New-Onset Gout as an Independent Risk Factor for Returning to Dialysis After Kidney Transplantation.

BACKGROUND: The causal relationship between gout and renal transplant outcomes is difficult to assess due to multiple interacting covariates. This study sought to estimate the independent effect of new-onset gout on renal transplant outcomes using a methodology that accounted for these interactions. METHODS: This study analyzed data on patients in the US Renal Data System (USRDS) who received a primary kidney transplant between 2008 and 2015. The exposure was new-onset gout, and the primary endpoint was returning to dialysis >12 months postindex date (transplant date). A marginal structural model (MSM) was fitted to determine the relative risk of new-onset gout on return to dialysis. RESULTS: 18 525 kidney transplant recipients in the USRDS met study eligibility. One thousand three hundred ninety-nine (7.6%) patients developed new-onset gout, and 1420 (7.7%) returned to dialysis >12 months postindex. Adjusting for baseline and time-varying confounders via the MSM showed new-onset gout was associated with a 51% increased risk of return to (RR, 1.51; 95% CI, 1.03-2.20). CONCLUSIONS: This finding suggests that new onset gout after kidney transplantation could be a harbinger for poor renal outcomes, and to our knowledge is the first study of kidney transplant outcomes using a technique that accounted for the dynamic relationship between renal dysfunction and gout.

A Retrospective Cohort Study of the Effect of Gout on Mortality Among Patients with a History of Kidney Transplantation.

BACKGROUND Kidney transplantation is associated with increased prevalence of gout. However, evidence of the effect of gout on long-term kidney transplantation outcomes is mixed. This study examined mortality risk among patients with a history of kidney transplantation with vs. without gout. MATERIAL AND METHODS A retrospective study was conducted using Medicare Fee-for-Service administrative claims of patients with a history of kidney transplantation. Cox proportional hazards models determined the effect of gout on all-cause mortality, controlling for confounders, including comorbid mortality risk, via the Charlson Comorbidity Index. Because the relationships between gout and components of the Charlson Comorbidity Index are also debated, 3 different model assumptions were used: 1) gout shares a common cause with these comorbidities, 2) gout is upstream of these comorbidities, 3) the effect of gout on mortality is modified by these comorbidities. RESULTS Gout increased the risk of all-cause mortality in the unadjusted model (hazard ratio: 1.44, 95% CI 1.27-1.63) and after adjustment for demographics and transplant vintage (hazard ratio: 1.16, 95% CI 1.02-1.32). Gout was not a significant risk after adjustment for baseline Charlson Comorbidity Index (hazard ratio: 1.03, 95% CI 0.90-1.17). Gout was associated with greater mortality among patients without baseline comorbidities (Charlson Comorbidity Index=0; hazard ratio: 3.48, 95% CI 1.27-9.57) in the stratified model. CONCLUSIONS Among patients with a history of kidney transplantation, gout did not have an independent effect on all-cause mortality. However, gout was a predictor of mortality among patients with no comorbidities, suggesting that gout is an early warning sign of poor health in kidney transplantation patients.

Immunosuppressant Use and Gout in the Prevalent Solid Organ Transplantation Population.

INTRODUCTION: Gout is a common comorbidity among solid organ transplantation patients and is usually attributed to the use of cyclosporine. This study aims to evaluate the prevalence of gout among solid organ transplantation patients to determine the prevalence in the tacrolimus era. RESEARCH QUESTIONS: To what degree is cyclosporine still used among prevalent solid organ transplantation patients? How prevalent is gout in the solid organ transplantation population not being treated by cyclosporine? METHODS: Immunosuppressant regimens and gout prevalence among prevalent solid organ transplantation patients were assessed using retrospective claims data for a representative sample of commercially insured patients. For comparison to the prevalent solid organ transplantation population, immunosuppressant use at time of transplantation was compiled from published reports. RESULTS: Between 2012 and 2016, the use of cyclosporine declined while use of tacrolimus increased, with greater cyclosporine use among prevalent versus incident solid organ transplantation patients. The prevalence of gout was 18.3%, 9.3%, and 9.1% for solid organ transplantation patients on cyclosporine, tacrolimus, and neither, respectively. Among all solid organ transplantation patients with gout, 66.6% and 21.5% were on tacrolimus versus cyclosporine. The prevalence of gout among noncyclosporine solid organ transplantation patients was significantly higher than in the general population without solid organ transplantation. DISCUSSION: Despite declining cyclosporine use, gout prevalence remains high, with the majority of patients with gout receiving tacrolimus rather than cyclosporine. In summary, gout remains a frequent comorbidity of solid organ transplantation.

