Publications by authors named "Laura Pieri"

34 Publications

Ridge Preservation Using an Innovative Enzyme-deantigenic Equine Bone Paste: A Case Report with 36-month Follow-up.

J Contemp Dent Pract 2019 Oct 1;20(10):1229-1234. Epub 2019 Oct 1.

Department of Health Sciences, University of Florence, Florence, Italy.

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, 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.
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October 2019

Increased Immune Activation by Pathologic α-Synuclein in Parkinson's Disease.

Ann Neurol 2019 10 15;86(4):593-606. Epub 2019 Aug 15.

Department of Neurology, Ulm University, Ulm, Germany.

Objective: Excessive inflammation in the central nervous system (CNS) and the periphery can result in neurodegeneration and parkinsonism. Recent evidence suggests that immune responses in Parkinson disease patients are dysregulated, leading to an increased inflammatory reaction to unspecific triggers. Although α-synuclein pathology is the hallmark of Parkinson disease, it has not been investigated whether pathologic α-synuclein is a specific trigger for excessive inflammatory responses in Parkinson disease.

Methods: We investigated the immune response of primary human monocytes and a microglial cell line to pathologic forms of α-synuclein by assessing cytokine release upon exposure.

Results: We show that pathologic α-synuclein (mutations, aggregation) results in a robust inflammatory activation of human monocytes and microglial BV2 cells. The activation is conformation- dependent, with increasing fibrillation and early onset mutations having the strongest effect on immune activation. We also found that activation of immune cells by extracellular α-synuclein is potentiated by extracellular vesicles, possibly by facilitating the uptake of α-synuclein. Blood extracellular vesicles from Parkinson disease patients induce a stronger activation of monocytes than blood extracellular vesicles from healthy controls. Most importantly, monocytes from Parkinson disease patients are dysregulated and hyperactive in response to stimulation with pathologic α-synuclein. Furthermore, we demonstrate that α-synuclein pathology in the CNS is sufficient to induce the monocyte dysregulation in the periphery of a mouse model.

Interpretation: Taken together, our data suggest that α-synuclein pathology and dysregulation of monocytes in Parkinson disease can act together to induce excessive inflammatory responses to α-synuclein. ANN NEUROL 2019;86:593-606.
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http://dx.doi.org/10.1002/ana.25557DOI Listing
October 2019

Chemical, Clinical and Histomorphometric Comparison between Equine Bone Manufactured through Enzymatic Antigen-Elimination and Bovine Bone Made Non-Antigenic Using a High-Temperature Process in Post-Extractive Socket Grafting. A Comparative Retrospective Clinical Study.

Dent J (Basel) 2019 Jul 1;7(3). Epub 2019 Jul 1.

Department of Health Sciences, Interdepartmental Forensic Medicine Section, University of Florence, Viale G. Pieraccini 6, 50139 Florence, Italy.

Enzyme-deantigenic equine bone (EDEB) and anorganic bovine bone (ABB) are two xenografts made non-antigenic through different processing methods. This study aimed to characterize them for the presence of native bone collagen and other proteins and to compare their histomorphometric outcome when they were used to graft post-extractive sockets. The records of 46 patients treated with EDEB ( = 22) or ABB ( = 24) and followed-up for at least four months after delayed implant placement, were retrospectively collected. Samples of EDEB and ABB were analyzed using Attenuated Total Reflection Fourier Transform Infrared and Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis for the presence of collagen and other proteins. For histomorphometric analysis on bone specimens, newly formed bone and residual biomaterial percentages were calculated. Results of the present study show that EDEB contains type I bone collagen in its native conformation, while no proteins were detected in ABB. Grafting EDEB resulted in a significantly greater quantity of newly formed bone and less residual biomaterial. Our findings suggest that the manufacturing process can greatly affect the graft behavior and a process preserving collagen in its native form may favor bone tissue regeneration.
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http://dx.doi.org/10.3390/dj7030070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6784468PMC
July 2019

Clustering of Tau fibrils impairs the synaptic composition of α3-Na/K-ATPase and AMPA receptors.

EMBO J 2019 02 10;38(3). Epub 2019 Jan 10.

CEA, Institut François Jacob (MIRcen) and CNRS Laboratory of Neurodegenerative Diseases (UMR9199), Fontenay-aux-Roses, France

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.
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http://dx.doi.org/10.15252/embj.201899871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356061PMC
February 2019

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 Nov 1;17(11):890-896. Epub 2016 Nov 1.

