Search

Abstract Background Retinal degeneration results from disruptions in retinal homeostasis due to injury, disease, or aging and triggers peripheral leukocyte infiltration. Effective immune responses rely on coordinated actions of resident microglia and recruited macrophages, critical for tissue remodeling and repair. However, these phagocytes also contribute to chronic inflammation in degenerated retinas, yet the precise coordination of immune response to retinal damage remains elusive. Recent investigations have demonstrated that phagocytic cells can produce extracellular traps (ETs), which are a source of self-antigens that alter the immune response, which can potentially lead to tissue injury. Methods Innovations in experimental systems facilitate real-time exploration of immune cell interactions and dynamic responses. We integrated in vivo imaging with ultrastructural analysis, transcriptomics, pharmacological treatments, and knockout mice to elucidate the role of phagocytes and their modulation of the local inflammatory response through extracellular traps (ETs). Deciphering these mechanisms is essential for developing novel and enhanced immunotherapeutic approaches that can redirect a specific maladaptive immune response towards favorable wound healing in the retina. Results Our findings underscore the pivotal role of innate immune cells, especially macrophages/monocytes, in regulating retinal repair and inflammation. The absence of neutrophil and macrophage infiltration aids parenchymal integrity restoration, while their depletion, particularly macrophages/monocytes, impedes vascular recovery. We demonstrate that macrophages/monocytes, when recruited in the retina, release chromatin and granular proteins, forming ETs. Furthermore, the pharmacological inhibition of ETosis support retinal and vascular repair, surpassing the effects of blocking innate immune cell recruitment. Simultaneously, the absence of ETosis reshapes the inflammatory response, causing neutrophils, helper, and cytotoxic T-cells to be restricted primarily in the superficial capillary plexus instead of reaching the damaged photoreceptor layer. Conclusions Our data offer novel insights into innate immunity's role in responding to retinal damage and potentially help developing innovative immunotherapeutic approaches that can shift the immune response from maladaptive to beneficial for retinal regeneration.

Abstract We present a miniature oblique back-illumination microscope (mOBM) for imaging the microcirculation of human oral mucosa, enabling real-time, label-free phase contrast imaging of individual leukocytes circulating in the bloodstream, as well as their rolling and adhesion on vascular walls—the initial steps in leukocyte recruitment that is a hallmark of inflammation. Using the mOBM system, we studied the leukocyte-endothelial interactions in healthy and locally inflamed tissue and observed drastic changes in leukocyte movement (velocity and displacement profile). Our findings suggest that real-time imaging of leukocyte dynamics can provide new diagnostic insights (assessment of inflammation, temporal progression of disease, evaluation of therapeutic response, etc.) that are not available using conventional static parameters such as cell number and morphology.

Abstract Background Retinal degeneration is a disease affecting the eye, which is an immune-privileged site because of its anatomical and physiological properties. Alterations in retinal homeostasis—because of injury, disease, or aging—initiate inflammatory cascades, where peripheral leukocytes (PL) infiltrate the parenchyma, leading to retinal degeneration. So far, research on PL's role in retinal degeneration was limited to observing a few cell types at specific times or sectioning the tissue. This restricted our understanding of immune cell interactions and response duration. Methods In vivo microscopy in preclinical mouse models can overcome these limitations enabling the spatio-temporal characterization of PL dynamics. Through in vivo imaging, we assessed structural and fluorescence changes in response to a focal injury at a defined location over time. We also utilized minimally invasive techniques, pharmacological interventions, and knockout (KO) mice to determine the role of PL in local inflammation. Furthermore, we investigated PL abundance and localization during retinal degeneration in human eyes by histological analysis to assess to which extent our preclinical study translates to human retinal degeneration. Results We demonstrate that PL, especially T cells, play a detrimental role during retinal injury response. In mice, we observed the recruitment of helper and cytotoxic T cells in the parenchyma post-injury, and T cells also resided in the macula and peripheral retina in pathological conditions in humans. Additionally, we found that the pharmacological PL reduction and genetic depletion of T-cells reduced injured areas in murine retinas and rescued the blood–retina barrier (BRB) integrity. Both conditions promoted morphological changes of Cx3cr1+ cells, including microglial cells, toward an amoeboid phenotype during injury response. Interestingly, selective depletion of CD8+ T cells accelerated recovery of the BRB compared to broader depletions. After anti-CD8 treatment, the retinal function improved, concomitant to a beneficial immune response. Conclusions Our data provide novel insights into the adaptive immune response to retinal injury in mice and human retinal degeneration. Such information is fundamental to understanding retinal disorders and developing therapeutics to modulate immune responses to retinal degeneration safely.

Abstract Background Sepsis is an uncontrolled inflammatory response against a systemic infection that results in elevated mortality, mainly induced by bacterial products known as endotoxins, producing endotoxemia. Disseminated intravascular coagulation (DIC) is frequently observed in septic patients and is associated with organ failure and death. Sepsis activates endothelial cells (ECs), promoting a prothrombotic phenotype contributing to DIC. Ion channel-mediated calcium permeability participates in coagulation. The transient reception potential melastatin 7 (TRPM7) non-selective divalent cation channel that also contains an α-kinase domain, which is permeable to divalent cations including Ca2+, regulates endotoxin-stimulated calcium permeability in ECs and is associated with increased mortality in septic patients. However, whether endothelial TRPM7 mediates endotoxemia-induced coagulation is not known. Therefore, our aim was to examine if TRPM7 mediates coagulation during endotoxemia. Results The results showed that TRPM7 regulated endotoxin-induced platelet and neutrophil adhesion to ECs, dependent on the TRPM7 ion channel activity and by the α-kinase function. Endotoxic animals showed that TRPM7 mediated neutrophil rolling on blood vessels and intravascular coagulation. TRPM7 mediated the increased expression of the adhesion proteins, von Willebrand factor (vWF), intercellular adhesion molecule 1 (ICAM-1), and P-selectin, which were also mediated by the TRPM7 α-kinase function. Notably, endotoxin-induced expression of vWF, ICAM-1 and P-selectin were required for endotoxin-induced platelet and neutrophil adhesion to ECs. Endotoxemic rats showed increased endothelial TRPM7 expression associated with a procoagulant phenotype, liver and kidney dysfunction, increased death events and an increased relative risk of death. Interestingly, circulating ECs (CECs) from septic shock patients (SSPs) showed increased TRPM7 expression associated with increased DIC scores and decreased survival times. Additionally, SSPs with a high expression of TRPM7 in CECs showed increased mortality and relative risk of death. Notably, CECs from SSPs showed significant results from the AUROC analyses for predicting mortality in SSPs that were better than the Acute Physiology and Chronic Health Evaluation II (APACHE II) and the Sequential Organ Failure Assessment (SOFA) scores. Conclusions Our study demonstrates that sepsis-induced DIC is mediated by TRPM7 in ECs. TRPM7 ion channel activity and α-kinase function are required by DIC-mediated sepsis-induced organ dysfunction and its expression are associated with increased mortality during sepsis. TRPM7 appears as a new prognostic biomarker to predict mortality associated to DIC in SSPs, and as a novel target for drug development against DIC during infectious inflammatory diseases.

