NPCs of the fetus are able to react towards an in utero immune response, and thus, changes in the neuronal stem cell pool can contribute to the development of neurological diseases like schizophrenia.

Endocannabinoids and the brain immune system: new neurones at the horizon?

Whereas, in most brain compartments, neuronal cell renewal during early life is replaced by synaptic plasticity and the potentiation of existing pathways and connections, neurogenesis in the hippocampus occurs throughout adulthood. Neuronal progenitor cells in the dentate gyrus of the hippocampus are thought to be the gatekeepers of memory. Neural progenitor cell proliferation and differentiation depends on their intrinsic properties and local environment and is down-regulated in conditions associated with brain inflammation. Conversely, newly-formed neurones can survive despite chronic inflammation and, moreover, specifically arise within an inflammatory environment. Since the endocannabinoid system controls immune responses via multiple cellular and molecular targets and influences cell proliferation, fate decision and cell survival in the central nervous system, we summarise how neurogenesis might be regulated by brain cannabinoids, either directly or indirectly via the immune system. This review presents clear evidence that the cannabinoid system influences adult neurogenesis. However, there is considerable variability with regard to the strain, model and methods utilised and therefore it is difficult to compare studies investigating the cannabinoid system. As a result, it remains far from clear exactly how endocannabinoids regulate neurogenesis.

Cognitive decline as an important sign for an operable cause of dementia: chronic subdural haematoma.

BACKGROUND: Cognitive decline, slow psychomotor regression and confusion, especially in the elderly, often result in medical consultation. Frequently, these rather unspecific symptoms are interpreted as signs of beginning dementia. When mental regression is joined by tremor or motor deficits, neurodegenerative disease is commonly considered and the need for neuroimaging is underestimated. Chronic subdural haematoma (CSH) is known to be the most frequent type of intracranial bleeding, appearing mostly in the elderly after minor trauma with unspecific symptoms. The aim of this retrospective study was the identification of the leading clinical symptoms in patients with the diagnosis CSH who had been treated surgically in our Neurosurgical Department. PATIENTS AND METHOD: 356 patients with symptomatic CSH (225 male, 131 female; mean age 68.3 years), who were admitted to our Neurosurgical Department between 1992 and 2003, were included in the study. We reviewed the charts documenting preoperative clinical status, radiological signs, history of trauma, operative complications, postoperative clinical status, days of hospitalisation as well as gender and age. RESULTS: The primary surgical procedure performed in 343 patients (96.4%) was burr-hole trepanation. The leading preoperative symptoms were mnestic deficits (cognitive decline, confusion) in 192 patients (55.8%), followed by headache in 150 patients (45.5%) and motor deficit in 144 patients (41.1%). Furthermore, we found a statistically significant correlation (p<0.005) between the thickness of the left-sided haematoma and the symptoms aphasia and psychosyndrome. CONCLUSION: The leading clinical symptoms identified in our cohort were mnestic deficits, headache and motor deficit, signs that mostly appear at the beginning of demential diseases. Thus, CSH should be taken into account as an important differential diagnosis for demential and neurodegenerative diseases and neuroimaging should be demanded. Once a CSH is detected this way, the patient should be transferred to a neurosurgical department where an easy standard procedure may potentially lead to early recovery.

Close localization of DAP-kinase positive tumour-associated macrophages and apoptotic colorectal cancer cells.

The death-associated protein kinase (DAP-kinase) is a cytoskeleton-associated protein crucially involved in the induction of early apoptotic pathways. Aberrant hypermethylation of the DAP-kinase promoter plays a major role in tumorigenesis. We aimed to investigate the inactivation of DAP-kinase and its association with apoptotic cell death in 94 colorectal carcinomas. DAP-kinase promoter hypermethylation and mRNA expression were investigated using methylation-specific PCR and real-time RT-PCR, respectively. The expression of DAP-kinase, Fas, and Fas-ligand (FasL) proteins was studied by immunohistochemistry and immunofluorescence. Apoptosis of tumour cells was investigated using the TUNEL assay. DAP-kinase was expressed in tumour cells and tumour-invading macrophages and was closely associated with high numbers of apoptotic tumour cells. DAP-kinase expression co-localized with FasL overexpression in tumour-associated macrophages, and aberrant promoter hypermethylation was verified in more than 50% of carcinomas. There was a tendency for proximal tumours to show DAP-kinase promoter methylation more frequently (p = 0.07). Promoter methylation resulted in a decrease or loss of DAP-kinase protein expression in tumour cells and tumour-associated macrophages. Simultaneously, a decreased apoptotic count and loss of Fas/FasL expression was observed in tumour cells. Our study is the first to demonstrate DAP-kinase expression in invading tumour-associated macrophages in colorectal cancer. The presence of similar expression levels of DAP-kinase in tumour cells and associated macrophages, and their dependence on the promoter methylation status of the tumour cells, suggests cross talk between these cell types during apoptotic cell death.

