Polyclonal and monoclonal B lymphocytes response in HCV-infected patients treated with direct-acting antiviral agents

Hepatitis C virus (HCV) chronic infection can be associated with extrahepatic manifestations such as mixed cryoglobulinaemia and lymphoproliferative disorders that are endowed with increased rates of morbidity and all-cause mortality. In this study, we used flow cytometry to evaluate the effect of interferon-free antiviral treatment on peripheral blood lymphocytes in HCV-infected patients with or without associated lymphoproliferative disorders. Flow cytometry analysis of peripheral blood lymphocytes was performed at baseline and at the end of treatment. In HCV-infected patients with lymphoproliferative disorders, we evaluated immunoglobulin (Ig) light chain \u3ba/\u3bb ratio variations as a measure of monoclonal B-cell response to antiviral therapy. Healthy volunteers were enrolled as controls. A total of 29 patients were included, nine with and 20 without lymphoproliferative disorders. Sustained virological response was achieved in 29 of 29 patients. We observed a significant reduction in the B-cell compartment (39% global reduction) in eight of nine HCV-infected patients with lymphoproliferative disorders after viral clearance. We recognized the same trend, even if less pronounced, in HCV-infected patients without lymphoproliferative disorders (9% global reduction). Among HCV-infected patients with lymphoproliferative disorders, three showed an improvement/normalization of the immunoglobulin light chain ratio, whereas in the remaining six patients monoclonal B cells persisted to be clonally restricted even 1\ua0year after the end of treatment. Our data show that DAAs treatment can be effective in reducing the frequency of pathological B cells in the peripheral blood of HCV-infected patients affected by HCV-associated lymphoproliferative disorders; however, monoclonal populations can persist after viral eradication

The UCMD-Causing COL6A1 (c:930 + 189C > T) Intron Mutation Leads to the Secretion and Aggregation of Single Mutated Collagen VI α1 Chains

Collagen VI is a unique member of the collagen family. Its assembly is a complex multistep process and the vulnerability of the process is manifested in muscular diseases. Mutations in COL6A1, COL6A2, and COL6A3 lead to the severe Ullrich Congenital Muscular Dystrophy (UCMD) and a spectrum of disease of varying severity including the milder Bethlem muscular dystrophy. The recently identified dominant intronic mutation in COL6A1 ( c . 930 + 189 C > T ) leads to the partial in-frame insertion of a pseudoexon between exon 11 and exon 12. The pseudoexon is translated into 24 amino acid residues in the N-terminal region of the triple helix and results in the interruption of the typical G-X-Y motif. This recurrent de novo mutation leads to UCMD with a severe progression within the first decade of life. Here, we demonstrate that a mutation-specific antibody detects the mutant chain colocalizing with wild type collagen VI in the endomysium in patient muscle. Surprisingly, in the cell culture of patient dermal fibroblasts, the mutant chain is secreted as a single α chain, while in parallel, normal collagen VI tetramers are assembled with the wild-type α1 chain. The mutant chain cannot be incorporated into collagen VI tetramers but forms large aggregates in the extracellular matrix that may retain the ability to interact with collagen VI and potentially with other molecules. Also, α1 chain-deficient WI-26 VA4 cells transfected with the mutant α1 chain do not assemble collagen VI tetramers but, instead, form aggregates. Interestingly, both the wild type and the mutant single α1 chains form amorphous aggregates when expressed in HEK293 cells in the absence of α2 and α3 chains. The detection of aggregated, assembly incompetent, mutant collagen VI α1 chains provides novel insights into the disease pathophysiology of UCMD patients with the COL6A1 ( c . 930 + 189 C > T ) mutation

Mapping the current state of the medical specialties in laboratory medicine in Italy

