Synthetic control of mammalian-cell motility by engineering chemotaxis to an orthogonal bioinert chemical signal

Directed migration of diverse cell types plays a critical role in biological processes ranging from development and morphogenesis to immune response, wound healing, and regeneration. However, techniques to direct, manipulate, and study cell migration in vitro and in vivo in a specific and facile manner are currently limited. We conceived of a strategy to achieve direct control over cell migration to arbitrary user-defined locations, independent of native chemotaxis receptors. Here, we show that genetic modification of cells with an engineered G protein-coupled receptor allows us to redirect their migration to a bioinert drug-like small molecule, clozapine-N-oxide (CNO). The engineered receptor and small-molecule ligand form an orthogonal pair: The receptor does not respond to native ligands, and the inert drug does not bind to native cells. CNO-responsive migration can be engineered into a variety of cell types, including neutrophils, T lymphocytes, keratinocytes, and endothelial cells. The engineered cells migrate up a gradient of the drug CNO and transmigrate through endothelial monolayers. Finally, we demonstrate that T lymphocytes modified with the engineered receptor can specifically migrate in vivo to CNO-releasing beads implanted in a live mouse. This technology provides a generalizable genetic tool to systematically perturb and control cell migration both in vitro and in vivo. In the future, this type of migration control could be a valuable module for engineering therapeutic cellular devices.The ability of many cell types to migrate long distances within the body and specifically localize to target sites of action is critical for their proper function. For example, immune cells rapidly home to sites of infection, concentrating their powerful cytotoxic and proinflammatory activities for maximum efficacy while limiting damage to healthy tissue. In morphogenesis, cells undergo a complex stereotyped process involving migration as well as proliferation, differentiation, and programmed cell death to produce fully developed multicellular structures. In wound healing and regenerative processes, stem and progenitor cells home to injured tissues from nearby sites—as well as from distant locations including the bone marrow—to provide a stream of new cells to replenish and provide trophic support to old and damaged cells.Cell migration is also an important factor to consider in the use of cells as therapeutic agents. The use of cells for the treatment of a growing array of diseases including cancer, autoimmunity, and chronic wounds is currently being explored (1–6). The appropriate and efficient localization of therapeutic cells to sites of disease has been identified as an important factor for successful cell-based therapy (7–17). However, preclinical studies and clinical trials to date have shown that the homing to sites of disease of many cell types commonly used as therapeutics is frequently impaired or limited, especially after ex vivo expansion of cells in culture (7, 12, 18, 19).The ability to redirect the migration of cells to any user-specified location in the body would be a powerful enabling technology for basic research as well as for future applications, but there are currently few easily generalizable strategies to accomplish this goal. We conceived of an approach to direct cellular homing to small molecules by expressing, in motile cells, engineered G protein-coupled receptors (GPCRs) called receptors activated solely by a synthetic ligand (RASSLs) (20, 21).RASSLs are engineered to be unresponsive to endogenous ligands but can be activated by pharmacologically inert orthogonal small molecules (Fig. 1A). Versions of these receptors exist for the three major GPCR signaling pathways (Gαs-, Gαi-, and Gαq-coupled receptors), and the design of a new arrestin-biased variant has recently been reported (21, 22). Because GPCRs control many important physiological functions, including cell migration, we hypothesized that, by expressing these engineered receptors in motile cells, we could develop a general strategy for establishing user control over cell homing (Fig. 1B). Here, we use a family of second-generation RASSLs, known as designer receptors exclusively activated by a designer drug (DREADDs), that are activated only by the small molecule clozapine-N-oxide (CNO), an inert metabolite of the FDA-approved antipsychotic drug clozapine (Fig. S1) (20). CNO is highly bioavailable in rodents and humans, lacks affinity for any known receptors, channels, and transporters, and does not cause any appreciable physiological effects when systemically administered in normal mice (20, 23, 24).Open in a separate windowFig. 1.Engineered Gαi-coupled GPCRs Di3 and Di mediate cytoskeletal changes and chemotaxis of HL-60 neutrophils in response to CNO. (A) RASSLs are engineered GPCRs that interact orthogonally with a bioinert small-molecule drug. Natural ligands do not interact with the engineered receptors, and the bioinert drug that activates the engineered receptors does not interact with native receptors. (B) We tested whether certain second-generation RASSLs known as DREADDs could mediate cell motility. (C) Changes in electrical impedance that result from cell spreading in response to drug or ligand are detected by an electrode array. HL-60 neutrophils transiently transfected to express engineered GPCRs were plated on fibronectin-coated impedance assay plates and stimulated with vehicle control, 100 nM fMLP (positive control chemoattractant) or 100 nM CNO. All cells responded to fMLP whereas only Di3- or Di-expressing cells responded to CNO. Mean ± SEM for n = 3 replicates is shown. (D) Cell migration of HL-60 neutrophils transiently transfected with engineered GPCRs was quantitated in a porous transwell Boyden-chamber assay. All cells migrated in response to fMLP whereas only Di3- or Di-expressing cells migrated in response to CNO. Drug concentrations used: 100 nM CNO, 100 nM fMLP. Mean ± SEM for n = 3 replicates is shown. (E) Polarization and cell migration in neutrophils involves Rac and PI3K activation. Di-expressing HL-60 neutrophils were treated with 100 nM fMLP or 100 nM CNO before immunoblotting for phosphorylated Akt and phosphorylated PAK as readouts for PI3K and Rac activity, respectively. Peak levels of phospho-Akt and phospho-PAK are shown for each condition. Both were increased by CNO stimulation in Di cells but not in control cells (P < 0.01 by Student t test). Stimulation with fMLP increased phospho-Akt and phospho-PAK levels in both Di and control cells (P < 0.01 by Student t test), but Di cells showed higher peak levels of phospho-Akt than did control cells (P < 0.01 by Student t test). Three (for CNO) or four (for fMLP) independent experiments were performed and mean ± SEM are shown.     