Comparison of the Claassen and Fisher CT classification scale to predict ischemia after aneurysmatic SAH?

BACKGROUND: Delayed cerebral ischemia (DCI) is an important cause of morbidity and mortality after aneurysmatic subarachnoid hemorrhage (SAH). The severity of SAH, reflected by the amount of blood in the initial CCT, is a well-established predictor of DCI and infarction. The Fisher CT scale is widely used to predict DCI, but recent studies criticised the scale due to the fact that this scale does not differentiate between intracerebral blood clots and intraventricular hemorrhage. Thus Claasen et al. recently proposed a new grading scale to predict DCI. The aim of this study was to compare clinical scales with the CT findings and to verify this newly developed scale in a different population in order to predict DCI.[nl] PATIENTS AND METHODS: We selected from our databank of patients suffering from aneurysmatic SAH 292 cases who had been treated between 1995 and 2000. The data acquisition included clinical data, radiological diagnostic data, the postoperative surgical course as well as a follow-up according to the Glasgow outcome scale.[nl] RESULTS: 83 out of 292 patients (28.5 %) developed ischemic lesions on the CT scans reflecting DCI. The severity of SAH according to the Hunt and Hess grading, the Fisher CT scale and the Claassen CT scale correlated statistically significant to DCI. All three scales showed an increasing odds ratio, but the most consistent increase was demonstrated by the Fisher scale.[nl] CONCLUSIONS: The newly proposed Claassen CT scale provides no additional information and seems not to be superior compared to the well-established Fisher scale to predict DCI.

Multiple receptors mediate apoJ-dependent clearance of cellular debris into nonprofessional phagocytes.

Phagocytosis of apoptotic, senescent, and dying cells by macrophages is a well characterized process. More recently it has been shown that in addition to macrophages vital neighboring cells in the affected tissue participate in the cellular clearance. While scavenger receptors have been shown to mediate uptake into macrophages, it is poorly understood how cellular debris is internalized by nonprofessional phagocytes. We here analyze the endocytic activity of vital fibroblasts and epithelial cells exposed to cellular debris and membrane remnants. We show a mutual stimulation in the endocytosis of debris and apolipoproteinJ (clusterin) in these cells. Experiments using RAP (receptor-associated protein) to block ligand binding to LRP and megalin as well as studies in LRP- and megalin-deficient cells suggest that the uptake of apoJ and cellular debris is mediated by megalin, LRP, and yet unidentified internalization mechanisms.

Proteasomal degradation of oxidatively damaged endogenous histones in K562 human leukemic cells.

A number of antitumor drugs act via the oxidation of nuclear material in the tumor cell. It is therefore important to know if tumor cells can effectively and precisely cope not only with oxidatively induced DNA damage, but also with nuclear protein oxidation. In this study, we investigated the endogenous degradation of oxidatively damaged histones in K562 human leukemic cells after oxidative challenge and demonstrated a link to the overall cellular stress response pathways by poly-ADP-ribose-polymerase (PARP). After an oxidative challenge, endogenous nuclear protein degradation, as well as histone degradation, was enhanced. Among the histone fractions, histone H1 revealed the highest degradation rate, and more than 85% of the total degraded H1 disappeared in the first 30 min after oxidative challenge. Short-term degradation of histones up to 30 min, as well as long-term degradation up to 48 h after oxidative challenge, was significantly reduced in the presence of the PARP inhibitor 3-aminobenzamide, and nearly completely abrogated by the selective proteasome inhibitor lactacystin. Immunoprecipitation experiments indicated that the proteasome specifically degraded oxidized histones. Thus, we show that the nuclear proteosome system in tumor cells is capable of preventing the accumulation of oxidized proteins in this compartment and may suggest further treatment strategies to effectively interfere with the protein "repair" and replacement strategies of tumor cells.

