Forty-Eight-Month Monitoring of Disease Activity in Patients with Long-Standing Rheumatoid Arthritis Treated with TNF-α Inhibitors: Time for Clinical Outcome Prediction and Biosimilar vs Biologic Originator Performance.

BACKGROUND AND OBJECTIVES: Long-term treatment of patients with rheumatoid arthritis with tumor necrosis factor-α inhibitors leads to initial changes in disease activity that can predict a late treatment response. This observational and retrospective study aimed to determine when it is possible to foresee the response to therapy in the case of long-standing rheumatoid arthritis comparing also the efficacy of the original biologics with their biosimilars. METHODS: A total of 1598 patients were recruited and treated with the original biologics, adalimumab and etanercept, or with biosimilars. Patients were monitored over a period of 48 months and disease activity scores (28-Joint Disease Activity Score, Simplified Disease Activity Index, and Clinical Disease Activity Index) were measured every 6 months. RESULTS: No differences in disease activity levels were observed in etanercept versus biosimilars (GP2015/SB4) and adalimumab versus biosimilar (GP2017) patient groups. All scores significantly decreased in all treatments during the first 18 months of therapy, and after 24 months reached a minimum that lasted up to 48 months. CONCLUSIONS: We conclude that biosimilars of adalimumab and etanercept have equivalent effectiveness over a long period of time compared to their originator drugs, and also that the levels of disease activity after 6 months of tumor necrosis factor-α inhibitors (originator drugs and biosimilars) might predict the response to therapy at 4 years in patients with long-standing rheumatoid arthritis.

Real life use of prostacyclin analog (iloprost), a multi-centric survey data from the Scleroderma study group Emilia Romagna (Sclero-RER) and review of the literature.

BACKGROUND AND AIM: Iloprost is recommend worldwide for the treatment of RP and the healing of DUs. The aim of this study is to report the regimens of Iloprost administered in different rheumatological centers within the same regional Health System Methods: A questionnaire exploring different items related to the use of Iloprost was developed and reviewed by three expert rheumatologists. The questionnaire was distributed as an online survey to all local SSc referral centers in Emilia-Romagna (Italy). Data are reported as percentage or median with interquartile range (IQR), as appropriate. An updated review of world literature on this topic was also carried out. RESULTS: All the invited centers completed the survey. There were both local (8) and university hospitals (4). The majority (58%) had a rheumatologist as head physician. All centers used Iloprost: a single monthly administration was the most common treatment (75%). The cycle lasted 1 [IQR 1-2] days with a 0.5-2.0 ng/Kg/min dose according to the drug tolerance of the patients. There were overall 68 spots (beds, reclining armchair, or simple armchair); 2.0 [1.5-4.0] patients were able to receive Iloprost at the same time. University Hospitals had more physicians at their disposal than local hospitals but less paramedic personnel (respectively: 1.8 vs 1.2 physicians, 1.5 vs 2.1 nurses). CONCLUSIONS: These observations were in line with the majority of previous studies reporting different regimens, comparing similar (but not identical) dose and schedule administration, however, despite differences being at times substantial, no standard infusion method is yet available.

An innovative and simple all electrochemical approach to functionalize electrodes with a carbon nanotubes/polypyrrole molecularly imprinted nanocomposite and its application for sulfamethoxazole analysis.

Sulfamethoxazole (SMX) is a commonly used antibiotic which accumulation can favor the development of antimicrobial resistance. Therefore, easy and cheap system to monitor the presence of SMX are needed for human health protection. Herein we present a straightforward all electrochemical approach to fabricate a sensor based on a nanocomposite molecularly imprinted polymer (nanoMIP) for the determination of SMX. Firstly, oxidized multiwalled carbon nanotubes (oxMWCNTs) were electrochemically deposited on a polarized electrode to increase electrodic surface area up to 350%. Then, ultrathin overoxidized polypyrrole MIP in presence of SMX was electropolymerized on oxMWCNTs surface (nanoMIP). Finally, antibiotic was electrochemically removed. The obtained nanoMIP was characterized by atomic force microscopy, X-ray photoelectron spectroscopy and electrochemical techniques. The nanoMIP was used for the electrochemical detection of SMX evidencing a lower limit of detection (413 nM) and a wider linear range (1.99-10.88 µM) with respect a non-nanostructured film. The nanoMIP evidenced also good affinity and a highly reproducible response (RSD = 1.2%). The sensor was able to determine SMX in milk samples evidencing good recovery values. The proposed approach can be also used in future to easily prepare different nanoMIP based sensors with improved performances for different target molecules thus overcoming current fabrication limits.