Department of Health Sciences, Interdepartmental Forensic Medicine Section, University of Florence, Florence, Italy.

Aim: To histologically assess the effectiveness of a socket-preservation 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.
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http://dx.doi.org/10.5005/jp-journals-10024-1949DOI Listing
November 2016

Tunneling nanotubes spread fibrillar α-synuclein by intercellular trafficking of lysosomes.

EMBO J 2016 10 22;35(19):2120-2138. Epub 2016 Aug 22.

Institut Pasteur, Unité Trafic Membranaire et Pathogénèse, Paris Cedex 15, France

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5048354PMC
http://dx.doi.org/10.15252/embj.201593411DOI Listing
October 2016

Structural and functional properties of prefibrillar α-synuclein oligomers.

Sci Rep 2016 Apr 14;6:24526. Epub 2016 Apr 14.

Paris-Saclay Institute of Neuroscience, Centre National de la Recherche Scientifique, Université Paris-Saclay, 91190 Gif-sur-Yvette, France.

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.
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http://dx.doi.org/10.1038/srep24526DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4830946PMC
April 2016

Bone Formation Following Sinus Augmentation with an Equine-Derived Bone Graft: A Retrospective Histologic and Histomorphometric Study with 36-Month Follow-up.

Int J Oral Maxillofac Implants 2016 Mar-Apr;31(2):406-12

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.
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http://dx.doi.org/10.11607/jomi.4373DOI Listing
November 2016

Data in support of the identification of neuronal and astrocyte proteins interacting with extracellularly applied oligomeric and fibrillar α-synuclein assemblies by mass spectrometry.

Data Brief 2016 Jun 12;7:221-8. Epub 2016 Feb 12.

École Normale Supérieure, Institut de Biologie de l'ENS (IBENS), INSERM, CNRS, PSL Research University, 46 Rue d׳Ulm, Paris 75005, France.

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: PXD002256 to PRIDE: PXD002263 and doi: 10.6019/PXD002256 to 10.6019/PXD002263.
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http://dx.doi.org/10.1016/j.dib.2016.02.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4773484PMC
June 2016

Cellular response of human neuroblastoma cells to α-synuclein fibrils, the main constituent of Lewy bodies.

Biochim Biophys Acta 2016 Jan 22;1860(1 Pt A):8-19. Epub 2015 Oct 22.

Paris-Saclay Institute of Neuroscience, CNRS, Université Paris-Saclay, Avenue de la terrasse, 91190 Gif-sur-Yvette, France. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbagen.2015.10.007DOI Listing
January 2016

Histomorphometric Comparison of Enzyme-Deantigenic Equine Bone and Anorganic Bovine Bone in Sinus Augmentation: A Randomized Clinical Trial with 3-Year Follow-Up.

Int J Oral Maxillofac Implants 2015 Sep-Oct;30(5):1161-7

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.
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http://dx.doi.org/10.11607/jomi.4057DOI Listing
May 2016

α-synuclein assemblies sequester neuronal α3-Na+/K+-ATPase and impair Na+ gradient.

EMBO J 2015 Oct 31;34(19):2408-23. Epub 2015 Aug 31.

École Normale Supérieure, Institut de Biologie de l'ENS (IBENS) INSERM CNRS PSL Research University, Paris, France

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.
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http://dx.doi.org/10.15252/embj.201591397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4601662PMC
October 2015

Horizontal-guided Bone Regeneration using a Titanium Mesh and an Equine Bone Graft.

J Contemp Dent Pract 2015 02 1;16(2):154-62. Epub 2015 Feb 1.

Department of Health Sciences, Interdepartmental Forensic Medicine Section, University of Florence, Florence, Italy.

Aim: The present work describes a horizontal ridge augmentation in which a titanium mesh was preshaped by adapting it to a stereolithographic model of the patient's jaw that was fabricated from CT scans.

Background: Guided bone regeneration (GBR) involves covering the augmentation site with a long-lasting barrier to protect it from the invasion of surrounding soft tissues. Among barriers, titanium meshes may provide a successful outcome, but the intraoperatory time needed to shape them is a disadvantage.

