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In this study, we explored whether Nutlin-3a, a well-known, nontoxic small-molecule compound antagonizing the inhibitory interaction of MDM2 with the tumor suppressor p53, may restore ligands for natural killer (NK) cell–activating receptors (NK-AR) on neuroblastoma cells to enhance the NK cell–mediated killing. Neuroblastoma cell lines were treated with Nutlin-3a, and the expression of ligands for NKG2D and DNAM-1 NK-ARs and the neuroblastoma susceptibility to NK cells were evaluated. Adoptive transfer of human NK cells in a xenograft neuroblastoma-bearing NSG murine model was assessed. Two data sets of neuroblastoma patients were explored to correlate p53 expression with ligand expression. Luciferase assays and chromatin immunoprecipitation analysis of p53 functional binding on PVR promoter were performed. Primary neuroblastoma cells were also treated with Nutlin-3a, and neuroblastoma spheroids obtained from one high-risk patient were assayed for NK-cell cytotoxicity. We provide evidence showing that the Nutlin-3a–dependent rescue of p53 function in neuroblastoma cells resulted in (i) increased surface expression of ligands for NK-ARs, thus rendering neuroblastoma cell lines significantly more susceptible to NK cell–mediated killing; (ii) shrinkage of human neuroblastoma tumor masses that correlated with overall survival upon adoptive transfer of NK cells in neuroblastoma-bearing mice; (iii) and increased expression of ligands in primary neuroblastoma cells and boosting of NK cell–mediated disaggregation of neuroblastoma spheroids. We also found that p53 was a direct transcription factor regulating the expression of PVR ligand recognized by DNAM-1. Our findings demonstrated an immunomodulatory role of Nutlin-3a, which might be prospectively used for a novel NK cell–based immunotherapy for neuroblastoma.

Besides its structural properties in the nucleoskeleton, Lamin A/C is a mechanosensor protein involved in perceiving the elasticity of the extracellular matrix. In this study we provide evidence about Lamin A/C-mediated regulation of osteosarcoma cell adhesion and spreading on substrates with tissue-specific elasticities. Our working hypothesis is based on the observation that low-aggressive and bone-resident SaOS-2 osteosarcoma cells express high level of Lamin A/C in comparison to highly metastatic, preferentially to the lung, osteosarcoma 143B cells, thereby suggesting a role for Lamin A/C in tumor cell tropism. Specifically, LMNA gene over-expression in 143B cells induced a reduction in tumor cell aggressiveness in comparison to parental cells, with decreased proliferation rate and reduced migration capability. Furthermore, LMNA reintegration into 143B cells changed the adhesion properties of tumor cells, from a preferential tropism toward the 1.5 kPa PDMS substrate (resembling normal lung parenchyma) to the 28 kPa (resembling pre-mineralized bone osteoid matrix). Our study suggests that Lamin A/C expression could be involved in the organ tropism of tumor cells, thereby providing a rationale for further studies focused on the definition of cancer mechanism of metastatization.

Vesicle biogenesis, trafficking and signaling via the ER-Golgi network support essential processes during development and their disruption can lead to neurodevelopmental disorders and neurodegeneration. We report that de novo missense variants in ARF3, encoding a small GTPase regulating Golgi structure and function, cause a neurodevelopmental disease showing microcephaly and progressive cortical atrophy, with microsomia and rib anomalies in severely affected subjects, suggesting a pleiotropic effect. All microcephaly-associated variants clustered in the guanine nucleotide binding pocket and perturbed the biochemical behavior of the protein by stabilizing it in a GTP-bound state. Functional analysis proved the disruptive consequences of the variants on Golgi integrity, and brain and body plan formation. In-depth analysis in zebrafish embryos expressing ARF3 mutants traced back the developmental alterations to defective gastrulation cell movements as the earliest detectable effect. Our findings document a role of ARF3 in Golgi homeostasis and demonstrate an obligate dependence for early development.

Mitochondria are highly dynamic organelles, undergoing continuous fission and fusion. The DNM1L (dynamin-1 like) gene encodes for the DRP1 protein, an evolutionary conserved member of the dynamin family, responsible for fission of mitochondria, and having a role in the division of peroxisomes, as well. DRP1 impairment is implicated in several neurological disorders and associated with either de novo dominant or compound heterozygous mutations. In five patients presenting with severe epileptic encephalopathy, we identified five de novo dominant DNM1L variants, the pathogenicity of which was validated in a yeast model. Fluorescence microscopy revealed abnormally elongated mitochondria and aberrant peroxisomes in mutant fibroblasts, indicating impaired fission of these organelles. Moreover, a very peculiar finding in our cohort of patients was the presence, in muscle biopsy, of core like areas with oxidative enzyme alterations, suggesting an abnormal distribution of mitochondria in the muscle tissue.

PCDH19-related epilepsy is a rare genetic disease caused by defective function of PCDH19, a calcium-dependent cell–cell adhesion protein of the cadherin superfamily. This disorder is characterized by a heterogeneous phenotypic spectrum, with partial and generalized febrile convulsions that are gradually increasing in frequency. Developmental regression may occur during disease progression. Patients may present with intellectual disability (ID), behavioral problems, motor and language delay, and a low motor tone. In most cases, seizures are resistant to treatment, but their frequency decreases with age, and some patients may even become seizure-free. ID generally persists after seizure remission, making neurological abnormalities the main clinical issue in affected individuals. An effective treatment is lacking. In vitro studies using patient-derived induced pluripotent stem cells (iPSCs) reported accelerated neural differentiation as a major endophenotype associated with PCDH19 mutations. By using this in vitro model system, we show that accelerated in vitro neurogenesis is associated with a defect in the cell division plane at the neural progenitors stage. We also provide evidence that altered PCDH19 function affects proper mitotic spindle orientation. Our findings identify an altered equilibrium between symmetric versus asymmetric cell division as a previously unrecognized mechanism contributing to the pathogenesis of this rare epileptic encephalopathy.

Leukodystrophies are a heterogeneous group of rare inherited disorders that mostly involve the white matter of the CNS. These conditions are characterized by primary glial cell and myelin sheath pathology of variable aetiology, which causes secondary axonal degeneration, generally emerging with disease progression. Whole exome sequencing performed in five large consanguineous nuclear families allowed us to identify homozygosity for two recurrent missense variants affecting highly conserved residues of RNF220 as the causative event underlying a novel form of leukodystrophy with ataxia and sensorineural deafness. We report these two homozygous missense variants (p.R363Q and p.R365Q) in the ubiquitin E3 ligase RNF220 as the underlying cause of this novel form of leukodystrophy with ataxia and sensorineural deafness that includes fibrotic cardiomyopathy and hepatopathy as associated features in seven consanguineous families. Mass spectrometry analysis identified lamin B1 as the RNF220 binding protein and co-immunoprecipitation experiments demonstrated reduced binding of both RNF220 mutants to lamin B1. We demonstrate that RNF220 silencing in Drosophila melanogaster specifically affects proper localization of lamin Dm0, the fly lamin B1 orthologue, promotes its aggregation and causes a neurodegenerative phenotype, strongly supporting the functional link between RNF220 and lamin B1. Finally, we demonstrate that RNF220 plays a crucial role in the maintenance of nuclear morphology; mutations in primary skin fibroblasts determine nuclear abnormalities such as blebs, herniations and invaginations, which are typically observed in cells of patients affected by laminopathies. Overall, our data identify RNF220 as a gene implicated in leukodystrophy with ataxia and sensorineural deafness and document a critical role of RNF220 in the regulation of nuclear lamina. Our findings provide further evidence on the direct link between nuclear lamina dysfunction and neurodegeneration.

