A Long-Term Follow-Up Study in Immune-Mediated Thrombotic Thrombocytopenic Purpura: What Are the Outcomes?

Endothelium damage triggers the multimeric protein von Willebrand factor (VWF) release and subsequent binding to platelets, which are recruited at sites of vascular injury. A complex and fragile equilibrium between circulating levels of von Willebrand factor and its metalloprotease, ADAMTS13, is responsible for the hemostatic balance. However, the presence of autoantibodies targeting ADAMTS13 results in an increase in von Willebrand factor, mainly in its ultra-large multimers. The latter lead to platelet aggregation, the formation of thrombi and microangiopathic hemolytic anemia. This pathologic condition, known as immune-mediated thrombotic thrombocytopenic purpura (iTTP), occurs with high morbidity and a high rate of relapses. In this work, the long-term follow-up of 40 patients with iTTP is reported. We assessed ADAMTS13 activity, plasmatic VWF levels and the ADAMTS13/VWF ratio, comparing iTTP relapsing patients with remitting ones. A decrease in the ADAMTS13/VWF ratio, along with a reduced ADAMTS13 activity, could serve as predictive and sensitive biomarkers of incoming relapses.

Anti-Phosphatidylserine, Anti-Prothrombin, and Anti-Annexin V Autoantibodies in Antiphospholipid Syndrome: A Real-Life Study.

The antiphospholipid antibodies (aPL) increase the risk of developing thrombotic events and may coexist with a variety of autoimmune diseases. They can be detected chronically or temporarily in patients with infectious diseases, during drug therapy, or in cases of cancer. A thrombotic event with aPL detection is known as antiphospholipid syndrome (APS) and the diagnostic criteria include the presence of lupus anticoagulant (LA), anticardiolipin (aCL) and ß2-glycoprotein-1(aß2GPI) antibodies. Other autoantigens recognized in APS are phosphatidylserine (aPS), prothrombin (aPT) and Annexin-5 (aA5). This real life study aimed to explore the connections between laboratory criteria and the prevalence of "non-criteria aPL" in APS. This study followed 300 patients with thrombosis and employed two phospholipid sensitivity assays for LA detection, chemiluminescence assays for aCL and aß2GPI and enzyme-linked immunoassays for aPS, aPT and aA5. A significant association was found between aPS and aCL (r = 0.76) as well as aß2GPI (r = 0.77), while the association with LA was less significant (r = 0.33). The results of the aPT and aA5 test did not correlate with criteria-antiphospholipid antibodies (r < 0.30). Since the risk of thrombotic complications increases with the intensity and the number of positive autoantibodies, measuring aPT and aA5 autoantibodies may be useful, particularly in aCL/aß2GPI-negative patients or in cases of isolated LA positivity.

New Trends in Pathology: From Cell Morphology to Molecular Medicine.

After Rudolf Virchow's pioneering works, technological advances boosted the scientific interest in this research field, which nowadays is still far from extinguished [...].

In Vitro Evaluation of the Effects of Hyaluronic Acid and an Aminoacidic Pool on Human Osteoblasts.

The treatment of bone injuries must be timely and effective to improve the chances of full recovery. In this respect, a mix of hyaluronic acid and an amino acidic pool has been marketed to promote soft tissue healing, fastening recovery times. Several studies have reported the in vitro and in vivo influence of hyaluronic acid and amino acids on fibroblasts and keratinocytes, highlighting the enhancement of cell proliferation, motility and cytokines synthesis. Even though the effectiveness of this combination of molecules on bone repair has been described in vivo, to the best of our knowledge, its in vitro effects on osteoblasts still need to be investigated. Therefore, this work describes for the first time osteoblast metabolism, proliferation and in vitro differentiation in the presence of hyaluronic acid and amino acids, aiming at understanding the mechanisms underlying their effectiveness in injured tissue repair. The reported results demonstrate the enhancement of osteoblasts' metabolic activity and the fastening of cell cycle progression. Furthermore, gene expression studies show a significant increase in differentiation markers, i.e., osteoprotegerin and osteonectin. Finally, alkaline phosphatase activity is also boosted by the combination of hyaluronic acid and aminoacids, confirming the ability of in vitro cultured cells to properly differentiate through the osteogenic lineage.

Correlation between In Vitro Neutralization Assay and Serological Tests for Protective Antibodies Detection.

Serological assays are useful in investigating the development of humoral immunity against SARS-CoV-2 in the context of epidemiological studies focusing on the spread of protective immunity. The plaque reduction neutralization test (PRNT) is the gold standard method to assess the titer of protective antibodies in serum samples. However, to provide a result, the PRNT requires several days, skilled operators, and biosafety level 3 laboratories. Therefore, alternative methods are being assessed to establish a relationship between their outcomes and PRNT results. In this work, four different immunoassays (Roche Elecsys® Anti SARS-CoV-2 S, Snibe MAGLUMI® SARS-CoV-2 S-RBD IgG, Snibe MAGLUMI® 2019-nCoV IgG, and EUROIMMUN® SARS-CoV-2 NeutraLISA assays, respectively) have been performed on individuals healed after SARS-CoV-2 infection. The correlation between each assay and the reference method has been explored through linear regression modeling, as well as through the calculation of Pearson's and Spearman's coefficients. Furthermore, the ability of serological tests to discriminate samples with high titers of neutralizing antibodies (>160) has been assessed by ROC curve analyses, Cohen's Kappa coefficient, and positive predictive agreement. The EUROIMMUN® NeutraLISA assay displayed the best correlation with PRNT results (Pearson and Spearman coefficients equal to 0.660 and 0.784, respectively), as well as the ROC curve with the highest accuracy, sensitivity, and specificity (0.857, 0.889, and 0.829, respectively).

