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A number of studies have shown that palladium nanoparticles are able to exert some adverse health effects, such as concentration-dependent cytotoxicity, induction of apoptosis and alterations of the release and expression of numerous cytokines. Nevertheless, our current knowledge of the potential toxic effects induced by exposure to these nanoparticles is far from being complete. For this reason, the present study assessed the possible renal toxicity of palladium nanoparticles by investigating urinary excretion of retinol binding protein, β2-microglobulin and albumin in female Wistar rats intravenously exposed to different nanoparticle concentrations (0.012, 0.12, 1.2 and 12 µg/kg) and by carrying out a morphological observation of the kidneys of treated animals. Results showed a significant increase in urinary retinol binding protein and β2-microglobulin levels in rats that were administered 12 µg/kg compared to controls. Moreover, an ultrastructural study of the kidneys revealed significant alterations in the proximal and distal tubular epithelium were observed, with a range of severity, in all experimental conditions. Collectively, our findings suggest that exposure to palladium nanoparticles is able to induce a significant renal tubular dysfunction, whereas it does not seem to affect kidney glomerular filtration. However, further studies are needed to confirm our results, to understand the molecular mechanisms of toxic action and to evaluate the potential adverse effects of these nanoparticles also on the glomerular section of the kidney.

Paragangliomas (PGs) of the head and neck region are typically benign, slow-growing neuroendocrine tumours. At times, they may exhibit unusual histological features, such as prominent stromal sclerosis (sclerosing PG), which may raise concerns of malignancy. We describe a case of sclerosing PG of the carotid body, emphasizing the value of immunohistochemical stains for differential diagnosis. A 43-year-old woman presented with a painless lump on the neck. A magnetic resonance imaging scan demonstrated a hypervascular lesion of the carotid body, which was surgically excised. Grossly, the lesion measured 1.8 cm at maximum diameter. On microscopic examination, irregular nests and tiny bundles of neoplastic cells were found between thick bands of fibrous tissue. Focal nuclear cytomegaly and marked pleomorphism were noted. Neoplastic cells proved to be immunoreactive for chromogranin, synaptophysin and neuron specific enolase, but negative for cytokeratins, smooth muscle actin and CD34. Ultrastructurally, numerous mitochondria, rough endoplasmic reticulum structures and endocrine granules were seen in the cytoplasm of the tumour cells. On consideration of the above-mentioned clinico-pathological and ultrastructural findings a diagnosis of sclerosing PG was established. Sclerosing PG is a rare entity which may mimic a malignant neoplasm. The recognition of this unusual morphological variant of PG, together with appropriate immunostains, leads to the correct diagnosis.

A missense mutation in the calsequestrin-1 gene (CASQ1) was found in a group of patients with a my- opathy characterized by weakness, fatigue, and the pres- ence of large vacuoles containing characteristic inclusions resulting from the aggregation of sarcoplasmic reticulum (SR) proteins. The mutation affects a conserved aspar- tic acid in position 244 (p.Asp244Gly) located in one of the high-affinity Ca 2+ -binding sites of CASQ1 and alters the kinetics of Ca 2+ release in muscle fibers. Expression of the mutated CASQ1 protein in COS-7 cells showed a markedly reduced ability in forming elongated polymers, whereas both in cultured myotubes and in in vivo mouse fibers induced the formation of electron-dense SR vac- uoles containing aggregates of the mutant CASQ1 protein that resemble those observed in muscle biopsies of pa- tients. Altogether, these results support the view that a single missense mutation in the CASQ1 gene causes the formation of abnormal SR vacuoles containing aggregates of CASQ1, and other SR proteins, results in altered Ca 2+ release in skeletal muscle fibers, and, hence, is responsible for the clinical phenotype observed in these patients.

CD99, a transmembrane protein encoded by MIC2 gene is involved in multiple cellular events including cell adhesion and migration, apoptosis, cell differentiation and regulation of protein trafficking either in physiological or pathological conditions. In osteosarcoma, CD99 is expressed at low levels and functions as a tumour suppressor. The full-length protein (CD99wt) and the short-form harbouring a deletion in the intracytoplasmic domain (CD99sh) have been associated with distinct functional outcomes with respect to tumour malignancy. In this study, we especially evaluated modulation of cell-cell contacts, reorganisation of the actin cytoskeleton and modulation of signalling pathways by comparing osteosarcoma cells characterised by different metastasis capabilities and CD99 expression, to identify molecular mechanisms responsible for metastasis. Our data indicate that forced expression of CD99wt induces recruitment of N-cadherin and β-catenin to adherens junctions. In addition, transfection of CD99wt inhibits the expression of several molecules crucial to the remodelling of the actin cytoskeleton, such as ACTR2, ARPC1A, Rho-associated, coiled-coil containing protein kinase 2 (ROCK2) as well as ezrin, an ezrin/radixin/moesin family member that has been clearly associated with tumour progression and metastatic spread in osteosarcoma. Functional studies point to ROCK2 as a crucial intracellular mediator regulating osteosarcoma migration. By maintaining c-Src in an inactive conformation, CD99wt inhibits ROCK2 signalling and this leads to ezrin decrease at cell membrane while N-cadherin and β-catenin translocate to the plasma membrane and function as main molecular bridges for actin cytoskeleton. Taken together, we propose that the re-expression of CD99wt, which is generally present in osteoblasts but lost in osteosarcoma, through inhibition of c-Src and ROCK2 activity, manages to increase contact strength and reactivate stop-migration signals that counteract the otherwise dominant promigratory action of ezrin in osteosarcoma cells.

The centrally localized alpha heating that allows the attainment of ignition in a toroidal plasma is shown to lead to a consistent plasma pressure profile. Such a profile turns out to depend much more on the achievement of ignition than on the transport assumptions. This feature is observed in a series of dynamic simulations relevant to the Ignitor experiment.

