Effect of Plasma Unit Weight and Donor Sex on Post-Donation Citrate Level: An Experimental Study on Plasmapheresis Donors.

Introduction: Plasmapheresis donation is considered safe and well tolerated, although long-term effects need to be clarified. The volumes of anticoagulant (ACD-A) used are variable and depend primarily on hematocrit (HCT), total blood processed, amount of plasma collected, and donor characteristics. To elucidate the effect of the plasma unit weight setting on plasmapheresis efficiency and ACD-A distribution, we enrolled male donors undergoing a controlled apheresis process donating 700 g and 720 g in two different sessions. In parallel, we investigated a possible effect of sex, recruiting women donating 700 g of plasma. Methods: The study was conducted on men donating 720 g and (12 months later) 700 g of plasma, and on women donating 700 g of plasma. The main outcomes were pre-/post-donation delta (Δ) citrate concentration in donor plasma and ACD-A reinfused to the donor. Information concerning the annual check-up and the procedure was also collected. Intergroup comparisons (men donating 720 g vs. men donating 700 g and men vs. women both donating 700 g) and intragroup associations with donor and procedural characteristics were reported. Results: With the procedure set at 720 g, the machine processed around 44 mL more whole blood to collect 20 g more plasma, and 720 g donors received around 12 mL more anticoagulant than 700 g donors. Accordingly, Δ citrate concentration was 1.5 times higher (12 µm), with a greater variability observed for 720 g donations. Citrate concentration in the plasma unit was lower in the 720 g group, although not significantly. Comparing outcomes between women and men donating 700 g, we observed higher (and highly variable) Δ citrate and reinfused ACD-A in women, accompanied by lower anticoagulant levels in the unit. Increased Δ citrate is inversely associated with HCT and age in men and with HCT and triglycerides in women. Reinfused ACD-A correlates with HCT in women but not in men. Conclusion: Unit weight setting and sex influence an ACD-A shift from the estimated values toward an increased reinfusion to donor. In parallel, we observed an impact of age and sex on post-donation citrate metabolism. Altogether, these elements should be taken into account for the development of tailored approaches aimed at maintaining similar safety profiles for all donors using different plasmapheresis settings.

Cryopreservation affects platelet macromolecular composition over time after thawing and differently impacts on cancer cells behavior in vitro.

Cryopreservation affects platelets' function, questioning their use for cancer patients. We aimed to investigate the biochemical events that occur over time after thawing to optimize transfusion timing and evaluate the effect of platelet supernatants on tumor cell behavior in vitro. We compared fresh (Fresh-PLT) with Cryopreserved platelets (Cryo-PLT) at 1 h, 3 h and 6 h after thawing. MCF-7 and HL-60 cells were cultured with Fresh- or 1 h Cryo-PLT supernatants to investigate cell proliferation, migration, and PLT-cell adhesion. We noticed a significant impairment of hemostatic activity accompanied by a post-thaw decrease of CD42b+ , which identifies the CD62P--population. FTIR spectroscopy revealed a decrease in the total protein content together with changes in their conformational structure, which identified two sub-groups: 1) Fresh and 1 h Cryo-PLT; 2) 3 h and 6 h cryo-PLT. Extracellular vesicle shedding and phosphatidylserine externalization (PS) increased after thawing. Cryo-PLT supernatants inhibited cell proliferation, impaired MCF-7 cell migration, and reduced ability to adhere to tumor cells. Within the first 3 hours after thawing, irreversible alterations of biomolecular structure occur in Cryo-PLT. Nevertheless, Cryo-PLT should be considered safe for the transfusion of cancer patients because of their insufficient capability to promote cancer cell proliferation, adhesion, or migration.