The molecular circuitry that drives dendrite formation during osteocytogenesis remains poorly understood. Here the authors show that deletion of Sp7, a gene linked to rare and common skeletal disease, in mature osteoblasts and osteocytes causes severe defects in osteocyte dendrites.

Abstract Conditional creER-mediated gene inactivation or gene induction has emerged as a robust tool for studying gene functions in mouse models of tissue development, homeostasis, and regeneration. Here, we present a method to conditionally induce cre recombination in the mouse calvarial bone while avoiding systemic recombination in distal bones. To test our method, we utilized Prx1creER-egfp;td-Tomato mice and delivered 4-hydroxytamoxifen (4-OHT) to the mouse calvaria, subperiosteally. First, we showed that two calvaria subperiosteal injections of 10 µg of 4-OHT (3.3 mg of 4-OHT/kg of body weight) can induce local recombination as efficiently as two intraperitoneal systemic injections of 200 μg of tamoxifen (70 mg of tamoxifen/kg of body weight). Then, we studied the recombination efficiency of various subperiosteal calvaria dosages and found that two subperiosteal injections of 5 µg 4-OHT (1.65 mg of 4-OHT/kg of body weight) uphold the same recombination efficiency observed with higher dosages. Importantly, the result indicated that the low dosage does not induce significant systemic recombination in remote skeletal tissues. With the proposed local low dosage protocol, the recombination efficiency at the injection site (calvarial bone) reached 94%, while the recombination efficiency at the mandible and the digits was as low as the efficiency measured in control animals.

The role of mTORC1 in AML has not yet been proven due to the mixed results of its inhibitors in clinical trials. Here the authors show the real-time dynamics of the mTORC1 pathway in association with AML growth and response to chemotherapy with fluorescent markers, providing guidance for timed intervention with pathway-specific inhibitors.

Advances in intravital microscopy (IVM) have enabled the studies of cellular organization and dynamics in the native microenvironment of intact organisms with minimal perturbation. The abilities to track specific cell populations and monitor their interactions have opened up new horizons for visualizing cell biology in vivo, yet the success of standard fluorescence cell labeling approaches for IVM comes with a “dark side” in that unlabeled cells are invisible, leaving labeled cells or structures to appear isolated in space, devoid of their surroundings and lacking proper biological context. Here we describe a novel method for “filling in the void” by harnessing the ubiquity of extracellular (interstitial) fluid and its ease of fluorescence labelling by commonly used vascular and lymphatic tracers. We show that during routine labeling of the vasculature and lymphatics for IVM, commonly used fluorescent tracers readily perfuse the interstitial spaces of the bone marrow (BM) and the lymph node (LN), outlining the unlabeled cells and forming negative contrast images that complement standard (positive) cell labeling approaches. The method is simple yet powerful, offering a comprehensive view of the cellular landscape such as cell density and spatial distribution, as well as dynamic processes such as cell motility and transmigration across the vascular endothelium. The extracellular localization of the dye and the interstitial flow provide favorable conditions for prolonged Intravital time lapse imaging with minimal toxicity and photobleaching.

Hematopoietic stem cells (HSCs) migrate from the fetal liver to the bone marrow (BM) during embryogenesis. Here the authors show that the WAVE2 complex scaffold Hem1 is required for engraftment of HSCs in BM, not through its canonical role regulating actin polymerization, but through c-Abl survival signaling.

Abstract Transplantation of a single hematopoietic stem cell is an important method for its functional characterization, but the standard transplantation protocol relies on cell homing to the bone marrow after intravenous injection. Here, we present a method to transplant single cells directly into the bone marrow of live mice. We developed an optical platform that integrates a multiphoton microscope with a laser ablation unit for microsurgery and an optical tweezer for cell micromanipulation. These tools allow image-guided single cell transplantation with high spatial control. The platform was used to deliver single hematopoietic stem cells. The engraftment of transplants was tracked over time, illustrating that the technique can be useful for studying both normal and malignant stem cells in vivo.

Summary: Post-natal skeletal stem cells expressing PRX1 (pnPRX1+) have been identified in the calvaria and in the axial skeleton. Here we characterize the location and functional capacity of the calvarial pnPRX1+ cells. We found that pnPRX1+ reside exclusively in the calvarial suture niche and decrease in number with age. They are distinct from preosteoblasts and osteoblasts of the sutures, respond to WNT signaling in vitro and in vivo by differentiating into osteoblasts, and, upon heterotopic transplantation, are able to regenerate bone. Diphtheria toxin A (DTA)-mediated lineage ablation of pnPRX1+ cells and suturectomy perturb regeneration of calvarial bone defects and confirm that pnPRX1+ cells of the sutures are required for bone regeneration. Orthotopic transplantation of sutures with traceable pnPRX1+ cells into wild-type animals shows that pnPRX1+ cells of the suture contribute to calvarial bone defect regeneration. DTA-mediated lineage ablation of pnPRX1+ does not, however, interfere with calvarial development. : Lin, Intini, and colleagues identify the calvarial suture as the stem cell niche for PRX1-expressing postnatal skeletal stem cells by means of intravital microscopy, lineage-tracing, and lineage-ablation experiments. They show that PRX1-expressing cells of the calvaria are required for bone regeneration but are dispensable for calvarial development. Keywords: calvarial skeletal stem cells, stem cell niche, PRX1, PRRX1, calvarial bone regeneration, calvarial development