Chordoid glioma is a rare neoplasm occurring in the third ventricle and, as the name implies, having a chordoid appearance. It is currently considered a glial neoplasm of uncertain histogenesis with distinct clinicopathologic features. We report three cases of chordoid glioma with a focus on the ultrastructural appearance. The patients were two men and one woman aged, respectively, 34, 40, and 43 years. Immunohistochemically, all tumors showed strong and diffuse reactivity for glial fibrillary acidic protein and vimentin, whereas immunoreactivity for epithelial membrane antigen and cytokeratin was focal. Ultrastructurally, they showed features of ependymal differentiation for the presence of an apical pole with microvilli and a basal pole characterized, as in normal ependyma, by many hemidesmosomelike structures connecting cell membranes to the underlying basal lamina. Constant features were a submicroscopic cell body zonation (i.e., perinuclear, intermediate, subapical, and apical regions) and the presence of secretory granules. These findings were similar to those described for the secretory ependymal cells of the subcommissural organ, a small structure located in a dorsocaudal region of the third ventricle that undergoes regression after birth in humans. Our observations suggest that chordoid glioma may represent a subtype of ependymoma whose cells resemble the highly specialized ependyma of the subcommissural organ.

Medulloblastomas (MBs) associated with the Wnt activation represent a subgroup with a favorable prognosis, but it remains unclear whether Wnt activation confers a less aggressive phenotype and/or enhances radiosensitivity. To investigate this issue, we evaluated the biological behavior of an MB cell line, UW228-1, stably transfected with human β-catenin cDNA encoding a nondegradable form of β-catenin (UW-B) in standard culture conditions and after radiation treatment. We evaluated the expression, transcriptional activity, and localization of β-catenin in the stably transfected cells using immunofluorescence and WB. We performed morphological analysis using light and electron microscopy. We then analyzed changes in the invasiveness, growth, and mortality in standard culture conditions and after radiation. We demonstrated that (A) Wnt activation inhibited 97 % of the invasion capability of the cells, (B) the growth of the UW-B cells was statistically significantly lower than that of all the other control cells (p

BACKGROUND: Gastrointestinal stromal tumors (GISTs) encompass a large group of mesenchymal neoplasms that display common cytologic spindle-shaped morphology on light microscopy. Immunocytochemical and ultrastructural studies can demonstrate several patterns of differentiation. CASE: A 70-year-old male presented with two intraabdominal small bowel masses. The cytopathologic features of a fine needle aspiration biopsy (FNAB) included plump spindle cells in densely populated aggregates or in a a fasciculated pattern, without significant pleomorphism. An epithelioid component in a lobular arrangement with abundant, eosinophilic cytoplasm was also noted. The nuclei were vesicular, with a very evident, eosinophilic nucleolus and finely distributed chromatin. Groups of loosely cohesive cells with slender, dendritic-like cytoplasm were evident. Immunocytochemical study of the embedded, fine needle aspirated fragments of the neoplasm demonstrated immunoreactivity for vimentin and neuron-specific enolase. Cytokeratin immunoreactivity or muscular, vascular, neuroendocrine or nerve sheath differentiation failed to be demonstrated. The cytologic and immunocytochemical findings correlated well with the histologic features of the neoplasm. The morphologic diagnosis was confirmed by ultrastructural study. CONCLUSION: FNAB and immunocytochemistry can be valuable in making the correct diagnosis between gastrointestinal stromal tumors.

The scientific objectives of the Ignitor experiment can be achieved owing to the high magnetic field and plasma current planned. The physics projections are here supported by an analysis, carried out by the free boundary 1 1/2-D code JETTO, of the plasma evolution during the current ramp up and hat-top phases. The most advanced operating scenario that is envisaged for the machine is considered, while taking the technological constraints of the project into account. The plasma shape and position are checked to agree with the reference magnetic configurations. The density values are always much lower than the Greenwald limit, and the avoidance of disruption boundaries in the (l(i), q(Psi)) diagram is assured. The influence of the density profile growth on the overall performance is analysed under different assumptions. The results show that ohmic ignition could be reached even assuming transport diffusion coefficients that account for energy confinement times close to the 'ITER89P' scaling, provided that the ramp-up phase is carefully programmed. The density is the main parameter with which to control the path to ignition, but some other items need attention, such as the plasma shape and dimensions, the current density profile and the impurity content. Even when ignition is not achieved globally, a central ignited core is present.

We describe the case of a 46-year-old male patient who presented withpain in the left thigh, often accompanied by lumbar pain. These symptomswere sustained by a neoplasm, which was located in the sympathetic ganglia,at the level of the 3rd left lumbar spinal root and which was completelyexcised. Immunohistochemical positivity for S100, HMB45, and NSE antibodiessuggested that the lesion was a melanotic schwannoma (MS), with bothschwannian and melanocytic differentiations, the latter containingmelanosomes at ultrastructural examination. Non-recurrence after 16 monthsof follow-up further supports our diagnosis of MS.

Mitochondrial biogenesis is an orchestrated process that presides to the regulation of the organelles homeostasis within a cell. We show that γ-rays, at doses commonly used in the radiation therapy for cancer treatment, induce an increase in mitochondrial mass and function, in response to a genotoxic stress that pushes cells into senescence, in the presence of a functional p53. Although the main effector of the response to γ-rays is the p53-p21 axis, we demonstrated that mitochondrial biogenesis is only indirectly regulated by p53, whose activation triggers a murine double minute 2 (MDM2)-mediated hypoxia-inducible factor 1α (HIF1α) degradation, leading to the release of peroxisome-proliferator activated receptor gamma co-activator 1β inhibition by HIF1α, thus promoting mitochondrial biogenesis. Mimicking hypoxia by HIF1α stabilization, in fact, blunts the mitochondrial response to γ-rays as well as the induction of p21-mediated cell senescence, indicating prevalence of the hypoxic over the genotoxic response. Finally, we also show in vivo that post-radiotherapy mitochondrial DNA copy number increase well correlates with lack of HIF1α increase in the tissue, concluding this may be a useful molecular tool to infer the trigger of a hypoxic response during radiotherapy, which may lead to failure of activation of cell senescence.

