HIV-1 Neutralizing Antibodies with Limited Hypermutation from an Infant.

HIV-1 broadly neutralizing antibodies (bnAbs) develop in a subset of infected adults and exhibit high levels of somatic hypermutation (SHM) due to years of affinity maturation. There is no precedent for eliciting highly mutated antibodies by vaccination, nor is it practical to wait years for a desired response. Infants develop broad responses early, which may suggest a more direct path to generating bnAbs. Here, we isolated ten neutralizing antibodies (nAbs) contributing to plasma breadth of an infant at ∼1 year post-infection, including one with cross-clade breadth. The nAbs bind to envelope trimer from the transmitted virus, suggesting that this interaction may have initiated development of the infant nAbs. The infant cross-clade bnAb targets the N332 supersite on envelope but, unlike adult bnAbs targeting this site, lacks indels and has low SHM. The identification of this infant bnAb illustrates that HIV-1-specific neutralization breadth can develop without prolonged affinity maturation and extensive SHM.

An atlas of healthy and injured cell states and niches in the human kidney.

Understanding kidney disease relies on defining the complexity of cell types and states, their associated molecular profiles and interactions within tissue neighbourhoods1. Here we applied multiple single-cell and single-nucleus assays (>400,000 nuclei or cells) and spatial imaging technologies to a broad spectrum of healthy reference kidneys (45 donors) and diseased kidneys (48 patients). This has provided a high-resolution cellular atlas of 51 main cell types, which include rare and previously undescribed cell populations. The multi-omic approach provides detailed transcriptomic profiles, regulatory factors and spatial localizations spanning the entire kidney. We also define 28 cellular states across nephron segments and interstitium that were altered in kidney injury, encompassing cycling, adaptive (successful or maladaptive repair), transitioning and degenerative states. Molecular signatures permitted the localization of these states within injury neighbourhoods using spatial transcriptomics, while large-scale 3D imaging analysis (around 1.2 million neighbourhoods) provided corresponding linkages to active immune responses. These analyses defined biological pathways that are relevant to injury time-course and niches, including signatures underlying epithelial repair that predicted maladaptive states associated with a decline in kidney function. This integrated multimodal spatial cell atlas of healthy and diseased human kidneys represents a comprehensive benchmark of cellular states, neighbourhoods, outcome-associated signatures and publicly available interactive visualizations.

Minimizing habitat conflicts in meeting net-zero energy targets in the western United States.

The scale and pace of energy infrastructure development required to achieve net-zero greenhouse gas (GHG) emissions are unprecedented, yet our understanding of how to minimize its potential impacts on land and ocean use and natural resources is inadequate. Using high-resolution energy and land-use modeling, we developed spatially explicit scenarios for reaching an economy-wide net-zero GHG target in the western United States by 2050. We found that among net-zero policy cases that vary the rate of transportation and building electrification and use of fossil fuels, nuclear generation, and biomass, the "High Electrification" case, which utilizes electricity generation the most efficiently, had the lowest total land and ocean area requirements (84,000 to 105,000 km2 vs. 88,100 to 158,000 km2 across all other cases). Different levels of land and ocean use protections were applied to determine their effect on siting, environmental and social impacts, and energy costs. Meeting the net-zero target with stronger land and ocean use protections did not significantly alter the share of different energy generation technologies and only increased system costs by 3%, but decreased additional interstate transmission capacity by 20%. Yet, failure to avoid development in areas with high conservation value is likely to result in substantial habitat loss.

Removal of Small, Asymptomatic Kidney Stones and Incidence of Relapse.

BACKGROUND: The benefits of removing small (≤6 mm), asymptomatic kidney stones endoscopically is unknown. Current guidelines leave such decisions to the urologist and the patient. A prospective study involving older, nonendoscopic technology and some retrospective studies favor observation. However, published data indicate that about half of small renal stones left in place at the time that larger stones were removed caused other symptomatic events within 5 years after surgery. METHODS: We conducted a multicenter, randomized, controlled trial in which, during the endoscopic removal of ureteral or contralateral kidney stones, remaining small, asymptomatic stones were removed in 38 patients (treatment group) and were not removed in 35 patients (control group). The primary outcome was relapse as measured by future emergency department visits, surgeries, or growth of secondary stones. RESULTS: After a mean follow-up of 4.2 years, the treatment group had a longer time to relapse than the control group (P<0.001 by log-rank test). The restricted mean (±SE) time to relapse was 75% longer in the treatment group than in the control group (1631.6±72.8 days vs. 934.2±121.8 days). The risk of relapse was 82% lower in the treatment group than the control group (hazard ratio, 0.18; 95% confidence interval, 0.07 to 0.44), with 16% of patients in the treatment group having a relapse as compared with 63% of those in the control group. Treatment added a median of 25.6 minutes (interquartile range, 18.5 to 35.2) to the surgery time. Five patients in the treatment group and four in the control group had emergency department visits within 2 weeks after surgery. Eight patients in the treatment group and 10 in the control group reported passing kidney stones. CONCLUSIONS: The removal of small, asymptomatic kidney stones during surgery to remove ureteral or contralateral kidney stones resulted in a lower incidence of relapse than nonremoval and in a similar number of emergency department visits related to the surgery. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases and the Veterans Affairs Puget Sound Health Care System; ClinicalTrials.gov number, NCT02210650.).

