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 β-catenin and TGFβ 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

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

Comparison of two alternative procedures to obtain packed red blood cells for β-thalassemia major transfusion therapy

β-thalassemia major (βTM) patients require frequent blood transfusions, with consequences that span from allogenic reactions to iron overload. To minimize these effects, βTM 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 βTM 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 βTM patients, which often need transfusions for the entire lifespan

Influence of Hospitalization upon Diagnosis on the Risk of Tuberculosis Clustering

Abstract. Setting: Culture-positive tuberculosis (TB) diagnosed in the metropolitan area of Milan (Italy) over a 5-year period (1995-1999). Objective: To assess the impact of short-course hospitalization upon diagnosis on the overall risk of TB clustering. Design: Restriction fragment length polymorphism profiles with a similarity of 100% defined a cluster. Uni- and multivariable logistic regression models were performed to assess factors associated with clustering. Results: Among 1139 patients, 392 (34.4%) were hospitalized before or soon after diagnosis, 405 (35.6%) received domiciliary treatment since the diagnosis and 392 (30%) had no information about initial clinical management. One hundred fifteen molecular clusters involving 363 patients were identified. Using multivariable analysis, hospitalization was not significantly associated with clustering (OR 1.06, 95%CI 0.75-1.50, p=0.575). Subjects aged >65 years old (OR 0.60; 95CI%:0.37-0.95; p=0.016) and non-Italian born patients (OR 0.56; 95%CI:0.41-0.76; p<0.001) were running a lower risk of clustering. Conversely, HIV co-infected patients (OR 1.88, 95%CI:1.20-2.95, p=0.006) and those with MDR TB (OR 2.50, 95%CI:1.46-4.25, p=0.001) were significantly more likely to be involved in clusters. Conclusion: In our cohort, domiciliary treatment was not associated with TB clustering. Expanding domiciliary treatment upon diagnosis appears as an advisable measure to reduce unnecessary costs for the health care system

A novel role for CRIM1 in the corneal response to UV and pterygium development

Pterygium is a pathological proliferative condition of the ocular surface, characterised by formation of a highly vascularised, fibrous tissue arising from the limbus that invades the central cornea leading to visual disturbance and, if untreated, blindness. Whilst chronic ultraviolet (UV) light exposure plays a major role in its pathogenesis, higher susceptibility to pterygium is observed in some families, suggesting a genetic component. In this study, a Northern Irish family affected by pterygium but reporting little direct exposure to UV was identified carrying a missense variant in CRIM1 NM_016441.2: c.1235 A &gt; C (H412P) through whole-exome sequencing and subsequent analysis. CRIM1 is expressed in the developing eye, adult cornea and conjunctiva, having a role in cell differentiation and migration but also in angiogenesis, all processes involved in pterygium formation. We demonstrate elevated CRIM1 expression in pterygium tissue from additional individual Northern Irish patients compared to unaffected conjunctival controls. UV irradiation of HCE-S cells resulted in an increase in ERK phosphorylation and CRIM1 expression, the latter further elevated by the addition of the MEK1/2 inhibitor, U0126. Conversely, siRNA knockdown of CRIM1 led to decreased UV-induced ERK phosphorylation and increased BCL2 expression. Transient expression of the mutant H412P CRIM1 in corneal epithelial HCE-S cells showed that, unlike wild-type CRIM1, it was unable to reduce the cell proliferation, increased ERK phosphorylation and apoptosis induced through a decrease of BCL2 expression levels. We propose here a series of intracellular events where CRIM1 regulation of the ERK pathway prevents UV-induced cell proliferation and may play an important role in the in the pathogenesis of pterygium

Keratin 12 missense mutation induces the unfolded protein response and apoptosis in meesmann epithelial corneal dystrophy

Meesmann epithelial corneal dystrophy (MECD) is a rare autosomal dominant disorder caused by dominant-negative mutations within the KRT3 or KRT12 genes, which encode the cytoskeletal protein keratins K3 and K12, respectively. To investigate the pathomechanism of this disease, we generated and phenotypically characterized a novel knock-in humanized mouse model carrying the severe, MECD-associated, K12-Leu132Pro mutation. Although no overt changes in corneal opacity were detected by slit-lamp examination, the corneas of homozygous mutant mice exhibited histological and ultrastructural epithelial cell fragility phenotypes. An altered keratin expression profile was observed in the cornea of mutant mice, confirmed by western blot, RNA-seq and quantitative real-time polymerase chain reaction. Mass spectrometry (MS) and immunohistochemistry demonstrated a similarly altered keratin profile in corneal tissue from a K12-Leu132Pro MECD patient. The K12-Leu132Pro mutation results in cytoplasmic keratin aggregates. RNA-seq analysis revealed increased chaperone gene expression, and apoptotic unfolded protein response (UPR) markers, CHOP and Caspase 12, were also increased in the MECD mice. Corneal epithelial cell apoptosis was increased 17-fold in the mutant cornea, compared with the wild-type (P &lt; 0.001). This elevation of UPR marker expression was also observed in the human MECD cornea. This is the first reporting of a mouse model for MECD that recapitulates the human disease and is a valuable resource in understanding the pathomechanism of the disease. Although the most severe phenotype is observed in the homozygous mice, this model will still provide a test-bed for therapies not only for corneal dystrophies but also for other keratinopathies caused by similar mutations