Background Although progress has been made in the treatment of articular cartilage lesions, they are still a major challenge because current techniques do not provide satisfactory long-term outcomes. Tissue engineering and the use of functional biomaterials might be an alternative regenerative strategy and fulfill clinical needs. Decellularized extracellular matrices have generated interest as functional biologic scaffolds, but there are few studies on cartilage regeneration. The aim of this study was to evaluate in vitro the biological influence of a newly developed decellularized human dermal extracellular matrix on two human primary cultures. Methods Normal human articular chondrocytes (NHAC-kn) and human mesenchymal stromal cells (hMSC) from healthy donors were seeded in polystyrene wells as controls (CTR), and on decellularized human dermis batches (HDM_derm) for 7 and 14 days. Cellular proliferation and differentiation, and anabolic and catabolic synthetic activity were quantified at each experimental time. Histology and scanning electron microscopy were used to evaluate morphology and ultrastructure. Results Both cell cultures had a similar proliferation rate that increased significantly (p < 0.0005) at 14 days. In comparison with CTR, at 14 days NHAC-kn enhanced procollagen type II (CPII, p < 0.05) and aggrecan synthesis (p < 0.0005), whereas hMSC significantly enhanced aggrecan synthesis (p < 0.0005) and transforming growth factor-beta1 release (TGF-β1, p < 0.0005) at both experimental times. Neither inflammatory stimulus nor catabolic activity induction was observed. By comparing data of the two primary cells, NHAC-kn synthesized significantly more CPII than did hMSC at both experimental times (p < 0.005), whereas hMSC synthesized more aggrecan at 7 days (p < 0.005) and TGF-β1 at both experimental times than did NHAC-kn (p < 0.005). Conclusions The results obtained showed that in in vitro conditions HDM_derm behaves as a suitable scaffold for the growth of both well-differentiated chondrocytes and undifferentiated mesenchymal cells, thus ensuring a biocompatible and bioactive substrate. Further studies are mandatory to test the use of HDM_derm with tissue engineering to assess its therapeutic and functional effectiveness in cartilage regeneration.

The scientific goal of the Ignitor experiment is to approach, for the first time, the ignition conditions of a magnetically confined D-T plasma. The IGNIR collaboration between Italy and Russia is centred on the construction of the core of the Ignitor machine in Italy and its installation and operation within the Triniti site (Troitsk). A parallel initiative has developed that integrates this programme, involving the study of plasmas in which high-energy populations are present, with ongoing research in high-energy astrophysics, with a theory effort involving the National Institute for High Mathematics, and with INFN and the University of Pisa for the development of relevant nuclear and optical diagnostics. The construction of the main components of the machine core has been fully funded by the Italian Government. Therefore, considerable attention has been devoted towards identifying the industrial groups having the facilities necessary to build these components. An important step for the Ignitor programme is the adoption of the superconducting MgB2 material for the largest poloidal field coils (P14) that is compatible with the He-gas cooling system designed for the entire machine. The progress made in the construction of these coils is described. An important advance has been made in the reconfiguration of the cooling channels of the toroidal magnet that can double the machine duty cycle. A facility has been constructed to test the most important components of the ICRH system at full scale, and the main results of the tests carried out are presented. The main physics issues that the Ignitor experiment is expected to face are analysed considering the most recent developments in both experimental observations and theory for weakly collisional plasma regimes. Of special interest is the I-regime that has been investigated in depth only recently and combines advanced confinement properties with a high degree of plasma purity. This is a promising alternative to the high-density L-regime that had been observed by the Alcator experiment and whose features motivated the Ignitor project. The provisions that are incorporated in the machine design, and in that of the plasma chamber in particular, in order to withstand or prevent the development of macroscopic instabilities with deleterious amplitudes are presented together with relevant analyses. © 2013 IAEA, Vienna.

Neutral lipid storage disease with myopathy (NLSDM) is a rare autosomal recessive disorder of neutral lipid metabolism. Clinical manifestations include progressive skeletal myopathy, cardiomyopathy, and liver dysfunction. Clinical severity is variable and additional symptoms may include diabetes mellitus, chronic pancreatitis, hypothyroidism, neurosensory hearing loss, and short stature. We report a 79-year-old man with progressive proximal arm weakness, lipid storage myopathy, dropped head syndrome, and peripheral nervous system involvement. He harboured a novel homozygous missense mutation, c.570A>C (p.S191R) in the patatin-like phospholipase domain containing 2 (PNPLA2) gene, confirming the diagnosis of NLSDM. The S191R mutation causes late-onset NLSDM without cardiac dysfunction. The previously unreported association with dropped head syndrome expands the clinical spectrum of NLSDM.

Cells with uniform, small-round nucleus and clear cytoplasm (oligodendroglial-like cell, OLC) are commonly observed in central nervous system (CNS) neoplasm of glial and neuronal lineage, such as oligodendroglioma, clear-cell ependymoma, and central neurocytoma. Immunohistochemistry does not always contribute to the characterization of OLC because of (1) loss of antigen expression; (2) lack of specific markers for oligodendrogliomas; and (3) occasional coexpression of neuronal and glial antigens. An ultrastructural analysis associated with an immunohistochemical study of 20 cases of CNS tumors largely constituted by OLCs has been performed. Neurocytomas (12 cases), medullocytomas (2 cases), cerebral neuroblastoma (1 case), and ganglioglioma (1 case) showed OLCs with ultrastructural features of neuronal differentiation (neuritic processes, dense-core granules, synaptic structures). Oligodendroglioma (3 cases) OLCs were characterized by mitochondrial-rich cytoplasm, and ependymoma (1 case) OLCs showed microrosettes and scattered cilia. The electron microscopic analysis can provide a more precise diagnosis of these OLC-containing tumors despite their uniform morphological appearance.

