Lupus anticoagulant laboratory diagnosis by applying the 2020 ISTH-SSC guidelines.

BACKGROUND: The ISTH-SSC guidelines for lupus anticoagulant (LA) testing recommend using in-house determined cut-off values, pooled normal plasma (PNP) for ratio normalization, and a ratio for the mixing test interpretation. They strongly support the mixing step role in the diagnostic process. OBJECTIVES: To investigate and compare the LA testing results and interpretations obtained following the ISTH-SSC guidelines or the available alternatives. PATIENTS/METHODS: Blood samples for LA testing from 462 consecutive patients were evaluated for screening, mixing and confirmatory tests. The analysis focused on the interpretation differences between using (1) the in-house cut-off values versus the manufacturer's cut-off values, (2) a normalized ratio calculated using PNP at each run versus the mean of the reference interval, (3) a normalized ratio versus the index of circulating anticoagulant to interpret the mixing step, and (4) a two-step versus three-step procedure. RESULTS: LA testing outcomes were comparable when using the in-house and manufacturer's cut-off values. More positive dilute Russell's viper venom (DRVV) time results were obtained with the normalized ratio based on PNP than with the mean of the reference interval. Overall, the mixing test results obtained with the normalized ratio and the index of circulating anticoagulant showed a good agreement. Among the 97 DRVV Screen test-positive samples, 33 and 89 were classified as LA-positive with the 3-step and the 2-step procedure, respectively. CONCLUSIONS: The cut-off value used and the way to normalize ratios had a limited impact. Conversely, it is important to understand the mixing test characteristics to maximize its diagnostic potential.

The management of the slow transit constipation in the laparoscopic era.

The slow transit constipation (STC) is a functional bowel pathology with slow total gut transit time with normal calibre colon in addition to a variety of other systemic symptoms. Patients with an abnormal colonic motility refractory to conservative treatment are regarded as appropriate candidates for surgery. Laparoscopic total colectomy with ileum-rectum anastomosis represents the commonest surgical operation in the treatment of STC, in well selected patients, after failure of conservative treatment. From 2012 to 2016, 8 patients suffering constipation according to Roma III criteria and diagnosed as STC were submitted to a total colectomy in our O.U. We evaluated the long-term post-operative quality of life and the bowel function, specifically the persistence of constipation and the number of daily bowel movements. Based on our results, we consider that the use of minimally invasive total colectomy with an ileum-rectal anastomosis is the procedure of choice in patients with colonic inertia, and should be performed by experts in laparoscopic colorectal surgery offering a satisfying post-operative quality of life with low morbidity and mortality rates.

Hydrogels for biomedical applications from glycol chitosan and PEG diglycidyl ether exhibit pro-angiogenic and antibacterial activity.

We aimed at producing a hydrogel from a chitosan (CS) derivative soluble in physiological conditions to avoid any purification step thus allowing to use the materials also as an in-situ forming material. So, we crosslinked glycol chitosan (GCS) with poly(ethylene glycol) diglycidyl ether (PEGDE) in water at 37 °C. The scaffolds, referred as GCS-PEG, were specifically designed to be used as wound dressing materials as such (after crosslinking) or as in-situ forming materials. Different amounts of PEGDE were tested. The obtained scaffolds showed macroscopic pores and a tailorable swelling in water by controlling the crosslinking degree. Moreover, GCS-PEG scaffolds displayed a significant antimicrobial activity against Staphylococcus aureus. In-vivo study using the chick embryo choriallantoic membrane resulted in a highly pronounced pro-angiogenic activity suggesting important tissue regeneration properties. Moreover, the employed materials are commercially available, no organic solvents are required and the scaling up is quite predictable.

Protection of dopamine towards autoxidation reaction by encapsulation into non-coated- or chitosan- or thiolated chitosan-coated-liposomes.