Sonographic parenchymal and brain perfusion imaging: preliminary results in four patients following decompressive surgery for malignant middle cerebral artery infarct.

To investigate new methods of diagnostic transcranial sonography for brain parenchymal, vascular and perfusion imaging, we performed 3-D native tissue harmonic transcranial sonography (3D-nthTCS), 3-D transcranial color-coded duplex sonography (3D-TCCS), and "loss-of-correlation" imaging (LOC-TCCS) in four patients following early hemicraniectomy due to space-occupying "malignant" middle cerebral artery infarction (MMCAI). Three-dimensional datasets, utilizing 3D-nthTCS and 3D-TCCS, were created and up to 10 axial 2-D B-mode image planes, similar to CCT, reconstructed in each patient. Three-dimensional reconstructions of the circle of Willis documented one persistent carotid-T occlusion and three recanalizations of the MCA. LOC-TCCS, based on stimulated acoustic emission from an ultrasound (US) contrast agent, demonstrated a perfusion deficit in 2 of 3 patients, with regard to their infarcts. Concluding, 3D-nthTCS, 3D-TCCS and LOC-TCCS are promising tools for bedside monitoring, early prognosis and treatment evaluation for MMCAI in the postoperative period. Further studies should be performed to standardize these new methods and evaluate their applications through the intact calvarina.

Regulation of the nuclear proteasome activity in myelomonocytic human leukemia cells after adriamycin treatment.

Treatment of different human leukemia cell variants with the anthracycline adriamycin was associated with a rapid activation of the proteasome. Thus, proliferating U937, TUR, and retrodifferentiated U937 cells exhibited a 4.3-fold, 5.8-fold, and 4.3-fold proteasome activation within 15 minutes after adriamycin treatment, respectively. In contrast, little if any proteasome activation was detectable in a growth-arrested differentiated U937 population following adriamycin treatment. Further analysis of this mechanism revealed a significant reduction of adriamycin-induced proteasome activity after inhibition of poly(ADP-ribose) polymerase (PARP) by 3-aminobenzamide (3-ABA) in the proliferating leukemic cell types. These findings suggested that PARP is involved in the regulation of drug-induced proteasome activation. Indeed, anti-PARP immunoprecipitation experiments of adriamycin-treated cells revealed increasing levels of coprecipitated, enzymatically active proteasome particularly in the proliferating cell variants in contrast to the differentiated U937 cells, with a maximum after 15 minutes, and sensitivity to PARP inhibition by 3-ABA. The specific role of the PARP was investigated in U937 and TUR cell clones stably transfected with a constitutively active antisense PARP (asPARP) vector. Thus, asPARP-TUR cells developed a 25-fold increased sensitivity to adriamycin treatment. Furthermore, we investigated leukemic blasts isolated from acute myelogenous leukemia patients and obtained a similarly enhanced proteasome activity after adriamycin treatment, which was dependent on the PARP and thus could be coprecipitated with anti-PARP antibodies. Transient transfection of leukemic blasts with the asPARP vector significantly reduced the adriamycin-induced proteasome activation. These data suggest that the PARP-associated nuclear proteasome activation represents a potential target within chemotherapeutic defense mechanisms developed by leukemia cells.

Dynamic and three-dimensional transcranial ultrasonography of an arachnoid cyst in the cerebral convexity. Technical note.