Cancer cells are able to release high amounts of extracellular vesicles, thereby conditioning the normal cells in the surrounding tissue and/or in distant target organs. In the context of bone cancers, previous studies suggested that osteosarcoma cancer cells produce transforming extracellular vesicles able to induce a tumour-like phenotype in normal recipient cells. Indeed, phosphoinositide-specific phospholipase C (PI-PLC) enzymes are differentially expressed in osteosarcoma cell lines with increasing aggressiveness, thus providing helpful insights to better define their role and functions in this bone tumour. By confocal microscopy analysis, we demonstrated that osteosarcoma-derived extracellular vesicles convey all the assessed PI-PLC isoforms, and that they localize into cell membrane bubble-like structures, resembling extracellular vesicles about to be released, as conveyed and/or membrane protein. Cytofluorimetric analysis confirmed the presence of PI-PLC isoforms in the extracellular vesicles collected from conditioned media of osteosarcoma cells. These findings suggest the feasibility to use circulating extracellular vesicles as biomarkers of osteosarcoma progression and/or the monitoring of this distressing disease.

Tubulinopathies are rare neurological disorders caused by alterations in tubulin structure and function, giving rise to a wide range of brain abnormalities involving neuronal proliferation, migration, differentiation and axon guidance. TUBB is one of the ten &beta, tubulin encoding genes present in the human genome and is broadly expressed in the developing central nervous system and the skin. Mutations in TUBB are responsible for two distinct pathological conditions: the first is characterized by microcephaly and complex structural brain malformations and the second, also known as &ldquo, circumferential skin creases Kunze type&rdquo, (CSC-KT), is associated to neurological features, excess skin folding and growth retardation. We used a combination of immunocytochemical and cellular approaches to explore, on patients&rsquo, derived fibroblasts, the functional consequences of two TUBB variants: the novel mutation (p.N52S), associated with basal ganglia and cerebellar dysgenesis, and the previously reported variant (p.M73T), linked to microcephaly, corpus callosum agenesis and CSC-KT skin phenotype. Our results demonstrate that these variants impair microtubule (MT) function and dynamics. Most importantly, our studies show an altered epidermal growth factor (EGF) and transferrin (Tf) intracellular vesicle trafficking in both patients&rsquo, fibroblasts, suggesting a specific role of TUBB in MT-dependent vesicular transport.

The nuclear lamina is essential for the maintenance of nuclear shape and mechanics. Mutations in lamin genes have been identified in a heterogeneous spectrum of human diseases known as &ldquo, laminopathies&rdquo, associated with nuclear envelope defects and deregulation of cellular functions. Interestingly, osteosarcoma is the only neoplasm described in the literature in association with laminopathies. This study aims characterized the expression of A-type and B-type lamins and emerin in osteosarcoma, revealing a higher percentage of dysmorphic nuclei in osteosarcoma cells in comparison to normal osteoblasts and all the hallmarks of laminopathic features. Both lamins and emerin were differentially expressed in osteosarcoma cell lines in comparison to normal osteoblasts and correlated with tumor aggressiveness. We analysed lamin A/C expression in a tissue-microarray including osteosarcoma samples with different prognosis, finding a positive correlation between lamin A/C expression and the overall survival of osteosarcoma patients. An inefficient MKL1 nuclear shuttling and actin depolymerization, as well as a reduced expression of pRb and a decreased YAP nuclear content were observed in A-type lamin deficient 143B cells. In conclusion, we described for the first time laminopathic nuclear phenotypes in osteosarcoma cells, providing evidence for an altered lamins and emerin expression and a deregulated nucleoskeleton architecture of this tumor.

Bone marrow (BM) is the major target organ for neuroblastoma (NB) metastasis and its involvement is associated with poor outcome. Yet, the mechanism by which NB cells invade BM is largely unknown. Tumour microenvironment represents a key element in tumour progression and mesenchymal stromal cells (MSCs) have been recognized as a fundamental part of the associated tumour stroma. Here, we show that BM-MSCs isolated from NB patients with BM involvement exhibit a greater osteogenic potential than MSCs from non-infiltrated BM. We show that BM metastasis-derived NB-cell lines secrete higher levels of exosomal miR-375, which promotes osteogenic differentiation in MSCs. Of note, clinical data demonstrate that high level of miR-375 correlates with BM metastasis in NB patients. Our findings suggest, indeed, a potential role for exosomal miR-375 in determining a favourable microenvironment in BM to promote metastatic progression. MiR-375 may, thus, represent a novel biomarker and a potential target for NB patients with BM involvement.

Microtubules participate in fundamental cellular processes, including chromosomal segregation and cell division, migration and intracellular trafficking. Their proper function is required for correct central nervous system development and operative preservation, and mutations in genes coding tubulins, the constituting units of microtubules, underlie a family of neurodevelopmental and neurodegenerative diseases, collectively known as 'tubulinopathies', characterized by a wide range of neuronal defects resulting from defective proliferation, migration and function. Here, we causally link a previously unreported missense mutation in TUBB2A (c.1249G>A, p.D417N), encoding one of the neuron-specific β-tubulin isotype II, to a disorder characterized by progressive spastic paraplegia, peripheral sensory-motor polyneuropathy and ataxia. Asp417 is a highly conserved solvent-exposed residue at the site mediating binding of kinesin superfamily motors. Impaired binding to KIF1A, a neuron-specific kinesin required for transport of synaptic vesicle precursors of the disease-associated TUBB2A mutant, was predicted by structural analyses and confirmed experimentally in vitro. We show that overexpression of TUBB2AD417N disrupts the mitotic spindle bipolarity and morphology and affects the M phase entry and length. Differently from the TUBB2AN247K and TUBB2AA248V, two mutants previously identified to affect neurodevelopment, TUBB2AD417N retains the ability to assemble into microtubules. Consistent with the differential clinical and structural impact, TUBB2AA248V does not drastically affect TUBB2A binding to KIF1A, nor mitotic spindle bipolarity. Overall, our data demonstrate a pathogenic role of the p.D417N substitution that is different from previously reported TUBB2A mutations and expand the phenotypic spectrum associated with mutations in this gene.

Osteosarcoma is the most common malignant bone tumor in children and young adults. The identification of proteins which exhibit different subcellular localization in low- versus high-risk osteosarcoma can be instrumental to obtain prognostic information and to develop innovative therapeutic strategies. Beside the well-characterized membrane and cytoplasmic localization of Src protein, this study evaluated the prognostic relevance of its so-far unknown nuclear compartmentalization. We analyzed the subcellular distribution of total and activated (pY418) Src in a tissue microarray including 60 osteosarcoma samples. Immunohistochemical analyses revealed a variable pattern of Src expression and localization, ranging from negative to high-stained nuclei combined with a substantial cytoplasmic staining for total and activated forms. The analysis of Kaplan-Meier survival curves in relationship to the diverse permutations of cytoplasmic and nuclear staining suggested a correlation between Src subcellular localization and the overall survival (OS) of osteosarcoma patients. In order to explain this different subcellular localization, normal osteoblasts and three osteosarcoma cell lines were used to investigate the molecular mechanism. Once confirmed a variable Src localization also in these cell lines, we demonstrated a correlation between the N-myristoyltransferase enzymes expression and activity and the Src nuclear content. In conclusion, these results described a so-far unknown Src nuclear localization in osteosarcoma cells, suggesting that the combined detection of nuclear and cytoplasmic Src levels can be used as a prognostic marker for osteosarcoma patient survival. A correlation between the N-myristoyltransferase enzymes and the Src subcellular localization was described as well.