Observational study on the QUality of life of Italian Axial SpondyloARthritis patients (QUASAR): baseline data.

OBJECTIVES: To describe the baseline characteristics of the patients enrolled in the QUality of life in patients with Axial SpondyloARthritis (QUASAR) study in terms of quality of life (QoL), disease activity, therapy adherence, and work ability in a real-world setting. METHODS: QUASAR is an Italian multicentre, prospective 12-month observational study, including consecutive adult patients classified as axial spondyloarthritis (axSpA) according to the Assessment of SpondyloArthritis international Society criteria for axSpA. RESULTS: Of 512 patients enrolled in 23 rheumatology centres, 80.7% had ankylosing spondylitis (AS) and 19.3% had non-radiographic axSpA (nr-axSpA). Mean ages were 34.1±13.3 years at axSpA symptoms onset and 39.5±13.0 years at diagnosis. Of the patients, 51.4% presented with ≥1 extra articular manifestation (EAM); the most common were psoriasis (17.8%) and uveitis (16.4%). Patients with nr-axSpA and AS had similar EAM rates, disease activity, and QoL. Biologic disease-modifying anti-rheumatic drugs (bDMARDs; 83.2%) were the most commonly received medication, followed by conventional synthetic DMARDs (22.9%) and non-steroidal anti-inflammatory drugs (NSAIDs; 16.6%). At baseline, higher treatment satisfaction was reported with bDMARDs which, together with NSAIDs, were associated with the best overall scores for disease activity, function, and QoL in the overall population and AS subgroup. CONCLUSIONS: QUASAR is the first Italian prospective study that comprehensively evaluated a large axSpA patient sample in a real-world setting. This interim analysis at baseline confirmed that i) patients with AS and nr-axSpA have similar QoL and disease burden, ii) nearly all axSpA patients receive treatment, and iii) bDMARDs and NSAIDs, overall, yield better disease activity and QoL.

Sputtering-Enabled Intracellular X-ray Photoelectron Spectroscopy: A Versatile Method To Analyze the Biological Fate of Metal Nanoparticles.

The investigation of the toxicological profile and biomedical potential of nanoparticles (NPs) requires a deep understanding of their intracellular fate. Various techniques are usually employed to characterize NPs upon cellular internalization, including high-resolution optical and electron microscopies. Here, we show a versatile method, named sputtering-enabled intracellular X-ray photoelectron spectroscopy, proving that it is able to provide valuable information about the behavior of metallic NPs in culture media as well as within cells, directly measuring their internalization, stability/degradation, and oxidation state, without any preparative steps. The technique can also provide nanoscale vertical resolution along with semiquantitative information about the cellular internalization of the metallic species. The proposed approach is easy-to-use and can become a standard technique in nanotoxicology/nanomedicine and in the rational design of metallic NPs. Two model cases were investigated: silver nanoparticles (AgNPs) and platinum nanoparticles (PtNPs) with the same size and coating. We observed that, after 48 h incubation, intracellular AgNPs were almost completely dissolved, forming nanoclusters as well as AgO, AgS, and AgCl complexes. On the other hand, PtNPs were resistant to the harsh endolysosomal environment, and only some surface oxidation was detected after 48 h.

Microenvironmental Stiffness of 3D Polymeric Structures to Study Invasive Rates of Cancer Cells.

