Diaminobenzidine photoconversion is a suitable tool for tracking the intracellular location of fluorescently labelled nanoparticles at transmission electron microscopy.

Chitosan-based nanoparticles (NPs) deserve particular attention as suitable drug carriers in the field of pharmaceutics, since they are able to protect the encapsulated drugs and/or improve their efficacy by making them able to cross biological barriers (such as the blood-brain barrier) and reach their intracellular target sites. Understanding the intracellular location of NPs is crucial for designing drug delivery strategies. In this study, fluorescently-labelled chitosan NPs were administered in vitro to a neuronal cell line, and diaminobenzidine (DAB) photoconversion was applied to correlate fluorescence and transmission electron microscopy to precisely describe the NPs intracellular fate. This technique allowed to demonstrate that chitosan NPs easily enter neuronal cells, predominantly by endocytosis; they were found both inside membrane-bounded vesicles and free in the cytosol, and were observed to accumulate around the cell nucleus

Effect of adapted physical exercise on satellite cells from skeletal muscles of old mice: ex vivo and in vitro analyses

Aging is characterized by loss of skeletal muscle mass termed sarcopenia, which contributes to frailty, disability and premature death (1). The mechanisms leading to sarcopenia are manifold, the decline in muscle regeneration efficiency playing a crucial role. In the absence of specific therapies, studies have stressed the importance of physical exercise as an effective approach to prevent/limit the sarcopenic drive (2,3). We investigated the effect of adapted exercise on the number and myogenic properties of satellite cells (SCs) in the quadriceps femoris and gastrocnemius muscles of old mice (28 months). Both muscles contain about 90% of type II fibers, which are especially affected by sarcopenia (4). We compared old exercised mice with old sedentary mice, adult sedentary mice (12 months) being the control. SCs were identified and quantified ex vivo; the proliferation and differentiation potential of SC-derived myoblasts from the three groups of mice was studied in vitro. Ultrastructural morphology and immunocytochemistry at light and electron microscopy level localized molecular markers of SC activation and protein factors involved in RNA transcription and splicing. Results showed that: 1) physical exercise induces an increase in the total number as well as activated fraction of SCs compared with sedentary old specimens; 2) myoblasts from exercised old muscles show morphological features and nuclear activity quite similar to myoblasts from adult subjects, whereas myoblasts from non-exercised old muscles exhibit structural and functional alterations suggestive of a reduced metabolic activity; 3) myotubes differentiated from myoblasts of exercised old muscles resemble those from adult myoblasts, whereas myotubes from non-exercised old muscles show marked structural alterations. Physical exercise induces numerical increase and activation of SCs in old muscles and improves their capability to differentiate into structurally and functionally complete myotubes. Adapted physical exercise may represent a non-pharmacological approach to stimulate SCs, enhancing muscle quality at very advanced age. MC is a PhD student of the Doctoral Program “Multimodal Imaging in Biomedicine” (University of Verona)

Clinical, radiologic, pathologic, and molecular characteristics of long-term survivors of diffuse intrinsic pontine glioma (DIPG): a collaborative report from the International and European Society for Pediatric Oncology DIPG registries

Purpose Diffuse intrinsic pontine glioma (DIPG) is a brainstem malignancy with a median survival of < 1 year. The International and European Society for Pediatric Oncology DIPG Registries collaborated to compare clinical, radiologic, and histomolecular characteristics between short-term survivors (STSs) and long-term survivors (LTSs). Materials and Methods Data abstracted from registry databases included patients from North America, Australia, Germany, Austria, Switzerland, the Netherlands, Italy, France, the United Kingdom, and Croatia. Results Among 1,130 pediatric and young adults with radiographically confirmed DIPG, 122 (11%) were excluded. Of the 1,008 remaining patients, 101 (10%) were LTSs (survival ≥ 2 years). Median survival time was 11 months (interquartile range, 7.5 to 16 months), and 1-, 2-, 3-, 4-, and 5-year survival rates were 42.3% (95% CI, 38.1% to 44.1%), 9.6% (95% CI, 7.8% to 11.3%), 4.3% (95% CI, 3.2% to 5.8%), 3.2% (95% CI, 2.4% to 4.6%), and 2.2% (95% CI, 1.4% to 3.4%), respectively. LTSs, compared with STSs, more commonly presented at age < 3 or > 10 years (11% v 3% and 33% v 23%, respectively; P < .001) and with longer symptom duration ( P < .001). STSs, compared with LTSs, more commonly presented with cranial nerve palsy (83% v 73%, respectively; P = .008), ring enhancement (38% v 23%, respectively; P = .007), necrosis (42% v 26%, respectively; P = .009), and extrapontine extension (92% v 86%, respectively; P = .04). LTSs more commonly received systemic therapy at diagnosis (88% v 75% for STSs; P = .005). Biopsies and autopsies were performed in 299 patients (30%) and 77 patients (10%), respectively; 181 tumors (48%) were molecularly characterized. LTSs were more likely to harbor a HIST1H3B mutation (odds ratio, 1.28; 95% CI, 1.1 to 1.5; P = .002). Conclusion We report clinical, radiologic, and molecular factors that correlate with survival in children and young adults with DIPG, which are important for risk stratification in future clinical trials