Nuclear integrity and mechanical stability of the nuclear envelope (NE) are conferred by the nuclear lamina, a meshwork of intermediate filaments composed of A- and B-type lamins, supporting the inner nuclear membrane and playing a pivotal role in chromatin organization and epigenetic regulation. During cell senescence, nuclear alterations also involving NE architecture are widely described. In the present study, we utilized induced pluripotent stem cells (iPSCs) upon prolonged in vitro culture as a model to study aging and investigated the organization and expression pattern of NE major constituents. Confocal and four-dimensional imaging combined with molecular analyses, showed that aged iPSCs are characterized by nuclear dysmorphisms, nucleoskeletal components (lamin A/C-prelamin isoforms, lamin B1, emerin, and nesprin-2) imbalance, leading to impaired nucleo-cytoplasmic MKL1 shuttling, actin polymerization defects, mitochondrial dysfunctions, SIRT7 downregulation and NF-kBp65 hyperactivation. The observed age-related NE features of iPSCs closely resemble those reported for premature aging syndromes (e.g., Hutchinson-Gilford progeria syndrome) and for somatic cell senescence. These findings validate the use of aged iPSCs as a suitable cellular model to study senescence and for investigating therapeutic strategies aimed to treat premature aging.

Oxidative stress is involved in the pathogenesis of Duchenne muscular dystrophy (DMD), an X-linked genetic disorder caused by mutations in the dystrophin gene and characterized by progressive, lethal muscle degeneration and chronic inflammation. In this study, we explored the expression and signaling pathway of a master player of the anti-oxidant and anti-inflammatory response, namely NF-E2-related Factor 2, in muscle biopsies of DMD patients. We classified DMD patients in two age groups (Class I, 0-2 years and Class II, 2-9 years), in order to evaluate the antioxidant pathway expression during the disease progression. We observed that altered enzymatic antioxidant responses, increased levels of oxidized glutathione and oxidative damage are differently modulated in the two age classes of patients and well correlate with the severity of pathology. Interestingly, we also observed a modulation of relevant markers of the inflammatory response, such as heme oxygenase 1 and Inteleukin-6 (IL-6), suggesting a link between oxidative stress and chronic inflammatory response. Of note, using a transgenic mouse model, we demonstrated that IL-6 overexpression parallels the antioxidant expression profile and the severity of dystrophic muscle observed in DMD patients. This study advances our understanding of the pathogenic mechanisms underlying DMD and defines the critical role of oxidative stress on muscle wasting with clear implications for disease pathogenesis and therapy in human.

SHOC2 is a scaffold protein mediating RAS-promoted activation of mitogen-activated protein kinase (MAPK) signaling in response to extracellular stimuli. A recurrent activating mutation in SHOC2 (p.Ser2Gly) causes Mazzanti syndrome, a RASopathy characterized by features resembling Noonan syndrome and distinctive ectodermal abnormalities. A second mutation (p.Met173Ile) supposed to cause loss-of-function was more recently identified in two individuals with milder phenotypes. Here, we report on the third RASopathy-causing SHOC2 mutation (c.807_808delinsTT, p.Gln269_His270delinsHisTyr), which was found associated with prenatal-onset hypertrophic cardiomyopathy. Structural analyses indicated a possible impact of the mutation on the relative orientation of the two SHOC2's leucine-rich repeat domains. Functional studies provided evidence of its activating role, revealing enhanced binding of the mutant protein to MRAS and PPP1CB, and increased signaling through the MAPK cascade. Differing from SHOC2 S2G , SHOC2 Q269_H270delinsHY is not constitutively targeted to the plasma membrane. These data document that diverse mechanisms in SHOC2 functional dysregulation converge toward MAPK signaling upregulation.

Background and objective In Duchenne Muscular Dystrophy (DMD) treatment, muscle fiber size can be considered as an indicator of muscle health and function. In particular, the statistical distribution of fibers cross-sectional areas (CSAs) has been used as quantitative efficacy endpoint. For each patient, assessment of treatment effect relies on the comparison of pre- and post-treatment biopsies. Since biopsies provide “distributional data”, i.e. empirical distributions of fibers CSA, the comparison must be carried out between the empirical pre- and post-treatment distributions. Methods Here, distributional fiber CSA data are analyzed by means of a hierarchical statistical model based on the population approach, considering both the single patient and the population level. Results The proposed method was used to assess the histological clinical effects of Givinostat, a compound under study for DMD treatment. At the single patient level, a two-component Gaussian mixture adequately represents pre- and post-treatment distributions of log-transformed CSAs; drug effect is described via a dose-dependent multiplicative increase of muscle fiber size. The single patient model was also validated via muscle composition data. At the patient population level, typical model parameters and inter-patient variabilities were obtained. Conclusions The proposed methodological approach completely characterizes fiber CSA distributions and quantifies drug effect on muscle fiber size, both at the single patient and at the patient population level. This approach might be applied also in other contexts, where outcomes measured in terms of distributional data are to be assessed.

Duchenne Muscular Dystrophy (DMD) is caused by mutations in the dystrophin gene leading to dystrophin deficiency, muscle fiber degeneration and progressive fibrotic replacement of muscles. Givinostat, a histone deacetylase (HDAC) inhibitor, significantly reduced fibrosis and promoted compensatory muscle regeneration in mdx mice. This study was conducted to evaluate whether the beneficial histological effects of Givinostat could be extended to DMD boys. Twenty ambulant DMD boys aged 7 to

Diffuse intrinsic pontine glioma (DIPG) and pediatric glioblastoma (pGBM) are heterogeneous brain tumors characterized by different anatomical and molecular subgroups and the presence of genetically and phenotypically distinct subclonal cell populations. It is recognized that exosomes mediate cross-talk among tumor cells. We hypothesize that there are different exosome-mediated paracrine signaling promoting tumour progression in DIPG and pGBM. Our aim was to determine the specific DIPG and pGBM-derived exosome oncogenic signatures. We used a panel of fifteen patient primary-derived cell lines, which included nine DIPG (seven H3.3 K27M, one H3.3 K27M/ACVR1 and one H3.1 K27M/ACVR1), one diffuse midline glioma H3.3 K27M and three GBM (one H3.3 G34R and two histone WT). Conditioned medium was collected from cells maintained under stem-cell culture condition, adherent on laminin and/or as neurospheres (NS), and exosomes harvested through serial centrifugations. Electron microscopy demonstrated that the isolated microvescicles are exosomes sized between 50–80 nm. DIPG derived-exosomes appeared to have a variable cargo of total protein (µg)/10(6) cells, which was higher than for pGBM-exosomes. Proteomic analysis revealed that proteins associated with vesicle docking, exocytosis and synaptic transmission were exclusively enriched in pontine-derived exosomes, while cell-cell and cell-matrix interaction proteinswere exclusive tohemispheric ones. Proteins in common to the two locations were involved in metabolism and energy pathways. Interestingly, principle component analysis on the different molecular subgroups suggests that ACVR1 may be not implicated in the exosomal proteomic signature. Exosomal miRNA profile appeared to be driven by the two main histone mutated subgroups H3.3 K27M and H3.1 K27M with the latter overexpressing hypoxia and angiogenic-associated miRNAs, leading to distinct oncogenic programs with different specific potential therapeutic targets. This study aimed to development new diagnostic/prognostic tools for DIPG and pGBM patients. Further investigations are aimed to identify new therapeutic strategies to inhibit the cross-talk among glioma subpopulations.