A large Italo-Spanish kindred with autosomal-dominant inheritance has been reported with proximal limb and axial muscle weakness. Clinical, histological and genetic features have been described. A limb girdle muscular dystrophy 1F (LGMD1F) disease locus at chromosome 7q32.1-32.2 has been previously identified. We report a muscle pathological study of two patients (mother and daughter) from this family. Muscle morphologic findings showed increased fiber size variability, fiber atrophy, and acid-phosphatase-positive vacuoles. Immunofluorescence against desmin, myotilin, p62 and LC3 showed accumulation of myofibrils, ubiquitin binding protein aggregates and autophagosomes. The ultrastructural study confirmed autophagosomal vacuoles. Many alterations of myofibrillar component were detected, such as prominent disarray, rod-like structures with granular aspect, and occasionally, cytoplasmic bodies. Our ultrastructural data and muscle pathological features are peculiar to LGMD1F and support the hypothesis that the genetic defect leads to a myopathy phenotype associated with disarrangement of the cytoskeletal network.

Ignitor was proposed as a breakthrough in reaching ignition in a high magnetic field. The evolution of the plasma parameters is analyzed. Different growth scenarios for the plasma current and density are considered to optimize the global plasma performance while satisfying stability requirements. The toroidal field is consistent with the current increase adopted. The alpha particles produced are considered only as a power source for electron and ion thermal energy. The effects of the current ramp rate are discussed, and the importance of the density profile growth is indicated. The results show that stable trajectories through the (l[sub i],q[psi]) plane can be obtained, assuming the inward pinch is not too large, a (65/35) deuterium-tritium mixture still yields acceptable performance, and a 10% prompt loss of alpha particles is allowed. 25 refs., 7 figs., 2 tabs.

We report two brothers affected by a dominantly inherited form of hereditary sensory and autonomic neuropathy (HSAN), characterized by clinical features of sensory ataxia, and by late onset in the 6th decade. Sural nerve biopsy in the proband showed almost complete loss of myelinated fibers, and relative sparing of unmyelinated fibers. This family showed an atypical presentation of HSAN, which is usually characterized by acrodystrophic manifestations of infantile or juvenile onset. Although a few reports of HSAN presenting with late onset and/or ataxia appeared, this is the first report of a family with dominant HSAN characterized by late onset sensory ataxia.

Objective The inflammatory myopathies (IMs) are a group of disorders characterised by weakness and inflammation of the skeletal muscles. Muscle biopsy is the most crucial test to confirm the clinical diagnosis, but also the most common cause of misdiagnosis. There are currently no markers specific or sensitive enough to distinguish IMs from other diseases with similar clinical and morphological features, and an international multidisciplinary effort is under way to develop new classification criteria for IMs. Methods Standards for Reporting of Diagnostic Accuracy recommendations to validate a diagnostic test based on the quantification of internal major histocompatibility complex class I (MHC-I) positive fibres were adopted. MHC-I immunostained specimens from 64 patients were scored by two independent blinded investigators, and the percentage of positive fibres was determined. Agreement between investigators was evaluated with the k-weighted statistic. The receiver operating characteristic curve, area under the curve, sensitivity, specificity, and positive and negative predictive values of each percentage range of positive fibres versus the diagnosis of IM were calculated. Results The main difference between IM and non-inflammatory samples was the number of internal MHC-I positive fibres. The k-weighted value was 0.89 for a percentage of MHC-I positive fibres above 50%; the positive predictive value was 100%, and the negative predictive value was 94%. Conclusions This is the first study on the validity of a quantitative analysis of internal MHC-I positive fibres for an IM diagnosis performed according to Standards for Reporting of Diagnostic Accuracy recommendations. The interobserver agreement was almost perfect, thus making the method reproducible. Applying an MHC-I cut-off above 50% is an optimal marker for polymyositis (PM) and dermatomyositis (DM) diagnosis.

Background Regarding regenerative medicine for diabetes, accessible sources of Mesenchymal Stem Cells (MSCs) for induction of insular beta cell differentiation may be as important as mastering the differentiation process itself. Methodology/Principal Findings In the present work, stem cells from pancreatic islets (human islet-mesenchymal stem cells, HI-MSCs) and from human bone marrow (bone marrow mesenchymal stem cells, BM-MSCs) were cultured in custom-made serum-free medium, using suitable conditions in order to induce differentiation into Islet-like Cells (ILCs). HI-MSCs and BM-MSCs were positive for the MSC markers CD105, CD73, CD90, CD29. Following this induction, HI-MSC and BM-MSC formed evident islet-like structures in the culture flasks. To investigate functional modifications after induction to ILCs, ultrastructural analysis and immunofluorescence were performed. PDX1 (pancreatic duodenal homeobox gene-1), insulin, C peptide and Glut-2 were detected in HI-ILCs whereas BM-ILCs only expressed Glut-2 and insulin. Insulin was also detected in the culture medium following glucose stimulation, confirming an initial differentiation that resulted in glucose-sensitive endocrine secretion. In order to identify proteins that were modified following differentiation from basal MSC (HI-MSCs and BM-MSCs) to their HI-ILCs and BM-ILCs counterparts, proteomic analysis was performed. Three new proteins (APOA1, ATL2 and SODM) were present in both ILC types, while other detected proteins were verified to be unique to the single individual differentiated cells lines. Hierarchical analysis underscored the limited similarities between HI-MSCs and BM-MSCs after induction of differentiation, and the persistence of relevant differences related to cells of different origin. Conclusions/Significance Proteomic analysis highlighted differences in the MSCs according to site of origin, reflecting spontaneous differentiation and commitment. A more detailed understanding of protein assets may provide insights required to master the differentiation process of HI-MSCs to functional beta cells based only upon culture conditioning. These findings may open new strategies for the clinical use of BM-MSCs in diabetes.

