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Purpose: Computer-aided oral implant surgery offers several advantages over the traditional approach. The purpose of this study was to evaluate the in vivo accuracy of computer-aided, template-guided oral implant surgery by comparing the three-dimensional positions of planned and placed implants. Materials and Methods: Oral implant therapy was performed in two treatment centers on eligible patients using computerized tomography (CT)-based software planning and computer-aided design/computer-assisted manufacture stereolithographic templates. A second CT scan was obtained after surgery. Preoperative and postoperative CT images were compared (planned vs actual implant positions), and the accuracy of this type of image-guided therapy was assessed. Results: Twenty-five adult patients were included in this retrospective study; 17 (11 partially and eight fully edentulous arches) were treated in center 1, and eight (six partially and two fully edentulous arches) in center 2. Of the 104 implants inserted with the computer-aided method, 100 integrated, giving a cumulative survival rate of 96% (mean follow-up, 36 months). There were no major surgical complications. With regard to accuracy, 89 implants were available for comparison; mean lateral deviations at the coronal and apical ends of the implants were 1.4 mm and 1.6 mm, respectively. Mean depth deviation was 1.1 mm and mean angular deviation was 7.9 degrees. There was a statistically significant correlation in the accuracy of any implants placed with the same guide. There was no difference in accuracy data from the two private centers; nor could a learning curve be demonstrated. Conclusions: Based upon this clinical study of 25 patients, the following observations were made: (1) computer-aided oral implant surgery used in two treatment centers provided a high likelihood (96%) of implant survival, and (2) deviations from planned implant positions existed in the coronal and apical portions of the implants as well as with implant angulation. Mean deviations were less than 2 mm in any direction and less than 8 degrees. INT J ORAL MAXILLOFAC IMPLANTS 2009;24:234-242 [ABSTRACT FROM AUTHOR]

Hematopoietic stem cell (HSC) mutations can result in clonal hematopoiesis (CH) with heterogeneous clinical outcomes. Here, we investigate how the cell state preceding Tet2 mutation impacts the pre-malignant phenotype. Using an inducible system for clonal analysis of myeloid progenitors, we find that the epigenetic features of clones at similar differentiation status are highly heterogeneous and functionally respond differently to Tet2 mutation. Cell differentiation stage also influences Tet2 mutation response indicating that the cell of origin's epigenome modulates clone-specific behaviors in CH. Molecular features associated with higher risk outcomes include Sox4 that sensitizes cells to Tet2 inactivation, inducing dedifferentiation, altered metabolism and increasing the in vivo clonal output of mutant cells, as confirmed in primary GMP and HSC models. Our findings validate the hypothesis that epigenetic features can predispose specific clones for dominance, explaining why identical genetic mutations can result in different phenotypes., (© 2024. The Author(s).)

Myelodysplastic syndromes (MDSs) are a heterogenous group of diseases affecting the hematopoietic stem cell that are curable only by stem cell transplantation. Both hematopoietic cell intrinsic changes and extrinsic signals from the bone marrow (BM) niche seem to ultimately lead to MDS. Animal models of MDS indicate that alterations in specific mesenchymal progenitor subsets in the BM microenvironment can induce or select for abnormal hematopoietic cells. Here, we identify a subset of human BM mesenchymal cells marked by the expression of CD271, CD146, and CD106. This subset of human mesenchymal cells is comparable with mouse mesenchymal cells that, when perturbed, result in an MDS-like syndrome. Its transcriptional analysis identified Osteopontin (SPP1) as the most overexpressed gene. Selective depletion of Spp1 in the microenvironment of the mouse MDS model, Vav-driven Nup98-HoxD13, resulted in an accelerated progression as demonstrated by increased chimerism, higher mutant myeloid cell burden, and a more pronounced anemia when compared with that in wild-type microenvironment controls. These data indicate that molecular perturbations can occur in specific BM mesenchymal subsets of patients with MDS. However, the niche adaptations to dysplastic clones include Spp1 overexpression that can constrain disease fitness and potentially progression. Therefore, niche changes with malignant disease can also serve to protect the host., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Myeloid cell heterogeneity is known, but whether it is cell-intrinsic or environmentally-directed remains unclear. Here, an inducible/reversible system pausing myeloid differentiation allowed the definition of clone-specific functions that clustered monocytes into subsets with distinctive molecular features. These subsets were orthogonal to the classical/nonclassical categorization and had inherent, restricted characteristics that did not shift under homeostasis, after irradiation, or with infectious stress. Rather, their functional fate was constrained by chromatin accessibility established at or before the granulocyte-monocyte or monocyte-dendritic progenitor level. Subsets of primary monocytes had differential ability to control distinct infectious agents in vivo. Therefore, monocytes are a heterogeneous population of functionally restricted subtypes defined by the epigenome of their progenitors that are differentially selected by physiologic challenges with limited plasticity to transition from one subset to another., (© 2023 by The American Society of Hematology.)