A crystallin mutant cataract with mineral deposits.

Connexin mutant mice develop cataracts containing calcium precipitates. To test whether pathologic mineralization is a general mechanism contributing to the disease, we characterized the lenses from a nonconnexin mutant mouse cataract model. By cosegregation of the phenotype with a satellite marker and genomic sequencing, we identified the mutant as a 5-bp duplication in the γC-crystallin gene (Crygcdup). Homozygous mice developed severe cataracts early, and heterozygous animals developed small cataracts later in life. Immunoblotting studies showed that the mutant lenses contained decreased levels of crystallins, connexin46, and connexin50 but increased levels of resident proteins of the nucleus, endoplasmic reticulum, and mitochondria. The reductions in fiber cell connexins were associated with a scarcity of gap junction punctae as detected by immunofluorescence and significant reductions in gap junction-mediated coupling between fiber cells in Crygcdup lenses. Particles that stained with the calcium deposit dye, Alizarin red, were abundant in the insoluble fraction from homozygous lenses but nearly absent in wild-type and heterozygous lens preparations. Whole-mount homozygous lenses were stained with Alizarin red in the cataract region. Mineralized material with a regional distribution similar to the cataract was detected in homozygous lenses (but not wild-type lenses) by micro-computed tomography. Attenuated total internal reflection Fourier-transform infrared microspectroscopy identified the mineral as apatite. These results are consistent with previous findings that loss of lens fiber cell gap junctional coupling leads to the formation of calcium precipitates. They also support the hypothesis that pathologic mineralization contributes to the formation of cataracts of different etiologies.

Epidemiology, Comorbidities, and Treatment of Cyclic Vomiting Syndrome in the United States.

INTRODUCTION: Cyclic vomiting syndrome (CVS) imposes a substantial burden, but epidemiological data are scarce. This study aimed to estimate the incidence and prevalence of CVS, comorbid conditions, and treatment patterns, using administrative databases in the United States. METHODS: This cross-sectional study used claims data from Merative MarketScan Commercial/Medicare Supplemental and Medicaid databases in all health care settings. Incidence and prevalence rates for 2019 were calculated and stratified by age, sex, region, and race/ethnicity. Patient characteristics were reported among newly diagnosed patients with CVS (i.e., no documented claims for CVS before 2019). CVS was defined as having 1+ inpatient and/or 2+ outpatient CVS claims that were 7+ days apart. RESULTS: The estimated prevalence of CVS was 16.7 (Commercial/Medicare) and 42.9 (Medicaid) per 100,000 individuals. The incidence of CVS was estimated to be 10.6 (Commercial/Medicare) and 26.6 (Medicaid) per 100,000 individuals. Both prevalence and incidence rates were higher among female individuals (for both Commercial/Medicare and Medicaid). Comorbid conditions were common and included abdominal pain (56%-64%), anxiety (32%-39%), depression (26%-34%), cardiac conditions (39%-42%), and gastroesophageal reflux disease (30%-40%). Despite a diagnosis of CVS, only 32%-35% had prescriptions for prophylactic treatment and 47%-55% for acute treatment within the first 30-day period following diagnosis. DISCUSSION: This study provides the first population-level estimates of CVS incidence and prevalence in the United States. Comorbid conditions are common, and most patients with CVS do not receive adequate treatment. These findings underscore the need for improving disease awareness and developing better screening strategies and effective treatments.

Comparative Analyses and Ablation Efficiency of Thulium Fiber Laser by Stone Composition.