The aim of this work is to evaluate the potential of non-coated-, chitosan-(CS)- or chitosan-glutathione conjugate- (CS-GSH)-coated liposomes to protect the neurotransmitter Dopamine (DA) from the autoxidation reaction in neutral/alkaline conditions. This may be of interest in the development of nanotechnology-based approaches to improve Parkinson's disease treatment because decreased ROS production and reduced DA associated neurotoxicity are expected. For the mentioned purposes, DA-loaded vesicles were prepared by the Dried Reconstituted Vesicles (DRV) method, and were subsequently coated using solutions of polycations. As for the mean diameters of liposomes so prepared, the CS-GSH coated liposomes showed a significant decrease in size compared to the corresponding non-coated and CS-coated vesicles. The surface charge of DA-loaded non-coated liposomes was -10.8 mV, whereas the CS or CS-GSH coated vesicles showed a slightly positive ζ-potential. The capability of the herein studied vesicles to prevent DA autoxidation was evaluated by visual inspection, monitoring DA/lipid ratio as such and under stressed conditions. The results suggest that liposome formulations partially protect the neurotransmitter from the autoxidation reaction. In particular, the CS-GSH coated liposomes were more stable than the corresponding CS-coated and non-coated ones against the oxidative damage and were found to deliver the neurotransmitter in a sustained manner. Probably, this is due to the localization of the neurotransmitter in the core of the vesicles as indicated by XPS which confirmed the absence of the neurotransmitter on the surface of these vesicles.

Nanoparticle delivery of grape seed-derived proanthocyanidins to airway epithelial cells dampens oxidative stress and inflammation.

BACKGROUND: Chronic respiratory diseases, whose one of the hallmarks is oxidative stress, are still incurable and need novel therapeutic tools and pharmaceutical agents. The phenolic compounds contained in grape are endowed with well-recognized anti-oxidant, anti-inflammatory, anti-cancer, and anti-aging activities. Considering that natural anti-oxidants, such as proanthocyanidins, have poor water solubility and oral bioavailability, we have developed a drug delivery system based on solid lipid nanoparticles (SLN), apt to encapsulate grape seed extract (GSE), containing proanthocyanidins. METHODS: Plain, 6-coumarin (6-Coum), DiR- and GSE-loaded SLN were produced with the melt-emulsion method. Physicochemical characterization of all prepared SLN was determined by photon correlation spectroscopy and laser Doppler anemometry. MTT assay (spectrophotometry) and propidium iodide (PI) assay (cytofluorimetry) were used to assess cell viability. Flow cytometry coupled with cell imaging was performed for assessing apoptosis and necrosis by Annexin V/7-AAD staining (plain SLE), cell internalization (6-Coum-SLN) and reactive oxygen species (ROS) production (SLN-GSE). NF-κB nuclear translocation was studied by immunofluorescence. In vivo bio-imaging was used to assess lung deposition and persistence of aerosolized DiR-loaded SLN. RESULTS: Plain SLN were not cytotoxic when incubated with H441 airway epithelial cells, as judged by both PI and MTT assays as well as by apoptosis/necrosis evaluation. 6-Coum-loaded SLN were taken up by H441 cells in a dose-dependent fashion and persisted into cells at detectable levels up to 16 days. SLN were detected in mice lungs up to 6 days. SLN-GSE possessed 243 nm as mean diameter, were negatively charged, and stable in size at 37 °C in Simulated Lung Fluid up to 48 h and at 4 °C in double distilled water up to 2 months. The content of SLN in proanthocyanidins remained unvaried up to 2 months. GSE-loaded SLN determined a significant reduction in ROS production when added 24-72 h before the stimulation with hydrogen peroxide. Interestingly, while at 24 h free GSE determined a higher decrease of ROS production than SLN-GSE, the contrary was seen at 48 and 72 h. Similar results were observed for NF-κB nuclear translocation. CONCLUSIONS: SLN are a biocompatible drug delivery system for natural anti-oxidants obtained from grape seed in a model of oxidative stress in airway epithelial cells. They feature stability and long-term persistence inside cells where they release proanthocyanidins. These results could pave the way to novel anti-oxidant and anti-inflammatory therapies for chronic respiratory diseases.