Prevalence of Gout in the Surviving United States Solid Organ Transplantation Population.

PURPOSE: Although incidence and survival are frequent topics within the solid organ transplantation (SOT) literature, the size of the surviving SOT population is not well known. Existing studies of gout in patients with SOT have focused on the incident SOT population. This analysis was performed to characterize the prevalent SOT population and the prevalence of gout within it. METHODS: This study includes the 2017 United States (US) population size of recipients of kidney, heart, liver, and lung transplants that was estimated by combining primary transplant recipient cohort sizes (1988-2017) with previously published survival rates for each annual cohort's time since transplantation (0-29 years). Gout among prevalent patients with SOT was assessed using Medicare and commercial claims. RESULTS: A total of 637,231 US patients received a primary kidney (393,953), liver (142,186), heart (66,637), or lung (34,455) transplant between 1988 and 2017. An estimated 356,000 (55.8%) recipients were alive in 2017 (233,000 kidney; 78,700 liver; 29,300 heart; 14,700 lung). Gout was identified in 11% of prevalent patients with SOT in 2016. Higher rates of gout were seen in recipients of kidney (13.1%) and heart (12.7%) compared to recipients of liver (6.7%) and lung (5.6%) (P < .0001 in both datasets). Active diagnosed gout prevalence in the US population without a SOT history was 1.1% in 2016. CONCLUSIONS: Hundreds of thousands of US patients are living with a transplanted organ today and these numbers are likely to increase. In patients with SOT, gout is a frequent comorbidity of which physicians should be aware. This study suggests a markedly higher rate of gout among transplant recipients compared to the general US population.

Gout Severity in Recipients of Kidney Transplant.

PURPOSE: This retrospective analysis of medical chart data was performed to compare severity and treatment of gout in patients with or without a history of kidney transplantation (KT). METHODS: Via an online survey, a panel of board-certified US nephrologists (N = 104) provided the following deidentified chart data for their 3 most recent patients with gout: age, sex, serum uric acid, numbers of swollen or tender joints, visible tophi, gout flare events (prior 12 months), gout drug treatment history, and KT history. The presence of "severe, uncontrolled gout" was defined as: serum uric acid ≥ 7.0 mg/dL, ≥1 tophi and ≥2 flares in the last 12 months, and history of xanthine oxidase inhibitor treatment. RESULTS: Twenty-five out of 312 (8.0%) gout patients had a history of KT. Univariate analysis found that patients with gout and history of kidney transplants had: greater prevalence of severe uncontrolled gout (27% vs 8%, P = .007) and tophi (36% vs 17%, P = .030), and higher rates of failure or physician perceived contraindication to allopurinol (44% vs 23%, P = .028). CONCLUSION: This study provides preliminary evidence that gout in patients with history of KT is more severe and poses greater challenges to pharmacologic management. Although gout has been linked to worse outcomes among kidney recipients in the literature, there are presently no publications on gout severity among patients with KT in comparison to other patients with gout. Further investigation of disease severity and appropriate, effective treatment options in recipients of kidney transplant with a diagnosis of gout, especially prior to the transplant, is warranted.

Author Correction: Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening.

In the published version of this paper, Step 64 of the Procedure reads, "Refer to Steps 37-39 for NGS analysis of the sgRNA distribution." This step should refer the reader to Steps 35-39. This text has not been corrected in the original paper.

Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening.

Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom- or ready-made guide RNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified by the screen, we further describe strategies for confirming the screening phenotype, as well as genetic perturbation, through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 9-15 weeks, followed by 4-5 weeks of validation.

Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex.

Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology.

Optical control of mammalian endogenous transcription and epigenetic states.

