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Objective: The study assesses the safety and efficacy of a 675-nm laser source on melasma. Background: Melasma is a frequent acquired skin disease defined by the presence of hyperpigmented macules ...

Significance The spectacular developments in cryoelectron microscopy involving new cameras, new microscopes, and new software make it possible today to routinely determine the atomic structures of a large range of molecular assemblies. This has allowed us to solve the atomic structure of nanotubes formed from a peptide, Lanreotide. Its gel, Somatuline, is used as a synthetic growth hormone inhibitor in the treatment of both acromegaly and cancers. The self-assembled nanotube results in a slow release form of the peptide, important pharmacologically. The nanotube structure shows an unexpected complexity and highlights the still unpredictable chemical and physicochemical determinants driving peptide self-assembly., Functional and versatile nano- and microassemblies formed by biological molecules are found at all levels of life, from cell organelles to full organisms. Understanding the chemical and physicochemical determinants guiding the formation of these assemblies is crucial not only to understand the biological processes they carry out but also to mimic nature. Among the synthetic peptides forming well-defined nanostructures, the octapeptide Lanreotide has been considered one of the best characterized, in terms of both the atomic structure and its self-assembly process. In the present work, we determined the atomic structure of Lanreotide nanotubes at 2.5-Å resolution by cryoelectron microscopy (cryo-EM). Surprisingly, the asymmetric unit in the nanotube contains eight copies of the peptide, forming two tetramers. There are thus eight different environments for the peptide, and eight different conformations in the nanotube. The structure built from the cryo-EM map is strikingly different from the molecular model, largely based on X-ray fiber diffraction, proposed 20 y ago. Comparison of the nanotube with a crystal structure at 0.83-Å resolution of a Lanreotide derivative highlights the polymorphism for this peptide family. This work shows once again that higher-order assemblies formed by even well-characterized small peptides are very difficult to predict.

Objective: To evaluate the histological effects of a new 675-nm laser device on the skin. Background: Innovative technologies based on physical principles have been proposed in recent years to impr...

Dura mater repair represents a final and crucial step in neurosurgery: an inadequate dural reconstruction determines dreadful consequences that significantly increase morbidity and mortality rates. Different dural substitutes have been used with suboptimal results. To overcome this issue, in previous studies, we proposed a laser-based approach to the bonding of porcine dura mater, evidencing the feasibility of the laser-assisted procedure. In this work, we present the optimization of this approach in ex vivo experiments performed on porcine dura mater. An 810-nm continuous-wave AlGaAs (Aluminium Gallium Arsenide) diode laser was used for welding Indocyanine Green-loaded patches (ICG patches) to the dura. The ICG-loaded patches were fabricated using chitosan, a resistant, pliable and stable in the physiological environment biopolymer, moreover, their absorption peak was very close to the laser emission wavelength. Histology, thermal imaging and leak pressure tests were used to evaluate the bonding effect. We demonstrated that the application of 3 watts (W), pulsed mode (Ton 30 ms, Toff 3.5 ms) laser light induces optimal welding of the ICG patch to the dura mater, ensuring an average fluid leakage pressure of 216 ± 105 mmHg, falling within the range of physiological parameters. This study demonstrated that the thermal effect is limited and spatially confined and that the laser bonding procedure can be used to close the dura mater. Our results showed the effectiveness of this approach and encourage further experiments in in vivo models.

AIM This study aimed to report a successful clinical, histological, and histomorphometric outcome of a novel equine-derived bone paste for a ridge preservation surgery involving a single post-extractive socket. BACKGROUND After tooth avulsion, unless the implant position is not carried out straightforwardly, the alveolar process undergoes resorption: to limit it, post-extractive sockets may be grafted according to the ridge preservation principles. Grafting materials should display proper biological properties and optimal handling characteristics. Bone pastes may facilitate grafting operations, avoid granules' dispersion, and maximize the contact of the graft with the surrounding bone. An innovative equine-derived bone paste has been recently introduced on the market, but its use has never been documented in the medical literature. CASE DESCRIPTION This report describes the treatment of a patient who received the equine-derived bone paste in a post-extractive socket to allow the preservation of the alveolar ridge and was later rehabilitated with a crown supported by a single implant. CONCLUSION The handling properties of the equine-derived bone paste were excellent. At the 36-month follow-up, the peri-implant bone levels had been maintained, with the implant being successful according to the Albrektsson and Zarb criteria. Histologic outcome showed that the bone paste was fully biocompatible; histomorphometric analysis showed that a significant amount of newly formed bone could be observed in the grafted socket. CLINICAL SIGNIFICANCE Alveolar ridge preservation using bone grafts is a well-known approach, yet there is still no agreement about which bone graft might be considered the most suitable for this indication. The novel equine-derived bone paste used in the present study appears a promising option for effective socket preservation and may promote secondary intention healing. How to cite this article: Di Stefano DA, Arosio P, Cinci L, et al. Ridge Preservation Using an Innovative Enzyme-deantigenic Equine Bone Paste: A Case Report with 36-month Follow-up. J Contemp Dent Pract 2019;20(10):1229-1234.