Laboratory medicine is the single highest volume medical activity and it plays an increasingly essential role in the modern healthcare systems. In vitro diagnostic tests are now used in virtually every aspect of the patient care, including disease prevention, diagnosis, disease monitoring as well as personalised medicine. Nevertheless, the profession of laboratory medicine differs between countries in many respects, such as specialist training (medical or scientific), fields of interest, responsibilities and professional organisation. Many attempts have been made to quantify the role of laboratory medicine in patient outcomes, but the precise figures are still not clear. Moreover, the relative contribution of medical specialists in laboratory medicine is not well known and somehow controversial. To start exploring these aspects, we studied the current state of the two medical specialties that make up the majority of laboratory medicine in Italy: clinical pathology and medical microbiology. Our analysis revealed that both specialties suffer from a low attractivity among postgraduate physicians, and suggest that a restructuring of the training programme and professional reorganisation should be considered

Expression and functional role of Emilin-3, a peculiar member of the Emilin/Multimerin family

Emilin-3 is a glycoprotein of the extracellular matrix belonging to a family of proteins that contain a characteristic N-terminal cystein-rich region, the EMI domain. Knockout mice for Emilin-1, the prototypic protein of the family, display hypertension and reduced diameter of blood vessels due to increased bioavailability of active TGF-β1 that reduces the proliferation rate of vascular smooth muscle cells. This finding is in agreement with the distribution of Emilin-1, which is mainly present in the cardiovascular system. In vitro studies demonstrated that Emilin-1 through its EMI domain acts as an extracellular inhibitor of TGF-β1 processing. At difference from other emilins/multimerins, Emilin-3 has a unique protein structure, lacking the C-terminal C1q domain. Emilin-3 has a peculiar pattern of expression, as revealed by our studies in mouse and zebrafish. The gene is not expressed in the cardiovascular system, while it is present in tissues playing key roles during the development. Emilin-3 mRNA is abundant in the tail bud, notochord and chordoneural hinge, in the subventricular zone of the midbrain, around the osteogenic mesenchyme and in the developing gonads. In the perspective of studying Emilin-3 properties and unveiling its function, in this PhD work I carried out different in vitro and in vivo studies. In summary, the data reported in this thesis provide the first detailed characterization, with both structural and functional insights, of the extracellular glycoprotein Emilin-3.Emilina-3 è una glicoproteina della matrice extracellulare appartenente ad una famiglia di proteine caratterizzate dalla presenza nella loro porzione N-terminale di un domino ricco di cisteine, il dominio EMI. Topi knockout per Emilina-1, la proteina capostipite di questa famiglia, presentano ipertensione arteriosa e una riduzione del calibro dei vasi sanguigni, alterazioni che sono state attribuite ad una disregolazione del signaling del TGF-β. Queste osservazioni sono in accordo con la distribuzione proteica di Emilina-1, che è abbondantemente presente a livello del sistema cardiovascolare. Esperimenti in vitro hanno dimostrato che Emilina-1, attraverso il suo dominio EMI, agisce come inibitore extracellulare della maturazione del precursore del TGF-β in citochina matura. Emilina-3 possiede una peculiare struttura proteica in quanto è priva del dominio gC1q al suo C-terminale, che è invece presente in tutte le altre Emiline/Multimerine. Inoltre Emilina-3 presenta anche un pattern di espressione unico, come dimostrato con i nostri studi di espressione in topo e zebrafish. Il gene infatti non è espresso nel sistema cardiovascolare come gli altri geni della famiglia, ma è espresso durante la vita embrionale in distretti importanti per i processi di sviluppo. Il messaggero di Emilina-3 è abbondante nel tail bud, nella cerniera cordoneurale, nella zona subventricolare del mesencefalo, nel pericondrio delle ossa lunghe in formazione e nei primordi delle gonadi. Con la finalità di studiare le caratteristiche e la funzione di Emilina-3, durante il mio ciclo di dottorato ho intrapreso diversi studi sia in vitro che in vivo. In conclusione, gli esperimenti riportati in questa tesi rappresentano la prima descrizione della glicoproteina Emilina-3, sia dal punto di vista strutturale che funzionale