Total Hip Arthroplasty Using Large-Diameter Metal-On-Metal Articulation in Patients With Neuromuscular Weakness

The purpose of this prospective study was to evaluate the clinical and functional outcomes of THA using large-diameter metal-on-metal articulation in patients with neuromuscular weakness. Nineteen consecutive patients (19 hips) with neuromuscular weakness and displaced femoral neck fractures were enrolled. Functional improvement and recovery, radiological evaluation of THA and surgical morbidity were assessed. Mean Harris hip and WOMAC scores at final follow-up were 81.0 and 42.9, respectively. At final follow-up, no dislocation, metal hypersensitivity, or osteolysis was observed and no patient required revision of THA. The findings of this study indicate that the functional results of THA using large-diameter metal-on-metal articulation in patients with neuromuscular weakness can produce satisfactory outcomes with early functional recovery and a low dislocation rate.     

Familial interactions and physical,lifestyle, and dietary factors to affect bone mineral density of children in the KNHANES 2009–2010

We examined familial bone mineral density (BMD) interactions between parents and children and lifestyle factors affecting BMD in the Korean general population of children under 20 and parents under 50 years of age. This cross-sectional study included 2,453 participants (667 daughters, 705 sons, 719 mothers, and 362 fathers) in the 2009–2010 Korean National Health and Nutrition Examination Survey. We calculated prevalence ratios and 95 % confidence intervals for BMD values of whole femur, femur neck, lumbar spine, and whole body excluding the head being in the low tertile in adolescents according to parental BMD tertile after adjusting for physical, lifestyle, and dietary factors. For daughters and sons, there were significant differences in BMD at the four bone sites according to age group, body fat percentage, regular walking and exercise, and milk consumption compared to the reference value for each classification category. Surprisingly, there were no differences in BMD according to serum 25-OH-D levels. Birth order affected BMD of only whole body except head, but its impact was less than that of lifestyle factors. The mean differences in BMD between daughters and sons in the first and third parental BMD tertiles were statistically significant. Notably, the prevalence ratio of whole body without head BMD being in the low tertile increased eight and ten-folds in adolescent daughters and sons, respectively, when parents were in the low BMD tertile. In specific bone regions, parental BMD had a greater effect on total femur in daughters but in the lumbar spine in sons. In conclusion, parental BMD positively influences BMD in daughters and sons after adjustment for environmental parameters. This suggests that the children from parents with low BMD need to make an extra effort to increase BMD through dietary and lifestyle changes.     

Construct validity of individual and summary performance metrics associated with a computer-based laparoscopic simulator

Background

Computer-based surgical simulators capture a multitude of metrics based on different aspects of performance, such as speed, accuracy, and movement efficiency. However, without rigorous assessment, it may be unclear whether all, some, or none of these metrics actually reflect technical skill, which can compromise educational efforts on these simulators. We assessed the construct validity of individual performance metrics on the LapVR simulator (Immersion Medical, San Jose, CA, USA) and used these data to create task-specific summary metrics.

Methods

Medical students with no prior laparoscopic experience (novices, N = 12), junior surgical residents with some laparoscopic experience (intermediates, N = 12), and experienced surgeons (experts, N = 11) all completed three repetitions of four LapVR simulator tasks. The tasks included three basic skills (peg transfer, cutting, clipping) and one procedural skill (adhesiolysis).

Results

We selected 36 individual metrics on the four tasks that assessed six different aspects of performance, including speed, motion path length, respect for tissue, accuracy, task-specific errors, and successful task completion. Four of seven individual metrics assessed for peg transfer, six of ten metrics for cutting, four of nine metrics for clipping, and three of ten metrics for adhesiolysis discriminated between experience levels. Time and motion path length were significant on all four tasks. We used the validated individual metrics to create summary equations for each task, which successfully distinguished between the different experience levels.