Analytical Assessment of the Vela Diagnostics NGS Assay for HIV Genotyping and Resistance Testing: The Apulian Experience.

Drug-resistance monitoring is one of the hardest challenges in HIV management. Next-generation sequencing (NGS) technologies speed up the detection of drug resistance, allowing the adjustment of antiretroviral therapy and enhancing the quality of life of people living with HIV. Recently, the NGS Sentosa® SQ HIV Genotyping Assay (Vela Diagnostics) received approval for in vitro diagnostics use. This work is the first Italian evaluation of the performance of the Vela Diagnostics NGS platform, assessed with 420 HIV-1 clinical samples. A comparison with Sanger sequencing performance is also reported, highlighting the advantages and disadvantages of the Sentosa® NGS assay. The precision of the technology was studied with reference specimens, while intra- and inter-assay reproducibility were evaluated for selected clinical samples. Vela Diagnostics' NGS assay reached an 87% success rate through 30 runs of analysis in a real-world clinical context. The concordance with Sanger sequencing outcomes was equal to 97.2%. Several detected mismatches were due to NGS's superior sensitivity to low-frequency variants. A high accuracy was observed in testing reference samples. Repeatability and reproducibility assays highlighted the good performance of the NGS platform. Beyond a few technical issues that call for further optimization, the key improvement will be a better balance between costs and processing speed. Once these issues have been solved, the Sentosa® SQ HIV Genotyping Assay will be the way forward for HIV resistance testing.

Dopamine-loaded lipid based nanocarriers for intranasal administration of the neurotransmitter: A comparative study.

Both dopamine (DA) loaded Solid Lipid Nanoparticles (SLN) and liposomes (Lip), designed for intranasal administration of the neurotransmitter as an innovative Parkinson disease treatment, were already characterized in vitro in some extent by us (Trapani et al., 2018a and Cometa et al., 2020, respectively). Herein, to gain insight into the structure of SLN, X-ray Photoelectron Spectroscopy Analysis was carried out and DA-SLN (SLN 1) were found to exhibit high amounts of the neurotransmitter on the surface, whereas the external side of Glycol Chitosan (GCS) containing SLN (SLN 2) possessed only few amounts. However, SLN 2 were characterized by the highest encapsulation DA efficiency (i.e., 81%). Furthermore, in view of intranasal administration, mucoadhesion tests in vitro were also conducted for SLN and Lip formulations, evidencing high muchoadesive effect exerted by SLN 2. Concerning ex-vivo studies, SLN and Lip were found to be safe for Olfactory Ensheathing Cells and fluorescent SLN 2 were taken up in a dose-dependent manner reaching the 100% of positive cells, while Lip 2 (chitosan-glutathione-coated) were internalised by 70% OECs with six-times more lipid concentration. Hence, SLN 2 formulation containing DA and GCS may constitute interesting formulations for further studies and promising dosage form for non-invasive nose-to-brain neurotransmitter delivery.

Innovative Eco-Friendly Hydrogel Film for Berberine Delivery in Skin Applications.

Hydrogel formulations (masks or patches, without tissue support) represent the new frontier for customizable skin beauty and health. The employment of these materials is becoming popular in wound dressing, to speed up the healing process while protecting the affected area, as well as to provide a moisturizing reservoir, control the inflammatory process and the onset of bacterial development. Most of these hydrogels are acrylic-based at present, not biodegradable and potentially toxic, due to acrylic monomers residues. In this work, we selected a new class of cellulose-derived and biodegradable hydrogel films to incorporate and convey an active compound for dermatological issues. Films were obtained from a combination of different polysaccharides and clays, and berberine hydrochloride, a polyphenolic molecule showing anti-inflammatory, immunomodulatory, antibacterial and antioxidant properties, was chosen and then embedded in the hydrogel films. These innovative hydrogel-based systems were characterized in terms of water uptake profile, in vitro cytocompatibility and skin permeation kinetics by Franz diffusion cell. Berberine permeation fitted well to Korsmeyer-Peppas kinetic model and achieved a release higher than 100 µg/cm2 within 24 h. The latter study, exploiting a reliable skin model membrane, together with the biological assessment, gained insights into the most promising formulation for future investigations.

A 3D Printed Composite Scaffold Loaded with Clodronate to Regenerate Osteoporotic Bone: In Vitro Characterization.