The dynamic nature of gene expression enables cellular programming, homeostasis and environmental adaptation in living systems. Dissection of causal gene functions in cellular and organismal processes therefore necessitates approaches that enable spatially and temporally precise modulation of gene expression. Recently, a variety of microbial and plant-derived light-sensitive proteins have been engineered as optogenetic actuators, enabling high-precision spatiotemporal control of many cellular functions. However, versatile and robust technologies that enable optical modulation of transcription in the mammalian endogenous genome remain elusive. Here we describe the development of light-inducible transcriptional effectors (LITEs), an optogenetic two-hybrid system integrating the customizable TALE DNA-binding domain with the light-sensitive cryptochrome 2 protein and its interacting partner CIB1 from Arabidopsis thaliana. LITEs do not require additional exogenous chemical cofactors, are easily customized to target many endogenous genomic loci, and can be activated within minutes with reversibility. LITEs can be packaged into viral vectors and genetically targeted to probe specific cell populations. We have applied this system in primary mouse neurons, as well as in the brain of freely behaving mice in vivo to mediate reversible modulation of mammalian endogenous gene expression as well as targeted epigenetic chromatin modifications. The LITE system establishes a novel mode of optogenetic control of endogenous cellular processes and enables direct testing of the causal roles of genetic and epigenetic regulation in normal biological processes and disease states.

Muscle on a chip: in vitro contractility assays for smooth and striated muscle.

INTRODUCTION: To evaluate the viability of a muscle tissue, it is essential to measure the tissue's contractile performance as well as to control its structure. Accurate contractility data can aid in development of more effective and safer drugs. This can be accomplished with a robust in vitro contractility assay applicable to various types of muscle tissue. METHODS: The devices developed in this work were based on the muscular thin film (MTF) technology, in which an elastic film is manufactured with a 2D engineered muscle tissue on one side. The tissue template is made by patterning extracellular matrix with microcontact printing. When muscle cells are seeded on the film, they self-organize with respect to the geometric cues in the matrix to form a tissue. RESULTS: Several assays based on the "MTF on a chip" technology are demonstrated. One such assay incorporates the contractility assay with striated muscle into a fluidic channel. Another assay platform incorporates the MTFs in a multi-well plate, which is compatible with automated data collection and analysis. Finally, we demonstrate the possibility of analyzing contractility of both striated and smooth muscle simultaneously on the same chip. DISCUSSION: In this work, we assembled an ensemble of contractility assays for striated and smooth muscle based on muscular thin films. Our results suggest an improvement over current methods and an alternative to isolated tissue preparations. Our technology is amenable to both primary harvests cells and cell lines, as well as both human and animal tissues.

Nanowired three-dimensional cardiac patches.

Engineered cardiac patches for treating damaged heart tissues after a heart attack are normally produced by seeding heart cells within three-dimensional porous biomaterial scaffolds. These biomaterials, which are usually made of either biological polymers such as alginate or synthetic polymers such as poly(lactic acid) (PLA), help cells organize into functioning tissues, but poor conductivity of these materials limits the ability of the patch to contract strongly as a unit. Here, we show that incorporating gold nanowires within alginate scaffolds can bridge the electrically resistant pore walls of alginate and improve electrical communication between adjacent cardiac cells. Tissues grown on these composite matrices were thicker and better aligned than those grown on pristine alginate and when electrically stimulated, the cells in these tissues contracted synchronously. Furthermore, higher levels of the proteins involved in muscle contraction and electrical coupling are detected in the composite matrices. It is expected that the integration of conducting nanowires within three-dimensional scaffolds may improve the therapeutic value of current cardiac patches.

Blast-induced phenotypic switching in cerebral vasospasm.

Vasospasm of the cerebrovasculature is a common manifestation of blast-induced traumatic brain injury (bTBI) reported among combat casualties in the conflicts in Afghanistan and Iraq. Cerebral vasospasm occurs more frequently, and with earlier onset, in bTBI patients than in patients with other TBI injury modes, such as blunt force trauma. Though vasospasm is usually associated with the presence of subarachnoid hemorrhage (SAH), SAH is not required for vasospasm in bTBI, which suggests that the unique mechanics of blast injury could potentiate vasospasm onset, accounting for the increased incidence. Here, using theoretical and in vitro models, we show that a single rapid mechanical insult can induce vascular hypercontractility and remodeling, indicative of vasospasm initiation. We employed high-velocity stretching of engineered arterial lamellae to simulate the mechanical forces of a blast pulse on the vasculature. An hour after a simulated blast, injured tissues displayed altered intracellular calcium dynamics leading to hypersensitivity to contractile stimulus with endothelin-1. One day after simulated blast, tissues exhibited blast force dependent prolonged hypercontraction and vascular smooth muscle phenotype switching, indicative of remodeling. These results suggest that an acute, blast-like injury is sufficient to induce a hypercontraction-induced genetic switch that potentiates vascular remodeling, and cerebral vasospasm, in bTBI patients.