What is the context? Transfusion of Fresh platelets (Fresh-PLT) with prophylaxis purposes is common in onco-hematological patients.Cryopreservation is an alternative storage method that allows to extend platelet component shelf life and build supplies usable in case of emergency.It is well established that cryopreservation affects platelet function questioning their use in onco-hematological patients.It is still unknown how platelet impairment, induced by cryopreservation, occurs over time after thawing, nor how the by-products of PLT deterioration may impact on cancer cell behavior.What is new? In this study, we deeply characterized the functional and morphological changes induced by cryopreservation on platelets by comparing Fresh-PLT with Cryo-PLT at 1 h, 3 h and 6 h after thawing. Afterwards, we evaluated the effect of PLT supernatants on cancer cell behavior in vitro.The data presented show that within 3 hours after thawing Cryo-PLT undergo to irreversible macromolecular changes accompanied by increase of peroxidation processes and protein misfolding.After thawing the clot formation is reduced but still supported at all-time points measured, combined with unchanged phosphatidylserine expression and extracellular vesicles release over time.Cryo-PLT supernatants do not sustain proliferation and migration of cancer cells.WHAT is the impact? Cryo-PLT may be considered a precious back-up product to be used during periods of Fresh-PLT shortage to prevent bleeding in non-hemorrhagic patients.It is desirable to make it logistically feasible to transfuse cryopreserved platelets within 1 hour of thawing to maintain the platelets in their best performing condition.

A cohort study on the biochemical and haematological parameters of Italian blood donors as possible risk factors of COVID-19 infection and severe disease in the pre- and post-Omicron period.

To investigate the association between biochemical and blood parameters collected before the pandemic in a large cohort of Italian blood donors with the risk of infection and severe disease. We also focused on the differences between the pre- and post-Omicron spread in Italy (i.e., pre- and post-January 01, 2022) on the observed associations. We conducted an observational cohort study on 13750 blood donors was conducted using data archived up to 5 years before the pandemic. A t-test or chi-squared test was used to compare differences between groups. Hazard ratios with 95% confidence intervals for SARS-CoV-2 infection and severe disease were estimated using Cox proportional hazards models. Subgroup analyses stratified by sex, age and epidemic phase of first infection (pre- and post-Omicron spread) were examined. We confirmed a protective effect of groups B and O, while groups A and AB had a higher likelihood of infection and severe disease. However, these associations were only significant in the pre-Omicron period. We found an opposite behavior after Omicron spread, with the O phenotype having a higher probability of infection. When stratified by variant, A antigen appeared to protect against Omicron infection, whereas it was associated with an increased risk of infection by earlier variants. We were able to stratify for the SARS CoV-2 dominant variant, which revealed a causal association between blood group and probability of infection, as evidenced by the strong effect modification observed between the pre- and post-Omicron spread. The mechanism by which group A acts on the probability of infection should consider this strong effect modification.

Homologous platelet gel on radiation-induced dermatitis in a patient receiving head and neck radiotherapy plus cetuximab: A case report.

INTRODUCTION: Acute radiodermatitis is a significant complication of cancer radiotherapy, and platelet-based therapies are emerging as potential new treatments. MAIN SYMPTOMS AND IMPORTANT CLINICAL FINDINGS: In this report, we present the case of a patient with head and neck cancer undergoing radiotherapy combined with the monoclonal antibody cetuximab. After 4 weeks of this treatment, the patient developed cutaneous radiation dermatitis. Despite receiving standard treatment with corticosteroids and emollient cream, the lesion did not improve. MAIN DIAGNOSIS: cutaneous radiation dermatitis on head and neck cancer patient. THERAPEUTIC INTERVENTIONS: Topical application of platelet gel was initiated on the wound. From the second week of radiotherapy to the 4th week, homologous platelet-rich plasma was applied on the dermatitis using a bandage, 4 times a day. OUTCOMES: The topical treatment with homologous platelet gel resulted in complete healing of the radiodermatitis, including restoration of the epidermis, reepithelialization, and reduction in associated pain. CONCLUSION: homologous platelet gel might be an alternative to standard treatment of radiation dermatitis.

Reduction of EpCAM-Positive Cells from a Cell Salvage Product Is Achieved by Leucocyte Depletion Filters Alone.