Additive manufacturing (AM) is changing our current approach to the clinical treatment of bone diseases, providing new opportunities to fabricate customized, complex 3D structures with bioactive materials. Among several AM techniques, the BioCell Printing is an advanced, integrated system for material manufacture, sterilization, direct cell seeding and growth, which allows for the production of high-resolution micro-architectures. This work proposes the use of the BioCell Printing to fabricate polymer-based scaffolds reinforced with ceramics and loaded with bisphosphonates for the treatment of osteoporotic bone fractures. In particular, biodegradable poly(ε-caprolactone) was blended with hydroxyapatite particles and clodronate, a bisphosphonate with known efficacy against several bone diseases. The scaffolds' morphology was investigated by means of Scanning Electron Microscopy (SEM) and micro-Computed Tomography (micro-CT) while Energy Dispersive X-ray Spectroscopy (EDX) and X-ray Photoelectron Spectroscopy (XPS) revealed the scaffolds' elemental composition. A thermal characterization of the composites was accomplished by Thermogravimetric analyses (TGA). The mechanical performance of printed scaffolds was investigated under static compression and compared against that of native human bone. The designed 3D scaffolds promoted the attachment and proliferation of human MSCs. In addition, the presence of clodronate supported cell differentiation, as demonstrated by the normalized alkaline phosphatase activity. The obtained results show that the BioCell Printing can easily be employed to generate 3D constructs with pre-defined internal/external shapes capable of acting as a temporary physical template for regeneration of cancellous bone tissues.

Unravelling the Antifungal Effect of Red Thyme Oil (Thymus vulgaris L.) Compounds in Vapor Phase.

The aim of this work was to evaluate the antifungal activity in vapor phase of thymol, p-cymene, and γ-terpinene, the red thyme essential oil compounds (RTOCs). The Minimum Inhibitory Concentration (MIC) of RTOCs was determined against postharvest spoilage fungi of the genera Botrytis, Penicillium, Alternaria, and Monilinia, by measuring the reduction of the fungal biomass after exposure for 72 h at 25 °C. Thymol showed the lowest MIC (7.0 µg/L), followed by γ-terpinene (28.4 µg/L) and p-cymene (40.0 µg/L). In the case of P. digitatum ITEM 9569, resistant to commercial RTO, a better evaluation of interactions among RTOCs was performed using the checkerboard assay and the calculation of the Fractional Inhibitory Concentration Index (FICI). During incubation, changes in the RTOCs concentration were measured by GC-MS analysis. A synergistic effect between thymol (0.013 ± 0.003 L/L) and γ-terpinene (0.990 ± 0.030 L/L) (FICI = 0.50) in binary combinations, and between p-cymene (0.700 ± 0.010 L/L) and γ-terpinene (0.290 ± 0.010 L/L) in presence of thymol (0.008 ± 0.001 L/L) (FICI = 0.19), in ternary combinations was found. The synergistic effect against the strain P. digitatum ITEM 9569 suggests that different combinations among RTOCs could be defined to control fungal strains causing different food spoilage phenomena.

In vitro investigations on dopamine loaded Solid Lipid Nanoparticles.

The progressive degeneration of nigrostriatal neurons leads to depletion of the neurotransmitter dopamine (DA) in Parkinson's disease (PD). The hydrophilicity of DA, hindering its cross of the Blood Brain Barrier, makes impossible its therapeutic administration. This work aims at investigating some physicochemical features of novel Solid Lipid Nanoparticles (SLN) intended to enhance DA brain delivery for PD patients by intranasal administration. For this aim, novel SLN were formulated in the presence of Glycol Chitosan (GCS), and it was found that SLN containing GCS and DA were smaller than DA-loaded SLN, endowed with a slightly positive zeta potential value and, remarkably, incorporated 81 % of the initial DA content. The formulated SLN were accurately characterized by Infrared Spectroscopy in Attenuated Total Reflectance mode (FT-IT/ATR) and Thermogravimetric Analysis (TGA) to highlight SLN solid-state properties as a preliminary step forward biological assay. Overall, in vitro characterization shows that SLN are promising for DA incorporation and stable from a thermal viewpoint. Further studies are in due course to test their potential for PD treatment.

Data from in vivo functionalization of diatom mesoporous biosilica with bisphosphonates.

Diatoms are unicellular photosynthetic microalgae that produce a sophisticated mesoporous biosilica shell called frustule. Easy to achieve and extract, diatom frustules represent a low-cost source of mesoporous biocompatible biosilica. In this paper, the possibility to in vivo functionalize the diatom biosilica with bisphosphonates (BPs) was investigated. In particular, two BPs were tested: the amino-containing sodium alendronate (ALE) and the amino-lacking sodium etidronate (ETI). According to first SEM-EDX analysis, the presence of the amino-moiety in ALE structure allowed a better incorporation of this BP into living diatom biosilica, compared to ETI. Then, diatom growth was deeply investigated in presence of ALE. After extraction of functionalized frustules, ALE-biosilica was further characterized by XPS and microscopy, and ALE release was evaluated by ferrochelation assay. Moreover, the bone regeneration performances of ALE-functionalized frustules were preliminarily investigated on bone osteoblast-like cells, via Comassie staining. Data are related to the research article "In vivo functionalization of diatom biosilica with sodium alendronate as osteoactive material".