Dura mater repair represents a final and crucial step in cranial surgery: an inadequate dural reconstruction determines dreadful consequences that significantly increase morbidity and mortality rates. Different dural substitutes have been used with poor results. To overcome this issue, in previous studies we proposed a laser-based approach to the bonding of porcine dura mater, evidencing the feasibility of the laser assisted procedure. In this work, we present the optimization of the laser bonding approach ex vivo in porcine dura mater and in vivo in rats. An 810 nm cw laser was used to weld the ICG stained chitosan patch to the dura. The ex vivo tests enabled to optimize the laser parameters, using histology and leak pressure evaluation to study the bonding effect. The in vivo tests were performed on 32 adult Wistar rats: laser bonding was carried out in 16 rats, while a collagen matrix was used for duroplasty in the control group. After the treatment, the animals were left to recover and were observed in a 15 and 90 days follow up study. At sacrifice, the rats were anesthetized for fluid leakage pressure test; treated tissue was harvested and underwent standard histology. The results of this study pointed out that the laser bonding procedure can be used to close the dura mater, both ex vivo and in vivo. The thermal effect is limited and spatially confined. The technique can thus be proposed as a valid alternative to standard method for the closuring of dura mater in cranial surgery.

This study aimed to report a successful clinical, histological, and histomorphometric outcome of a novel equine-derived bone paste for a ridge preservation surgery involving a single post-extractive socket.After tooth avulsion, unless the implant position is not carried out straightforwardly, the alveolar process undergoes resorption: to limit it, post-extractive sockets may be grafted according to the ridge preservation principles. Grafting materials should display proper biological properties and optimal handling characteristics. Bone pastes may facilitate grafting operations, avoid granules' dispersion, and maximize the contact of the graft with the surrounding bone. An innovative equine-derived bone paste has been recently introduced on the market, but its use has never been documented in the medical literature.This report describes the treatment of a patient who received the equine-derived bone paste in a post-extractive socket to allow the preservation of the alveolar ridge and was later rehabilitated with a crown supported by a single implant.The handling properties of the equine-derived bone paste were excellent. At the 36-month follow-up, the peri-implant bone levels had been maintained, with the implant being successful according to the Albrektsson and Zarb criteria. Histologic outcome showed that the bone paste was fully biocompatible; histomorphometric analysis showed that a significant amount of newly formed bone could be observed in the grafted socket.Alveolar ridge preservation using bone grafts is a well-known approach, yet there is still no agreement about which bone graft might be considered the most suitable for this indication. The novel equine-derived bone paste used in the present study appears a promising option for effective socket preservation and may promote secondary intention healing. How to cite this article: Di Stefano DA, Arosio P, Cinci L

α-Synuclein (α-syn) is the principal component of Lewy bodies, the pathophysiological hallmark of individuals affected by Parkinson disease (PD). This neuropathologic form of α-syn contributes to PD progression and propagation of α-syn assemblies between neurons. The data we present here support the proteomic analysis used to identify neuronal proteins that specifically interact with extracellularly applied oligomeric or fibrillar α-syn assemblies (conditions 1 and 2, respectively) (doi: 10.15252/embj.201591397 [1]). α-syn assemblies and their cellular partner proteins were pulled down from neuronal cell lysed shortly after exposure to exogenous α-syn assemblies and the associated proteins were identified by mass spectrometry using a shotgun proteomic-based approach. We also performed experiments on pure cultures of astrocytes to identify astrocyte-specific proteins interacting with oligomeric or fibrillar α-syn (conditions 3 and 4, respectively). For each condition, proteins interacting selectively with α-syn assemblies were identified by comparison to proteins pulled-down from untreated cells used as controls. The mass spectrometry data, the database search and the peak lists have been deposited to the ProteomeXchange Consortium database via the PRIDE partner repository with the dataset identifiers PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002256 to PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002263 and doi: 10.6019/http://www.ebi.ac.uk/pride/archive/projects/PXD002256 to 10.6019/http://www.ebi.ac.uk/pride/archive/projects/PXD002263. Keywords: Parkinson׳s disease, Alpha-synuclein assemblies, Proteomic Analysis, Pull down

Tau assemblies have prion‐like properties: they propagate from one neuron to another and amplify by seeding the aggregation of endogenous Tau. Although key in prion‐like propagation, the binding of exogenous Tau assemblies to the plasma membrane of naïve neurons is not understood. We report that fibrillar Tau forms clusters at the plasma membrane following lateral diffusion. We found that the fibrils interact with the Na+/K+‐ATPase (NKA) and AMPA receptors. The consequence of the clustering is a reduction in the amount of α3‐NKA and an increase in the amount of GluA2‐AMPA receptor at synapses. Furthermore, fibrillar Tau destabilizes functional NKA complexes. Tau and α‐synuclein aggregates often co‐exist in patients’ brains. We now show evidences for cross‐talk between these pathogenic aggregates with α‐synuclein fibrils dramatically enhancing fibrillar Tau clustering and synaptic localization. Our results suggest that fibrillar α‐synuclein and Tau cross‐talk at the plasma membrane imbalance neuronal homeostasis.

Extracellular α-synuclein (α-syn) assemblies can be up-taken by neurons; however, their interaction with the plasma membrane and proteins has not been studied specifically. Here we demonstrate that α-syn assemblies form clusters within the plasma membrane of neurons. Using a proteomic-based approach, we identify the α3-subunit of Na+/K+-ATPase (NKA) as a cell surface partner of α-syn assemblies. The interaction strength depended on the state of α-syn, fibrils being the strongest, oligomers weak, and monomers none. Mutations within the neuron-specific α3-subunit are linked to rapid-onset dystonia Parkinsonism (RDP) and alternating hemiplegia of childhood (AHC). We show that freely diffusing α3-NKA are trapped within α-syn clusters resulting in α3-NKA redistribution and formation of larger nanoclusters. This creates regions within the plasma membrane with reduced local densities of α3-NKA, thereby decreasing the efficiency of Na+ extrusion following stimulus. Thus, interactions of α3-NKA with extracellular α-syn assemblies reduce its pumping activity as its mutations in RDP/AHC.