Previous studies have shown that post-natal skeletal stem cells expressing Paired-related homeobox 1 (PRX1 or PRRX1) are present in the periosteum of long bones where they contribute to post-natal bone development and regeneration. Our group also identified post-natal PRX1 expressing cells (pnPRX1+ cells) in mouse calvarial synarthroses (sutures) and showed that these cells are required for calvarial bone regeneration. Since calvarial synarthroses are similar to dentoalveolar gomphosis (periodontium) and since there is no information available on the presence or function of pnPRX1+ cells in the periodontium, the present study aimed at identifying and characterizing pnPRX1+ cells within the mouse periodontium and assess their contribution to periodontal development and regeneration. Here we demonstrated that pnPRX1+ cells are present within the periodontal ligament (PDL) of the mouse molars and of the continuously regenerating mouse incisor. By means of diphtheria toxin (DTA)-mediated conditional ablation of pnPRX1+ cells, we show that pnPRX1+ cells contribute to post-natal periodontal development of the molars and the incisor, as ablation of pnPRX1+ cells in 3-days old mice resulted in a significant enlargement of the PDL space after 18 days. The contribution of pnPRX1+ cells to periodontal regeneration was assessed by developing a novel non-critical size periodontal defect model. Outcomes showed that DTA-mediated post-natal ablation of pnPRX1+ cells results in lack of regeneration in periodontal non-critical size defects in the regeneration competent mouse incisors. Importantly, gene expression analysis of these cells shows a profile typical of quiescent cells, while gene expression analysis of human samples of periodontal stem cells (PDLSC) confirmed that Prx1 is highly expressed in human periodontium. In conclusion, pnPRX1+ cells are present within the continuously regenerating PDL of the mouse incisor, and at such location they contribute to post-natal periodontal development and regeneration. Since this study further reports the presence of PRX1 expressing cells within human periodontal ligament, we suggest that studying the mouse periodontal pnPRX1+ cells may provide significant information for the development of novel and more effective periodontal regenerative therapies in humans.

Poor homing of systemically infused cells to disease sites may limit the success of exogenous cell-based therapy. In this study, we screened 9,000 signal-transduction modulators to identify hits that increase mesenchymal stromal cell (MSC) surface expression of homing ligands that bind to intercellular adhesion molecule 1 (ICAM-1), such as CD11a. Pretreatment of MSCs with Ro-31-8425, an identified hit from this screen, increased MSC firm adhesion to an ICAM-1-coated substrate in vitro and enabled targeted delivery of systemically administered MSCs to inflamed sites in vivo in a CD11a- (and other ICAM-1-binding domains)-dependent manner. This resulted in a heightened anti-inflammatory response. This represents a new strategy for engineering cell homing to enhance therapeutic efficacy and validates CD11a and ICAM-1 as potential targets. Altogether, this multi-step screening process may significantly improve clinical outcomes of cell-based therapies.

Introduction It remains uncertain whether Descemet membrane endothelial keratoplasty (DMEK) or Descemet stripping only (DSO) yields better outcomes in patients with symptomatic Fuchs endothelial corneal dystrophy (FECD). This paper presents the protocol for the Descemet Endothelial Thickness Comparison Trial II (DETECT II), a multicentre, outcome-masked, randomised, placebo-controlled, clinical trial comparing DMEK to DSO with ripasudil (DSO-R) for this patient population.Methods and analysis A total of 60 patients with endothelial dysfunction due to symptomatic FECD will be enrolled from seven participating sites in the USA. The patients will be randomly assigned in a 1:1 ratio to one of the following treatment groups: group 1—DMEK plus topical placebo and group 2—DSO plus topical ripasudil 0.4%. The enrolment period is 24 months. The primary outcome is best spectacle-corrected visual acuity at 12 months. Secondary outcomes include peripheral and central endothelial cell density, visual acuity, vision-related quality of life and Pentacam Scheimpflug tomography. Study outcomes will be analysed using mixed effects linear regression. Adverse events, including rebubble procedures, endothelial failure and graft rejection, will be documented and analysed using appropriate statistical methods. DETECT II aims to provide evidence on the comparative effectiveness of DMEK and DSO-R. The results of this trial will contribute to optimising the treatment of FECD, while also exploring the cost-effectiveness of these interventions. Dissemination of findings through peer-reviewed publications and national/international meetings will facilitate knowledge translation and guide clinical practice in the field of corneal transplantation.Ethics and dissemination A data and safety monitoring committee has been empanelled by the National Eye Institute. All study protocols will be subject to review and approval by WCG IRB as the single IRB of record. This study will comply with the National Institute of Health (NIH) Data Sharing Policy and Policy on the Dissemination of NIH-Funded Clinical Trial Information and the Clinical Trials Registration and Results Information Submission rule. Data from the trial will be made available on reasonable request.Trial registration number NCT05275972.

Atherosclerosis has been recognized as a chronic inflammation disease, in which many types of cells participate in this process, including lymphocytes, macrophages, dendritic cells (DCs), mast cells, vascular smooth muscle cells (SMCs). Developments in imaging technology provide the capability to observe cellular and tissue components and their interactions. The knowledge of the functions of immune cells and their interactions with other cell and tissue components will facilitate our discovery of biomarkers in atherosclerosis and prediction of the risk factor of rupture-prone plaques. Nonlinear optical microscopy based on two-photon excited autofluorescence and second harmonic generation (SHG) were developed to image mast cells, SMCs and collagen in plaque ex vivo using endogenous optical signals. Mast cells were imaged with two-photon tryptophan autofluorescence, SMCs were imaged with two-photon NADH autofluorescence, and collagen were imaged with SHG. This development paves the way for further study of mast cell degranulation, and the effects of mast cell derived mediators such as induced synthesis and activation of matrix metalloproteinases (MMPs) which participate in the degradation of collagen.