Children with Down Syndrome (DS) suffer from immune deficiency with a severe reduction in switched memory B cells (MBCs) and poor response to vaccination. Chromosome 21 (HSA21) encodes two microRNAs (miRs), miR-125b, and miR-155, that regulate B-cell responses. We studied B- and T- cell subpopulations in tonsils of DS and age-matched healthy donors (HD) and found that the germinal center (GC) reaction was impaired in DS. GC size, numbers of GC B cells and Follicular Helper T cells (TFH) expressing BCL6 cells were severely reduced. The expression of miR-155 and miR-125b was increased in tonsillar memory B cells and miR-125b was also higher than expected in plasma cells (PCs). Activation-induced cytidine deaminase (AID) protein, a miR-155 target, was significantly reduced in MBCs of DS patients. Increased expression of miR-155 was also observed in vitro. MiR-155 was significantly overexpressed in PBMCs activated with CpG, whereas miR-125b was constitutively higher than normal. The increase of miR-155 and its functional consequences were blocked by antagomiRs in vitro. Our data show that the expression of HSA21-encoded miR-155 and miR-125b is altered in B cells of DS individuals both in vivo and in vitro. Because of HSA21-encoded miRs may play a role also in DS-associated dementia and leukemia, our study suggests that antagomiRs may represent pharmacological tools useful for the treatment of DS.

Duchenne muscular dystrophy (DMD) is a lethal disease. The outcome measures used in numerous therapeutic trials include skeletal muscle biopsy. We studied the natural history of DMD from the standpoint of muscle histology with the aim of providing a reproducible tool for use in evaluating and comparing any histologic changes occurring in patients with DMD undergoing treatment and hence be able to determine how therapy modulates the histologic evolution of the disease.Three independent operators analyzed 56 muscle biopsies from 40 patients not treated with steroids, aged 1 to 10 years and 16 individuals treated with steroids, aged 7 to 10 years. We analyzed morphologic measures, normalized every measure for the average number of fibers observed for each year of age, and calculated intraclass correlation coefficients.The average proportion of connective tissue in patients not treated with steroids was 16.98% from ages 1 to 6 years and 30% from ages 7 to 10 years (p0.0001). The average proportion in patients treated with steroids was 24.90%. Muscle fiber area mirrored that of connective tissue in both groups.Having provided a reproducible tool for evaluation and comparison of histologic changes occurring in patients undergoing clinical trials, it was observed that at ages 6 to 7 years, fibrotic tissue rapidly peaks to 29.85%; this is a crucial moment when muscle tissue loses its self-regeneration ability, veering toward fibrotic degeneration. These data should be considered when deciding the most suitable time to begin therapy.

Lipopolysaccharide (LPS) is currently considered one of the major players in non-alcoholic fatty liver disease (NAFLD) pathogenesis and progression. Here, we aim to investigate the possible role of LPS-induced TNF-α factor (LITAF) in inducing a pro-inflammatory and pro-fibrogenic phenotype of non-alcoholic steatohepatitis (NASH). We found that children with NAFLD displayed, in different liver-resident cells, an increased expression of LITAF which correlated with histological traits of hepatic inflammation and fibrosis. Total and nuclear LITAF expression increased in mouse and human hepatic stellate cells (HSCs). Moreover, LPS induced LITAF-dependent transcription of IL-1β, IL-6 and TNF-α in the clonal myofibroblastic HSC LX-2 cell line, and this effect was hampered by LITAF silencing. We showed, for the first time in HSCs, that LITAF recruitment to these cytokine promoters is LPS dependent. However, preventing LITAF nuclear translocation by p38MAPK inhibitor, the expression of IL-6 and TNF-α was significantly reduced with the aid of p65NF-ĸB, while IL-1β transcription exclusively required LITAF expression/activity. Finally, IL-1β levels in plasma mirrored those in the liver and correlated with LPS levels and LITAF-positive HSCs in children with NASH. In conclusion, a more severe histological profile in paediatric NAFLD is associated with LITAF over-expression in HSCs, which in turn correlates with hepatic and circulating IL-1β levels outlining a panel of potential biomarkers of NASH-related liver damage. The in vitro study highlights the role of LITAF as a key regulator of the LPS-induced pro-inflammatory pattern in HSCs and suggests p38MAPK inhibitors as a possible therapeutic approach against hepatic inflammation in NASH.

Vici syndrome is a human inherited multi-system disorder caused by recessive mutations in EPG5, encoding the EPG5 protein that mediates the fusion of autophagosomes with lysosomes. Immunodeficiency characterized by lack of memory B cells and increased susceptibility to infection is an integral part of the condition, but the role of EPG5 in the immune system remains unknown. Here we show that EPG5 is indispensable for the transport of the TLR9 ligand CpG to the late endosomal-lysosomal compartment, and for TLR9-initiated signaling, a step essential for the survival of human memory B cells and their ultimate differentiation into plasma cells. Moreover, the predicted structure of EPG5 includes a membrane remodeling domain and a karyopherin-like domain, thus explaining its function as a carrier between separate vesicular compartments. Our findings indicate that EPG5, by controlling nucleic acids intracellular trafficking, links macroautophagy/autophagy to innate and adaptive immunity.

Osteosarcoma is the most common primary bone cancer and the most frequent cause of bone cancer-related deaths in children and adolescents. Osteosarcoma cells are able to establish a crosstalk with resident bone cells leading to the formation of a deleterious vicious cycle. We hypothesized that osteosarcoma cells can release, in the bone microenvironment, transforming Extracellular Vesicles (EVs) involved in regulating bone cell proliferation and differentiation, thereby promoting tumor growth. We assessed EV production by three osteosarcoma cell lines with increasing aggressiveness in order to investigate their roles in the communication between osteosarcoma cells and normal recipient cells. Osteosarcoma-derived EVs were used to treat the murine fibroblast cell line NIH3T3 and to study the induction of tumor-like phenotypes. Our results showed that osteosarcoma cell lines are able to produce EVs that fuse to recipient cells, with a very high uptake efficiency. The treatment of recipient NIH3T3 with osteosarcoma-derived EVs induced substantial biological and functional effects, as an enhanced proliferation and survival capability under starved conditions, high levels of activated survival pathways, an increased migration, adhesion, and 3D sphere formation and the acquired capability to grow in an anchorage-independent manner. Moreover, in murine NIH3T3 we found human mRNAs of TNF-α, IL-6, and TGF-β, as well as a de novo expression of murine MMP-9 and TNF-α following the treatment of human osteosarcoma-derived EVs.