The aggregation of Huntingtin (HTT) protein and of its moiety encoded by its Exon1 (HTTExon1) into fibrillar structures inside neurons is the molecular hallmark of Huntington's disease. Prion-like transmission of these aggregates between cells has been demonstrated. The cell-to-cell transmission mechanisms of these protein aggregates and the susceptibility of different kinds of neuronal cells to these toxic assemblies still need assessment. Here we documented the binding to and internalization by differentiated and undifferentiated neuroblastoma cells of exogenous fibrillar HTTExon1 and polyglutamine (polyQ) polypeptides containing the same number of glutamines. We assessed the contribution of endocytosis to fibrillar HTTExon1 uptake, their intracellular localization and fate. We observed that undifferentiated neuroblastoma cells were more susceptible to fibrillar HTTExon1 and polyQ than their differentiated counterparts. Furthermore, we demonstrated that exogenous HTTExon1 aggregates are mainly taken up by endocytosis and directed to lysosomal compartments in both mitotic and quiescent cells. These data suggest that the rates of endocytic processes that differ in mitotic and quiescent cells strongly impact the uptake of exogenous HTTExon1 and polyQ fibrils. This may be either the consequence of distinct metabolisms or distributions of specific protein partners for amyloid-like assemblies at the surface of highly dividing versus quiescent cells. Our results highlight the importance of endocytic processes in the internalization of exogenous HTTExon1 fibrils and suggest that a proportion of those assemblies reach the cytosol where they can amplify by recruiting the endogenous protein after escaping, by yet an unknown process, from the endo-lysosomal compartments.

The response to wounds until healing requires the activity of many cell types coordinate in space and time, so that the types of cells in a wound and their localization may be of help to date lesions with respect to death, which would be useful in forensic pathology. Cells reacting to injury include dendritic cells; the early reaction of these cells to skin wounding has not yet been investigated in humans, which was the aim of this study. Samples of wounded and control skin were taken at autopsy and analyzed by affinity histochemistry. Both epidermal and dermal MHC-II+ cells increased transiently in number within the first hour after wounding, then decreased. In the epidermis the increase affected also CD1a+ cells, i.e. well differentiated Langherhans cells, which however increased less, earlier and for a shorter time period than MHC-II+ cells. Dermal MHC-II+ cells became part of a perivascular mononuclear cell infiltrate visible in the subpapillary dermis by 60 min after wounding, which contained also mast cells. The immediately perivascular MHC-II+ cells were DC-SIGN- and CD11c-, while MHC-II+, DC-SIGN+, CD11c+ dendritic cells were predominantly located at the periphery of infiltrates and some were near the epidermis. Mast cells underwent degranulation, besides increase in number, in the first hours after wounding. The results suggest that skin dendritic cells, including Langerhans cells, participate to the early response to wounding in concert with mast cells, and that subpapillary blood vessels are primary sites of cell infiltration during that response in humans. The results show that the ratio between CD1a positive and MHC-II positive cells in the epidermis, the degranulation index of mast cells and the relative volume of MHC-II positive cells in the dermis can be added to the tools useful to distinguish vital from post mortem lesions and, the first two of them, to estimate the interval between a lesion and death.

Aim To histologically assess the effectiveness of a socketpreservation technique using enzyme-treated equine bone granules as a bone-graft material in combination with an equine collagen matrix as a scaffold for soft-tissue regeneration. Background Enzyme-treated equine bone granules and equine collagen matrix recently have been developed to help overcome alveolar bone deficiencies that develop in the wake of edentulism. Case report The patient had one mandibular molar extracted and the socket grafted with equine bone granules. The graft was covered with the equine collagen matrix, placed in a double layer. No flap was prepared, and the gingival margins were stabilized with a single stitch, leaving the matrix partially exposed and the site to heal by secondary intention. The adjacent molar was extracted 1 month later, and that socket was left to heal by secondary intention without any further treatment. Three months after each surgery, an implant was placed and a biopsy was collected. The two biopsies underwent histological processing and qualitative evaluation. Histomorphometric analysis was also performed to calculate the percentage of newly formed bone (NFB) in the two cores. Healing at both sites was uneventful, and no inflammation or other adverse reactions were observed in the samples. Soft-tissue healing by secondary intention appeared to occur faster at the grafted site. The corresponding core showed a marked separation between soft and hard tissue that was not observed in the core from the nongrafted site, where soft-tissue hypertrophy could be observed. Newly formed bone at the grafted and nongrafted sites was not significantly different (27.2 ± 7.1 and 29.4 ± 6.2% respectively, p=0.45). Conclusion The surgical technique employed in this case appeared to facilitate postextraction soft-tissue healing by second intention and simplify soft-tissue management. Clinical significance Using a collagen-based matrix to cover a postextraction grafted site may facilitate second intention soft-tissue healing and proper soft-tissue growth. How to cite this article Leonida A, Todeschini G, Lomartire G, Cinci L, Pieri L. Socket Preservation using Enzyme-treated Equine Bone Granules and an Equine Collagen Matrix: A Case Report with Histological and Histomorphometrical Assessment. J Contemp Dent Pract 2016;17(11):890-896.