The effect of intralysosomal proteolysis of near-infrared fluorescent (NIRF) self-quenched macromolecular probe (PGC-Cy5.5) has been previously reported and used for tumor imaging. Here we demonstrate that proteolysis can be detected noninvasively in vivo at the cellular level. A codetection of GFP fluorescence (using two-photon excitation) and NIRF was performed in tumor-bearing animals injected with PGC-Cy5.5. In vivo microscopy of tumor cells in subdermal tissue layers (up to 160 μm) showed a strong Cy5.5 dequenching effect in GFP-negative cells. This observation was corroborated by flow cytometry, sorting, and reverse transcription polymerase chain reaction analysis of tumor-isolated cells. Both GFP-positive (81% total) and GFP-negative (19% total) populations contained Cy5.5-positive cells. The GFP-negative cells were confirmed to be host mouse cells by the absence of rat cathepsin mRNA signal. The subfraction of GFPnegative cells (2.5-3.0%) had seven times higher NIRF intensity than the majority of GFP-positive or GFPnegative cells (372 and 55 AU, respectively). Highly NIRF-positive, FP-negative cells were CD45-and MAC3-positive. Our results indicate that: 1) intracellular proteolysis can be imaged in vivo at the cellular level using cathepsin-sensitive probes; 2) tumor-recruited cells of hematopoetic origin participate most actively in uptake and degradation of long-circulating macromolecular probes.

Expression of the immediate early response gene X-1 (IEX-1, IER3) is diminished significantly in hematopoietic stem cells in a subgroup of patients with early stage myelodysplastic syndromes, but it is not clear whether the deregulation contributes to the disease. The current study demonstrates increased apoptosis and a concomitant decrease in the number of hematopoietic stem cells lacking this early response gene. Null mutation of the gene also impeded platelet differentiation and shortened a lifespan of red blood cells. When bone marrow cells deficient in the gene were transplanted into wild-type mice, the deficient stem cells produced significantly fewer circulating platelets and red blood cells, despite their enhanced repopulation capability. Moreover, after exposure to a non-myeloablative dose of radiation, absence of the gene predisposed to thrombocytopenia, a significant decline in red blood cells, and dysplastic bone marrow morphology, typical characteristics of myelodysplastic syndromes. These findings highlight a previously unappreciated role for this early response gene in multiple differentiation steps within hematopoiesis, including thrombopoiesis, erythropoiesis and in the regulation of hematopoietic stem cell quiescence. The deficient mice offer a novel model for studying the initiation and progression of myelodysplastic syndromes as well as strategies to prevent this disorder.

Molecular expression on the vascular endothelium is critical in regulating the interaction of circulating cells with the blood vessel wall. Leukocytes as well as circulating cancer cells gain entry into tissue by interacting with adhesion molecules on the endothelial cells (EC). Molecular targets on the EC are increasingly being explored for tissue-specific delivery of therapeutic and imaging agents. Here we use in vivo immunofluorescence microscopy to visualize the endothelial molecular expression in the vasculature of live animals. High-resolution images are obtained by optical sectioning through the intact skin using in vivo confocal and multiphoton microscopy after in situ labeling of EC surface markers with fluorescent antibodies. Other vascular beds such as the bone marrow and ocular blood vessels can be imaged with little or no tissue manipulation. Live imaging is particularly useful for following the dynamic expression of inducible molecules such as E-selectin during an inflammatory response.

Background: The purpose of this study was to investigate temporal trends in gunshot wound (GSW)-associated orthopedic injuries among children, adolescents, and young adults in New York State, and to determine the impact of the onset of the COVID-19 pandemic on the incidence of these injuries. Methods: The New York Statewide Planning and Research Cooperative System (SPARCS) inpatient database was reviewed to identify patients ≤ 21 years of age who presented to a hospital with GSW-associated injuries from January 2016 to December 2020. Patient diagnosis codes were cross-referenced with the list of the International Classification of Diseases Version 10 Clinical Modification (ICD-10-CM) codes for orthopedic injuries to determine the incidence of GSW-associated orthopedic injuries among this cohort. The number of cases was cross-referenced with New York State census population estimates to calculate incidence per million. The geographic incidence was plotted over a map of New York State with sub-division based on facility Zone Improvement Plan (ZIP) codes. Poisson regression was used to compare the injury incidence in 2020 (pandemic onset) versus the preceding years (pre-pandemic). Results: Between 2016 and 2020, there were 548 inpatient admissions for GSW-associated orthopedic injuries, representing an incidence of 5.6 cases per million. Injury incidence decreased from 2016 to 2019, with an increase in 2020 representing almost 28% of the total cases identified. There was a statistically significant difference in the incidence rate ratio for 2020 compared to 2016–2019 (p < 0.001). The majority of patients were male (94%), African–American (73%), and covered by either Medicare (49%) or Managed Care (47%). Most cases were clustered around large metropolitan areas with low incidence in suburban and rural regions of the state. Conclusions: There was a two-fold increase in the incidence of GSW-associated orthopedic injuries among patients ≤ 21 years old in New York State during the onset of the COVID-19 pandemic.

There are many diseases and biological processes that involve circulating cells in the bloodstream, such as cancer metastasis, immunology, reproductive medicine, and stem cell therapies. This has driven significant interest in new technologies for the study of circulating cells in small animal research models and clinically. Most currently used methods require drawing and enriching blood samples from the body, but these suffer from a number of limitations. In contrast, ‘in vivo flow cytometry’ (IVFC) refers to set of technologies that allow study of cells directly in the bloodstream of the organism in vivo. In recent years the IVFC field has grown significantly and new techniques have been developed, including fluorescence microscopy, multi-photon, photo-acoustic, and diffuse fluorescence IVFC. In this paper we review recent technical advances in IVFC, with emphasis on instrumentation, contrast mechanisms, and detection sensitivity. We also describe key applications in biomedical research, including cancer research and immunology. Last, we discuss future directions for IVFC, as well as prospects for broader adoption by the biomedical research community and translation to humans clinically. [ABSTRACT FROM AUTHOR]