Dominant optic atrophy (DOA) is genetically heterogeneous and pathogenic mutations have been identified in the OPA1 and OPA3 genes, both encoding for mitochondrial proteins. We characterized clinical and laboratory features in a large OPA1-negative family with complicated DOA. Search for mitochondrial dysfunction was performed by studying muscle biopsies, fibroblasts, platelets and magnetic resonance (MR) spectroscopy. Genetic investigations included mitochondrial DNA (mtDNA) analysis, linkage analysis, copy number variation (CNV) analysis and candidate gene screening. Optic neuropathy was undistinguishable from that in OPA1-DOA and frequently associated with late-onset sensorineural hearing loss, increases of central conduction times at somato-sensory evoked potentials and various cardiac abnormalities. Serum lactic acid after exercise, platelet respiratory complex activities, adenosine triphosphate (ATP) content in fibroblasts and muscle phosphorus MR spectroscopy all failed to reveal a mitochondrial dysfunction. However, muscle biopsies and their mtDNA analysis showed increased mitochondrial biogenesis. Furthermore, patient's fibroblasts grown in the galactose medium were unable to increase ATP content compared with controls, and exhibited abnormally high rate of fusion activity. Genome-wide linkage revealed a locus on chromosome 16q21-q22 with a maximum two-point LOD score of 8.84 for the marker D16S752 and a non-recombinant interval of ∼ 6.96 cM. Genomic screening of 45 genes in this interval including several likely candidate genes (CALB2, CYB5B, TK2, DHODH, PLEKHG4) revealed no mutation. Moreover, we excluded the presence of CNVs using array-based comparative genome hybridization. The identification of a new OPA locus (OPA8) in this pedigree demonstrates further genetic heterogeneity in DOA, and our results indicate that the pathogenesis may still involve mitochondria.

Acute kidney injury (AKI) is a major health care condition with limited current treatment options. Within this context, stem cells may provide a clinical approach for AKI. Moreover, a synthetic compound previously developed, hyaluronan monoesters with butyric acid (HB), able to induce metanephric differentiation, formation of capillary-like structures, and secretion of angiogenic cytokines, was tested in vitro. Thereafter, we investigated the effects of human mesenchymal stem cells from fetal membranes (FMhMSCs), both treated and untreated with HB, after induction of ischemic AKI in a rat model. At reperfusion following 45-min clamping of renal pedicles, each rat was randomly assigned to one of four groups: CTR, PBS, MSC, and MSC-HB. Renal function at 1, 3, 5, and 7 days was assessed. Histological samples were analyzed by light and electron microscopy and renal injury was graded. Cytokine analysis on serum samples was performed. FMhMSCs induced an accelerated renal functional recovery, demonstrated by biochemical parameters and confirmed by histology showing that histopathological alterations associated with ischemic injury were less severe in cell-treated kidneys. HB-treated rats showed a minor degree of inflammation, both at cytokine and TEM analyses. Better functional and morphological recovery were not associated to stem cells' regenerative processes, but possibly suggest paracrine effects on microenvironment that induce retrieval of renal damaged tissues. These results suggest that FMhMSCs could be useful in the treatment of AKI and the utilization of synthetic compounds could enhance the recovery induction ability of cells.

We describe a new polychrome stain and simultaneous methods of histological, histochemical and immunocytochemical staining performed on sections from human tissues embedded in the new hydrophilic resin Bioacryl. The polychrome stain involves the sequential use of Harris' Haematoxylin, silver methenamine, Light Green and Eosin or Safranin dyes and provides a highly specific visualization of the overall cytological tissue architecture. When histochemical, immunocytochemical, and polychrome stains are performed together on the same section, crisp images are obtained, yielding simultaneous data of histochemical and immunological reactivities with clear tissue architecture.

One of the most important aspects of Behcet's disease is aneurysmal arteriopathy. The major problem of this complication is its tendency to develop recurrent false aneurysms at anastomotic and traumatic sites, such as angiographic punctures. We present a clinical case in which five aneurysms, some true, some false, were operated on during a period of 6 years, with the aid of ultrastructural observations of the wall of a true and a false aneurysm. One of the true aneurysms, localized in the aorta, was treated by direct aneurysmorrhaphy, and the 6-year follow-up demonstrated the absence of recurrences. Based on both this experience and the data in the literature, we suggest that the most appropriate surgical approach would, when possible, be direct aneurysmorrhaphy.

Patients with myasthenia gravis (MG) with antibodies to muscle-specific receptor tyrosine kinase (MuSK) differ from acetylcholine receptor (AChR)-positive MG patients, as they frequently present with severe oculobulbar muscle weakness or with neck, shoulder, and respiratory muscle involvement. The neuromuscular junction (NMJ) has been confirmed to be the main target of both AChR- and MuSK-MG. However, histopathological investigation disclosed that muscle fiber atrophy was prevalent in AChR-MG, whereas mild myopathic changes and mitochondrial abnormalities were more frequently observed in MuSK-MG. As the pathogenetic mechanism in MuSK-MG remains unclear, this study investigated the submicroscopic pattern of muscle histopathology to establish a possible correlation between clinical involvement and subcellular morphological findings. Muscle biopsies from seven MuSK-MG patients and from seven patients with AChR-MG were analyzed by transmission electron microscopy. Myopathic and mitochondrial abnormalities were more prominent in MuSK-MG and show giant, swollen, and degenerated mitochondria with fragmented cristae. The most common changes in AChR-MG muscles were fiber atrophy, myofibrillar disarray, and Z-line streaming, consistent with mild neurogenic abnormalities. A different pathogenetic mechanism is emerging in MuSK-MG compared to AChR-MG. Mitochondrial abnormalities seem to be more prominent in MuSK-MG, whereas neurogenic atrophy is observed in AChR-MG.