EPEN-04. SIOP EPENDYMOMA I: FINAL RESULTS, LONG TERM FOLLOW-UP AND MOLECULAR ANALYSIS OF THE TRIAL COHORT: A BIOMECA CONSORTIUM STUDY

IntroductionSurgery and radiotherapy are established childhood ependymoma treatments. The efficacy of chemotherapy has been debated. We report final results of the SIOP Ependymoma I trial, with 12-year follow-up, in the context of a post-hoc analysis of more recently described biomarkers.Aims and MethodsThe trial assessed event free (EFS) and overall survival (OS) of patients aged three to 21 years with non-metastatic intracranial ependymoma, treated with a staged management strategy targeting maximum local control. The study also assessed: the response rate (RR) of subtotally resected (STR) disease to vincristine, etoposide and cyclophosphamide (VEC); and surgical operability. Children with gross total resection (GTR) received radiotherapy of 54 Gy in 30 daily fractions over six weeks, whilst those with STR received VEC before radiotherapy. We retrospectively assessed methylation and 1q status alongside hTERT, RELA, Tenascin C, H3K27me3 and pAKT expression.ResultsBetween 1999 and 2007, 89 participants were enrolled, 15 were excluded with metastatic (n=4) or non-ependymoma tumours (n=11) leaving a final cohort of 74. Five- and ten-year EFS was 49.5% and 46.7%, OS was 69.3% and 60.5%. 1q gain was associated with poorer EFS (p=0.002, HR=3.00, 95%CI 1.49–6.10). hTERT expression was associated with worse five-year EFS (20.0% Vs 83.3%, p=0.014, HR=5.8). GTR was achieved in 33/74 (44.6%) and associated with improved EFS (p=0.006, HR=2.81, 95% confidence interval 1.35–5.84). There was an improvement in GTR rates in the latter half of the trial (1999-2002 32.4% versus 2003-2007 56.8%). Despite the protocol, 12 participants with STR did not receive chemotherapy. However, chemotherapy RR was 65.5% (19/29, 95%CI 45.7–82.1).ConclusionsVEC exceeded the pre-specified RR of 45% in children over three years with STR intracranial ependymoma. However, cases of inaccurate stratification at treating centres highlights the need for rapid central review. We also confirmed associations between 1q gain, hTERT expression and outcome

Nuclear ribonucleoprotein-containing foci increase in size in non-dividing cells from patients with myotonic dystrophy type 2

Myotonic dystrophies (DM) are genetically based neuromuscular disorders characterized by the accumulation of mutant transcripts into peculiar intranuclear foci, where different splicing factors (among which the alternative splicing regulator muscleblind-like 1 protein, MBNL1) are ectopically sequestered. The aim of the present investigation was to describe the dynamics of the DM-specific intranuclear foci in interphase nuclei and during mitosis, as well as after the exit from the cell cycle. Primary cultures of skin fibroblasts from DM2 patients were used, as a model system to reproduce in vitro, as accurately as possible, the in vivo conditions. Cycling and resting fibroblasts were investigated by immunocytochemical and morphometric techniques, and the relative amounts of MBNL1 were also estimated by western blotting. MBNL1-containing foci were exclusively found in the nucleus during most of the interphase, while being observed in the cytoplasm during mitosis when they never associate with the chromosomes; the foci remained in the cytoplasm at cytodieresis, and underwent disassembly in early G1 to be reformed in the nucleus at each cell cycle. After fibroblasts had stopped dividing in late-passage cultures, the nuclear foci were observed to progressively increase in size. Interestingly, measurements on muscle biopsies taken from the same DM2 patients at different ages demonstrated that, in the nuclei of myofibers, the MBNL1-containing foci become larger with increasing patient's age. As a whole, these results suggest that in non-dividing cells of DM2 patients the sequestration in the nuclear foci of factors needed for RNA processing would be continuous and progressive, eventually leading to the onset (and the worsening with time) of the pathological traits. This is consistent with the evidence that in DM patients the most affected organs or tissues are those where non-renewing cells are mainly present, i.e., the central nervous system, heart and skeletal muscle