Background: Collagen VI-related disorders (COL6-RD) are a group of heterogenous muscular diseases due to mutations in the COL6A1, COL6A2 and COL6A3 genes, encoding for collagen VI, a critical component of the extracellular matrix. Ullrich congenital muscle disorder and Bethlem myopathy represent the ends of a clinical spectrum that includes intermediate phenotypes of variable severity. UCMD are caused by recessive loss of function mutations or de-novo dominant-negative mutations. The intermediate phenotype and BM are more commonly caused by dominantly acting mutations, and less commonly by recessive mutations. Recently parental mosaicism for dominant mutations in COL6 have been reported in four COL6-RD families and germinal mosaicism has been also identified in a family with recurrence of UCMD in two half-sibs. Methods and results: Here we report three unrelated patients affected by a COL6-RD who carried de novo mosaic mutations in COL6A genes. These mutations, missed by Sanger sequencing, were identified by next generation sequencing. Conclusions: This report highlights the importance of a complete diagnostic workup when clinical and histological finding are consistent with a COL6-RD and strengthen the impression that mosaicisms are underestimated events underlying COL6-RD. (C) 2017 The Authors. Published by Elsevier Ltd on behalf of European Paediatric Neurology Society.

Post-translational modulation of peptidylprolyl isomerase Pin1 might link impaired glucose metabolism and neurodegeneration, being Pin1 effectors target for the glucagon-Like-Peptide1 analog liraglutide. We tested the hypotheses in Pin1 silenced cells (SH-SY5Y) treated with 2-deoxy-d-glucose (2DG) and methylglyoxal (MG), stressors causing altered glucose trafficking, glucotoxicity and protein glycation. Rescue by liraglutide was investigated. Pin1 silencing caused increased levels of reactive oxygen species, upregulated energy metabolism as suggested by raised levels of total ATP content and mRNA of SIRT1, PGC1&alpha, NRF1, enhanced mitochondrial fission events as supported by raised protein expression of FIS1 and DRP1. 2DG and MG reduced significantly cell viability in all the cell lines. In Pin1 KD clones, 2DG exacerbated altered mitochondrial dynamics causing higher rate of fission events. Liraglutide influenced insulin signaling pathway (GSK3b/Akt), improved cell viability also in cells treated with 2DG, but it did not revert mitochondrial dysfunction in Pin1 KD model. In cells treated with MG, liraglutide enhanced cell viability, reduced ROS levels and cell death (AnnexinV/PI), and trended to reduce anti-apoptotic signals (BAX, BCL2, CASP3). Pin1 silencing mimics neuronal metabolic impairment of patients with impaired glucose metabolism and neurodegeneration. Liraglutide rescues to some extent cellular dysfunctions induced by Pin1 silencing.

Pediatric diffuse lung diseases are rare disorders with an onset in the neonatal period or in infancy, characterized by chronic respiratory symptoms and diffuse interstitial changes on imaging studies. Genetic disorders of surfactant homeostasis represent the main etiology. Surfactant protein B and ABCA3 deficiencies typically cause neonatal respiratory failure, which is often lethal within a few weeks or months. Although heterozygous ABCA3 mutation carriers are mostly asymptomatic, there is growing evidence that monoallelic mutations may affect surfactant homeostasis. Surfactant protein C mutations are dominant or sporadic disorders leading to a broad spectrum of manifestations from neonatal respiratory distress syndrome to adult pulmonary fibrosis. The authors performed pathology and ultrastructural studies in 12 infants who underwent clinical lung biopsy. One carried a heterozygous SP-B mutation, 3 carried SP-C mutations, and 7 carried ABCA3 mutations (5 biallelic and 2 monoallelic). Optical microscopy made it possible to distinguish between surfactant-related disorders and other forms. One of the ABCA3 monoallelic carriers had morphological features of alveolar capillary dysplasia, a genetic disorder of lung alveolar, and vascular development. One patient showed no surfactant-related anomalies but had pulmonary interstitial glycogenosis, a developmental disorder of unknown origin. Electron microscopy revealed specific lamellar bodies anomalies in all SP-B, SP-C, and ABCA3 deficiency cases. In addition, the authors showed that heterozygous ABCA3 mutation carriers have an intermediate ultrastructural phenotype between homozygous carriers and normal subjects. Lung biopsy is an essential diagnostic procedure in unexplained diffuse lung disorders, and electron microscopy should be performed systematically, since it may reveal specific alterations in genetic disorders of surfactant homeostasis.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare congenital leukodystrophy characterized by macrocephaly, subcortical cysts and demyelination. The majority of patients harbor mutations in the MLC1 gene encoding for a membrane protein with largely unknown function. Mutations in MLC1 hamper its normal trafficking and distribution in cell membranes, leading to enhanced degradation. MLC1 protein is highly expressed in brain astrocytes and in circulating blood cells, particularly monocytes. We used these easily available cells and monocyte-derived macrophages from healthy donors and MLC1-mutated patients to study MLC1 expression and localization, and to investigate how defective MLC1 mutations may affect macrophage functions. RT-PCR, western blot and immunofluorescence analyses show that MLC1 is expressed in both monocytes and macrophages, and its biosynthesis follows protein trafficking between endoplasmic reticulum and trans-Golgi network and the secretory pathway to the cell surface. MLC1 is transported along the endosomal recycling pathway passing through Rab5+ and Rab11A+vesicles before lysosomal degradation. Alterations in MLC1 trafficking and distribution were observed in macrophages from MLC1-mutated patients, which also showed changes in the expression and localization of several proteins involved in plasma membrane permeability, ion and water homeostasis and ion-regulated exocytosis. As a consequence of these alterations, patient-derived macrophages show abnormal cell morphology and intracellular calcium influx and altered response to hypo-osmotic stress. Our results suggest that blood-derived macrophages may give relevant information on MLC1 function and may be considered as valid biomarkers for MLC diagnosis and for investigating therapeutic strategies aimed to restore MLC1 trafficking in patient cells.

Nephropathic cystinosis is a lysosomal storage disease that is caused by mutations in the CTNS gene encoding a cystine/proton symporter cystinosin and an isoform cystinosin-LKG which is generated by an alternative splicing of exon 12. We have investigated the physiological role of the cystinosin-LKG that is widely expressed in epithelial tissues. We have analyzed the intracellular localization and the function of the cystinosin-LKG conjugated with DsRed (cystinosin-LKG-RFP) in Madin-Darby canine kidney cells (MDCK II) and in proximal tubular epithelial cells carrying a deletion of the CTNS gene (cystinotic PTEC), respectively. Cystinosin-LKG-RFP colocalized with markers of lysosomes, late endosomes and was also expressed on the apical surface of polarized MDCK II cells. Moreover, immune-electron microscopy images of MDCK II cells overexpressing cystinosin-LKG-RFP showed stacked lamellar membranes inside perinuclear lysosomal structures. To study the role of LKG-isoform, we have investigated cystine accumulation and apoptosis that have been described in cystinotic cells. Cystinosin-LKG decreased cystine levels by approximately 10-fold similarly to cystinosin-RFP. The levels of TNFα- and actinomycin D-inducted apoptosis dropped in cystinotic cells expressing LKG-isoform. This effect was also similar to the main isoform. Our results suggest that cystinosin-LKG and cystinosin move similar functional activities in cells.