Background It remains uncertain which endothelial keratoplasty (EK) technique yields the best outcomes while maintaining safety, particularly in eyes with coexisting ocular conditions. Moreover, the impact of endothelial cell loss (ECL) on long-term graft survival requires further investigation. Adjuvant ripasudil, a rho kinase inhibitor, may address the challenge of ECL in corneal transplantation. This paper presents the protocol for the Descemet Endothelial Thickness Comparison Trial 1 (DETECT 1), a multicentre, outcome-masked, randomised, placebo-controlled, four-arm clinical trial.Methods A total of 160 eligible patients with endothelial dysfunction will be enrolled from five participating sites in the USA. The patients will be randomly assigned in a 2×2 factorial design to one of the following treatment groups: group 1—ultrathin Descemet stripping endothelial keratoplasty (UT-DSAEK) plus topical ripasudil 0.4%; group 2—UT-DSAEK plus topical placebo; group 3—Descemet membrane endothelial keratoplasty (DMEK) plus topical ripasudil 0.4% and group 4—DMEK plus topical placebo. Primary outcomes include the best spectacle-corrected visual acuity at 12 months and ECL at 12 months. Secondary outcomes include visual acuity at different time points, vision-related quality of life, endothelial cell morphology and cost-effectiveness.Results The study outcomes will be analysed using mixed effects linear regression models, taking into account the treatment arms and relevant covariates. Adverse events, including rebubble procedures, graft failure and graft rejection, will be documented and analysed using appropriate statistical methods.Conclusion DETECT I aims to provide evidence on the comparative effectiveness of UT-DSAEK and DMEK, as well as the potential benefits of adjuvant topical ripasudil in reducing ECL. The results of this trial will contribute to optimising corneal transplantation techniques and improving long-term graft survival, while also exploring the cost-effectiveness of these interventions. Dissemination of findings through peer-reviewed publications and national/international meetings will facilitate knowledge translation and guide clinical practice in the field of corneal transplantation.Ethics and dissemination A data and safety monitoring committee (DSMC) has been empaneled by the NEI.All study protocols will be subject to review and approval by WCG IRB as the single IRB of record.This study will comply with the National Institute of Health (NIH) Data Sharing Policy and Policy on the Dissemination of NIH-Funded Clinical Trial Information and the Clinical Trials Registration and Results Information Submission rule. Data from the trial will be made available on reasonable request.

Introduction: Ski jumping (SJ) and Nordic combined (NC) athletes are at higher risk for femoroacetabular impingement (FAI) compared to the general population. Objectives: The purpose of this study was to determine the relationship between force plate testing metrics, Functional Movement Screen (FMS) scores, and radiographic signs of FAI and SJ performance. Methods: Elite SJ and NC athletes were prospectively recruited. FMS testing was performed to assess muscular imbalance, instability, and immobility. Vertical jump testing was performed using a commercially available force plate device. Ski jump score and distance score were measured on a single jump for each athlete. Results: Twenty-one athletes (10 SJ, 11 NC) with competition scores were included. On FMS testing, most SJ (67%) and NC (73%) athletes demonstrated core instability. There were higher rates of hip instability and ankle stiffness among SJ athletes, although not statistically significant (P > .05). Ski jump distance score was significantly positively correlated with force plate vertical jump height, maximum acceleration, maximum velocity, concentric impulse, counter-movement depth, and flight time (P

Gauging stromal dissection depth is crucial to successfully perform deep anterior lamellar keratoplasty (DALK) surgery. Intraoperative optical coherence tomography (iOCT) offers a promising tool to aid DALK surgery but visualization of surgical maneuvers is impaired due to artifacts from metallic instruments. We describe a novel surgical technique utilizing suture-assisted iOCT guidance that facilitates clear visualization of corneal dissection planes during DALK. A stromal dissection tunnel is performed with a Fogla probe and its depth is subsequently identified by threading a 1 cm segment of 8-0 nylon into the tunnel. In contrast to the Fogla probe, the 8-0 nylon is conspicuously highlighted on iOCT. If the tunnel is too superficial, a separate, deeper stromal tunnel can be created and visualized again with the 8-0 nylon suture and iOCT. This iterative process facilitates a deep stromal dissection, increasing the probability of successful big-bubble formation and Descemet baring DALK surgery. This technique was utilized for a successful big-bubble DALK in a patient with severe keratoconus.

Intra-oral stents (including mouth-pieces and bite blocks) can be used to displace adjacent non-involved oral tissue and reduce radiation side effects from radiotherapy treatments for head-and-neck cancer. In this study, a modular and customisable 3D printed intra-oral stent was designed, fabricated and evaluated, to utilise the advantages of the 3D printing process without the interruption of clinical workflow associated with printing time. The stent design used a central mouth-opening and tongue-depressing main piece, with optional cheek displacement pieces in three different sizes, plus an anchor point for moulding silicone to fit individual patients’ teeth. A magnetic resonance imaging (MRI) study of one healthy participant demonstrated the tissue displacement effects of the stent, while providing a best-case indication of its comfort.

Myelin loss and axonal degeneration predominate in many neurological disorders; however, methods to visualize them simultaneously in live tissue are unavailable. We describe a new imaging strategy combining video rate reflectance and fluorescence confocal imaging with coherent anti-Stokes Raman scattering (CARS) microscopy tuned to CH2vibration of myelin lipids, applied in live tissue of animals with chronic experimental autoimmune encephalomyelitis (EAE). Our method allows monitoring over time of demyelination and neurodegeneration in brain slices with high spatial resolution and signal-to-noise ratio. Local areas of severe loss of lipid signal indicative of demyelination and loss of the reflectance signal from axons were seen in the corpus callosum and spinal cord of EAE animals. Even in myelinated areas of EAE mice, the intensity of myelin lipid signals is significantly reduced. Using heterozygous knock-in mice in which green fluorescent protein replaces the CX3CR1 coding sequence that labels central nervous system microglia, we find areas of activated microglia colocalized with areas of altered reflectance and CARS signals reflecting axonal injury and demyelination. Our data demonstrate the use of multimodal CARS microscopy for characterization of demyelinating and neurodegenerative pathology in a mouse model of multiple sclerosis, and further confirm the critical role of microglia in chronic inflammatory neurodegeneration. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM), the second most common hematological malignancy, initiates from a single site and spreads via circulation to multiple sites in the bone marrow (BM). Methods to track MM cells both in the BM and circulation would be useful for developing new therapeutic strategies to target MM cell spread. We describe the use of complementary optical techniques to track human MM cells expressing both bioluminescent and fluorescent reporters in a mouse xenograft model. Long-term tumor growth and response to therapy are monitored using bioluminescence imaging (BLI), while numbers of circulating tumor cells are detected by in-vivoflow cytometry. Intravital microscopy is used to detect early seeding of MM cells to the BM, as well as residual cancer cells that remain in the BM after the bulk of the tumor is eradicated following drug treatment. Thus, intravital microscopy provides a powerful, albeit invasive, means to study cellular processes in vivoat the very early stage of the disease process and at the very late stage of therapeutic intervention when the tumor burden is too small to be detected by other imaging methods. [ABSTRACT FROM AUTHOR]