Adapted physical exercise enhances activation and differentiation potential of satellite cells in the skeletal muscle of old mice

During ageing, a progressive loss of skeletal muscle mass and a decrease in muscle strength and endurance take place, in the condition termed sarcopenia. The mechanisms of sarcopenia are complex and still unclear; however, it is known that muscle atrophy is associated with a decline in the number and/or efficiency of satellite cells, the main contributors to muscle regeneration. Physical exercise proved beneficial in sarcopenia; however, knowledge of the effect of adapted physical exercise on the myogenic properties of satellite cells in aged muscles is limited. In this study the amount and activation state of satellite cells as well as their proliferation and differentiation potential were assessed in situ by morphology, morphometry and immunocytochemistry at light and transmission electron microscopy on 28-month-old mice submitted to adapted aerobic physical exercise on a treadmill. Sedentary age-matched mice served as controls, and sedentary adult mice were used as a reference for an unperturbed control at an age when the capability of muscle regeneration is still high. The effect of physical exercise in aged muscles was further analysed by comparing the myogenic potential of satellite cells isolated from old running and old sedentary mice using an in vitro system that allows observation of the differentiation process under controlled experimental conditions. The results of this ex vivo and in vitro study demonstrated that adapted physical exercise increases the number and activation of satellite cells as well as their capability to differentiate into structurally and functionally correct myotubes (even though the age-related impairment in myotube formation is not fully reversed): this evidence further supports adapted physical exercise as a powerful, non-pharmacological approach to counteract sarcopenia and the age-related deterioration of satellite cell capabilities even at very advanced age

Ultrastructural detection of photosensitizing molecules by fluorescence photoconversion of diaminobenzidine

Photodynamic therapy is a moderately invasive therapeutic procedure based on the action of photosensitizers (PSs). These compounds are able to absorb light, and dissipate energy through photochemical processes leading to the production of oxidizing chemical species (singlet oxygen, free radicals or reactive oxygen species) which can damage the cell molecular structures eventually inducing cell death. To increase the entering through the plasma membrane, a PS with suitable chemical structure can be modified by addition of chemical groups (e.g., acetate or phosphate): this affects both the fluorescence emission and of the photosensitizing properties of the native PS. The modified compounds behave as fluorogenic substrates (FSs), since inside the cell the bound groups can be enzymatically removed and the fluorescence and photosensitizing properties of the native molecules are restored. With the aim to detect the subcellular localization of photoactive molecules at transmission electron microscopy, we loaded cultured HeLa cells with two different FSs, Rose Bengal acetate (RB-Ac) or Hypocrellin B acetate (HypB-Ac), and took advantage of the photophysical properties of the intracellularly restored PS molecules to obtain the photoconversion of diaminobenzidine (DAB) into an electrondense product. We demonstrated that RB-Ac and HypB-Ac are mostly internalized by endocytosis, and are converted into the native PSs already at the cell surface. Endocytosed PS molecules apparently follow the endosomes-lysosome route, being found in endosomes, lysosomes and multivescicular bodies; PS molecules were also detected in the cytosol. This ultrastructural localization of the photoactive molecules is fully consistent with the multiorganelle photodamage observed after irradiation in culture of RB-Ac- or HypB-Ac-loaded cells. Due to the very short half-life of the oxidizing chemical species and their limited mobility, DAB deposits do localize in close proximity of the very place where photoactive molecules elicited the production of reactive oxygen species upon light irradiation. Therefore, DAB photoconversion promises to be a suitable tool for directly visualizing in single cells the PS molecules at high resolution, helping to elucidate their mode of penetration into the cell as well as their dynamic intracellular redistribution and organelle targeting

Retrospective analysis on the consistency of MRI features with histological and molecular markers in diffuse intrinsic pontine glioma (DIPG )