Cells are highly dynamic elements, continuously interacting with the extracellular environment. Mechanical forces sensed and applied by cells are responsible for cellular adhesion, motility, and deformation, and are heavily involved in determining cancer spreading and metastasis formation. Cell/extracellular matrix interactions are commonly analyzed with the use of hydrogels and 3D microfabricated scaffolds. However, currently available techniques have a limited control over the stiffness of microscaffolds and do not allow for separating environmental properties from biological processes in driving cell mechanical behavior, including nuclear deformability and cell invasiveness. Herein, a new approach is presented to study tumor cell invasiveness by exploiting an innovative class of polymeric scaffolds based on two-photon lithography to control the stiffness of deterministic microenvironments in 3D. This is obtained by fine-tuning of the laser power during the lithography, thus locally modifying both structural and mechanical properties in the same fabrication process. Cage-like structures and cylindric stent-like microscaffolds are fabricated with different Young's modulus and stiffness gradients, allowing obtaining new insights on the mechanical interplay between tumor cells and the surrounding environments. In particular, cell invasion is mostly driven by softer architectures, and the introduction of 3D stiffness "weak spots" is shown to boost the rate at which cancer cells invade the scaffolds. The possibility to modulate structural compliance also allowed estimating the force distribution exerted by a single cell on the scaffold, revealing that both pushing and pulling forces are involved in the cell-structure interaction. Overall, exploiting this method to obtain a wide range of 3D architectures with locally engineered stiffness can pave the way for unique applications to study tumor cell dynamics.

Boosting the therapeutic efficiency of nanovectors: exocytosis engineering.

In this work, we developed a new general strategy, which we named "exocytosis engineering", to strongly increase the intracellular persistence of nanocarriers and thus the effective dose of transported drugs. The strategy is based on the co-loading of a drug and an exocytosis inhibitor in the nanocarrier, to hinder the high tendency of cells to remove internalized nanocarriers, limiting the pharmacological efficiency of the nanoformulation. In particular, by using a well-known chemotherapeutic drug (doxorubicin) and an efficient exocytosis inhibitor (dimethilamyloride) co-loaded in mesoporous silica nanocarriers, we demonstrated a >6-fold increase in the intracellular dose of the drug (for the same administered dose), achieving a great improvement in its therapeutic action. A strong gain in the cytotoxic effect of the drug was, in fact, observed both in several tumor cell lines and in 3D tumor spheroids. The proposed approach is versatile and broadly applicable to several classes of nanocarriers and drugs, thus opening a fascinating outlook in nanomedicine.

Easy fabrication of aligned PLLA nanofibers-based 2D scaffolds suitable for cell contact guidance studies.

An easy, low-cost and fast wet processing-based method named ASB-SANS (Auxiliary Solvent-Based Sublimation-Aided NanoStructuring) has been used to fabricate poly(l-lactic acid) (PLLA) highly ordered and hierarchically organized 2D fibrillar patterns, with fiber widths between 40 and 500 nm and lengths exceeding tens of microns. A clear contact guidance effect of these nanofibrillar scaffolds with respect to HeLa and NIH-3T3 cells growth has been observed, on top of an overall good viability. For NIH-3T3 pronounced elongation of the cells was observed, as well as a remarkable ability of the patterns to guide the extension of pseudopodia. Moreover, SEM imaging revealed filopodia stemming from both sides of the pseudopodia and aligned with the secondary PLLA nanofibrous structures created by the ASB-SANS procedure. These results validate ASB-SANS as a technique capable to provide biocompatible 2D nanofibrillar patterns suitable for studying phenomena of contact guidance (and, more in general, the behavior of cells onto nanofibrous scaffolds), at very low costs and in an extremely easy way, accessible to virtually any laboratory.

Electrochemical sensor for sulfadimethoxine based on molecularly imprinted polypyrrole: study of imprinting parameters.

