An apolipoprotein-enriched biomolecular corona switches the cellular uptake mechanism and trafficking pathway of lipid nanoparticles.

Following exposure to biological milieus (e.g. after systemic administration), nanoparticles (NPs) get covered by an outer biomolecular corona (BC) that defines many of their biological outcomes, such as the elicited immune response, biodistribution, and targeting abilities. In spite of this, the role of BC in regulating the cellular uptake and the subcellular trafficking properties of NPs has remained elusive. Here, we tackle this issue by employing multicomponent (MC) lipid NPs, human plasma (HP) and HeLa cells as models for nanoformulations, biological fluids, and target cells, respectively. By conducting confocal fluorescence microscopy experiments and image correlation analyses, we quantitatively demonstrate that the BC promotes a neat switch of the cell entry mechanism and subsequent intracellular trafficking, from macropinocytosis to clathrin-dependent endocytosis. Nano-liquid chromatography tandem mass spectrometry identifies apolipoproteins as the most abundant components of the BC tested here. Interestingly, this class of proteins target the LDL receptors, which are overexpressed in clathrin-enriched membrane domains. Our results highlight the crucial role of BC as an intrinsic trigger of specific NP-cell interactions and biological responses and set the basis for a rational exploitation of the BC for targeted delivery.

Mechanistic evaluation of the transfection barriers involved in lipid-mediated gene delivery: interplay between nanostructure and composition.

Here we present a quantitative mechanism-based investigation aimed at comparing the cell uptake, intracellular trafficking, endosomal escape and final fate of lipoplexes and lipid-protamine/deoxyribonucleic acid (DNA) (LPD) nanoparticles (NPs) in living Chinese hamster ovary (CHO) cells. As a model, two lipid formulations were used for comparison. The first formulation is made of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic lipid dioleoylphosphocholine (DOPC), while the second mixture is made of the cationic 3ß-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine (DOPE). Our findings indicate that lipoplexes are efficiently taken up through fluid-phase macropinocytosis, while a less efficient uptake of LPD NPs occurs through a combination of both macropinocytosis and clathrin-dependent pathways. Inside the cell, both lipoplexes and LPD NPs are actively transported towards the cell nucleus, as quantitatively addressed by spatio-temporal image correlation spectroscopy (STICS). For each lipid formulation, LPD NPs escape from endosomes more efficiently than lipoplexes. When cells were treated with DOTAP-DOPC-containing systems the majority of the DNA was trapped in the lysosome compartment, suggesting that extensive lysosomal degradation was the rate-limiting factors in DOTAP-DOPC-mediated transfection. On the other side, escape from endosomes is large for DC-Chol-DOPE-containing systems most likely due to DOPE and cholesterol-like molecules, which are able to destabilize the endosomal membrane. The lipid-dependent and structure-dependent enhancement of transfection activity suggests that DNA is delivered to the nucleus synergistically: the process requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability.

Mechanistic understanding of gene delivery mediated by highly efficient multicomponent envelope-type nanoparticle systems.

We packaged condensed DNA/protamine particles in multicomponent envelope-type nanoparticle systems (MENS) combining different molar fractions of the cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3ß-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipids dioleoylphosphocholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE). Dynamic light scattering (DLS) and microelectrophoresis allowed us to identify the cationic lipid/DNA charge ratio at which MENS are small sized and positively charged, while synchrotron small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) revealed that MENS are well-shaped DNA/protamine particles covered by a lipid monobilayer. Transfection efficiency (TE) experiments indicate that a nanoparticle formulation, termed MENS-3, was not cytotoxic and highly efficient to transfect Chinese hamster ovary (CHO) cells. To rationalize TE, we performed a quantitative investigation of cell uptake, intracellular trafficking, endosomal escape, and final fate by laser scanning confocal microscopy (LSCM). We found that fluid-phase macropinocytosis is the only endocytosis pathway used by MENS-3. Once taken up by the cell, complexes that are actively transported by microtubules frequently fuse with lysosomes, while purely diffusing systems do not. Indeed, spatiotemporal image correlation spectroscopy (STICS) clarified that MENS-3 mostly exploit diffusion to move in the cytosol of CHO cells, thus explaining the high TE levels observed. Also, MENS-3 exhibited a marked endosomal rupture ability resulting in extraordinary DNA release. The lipid-dependent and structure-dependent TE boost suggests that efficient transfection requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability.