Stimuli-responsive microwells for formation and retrieval of cell aggregates.

Generating cell aggregates is beneficial for various applications ranging from biotechnology to regenerative therapies. Previously, poly(ethylene glycol) (PEG) microwells have been demonstrated as a potentially useful method for generating controlled-size cell aggregates. In addition to controlling cell aggregate size and homogeneity, the ability to confine cell aggregates on glass adhesive substrates and subsequently retrieve aggregates from microwells for further experimentation and analysis could be beneficial for various applications. However, it is often difficult to retrieve cell aggregates from these microwells without the use of digestive enzymes. This study describes the stable formation of cell aggregates in responsive microwells with adhesive substrates and their further retrieval in a temperature dependent manner by exploiting the stimuli responsiveness of these microwells. The responsive polymer structure of the arrays can be used to thermally regulate the microwell diameters causing a mechanical force on the aggregates, subsequently facilitating the retrieval of cell aggregates from the microwells with high efficiency compared to PEG arrays. This approach can be potentially integrated into high-throughput systems and may become a versatile tool for various applications that require aggregate formation and experimentation, such as tissue engineering, drug discovery, and stem cell biology.

Microporous cell-laden hydrogels for engineered tissue constructs.

In this article, we describe an approach to generate microporous cell-laden hydrogels for fabricating biomimetic tissue engineered constructs. Micropores at different length scales were fabricated in cell-laden hydrogels by micromolding fluidic channels and leaching sucrose crystals. Microengineered channels were created within cell-laden hydrogel precursors containing agarose solution mixed with sucrose crystals. The rapid cooling of the agarose solution was used to gel the solution and form micropores in place of the sucrose crystals. The sucrose leaching process generated homogeneously distributed micropores within the gels, while enabling the direct immobilization of cells within the gels. We also characterized the physical, mechanical, and biological properties (i.e., microporosity, diffusivity, and cell viability) of cell-laden agarose gels as a function of engineered porosity. The microporosity was controlled from 0% to 40% and the diffusivity of molecules in the porous agarose gels increased as compared to controls. Furthermore, the viability of human hepatic carcinoma cells that were cultured in microporous agarose gels corresponded to the diffusion profile generated away from the microchannels. Based on their enhanced diffusive properties, microporous cell-laden hydrogels containing a microengineered fluidic channel can be a useful tool for generating tissue structures for regenerative medicine and drug discovery applications.

Cell confinement in patterned nanoliter droplets in a microwell array by wiping.

Cell patterning is useful for a variety of biological applications such as tissue engineering and drug discovery. In particular, the ability to localize cells within distinct fluids is beneficial for a variety of applications ranging from microencapsulation to high-throughput analysis. However, despite much progress, cell immobilization and maintenance within patterned microscale droplets remains a challenge. In particular, no method currently exists to rapidly seed cells into microwell arrays in a controllable and reliable manner. In this study, we present a simple wiping technique to localize cells within arrays of polymeric microwells. This robust method produces cell seeding densities that vary consistently with microwell geometry and cell concentration. Moreover, we develop a simple theoretical model to accurately predict cell seeding density and seeding efficiency in terms of the design parameters of the microwell array and the cell density. This short-term cell patterning approach is an enabling tool to develop new high-throughput screening technologies that utilize microwell arrays containing cells for screening applications.

Mechanically robust and bioadhesive collagen and photocrosslinkable hyaluronic acid semi-interpenetrating networks.

In this work, we present a class of hydrogels that leverage the favorable properties of the photo-cross-linkable hyaluronic acid (HA) and semi-interpenetrating collagen components. The mechanical properties of the semi-interpenetrating-network (semi-IPN) hydrogels far surpass those achievable with collagen gels or collagen gel-based semi-IPNs. Furthermore, the inclusion of the semi-interpenetrating collagen chains provides a synergistic mechanical improvement over unmodified HA hydrogels. Collagen-HA semi-IPNs supported fibroblast adhesion and proliferation and were shown to be suitable for cell encapsulation at high levels of cell viability. To demonstrate the utility of the semi-IPNs as a microscale tissue engineering material, cell-laden microstructures and microchannels were fabricated using soft lithographic techniques. Given their enhanced mechanical and biomimetic properties, we anticipate that these materials will be of value in tissue engineering and three-dimensional cell culture applications.