Selective laser targeting of the retinal pigment epithelium (RPE) is an attractive method for treating RPE-associated disorders. We are developing a method for optically detecting intracellular microcavitation that can potentially serve as an immediate feedback of the treatment outcome. Thermal denaturation or intracellular cavitation can kill RPE cells during selective targeting. We examined the cell damage mechanism for laser pulse durations from 1 to 40 μsex vivo. Intracellular cavitation was detected as a transient increase in the backscattered treatment beam. Cavitation and cell death were correlated for individual cells after single-pulse irradiation. The threshold radiant exposures for cell death (ED50,d) and cavitation (ED50,c) increased with pulse duration and were approximately equal for pulses of up to 10 μs. For 20 μs, the ED50,d was about 10% lower than the ED50,c; the difference increased with 40-μs pulses. Cells were killed predominantly by cavitation (up to 10-μs pulses); probability of thermally induced cell death without cavitation gradually increases with pulse duration. Threshold measurements are discussed by modeling the temperature distribution around laser-heated melanosomes and the scattering function from the resulting cavitation. Detection of intracellular cavitation is a highly sensitive method that can potentially provide real-time assessment of RPE damage during selective laser targeting. [ABSTRACT FROM AUTHOR]

In this study, Hsu et al. show that inhibition of CDK7/12/13 attenuates maladaptive transcriptional activation in cultured cardiomyocytes and a mouse model of heart failure, suggesting that targeting the transcription machinery might be a therapeutic approach to treat heart failure with reduced ejection fraction.

The specific functional properties of a tissue are distributed amongst its component cell types. The various cells act coherently, as an ensemble, in order to execute a physiologic response. Modern approaches for identifying and dissecting novel physiologic mechanisms would benefit from an ability to identify specific cell types in live tissues that could then be imaged in real time. Current techniques require the use of fluorescent genetic reporters that are not only cumbersome, but which only allow the study of three or four cell types at a time. We report a non-invasive imaging modality that capitalizes on the endogenous autofluorescence signatures of the metabolic cofactors NAD(P)H and FAD. By marrying morphological characteristics with autofluorescence signatures, all seven of the airway epithelial cell types can be distinguished simultaneously in mouse tracheal explants in real time. Furthermore, we find that this methodology for direct cell type-specific identification avoids pitfalls associated with the use of ostensibly cell type-specific markers that are, in fact, altered by clinically relevant physiologic stimuli. Finally, we utilize this methodology to interrogate real-time physiology and identify dynamic secretory cell associated antigen passages (SAPs) that form in response to cholinergic stimulus. The identical process has been well documented in the intestine where the dynamic formation of SAPs and goblet cell associated antigen passages (GAPs) enable luminal antigen sampling. Airway secretory cells with SAPs are frequently juxtaposed to antigen presenting cells, suggesting that airway SAPs, like their intestinal counterparts, not only sample antigen but convey their cargo for immune cell processing.

Purpose: To describe a case of corneal decompensation in the setting of a Descemet's membrane detachment that developed following aborted Hydrus minimally invasive glaucoma surgery (MIGS) that was successfully treated with a Descemet's stripping only procedure. Observations: A 75 year-old female patient presented with symptomatic corneal decompensation following complicated Hydrus MIGS surgery. Ocular coherence tomography demonstrated an inferonasal Descemet's detachment with overlying edema. Specular microscopy revealed undetectable cells centrally but a peripheral cell density of 1446 cells/mm2. The Descemet's detachment did not respond to an intracameral air injection and a subsequent Descemet's stripping only procedure was performed. The corneal edema resolved by postoperative week 6. At postoperative month 2, best corrected visual acuity was 20/40 and specular microscopy demonstrated central cell density of 975 cells/mm2. Conclusions and Importance: Descemet's stripping only can be an effective treatment for some cases of corneal decompensation that occur in the setting of a Descemet's membrane detachment from complicated intraocular surgery, such as in this case with MIGS surgery.

Clinical application of somatic genome editing requires therapeutics that are generalizable to a broad range of patients. Targeted insertion of promoterless transgenes can ensure that edits are permanent and broadly applicable while minimizing risks of off-target integration. In the liver, the Albumin (Alb) locus is currently the only well-characterized site for promoterless transgene insertion. Here, we target the Apoa1 locus with adeno-associated viral (AAV) delivery of CRISPR-Cas9 and achieve rates of 6% to 16% of targeted hepatocytes, with no evidence of toxicity. We further show that the endogenous Apoa1 promoter can drive robust and sustained expression of therapeutic proteins, such as apolipoprotein E (APOE), dramatically reducing plasma lipids in a model of hypercholesterolemia. Finally, we demonstrate that Apoa1-targeted fumarylacetoacetate hydrolase (FAH) can correct and rescue the severe metabolic liver disease hereditary tyrosinemia type I. In summary, we identify and validate Apoa1 as a novel integration site that supports durable transgene expression in the liver for gene therapy applications.