A major goal of biological imaging is localization of biomolecules inside a cell. Fluorescence microscopy can localize biomolecules inside whole cells and tissues, but its ability to count biomolecules and accuracy of the spatial coordinates is limited by the wavelength of visible light. Cryo-electron microscopy (cryo-EM) provides highly accurate position and orientation information of biomolecules but is often confined to small fields of view inside a cell, limiting biological context. In this study, we use a new data-acquisition scheme called Defocus-Corrected Large-Area cryo-EM (DeCo-LACE) to collect high-resolution images of entire sections (100- to 250-nm-thick lamellae) of neutrophil-like mouse cells, representing 1-2% of the total cellular volume. We use 2D template matching (2DTM) to determine localization and orientation of the large ribosomal subunit in these sections. These data provide maps of ribosomes across entire sections of mammalian cells. This high-throughput cryo-EM data collection approach together with 2DTM will advance visual proteomics and provide biological insight that cannot be obtained by other methods., Competing Interests: JE, GS, DS No competing interests declared, NG Reviewing editor, eLife, (© 2022, Elferich et al.)

A sleepless night may feel awful in its aftermath, but sleep's revitalizing powers are substantial, perpetuating the idea that convalescent sleep is a consequence-free physiological reset. Although recent studies have shown that catch-up sleep insufficiently neutralizes the negative effects of sleep debt, the mechanisms that control prolonged effects of sleep disruption are not understood. Here, we show that sleep interruption restructures the epigenome of hematopoietic stem and progenitor cells (HSPCs) and increases their proliferation, thus reducing hematopoietic clonal diversity through accelerated genetic drift. Sleep fragmentation exerts a lasting influence on the HSPC epigenome, skewing commitment toward a myeloid fate and priming cells for exaggerated inflammatory bursts. Combining hematopoietic clonal tracking with mathematical modeling, we infer that sleep preserves clonal diversity by limiting neutral drift. In humans, sleep restriction alters the HSPC epigenome and activates hematopoiesis. These findings show that sleep slows decay of the hematopoietic system by calibrating the hematopoietic epigenome, constraining inflammatory output, and maintaining clonal diversity., (© 2022 McAlpine et al.)

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., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Gene targeting constitutes a new step in the development of gene therapy for inherited diseases. Although previous studies have shown the feasibility of editing fibroblasts from Fanconi anemia (FA) patients, here we aimed at conducting therapeutic gene editing in clinically relevant cells, such as hematopoietic stem cells (HSCs). In our first experiments, we showed that zinc finger nuclease (ZFN)-mediated insertion of a non-therapeutic EGFP-reporter donor in the AAVS1 "safe harbor" locus of FA-A lymphoblastic cell lines (LCLs), indicating that FANCA is not essential for the editing of human cells. When the same approach was conducted with therapeutic FANCA donors, an efficient phenotypic correction of FA-A LCLs was obtained. Using primary cord blood CD34 + cells from healthy donors, gene targeting was confirmed not only in in vitro cultured cells, but also in hematopoietic precursors responsible for the repopulation of primary and secondary immunodeficient mice. Moreover, when similar experiments were conducted with mobilized peripheral blood CD34 + cells from FA-A patients, we could demonstrate for the first time that gene targeting in primary hematopoietic precursors from FA patients is feasible and compatible with the phenotypic correction of these clinically relevant cells., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