PURPOSE: There are limited data on ablation effects of thulium fiber laser (TFL) settings with varying stone composition. Similarly, little is known surrounding the photothermal effects of TFL lithotripsy regarding the chemical and structural changes after visible char formation. We aim to understand the TFL's ablative efficiency across various stone types and laser settings, while simultaneously investigating the photothermal effects of TFL lithotripsy. MATERIALS AND METHODS: Human specimens of calcium oxalate monohydrate, calcium oxalate dihydrate, uric acid, struvite, cystine, carbonate apatite, and brushite stones were ablated using 13 prespecified settings with the Coloplast TFL Drive. Pre- and postablation mass, ablation time, and total energy were recorded. Qualitative ablative observations were recorded at 1-minute intervals with photographs and gross description. Samples were analyzed with Fourier-transform infrared spectroscopy pre- and postablation and electron microscopy postablation to assess the photothermal effects of TFL. RESULTS: Across all settings and stone types, 0.05 J × 1000 Hz was the best numerically efficient ablation setting. When selected for more clinically relevant laser settings (ie, 10-20 W), 0.2 J × 100 Hz, short pulse was the most numerically efficient setting for calcium oxalate dihydrate, cystine, and struvite stones. Calcium oxalate monohydrate ablated with the best numerical efficiency at 0.4 J × 40 Hz, short pulse. Uric acid and carbonate apatite stones ablated with the best numerical efficiency at 0.3 J × 60 Hz, short pulse. Brushite stones ablated with the best numerical efficiency at 0.5 J × 30 Hz, short pulse. Pulse duration impacted ablation effectiveness greatly with 6/8 (75%) of inadequate ablations occurring in medium or long pulse settings. The average percent of mass lost during ablation was 57%; cystine stones averaged the highest percent mass lost at 71%. Charring was observed in 36/91 (40%) specimens. Charring was most often seen in uric acid, cystine, and brushite stones across all laser settings. Electron microscopy of char demonstrated a porous melting effect different to that of brittle fracture. Fourier-transform infrared spectroscopy of brushite char demonstrated a chemical composition change to amorphous calcium phosphate. CONCLUSIONS: We describe the optimal ablation settings based on stone composition, which may guide urologists towards more stone-specific care when using thulium laser for treating renal stones (lower energy settings would be safer for ureteral stones). For patients with unknown stone composition, lasers can be preset to target common stone types or adjusted based on visual cues. We recommend using short pulse for all TFL lithotripsy of calculi and altering the settings based on visual cues and efficiency to minimize the charring, an effect which can make the stone refractory to further dusting and fragmentation.

Glacial vicariance and secondary contact shape demographic histories in a freshwater mussel species complex.

Characterizing the mechanisms influencing the distribution of genetic variation in aquatic species can be difficult due to the dynamic nature of hydrological landscapes. In North America's Central Highlands, a complex history of glacial dynamics, long-term isolation, and secondary contact have shaped genetic variation in aquatic species. Although the effects of glacial history have been demonstrated in many taxa, responses are often lineage- or species-specific and driven by organismal ecology. In this study, we reconstruct the evolutionary history of a freshwater mussel species complex using a suite of mitochondrial and nuclear loci to resolve taxonomic and demographic uncertainties. Our findings do not support Pleurobema rubrum as a valid species, which is proposed for listing as threatened under the U.S. Endangered Species Act. We synonymize P. rubrum under Pleurobema sintoxia-a common and widespread species found throughout the Mississippi River Basin. Further investigation of patterns of genetic variation in P. sintoxia identified a complex demographic history, including ancestral vicariance and secondary contact, within the Eastern Highlands. We hypothesize these patterns were shaped by ancestral vicariance driven by the formation of Lake Green and subsequent secondary contact after the last glacial maximum. Our inference aligns with demographic histories observed in other aquatic taxa in the region and mirrors patterns of genetic variation of a freshwater fish species (Erimystax dissimilis) confirmed to serve as a parasitic larval host for P. sintoxia. Our findings directly link species ecology to observed patterns of genetic variation and may have significant implications for future conservation and recovery actions of freshwater mussels.

Comparison of 16 Pediatric Acute Respiratory Distress Syndrome-Associated Plasma Biomarkers With Changing Lung Injury Severity.