Case Description: The 54-year-old patient, missing the right mandibular second bicuspid, first molar, and second molar, had her atrophic ridge augmented with a 30:70 mixture of autogenous bone and equine, enzyme-deantigenic collagen-preserved bone substitute. Two conical implants were inserted concomitantly in the second bicuspid and first molar positions, and the site was protected with the preshaped mesh. Four months later, the titanium mesh was retrieved, a bone sample was collected, and histological and histomorphometric analyses were performed. Provisional and definitive prostheses were then delivered, and follow-up controls were performed for up to 24 months.

Conclusion: Preshaping the mesh on a model of the patient's mandible shortened the surgical time and enabled faster mesh placement. Two years after surgery, the implants were perfectly functional, and the bone width was stable over time as shown by radiographic controls. Histological analysis of the bone sample showed the heterologous biomaterial to be biocompatible and undergoing advanced remodeling and replacement with newly formed bone.

Clinical Significance: Preshaping a titanium mesh over a stereolithographic model of the patient's jaw allowed for a significant reduction of the intraoperative time and may be therefore, advisable in routine practice.
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http://dx.doi.org/10.5005/jp-journals-10024-1653DOI Listing
February 2015

Overexpression of the calpain-specific inhibitor calpastatin reduces human alpha-Synuclein processing, aggregation and synaptic impairment in [A30P]αSyn transgenic mice.

Hum Mol Genet 2014 Aug 11;23(15):3975-89. Epub 2014 Mar 11.

Institute of Medical Genetics and Applied Genomics, University of Tuebingen, Tuebingen 72076, Germany, Department of Neurosciences, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92003-0624, USA

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.
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http://dx.doi.org/10.1093/hmg/ddu112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4110482PMC
August 2014

Structural and functional characterization of two alpha-synuclein strains.

Nat Commun 2013 ;4:2575

Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France.

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.
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http://dx.doi.org/10.1038/ncomms3575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3826637PMC
May 2014

G51D α-synuclein mutation causes a novel parkinsonian-pyramidal syndrome.

Ann Neurol 2013 Apr;73(4):459-71

Pierre and Marie Curie University-Paris 6, Research Center of the Institute for Brain and Spinal Cord, National Institute of Health and Medical Research, Paris.

Objective: 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.

Methods: 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.

Results: 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.

Interpretation: We have identified a novel SNCA G51D mutation that causes a form of PD with unusual clinical, neuropathological, and biochemical features.
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http://dx.doi.org/10.1002/ana.23894DOI Listing
April 2013

Fibrillar α-synuclein and huntingtin exon 1 assemblies are toxic to the cells.

Biophys J 2012 Jun 19;102(12):2894-905. Epub 2012 Jun 19.

Laboratoire d'Enzymologie et Biochimie Structurales, Centre Nationale de la Recherche Scientifique, Gif-sur-Yvette, France.

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.
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http://dx.doi.org/10.1016/j.bpj.2012.04.050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3379023PMC
June 2012

Toxic effects of amyloid fibrils on cell membranes: the importance of ganglioside GM1.

FASEB J 2012 Feb 9;26(2):818-31. Epub 2011 Nov 9.

Department of Biochemical Sciences, University of Florence, Florence, Italy.

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.
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http://dx.doi.org/10.1096/fj.11-189381DOI Listing
February 2012

Hsc70 protein interaction with soluble and fibrillar alpha-synuclein.

J Biol Chem 2011 Oct 10;286(40):34690-9. Epub 2011 Aug 10.

Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 91198 Gif-sur-Yvette, France.

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.
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http://dx.doi.org/10.1074/jbc.M111.261321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3186418PMC
October 2011

Dendritic cells with lymphocyte-stimulating activity differentiate from human CD133 positive precursors.

Blood 2011 Apr 8;117(15):3983-95. Epub 2011 Feb 8.

Department of Anatomy, Histology and Forensic Medicine, University of Florence, Florence, Italy.

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.
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http://dx.doi.org/10.1182/blood-2010-08-299735DOI Listing
April 2011

α-Synuclein propagates from mouse brain to grafted dopaminergic neurons and seeds aggregation in cultured human cells.

J Clin Invest 2011 Feb 18;121(2):715-25. Epub 2011 Jan 18.

Neuronal Survival Unit, Wallenberg Neuroscience Center, Lund University, Lund, Sweden.