Satellite cells (SCs) are skeletal muscle progenitor cells located between the basal lamina and the sarcolemma of muscle fibers. They are responsible for muscle growth and repair. In humans, aging results in the depletion of the SC population and in its proliferative activity, but not in its function. It has not yet been determined whether under conditions of massive muscle fiber death in vivo, the regenerative potential of SCs is totally or partially compromised in old muscle. No studies have yet tested whether advanced age is a factor that restrains the response of SCs to muscle denervation in humans; this is also due to difficulties in the isolation and in the culture of SCs from a small human surgery fragment. The aim of this study was to study in depth muscle regeneration analysing the SC ability of SCs to proliferate and differentiate in aging human patients.In order to study in more detail the molecular mechanism, the proliferative and differentiative ability of aging SCs, we isolated SCs from aging human muscle biopsies and analysed their morphology by transmission electron microscopy and immunocytochemical analysis (antibodies against desmin, N-CAM and M-cadherin) and their capacity to grow and to expand in vitro. Moreover, in order to evaluate gene expression of myogenic regulatory factors Myf5, MyoD and myogenin (Myf4), RT-PCR was performed.SCs isolated from aging human muscle biopsies and plated into favorable proliferation and differentiation conditions were able to proceed through the myogenic program and actively form myotubes, although taking longer than the young control sample. The RT-PCR analysis together with the ultrastructural SC features showed that the myogenic potential seemed to be compromised during the aging human muscle proliferation in vitro.

Storage diseases (SDs) are rare metabolic disorders characterized by the intra- or extralysosomal accumulation of unmetabolized compounds. Different causes determine the buildup of undigested material, resulting in typical histochemical and ultrastructural changes. Ultrastructural examination of tissue from patients with clinically suspected SDs may disclose pathognomonic alterations or suggest a differential diagnosis even in the absence of clinically evident involvement of the biopsied tissue. Accurate diagnosis of SDs requires a continuous integration of clinical, biochemical, ultrastructural, and, when available, molecular data. It is also important for the pathologist to be familiar with the morphological variability characterizing each SD, because some morphologies are often the early stages of undeveloped forms and morphologically similar diseases are easily confused. The major advantages of transmission electron microscopy (TEM) techniques are discussed, emphasizing the current role of TEM as a rapid, cost-effective, and efficient diagnostic tool.

Lithium is the main therapeutic agent for the treatment of bipolar disorders but nerve cells are not the sole target of this drug. Indeed, lithium has been reported to target numerous cell types and to affect cell proliferation, differentiation and death. Lithium targets a variety of enzymes among which there is GSK-3beta and a number of cell responses elicited by lithium are mediated by the Wnt pathway that is involved in medulloblastoma (MB) pathogenesis. We studied the in vitro effects of lithium on two different MB cell lines: D283MED and DAOY. High doses of lithium inhibited GSK3-beta, decreased cell proliferation and induced non-apoptotic cell death in both cell lines independently by intracellular levels of beta-catenin that is consistently high only in D283MED. At clinical doses, the anti-neoplastic effects were observed only in this cell line, highlighting the importance of a specific molecular background in determining the target therapy response. In conclusion, lithium could be a promising drug in MB, but an accurate molecular profile predictive of drug response still needs to be clarified.

A case of primary neoplasm of the meninges with unusual histological and clinical features, which occurred in a 61-year-old man is described. Although conventional light microscopy revealed an undifferentiated tumor consisting of small- to medium-sized elements, it did not allow any definitive histogenetic interpretation. Immunohistochemistry showed that the neoplastic cells were positive for actin, desmin, myoglobin, and vimentin, thus leading to the diagnosis of meningeal sarcoma with rhabdomyoblastic differentiation. This interpretation was also supported by electron microscopy, in dewaxed tissue samples, which revealed the presence of abortive Z-lines. It is noteworthy that retrospective microscopic examination of all the slides obtained both at initial presentation and at relapses demonstrated in some fields that the undifferentiated population was associated with typical meningiomatous features, as was also shown by the staining for the epithelial membrane antigen. Clinically, the tumor was characterized by an exceedingly long course (10 years). The histogenesis of the tumor and the diagnostic relevance of immunohistochemical techniques are discussed.

Peroneal muscular atrophy (PMA) associated with hereditary spastic paraparesis (HSP) is a nosologically ill-defined disease, which has been classified by Dyck as hereditary motor and sensory neuropathy type V (HMSN V). Nerve biopsy has been rarely reported in this condition. We examined sural nerve biopsies in four patients, demonstrating the following: severe myelinated fiber loss especially of large fibers, with moderate (one case) or prominent (one case) onion bulb formation; selective decrease of large fibers with moderate Schwann cell hyperplasia (one case); normal myelinated fiber population with minimal changes (one case). After reviewing previously reported cases we, conclude that in PMA with HSP sural nerve biopsy may show features either of hypertrophic type of PMA, of neuronal type, or of spinal type; thus, it seems inappropriate to allocate PMA with HSP in a unique subtype of HMSN. In addition, HSP may be not associated with peripheral neuropathy, and thus the classification in the HMSN group may be incongruous. A proper classification of PMA with HSP may be in the "complicated" forms of HSP according to Harding [Lancet I: 1151-1155 (1983)]; however, the nosology of this condition needs to be further elucidated, possibly on the basis of the underlying molecular genetic mechanisms of HSP and PMA.

We previously showed that disruptive complex I mutations in mitochondrial DNA are the main genetic hallmark of oncocytic tumors of the thyroid and kidney. We here report a high frequency of homoplasmic disruptive mutations in a large panel of oncocytic pituitary and head-and-neck tumors. The presence of such mutations implicates disassembly of respiratory complex I in vivo which in turn contributes to the inability of oncocytic tumors to stabilize HIF1alpha and to display pseudo-hypoxia. By utilizing transmitochondrial cytoplasmic hybrids (cybrids), we induced the shift to homoplasmy of a truncating mutation in the mitochondria-coded MTND1 gene. Such shift is associated with a profound metabolic impairment leading to the imbalance of alpha-ketoglutarate and succinate, the Krebs cycle metabolites which are the main responsible for HIF1alpha stabilization. We conclude that the main hallmarks of oncocytic transformation, namely the occurrence of homoplasmic disruptive mutations and complex I disassembly, may explain the benign nature of oncocytic neoplasms through lack of HIF1alpha stabilization.

