Nano-bio interactions of upconversion nanoparticles at subcellular level: biodistribution and cytotoxicity.

Background: Modern medicine requires intensive research to find new diagnostic and therapeutic solutions. Recently, upconverting nanoparticles (UCNPs) doped with lanthanide ions have attracted significant attention. Methods: The efficient internalization of UCNPs by cells was confirmed, and their precise cellular localization was determined by electron microscopy and confocal studies. Results: UCNPs colocalized only with specific organelles, such as early endosomes, late endosomes and lysosomes. Furthermore, experiments with chemical inhibitors confirmed the involvement of endocytosis in UCNPs internalization and helped select several mechanisms involved in internalization. Exposure to selected UCNPs concentrations did not show significant cytotoxicity, induction of oxidative stress or ultrastructural changes in cells. Conclusion: This study suggests that UCNPs offer new diagnostic options for biomedical infrared imaging.

Molecular subgroups of atypical teratoid rhabdoid tumours in children: an integrated genomic and clinicopathological analysis.

BACKGROUND: Rhabdoid brain tumours, also called atypical teratoid rhabdoid tumours, are lethal childhood cancers with characteristic genetic alterations of SMARCB1/hSNF5. Lack of biological understanding of the substantial clinical heterogeneity of these tumours restricts therapeutic advances. We integrated genomic and clinicopathological analyses of a cohort of patients with atypical teratoid rhabdoid tumours to find out the molecular basis for clinical heterogeneity in these tumours. METHODS: We obtained 259 rhabdoid tumours from 37 international institutions and assessed transcriptional profiles in 43 primary tumours and copy number profiles in 38 primary tumours to discover molecular subgroups of atypical teratoid rhabdoid tumours. We used gene and pathway enrichment analyses to discover group-specific molecular markers and did immunohistochemical analyses on 125 primary tumours to evaluate clinicopathological significance of molecular subgroup and ASCL1-NOTCH signalling. FINDINGS: Transcriptional analyses identified two atypical teratoid rhabdoid tumour subgroups with differential enrichment of genetic pathways, and distinct clinicopathological and survival features. Expression of ASCL1, a regulator of NOTCH signalling, correlated with supratentorial location (p=0·004) and superior 5-year overall survival (35%, 95% CI 13-57, and 20%, 6-34, for ASCL1-positive and ASCL1-negative tumours, respectively; p=0·033) in 70 patients who received multimodal treatment. ASCL1 expression also correlated with superior 5-year overall survival (34%, 7-61, and 9%, 0-21, for ASCL1-positive and ASCL1-negative tumours, respectively; p=0·001) in 39 patients who received only chemotherapy without radiation. Cox hazard ratios for overall survival in patients with differential ASCL1 enrichment treated with chemotherapy with or without radiation were 2·02 (95% CI 1·04-3·85; p=0·038) and 3·98 (1·71-9·26; p=0·001). Integrated analyses of molecular subgroupings with clinical prognostic factors showed three distinct clinical risk groups of tumours with different therapeutic outcomes. INTERPRETATION: An integration of clinical risk factors and tumour molecular groups can be used to identify patients who are likely to have improved long-term radiation-free survival and might help therapeutic stratification of patients with atypical teratoid rhabdoid tumours. FUNDING: C17 Research Network, Genome Canada, b.r.a.i.n.child, Mitchell Duckman, Tal Doron and Suri Boon foundations.

Exome sequencing identifies NFS1 deficiency in a novel Fe-S cluster disease, infantile mitochondrial complex II/III deficiency.

Iron-sulfur (Fe-S) clusters are a class of highly conserved and ubiquitous prosthetic groups with unique chemical properties that allow the proteins that contain them, Fe-S proteins, to assist in various key biochemical pathways. Mutations in Fe-S proteins often disrupt Fe-S cluster assembly leading to a spectrum of severe disorders such as Friedreich's ataxia or iron-sulfur cluster assembly enzyme (ISCU) myopathy. Herein, we describe infantile mitochondrial complex II/III deficiency, a novel autosomal recessive mitochondrial disease characterized by lactic acidemia, hypotonia, respiratory chain complex II and III deficiency, multisystem organ failure and abnormal mitochondria. Through autozygosity mapping, exome sequencing, in silico analyses, population studies and functional tests, we identified c.215G>A, p.Arg72Gln in NFS1 as the likely causative mutation. We describe the first disease in man likely caused by deficiency in NFS1, a cysteine desulfurase that is implicated in respiratory chain function and iron maintenance by initiating Fe-S cluster biosynthesis. Our results further demonstrate the importance of sufficient NFS1 expression in human physiology.

A multiplex human syndrome implicates a key role for intestinal cell kinase in development of central nervous, skeletal, and endocrine systems.