Intraoperative cell salvage reduces the need for allogeneic blood transfusion in complex cancer surgery, but concerns about the possibility of it re-infusing cancer cells have hindered its application in oncology. We monitored the presence of cancer cells on patient-salvaged blood by means of flow cytometry; next, we simulated cell salvage, followed by leucodepletion and irradiation on blood contaminated with a known amount of EpCAM-expressing cancer cells, assessing also residual cancer cell proliferation as well as the quality of salvaged red blood cell concentrates (RBCs). We observed a significant reduction of EpCAM-positive cells in both cancer patients and contaminated blood, which was comparable to the negative control after leucodepletion. The washing, leucodepletion and leucodepletion plus irradiation steps of cell salvage were shown to preserve the quality of RBCs in terms of haemolysis, membrane integrity and osmotic resistance. Finally, cancer cells isolated from salvaged blood lose their ability to proliferate. Our results confirm that cell salvage does not concentrate proliferating cancer cells, and that leucodepletion allows for the reduction of residual nucleated cells, making irradiation unnecessary. Our study gathers pieces of evidence on the feasibility of this procedure in complex cancer surgery. Nevertheless, it highlights the necessity of finding a definitive consensus through prospective trials.

GSK-3 inhibition reverts mesenchymal transition in primary human corneal endothelial cells.

Human corneal endothelial cells are organized in a tight mosaic of hexagonal cells and serve a critical function in maintaining corneal hydration and clear vision. Regeneration of the corneal endothelial tissue is hampered by its poor proliferative capacity, which is partially retrieved in vitro, albeit only for a limited number of passages before the cells undergo mesenchymal transition (EnMT). Although different culture conditions have been proposed in order to delay this process and prolong the number of cell passages, EnMT has still not been fully understood and successfully counteracted. In this perspective, we identified herein a single GSK-3 inhibitor, CHIR99021, able to revert and avoid EnMT in primary human corneal endothelial cells (HCEnCs) from old donors until late passages in vitro (P8), as shown from cell morphology analysis (circularity). In accordance, CHIR99021 reduced expression of α-SMA, an EnMT marker, while restored endothelial markers such as ZO-1, Na+/K+ ATPase and N-cadherin, without increasing cell proliferation. A further analysis on RNA expression confirmed that CHIR99021 induced downregulation of EnMT markers (α-SMA and CD44), upregulation of the proliferation repressor p21 and revealed novel insights into the ß-catenin and TGFß pathways intersections in HCEnCs. The use of CHIR99021 sheds light on the mechanisms involved in EnMT, providing a substantial advantage in maintaining primary HCEnCs in culture until late passages, while preserving the correct morphology and phenotype. Altogether, these results bring crucial advancements towards the improvement of the corneal endothelial cells based therapy.

Mother-newborn ABO group discrepancy caused by a rare BW.17 variant.

Several ABO gene mutations are known to determine rare subgroups: these ABO variants are often responsible for weak or null phenotypes and may cause an incorrect determination of the serotype. Here we describe for the first time the phenotypic discrepancy of a rare B allele within the same Caucasian family that depends on the co-inheritance with A or H antigen. Blood samples from newborns, mothers, and grandmothers were analysed through routine serotype and genotype testing. Blood compatibility test was performed for red blood cells or serum of the grandmother. ABO exons were investigated through PCR and sanger sequencing. According to serology, the phenotype of the mother was AB, while it was O for the newborn. Genotype analysis confirmed that the mother was AB, while the newborn was found to be B. Sanger sequencing revealed the presence of a rare mutation in both individuals (784 G>A, D262N), corresponding to the ABO*BW.17 allele. The grandmother was found to have the same genotype/serotype of the newborn. Crossmatch testing suggested that subjects with this genotype/serotype might be considered O donors and recipients.

Nanoneedles Induce Targeted siRNA Silencing of p16 in the Human Corneal Endothelium.