Background and purpose: Radiopacifiers are introduced to bone cements to provide the appearance of bone in kilovoltage (kV) radiographic images. For higher energy megavoltage (MV) radiotherapy treatment beams, however, these radiopacifiers do not cause a bone-like perturbation of dose. This study therefore aimed to determine the impact of the barium-contrasted plastic-based cement materials on radiotherapy dose calculations. Materials and methods: The radiological properties of a physical sample of bone cement were characterised by computed tomography (CT) imaging and transmission measurements. Monte Carlo simulations of percentage depth-dose profiles were performed to determine the possible dose error for MV treatment beams. Dose differences were then investigated for clinical volumetric modulated radiotherapy treatment plans, with and without density overrides applied. Results: Differences of up to 7% were observed at the downstream interface of a 0.6 cm thick bone cement layer, compared to bone. Differences in planning target volume dose-volume metrics varied between −0.5% and 2.0%. Conclusion: Before planning radiotherapy treatments for patients who have undergone cranioplasty, every effort should be made to identify whether a radiopacified bone cement has been implanted. Density overrides should be applied to minimise dose calculation errors, whenever bone cement is used.

Abstract Residual knee instability and low rates of return to previous sport are major concerns after anterior cruciate ligament (ACL) reconstruction. To improve outcomes, surgical methods, such as the anatomical single-bundle technique or the double-bundle technique, were developed. However, these reconstruction techniques failed to adequately overcome these problems, and, therefore, new potential answers continue to be of great interest. Based on recent anatomical and biomechanical studies emphasizing the role of the anterolateral ligament (ALL) in rotational stability, novel surgical methods including ALL reconstruction and anterolateral tenodesis have been introduced with the possibility of resolving residual instability after ACL reconstruction. However, there is still little consensus on many aspects of the ALL, including: several anatomical issues, appropriate indications for ALL surgery, and the optimal surgical method and graft choice for reconstruction surgery. Therefore, further studies are necessary to advance our knowledge of the ALL and its contribution to knee stability.

Epidermal homeostasis requires long term stem cell function. Here, the authors apply transcriptional circuitry analysis based on integrated epigenomic profiling of primary human keratinocytes with high and low stem cell function to identify IRF2 as a negative regulator of stemness.

Prostate cancer progression may be enhanced by a high-fat diet. Here the authors show that a diet high in saturated fats enhance the MYC-driven transcriptional program, a feature that independently predicts prostate cancer progression and death.

Transcription factors are critical regulators of cell identity. Here, the authors use computational and functional genomic approaches to show an oncogenic role of PAX8 in renal cancer. Mechanistic dissection of PAX8 functions reveal its role in activating genes associated with metabolic pathways.

Purpose: To report two cases of severe acute corneal hydrops that were resolved by intracameral gas injection alone. Observations: Case 1 is a 27-year-old woman with bilateral severe keratoconus who developed sequential acute corneal hydrops in the right eye followed by the left eye that were each successfully treated using intracameral 20% sulfur hexafluoride gas injection. Case 2 is a 62-year-old man that developed a large fluid cleft beneath a pre-existing LASIK flap, which resolved with intracameral 20% sulfur hexafluoride gas injection without the need for corneal transplantation. Conclusions and importance: In acute corneal hydrops, intracameral gas injection to tamponade Descemet's membrane tears with decompression of stromal fluid can be an effective intervention to delay or avoid keratoplasty in individuals whose corneal hydrops does not improve with conventional medical management.

N/A

Purpose: To characterize the volume and variation in orthopedic consults and surgeries that took place during a period of social distancing and pandemic. Methods: All orthopedic consults and surgeries at an urban level 1 trauma center from 3/22/20-4/30/2020 were retrospectively reviewed (the social distancing period). Data from the same dates in 2019 were reviewed for comparison. Age, gender, Score for Trauma Triage in the Geriatric and Middle Aged (STTGMA) score and injury type were queried. Operating room data collected included: type of surgery performed, inpatient or outpatient status, and if the cases were categorized as elective, trauma or infectious cases. Results: Compared to 2019, there was a 48.3% decrease in consult volume in 2020. The 2020 population was significantly older (44.0 vs 52.6 years-old, p = 0.001) and more male (65% vs 35%, p = 0.021). There were 23 COVID positive patients, 10 of which died within the collection period. Consult distribution dramatically changed, with decreases in ankle fractures, distal radius fractures and proximal humerus fractures of 76.5%, 77.4% and 55.0%, respectively. However, there was no significant difference in volume of hip, tibial shaft and femoral shaft fractures (p > 0.05). In 2020, there was a 41.4% decrease in operating room volume, no elective cases were performed, and cases were primarily trauma related. Conclusions: During a period of pandemic and social distancing, the overall volume of orthopedic consults and surgeries significantly declined. However, hip fracture volume remained unchanged. Patients presenting with orthopedic injuries were older, and at higher risk for inpatient mortality.

Purpose: To describe a novel surgical technique to rescue a partially expulsed DMEK graft after insertion. Observations: We present a case of a 66-year-old woman with visually significant Fuchs endothelial dystrophy who underwent a DMEK surgery complicated by partial expulsion of the DMEK graft during insertion. To rescue the graft, MicroSurgical Technology (MST) forceps were inserted through a nasal paracentesis to grasp the DMEK graft and redirect it back into the anterior chamber. The surgical technique and postoperative outcomes are described. The partially expulsed graft was centered and attached successfully during surgery. Postoperatively, the patient required two rebubbling procedures, but achieved best-corrected visual acuity of 20/20 vision 1 month after surgery. Conclusions: and Importance: We describe an underreported complication and present a novel rescue technique for a partially expulsed DMEK graft that is effective and minimizes manipulation of the graft. Keywords: DMEK, Expulsed graft, Rescue technique

Purpose: To report the first case of Acanthamoeba keratitis treated with oral miltefosine in the United States. Observations: A 17-year-old female with a history of orthokeratology contact lens wear presented after five months of left eye pain, redness, and photophobia. She was previously treated with antivirals and topical corticosteroids for presumed herpetic disease. She was found to have a large central ring infiltrate and corneal cultures were positive for Acanthamoeba. The infection progressed despite hourly PHMB 0.02% and chlorhexidine 0.02%, and oral vorizonazole. The patient was started on oral miltefosine 50 mg 3 times per day. Following one week of treatment, repeat cultures were positive for Acanthamoeba and therefore, the concentration of chlorhexidine was increased from 0.02% to 0.06% and PHMB was changed to propamidine isetionate (Brolene 0.1%). There was definite clinical improvement after five weeks of treatment with oral miltefosine, topical chlorhexidine 0.06% and propamidine isetionate 0.1%. Conclusions and importance: Acanthamoeba keratitis is a challenging entity to treat and often associated with a poor prognosis. Oral miltefosine may offer additional therapeutic benefit in cases of refractory Acanthamoeba keratitis. Keywords: Miltefosine, Acanthamoeba keratitis, Amoebic keratitis