Realization of polyaspartamide-based nanoparticles and in vivo lung biodistribution evaluation of a loaded glucocorticoid after aerosolization in mice.

In this study, novel polymeric nanoparticles (NPs) were developed and their potential as carriers for beclomethasone dipropionate (BDP) into the lung after aerosolization was demonstrated by in vivo studies in mice. In particular, these NPs were obtained starting from two polyaspartamide-based copolymers which were synthesized by chemical reaction of α,ß-poly(N-2-hydroxyethyl)-dl-aspartamide (PHEA) and its pegylated derivative (PHEA-PEG2000) with poly(lactic acid) (PLA). To obtain nanosized particles, the high pressure homogenization (HPH)-solvent evaporation method was followed by using an organic phase containing both PHEA-PLA and PHEA-PEG2000-PLA (at a weight ratio equal to 1:1), lactose as cryoprotectant and no surfactant was adopted. PHEA-PLA/PHEA-PEG2000-PLA NPs were characterized by a quite spherical shape, ζ potential slightly negative, and size lower than 50 and 200nm, respectively, for empty and BDP-loaded NPs. In vivo biodistribution of BDP and its metabolites in various lung compartments, i.e. bronchoalveolar lavage fluid (BALF), alveolar macrophages (MPG) obtained from BALF, and lung tissue, was carried out at 3h post-administration in mice by aerosolization of BDP-loaded NPs or free BDP (commercial formulation, Clenil(®)) at the dose of 0.5mg/kg BDP. Results demonstrated that BDP entrapped into NPs reached all analyzed lung compartments and were internalized by both alveolar MPG and respiratory epithelial cells, and detected amounts were comparable to those of Clenil-treated mice. Moreover, the entrapment into NPs protects the drug from the enzymatic hydrolysis, allowing a significant lower amount of beclomethasone (BOH) into the lung tissue and BALF than that obtained after Clenil administration.

Comparison between transdermal nitroglycerin and sildenafil citrate in intrauterine growth restriction: effects on uterine, umbilical and fetal middle cerebral artery pulsatility indices.

OBJECTIVES: To evaluate the effects of transdermal nitroglycerin (GTN) and sildenafil citrate on Doppler velocity waveforms of the uterine (UtA), umbilical (UA) and fetal middle cerebral (MCA) arteries in pregnancies with intrauterine growth restriction (IUGR). METHODS: This was a prospective study of 35 singleton pregnancies (gestational age, 24-31 weeks) with IUGR and abnormal UtA and UA Doppler waveforms. We compared maternal arterial blood pressure and Z-scores of the pulsatility index (PI) of UtA, UA and fetal MCA before and after application of a transdermal GTN patch (average dose, 0.4 mg/h), oral sildenafil citrate (50 mg) or placebo. Statistical analysis was performed by ANOVA for paired samples. RESULTS: There was a significant decrease in UtA-PI after application of GTN (21.0%) and sildenafil citrate (20.4%). A significant reduction in UA-PI was also observed for both GTN (19.1%) and sildenafil citrate (18.2%). There was no difference in UtA- and UA-PI when the GTN and sildenafil groups were compared. No changes in Doppler velocimetry were observed in the placebo group and no significant change in MCA-PI was observed in any group. Maternal arterial blood pressure decreased with administration of both GTN and sildenafil citrate in those with pre-eclampsia. CONCLUSION: The use of transdermal GTN or sildenafil citrate in pregnancies with IUGR is associated with a significant reduction in both UtA and UA Doppler PI, as well as maternal arterial blood pressure. Neither drug affected the MCA-PI. Copyright © 2015 ISUOG. Published by John Wiley & Sons Ltd.

Enhanced solubility and antibacterial activity of lipophilic fluoro-substituted N-benzoyl-2-aminobenzothiazoles by complexation with ß-cyclodextrins.