Background: The diagnosis of diffuse intrinsic pontine glioma (DIPG) is based largely on a combination of clinical and radiological findings due to the difficulty of obtaining a biopsy. An accurate evaluation of magnetic resonance imaging (MRI) scans is consequently essential. Recent analyses on the genomic landscape of DIPG revealed recurrent mutations in the H3F3A and HIST1H3B histone genes. We reviewed cases with available tumor tissue from institutional DIPG series to ascertain the consistency between their histo-molecular findings and clinical-radiological features. Methods: We conducted a radiological and pathological review of 22 cases of DIPG. We performed immunohistochemical analyses to detect H3F3A/HIST1H3B K27M mutations, histone tri-methylation and EZH2 expression. Mutational analysis was performed for ACVR1, H3F3A and HIST1H3B genes. Results: Patients’ median age at diagnosis was 8 years, and their median overall survival was 11 months. Nineteen/22 cases (86%) showed evidence of K27M mutation on immunohistochemistry and/or mutation analysis. Histone tri-methylation expression was low or lacking in these mutated cases. Sequence analysis revealed 13 cases with H3F3A and one case with HIST1H3B K27M mutation. There was no significant difference in EZH2 expression between the K27M-mutant and wild-type DIPGs. On external, blinded MRI re-evaluation, all but one lesion were consistent with DIPG; the one lesion that was not showed no evidence of K27M mutation and retained histone trimethylation expression. Conclusion: In conclusion, our study demonstrates a high frequency of histone K27M mutations in DIPG when MRI features are carefully assessed, thus confirming the consistency of imaging with biological markers in our institutional series of DIPG

A Peculiar CLL Case with Complex Chromosome 6 Rearrangements and Refinement of All Breakpoints at the Gene Level by Genomic Array: A Case Report

Introduction: Chronic lymphocytic leukemia (CLL), the most common leukemia in Western countries, is a mature B-cell chronic lymphoproliferative disorder characterized by the accumulation of neoplastic CD5+ B lymphocytes, functionally incompetent and usually monoclonal in origin, in bone marrow, lymph nodes and blood. Diagnosis occurs predominantly in elderly patients, with a median age reported between 67 and 72 years. CLL has a heterogeneous clinical course, which can vary from indolent to, less frequently, aggressive forms. Early-stage asymptomatic CLL patients do not require immediate therapeutic intervention, but only observation; treatment is necessary for patients with advanced disease or when “active disease” is observed. The most frequent autoimmune cytopenia (AIC) is autoimmune haemolytic anaemia (AHIA). The main mechanisms underlying the appearance of AIC in CLL are not fully elucidated, the predisposition of patients with CLL to suffering autoimmune complications is variable and autoimmune cytopenia can precede, be concurrent, or follow the diagnosis of CLL. Case presentation: A 74-year-old man was admitted to the emergency room following the finding of severe macrocytic anaemia during blood tests performed that same day, in particular the patient showed a profound asthenia dating back several months. The anamnesis was silent and the patient was not taking any medications. The blood examination showed an extremely high White Blood Cell count and findings of AIHA in CLL-type mature B-cell lymphoproliferative neoplasia. Genetic investigations: Conventional karyotyping was performed and it obtained a trisomy 8 and an unbalanced translocation between the short arm of chromosome 6 and the long arm of chromosome 11, concurrent with interstitial deletions in chromosomes 6q and 11q that could not be defined in detail. Molecular cytogenetics (FISH) analyses revealed Ataxia Telangiectasia Mutated (ATM) monoallelic deletion (with loss of ATM on derivative chromosome 11) and retained signals for TP53, 13q14 and centromere 12 FISH probes. TP53 and IGHV were not mutated. Array-CGH confirmed trisomy of the entire chromosome 8 and allowed us to resolve in detail the nature of the unbalanced translocation, revealing multiple regions of genomic losses on chromosomes 6 and 11. Discussion: The present case report is an unusual CLL case with complex karyotype and refinement of all breakpoints at the gene level by the genomic array. From a genetic point of view, the case under study presented several peculiarities. Conclusions: We report the genetic findings of a CLL patient with abrupt disease onset, so far responding properly to treatments despite the presence of distinct genetic adverse traits including ATM deletion, complex karyotype and chromosome 6q chromoanagenesis event. Our report confirms that interphase FISH alone is not able to provide an overview of the whole genomic landscape in selected CLL cases and that additional techniques are required to reach an appropriate cytogenetic stratification of patients