Cyclosporine H Overcomes Innate Immune Restrictions to Improve Lentiviral Transduction and Gene Editing In Human Hematopoietic Stem Cells

Innate immune factors may restrict hematopoietic stem cell (HSC) genetic engineering and contribute to broad individual variability in gene therapy outcomes. Here, we show that HSCs harbor an early, constitutively active innate immune block to lentiviral transduction that can be efficiently overcome by cyclosporine H (CsH). CsH potently enhances gene transfer and editing in human long-term repopulating HSCs by inhibiting interferon-induced transmembrane protein 3 (IFITM3), which potently restricts VSV glycoprotein-mediated vector entry. Importantly, individual variability in endogenous IFITM3 levels correlated with permissiveness of HSCs to lentiviral transduction, suggesting that CsH treatment will be useful for improving ex vivo gene therapy and standardizing HSC transduction across patients. Overall, our work unravels the involvement of innate pathogen recognition molecules in immune blocks to gene correction in primary human HSCs and highlights how these roadblocks can be overcome to develop innovative cell and gene therapies

現地観測に基づく河川流の乱流特性に関する研究

博士（工学）神戸大

Modeling, optimization, and comparable efficacy of T cell and hematopoietic stem cell gene editing for treating hyper-IgM syndrome

Precise correction of the CD40LG gene in T cells and hematopoietic stem/progenitor cells (HSPC) holds promise for treating X-linked hyper-IgM Syndrome (HIGM1), but its actual therapeutic potential remains elusive. Here, we developed a one-size-fits-all editing strategy for effective T-cell correction, selection, and depletion and investigated the therapeutic potential of T-cell and HSPC therapies in the HIGM1 mouse model. Edited patients' derived CD4 T cells restored physiologically regulated CD40L expression and contact-dependent B-cell helper function. Adoptive transfer of wild-type T cells into conditioned HIGM1 mice rescued antigen-specific IgG responses and protected mice from a disease-relevant pathogen. We then obtained similar to 25% CD40LG editing in long-term repopulating human HSPC. Transplanting such proportion of wild-type HSPC in HIGM1 mice rescued immune functions similarly to T-cell therapy. Overall, our findings suggest that autologous edited T cells can provide immediate and substantial benefits to HIGM1 patients and position T-cell ahead of HSPC gene therapy because of easier translation, lower safety concerns and potentially comparable clinical benefits.Transplantation and immunomodulatio

Zygomatic implant placement with flapless computer-guided surgery: A Proposed clinical protocol

PURPOSE: The aim of the present report is to describe a procedure for zygomatic implant placement using image-guided implant surgery. This is an innovative technique and includes a new clinical approach to provide the direction to guide drilling. MATERIALS AND METHODS: The ethical committee of the University of Genoa approved the study. All patients had clinical indications of severe maxillary atrophy (Class 4 Cadwood-Howell). A total of 25 implants were placed, of which 17 were in the premaxilla, 7 in the zygomatic area, and 1 in the pterygoid bone. The treatment was performed in 2 phases. The first phase included cone-beam acquisition to exclude sinus disease and evaluate the anatomy of the residual premaxillary bone in 3 dimensions. The success of osseointegration achieved by the primary implants (PIs) was confirmed after temporary loading and before proceeding with the second phase, in which all patients were scheduled for zygomatic implants. A total of 3 or 4 regular platform MK III implants (17 in all) were placed in the premaxilla using conventional implant surgery. A model within the analogs of the PIs was prepared (master model), taking a conventional impression. Next, a radiologic template was prepared on the same master model and stabilized on the PIs, using dedicated prosthetic components. The guide was screwed onto the PIs during computed tomography acquisition to determine a fixed and repeatable position of the guide. In the second phase, after routine planning, a mucosa-supported stereolithographic SurgiGuide with sleeves for the zygomatic implants and the corresponding stereolithographic model, including the mucosa, were received from the manufacturers. The guide was repositioned on the master model to replace the sleeves for the PIs in the same position. With an original customized surgical kit, including an innovative intrasinus device, we next simulated surgery on the stereolithographic model to determine and control the direction of the osteotomies and the final depth of drilling. The SurgiGuide was anchored onto the PIs before the zygomatic osteotomy, after which flapless surgery was performed to place the zygomatic implants according to the plan. RESULTS: Two PIs in the premaxillary area failed (and were replaced before the zygomatic step). No zygomatic implants failed. The follow-up examinations at 4 to 39 months showed good esthetic, phonetic, and functional results. CONCLUSIONS: The results of the proposed surgical procedure appear to be encouraging. Although it is difficult to achieve the correct driven angle of osteotomies for zygomatic implants, in all patients we achieved correct zygomatic positioning, in agreement with previous planning. Additional research and randomized clinical trials are needed to assess the predictability of the procedure