Nephropathic cystinosis is multisystemic progressive disorder caused by mutations of CTNS gene that encodes for the lysosomal cystine co-transporter cystinosin, and for a less abundant isoform termed cystinosin-LKG, which is expressed in not only lysosomes but also other cell compartments. To overcome the absence of high-quality antibodies against cystinosin, we have obtained a rabbit antiserum against cystinosin-LKG and have analyzed in human tissues the expression of the two known cystinosin isoforms by RT-PCR, and the expression of cystinosin-LKG by immunohistochemistry. In most tissues, CTNS-LKG represents 5-20 % of CTNS transcripts, with the exception of the testis that expresses both isoforms in equal proportions. Cystinosin-LKG was found to be highly expressed in renal tubular cells, pancreatic islets of Langerhans, Leydig cells of the testis, mucoserous glands of the bronchial wall, melanocytes and keratinocytes. These results are parallel with many features of cystinosis, such as early onset Fanconi syndrome, male infertility, diabetes mellitus and hypopigmentation. Intermediate expression levels were of the LKG isoform observed in the gastro-intestinal tract and thyroid glands; low levels of expression were observed in the brain, skeletal and cardiac muscles.

Objectives: Duchenne muscular dystrophy (DMD) is a degenerative muscle wasting disease caused by mutations in the dystrophin gene. Dystrophic muscle is characterized by chronic inflammation, and inflammatory mediators could be promising targets for innovative therapeutic interventions. We analyzed muscle biopsy samples of DMD-affected children to characterize interleukin (IL)-17 and Forkhead box P3 (Foxp3) expression levels and to identify possible correlations with clinical status. Methods: Expression levels of IL-17, Foxp3, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), IL-6, and transforming growth factor-β (TGF-β) were analyzed by real-time PCR in muscle biopsy samples from patients with DMD (n = 27) and juvenile dermatomyositis (JDM) (n = 8). Motor outcome of patients with DMD was evaluated by North Star Ambulatory Assessment score. Results: In DMD, we found higher levels of IL-17 and lower levels of Foxp3 mRNA compared with those for a typical inflammatory myopathy, JDM. Moreover, the IL-17/Foxp3 ratio was higher in DMD than in JDM biopsy samples. IL-17 mRNA levels appeared to be related to the expression levels of other proinflammatory cytokines (TNF-α and MCP-1) and significantly associated with clinical outcome of patients. Conclusions: The association of IL-17 expression with levels of other inflammatory cytokines and with the clinical course of DMD suggests a possible pathogenic role of IL-17.

Ullrich congenital muscular dystrophy (UCMD) is clinically characterized by muscle weakness, proximal contractures and distal hyperlaxity and morphologically branded by absence or reduction of collagen VI (ColVI), in muscle and in cultured fibroblasts. The ColVI defect is generally related to COL6 genes mutations, however UCDM patients without COL6 mutations have been recently reported, suggesting genetic heterogeneity. We report comparative morphological findings between a UCMD patient harboring a homozygous COL6A2 mutation and a patient with a typical UCMD phenotype in which mutations in COL6 genes were excluded. The patient with no mutations in COL6 genes exhibited a partial ColVI defect, which was only detected close to the basal membrane of myofibers. We describe how confocal microscopy and rotary-shadowing electron microscopy may be useful to identify a secondary ColVI defect.

© 2015 Macmillan Publishers Limited Genetic defects of surfactant metabolism are associated with a broad range of clinical manifestations, from neonatal respiratory distress syndrome to adult interstitial lung disease. Early therapies may improve symptoms but diagnosis is often delayed owing to phenotype and genotype variability. Our objective was to characterize the cellular/ultrastructural correlates of surfactant protein C (SP-C) mutations in children with idiopathic diffuse lung diseases. We sequenced SFTPC – the gene encoding SP-C – SFTPB and ABCA3, and analyzed morphology, ultrastructure and SP expression in lung tissue when available. We identified eight subjects who were heterozygous for SP-C mutations. Median age at onset and clinical course were variable. None of the mutations were located in the mature peptide-encoding region, but were either in the pro-protein BRICHOS or linker C-terminal domains. Although lung morphology was similar to other genetic surfactant metabolism disorders, electron microscopy studies showed specific anomalies, suggesting surfactant homeostasis disruption, plus trafficking defects in the four subjects with linker domain mutation and protein misfolding in the single BRICHOS mutation carrier in whom material was available. Immunolabeling studies showed increased proSP-C staining in all cases. In two cases, amyloid deposits could be identified. Immunochemistry and ultrastructural studies may be useful for diagnostic purposes and for genotype interpretation.European Journal of Human Genetics advance online publication, 18 March 2015; doi:10.1038/ejhg.2015.45.

In this study, we characterized five Ullrich scleroatonic muscular dystrophy patients (two Italians, one Belgian, and two Turks) with a clinical phenotype showing different degrees of severity, all carrying mutations localized in COL6A1. We sequenced the three entire COL6 complementary DNA. Three of five patients have recessive mutations: two patients (P1and P3) have homozygous single-nucleotide deletions, one in exon 9 and one in exon 22; one patient (P2) has a homozygous single-nucleotide substitution leading to a premature termination codon in exon 31. The nonsense mutation of P2 also causes a partial skipping of exon 31 with the formation of a premature termination codon in exon 32 in 15% of the total COL6A1 messenger RNA. The remaining two patients carry a heterozygous glycine substitution in exons 9 and 10 inside the triple-helix region; both are dominant mutations because the missense mutations are absent in the DNA of their respective parents. As for the three homozygous recessive mutations, the apparently healthy consanguineous parents all carry a heterozygous mutated allele. Here, for the first time, we report a genotype–phenotype correlation demonstrating that heterozygous glycine substitutions in the triple-helix domain of COL6A1 are dominant and responsible for a milder Ullrich scleroatonic muscular dystrophy phenotype, and that recessive mutations in COL6A1 correlate with more severe clinical and biochemical Ullrich scleroatonic muscular dystrophy phenotypes. Ann Neurol 2005;58:400 – 410

NG2 is the rat homologue of the human melanoma chondroitin sulfate proteoglycan (MCSP) preferentially expressed in dividing progenitor cells of the glial and mesenchymal lineage but downregulated after differentiation. It has recently been demonstrated that MCSP/NG2 expression is not restricted to mitotic or malignant cells. We show that MCSP/NG2 expression is detectable in the sarcolemma, and in the neuromuscular junction of human postnatal skeletal muscle, and it gradually reduces with advancing age. In human and murine myogenic cell lines, we found no clear differences in MCSP/NG2 expression between myoblasts and myotubes. Reduced levels of the core protein were found in merosin-negative congenital muscular dystrophy (MDC1A). Duchenne muscular dystrophy patients muscles exhibited an overexpression of the MCSP/NG2 core protein. In γ-sarcoglycanopathy and calpainopathy, MCSP/NG2 upregulation was restricted to regenerating myofibers. We demonstrate that MCSP/NG2 is expressed in differentiated myofibers, and appears to have a role in the pathogenesis of MDC1A and severe dystrophinopathies.