Post-mortem analyses of brains from patients with Parkinson disease who received fetal mesencephalic transplants show that α-synuclein-containing (α-syn-containing) Lewy bodies gradually appear in grafted neurons. Here, we explored whether intercellular transfer of α-syn from host to graft, followed by seeding of α-syn aggregation in recipient neurons, can contribute to this phenomenon. We assessed α-syn cell-to-cell transfer using microscopy, flow cytometry, and high-content screening in several coculture model systems. Coculturing cells engineered to express either GFP- or DsRed-tagged α-syn resulted in a gradual increase in double-labeled cells. Importantly, α-syn-GFP derived from 1 neuroblastoma cell line localized to red fluorescent aggregates in other cells expressing DsRed-α-syn, suggesting a seeding effect of transmitted α-syn. Extracellular α-syn was taken up by cells through endocytosis and interacted with intracellular α-syn. Next, following intracortical injection of recombinant α-syn in rats, we found neuronal uptake was attenuated by coinjection of an endocytosis inhibitor. Finally, we demonstrated in vivo transfer of α-syn between host cells and grafted dopaminergic neurons in mice overexpressing human α-syn. In summary, intercellularly transferred α-syn interacts with cytoplasmic α-syn and can propagate α-syn pathology. These results suggest that α-syn propagation is a key element in the progression of Parkinson disease pathology.
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http://dx.doi.org/10.1172/JCI43366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3026723PMC
February 2011

Human mesenchymal stromal cells preserve their stem features better when cultured in the Dulbecco's modified Eagle medium.

Cytotherapy 2011 May 3;13(5):539-48. Epub 2011 Jan 3.

Department of Anatomy, Histology and Forensic Medicine, Section of Histology, University of Florence, Florence, Italy.

Background Aims: 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.

Methods: 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.

Results And Conclusions: 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.
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http://dx.doi.org/10.3109/14653249.2010.542459DOI Listing
May 2011

Inhibition of immune synapse by altered dendritic cell actin distribution: a new pathway of mesenchymal stem cell immune regulation.

J Immunol 2010 Nov 1;185(9):5102-10. Epub 2010 Oct 1.

Department of Neurological Sciences, University of Florence, Florence, Italy.

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.
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http://dx.doi.org/10.4049/jimmunol.1001332DOI Listing
November 2010

Proteomic analysis of cells exposed to prefibrillar aggregates of HypF-N.

Biochim Biophys Acta 2009 Aug 3;1794(8):1243-50. Epub 2009 May 3.

Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Italy.

Several human diseases are associated with the deposition of stable ordered protein aggregates known as amyloid fibrils. In addition, a large wealth of data shows that proteins not involved in amyloidoses, are able to form, in vitro, amyloid-like prefibrillar and fibrillar assemblies indistinguishable from those grown from proteins associated with disease. Previous studies showed that early prefibrillar aggregates of the N-terminal domain of the prokaryotic hydrogenase maturation factor HypF (HypF-N) are cytotoxic, inducing early mitochondria membrane depolarization, activation of caspase 9 and eventually cell death. To gain knowledge on the molecular basis of HypF-N aggregate cytotoxicity, we performed a differential proteomic analysis of NIH-3T3 cells exposed to HypF-N prefibrillar aggregates in comparison with control cells. Two-dimensional gel electrophoresis followed by protein identification by MALDI-TOF MS, allowed us to identify 21 proteins differentially expressed. The changes of the expression level of proteins involved in stress response (Hsp60 and 78 kDa glucose-regulated protein) and in signal transduction (Focal adhesion kinase1) appear particularly interesting as possible determinants of the cell fate. The levels of some of the differently expressed proteins were modified also in similar studies carried out on cells exposed to Abeta or alpha-synuclein aggregates, supporting the existence of shared features of amyloid cytotoxicity.
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http://dx.doi.org/10.1016/j.bbapap.2009.04.009DOI Listing
August 2009

Synthetic lipid vesicles recruit native-like aggregates and affect the aggregation process of the prion Ure2p: insights on vesicle permeabilization and charge selectivity.

Biophys J 2009 Apr;96(8):3319-30

Department of Biochemical Sciences, University of Florence, Italy; Research Centre on the Molecular Basis of Neurodegeneration, University of Florence, Italy.