While the multiple endocrine neoplasia type 1 (MEN1) gene functions as a tumor suppressor in a variety of cancer types, we explored its oncogenic role in breast tumorigenesis. The MEN1 gene product menin is involved in H3K4 trimethylation and co-activates transcription. We integrated ChIP-seq and RNA-seq data to identify menin target genes. Our analysis revealed that menin-dependent target gene promoters display looping to distal enhancers that are bound by menin, FOXA1 and GATA3. In this fashion, MEN1 co-regulates a proliferative breast cancer-specific gene expression program in ER+ cells. In primary mammary cells, MEN1 exerts an anti-proliferative function by regulating a distinct expression signature. Our findings clarify the cell-type-specific functions of MEN1 and inform the development of menin-directed treatments for breast cancer.

Summary: The relationship between promoter proximal transcription factor-associated gene expression and super-enhancer-driven transcriptional programs are not well defined. However, their distinct genomic occupancy suggests a mechanism for specific and separable gene control. We explored the transcriptional and functional interrelationship between E2F transcription factors and BET transcriptional co-activators in multiple myeloma. We found that the transcription factor E2F1 and its heterodimerization partner DP1 represent a dependency in multiple myeloma cells. Global chromatin analysis reveals distinct regulatory axes for E2F and BETs, with E2F predominantly localized to active gene promoters of growth and/or proliferation genes and BETs disproportionately at enhancer-regulated tissue-specific genes. These two separate gene regulatory axes can be simultaneously targeted to impair the myeloma proliferative program, providing an important molecular mechanism for combination therapy. This study therefore suggests a sequestered cellular functional control that may be perturbed in cancer with potential for development of a promising therapeutic strategy. : Uncontrolled proliferation is a hallmark of tumorigenesis and is associated with perturbed transcriptomic profile. Fulciniti et al. explored the interrelationship between E2F transcription factors and BET transcriptional co-activators in multiple myeloma, reporting the existence of two distinct regulatory axes that can be synergistically targeted to impact myeloma growth and survival. Keywords: multiple myeloma, transcription factor, transcriptional regulation, super-enhancers

Summary: Down syndrome (DS, trisomy 21) is associated with developmental abnormalities and increased leukemia risk. To reconcile chromatin alterations with transcriptome changes, we performed paired exogenous spike-in normalized RNA and chromatin immunoprecipitation sequencing in DS models. Absolute normalization unmasks global amplification of gene expression associated with trisomy 21. Overexpression of the nucleosome binding protein HMGN1 (encoded on chr21q22) recapitulates transcriptional changes seen with triplication of a Down syndrome critical region on distal chromosome 21, and HMGN1 is necessary for B cell phenotypes in DS models. Absolute exogenous-normalized chromatin immunoprecipitation sequencing (ChIP-Rx) also reveals a global increase in histone H3K27 acetylation caused by HMGN1. Transcriptional amplification downstream of HMGN1 is enriched for stage-specific programs of B cells and B cell acute lymphoblastic leukemia, dependent on the developmental cellular context. These data offer a mechanistic explanation for DS transcriptional patterns and suggest that further study of HMGN1 and RNA amplification in diverse DS phenotypes is warranted. : How trisomy 21 contributes to Down syndrome phenotypes, including increased leukemia risk, is not well understood. Mowery et al. use per-cell normalization approaches to reveal global transcriptional amplification in Down syndrome models. HMGN1 overexpression is sufficient to induce these alterations and promotes lineage-associated transcriptional programs, signaling, and B cell progenitor phenotypes. Keywords: Down syndrome, leukemia, HMGN1, spike-in normalization, RNA sequencing, ChIP-Rx, transcriptional amplification, trisomy 21, Down syndrome critical region, DSCR, B cells

High-grade serous ovarian cancer is characterized by extensive copy number alterations, among which the amplification of MYC oncogene occurs in nearly half of tumors. We demonstrate that ovarian cancer cells highly depend on MYC for maintaining their oncogenic growth, indicating MYC as a therapeutic target for this difficult-to-treat malignancy. However, targeting MYC directly has proven difficult. We screen small molecules targeting transcriptional and epigenetic regulation, and find that THZ1 - a chemical inhibiting CDK7, CDK12, and CDK13 - markedly downregulates MYC. Notably, abolishing MYC expression cannot be achieved by targeting CDK7 alone, but requires the combined inhibition of CDK7, CDK12, and CDK13. In 11 patient-derived xenografts models derived from heavily pre-treated ovarian cancer patients, administration of THZ1 induces significant tumor growth inhibition with concurrent abrogation of MYC expression. Our study indicates that targeting these transcriptional CDKs with agents such as THZ1 may be an effective approach for MYC-dependent ovarian malignancies.

BRD4 governs pathological cardiac gene expression by binding acetylated chromatin, resulting in enhanced RNA polymerase II (Pol II) phosphorylation and transcription elongation. Here, we describe a signal-dependent mechanism for the regulation of BRD4 in cardiomyocytes. BRD4 expression is suppressed by microRNA-9 (miR-9), which targets the 3′ UTR of the Brd4 transcript. In response to stress stimuli, miR-9 is downregulated, leading to derepression of BRD4 and enrichment of BRD4 at long-range super-enhancers (SEs) associated with pathological cardiac genes. A miR-9 mimic represses stimulus-dependent targeting of BRD4 to SEs and blunts Pol II phosphorylation at proximal transcription start sites, without affecting BRD4 binding to SEs that control constitutively expressed cardiac genes. These findings suggest that dynamic enrichment of BRD4 at SEs genome-wide serves a crucial role in the control of stress-induced cardiac gene expression and define a miR-dependent signaling mechanism for the regulation of chromatin state and Pol II phosphorylation.