The present work describes the development of a simple and cost-effective electrochemical sensor for sulfadimethoxine (SDM) based on molecularly imprinted overoxidized polypyrrole (PPy). An all electrochemical approach is used for sensor fabrication and application consisting in molecularly imprinted polymer (MIP) galvanostatic deposition on a gold electrode and its overoxidation under different experimental conditions and in SDM amperometric detection. Several parameters influencing the imprinting effect are critically discussed and evaluated. A key role of the electrolyte used in electropolymerization (tetrabuthylammonium perchlorate and lithium perchlorate) has emerged demonstrating its effect on sensing performances of imprinted PPy and, related to this, on its morphology, as highlighted by atomic force microscopy (AFM). The effect of different overoxidation conditions in removing template is evaluated by analyzing MIP films before and after the treatment by X-ray photoelectron spectroscopy (XPS) also evidencing the correlation between MIP chemical structure and its rebinding ability. MIP-template interaction is verified also by Fourier Transform Infrared (FT-IR) spectroscopy. Under the selected optimal conditions, MIP sensor shows a linear range from 0.15 to 3.7 mM SDM, a limit of detection of 70 µM, a highly reproducible response (RSD 4.2%) and a good selectivity in the presence of structurally related molecules. SDM was determined in milk samples spiked at two concentration levels: 0.2 mM and 0.4 mM obtaining a satisfactory recovery of (97±3)% and (96±8)%, respectively.

A DNA-based nano-immunoassay for the label-free detection of glial fibrillary acidic protein in multicell lysates.

We have developed a quantitative approach to eventually enable precise and multiplexing protein analysis of very small systems, down to a single or a few cells. Through DNA-directed immobilization of DNA-protein conjugates we immobilized antibodies specific for a certain protein of interest, on a complementary DNA nanoarray fabricated by means of nanografting, a nanolithography technique based on atomic force microscopy (AFM). The proof of concept was realized for glial fibrillary acidic protein (GFAP), a biomarker crucial in cell's differentiation of astrocytes, and functional to grade classification of gliomas, the most common of primary malignant brain tumors. The efficiency of the nano-immuno sensing was tested by obtaining the immobilization of purified recombinant GFAP protein at different concentration in a standard solution then in a cellular lysate. A comparison of sensitivity between our technique and conventional ELISA assays is provided at the end of the paper. FROM THE CLINICAL EDITOR: This team developed a quantitative approach to enable precise and multiplexing protein analysis of very small systems, down to a single or a few cells, demonstrating the utility of this DNA-based nano-immunoassay in the detection of GFAP.

Atomic force microscopy based nanoassay: a new method to study α-Synuclein-dopamine bioaffinity interactions.

Intrinsically Disordered Proteins (IDPs) are characterized by the lack of well-defined 3-D structure and show high conformational plasticity. For this reason, they are a strong challenge for the traditional characterization of structure, supramolecular assembly and biorecognition phenomena. We show here how the fine tuning of protein orientation on a surface turns useful in the reliable testing of biorecognition interactions of IDPs, in particular α-Synuclein. We exploited atomic force microscopy (AFM) for the selective, nanoscale confinement of α-Synuclein on gold to study the early stages of α-Synuclein aggregation and the effect of small molecules, like dopamine, on the aggregation process. Capitalizing on the high sensitivity of AFM topographic height measurements we determined, for the first time in the literature, the dissociation constant of dopamine-α-Synuclein adducts.

Defined α-synuclein prion-like molecular assemblies spreading in cell culture.

BACKGROUND: α-Synuclein (α-syn) plays a central role in the pathogenesis of synucleinopathies, a group of neurodegenerative disorders that includes Parkinson disease, dementia with Lewy bodies and multiple system atrophy. Several findings from cell culture and mouse experiments suggest intercellular α-syn transfer. RESULTS: Through a methodology used to obtain synthetic mammalian prions, we tested whether recombinant human α-syn amyloids can promote prion-like accumulation in neuronal cell lines in vitro. A single exposure to amyloid fibrils of human α-syn was sufficient to induce aggregation of endogenous α-syn in human neuroblastoma SH-SY5Y cells. Remarkably, endogenous wild-type α-syn was sufficient for the formation of these aggregates, and overexpression of the protein was not required. CONCLUSIONS: Our results provide compelling evidence that endogenous α-syn can accumulate in cell culture after a single exposure to exogenous α-syn short amyloid fibrils. Importantly, using α-syn short amyloid fibrils as seed, endogenous α-syn aggregates and accumulates over several passages in cell culture, providing an excellent tool for potential therapeutic screening of pathogenic α-syn aggregates.