The yeast prion Ure2p polymerizes into native-like fibrils, retaining the overall structure and binding properties of the soluble protein. Recently we have shown that, similar to amyloid oligomers, the native-like Ure2p fibrils and their precursor oligomers are highly toxic to cultured mammalian cells when added to the culture medium, whereas Ure2p amyloid fibrils generated by heating the native-like fibrils are substantially harmless. We show here that, contrary to the nontoxic amyloid fibrils, the toxic, native-like Ure2p assemblies induce a significant calcein release from negatively charged phosphatidylserine vesicles. A minor and less-specific effect was observed with zwitterionic phosphatidylcholine vesicles, suggesting that the toxic aggregates preferentially bind to negatively charged sites on lipid membranes. We also found that cholesterol-enriched phospholipid membranes are protected against permeabilization by native-like Ure2p assemblies. Moreover, vesicle permeabilization appears charge-selective, allowing calcium, but not chloride, influx to be monitored. Finally, we found that the interaction with phosphatidylserine membranes speeds up Ure2p polymerization into oligomers and fibrils structurally and morphologically similar to the native-like Ure2p assemblies arising in free solution, although less cytotoxic. These data suggest that soluble Ure2p oligomers and native-like fibrils, but not amyloid fibrils, interact intimately with negatively charged lipid membranes, where they allow selective cation influx.
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http://dx.doi.org/10.1016/j.bpj.2008.12.3958DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2718290PMC
April 2009

Rosiglitazone reduces the inflammatory response in a model of vascular injury in rats.

Shock 2009 Dec;32(6):638-44

Department of Experimental Medicine, Excellence Centre for Cardiovascular Diseases, Second University of Naples, Naples, Italy.

Thiazolidinediones are ligands that bind to and activate the nuclear peroxisome proliferator-activated receptor gamma. They are widely used as insulin sensitizers for the treatment of type 2 diabetes. Several studies have implicated the peroxisome proliferator-activated receptor gamma agonists rosiglitazone and pioglitazone in inflammatory events. To assess the anti-inflammatory properties of rosiglitazone, we investigated its effects on the molecular and cellular inflammatory response induced by a carotid injury in the rat. Male Wistar rats were randomized into a rosiglitazone-treated group (10 mg kg(-1) day(-1)) and a control group (0.9% w/v NaCl). The drug or vehicle was administered by gavage for 7 days before carotid injury and for up to 21 days after injury. The inflammatory markers p38 mitogen-activated protein kinase, cyclooxygenase 2, nuclear factor-kappaB, and heat shock protein 47 and the influx and activity of cells in response to injury were measured. Rosiglitazone treatment significantly reduced the expression of the inflammatory markers compared with control group. p38 mitogen-activated protein kinase and nuclear factor-kappaB started to decrease a few hours after injury, whereas cyclooxygenase 2 and heat shock protein 47 expression decreased 7 and 14 days, respectively, after injury. Rosiglitazone also reduced neointima formation and inflammatory cell infiltration. In conclusion, rosiglitazone negatively regulated the inflammatory events involved in tissue repair at molecular and cellular levels. These results suggest that rosiglitazone plays a protective role in inflammatory vascular diseases.
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http://dx.doi.org/10.1097/SHK.0b013e3181a5a377DOI Listing
December 2009

Histochemical and ultrastructural characteristics of an interstitial cell type different from ICC and resident in the muscle coat of human gut.

J Cell Mol Med 2008 Oct;12(5B):1944-55

Department of Anatomy, Histology and Forensic Medicine, University of Florence, Florence, Italy.

CD117 (or c-kit) is expressed by the interstitial cells of Cajal (ICC), which are located within the gastrointestinal (GI) muscle coat and directly involved in its motility. CD34 is expressed by several cell types some of which have features and location resembling the ICC; however, a sure identification of these cells is still lacking. In order to establish whether the CD34-positive cells of the human GI tract are to be considered as ICC subpopulation or a novel independent cell type, and to hypothesize their nature and role, we verified CD34 and CD117 receptor expression under light and fluorescence microscope and performed a routine and a CD34-immuno-electron microscopy. CD34-positive cells were seen in the entire human GI tract. In the muscularis propria, shared morphologies similar to the c-kit-positive cells, in the submucosa, resembled fibroblasts. Their ultrastructure resembled that of the fibrocytes/fibroblasts and of the interstitial Cajal-like cells (ICLC). Double labelling and immunoelectro-microscopy demonstrated that they are unequivocally different to the ICC and, due to the similarities with the ICLC, we identified them as ICLC. The novelty of these results is that two types of interstitial cells are present in the GI muscle coat of humans: the ICC and the ICLC. We hypothesize a mechanical role for the septal ICLC, those at the myenteric plexus level and those bordering the muscle layers; a helping role in neurotransmission is proposed for the ICLC intercalated with the intramuscular ICC, possibly in spreading the slow waves generated by the ICC. Furthermore, the possibility that the ICLC represent the adult mesenchymal stromal cells able to guarantee the ICC renewal deserves to be considered.
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http://dx.doi.org/10.1111/j.1582-4934.2008.00461.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4506162PMC
October 2008

Smooth muscle cells, dendritic cells and mast cells are sources of TNFalpha and nitric oxide in human carotid artery atherosclerosis.