Conclusion

Educators should maintain some skepticism when reviewing the plethora of metrics captured by computer-based simulators, as some but not all are valid. We showed the construct validity of a limited number of individual metrics and developed summary metrics for the LapVR. The summary metrics provide a succinct way of assessing skill with a single metric for each task, but require further validation.     

Clinical outcomes of second-look endoscopy after gastric endoscopic submucosal dissection: predictive factors with high risks of bleeding

Background

A second-look endoscopy is routinely performed after endoscopic submucosal dissection (ESD) in many institutions, although the need is questionable. Additional hemostatic procedures might be necessary for the post-ESD ulcer with a high risk of bleeding. We investigated the predictive factors for post-ESD ulcers with a high risk of bleeding.

Methods

Second-look endoscopy was performed on the day following ESD. The post-ESD ulcers were categorized into two risk groups according to the Forrest classification: high-risk ulcer stigma (type I and IIa) and low-risk ulcer stigma. We analyzed the risk factors associated with high-risk ulcer stigma and late delayed bleeding.

Results

During the study period, 616 ESD procedures were performed. Second-look endoscopy revealed that the incidence of high-risk ulcer stigma post-ESD was 15.1 %. Early and late delayed bleeding rates were 3.7 and 1.9 %, respectively. Multivariate analysis revealed that submucosal fibrosis and nausea were significantly related to high-risk ulcer stigma after ESD. Multivariate analysis revealed that surface erosion, location of the lesion, and high-risk ulcer stigma identified by second-look endoscopy were significantly associated with late delayed bleeding.

Conclusions

The effective use of selective second-look endoscopy will help limit unnecessary procedures. Submucosal fibrosis and nausea were risk factors associated with high-risk ulcer stigma after ESD.     

Cross‐reactivity between decellularized porcine corneal lamellae for corneal xenobridging and subsequent corneal allotransplants

The purpose of this study is to investigate cross‐reactivity between hypertonic saline‐treated decellularized porcine corneal lamellae for corneal xenobridging and subsequent corneal allotransplants. Five Chinese rhesus macaques, which had undergone anterior partial thickness corneal transplantation using hypertonic saline‐treated decellularized porcine corneal lamellae in preceding experiments, were used as recipients for subsequent full‐thickness corneal allografts. To determine whether sensitization of recipients to xenoantigens leads to cross‐reactivity against alloantigens, we compared; (i) allogeneic one‐way mixed lymphocyte reaction (MLR) of peripheral blood mononuclear cells (PBMCs) from xeno‐sensitized recipients with that of PBMCs from naïve rhesus macaques, and (ii) amounts of IgG antibodies that bound to the PBMCs of a rhesus panel (five monkeys) before and after xeno‐sensitization. Graft survival and immunologic profiles including memory T‐cell subsets and donor rhesus‐specific antibodies were also evaluated. No hyperacute or acute rejection was observed within a month of subsequent allotransplantation in any recipient. Alloreactivity by MLR was not different between xeno‐sensitized rhesus recipients and naïve rhesus monkeys. Panel‐reactive IgG antibodies were unchanged after xeno‐sensitization, and no change in donor rhesus‐specific antibodies was observed in any recipient. No significant changes in memory T‐cell subsets were observed during the early post‐operative period in any recipient. Decellularized porcine corneal lamellae may not increase cross‐reactivity to alloantigens, and thus, porcine corneal lamellae may be used as a bridge to subsequent corneal allografting.     

Determination of in vitro susceptibility of ocular Fusarium spp. isolates from keratitis cases and comparison of Clinical and Laboratory Standards Institute M38-A2 and E test methods

We evaluated the susceptibility of 85 Fusarium spp. isolates from cases of fungal keratitis with 8 antifungal drugs using the standard Clinical and Laboratory Standards Institute broth microdilution and E test methods. Members of the Fusarium solani species complex showed consistently higher MICs to the triazole drugs itraconazole, voriconazole, and posaconazole than did members of other species complexes (Fusarium oxysporum and other minor species). High MICs to amphotericin B, natamycin, and echinocandins were consistently obtained with no discrimination based on species or method. Further work is required to determine any potential correlation between MIC and clinical outcome in keratitis.     

Isolated cricoid fracture associated with blunt neck trauma

A 32-year-old woman without a remarkable history presented at the emergency department with strangulation of the neck. CT scans of the neck revealed a displaced cricoid fracture. Six days after admission to hospital, hoarseness and dyspnoea disappeared. On the 10th day, the patient was discharged without complications. The traditional treatment guidelines for laryngeal trauma have recommended an early surgical intervention after immediate tracheotomy in cases of displaced fractures of the cricoid cartilage. The patient could be treated successfully through continuous monitoring of airway obstruction without surgical management.