Six infants in an Old Order Amish pedigree were observed to be affected with endocrine-cerebro-osteodysplasia (ECO). ECO is a previously unidentified neonatal lethal recessive disorder with multiple anomalies involving the endocrine, cerebral, and skeletal systems. Autozygosity mapping and sequencing identified a previously unknown missense mutation, R272Q, in ICK, encoding intestinal cell kinase (ICK). Our results established that R272 is conserved across species and among ethnicities, and three-dimensional analysis of the protein structure suggests protein instability due to the R272Q mutation. We also demonstrate that the R272Q mutant fails to localize at the nucleus and has diminished kinase activity. These findings suggest that ICK plays a key role in the development of multiple organ systems.

Coma with diffuse white matter hemorrhages in juvenile diabetic ketoacidosis.

Cerebral edema is the most common neurologic complication of diabetic ketoacidosis in children. A minority of young patients with intracerebral crises in diabetic ketoacidosis present with cerebrovascular accidents. We report 2 adolescent patients with diabetic ketoacidosis who presented with coma and diffuse white matter hemorrhages in the absence of either cerebral edema or cerebrovascular accidents. These 2 cases illustrate a novel clinical and neuropathologic description of diffuse white matter hemorrhages, possibly related to a cytotoxic process as the underlying mechanism. These case descriptions emphasize that pediatric patients with diabetic ketoacidosis and coma can present with pathology not related to either cerebral edema or cerebrovascular accidents.

Polyhydramnios, megalencephaly and symptomatic epilepsy caused by a homozygous 7-kilobase deletion in LYK5.

We used single nucleotide polymorphism (SNP) microarrays to investigate the cause of a symptomatic epilepsy syndrome in a group of seven distantly related Old Order Mennonite children. Autozygosity mapping was inconclusive, but closer inspection of the data followed by formal SNP copy number analyses showed that all affected patients had homozygous deletions of a single SNP (rs721575) and their parents were hemizygous for this marker. The deleted SNP marked a larger deletion encompassing exons 9-13 of LYK5, which encodes STE20-related adaptor protein, a pseudokinase necessary for proper localization and function of serine/threonine kinase 11 (a.k.a. LKB1). Homozygous LYK5 deletions were associated with polyhydramnios, preterm labour and distinctive craniofacial features. Affected children had large heads, infantile-onset intractable multifocal seizures and severe psychomotor retardation. We designated this condition PMSE syndrome (polyhydramnios, megalencephaly and symptomatic epilepsy). Thirty-eight percent (N = 16) of affected children died during childhood (ages 7 months to 6 years) from medical complications of the disorder, which included status epilepticus, congestive heart failure due to atrial septal defect and hypernatremic dehydration due to diabetes insipidus. A single post-mortem neuropathological study revealed megalencephaly, ventriculomegaly, cytomegaly and extensive vacuolization and astrocytosis of white matter. There was abundant anti-phospho-ribosomal S6 labelling of large cells within the frontal cortex, basal ganglia, hippocampus and spinal cord, consistent with constitutive activation of the mammalian target of rapamycin (mTOR) signalling pathway in brain.

The cellular inflammatory response in human spinal cords after injury.

Spinal cord injury (SCI) provokes an inflammatory response that generates substantial secondary damage within the cord but also may contribute to its repair. Anti-inflammatory treatment of human SCI and its timing must be based on knowledge of the types of cells participating in the inflammatory response, the time after injury when they appear and then decrease in number, and the nature of their actions. Using post-mortem spinal cords, we evaluated the time course and distribution of pathological change, infiltrating neutrophils, monocytes/macrophages and lymphocytes, and microglial activation in injured spinal cords from patients who were 'dead at the scene' or who survived for intervals up to 1 year after SCI. SCI caused zones of pathological change, including areas of inflammation and necrosis in the acute cases, and cystic cavities with longer survival (Zone 1), mantles of less severe change, including axonal swellings, inflammation and Wallerian degeneration (Zone 2) and histologically intact areas (Zone 3). Zone 1 areas increased in size with time after injury whereas the overall injury (size of the Zones 1 and 2 combined) remained relatively constant from the time (1-3 days) when damage was first visible. The distribution of inflammatory cells correlated well with the location of Zone 1, and sometimes of Zone 2. Neutrophils, visualized by their expression of human neutrophil alpha-defensins (defensin), entered the spinal cord by haemorrhage or extravasation, were most numerous 1-3 days after SCI, and were detectable for up to 10 days after SCI. Significant numbers of activated CD68-immunoreactive ramified microglia and a few monocytes/macrophages were in injured tissue within 1-3 days of SCI. Activated microglia, a few monocytes/macrophages and numerous phagocytic macrophages were present for weeks to months after SCI. A few CD8(+) lymphocytes were in the injured cords throughout the sampling intervals. Expression by the inflammatory cells of the oxidative enzymes myeloperoxidase (MPO) and nicotinamide adenine dinucleotide phosphate oxidase (gp91(phox)), and of the pro-inflammatory matrix metalloproteinase (MMP)-9, was analysed to determine their potential to cause oxidative and proteolytic damage. Oxidative activity, inferred from MPO and gp91(phox) immunoreactivity, was primarily associated with neutrophils and activated microglia. Phagocytic macrophages had weak or no expression of MPO or gp91(phox). Only neutrophils expressed MMP-9. These data indicate that potentially destructive neutrophils and activated microglia, replete with oxidative and proteolytic enzymes, appear within the first few days of SCI, suggesting that anti-inflammatory 'neuroprotective' strategies should be directed at preventing early neutrophil influx and modifying microglial activation.