OBJECTIVES: Pediatric acute respiratory distress syndrome (PARDS) is a source of substantial morbidity and mortality in the PICU, and different plasma biomarkers have identified different PARDS and ARDS subgroups. We have a poor understanding of how these biomarkers change over time and with changing lung injuries. We sought to determine how biomarker levels change over PARDS course, whether they are correlated, and whether they are different in critically ill non-PARDS patients. DESIGN: Two-center prospective observational study. SETTING: Two quaternary care academic children's hospitals. PATIENTS: Subjects under 18 years of age admitted to the PICU who were intubated and met the Second Pediatric Acute Lung Injury Consensus Conference-2 PARDS diagnostic criteria and nonintubated critically ill subjects without apparent lung disease. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Plasma samples were obtained on study days 1, 3, 7, and 14. The levels of 16 biomarkers were measured using a fluorometric bead-based assay. Compared with non-PARDS subjects, on day 1 PARDS subjects had increased concentrations of tumor necrosis factor-alpha, interleukin (IL)-8, interferon-γ, IL17, granzyme B, soluble intercellular adhesion molecule-1 (sICAM1), surfactant protein D, and IL18 but reduced matrix metalloproteinase 9 (MMP-9) concentrations (all p < 0.05). Day 1 biomarker concentrations and PARDS severity were not correlated. Over PARDS course, changes in 11 of the 16 biomarkers positively correlated with changing lung injury with sICAM1 ( R = 0.69, p = 2.2 × 10 -16 ) having the strongest correlation. By Spearman rank correlation of biomarker concentrations in PARDS subjects, we identified two patterns. One had elevations of plasminogen activator inhibitor-1, MMP-9, and myeloperoxidase, and the other had higher inflammatory cytokines. CONCLUSIONS: sICAM1 had the strongest positive correlation with worsening lung injury across all study time points suggesting that it is perhaps the most biologically relevant of the 16 analytes. There was no correlation between biomarker concentration on day 1 and day 1 PARDS severity; however, changes in most biomarkers over time positively correlated with changing lung injury. Finally, in day 1 samples, 7 of the 16 biomarkers were not significantly different between PARDS and critically ill non-PARDS subjects. These data highlight the difficulty of using plasma biomarkers to identify organ-specific pathology in critically ill patients.

Evaluation of enoxaparin for bridging of warfarin in outpatients with left ventricular assist devices (LVADs).

BACKGROUND: Patients with left ventricular assist devices (LVADs) require systemic anticoagulation. The use of enoxaparin for bridging to warfarin remains understudied in this population. METHODS: This single-center retrospective study was performed to characterize enoxaparin use and associated thrombotic and bleeding outcomes in adult outpatients with LVADs from January 2018 to July 2021. RESULTS: Fifty-four enoxaparin bridging events were evaluated in 49 patients. Most patients with HeartMate II (HM2) and HeartWare (HVAD) devices received enoxaparin dosed 1 mg/kg every 12 h. In patients with HeartMate 3 (HM3) devices, an equal number of patients received 0.5 mg/kg every 12 h and 1 mg/kg every 12 h, with a smaller subset receiving intermediate doses. The median duration of bridging was 6 days (4-8 [IQR]). One major bleeding event required discontinuation of enoxaparin and hospitalization in a patient with an HM3 device. Thrombotic events occurred in four patients with two incidents of pump thrombosis requiring pump exchange and two ischemic strokes. All thrombotic events occurred in patients with HVAD or HM2 devices. CONCLUSION: These results suggest that enoxaparin bridging in LVAD patients was well-tolerated with low bleeding and thrombotic rates, particularly with the HM3 device.

Adapting an Alcohol Care Linkage Intervention to US Military Veterans Presenting to Primary Care with Hazardous Drinking and PTSD and/or Depression Symptoms: A Qualitative Study.

There is a critical need to improve linkage to alcohol care for veterans in primary care with hazardous drinking and PTSD and/or depression symptoms (A-MH). We adapted an alcohol care linkage intervention, "Connect to Care" (C2C), for this population. We conducted separate focus groups with veterans with A-MH, providers, and policy leaders. Feedback centered on how psychologists and other providers can optimally inform veterans about their care options and alcohol use, and how to ensure C2C is accessible. Participants reported that veterans with A-MH may not view alcohol use as their primary concern but rather as a symptom of a potential co-occurring mental health condition. Veterans have difficulty identifying and accessing existing alcohol care options within the Veterans Health Administration. C2C was modified to facilitate alcohol care linkage for this population specific to their locality, provide concrete support and education, and offer care options to preserve privacy.

Integrated Cytometry With Machine Learning Applied to High-Content Imaging of Human Kidney Tissue for In Situ Cell Classification and Neighborhood Analysis.