Nanoneedles can target nucleic acid transfection to primary cells at tissue interfaces with high efficiency and minimal perturbation. The corneal endothelium is an ideal target for nanoneedle-mediated RNA interference therapy aimed at enhancing its proliferative capacity, necessary for tissue regeneration. This work develops a strategy for siRNA nanoninjection to the human corneal endothelium. Nanoneedles can deliver p16-targeting siRNA to primary human corneal endothelial cells in vitro without toxicity. The nanoinjection of siRNA induces p16 silencing and increases cell proliferation, as monitored by ki67 expression. Furthermore, siRNA nanoinjection targeting the human corneal endothelium is nontoxic ex vivo, and silences p16 in transfected cells. These data indicate that nanoinjection can support targeted RNA interference therapy for the treatment of endothelial corneal dysfunction.

Platelet-rich Plasma Lysate for Treatment of Eye Surface Diseases.

Various ocular surface diseases are treated with blood-derived eye drops. Their use has been introduced in clinical practice because of their metabolite and growth factor content, which promotes eye surface regeneration. Blood-based eye drops can be prepared from different sources (i.e., whole blood or platelet apheresis donation), as well as with different protocols (e.g., different dilutions and freeze/thaw cycles). This variability hampers the standardization of clinical protocols and, consequently, the evaluation of their clinical efficacy. Detailing and sharing the methodological procedures may contribute to defining common guidelines. Over the last years, allogenic products have been diffusing as an alternative to the autologous treatments since they guarantee higher efficacy standards; among them, the platelet-rich plasma lysate (PRP-L) eye drops are prepared with simple manufacturing procedures. In the transfusion medicine unit at AUSL-IRCCS di Reggio Emilia, Italy, PRP-L is obtained from platelet-apheresis donation. This product is initially diluted to 0.3 x 109 platelets/mL (starting from an average concentration of 1 x 109 platelets/mL) in 0.9% NaCl. Diluted platelets are frozen/thawed and, subsequently, centrifuged to eliminate debris. The final volume is split into 1.45 mL aliquots and stored at -80 °C. Before being dispensed to patients, eye drops are tested for sterility. Patients may store platelet lysates at -15 °C for up to 1 month. The growth factor composition is also assessed from randomly selected aliquots, and the mean values are reported here.

Fluctuations in Corneal Endothelial LAP2 Expression Levels Correlate with Passage Dependent Declines in Their Cell Proliferative Activity.

The corneal endothelium is the inner corneal mono-layered epithelium, fundamental for preserving corneal hydration and transparency. However, molecular mechanisms that regulate corneal endothelial cells (CEnCs), in particular regarding their proliferative capacity, have been only partially elucidated. CEnCs are quiescent in vivo and they easily undergo endothelial to mesenchymal transition (EnMT) in vitro. This study aims to analyze CEnCs behavior and expression in vitro, either in sub-confluent growing (S) or confluent (C) CEnCs cultures. Primary rabbit and human CEnCs were cultured and used for RT-PCR, immunofluorescence or western blot analysis. These methods allowed identifying a novel molecular marker, LAP2, that is upregulated in S while downregulated in C human or rabbit CEnCs. Those results were observed for several subsequent passages in culture and this, together with the correlation between ki67 and LAP2 expression, suggested LAP2 as a novel possible indicator for culture ageing. Finally, treatment with FGF and TGFß in rCEnCs highlighted how LAP2 can vary as the cells regulate their proliferative state. In conclusion, we have identified a novel marker for CEnCs, LAP2, that regulates its expression depending on the cells sub/confluent state and that correlates with CEnCs proliferation.

The impact of COVID-19 outbreak on the Transfusion Medicine Unit of a Northern Italy Hospital and Cancer Centre.