To date, 3 rare missense mutations in the SNCA (α-synuclein) gene and the more frequent duplications or triplications of the wild-type gene are known to cause a broad array of clinical and pathological symptoms in familial Parkinson disease (PD). Here, we describe a French family with a parkinsonian-pyramidal syndrome harboring a novel heterozygous SNCA mutation. Whole exome sequencing of DNA from 3 patients in a 3-generation pedigree was used to identify a new PD-associated mutation in SNCA. Clinical and pathological features of the patients were analyzed. The cytotoxic effects of the mutant and wild-type proteins were assessed by analytical ultracentrifugation, thioflavin T binding, transmission electron microscopy, cell viability assay, and caspase-3 activation. We identified a novel SNCA G51D (c.152 G>A) mutation that cosegregated with the disease and was absent from controls. G51D was associated with an unusual PD phenotype characterized by early disease onset, moderate response to levodopa, rapid progression leading to loss of autonomy and death within a few years, marked pyramidal signs including bilateral extensor plantar reflexes, occasionally spasticity, and frequently psychiatric symptoms. Pathological lesions predominated in the basal ganglia and the pyramidal tracts and included fine, diffuse cytoplasmic inclusions containing phospho-α-synuclein in superficial layers of the cerebral cortex, including the entorhinal cortex. Functional studies showed that G51D α-synuclein oligomerizes more slowly and its fibrils are more toxic than those of the wild-type protein. We have identified a novel SNCA G51D mutation that causes a form of PD with unusual clinical, neuropathological, and biochemical features.

The deposition of fibrillar alpha-synuclein (α-syn) within inclusions (Lewy bodies and Lewy neurites) in neurons and glial cells is a hallmark of synucleinopathies. α-syn populates a variety of assemblies ranging from prefibrillar oligomeric species to fibrils whose specific contribution to neurodegeneration is still unclear. Here, we compare the specific structural and biological properties of distinct soluble prefibrillar α-syn oligomers formed either spontaneously or in the presence of dopamine and glutaraldehyde. We show that both on-fibrillar assembly pathway and distinct dopamine-mediated and glutaraldehyde-cross-linked α-syn oligomers are only slightly effective in perturbing cell membrane integrity and inducing cytotoxicity, while mature fibrils exhibit the highest toxicity. In contrast to low-molecular weight and unstable oligomers, large stable α-syn oligomers seed the aggregation of soluble α-syn within reporter cells although to a lesser extent than mature α-syn fibrils. These oligomers appear elongated in shape. Our findings suggest that α-syn oligomers represent a continuum of species ranging from unstable low molecular weight particles to mature fibrils via stable elongated oligomers composed of more than 15 α-syn monomers that possess seeding capacity.

Purpose: The aim of this study was to investigate bone formation over time following maxillary sinus augmentation with an enzyme-deantigenic, bone collagen-preserving equine bone graft by retrospective assessment of histomorphometric data. Materials and Methods: Records of patients with atrophic ridges who underwent maxillary sinus augmentation with the enzyme-deantigenic equine bone graft and two-step implant placement between 3 and 12 months after the sinus-augmentation surgery were assessed retrospectively. The histomorphometric data were clustered in three classes according to time of collection from the augmentation surgery and analyzed to assess newly formed bone deposition and residual biomaterial degradation rates. Data concerning the 36-month clinical follow-up were also assessed. Results: Records of 77 patients and 115 biopsy specimens were retrieved, and histomorphometric data were clustered (3 to 5 months, n = 33; 6 to 8 months, n = 57; 9 to 12 months, n = 25). Mean minimum atrophic ridge thickness was 4.9 ± 0.5 mm (range, 4.0 to 7.1 mm). The amount of newly formed bone and residual biomaterial did not significantly differ among the three clusters. Qualitative analysis showed a denser trabecular structure in late (> 8 months) samples. At the 36-month clinical follow-up, no differences were found among the implant success rates in the three groups, according to the Albrektsson and Zarb criteria for success. The overall implant success rate was 98.3%. Conclusion: Based upon this retrospective human study of 77 patients with 4 to 7 mm of residual bone, when enzyme-deantigenic equine bone is used for sinus augmentation, new bone formation occurs at an early time (< 3 months) after the grafting, and implant placement can be safely carried out as soon as 3 to 5 months after the augmentation surgery.

Background α-Synuclein (α-Syn) fibrils are the main constituent of Lewy bodies and a neuropathological hallmark of Parkinson's disease (PD). The propagation of α-Syn assemblies from cell to cell suggests that they are involved in PD progression. We previously showed that α-Syn fibrils are toxic because of their ability to bind and permeabilize cell membranes. Here, we document the cellular response in terms of proteome changes of SH-SY5Y cells exposed to exogenous α-Syn fibrils. Methods We compare the proteomes of cells of neuronal origin exposed or not either to oligomeric or fibrillar α-Syn using two dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry. Results Only α-Syn fibrils induce significant changes in the proteome of SH-SY5Y cells. In addition to proteins associated to apoptosis and toxicity, or proteins previously linked to neurodegenerative diseases, we report an overexpression of proteins involved in intracellular vesicle trafficking. We also report a remarkable increase in fibrillar α-Syn heterogeneity, mainly due to C-terminal truncations. Conclusions Our results show that cells of neuronal origin adapt their proteome to exogenous α-Syn fibrils and actively modify those assemblies. General significance Cells of neuronal origin adapt their proteome to exogenous toxic α-Syn fibrils and actively modify those assemblies. Our results bring insights into the cellular response and clearance events the cells implement to face the propagation of α-Syn assemblies associated to pathology.