Expanding the phenotype associated with FOXG1 mutations and in vivo FoxG1 chromatin-binding dynamics.

Mutations in the Forkhead box G1 (FOXG1) gene, a brain specific transcriptional factor, are responsible for the congenital variant of Rett syndrome. Until now FOXG1 point mutations have been reported in 12 Rett patients. Recently seven additional patients have been reported with a quite homogeneous severe phenotype designated as the FOXG1 syndrome. Here we describe two unrelated patients with a de novo FOXG1 point mutation, p.Gln46X and p.Tyr400X, respectively, having a milder phenotype and sharing a distinctive facial appearance. Although FoxG1 action depends critically on its binding to chromatin, very little is known about the dynamics of this process. Using fluorescence recovery after photobleaching, we showed that most of the GFP-FoxG1 fusion protein associates reversibly to chromatin whereas the remaining fraction is bound irreversibly. Furthermore, we showed that the two pathologic derivatives of FoxG1 described in this paper present a dramatic alteration in chromatin affinity and irreversibly bound fraction in comparison with Ser323fsX325 mutant (associated with a severe phenotype) and wild type Foxg1 protein. Our observations suggest that alterations in the kinetics of FoxG1 binding to chromatin might contribute to the pathological effects of FOXG1 mutations.

Sports-related dental injuries: knowledge of first aid and mouthguard use in a sample of Italian children and youngsters.

AIM: The purpose of this study was to evaluate the awareness of sports as risk factor of dental injuries, the emergency management when a tooth avulsion occurs and the compliance about mouthguards. MATERIALS AND METHODS: Two hundred children and youngsters 8- to 15-year-old (147 boys and 53 girls) attending Sports Societies in Isernia, a town in Southern Italy, participated to the investigation. The sports involved were soccer, martial arts, tennis, swimming, volleyball, basketball and cycling. The questionnaire was structured into three parts: 1) questions about age, sex, type and time of sports practice; 2) questions about dental injuries, particularly personal experience, awareness of first aid and procedure about tooth avulsion; 3) questions about knowledge and use of mouthguards. RESULTS: Sixty-five per cent of the athletes were aware of the possibility of oral injuries during sports practice and 8.5% referred an experience of dental trauma; 71.5% of the participants think that the immediate management of dental injuries by a dentist is very important to increase the rate of success; 31% know that the avulsed tooth may be reimplanted: 33.9% would reimplant the tooth within an hour and 62.9% would keep it in a wet storage medium. Finally, 80.5% of the athletes knew about mouthguards as protective devices, but only 5% actually used them; eight out of ten were provided by the dentist. CONCLUSION: Educational programs organized by the sports dentistry community are needed to inform coaches, teachers, athletes and parents about dental injuries and to promote the mouthguards use, especially in contact sports practice.

Erythrocyte and reticulocyte indices in iron deficiency in chronic kidney disease: comparison of two methods.