BACKGROUND AND OBJECTIVES: The first wave of coronavirus disease-2019 (COVID-19) dramatically affected the Transfusion Medicine Unit of the Azienda Unità Sanitari Locale - Istituto di Ricovero e Cura a Carattere Scientifico (AUSL-IRCCS) di Reggio Emilia, which faced a total rearrangement of the procedures for donors and patients. This study aims to assess the major implications of COVID-19 on our department, focusing on the blood transfusion chain and therapies, in order to support transfusion specialists in seeking efficient ways to face similar future emergencies. MATERIALS AND METHODS: This retrospective study compares our Transfusion Medicine Unit data collected between February and May 2020 with the same period in 2017-2019. Data on red blood cells and platelets donations, transfusions and clinical procedures were collected as aggregates from our internal electronic database. RESULTS: During the lockdown, donor centres were re-organized to reduce the risk of contagion and avoid unnecessary blood collection. Blood donations were re-scheduled to meet the decrease in elective surgery; consequently, plateletapheresis was implemented to supply the reduction of buffycoat-derived platelets. Transfusions significantly decreased together with orthopaedic and vascular surgery, while they were only marginally diminished for both cancer and onco-haematological patients. Reduced procedures for inpatients and outpatients were matched by remote medicine, addressing the need of a constant healthcare support for patients with chronic diseases. CONCLUSIONS: The described measures were adopted to avoid excessive blood collection and expiration, guarantee the safety of our ward (for both patients and staff) and supply the necessary transfusion therapies. These measures may support the development of appropriate risk management plans and safety procedures for other hospitals and transfusion services that have to face similar events.

Structure network-based landscape of rhodopsin misfolding by mutations and algorithmic prediction of small chaperone action.

Failure of a protein to achieve its functional structural state and normal cellular location contributes to the etiology and pathology of heritable human conformational diseases. The autosomal dominant form of retinitis pigmentosa (adRP) is an incurable blindness largely linked to mutations of the membrane protein rod opsin. While the mechanisms underlying the noxious effects of the mutated protein are not completely understood, a common feature is the functional protein conformational loss. Here, the wild type and 39 adRP rod opsin mutants were subjected to mechanical unfolding simulations coupled to the graph theory-based protein structure network analysis. A robust computational model was inferred and in vitro validated in its ability to predict endoplasmic reticulum retention of adRP mutants, a feature linked to the mutation-caused misfolding. The structure-based approach could also infer the structural determinants of small chaperone action on misfolded protein mutants with therapeutic implications. The approach is exportable to conformational diseases linked to missense mutations in any membrane protein.

Comparison of Two Alternative Procedures to Obtain Packed Red Blood Cells for ß-Thalassemia Major Transfusion Therapy.

ß-thalassemia major (ßTM) patients require frequent blood transfusions, with consequences that span from allogenic reactions to iron overload. To minimize these effects, ßTM patients periodically receive leucodepleted packed red blood cells (P-RBCs) stored for maximum 14 days. The aim of this study was to compare two alternative routine procedures to prepare the optimal P-RBCs product, in order to identify differences in their content that may somehow affect patients' health and quality of life (QoL). In method 1, blood was leucodepleted and then separated to obtain P-RBCs, while in method 2 blood was separated and leucodepleted after removal of plasma and buffycoat. Forty blood donors were enrolled in two independent centers; couples of phenotypically matched whole blood units were pooled, divided in two identical bags and processed in parallel following the two methods. Biochemical properties, electrolytes and metabolic composition were tested after 2, 7 and 14 days of storage. Units prepared with both methods were confirmed to have all the requirements necessary for ßTM transfusion therapy. Nevertheless, RBCs count and Hb content were found to be higher in method-1, while P-RBCs obtained with method 2 contained less K+, iron and storage lesions markers. Based on these results, both methods should be tested in a clinical perspective study to determine a possible reduction of transfusion-related complications, improving the QoL of ßTM patients, which often need transfusions for the entire lifespan.

Apheresis Platelet Rich-Plasma for Regenerative Medicine: An In Vitro Study on Osteogenic Potential.

Background: Platelet-Rich Plasma (PRP) induces bone regeneration; however, there is low evidence supporting its efficacy in bone healing. The lack of a standardized protocol of administration represents the main obstacle to its use in the clinical routine for bone defects' treatment. The purpose of this study was to characterize PRP and elucidate its osteogenic potential. Methods: Platelet count, fibrinogen levels, and growth factors concentration were measured in PRP obtained by four apheresis procedures. HOB-01-C1, a pre-osteocytic cell line, was used to examine the effects of different PRP dilutions (from 1% to 50%) on cell viability, growth, and differentiation. Gene expression of RUNX2, PHEX, COL1A1, and OCN was also assayed. Results: PRP showed a mean 4.6-fold increase of platelets amount compared to whole blood. Among the 36 proteins evaluated, we found the highest concentrations for PDGF isoforms, EGF, TGF-ß and VEGF-D. PDGF-AA positively correlated with platelet counts. In three of the four tested units, 25% PRP induced a growth rate comparable to the positive control (10% FBS); whereas, for all the tested units, 10% PRP treatment sustained differentiation. Conclusions: This study showed that PRP from apheresis stimulates proliferation and differentiation of pre-osteocyte cells through the release of growth factors from platelets.