Some lipophilic fluoro-substituted N-benzoyl-2-aminobenzothiazole antibacterial agents have been evaluated for their activity in the presence of cyclodextrins (CDs) containing aqueous solutions where CDs are adopted as solubilizing excipients for improving the poor water solubility of these compounds. For such purpose both the natural ß-CD and one of FDA/EMA approved CDs for parenteral use (i.e. HP-ß-CD) have been employed. The solubility rank order observed was accounted for by thermal analysis (Differential Scanning Calorimetry) and FT-IR spectroscopy. The most promising compound was subjected to further NMR spectroscopic studies and molecular modelling simulations to verify the interactions between the guest molecule and the CD cavity. The assessment of the antibacterial activity of such compounds against selected Gram positive and Gram negative bacterial strains clearly showed that their antimicrobial effectiveness may, quite in all instances, be positively affected by complexation with ß-CD and HP-ß-CD. These results, which are in some ways in contrast with those already reported in the literature, are herein discussed on the basis of plausible mechanisms. Moreover, this investigation also reveals that the described methodology of complexing both lipophilic and hydrophilic antimicrobial agents with CDs may be an useful approach to enhance their effectiveness as well as a promising strategy to overcome even the microbial resistance problem.

Cloning, expression in E. coli and immunological characterization of Par j 3.0201, a Parietaria pollen profilin variant.

Parietaria judaica pollen is one of the main sources of allergens in the Mediterranean area. Its allergenic composition has been studied in detail showing the presence of two major allergens (Par j 1 and Par j 2) and two minor allergens belonging to the profilin and calcium binding protein families of allergens (Par j 3 and Par j 4, respectively). Clinical reports support the hypothesis of a limited cross-reactivity between profilin from Parietaria and unrelated sources. We screened a P. judaica cDNA library to identify novel forms of profilins with allergenic activity. This strategy allowed us to isolate a 767 bp cDNA containing the information for a 131 amino acids protein with homology to profilins from unrelated sources greater than that observed with the already published Parietaria profilins. This profilin was expressed in Escherichia coli as a recombinant protein and its immunological prevalence was studied in a population of Parietaria allergic patients from Southern Europe. Immunoblotting analysis showed that the Parietaria profilin was recognized by IgE from 6.5% of the allergic population. Finally, a selected population of profilin allergic patients was enrolled to demonstrate the cross-reactivity of this novel variant with other profilins from grass and date palm. In conclusion, molecular cloning and immunological studies have allowed the isolation, expression and immunological characterization of a novel cross-reactive profilin allergen from P. judaica pollen named Par j 3.0201.

In vitro evaluation on a model of blood brain barrier of idebenone-loaded solid lipid nanoparticles.

The aim of this study was to evaluate in vitro the permeation of the antioxidant agent Idebenone (IDE) loaded into solid lipid nanoparticles (SLN) across MDCKII-MDR1 cell monolayer, selected as an in vitro model of the Blood Brain Barrier (BBB). SLN were prepared using cetyl palmitate as solid lipid and different non-ionic surfactants, oleth-20, ceteth-20 and isoceth-20, by the phase inversion temperature (PIT) technique. The resulting SLN showed physiological pH and osmolarity values, a mean particle diameter in the range of 33-63 nm, a single peak in size distribution, and a zeta potential ranging from +3.14 to -2.89 mV. When incubating these SLN in Simulated Body Fluid (SBF), the particle size was maintained for all samples throughout the study. IDE permeability across MDCKII-MDR1 cell monolayers from the SLN under investigation was 0.40-0.53 fold lower than free IDE and no significant difference was observed comparing IDE permeation from all the SLN tested. It is noteworthy that IDE loading into SLN avoided the use of an organic solvent to solubilize IDE, a poor water soluble compound, allowing the parenteral administration of this drug in aqueous vehicles. Furthermore, the results of in vitro transport studies, performed using fluorescein-dextran 4000 (FD4) and diazepam (DZ) as markers of the paracellular pathway and the transcellular pathway, respectively, pointed out that IDE could permeate via a transcellular pathway. Therefore, these novel nanocarriers could be regarded as a promising strategy to design delivery systems for IDE administration to the brain, deserving further investigations under in vivo conditions.