International audience; Synucleinopathies such as Parkinson's disease are characterized by the pathological deposition of misfolded α-synuclein aggregates into inclusions throughout the central and peripheral nervous system. Mounting evidence suggests that intercellular propagation of α-synuclein aggregates may contribute to the neuropathology; however, the mechanism by which spread occurs is not fully understood. By using quantitative fluorescence microscopy with co-cultured neurons, here we show that α-synuclein fibrils efficiently transfer from donor to acceptor cells through tunneling nanotubes (TNTs) inside lysosomal vesicles. Following transfer through TNTs, α-synuclein fibrils are able to seed soluble α-synuclein aggregation in the cytosol of acceptor cells. We propose that donor cells overloaded with α-synuclein aggregates in lysosomes dispose of this material by hijacking TNT-mediated intercellular trafficking. Our findings thus reveal a possible novel role of TNTs and lysosomes in the progression of synucleinopathies.

Periodate-lysine-paraformaldehyde (PLP) has been proposed as a fixative for glycoprotein antigens which should stabilize periodate oxidized polysaccharide chains through lysine mediated crosslinks, either directly or by the intermediation of formaldehyde. In spite of premises and attempts reported in the literature, this fixative has never become popular for the study of membrane antigens of immune system cells, which leads to doubts on its real efficacy. We have addressed this issue in biopsies of human skin and found that PLP followed by cryoprotection with 30% sucrose and cryosectioning, or PLP fixation of isolated epidermal sheets, consistently provided for good preservation of morphology and intense labeling of major histocompatibility complex class II molecules, CD1a, CD4, CD8, E-cadherin, cytokeratins in general, cytokeratin-18 in particular, and bromodeoxyuridine, incorporated by cycling cells in vitro, and for the demonstration of tyrosinase enzyme activity. PLP-fixed, osmicated and epon-embedded epidermal sheets proved as good as sheets fixed 365 with a mixture of formaldehyde and glutaraldehyde for electron microscopic morphological analysis. Also, these sheets were amenable to immunoperoxidase staining of Langerhans cell membrane antigen CD1a and keratinocyte membrane antigen E-cadherin before being osmicated and prepared for electron microscopy. In a parallel paper, we had also shown that oral mucosa biopsies fixed in PLP showed good morphology and immunolabeling of CD54, CD80, CD83 and CD86. Therefore, we conclude that PLP can be proposed as a multi-task fixative for light and electron microscopic analysis of membrane, cytoplasmic and nuclear antigens of immune system cells and keratinocytes.

Introduction In atherogenesis, dendritic cells, beside presenting antigens, may be sources of tumour necrosis factor (TNF)alpha and nitric oxide (NO), together with mast cells and smooth muscle cells. Material and Methods We have looked at the expression of TNFalpha and inducible NO synthase (iNOs) by these cells by affinity cytochemistry in autoptical specimens from normal carotid arteries and not ruptured, hemorrhagic or calcified atheromata. Results Round to dendritic, major histocompatibility complex class II molecules (MHC-II+) cells and avidin-labeled mast cells were rare in normal arteries and significantly more numerous in atheromata. Many MHC-II+ cells expressed S-100 antigen; while a few were positive for phalloidin; appreciable fractions of these cells were immunoreactive for TNFalpha and iNOs, both in control specimens and atheromata. The fraction of mast cells labeled for iNOs was significantly lower in atheromata than in controls. Phalloidin positive cells were the most abundant cell type in the normal intima and atheromata; the fractions of these cells labeled for TNFalpha and iNOs were significantly higher in atheromata than in controls. Very few of these cells were also labeled for MHC-II. Computerized image analysis confirmed that the amounts of iNOs and TNFalpha were higher in atheromata than in controls. The increase in TNFalpha in atheromata was also confirmed by western blot. Conclusions Dendritic cells and mast cells can participate to the generation of TNFalpha and NO in the normal arterial wall and in atheromata, but myointimal cells are candidates as major sources of these molecules.

Purpose: To conduct a histomorphometric investigation comparing the use of enzyme-deantigenic equine bone (EDEB) and anorganic bovine bone (ABB) for maxillary sinus augmentation. Materials and Methods: Forty patients with Cawood Class V atrophic ridges who required maxillary sinus augmentation randomly received EDEB (n = 20) or ABB (n = 20) granules. Six months later, biopsy specimens were obtained, and implants were placed. Bone specimens were subjected to histomorphometric analysis, and newly formed bone (NFB) and residual biomaterial (RB) percentages were calculated. Patients were followed up for 3 years after definitive prosthetic rehabilitation, and implant success and survival rates were determined according to the criteria of Albrektsson and Zarb. Results: All patients healed uneventfully. Histomorphometric results for the EDEB were as follows: NFB = 46.86% ± 12.81% and RB = 11.05% ± 9.27%. For ABB, they were: NFB = 25.12% ± 7.25% and RB = 28.65% ± 9.70%. The difference was significant at a .05 level of confidence both for NFB and RB. At the 3-year follow-up, the implant survival rate was identical in the two groups (100%). Conclusion: Grafting with EDEB resulted in a greater quantity of NFB at implant insertion. No significant clinical differences were observed between the two patient groups at the 3-year follow-up. EDEB was as effective as ABB for sinus augmentation. Int J Oral Maxillofac Implants 2015;30:1161–1167.