EMILIN proteins are novel extracellular constituents of the dentin-pulp complex

Odontoblasts and pulp stroma cells are embedded within supramolecular networks of extracellular matrix (ECM). Fibrillin microfibrils and associated proteins are crucial constituents of these networks, serving as contextual scaffolds to regulate tissue development and homeostasis by providing both structural and mechanical properties and sequestering growth factors of the TGF-beta superfamily. EMILIN-1, -2, and -3 are microfibril-associated glycoproteins known to modulate cell behaviour, growth factor activity, and ECM assembly. So far their expression in the various cells of the dentin-pulp complex during development, in the adult stage, and during inflammation has not been investigated. Confocal immunofluorescence microscopy and western blot analysis of developing and adult mouse molars and incisors revealed an abundant presence of EMILINs in the entire dental papilla, at early developmental stages. Later in development the signal intensity for EMILIN-3 decreases, while EMILIN-1 and -2 staining appears to increase in the pre-dentin and in the ECM surrounding odontoblasts. Our data also demonstrate new specific interactions of EMILINs with fibulins in the dentin enamel junction. Interestingly, in dentin caries lesions the signal for EMILIN-3 was significantly increased in inflamed odontoblasts. Overall our findings point for the first time to a role of EMILINs in dentinogenesis, pulp biology, and inflammation

Collagen type VI is the antigen recognized by the ER-TR7 antibody

The monoclonal antibody ER-TR7 was used in a great number of studies for detecting reticular fibroblasts and the ECM of lymphoid and non-lymphoid organs even if the protein recognized by the ER-TR7 antibody was not known. We have now identified native collagen VI microfibrils as its tissue antigen

Fibulin-4 deposition requires EMILIN-1 in the extracellular matrix of osteoblasts

Tissue microenvironments formed by extracellular matrix networks play an important role in regulating tissue structure and function. Extracellular microfibrillar networks composed of fibrillins and their associated ligands such as LTBPs, fibulins, and EMILINs are of particular interest in this regard since they provide a specialized cellular microenvironment guiding proper morphology and functional behavior of specialized cell types. To understand how cellular microenvironments composed of intricate microfibrillar networks influence cell fate decisions in a contextual manner, more information about the spatiotemporal localization, deposition, and function of their components is required. By employing confocal immunofluorescence and electron microscopy we investigated the localization and extracellular matrix deposition of EMILIN-1 and -2 in tissues of the skeletal system such as cartilage and bone as well as in in vitro cultures of osteoblasts. We found that upon RNAi mediated depletion of EMILIN-1 in primary calvarial osteoblasts and MC3T3-E1 cells only fibulin-4 matrix deposition was lost while other fibulin family members or LTBPs remained unaffected. Immunoprecipitation and ELISAstyle binding assays confirmed a direct interaction between EMILIN-1 and fibulin-4. Our data suggest a new function for EMILIN-1 which implies the guidance of linear fibulin-4 matrix deposition and thereby fibulin-4 fiber formation

Physical exercise stimulates autophagy in normal skeletal muscles but is detrimental for collagen VI deficient muscles.

Autophagy is a catabolic process that provides the degradation of altered/damaged organelles through the fusion between autophagosomes and lysosomes. Proper regulation of the autophagic flux is fundamental for the homeostasis of skeletal muscles in physiological conditions and in response to stress. Defective as well as excessive autophagy is detrimental for muscle health and has a pathogenic role in several forms of muscle diseases. Recently, we found that defective activation of the autophagic machinery plays a key role in the pathogenesis of muscular dystrophies linked to collagen VI. Impairment of the autophagic flux in collagen VI null (Col6a1\u2013/\u2013) mice causes accumulation of dysfunctional mitochondria and altered sarcoplasmic reticulum, leading to apoptosis and degeneration of muscle fibers. Here we show that physical exercise activates autophagy in skeletal muscles. Notably, physical training exacerbated the dystrophic phenotype of Col6a1\u2013/\u2013 mice, where autophagy flux is compromised. Autophagy was not induced in Col6a1\u2013/\u2013 muscles after either acute or prolonged exercise, and this led to a marked increase of muscle wasting and apoptosis. These findings indicate that proper activation of autophagy is important for muscle homeostasis during physical activity