Targeted genome editing in hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematological diseases. However, the limited efficiency of homology-directed editing in primitive HSPCs constrains the yield of corrected cells and might affect the feasibility and safety of clinical translation. These concerns need to be addressed in stringent preclinical models and overcome by developing more efficient editing methods. We generated a humanized X-linked severe combined immunodeficiency (SCID-X1) mouse model and evaluated the efficacy and safety of hematopoietic reconstitution from limited input of functional HSPCs, establishing thresholds for full correction upon different types of conditioning. Unexpectedly, conditioning before HSPC infusion was required to protect the mice from lymphoma developing when transplanting small numbers of progenitors. We then designed a one-size-fits-all IL2RG (interleukin-2 receptor common γ-chain) gene correction strategy and, using the same reagents suitable for correction of human HSPC, validated the edited human gene in the disease model in vivo, providing evidence of targeted gene editing in mouse HSPCs and demonstrating the functionality of the IL2RG -edited lymphoid progeny. Finally, we optimized editing reagents and protocol for human HSPCs and attained the threshold of IL2RG editing in long-term repopulating cells predicted to safely rescue the disease, using clinically relevant HSPC sources and highly specific zinc finger nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9). Overall, our work establishes the rationale and guiding principles for clinical translation of SCID-X1 gene editing and provides a framework for developing gene correction for other diseases., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Transfer of T-cell receptors (TCRs) specific for tumor-associated antigens is a promising approach for cancer immunotherapy. We developed the TCR gene editing technology that is based on the knockout of the endogenous TCR α and β genes, followed by the introduction of tumor-specific TCR genes, and that proved safer and more effective than conventional TCR gene transfer. Although successful, complete editing requires extensive cell manipulation and 4 transduction procedures. Here we propose a novel and clinically feasible TCR "single editing" (SE) approach, based on the disruption of the endogenous TCR α chain only, followed by the transfer of genes encoding for a tumor-specific TCR. We validated SE with the clinical grade HLA-A2 restricted NY-ESO-1 157-165 -specific TCR. SE allowed the rapid production of high numbers of tumor-specific T cells, with optimal TCR expression and preferential stem memory and central memory phenotype. Similarly to unedited T cells redirected by TCR gene transfer (TCR transferred [TR]), SE T cells efficiently killed NY-ESO-1 pos targets; however, although TR cells proved highly alloreactive, SE cells showed a favorable safety profile. Accordingly, when infused in NSG mice previously engrafted with myeloma, SE cells mediated tumor rejection without inducing xenogeneic graft-versus-host disease, thus resulting in significantly higher survival than that observed in mice treated with TR cells. Overall, single TCR gene editing represents a clinically feasible approach that is able to increase the safety and efficacy of cancer adoptive immunotherapy., (© 2017 by The American Society of Hematology.)

Background: This work evaluated the accuracy and safety of a protocol for minimally-invasive flapless zygomatic implant placement that uses computer-guided stereolithographic mucosa-supported surgical templates., Methods: A total of six zygomatic implants were placed in three formalin-fixed human cadaver heads, with edentulous and severely atrophic posterior maxillae. CT scans of each cadaver head were performed after zygomatic implant placement, to evaluate the lateral error (LE) at implant tip and base, and the angular error (AE) of the implant body., Results: Excluding the implant placed outside the drilling channel, the LE at the implant base was less than 1 mm in all cases. The average LE at the tip was 3.86 mm, and the AE was less than 6° in all cases, with an average of 4.5°., Conclusion: Computer-guided minimally-invasive flapless zygomatic implant surgery remains challenging. Careful planning and perfect stability of the surgical guide are essential., (Copyright © 2015 John Wiley & Sons, Ltd.)