Inhibiting effect of α(s1)-casein on Aß(1-40) fibrillogenesis.

BACKGROUND: α(s1)-Casein is one of the four types of caseins, the largest protein component of bovine milk. The lack of a compact folded conformation and the capability to form micelles suggest a relationship of α(s1)-casein with the class of the intrinsically disordered (or natively unfolded) proteins. These proteins are known to exert a stabilizing activity on biomolecules through specific interaction with hydrophobic surfaces. In the present work we focused on the effect of α(s1)-casein on the fibrillogenesis of 1-40 ß-amyloid peptide, involved in Alzheimer's disease. METHODS: The aggregation kinetics of ß-peptide in presence and absence of α(s1)-casein was followed under shear at 37°C by recording the Thioflavine fluorescence, usually taken as an indicator of fibers formation. Measurements of Static and Dynamic Light Scattering, Circular Dichroism, and AFM imaging were done to reveal the details of α(s1)-casein-Aß(1-40) interaction. RESULTS AND DISCUSSIONS: α(s1)-Casein addition sizably increases the lag-time of the nucleation phase and slows down the entire fibrillization process. α(s1)-Casein sequesters the amyloid peptide on its surface thus exerting a chaperone-like activity by means a colloidal inhibition mechanism. GENERAL SIGNIFICANCE: Insights on the working mechanism of natural chaperones in preventing or controlling the amyloid aggregation.

Transdermal nitroglycerin in patients with severe pre-eclampsia with placental insufficiency: effect on uterine, umbilical and fetal middle cerebral artery resistance indices.

OBJECTIVES: To evaluate the effect of transdermal nitroglycerin on Doppler velocity waveforms of the uterine, umbilical and fetal middle cerebral arteries in patients with severe pre-eclampsia. METHODS: This was a prospective study of 30 singleton pregnancies (gestational age range: 24-31 weeks) with severe pre-eclampsia and abnormal uterine and umbilical artery Doppler waveforms. We compared maternal blood pressure as well as the resistance index (RI) and the pulsatility index (PI) of the uterine, umbilical and fetal middle cerebral arteries before and after application of a transdermal nitroglycerin patch (average dose 0.4 mg/h) for a period of 3 days. Intra-day comparisons before and after administration of nitroglycerin and a comparison between days 0 (no patch) and 3 after administration of the first dose of nitroglycerin were performed using ANOVA for paired samples. RESULTS: A significant decrease in the PI and RI of the uterine (25.3 ± 4.9% and 21.2 ± 6.2%, respectively, P < 0.001) and umbilical (23.1 ± 6.9% and 19.7 ± 6.1%, respectively, P < 0.001) arteries was noted when comparing the first day without medication against the third day with the patch. No significant change in the PI and RI of the middle cerebral artery was observed. The mean arterial blood pressure decreased from 119.5 ± 4.5 mmHg to 114.8 ± 4.4 mmHg (P < 0.05). CONCLUSION: The use of transdermal nitroglycerin in patients with severe pre-eclampsia is associated with a significant reduction in the RI and PI of the uterine and umbilical arteries, as well as of maternal blood pressure. Transdermal nitroglycerin does not affect the RI and PI of the fetal middle cerebral artery.

Analytical characterization of chitosan nanoparticles for peptide drug delivery applications.

Chitosan-cyclodextrin hybrid nanoparticles (NPs) were obtained by the ionic gelation process in the presence of glutathione (GSH), chosen as a model drug. NPs were characterized by means of transmission electron microscopy and zeta-potential measurements. Furthermore, a detailed X-ray photoelectron spectroscopy study was carried out in both conventional and depth-profile modes. The combination of controlled ion-erosion experiments and a scrupulous curve-fitting approach allowed for the first time the quantitative study of the GSH in-depth distribution in the NPs. NPs were proven to efficiently encapsulate GSH in their inner cores, thus showing promising perspectives as drug carriers.