Copyright © 2014 by the American Society of Nephrology. Intralysosomal cystine crystal accumulation, due tomutations in the CTNS gene, is a hallmark of nephropathic cystinosis, but the role of these crystals in disease pathogenesis remains unclear. We hypothesized that, similar to other host-derived crystalline moieties, cystine crystals can induce IL-1β production through inflammasome activation. Thus, we investigated the proinflammatory effects of cystine crystals in primary human PBMCs. LPS-primed PBMCs stimulated with cystine crystals secreted IL-1β in a dose-dependent manner. Similarly to IL-1β secretion induced by other crystalline inflammasome activators, cystine crystal-induced IL-1β secretion required activation of caspase-1. Additionally, exogenous cystine crystals were internalized by monocytes, and inhibition of phagocytosis, cathepsin B leakage, generation of reactive oxygen species, and potassium efflux reduced cystine crystal-induced IL-1β secretion. Patients with cystinosis had higher levels of circulating IL-1β and IL-18 compared with controls. Analysis of inflammasome-related gene expression in PBMCs from patients with cystinosis revealed a significant increase in IL-1β and CASP-1 transcript levels compared with controls. Moreover, knockout of cystinosin in mice led to significant increases in serum IL-18 levels and kidney expression of inflammasome-related genes (Casp-1, Pycard, Il-18, Il18r1, Il1r1, and Il1rl2). Taken together, these data demonstrate that cystine crystals are endogenous inflammasome-activating stimuli, suggesting a novel role for cystine crystals in the pathogenesis of nephropathic cystinosis.

Emerin is an almost ubiquitous protein which is abnormal in X-linked Emery-Dreifuss muscular dystrophy (EMD), a syndrome characterized by muscle weakness, joint contractures and cardiac arrhythmia. Emerin is localized in the cells at the nuclear rim and its function is still unknown. In some models, emerin has also been described in the cytoplasm; however, its presence outside the nucleus is still matter of debate. We report the presence of emerin in circulating normal human platelets and its absence in platelets from X-linked EMD patients. Since platelets are cytoplasmic fragments derived from megakaryocytes, the presence of emerin in platelets confirms cytoplasmic localization of this protein, probably related to specific functions. We found also that emerin is present in the cytoplasm of megakaryocytes, while it is absent in circulating granulocytes.

Limb-girdle muscle dystrophy type 2I is associated with mutations in the gene encoding Fukutin-related protein. Clinical phenotypes are heterogeneous, ranging from isolated hyperCkemia to severe congenital muscular dystrophy. Affected patients frequently develop dilated cardiomyopathy, depending on evolution of their skeletal myopathy. We report on an 8 years-old boy presenting a severe dilated cardiomyopathy requiring heart transplantation. The child harbored a homozygous p.Leu276Ile mutation in Fukutin-related protein gene (FKRP). At the current age of 20 years, the patient shows persistent hyperCKemia but no clinical muscle weakness, CT scan showing very mild features of muscle involvement. Our findings add to the array of clinical presentations of FKRP mutations.

A previous study of two patients with Walker-Warburg syndrome (WWS) showed a severe deficiency of the extracellular matrix protein laminin beta2 chain and alpha-sarcoglycan (adhalin) in skeletal muscle fibers. More recently, however, other researchers have shown that in their WWS patients the expression of the laminin beta2 chain and alpha-sarcoglycan was normal. Here we describe a 1-year-old boy affected with WWS. We performed immunohistochemical studies on a muscle biopsy from this patient using monoclonal antibodies against dystrophin, dystrophin-associated glycoproteins and several proteins of the extracellular matrix. We confirm previously reports as far as the diminished expression of laminin beta2 chain and alpha-sarcoglycan is concerned. The expression of some other laminins was unusual, whereas the expression of collagen IV and VI was normal. These results suggest that complex syndromes like WWS are quite heterogeneous, although they might represent variant expressions of a single pathological entity.

Emery–Dreifuss muscular dystrophy (EMD) is an inherited myopathy characterised by muscle contractures, progressive muscle wasting and weakness, with humeroperoneal distribution. Cardiac arrhythmia and heart conduction block are also important characteristics of this disease. The X-linked form of EMD is caused by the absence of emerin, encoded by the STA gene (Xq28). Emerin is normally localised in muscle and other tissues at the nuclear rim. Currently, muscle and skin biopsies are used for the immunohistochemical diagnosis. We demonstrate that emerin is present in the cheek oral mucosa, in the exfoliating epithelial cells, and we propose the collection of these cells as a new method for the diagnosis of X-linked EMD patients and the detection of carriers by immunofluorescence techniques: smears from healthy subjects contained about 98% emerin-positive cells, those from X-linked EMD patients contained none and those from carriers contained about 45%. The technique is completely non-invasive, simple, repeatable and inexpensive.

Background Mutations within the C-terminal region of the COL6A1 gene are only detected in Ullrich/Bethlem patients on extremely rare occasions. Case presentation Herein we report two Brazilian brothers with a classic Ullrich phenotype and compound heterozygous for two truncating mutations in COL6A1 gene, expected to result in the loss of the α1(VI) chain C2 subdomain. Despite the reduction in COL6A1 RNA level due to nonsense RNA decay, three truncated alpha1 (VI) chains were produced as protein variants encoded by different out-of-frame transcripts. Collagen VI matrix was severely decreased and intracellular protein retention evident. Conclusion The altered deposition of the fibronectin network highlighted abnormal interactions of the mutated collagen VI, lacking the α1(VI) C2 domain, within the extracellular matrix, focusing further studies on the possible role played by collagen VI in fibronectin deposition and organization.

High-fat and high-carbohydrate diets may predispose to simple steatosis, alone or associated with necroinflammation and fibrosis (steatohepatitis). However, there are few reports about the real effect of these nutrients on hepatocyte redox homeostasis and consequent molecular derangement. Here, we investigated whether different diets would induce oxidative damage in primary rat hepatocytes and thereby affect the activity of phosphatase and tensin homolog (PTEN). We used Sprague–Dawley rats fed, for 14 weeks, a standard diet (SD), a high-fat/low-carbohydrate diet (HFD-LC), a normal-fat/high-fructose diet (NFD-HF), or a high-fat/high-fructose diet (HFD-HF). Metabolic and histological parameters were analyzed in blood and liver samples, while oxidative stress markers and related posttranscriptional modification of PTEN were analyzed in isolated hepatocytes. Our results indicate that different dietetic hypercaloric regimens caused liver damage and a significant increase of body and liver weight, as well as elevated plasma levels of alanine aminotransferase, triglycerides and insulin. Hepatocytes from NFD-HF and HFD-HF rats displayed a decrement of cell viability and proliferation rate. Hepatocytes from animals treated with hypercaloric regimens also exhibited oxidative stress greater than SD hepatocytes. Finally, NFD-HF and HFD-HF hepatocytes showed an increased PTEN phosphorylation and decreased PTEN activity, which seem strongly correlated to an increased glutathionylation of the protein. In conclusion, we demonstrate that fructose-enriched diets cause a tissue and hepatocyte damage that might exacerbate those observed in the presence of high-fat alone and might render, via redox homeostasis imbalance, the hepatocytes more prone to posttranslational modifications and activity alteration of PTEN.