Targeted genome editing by artificial nucleases has brought the goal of site-specific transgene integration and gene correction within the reach of gene therapy. However, its application to long-term repopulating haematopoietic stem cells (HSCs) has remained elusive. Here we show that poor permissiveness to gene transfer and limited proficiency of the homology-directed DNA repair pathway constrain gene targeting in human HSCs. By tailoring delivery platforms and culture conditions we overcame these barriers and provide stringent evidence of targeted integration in human HSCs by long-term multilineage repopulation of transplanted mice. We demonstrate the therapeutic potential of our strategy by targeting a corrective complementary DNA into the IL2RG gene of HSCs from healthy donors and a subject with X-linked severe combined immunodeficiency (SCID-X1). Gene-edited HSCs sustained normal haematopoiesis and gave rise to functional lymphoid cells that possess a selective growth advantage over those carrying disruptive IL2RG mutations. These results open up new avenues for treating SCID-X1 and other diseases.

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., (Copyright © 2011 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.)

This study reports on the first longitudinal results of an alumina (70%)-zirconia (30%) ceramic restorative system for implant-supported, single-tooth replacement in the anterior maxillary jaw. Eighteen patients (9 men, 9 women, mean age = 42.4 years) were treated with 22 implants placed into 19 extraction sockets and 3 residual ridges. Eight implants were immediately loaded with nonoccluding provisional prostheses. All implants were definitively loaded with fully occluding ceramic restorations after osseointegration and soft tissue maturation. Patients were monitored from 7 to 30 months (mean = 18.1 months) after loading. All implants osseointegrated and were successfully restored. One case of abutment screw loosening occurred because of patient parafunction (bruxing), and another patient reported chewing pain attributed to malocclusion. Both problems were successfully resolved without further incidence. Within the context of this study, single-tooth replacement in the anterior maxillary jaw was successfully achieved with alumina-zirconia ceramic single-tooth restorations with up to 30 months of clinical function.

The anterior maxillary jaw is frequently called the "esthetic zone" because of its high visibility and influence on facial appearance. Implant placement and single-tooth restoration in this region can present some esthetic challenges for the clinician. The underlying titanium abutment, for example, can diminish porcelain translucency and result in a darkened prosthesis. Subsequent changes in soft-tissue margins can also result in the visibility of titanium at the gingival crevice. In recent years, all-ceramic restorations have gained popularity in response to increasing patient demands for improved esthetics. This article describes the clinical use of a new, tooth-shaped, ceramic coping system in the restoration of single teeth in the esthetic zones of 3 patients. Four tapered screw implants were placed in the locations of the central incisors (n = 3) and bicuspid (n = 1). Conventional ceramic protocols were used to fabricate all-ceramic crowns that were cemented onto small core abutments attached to the implants. All prostheses restored the biomechanical needs of the patients and achieved excellent esthetic results.

Purpose: This clinical report describes an immediate tooth extraction, followed by placement and provisional restoration of a dental implant in the prepared socket of a right maxillary central incisor., Materials and Methods: The tooth was extracted with minimal hard and soft tissue trauma and without flap reflection. A flapless, transmucosal surgical approach was used to prepare the socket and insert a tapered implant. The implant was immediately restored with a provisional abutment and crown without occlusal contacts. An impression was made 22 days after implant insertion, and a definitive, all-ceramic restoration was placed 3 days later., Results: During the period of provisional progressive loading, no significant soft tissue contraction was observed related to noninvasive operating techniques and the immediate insertion of the provisional restoration. The patient exhibited no clinical or radiologic complications through 8 months of clinical monitoring after loading., Conclusion: The Tapered Screw-Vent implant and all-ceramic restoration provided the patient with immediate esthetics, function, and comfort without any complications during the postloading follow-up period.