Metabolomics comparison of cord and peripheral blood-derived serum eye drops for the treatment of dry eye disease.

Allogeneic peripheral blood-derived (PBS) serum eye drops have been largely used in the treatment of dry eye disease (DED). Recently, cord blood has emerged as an effective alternative serum source (cord blood serum, CBS), containing a higher amount of growth factors than PBS, it holds the promise of a better capability to stimulate corneal healing. However, the lack of a standardized method for preparation, dispensation, storage and a poor biochemical characterization still hamper the establishment of a clinical consensus. Here the metabolomes of the two different serum eye drop preparations were compared using proton nuclear magnetic resonance spectroscopy. We found that both PBS and CBS contained several organic compounds, the majority of them already detected in human tears and may be thereby considered lacrimal substitutes. Metabolites having in the multivariate statistical analysis Partial least squares discriminant analysis (PLS-DA) a VIP scores > 1.0 were considered to be significantly different. All the metabolites identified were found to have a p < 0.05 in the univariate analysis. CBS, in particular, showed the highest amount of choline, myo-inositol, glutamine, creatine and ß-hydroxybutyrate. These evidences constitute relevant advances towards serum eye drops characterization and confirm that cord blood is a valid alternative source of serum eye drops.

Imbalance of Mg Homeostasis as a Potential Biomarker in Colon Cancer.

BACKGROUND: Increasing evidences support a correlation between magnesium (Mg) homeostasis and colorectal cancer (CRC). Nevertheless, the role of Mg and its transporters as diagnostic markers in CRC is still a matter of debate. In this study we combined X-ray Fluorescence Microscopy and databases information to investigate the possible correlation between Mg imbalance and CRC. METHODS: CRC tissue samples and their non-tumoural counterpart from four patients were collected and analysed for total Mg level and distribution by X-Ray Fluorescence Microscopy. We also reviewed the scientific literature and the main tissue expression databases to collect data on Mg transporters expression in CRC. RESULTS: We found a significantly higher content of total Mg in CRC samples when compared to non-tumoural tissues. Mg distribution was also impaired in CRC. Conversely, we evidenced an uncertain correlation between Mg transporters expression and colon malignancies. DISCUSSION: Although further studies are necessary to determine the correlation between different cancer types and stages, this is the first report proposing the measurement of Mg tissue localisation as a marker in CRC. This study represents thus a proof-of-concept that paves the way for the design of a larger prospective investigation of Mg in CRC.

A fine-tuned ß-catenin regulation during proliferation of corneal endothelial cells revealed using proteomics analysis.

Corneal endothelial (CE) dysfunction is the main indication for corneal transplantation, an invasive procedure with several limitations. Developing novel strategies to re-activate CE regenerative capacity is, therefore, of fundamental importance. This goal has proved to be challenging as corneal endothelial cells (CEnC) are blocked in the G0/G1 phase of the cell cycle in vivo and, albeit retaining proliferative capacity in vitro, this is further hindered by endothelial-to-mesenchymal transition. Herein we investigated the mechanisms regulating CEnC proliferation in vitro. Comparing the proteome of non-proliferating (in vivo-G0/G1) and proliferating (in vitro-G2/M) rabbit CEnC (rCEnC), 77 proteins, out of 3,328 identified, were differentially expressed in the two groups (p < 0.005). Literature and Gene Ontology analysis revealed ß-catenin and transforming growth factor (TGF-ß) pathways to be correlated with the identified proteins. Treatment of rCEnC with a ß-catenin activator and inhibitor showed that ß-catenin activation was necessary during rCEnC proliferation, but not sufficient for its induction. Furthermore, both pro-proliferative activity of basic fibroblast growth factor and anti-proliferative effects of TGF-ß were regulated through ß-catenin. Overall, these results provide novel insights into the molecular basis underlying the proliferation process that CEnC re-activate in vitro, consolidating the role of ß-catenin and TGF-ß.