Thromb Res 2008 17;122(5):657-67. Epub 2008 Jun 17.

Department of Anatomy, Histology and Forensic Medicine, University of Florence, Florence, Italy.

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.
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http://dx.doi.org/10.1016/j.thromres.2008.04.013DOI Listing
December 2008

Delayed development of interstitial cells of Cajal in the ileum of a human case of gastroschisis.

J Cell Mol Med 2008 Apr 8;12(2):471-8. Epub 2008 Feb 8.

Department of Pediatric Surgery, University of Padua, Padua, Italy.

The Interstitial Cells of Cajal (ICC) are responsible for rhythmic electrical activity. A paralytic ileus is present in gastroschisis (GS), a malformation due to a defective closure of the abdominal wall through which part of the intestine herniates during pregnancy. In experimental GS, ICC morphological immaturity was shown in the rat foetus at-term but it could not be demonstrated whether differentiation is accomplished post-natally. For this purpose we morphologically investigated ICC, as well as enteric neurons and smooth muscle cells, in a case of human GS at birth and 1 month later when peristaltic activity had initiated. A 36 weeks gestation female was born by c/section with prenatal diagnosis of GS and possible volvulus of the herniated intestine. At birth, the necrotic intestine was resected and both ileostomy and colostomy were performed. The intestine continuity was restored after 4 weeks. Intestinal specimens, taken during both operations at the level of the proximal stoma, were immunostained with c-kit, neuron-specific-enolase and alpha-smooth-muscle-actin antibodies and some processed for electron microscopy. ICC were present at the myenteric plexus only. At birth, these cells were rare and ultrastructurally immature; 1 month later, when partial enteral feeding was tolerated, they formed rows or groups and many of them were ultrastructurally differentiated. Neurons and smooth muscle cells, immature at birth, had developed after 1 month. Therefore, ICC differentiation, as well as that of neurons and smooth muscle cells, is delayed at birth and this might explain the paralytic ileus in GS. One month later, differentiation quickly proceeded at all cellular levels paralleling the increasing tolerance of enteral nutrition.
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http://dx.doi.org/10.1111/j.1582-4934.2008.00277.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3822536PMC
April 2008

A role for transforming growth factor-beta1 in maintaining the differentiated state of Langerhans cells in human epidermis.

Ital J Anat Embryol 2006 Jul-Sep;111(3):133-49

Department of Anatomy, Histology and Forensic Medicine of the University of Florence, Florence, Italy.

Since during the generation of dendritic cells transforming growth factor (TGF)-beta1 is required to generate specifically Langerhans cells, we have addressed whether TGF-beta1 also affects the number and immunophenotype of Langerhans cells within the epidermis. Isolated human epidermal sheets were cultured as follows: serum free; with serum; serum free with TGF-beta1; with serum plus anti-TGF-betal; with serum plus an irrelevant antibody of the same isotype as anti-TGF-beta1. The expression of Langerhans cell antigens was analyzed by immunofluorescence and the preservation of epidermal structure and the expression of E-cadherin by electron microscopy. The number, surface area and perimeter length of Langerhans cells were measured and the results subjected to analysis of variance. Independent of serum, the architecture of the isolated epidermis was well preserved and E-cadherin was expressed for at least 48 h. In cultures without serum, Langerhans cells appeared well preserved when stained for MHC-II antigens. On the contrary, their number, surface area and perimeter length were significantly decreased upon labeling for CD1a and langerin, indicating altered expression and distribution of these differentiation specific antigens. These alterations were not accompanied by the expression of antigens of mature dendritic cells and were almost entirely prevented by serum. TGF-beta1, 1.0 ng/mL, had similar effects as serum on CD1a and langerin expression and distribution within cells, whereas anti-TGF-beta1 antibodies neutralized the effect of serum. The results indicate that Langerhans cells depend on soluble factors for the maintenance of the differentiated state within epidermis and that TGF-beta1 is a major one of such factors.
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April 2007