Structural imaging of the brain, such as cerebral computerized tomography (CT) and magnetic resonance (MR) imaging, is state-of-the-art. Dynamic transcranial (dTC) ultrasonography and three-dimensional (3D) transcranial color-coded duplex (TCC) ultrasonography are complementary, noninvasive procedures with the capacity for real-time imaging, which may aid in the temporary management of space-occupying lesions. A 16-year-old woman presented with recurrent tension-type headaches. A space-occupying arachnoid cyst in the cerebral convexity was demonstrated on MR images. The patient underwent an examination for raised intracranial pressure. which was performed using a standard color-coded duplex ultrasonography system attached to a personal computer-based system for 3D data acquisition. Transcranial ultrasonography was used to identify the outer arachnoid membrane of the cyst, which undulated freely in response to rotation of the patient's head (headshake maneuver). Three-dimensional data sets were acquired and, using a multiplanar reformatting reconstruction algorithm, the authors obtained high-resolution images that corresponded to the initial MR image and a follow-up cranial CT scan. No detectable differences were observed on dTC or 3D TC ultrasonograms obtained at follow-up examinations performed 9 and 28 months later. Three-dimensional TCC and dTC ultrasonography may complement conventional diagnostic procedures such as MR and CT imaging. This report represents evidence of the high resolution and good reproducibility of 3D TC methods. Ultrasonography is a mobile and inexpensive tool and may be used to improve management and therapeutic strategies for patients with space-occupying brain lesions in selected cases.

Hydrogen peroxide-induced structural alterations of RNAse A.

Proteins exposed to oxidative stress are degraded via proteolytic pathways. In the present study, we undertook a series of in vitro experiments to establish a correlation between the structural changes induced by mild oxidation of the model protein RNase A and the proteolytic rate found upon exposure of the modified protein toward the isolated 20 S proteasome. Fourier transform infrared spectroscopy was used as a structure-sensitive probe. We report here strong experimental evidence for oxidation-induced conformational rearrangements of the model protein RNase A and, at the same time, for covalent modifications of amino acid side chains. Oxidation-related conformational changes, induced by H(2)O(2) exposure of the protein may be monitored in the amide I region, which is sensitive to changes in protein secondary structure. A comparison of the time- and H(2)O(2) concentration-dependent changes in the amide I region demonstrates a high degree of similarity to spectral alterations typical for temperature-induced unfolding of RNase A. In addition, spectral parameters of amino acid side chain marker bands (Tyr, Asp) revealed evidence for covalent modifications. Proteasome digestion measurements on oxidized RNase A revealed a specific time and H(2)O(2) concentration dependence; at low initial concentration of the oxidant, the RNase A turnover rate increases with incubation time and concentration. Based on these experimental findings, a correlation between structural alterations detected upon RNase A oxidation and proteolytic rates of RNase A is established, and possible mechanisms of the proteasome recognition process of oxidatively damaged proteins are discussed.

Regulation of microglial expression of integrins by poly(ADP-ribose) polymerase-1.

Excitotoxic brain lesions initially result in the primary destruction of brain parenchyma, after which microglial cells migrate towards the sites of injury. At these sites, the cells produce large quantities of oxygen radicals and cause secondary damage that accounts for most of the loss of brain function. Here we show that this microglial migration is strongly controlled in living brain tissue by expression of the integrin CD11a, regulated by the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) through the formation of a nuclear PARP-NF-kappaB-protein complex. Downregulation of PARP or CD11a by transfection with antisense DNA abrogated microglial migration almost completely and prevented neurons from secondary damage.

Apical transport of osteopontin is independent of N-glycosylation and sialylation.

Studies of how epithelial surface polarity into apical and basolateral domains is generated and maintained have proposed that carbohydrate modifications serve as apical targeting signals for proteins by interacting with lectin sorters. However, the experimental evidence in support of N-glycans, O-glycans and sialic acids mediating apical transport is still very controversial. This could be partly due to the fact that in most studies exogenously expressed proteins were analysed. One has, therefore, examined the role of carbohydrate moieties in apical targeting of the endogenous secretory protein osteopontin in MDCK cells. It was found, however, that sorting of osteopontin does not require N-glycosylation of the protein itself nor that of other factors involved in the sorting process. Incubation of cells with the inhibitor of O-glycosylation benzyl-alpha-GaINAc reduced the molecular weight of osteopontin by blocking sialic acid addition to O-glycans. Interestingly, also impairment of sialylation had no effect on polar secretion of the protein. Thus, the results show that both N-glycans and sialic acids are not essential sorting signals, suggesting that inner core carbohydrates and/or a proteinaceous signal mediate apical targeting of osteopontin.

Proteasome activation by poly-ADP-ribose-polymerase in human myelomonocytic cells after oxidative stress.