Background Mutations of genes affecting surfactant homeostasis, such as SFTPB, SFTPC and ABCA3, lead to diffuse lung disease in neonates and children. Haploinsufficiency of NKX2.1, the gene encoding the thyroid transcription factor-1 (TTF-1) - critical for lung, thyroid and central nervous system morphogenesis and function - causes a rare form of progressive respiratory failure designated brain-lung-thyroid syndrome. Molecular mechanisms involved in this syndrome are heterogeneous and poorly explored. We report a novel TTF-1 molecular defect causing recurrent respiratory failure episodes in an infant. Methods The subject was an infant with severe neonatal respiratory distress syndrome followed by recurrent respiratory failure episodes, hypopituitarism and neurological abnormalities. Lung histology and ultrastructure were assessed by surgical biopsy. Surfactant-related genes were studied by direct genomic DNA sequencing and array chromatine genomic hybridization (aCGH). Surfactant protein expression in lung tissue was analyzed by confocal immunofluorescence microscopy. For kinetics studies, surfactant protein B and disaturated phosphatidylcholine (DSPC) were isolated from serial tracheal aspirates after intravenous administration of stable isotope-labeled 2H2O and 13C-leucine; fractional synthetic rate was derived from gas chromatography/mass spectrometry 2H and 13C enrichment curves. Six intubated infants with no primary lung disease were used as controls. Results Lung biopsy showed desquamative interstitial pneumonitis and lamellar body abnormalities suggestive of genetic surfactant deficiency. Genetic studies identified a heterozygous ABCA3 mutation, L941P, previously unreported. No SFTPB, SFTPC or NKX2.1 mutations or deletions were found. However, immunofluorescence studies showed TTF-1 prevalently expressed in type II cell cytoplasm instead of nucleus, indicating defective nuclear targeting. This pattern has not been reported in human and was not found in two healthy controls and in five ABCA3 mutation carriers. Kinetic studies demonstrated a marked reduction of SP-B synthesis (43.2 vs. 76.5 ± 24.8%/day); conversely, DSPC synthesis was higher (12.4 vs. 6.3 ± 0.5%/day) compared to controls, although there was a marked reduction of DSPC content in tracheal aspirates (29.8 vs. 56.1 ± 12.4% of total phospholipid content). Conclusion Defective TTF-1 signaling may result in profound surfactant homeostasis disruption and neonatal/pediatric diffuse lung disease. Heterozygous ABCA3 missense mutations may act as disease modifiers in other genetic surfactant defects.

Inherited rippling muscle disease is an autosomal dominant disorder usually associated with caveolin-3 mutations. Rare cases of acquired rippling muscle disease with abnormal caveolin-3 localisation have been reported, without primary caveolin-3 mutations and in association with myasthenia gravis and acetylcholine receptor autoantibodies, or thymoma. We present three new patients with electrically-silent muscle rippling and abnormal caveolin-3 localisation, but without acetylcholine receptor autoantibodies, or clinical or electrophysiological evidence of myasthenia gravis. An autoimmune basis for rippling muscle disease is supported by spontaneous recovery and normalisation of caveolin-3 staining in one patient and alleviation of symptoms in response to plasmapheresis and immunosuppression in another. These patients expand the autoimmune rippling muscle disease phenotype, and suggest that autoantibodies to additional unidentified muscle proteins result in autoimmune rippling muscle disease.

Background and Aim: Oxidative stress is an important pathophysiological mechanism in non-alcoholic steatohepatitis, where hepatocyte apoptosis is significantly increased correlating with disease severity. Protein glutathionylation occurs as a response to oxidative stress, where an increased concentration of oxidized glutathione modifies post-translational proteins by thiol disulfide exchange. In this study, we analyzed the protein glutathionylation in non-alcoholic fatty liver disease (NAFLD) and evaluated a potential association between glutathionylation, fibrosis, and vitamin E treatment. Methods: Protein glutathionylation was studied in the livers of 36 children (mean age 12.5 years, range 4–16 years) subdivided into three groups according to their NAFLD activity score (NAS) by Western blot analysis and immunohistochemistry, using a specific monoclonal antibody. In addition, we identified the hepatocyte ultrastructures involved in glutathionylation by immunogold electron microscopy. Results: Our findings showed that protein glutathionylation increases in the livers of patients with NAFLD and it is correlated with steatohepatitis and liver fibrosis. Its increase appears mainly in nuclei and cytosol of hepatocytes, and it is reversed by antioxidant therapy with reduced fibrosis. Conclusion: Protein glutathionylation significantly increases in livers with NAFLD, strongly suggesting that oxidative injury plays a crucial role in this disease. Furthermore, the marked increase of protein glutathionylation, in correlation with collagen VI immunoreactivity, suggests a link between the redox status of hepatic protein thiols and fibrosis.

Background Muscle-eye-brain disease is a congenital muscular dystrophy with eye and brain involvement due to POMGnT1 mutations. Objective To describe the clinical and molecular features of 3 Italian patients with POMGnT1 mutations. Design Case reports. Patients One patient had muscle and brain abnormalities without eye involvement. Two patients had a classic muscle-eye-brain disease phenotype with different levels of clinical severity. Results Brain magnetic resonance imaging showed cortical malformation and posterior fossa involvement. Immunofluorescence for glycosylated α-dystroglycan performed on muscle biopsy specimens demonstrated an absent signal in 1 patient and reduced staining in 2 patients. Molecular analysis identified 5 mutations, 2 of which are novel. Conclusion This article adds to what is known about the genotype-phenotype correlation and expands our awareness of the clinical spectrum associated with POMGnT1 mutations.

We report on a 2-year-old male child with both nemaline myopathy and hypertrophic cardiomyopathy (HCM). Sequencing of the ACTA1 gene showed a "de novo" missense heterozygous mutation a>g in exon 7 (Lys336Glu). Two-dimensional electrophoresis showed 28% mutant actin present in his muscle biopsy. Actin was isolated from the muscle biopsy and examined by in vitro motility assay. The sliding speed was 13+/-3% less than normal and the affinity of actin for the Z-line protein alpha-actinin was reduced 10 fold. This is the first report on a hypertrophic cardiomyopathy associated with nemaline myopathy and an ACTA1 mutation.

Danon disease, an X-linked cardioskeletal myopathy caused by primary deficiency of lysosome-associated membrane protein-2 (LAMP-2), is clinically characterized by cardiomyopathy, myopathy, and variable mental retardation. The pathological hallmark of the disease is the absence of LAMP-2 immunohistochemical staining in muscle. The LAMP-2 gene mutations reported thus far are generally private mutations. We describe two cases of Danon disease with different clinical presentation, in whom we identified the same exon skipping mutation c.928G>A in the LAMP-2 gene. The first patient was affected by an early onset myopathy and hypertrophic cardiomyopathy (HCM) that partially improved with drug treatment. A first muscle biopsy at age 4 months showed markedly increased glycogen, and acid maltase deficiency was ruled out biochemically. A second muscle biopsy, performed at age 3(1/2) years, showed very mild abnormalities. The second child at age 15 years had mild, diffuse muscle weakness and wasting, moderate mental deficiency, and HCM. Two serial biopsies performed at age 8 and 15 years showed similar findings of multiple esterase-positive vacuoles in type I myofibers. In both patients the immunohistochemical study demonstrated the absence of LAMP-2 in skeletal muscle.

The authors report on a family with dominantly inherited progressive external ophthalmoplegia and a diagnostic and statistical manual (fourth revised edition) diagnosis of bipolar psychiatric disorder in several members. Skeletal muscle biopsy from the proposita showed decreased cytochrome c oxidase staining, several ragged-red fibers, and multiple mtDNA deletions. The authors identified a missense mutation (leucine 98→proline) in the adenine nucleotide translocator 1 gene. The presence of bipolar affective disorder expands the phenotype of adenine nucleotide translocator 1 allelic variants.