OBJECTIVE: Anaemia is a common complication of chronic kidney disease (CKD), particularly in dialysis patients. The recent European guidelines for anaemia treatment in CKD indicate the percentage of hypochromic red cells (%HYPO) and reticulocyte haemoglobin content (CHr) calculated by Siemens ADVIA haematology analysers as a useful tool indicating iron deficiency. The aim of this study was to evaluate the agreement between CHr and %HYPO parameters and the reticulocyte haemoglobin equivalent (RET-He) and red blood cell haemoglobin equivalent (RBC-He) calculated by the Sysmex XE-2100 haematology analyser in a cohort of 200 dialysis patients referred to the Nephrology Unit of our hospital. Furthermore, we evaluated a new index, the DF-Hypo XE, obtained from haemoglobin (Hb), haematocrit (Hct) and RET-He, provided by the Sysmex XE-2100, as a new potential marker of %HYPO in dialysed patients. MATERIAL AND METHODS: Blood samples collected in EDTA anticoagulant from 200 CKD patients receiving erythropoietin and iron to maintain haemoglobin level between 10 and 12 mg/dL were analysed on both the Siemens ADVIA 2120 and the Sysmex XE-2100 within 2 h of collection. RESULTS: There was good correlation between CHr and RET-He (r = 0.88; p<0.0001), %HYPO and DF-Hypo XE (r = 0.89; p<0.0001) and between RBC-He and CH (r = 0.96; p<0.0001), but there was a lower correlation, even though statistically significant, between RBC-He and %HYPO (r = -0.59; p<0.0001). The Altman-Bland analysis showed a very good level of agreement between CHr and RET-He (mean bias = 1.04 pg), %HYPO and DF-Hypo XE (mean bias = 1.73). Using a cut-off value of 29.4 pg for the RET-He and of 10.2 for the DF-Hypo XE, 15 out 17 patients with a CHr <29.0 pg and 9 out 11 patients with a %Hypo <10.0% were respectively correctly identified. CONCLUSIONS: Our study shows good correlation and agreement between CHr and RET-He and between %HYPO and DF-Hypo XE in evaluating CKD patients needing iron support.

[A 'lucky' patient]. / Un caso 'fortunato'

A 72-year-old male diabetic patient admitted to our operative unit of nephrology and dialysis underwent hemodialytic treatment because of rapidly progressive renal failure. A moderate hypertensive state was associated to nephrotic proteinuria and microematuria. Renal angiography showed a severe stenosis of the right renal artery and a smaller left kidney. Right renal artery stenting induced a significant reduction in serum creatinine (Cr) and the patient discontinued with the dialytic treatment.

The problem of late allograft loss in kidney transplantation.

The 2 principal factors implicated in late kidney allograft failure are chronic rejection (also called chronic allograft nephropathy) and death of the patient with a functioning graft (mainly from cardiovascular causes). Despite lifelong immunosuppression of the recipient, immunological responses remain the leading factor in the pathogenesis of chronic rejection and both cellular and humoral immune mechanisms have been shown to play important roles. In this review, we highlight the relevance of humoral mechanisms of rejection to the pathogenesis of late allograft loss. Non immunological factors, such as donor organ quality, initial ischemic injury, calcineurin inhibitor (CNI) toxicity, hypertension, and hyperlipidemia, also contribute to progressive chronic allograft injury, but will not be reviewed in detail here. Possible strategies to stabilize or improve allograft function in patients with already established "chronic rejection/chronic allograft nephropathy" (CR/CAN) are the addition of mycophenolate mofetil (or sirolimus) with or without a reduction of cyclosporine dosage, or conversion from cyclosporine to tacrolimus. However, prospective randomized clinical trials are needed to test the efficacy of these strategies. A major current challenge for transplant physicians is to develop regimens that prevent CR/CAN, since, once established, the process typically progresses inexorably to renal allograft loss in most recipients. Evidence is now accumulating that new immunosuppressive regimens must control not only T cell but also B cell responses (i.e. limit antidonor antibody production) in order to prevent CR/CAN and improve long-term allograft survival.

Membranous glomerulonephritis after haematopoietic cell transplantation for multiple myeloma.

Renal involvement during graft-versus-host disease following haematopoietic cell transplantation for multiple myeloma has never been described. We report a case of a recipient who developed nephrotic syndrome and membranous glomerulonephritis 22 months after the graft and 6 months after cyclosporine withdrawal. Symptoms resolved when immunosuppressive therapy was reinstituted.