Cytotoxic action of a variety of antitumor drugs generate oxidatively modified proteins that are predominantly metabolized via the proteasome. In the present study, a differentiation-retrodifferentiation cell system was exposed to oxidative stress by hydrogen peroxide treatment. Thus, the activity of the nuclear proteasome in proliferating human U937 leukemic cells increased by 2.5-fold after hydrogen peroxide treatment. In contrast, growth-arrested differentiated U937 cells demonstrated 40% less constitutive proteasomal activity, which was not inducible after hydrogen peroxide exposure. After a retrodifferentiation process, however, in which differentiated U937 cells resume autonomous growth again, the proteasomal activity was indistinguishable from that in U937 control cells, both constitutively and after induction of oxidative stress. Moreover, cells of TUR, a differentiation-resistant U937 subclone, expressed an elevated constitutive proteasomal activity that increased by 2.5-fold after oxidative stress. Immunoblot analysis revealed that these differences in proteasomal activities did not correlate with proteasome protein expression but with protein levels of the nuclear enzyme poly-ADP-ribose-polymerase (PARP). Further studies using specific PARP inhibitors revealed that the noninducible proteasome activity in differentiated U937 cells was PARP independent, whereas the increased activity level in oxidatively stressed TUR cells was downregulated upon PARP inhibition. Immunoprecipitation experiments demonstrated a protein-protein interaction of the functional active PARP with the proteasome in correlation with the proteasome activity. Similar results were obtained by analyzing protein carbonyls after oxidative stress. Taken together, these data suggest that proliferating, rather than growth-arrested, cells metabolize oxidatively damaged nuclear proteins via the proteasome by expressing high levels of PARP.

Differential impairment of 20S and 26S proteasome activities in human hematopoietic K562 cells during oxidative stress.

The 20S proteasome and the 26S proteasome are major components of the cytosolic and nuclear proteasomal proteolytic systems. Since proteins are known to be highly susceptible targets for reactive oxygen species, the effect of H(2)O(2) treatment of K562 human hematopoietic cells toward the activities of 20S and 26S proteasomes was investigated. While the ATP-independent degradation of the fluorogenic peptide suc-LLVY-MCA was not affected by H(2)O(2) concentrations of up to 5 mM, the ATP-stimulated degradation of suc-LLVY-MCA by the 26S proteasome began to decline at 400 microM and was completely abolished at 1 mM oxidant treatment. A combination of nondenaturing electrophoresis and Western blotting let us believe that the high oxidant susceptibility of the 26S proteasome is due to oxidation of essential amino acids in the proteasome activator PA 700 which mediates the ATP-dependent proteolysis of the 26S-proteasome. The activity of the 26S-proteasome could be recovered within 24 h after exposure of cells to 1 mM H(2)O(2) but not after 2 mM H(2)O(2). In view of the specific functions of the 26S proteasome in cell cycle control and other important physiological functions, the consequences of the higher susceptibility of this protease toward oxidative stress needs to be considered.

Mobilization of late-endosomal cholesterol is inhibited by Rab guanine nucleotide dissociation inhibitor.

Cholesterol entering cells in low-density lipoproteins (LDL) via receptor-mediated endocytosis is transported to organelles of the late endocytic pathway for degradation of the lipoprotein particles. The fate of the free cholesterol released remains poorly understood, however. Recent observations suggest that late-endosomal cholesterol sequestration is regulated by the dynamics of lysobisphosphatidic acid (LBPA)-rich membranes [1]. Genetic studies have pinpointed a protein, Niemann-Pick C-1 (NPC-1), that is required for the mobilization of late-endosomal/lysosomal cholesterol by an unknown mechanism [2]. Here, we report the removal of accumulated cholesterol by overexpression of the NPC-1 protein in NPC-1-deficient fibroblasts from patients with Niemann-Pick disease, and in normal fibroblasts upon release of a progesterone-induced block of cholesterol transport. We show that late-endosomal/lysosomal cholesterol mobilization is specifically inhibited by microinjection of Rab GDP-dissociation inhibitor (Rab-GDI). Moreover, clearance of the cholesterol deposits by NPC-1 in patients' fibroblasts is accompanied by the redistribution of LBPA and of a lysosomal hydrolase that utilizes the mannose-6-phosphate receptor. Our results reveal, for the first time, the involvement of a specific molecular component of the membrane-trafficking machinery in cholesterol transport and the coupling of late-endosomal cholesterol egress to the trafficking of other lipid and protein cargo.