Rabbit antisera raised against the product of ORF UL 80 of human cytomegalovirus (CMV) genome ( Hin dIII L fragment of AD169 strain) as well as IgM from acutely infected patients recognize an antigen of M r 38 kDa. In the viral particle this antigen is bound via S-S bridge to a lower M r . compound to form a final complex of 62 kDa that is also recognized by rabbit antisera as well as patients' IgM. P38 is present both in the nucleus and in the cytoplasm of CMV-infected cells starting from 24 h p.i. and increasingly thereafter. Immunoelectron microscopy revealed that this antigen is mainly associated with the internal portion of viral capsids both in the nucleus and in the cytoplasm.

This paper analyzes the influence of a few parameters on the possibility of reaching ignition in the Ignitor device using a 1 {1/2}-dimensional equilibrium transport code. The models adopted for electron and ion thermal diffusivities scale with the net power input to the plasma components. No auxiliary heating is considered besides that due to alpha particles. In the operative scenario examined, the plasma current (I{sub p} = 10 MA) and the toroidal field (B{sub T} = 11.1 T) are maintained for 4s. The importance of density profile shaping is pointed out together with the influence of current density peaking. Sensitivity to specified assumptions is also analyzed and the limit values, still guaranteeing ignition, for different parameters are determined.

Autosomal dominant optic atrophy (ADOA), the commonest cause of inherited optic atrophy, is caused by mutations in the ubiquitously expressed gene optic atrophy 1 (OPA1), involved in fusion and biogenesis of the inner membrane of mitochondria. Bioenergetic failure, mitochondrial network abnormalities and increased apoptosis have all been proposed as possible causal factors. However, their relative contribution to pathogenesis as well as the prominent susceptibility of the retinal ganglion cell (RGC) in this disease remains uncertain. Here we identify a novel deletion of OPA1 gene in the GTPase domain in three patients affected by ADOA. Muscle biopsy of the patients showed neurogenic atrophy and abnormal morphology and distribution of mitochondria. Confocal microscopy revealed increased mitochondrial fragmentation in fibroblasts as well as in myotubes, where mitochondria were also unevenly distributed, with clustered organelles alternating with areas where mitochondria were sparse. These abnormalities were not associated with altered bioenergetics or increased susceptibility to pro-apoptotic stimuli. Therefore, changes in mitochondrial shape and distribution can be independent of other reported effects of OPA1 mutations, and therefore may be the primary cause of the disease. The arrangement of mitochondria in RGCs, which degenerate in ADOA, may be exquisitely sensitive to disturbance, and this may lead to bioenergetic crisis and/or induction of apoptosis. Our results highlight the importance of mitochondrial dynamics in the disease per se, and point to the loss of the fine positioning of mitochondria in the axons of RGCs as a possible explanation for their predominant degeneration in ADOA.

We describe magnetic resonance (MR) aspect of cardiac glycogenesis in a 49-years old man, presented a progressively declining cardiac function and negative coronary angiography. Delayed enhancement MR confirmed non-ischemic pattern with unusual diffuse distribution of Gadolinium. Cardiac biopsy revealed a Glycogen Storage Disease, extra-lysosomial type. Cardiac MR with analysis of delayed enhancement distribution is an emerging tool that can discriminate between ischemic and non-ischemic diseases; however to identify the precise aetiology of a non-ischemic distribution, myocardial biopsy is still needed.

A 66-year-old woman was referred with left hearing loss. A probable diagnosis of left secretory otitis media with effusion was formulated. A left myringotomy was performed to remove hyperplastic hard tissue from the tympanic cavity. A high resolution CT scan of the temporal bone disclosed a soft-tissue mass completely involving the mastoid and tympanic cavity, surrounding the ossicular chain which appeared spared with no signs of infiltration. The histopathologic, immunohistochemical and ultrastructural response was secretory meningioma, a rare variant of conventional meningothelial meningioma in atypical sites.

Medulloblastoma (MB) is the most common brain malignancy in children. Whole neural axis irradiation is the treatment of choice, but it often results in long-term neurocognitive and developmental impairment. Only insights into MB biology will lead to improved therapeutic outcome. Wingless (WNT) signalling deregulation occurs in up to 25% of sporadic tumors, but the specific role of nuclear beta-catenin and its involvement in the radioresponse remains unsettled. Therefore we studied the gamma-radiation response of two MB cell lines from cellular and molecular points of view. Our data show that the p53 wild-type cell line is more sensitive to ionizing radiations (IR) than the p53 mutated line, but apoptosis is also induced in p53-mutated cells, suggesting an alternative p53-independent mechanism. In addition, this study is the first to demonstrate that gamma-rays trigger the WNT system in our in vitro models. Further studies are required to test if this could explain the radiosensitivity of MB and the favorable prognostic value of nuclear beta-catenin in this tumor.