Calpain Activation Is the Major Cause of Cell Death in Photoreceptors Expressing a Rhodopsin Misfolding Mutation.

The majority of mutations in rhodopsin (RHO) cause misfolding of the protein and has been linked to degeneration of photoreceptor cells in the retina. A lot of attention has been set on targeting ER stress for the development of new therapies for inherited retinal degeneration caused by mutations in the RHO gene. Nevertheless, the cell death pathway activated by RHO misfolded protein is still debated. In this study, we analyzed the retina of the knock-in mouse expressing the P23H misfolded mutant RHO. We found persistent unfolded protein response (UPR) during degeneration. Interestingly, long-term stimulation of the PERK branch of ER stress had a protective effect by phosphorylating nuclear factor erythroid 2-related factor 2 (NRF2) transcription factor, associated with antioxidant responses. Otherwise, we provide evidence that increased intracellular calcium and activation of calpains strongly correlated with rod photoreceptor cell death. By blocking calpain activity, we significantly decreased the activation of caspase-7 and apoptosis-inducing factor (AIF), two cell death effectors, and cell demise, and effectively protected the retina from degeneration caused by the P23H dominant mutation in RHO.

Differential Contribution of Calcium-Activated Proteases and ER-Stress in Three Mouse Models of Retinitis Pigmentosa Expressing P23H Mutant RHO.

Autosomal dominant retinitis pigmentosa (adRP) is mainly caused by mutations responsible for rhodopsin (RHO) misfolding. Although it was previously proved that unfolded RHO is retained into the endoplasmatic reticulum (ER) eliciting ER-stress, consequent mechanisms underlying photoreceptor degeneration need to be further clarified. Several animal models of RHO mutants have been developed for this purpose and for development of neuroprotective treatments. Here, we compared two of the most used models of adRP, the P23H mutant RHO transgenic and knock-in mouse models, in order to define which are their limits and potentials. Although they were largely used, the differences on the activation of the cell death pathways occurring in these two models still remain to be fully characterized. We present data proving that activation of calpains is a mechanism of cell death shared by both models and that molecules targeting calpains are neuroprotective. Conversely, the role of ER-stress contribution to cell death appears to be divergent and remains controversial.

Effective In Vivo Topical Delivery of siRNA and Gene Silencing in Intact Corneal Epithelium Using a Modified Cell-Penetrating Peptide.

Autosomal dominantly inherited genetic disorders such as corneal dystrophies are amenable to allele-specific gene silencing with small interfering RNA (siRNA). siRNA delivered to the cornea by injection, although effective, is not suitable for a frequent long-term treatment regimen, whereas topical delivery of siRNA to the cornea is hampered by the eye surface's protective mechanisms. Herein we describe an attractive and innovative alternative for topical application using cell-penetrating peptide derivatives capable of complexing siRNA non-covalently and delivering them into the cornea. Through a rational design approach, we modified derivatives of a cell-penetrating peptide, peptide for ocular delivery (POD), already proved to diffuse into the corneal layers. These POD derivatives were able to form siRNA-peptide complexes (polyplexes) of size and ζ-potential similar to those reported able to undergo cellular internalization. Successful cytoplasmic release and gene silencing in vitro was obtained when an endosomal disruptor, chloroquine, was added. A palmitoylated-POD, displaying the best delivery properties, was covalently functionalized with trifluoromethylquinoline, an analog of chloroquine. This modified POD, named trifluoromethylquinoline-palmitoyl-POD (QN-Palm-POD), when complexed with siRNA and topically applied to the eye in vivo, resulted in up to 30% knockdown of luciferase reporter gene expression in the corneal epithelium. The methods developed within represent a valid standardized approach that is ideal for screening of a range of delivery formulations.