Extracellular α-synuclein (α-syn) assemblies can be up-taken by neurons; however, their interaction with the plasma membrane and proteins has not been studied specifically. Here we demonstrate that α-syn assemblies form clusters within the plasma membrane of neurons. Using a proteomic-based approach, we identify the α3-subunit of Na+/K+-ATPase (NKA) as a cell surface partner of α-syn assemblies. The interaction strength depended on the state of α-syn, fibrils being the strongest, oligomers weak, and monomers none. Mutations within the neuron-specific α3-subunit are linked to rapid-onset dystonia Parkinsonism (RDP) and alternating hemiplegia of childhood (AHC). We show that freely diffusing α3-NKA are trapped within α-syn clusters resulting in α3-NKA redistribution and formation of larger nanoclusters. This creates regions within the plasma membrane with reduced local densities of α3-NKA, thereby decreasing the efficiency of Na+ extrusion following stimulus. Thus, interactions of α3-NKA with extracellular α-syn assemblies reduce its pumping activity as its mutations in RDP/AHC.

The yeast prion Ure2p assembles in vitro into oligomers and fibrils retaining the alpha-helix content and binding properties of the soluble protein. Here we show that the different forms of Ure2p native-like assemblies (dimers, oligomers, and fibrils) are similarly toxic to murine H-END cells when added to the culture medium. Interestingly, the amyloid fibrils obtained by heat treatment of the toxic native-like fibrils appear harmless. Moreover, the Ure2p C-terminal domain, lacking the N-terminal segment necessary for aggregation but containing the glutathione binding site, is not cytotoxic. This finding strongly supports the idea that Ure2p toxicity depends on the structural properties of the flexible N-terminal prion domain and can therefore be considered as an inherent feature of the protein, unrelated to its aggregation state but rather associated with a basic toxic fold shared by all of the Ure2p native-like assemblies. Indeed, the latter are able to interact with the cell surface, leading to alteration of calcium homeostasis, membrane permeabilization, and oxidative stress, whereas the heat-treated amyloid fibrils do not. Our results support the idea of a general mechanism of toxicity of any protein/peptide aggregate endowed with structural features, making it able to interact with cell membranes and to destabilize them. This evidence extends the widely accepted view that the toxicity by protein aggregates is restricted to amyloid prefibrillar aggregates and provides new insights into the mechanism by which native-like oligomers compromise cell viability.

At least two populations of c-kit positive interstitial cells of Cajal (ICC) lie in the gastric wall, one located at the myenteric plexus level has a pace-making function and the other located intramuscularly is intermediary in the neurotransmission and regenerates the slow waves. Both of these ICC sub-types express full-length dystrophin. Mdx mice, an animal model lacking in full-length dystrophin and used to study Duchenne muscular dystrophy (DMD), show gastric dismotilities. The aim of the present study was to verify in mdx mice whether: (i) gastric ICC undergo morphological changes, through immunohistochemical and ultrastructural analyses; and (ii) there are alterations in the electrical activity, using intracellular recording technique. In control mice, ICC sub-types showed heterogeneous ultrastructural features, either intramuscularly or at the myenteric plexus level. In mdx mice, all of the ICC sub-types underwent important changes: coated vesicles were significantly more numerous and caveolae significantly fewer than in control; moreover, cytoskeleton and smooth endoplasmic reticulum were reduced and mitochondria enlarged. c-Kit-positivity and integrity of the ICC networks were maintained. In the circular muscle of normal mice slow waves, which consisted of initial and secondary components, occurred with a regular frequency. In mdx mice, slow waves occurred in a highly dysrhythmic fashion and they lacked a secondary component. We conclude that the lack of the full-length dystrophin is associated with ultrastructural modifications of gastric ICC, most of which can be interpreted as signs of new membrane formation and altered Ca2+ handling, and with defective generation and regeneration of slow wave activity. J. Cell. Physiol. 199: 293–309, 2004© 2003 Wiley-Liss, Inc.

International audience; Lewy bodies, a pathological hallmark of Parkinson's disease (PD), contain aggregated alpha-synuclein (αSyn), which is found in several modified forms and can be discovered phosphorylated, ubiquitinated and truncated. Aggregation-prone truncated species of αSyn caused by aberrant cleavage of this fibrillogenic protein are hypothesized to participate in its sequestration into inclusions subsequently leading to synaptic dysfunction and neuronal death. Here, we investigated the role of calpain cleavage of αSyn in vivo by generating two opposing mouse models. We crossed into human [A30P]αSyn transgenic (i) mice deficient for calpastatin, a calpain-specific inhibitor, thus enhancing calpain activity (SynCAST(-)) and (ii) mice overexpressing human calpastatin leading to reduced calpain activity (SynCAST(+)). As anticipated, a reduced calpain activity led to a decreased number of αSyn-positive aggregates, whereas loss of calpastatin led to increased truncation of αSyn in SynCAST(-). Furthermore, overexpression of calpastatin decreased astrogliosis and the calpain-dependent degradation of synaptic proteins, potentially ameliorating the observed neuropathology in [A30P]αSyn and SynCAST(+) mice. Overall, our data further support a crucial role of calpains, particularly of calpain 1, in the pathogenesis of PD and in disease-associated aggregation of αSyn, indicating a therapeutic potential of calpain inhibition in PD.