CADASIL (OMIM 125310) is an increasingly recognised adult-onset autosomal-dominant vascular disease that is characterised by recurrent transient ischaemic attacks and strokes (43% of patients), vascular dementia (6%), migraine with aura (40% of patients) and psychiatric disturbances (9% of patients); epilepsy has been reported in 2–10% of subjects.1 All patients revealed prominent signal abnormalities on brain magnetic resonance imaging (MRI)—leukoencephalopathy on T2- and small subcortical infarcts on T1-weighted images.2 The pathological hallmark of CADASIL is a non-amyloid and non-arteriosclerotic angiopathy, which predominantly affects the small penetrating brain arteries. Vascular lesions are characterised by degeneration and loss of smooth-muscle cells and by the presence of granular osmiophilic material (GOM) accumulating within the smooth-muscle-cell basement membrane and the surrounding extracellular matrix. Examination of several peripheral organs revealed vessel changes, including the presence of GOM deposits, providing evidence that CADASIL is a systemic arteriopathy.3 It has been reported that CADASIL is caused by single missense mutations, small in-frame deletions or splice-site mutations in the NOTCH3 gene encoding a transmembrane receptor (http://www.hgmd.cf.ac.uk/ac/gene.php?gene = NOTCH3). Almost all previously reported mutations resulted in an odd number of cysteine residues within one of the 34 epidermal growth factor (EGF)-like repeats in the …

Limb girdle muscular dystrophy type 2I (LGMD2I) is due to mutations in the fukutin-related protein gene (FKRP), encoding a putative glycosyltransferase involved in alpha-dystroglycan processing. To further characterize the molecular pathogenesis of LGMD2I, we conducted a histological, immunohistochemical, ultrastructural and molecular analysis of ten muscle biopsies from patients with molecularly diagnosed LGMD2I. Hypoglycosylation of alpha-dystroglycan was observed in all FKRP-mutated patients. Muscle histopathology was consistent with either severe muscular dystrophy or myopathy with a mild inflammatory response consisting of up-regulation of class I major histocompatibility complex in skeletal muscle fibers and small foci of mononuclear cells. At the ultrastructural level, muscle fibers showed focal thinning of basal lamina and swollen endoplasmic reticulum cisternae with membrane re-arrangement. The pathways of the unfolded protein response (UPR; glucose-regulated protein 78 and CHOP) were significantly activated in LGMD2I muscle tissue. Our data suggest that the UPR response is activated in LGMD2I muscle biopsies, and the observed histopathological and ultrastructural alterations may be related to sarcoplasmic structures involved in FKRP and alpha-dystroglycan metabolism and malfunctioning.

Background Erosive esophagitis is a frequent endoscopic feature in patients with gastro-oesophageal reflux disease. However, most of patients with heartburn/regurgitation have a non-erosive reflux disease. The reason for this heterogeneous impact of gastro-oesophageal reflux disease on oesophageal mucosa is unknown to date. Aim To evaluate the cell proliferation status of oesophageal epithelium in both healthy normal subjects and patients with gastro-oesophageal reflux disease with or without erosions. Materials and methods All the subjects underwent endoscopy and biopsies were taken at 5 cm from the squamo-columnar junction. Specimens were analysed both at histology and at transmission electron microscopy. Cell proliferation was evaluated by MIB1 immunostaining. Of the 85 subjects were studied, 10 were healthy controls with normal pH-testing and macroscopical, histological and ultrastructural patterns; 37were patients with erosive esophagitis, and 38 patients with non-erosive reflux disease. Results At histology, of the 37 patients affected by erosive esophagitis, 30 had normal mucosa and 7 showed mild oesophagitis. One patient with non-erosive reflux disease showed signs of oesophagitis at histology. At TEM, all patients with gastro-oesophageal reflux disease had ultrastructural patterns of damage i.e. dilations of intercellular spaces (DIS), and all controls had a normal ultrastructural pattern. The mean (±SD) MIB1-LI values of normal subjects and non-erosive reflux disease and erosive oesophagitis patients were 62.2% (±9.1), 29.7% (±7.2) and 16.2% (±5.2), respectively; there were significant differences among the three groups (p Conclusions Oesophageal mucosa of patients with reflux symptoms presents a decrease in MIB1 immunostaining of 50% and 25% in non-erosive reflux disease and erosive esophagitis patients with respect to normal subjects.

In order to characterize, at the clinical, molecular and imaging level, myopathies due to MYH7 gene mutations, MYH7 gene analysis was conducted by RT-PCR/SSCP/sequencing in two patients diagnosed with myosin storage myopathy and 17 patients diagnosed with scapulo-peroneal myopathy of unknown etiology. MYH7 gene studies revealed the 5533C>T mutation (Arg1845Trp) in both myosin storage myopathy and in 2 of the 17 scapulo-peroneal patients studied. 5533C>T segregation analysis in the mutation carrier families identified 11 additional patients. The clinical spectrum in our cohort of patients included asymptomatic hyperCKemia, scapulo-peroneal myopathy and proximal and distal myopathy with muscle hypertrophy. Muscle MRI identified a unique pattern in the posterior compartment of the thigh, characterized by early involvement of the biceps femoris and semimembranosus, with relative sparing of the semitendinosus. Muscle biopsy revealed hyaline bodies in only half of biopsied patients (2/4). In conclusion, phenotypic and histopathological variability may underlie MYH7 gene mutation and the absence of hyaline bodies in muscle biopsies does not rule out MYH7 gene mutations.

Gene mutations impairing the functions of the WNT signaling transduction pathway have been found in approximately 15% of human sporadic medulloblastomas. To understand the functional role of the WNT pathway in medulloblastoma, we have investigated the intracellular distribution of beta-catenin in a series of 17 human medulloblastomas to correlate such expression with neuronal differentiation and in cultured cell models following functional silencing of the APC gene by small-interference RNA (siRNA). Transient siRNA transfection resulted in a 50% reduction of the APC gene product levels in both DAOY and D283MED cell lines. In the former, less-differentiated cell line, beta-catenin levels remained unchanged or were slightly reduced, but beta-catenin translocated in the nucleus following APC gene siRNA silencing. In contrast, in the more differentiated D283MED cells, beta-catenin levels increased about twofold while mainly maintaining the cytoplasmic and cell membrane localization. Cytoplasmic/nuclear localization of beta-catenin was present in 12 of 17 cases of medulloblastoma with a prevalent distribution in the classic, 6/7 cases, and large cell/anaplastic variant, 4/4 cases. The nodular/desmoplastic lesions showed strongly positivity in the cell membrane mainly of intranodular cells with advanced neuronal differentiation. These observations support an important functional role of WNT/beta-catenin pathway in neuronal differentiation in medulloblastoma.