Fatal cerebral edema from late-onset ornithine transcarbamylase deficiency in a juvenile male patient receiving valproic acid.

OBJECTIVES: The aims of this report are to 1) present a rare case of fatal cerebral edema associated with late-onset ornithine transcarbamylase (OTC) deficiency in a juvenile male patient receiving valproic acid and 2) review the neuropathologic changes associated with the hyperammonemia. DESIGN: Case report. SETTING: A community hospital and a tertiary pediatric critical care unit. INTERVENTIONS: Carbohydrate administration, intravenous nitrogen excretion cocktail, and high-flux hemodialysis. MEASUREMENTS AND MAIN RESULTS: Despite aggressive therapy for presumed late-onset OTC deficiency, the patient rapidly developed fatal cerebral edema with tonsillar herniation. A liver biopsy confirmed OTC deficiency with approximately 3% of residual hepatic enzyme activity. Chromosomal analysis showed a normal male karyotype. A thorough molecular analysis of the coding region in the OTC gene Xp21.1 was completed, but mutations were not identified, suggesting an upstream or downstream abnormality. Severe brain swelling was evident on neuropathology, and histopathology showed Alzheimer type II astrocytes, neuronal cytoplasmic changes, and hypertrophy and eosinophilia of the small arterial walls. CONCLUSIONS: OTC deficiency is the most common urea cycle defect causing hyperammonemia. Late-onset presentations of OTC are infrequent, primarily affecting female patients. We present a rare case of a late-onset OTC deficiency in a juvenile male patient receiving valproic acid therapy who developed fatal cerebral edema. Valproic acid exacerbates acute elevations in ammonia and may contribute synergistically with ammonia to cerebral mitochondrial dysfunction.

High-dose chemotherapy with stem cell rescue as initial therapy for anaplastic oligodendroglioma: long-term follow-up.

We previously reported a phase 2 trial of 69 patients with newly diagnosed anaplastic or aggressive oligodendroglioma who were treated with intensive procarbazine, CCNU (lomustine), and vincristine (PCV) followed by high-dose thiotepa with autologous stem cell rescue. This report summarizes the long-term follow-up of the cohort of 39 patients who received high-dose thiotepa with autologous stem cell support. Thirty-nine patients with a median age of 43 (range, 18-67) and a median KPS of 100 (range, 70-100) were treated. Surviving patients now have a median follow-up of 80.5 months (range, 44-142). The median progression-free survival is 78 months, and median overall survival has not been reached. Eighteen patients (46%) have relapsed. Neither histology nor prior low-grade oligodendroglioma correlated with risk of relapse. Persistent nonenhancing tumor at transplant was identified in our initial report as a significant risk factor for relapse; however, long-term follow-up has not confirmed this finding. Long-term neurotoxicity has developed only in those patients whose disease relapsed and required additional therapy; no patient in continuous remission has developed a delayed neurologic injury. This treatment strategy affords long-term disease control to a subset of patients with newly diagnosed anaplastic oligodendroglioma without evidence of delayed neurotoxicity or myelodysplasia.

Cytokine and cytokine receptor mRNA expression in human glioblastomas: evidence of Th1, Th2 and Th3 cytokine dysregulation.

Immunotherapies, although promising in preclinical studies, have not yet enhanced the survival of patients with glioblastomas. To further understand the immunobiology of glioblastomas in clinical settings, we examined 53 cytokine or cytokine receptor transcripts in 12 human glioblastomas and 6 human glioblastoma cell lines and correlated the findings with the degree of inflammation. Multi-probe RNase protection assays were used to examine Th1, Th2, and Th3 cytokine and cytokine receptor expression. Th2 [interleukin (IL)-6, leukemia inhibitory factor and oncostatin M] and Th3 (transforming growth factor-beta1, 2, 3) cytokine and their receptor transcripts were strongly expressed in almost all glioblastomas and glioma cell lines. Two other Th2 cytokine receptor subunit transcripts (IL-4Ralpha and IL-13Ralpha) were also commonly detected. In contrast, although Th1 cytokine receptors tumor necrosis factor (TNF) RI, interferon (IFN)-gammaRalpha, IFN-gammaRbeta, were detected, their cytokines (IFN-gamma, TNF-alpha, lymphotoxin-alpha) were not. Transcripts for IL-2 family cytokine (IL-2, IL-7, IL-9, IL-15) and receptors (IL-2Ralpha, IL-2Rbeta, gammac, IL-7Ralpha, IL-9Ralpha, IL15Ralpha) and IL-12 family cytokine (IL-12p40) and receptors (IL-12Rbeta1 and IL-12beta2) were essentially absent in both tumors and cell lines. Immunohistochemical methods showed sparse T lymphocyte infiltrates and numerous microglia in the glioblastomas. This pattern indicates an 'immunosuppressive status' in glioblastomas and could account for the failure of immunotherapy in such tumors.