International audience; To date, 3 rare missense mutations in the SNCA (α-synuclein) gene and the more frequent duplications or triplications of the wild-type gene are known to cause a broad array of clinical and pathological symptoms in familial Parkinson disease (PD). Here, we describe a French family with a parkinsonian-pyramidal syndrome harboring a novel heterozygous SNCA mutation. Whole exome sequencing of DNA from 3 patients in a 3-generation pedigree was used to identify a new PD-associated mutation in SNCA. Clinical and pathological features of the patients were analyzed. The cytotoxic effects of the mutant and wild-type proteins were assessed by analytical ultracentrifugation, thioflavin T binding, transmission electron microscopy, cell viability assay, and caspase-3 activation. We identified a novel SNCA G51D (c.152 G>A) mutation that cosegregated with the disease and was absent from controls. G51D was associated with an unusual PD phenotype characterized by early disease onset, moderate response to levodopa, rapid progression leading to loss of autonomy and death within a few years, marked pyramidal signs including bilateral extensor plantar reflexes, occasionally spasticity, and frequently psychiatric symptoms. Pathological lesions predominated in the basal ganglia and the pyramidal tracts and included fine, diffuse cytoplasmic inclusions containing phospho-α-synuclein in superficial layers of the cerebral cortex, including the entorhinal cortex. Functional studies showed that G51D α-synuclein oligomerizes more slowly and its fibrils are more toxic than those of the wild-type protein. We have identified a novel SNCA G51D mutation that causes a form of PD with unusual clinical, neuropathological, and biochemical features.

α-synuclein aggregation is implicated in a variety of diseases including Parkinson’s disease, dementia with Lewy bodies, pure autonomic failure and multiple system atrophy. The association of protein aggregates made of a single protein with a variety of clinical phenotypes has been explained for prion diseases by the existence of different strains that propagate through the infection pathway. Here we structurally and functionally characterize two polymorphs of α-synuclein. We present evidence that the two forms indeed fulfil the molecular criteria to be identified as two strains of α-synuclein. Specifically, we show that the two strains have different structures, levels of toxicity, and in vitro and in vivo seeding and propagation properties. Such strain differences may account for differences in disease progression in different individuals/cell types and/or types of synucleinopathies., α-synuclein is implicated in neurodegenerative diseases. Bousset et al. generate two α-synuclein polymorphs and find differences in aggregation, function and toxicity, suggesting that these altered properties may be the cause for differences in disease progression.

International audience; The aggregation of alpha-synuclein (α-syn) and huntingtin (htt) into fibrillar assemblies in nerve and glial cells is a molecular hallmark of Parkinson's and Huntington's diseases. Within the aggregation process, prefibrillar and fibrillar oligomeric species form. Prefibrillar assemblies rather than fibrils are nowadays considered cytotoxic. However, recent reports describing spreading of fibrillar assemblies from one cell to another, in cell cultures, animal models, and brains of grafted patients suggest a critical role for fibrillar assemblies in pathogenesis. Here we compare the cytotoxic effect of defined and comparable particle concentrations of on-assembly pathway oligomeric and fibrillar α-syn and Htt fragment corresponding to the first exon of the protein (HttEx1). We show that homogeneous populations of α-syn and HttEx1 fibrils, rather than their precursor on-assembly pathway oligomers, are highly toxic to cultured cells and induce apoptotic cell death. We document the reasons that make fibrils toxic. We show that α-syn and HttEx1 fibrils bind and permeabilize lipid vesicles. We also show that fibrils binding to the plasma membrane in cultured cells alter Ca(2+) homeostasis. Overall, our data indicate that fibrillar α-syn and HttEx1, rather than their precursor oligomers, are highly cytotoxic, the toxicity being associated to their ability to bind and permeabilize the cell membranes.

The interaction of amyloid aggregates with the cell plasma membrane is currently considered among the basic mechanisms of neuronal dysfunction in amyloid neurodegeneration. We used amyloid oligomers and fibrils grown from the yeast prion Sup35p, responsible for the specific prion trait [PSI(+)], to investigate how membrane lipids modulate fibril interaction with the membranes of cultured H-END cells and cytotoxicity. Sup35p shares no homology with endogenous mammalian polypeptide chains. Thus, the generic toxicity of amyloids and the molecular events underlying cell degeneration can be investigated without interference with analogous polypeptides encoded by the cell genome. Sup35 fibrils bound to the cell membrane without increasing its permeability to Ca(2+). Fibril binding resulted in structural reorganization and aggregation of membrane rafts, with GM1 clustering and alteration of its mobility. Sup35 fibril binding was affected by GM1 or its sialic acid moiety, but not by cholesterol membrane content, with complete inhibition after treatment with fumonisin B1 or neuraminidase. Finally, cell impairment resulted from caspase-8 activation after Fas receptor translocation on fibril binding to the plasma membrane. Our observations suggest that amyloid fibrils induce abnormal accumulation and overstabilization of raft domains in the cell membrane and provide a reasonable, although not unique, mechanistic and molecular explanation for fibril toxicity.

International audience; The aggregation of α-synuclein (α-Syn), the primary component of Lewy bodies, into high molecular weight assemblies is strongly associated with Parkinson disease. This event is believed to result from a conformational change within native α-Syn. Molecular chaperones exert critical housekeeping functions in vivo including refolding, maintaining in a soluble state, and/or pacifying protein aggregates. The influence of the stress-induced heat shock protein 70 (Hsp70) on α-Syn aggregation has been notably investigated. The constitutively expressed chaperone Hsc70 acts as an antiaggregation barrier before cells are overwhelmed with α-Syn aggregates and Hsp70 expression induced. Here, we investigate the interaction between Hsc70 and α-Syn, the consequences of this interaction, and the role of nucleotides and co-chaperones Hdj1 and Hdj2 as modulators. We show that Hsc70 sequesters soluble α-Syn in an assembly incompetent complex in the absence of ATP. The affinity of Hsc70 for soluble α-Syn diminishes upon addition of ATP alone or together with its co-chaperones Hdj1 or Hdj2 allowing faster binding and release of client proteins thus abolishing α-Syn assembly inhibition by Hsc70. We show that Hsc70 binds α-Syn fibrils with a 5-fold tighter affinity compared with soluble α-Syn. This suggests that Hsc70 preferentially interacts with high molecular weight α-Syn assemblies in vivo. Hsc70 binding certainly has an impact on the physicochemical properties of α-Syn assemblies. We show a reduced cellular toxicity of α-Syn fibrils coated with Hsc70 compared with "naked" fibrils. Hsc70 may therefore significantly affect the cellular propagation of α-Syn aggregates and their spread throughout the central nervous system in Parkinson disease.

The human mesenchymal stromal cell (hMSC), a type of adult stem cell with a fibroblast-like appearance, has the potential to differentiate along the mesenchymal lineage and also along other cell lineages. These abilities make hMSC a promising candidate for use in regenerative medicine. As the hMSC represents a very rare population in vivo, in vitro expansion is necessary for any clinical use. hMSC characterization is commonly carried out through the expression of specific markers and by the capability of differentiating toward at least adipo-, osteo- and chondrocytic lineages. Commitment processes also result in significant changes in the ultrastructure in order to acquire new functional abilities; however, few studies have dealt with the ultrastructural characteristics of hMSC according to the time of incubation and type of media.The immunophenotype, functional characteristics and ultrastructural features of bone marrow (BM) hMSC cultured in two different media were investigated. The media chosen were Iscove's modified Dulbecco's medium (IMDM) and the Dulbecco's modified Eagle medium (DMEM). The latter has been recommended recently by two international transplantation and cytotherapy societies, the International Society of Cellular Therapy (ISCT) and European Group for Blood and Bone Marrow Transplantation (EBMT), for hMSC expansion for clinical applications.The present results indicate that culture conditions greatly influence hMSC ultrastructural features, proliferation, growth and differentiation. In particular, our findings demonstrate that DMEM preserves the hMSC stem features better. Furthermore, the results obtained in IMDM suggest that a small size does not always correlate with conditions of cell immaturity and a greater proliferative potential.

CD133 is a hallmark of primitive myeloid progenitors. We have addressed whether human cord blood cells selected for CD133 can generate dendritic cells, and Langerhans cells in particular, in conditions that promote that generation from CD34+ progenitors. Transforming growth factor-β1 (TGF-β1) and anti–TGF-β1 antibody, respectively, were added in some experiments. With TGF-β, monocytoid cells were recognized after 7 days. Immunophenotypically immature dendritic cells were present at day 14. After 4 more days, the cells expressed CD54, CD80, CD83, and CD86 and were potent stimulators in mixed lymphocyte reaction; part of the cells expressed CD1a and langerin, but not Birbeck granules. Without TGF-β, only a small fraction of cells acquired a dendritic shape and expressed the maturation-related antigens, and lymphocytes were poorly stimulated. With anti–TGF-β, the cell growth was greatly hampered, CD54 and langerin were never expressed, and lymphocytes were stimulated weakly. In conclusion, CD133+ progenitors can give rise in vitro, through definite steps, to mature, immunostimulatory dendritic cells with molecular features of Langerhans cells, although without Birbeck granules. Addition of TGF-β1 helps to stimulate cell growth and promotes the acquisition of mature immunophenotypical and functional features. Neither langerin nor Birbeck granules proved indispensable for lymphocyte stimulation.

Immune synapse formation between dendritic cells (DCs) and T cells is one of the key events in immune reaction. In immunogenic synapses, the presence of fully mature DCs is mandatory; consequently, the modulation of DC maturation may promote tolerance and represents a valuable therapeutic approach in autoimmune diseases. In the field of cell therapy, bone marrow mesenchymal stem cells (MSCs) have been extensively studied for their immunoregulatory properties, such as inhibiting DC immunogenicity during in vitro differentiation and ameliorating in vivo models of autoimmune diseases (e.g., experimental allergic encephalomyelitis). MSCs seem to play different roles with regard to DCs, depending on cell concentration, mechanism of stimulation, and accompanying immune cells. The aim of this work was to elucidate the immunogenic effects of MSC/DC interactions during DC activation (LPS stimulation or Ag loading). Human monocyte-derived DCs, bone marrow-derived MSCs, and circulating lymphocytes obtained from healthy donors, as well as the laboratory-generated influenza virus hemagglutinin-derived peptide, aa 306–318 peptide-specific T cell line were used for this study. We demonstrate that MSCs mediate inhibition of DC function only upon cell–cell contact. Despite no modification observed in cell phenotype or cytokine production, MSC-treated DCs were unable to form active immune synapses; they retained endocytic activity and podosome-like structures, typical of immature DCs. The transcriptional program induced by MSC–DC direct interaction supports at the molecular pathway level the phenotypical features observed, indicating the genes involved into contact-induced rearrangement of DC cytoskeleton.