The human kidney is a complex organ with various cell types that are intricately organized to perform key physiological functions and maintain homeostasis. New imaging modalities, such as mesoscale and highly multiplexed fluorescence microscopy, are increasingly being applied to human kidney tissue to create single-cell resolution data sets that are both spatially large and multidimensional. These single-cell resolution high-content imaging data sets have great potential to uncover the complex spatial organization and cellular makeup of the human kidney. Tissue cytometry is a novel approach used for the quantitative analysis of imaging data; however, the scale and complexity of such data sets pose unique challenges for processing and analysis. We have developed the Volumetric Tissue Exploration and Analysis (VTEA) software, a unique tool that integrates image processing, segmentation, and interactive cytometry analysis into a single framework on desktop computers. Supported by an extensible and open-source framework, VTEA's integrated pipeline now includes enhanced analytical tools, such as machine learning, data visualization, and neighborhood analyses, for hyperdimensional large-scale imaging data sets. These novel capabilities enable the analysis of mesoscale 2- and 3-dimensional multiplexed human kidney imaging data sets (such as co-detection by indexing and 3-dimensional confocal multiplexed fluorescence imaging). We demonstrate the utility of this approach in identifying cell subtypes in the kidney on the basis of labels, spatial association, and their microenvironment or neighborhood membership. VTEA provides an integrated and intuitive approach to decipher the cellular and spatial complexity of the human kidney and complements other transcriptomics and epigenetic efforts to define the landscape of kidney cell types.

Airborne! UAV delivery of blood products and medical logistics for combat zones.

BACKGROUND: Innovative solutions to resupply critical medical logistics and blood products may be required in future near-peer conflicts. Unmanned aerial vehicles (UAVs) are increasingly being used in austere environments and may be a viable platform for medical resupply and the transport of blood products. METHODS: A literature review on PubMed and Google Scholar up to March of 2022 yielded a total of 27 articles that were included in this narrative review. The objectives of this article are to discuss the current limitations of prehospital blood transfusion in military settings, discuss the current uses of UAVs for medical logistics, and highlight the ongoing research surrounding UAVs for blood product delivery. DISCUSSION: UAVs allow for the timely delivery of medical supplies in numerous settings and have been utilized for both military and civilian purposes. Investigations into the effects of aeromedical transportation on blood products have found minimal blood product degradation when appropriately thermoregulated and delivered in a manner that minimizes trauma. UAV delivery of blood products is now actively being explored by numerous entities around the globe. Current limitations surrounding the lack of high-quality safety data, engineering constraints over carrying capacity, storage capability, and distance traveled, as well as air space regulations persist. CONCLUSION: UAVs may offer a novel solution for the transport of medical supplies and blood products in a safe and timely manner for the forward-deployed setting. Further research on optimal UAV design, optimal delivery techniques, and blood product safety following transport should be explored prior to implementation.

Pooled Phase 2 and 3 Efficacy and Safety Data on Budesonide Oral Suspension in Adolescents with Eosinophilic Esophagitis.

OBJECTIVES: The objective of this study was to evaluate the efficacy and safety of budesonide oral suspension (BOS) in adolescents with eosinophilic esophagitis (EoE). METHODS: This post hoc analysis pooled data from two 12-week, randomized, double-blind, placebo-controlled studies of BOS 2.0 mg twice daily (b.i.d.) (phase 2, NCT01642212; phase 3, NCT02605837) in patients aged 11-17 years with EoE and dysphagia. Efficacy endpoints included histologic (≤6, ≤1, and <15 eosinophils per high-power field [eos/hpf]), dysphagia symptom (≥30% reduction in Dysphagia Symptom Questionnaire [DSQ] scores from baseline), and clinicopathologic (≤6 eos/hpf and ≥30% reduction in DSQ scores from baseline) responses at week 12. Change from baseline to week 12 in peak eosinophil counts, DSQ scores, EoE Histology Scoring System (EoEHSS) grade (severity) and stage (extent) total score ratios (TSRs), and total EoE Endoscopic Reference Scores (EREFS) were assessed. Safety outcomes were also examined. RESULTS: Overall, 76 adolescents were included (BOS, n = 45; placebo, n = 31). Significantly more patients who received BOS than placebo achieved histologic responses (≤6 eos/hpf: 46.7% vs 6.5%; ≤1 eos/hpf: 42.2% vs 0.0%; <15 eos/hpf: 53.3% vs 9.7%; P < 0.001) and a clinicopathologic response (31.1% vs 3.2%; P = 0.003) at week 12. More BOS-treated than placebo-treated patients achieved a dysphagia symptom response at week 12 (68.9% vs 58.1%; not statistically significant P = 0.314). BOS-treated patients had significantly greater reductions in EoEHSS grade and stage TSRs ( P < 0.001) and total EREFS ( P = 0.021) from baseline to week 12 than placebo-treated patients. BOS was well tolerated, with no clinically meaningful differences in adverse events versus placebo. CONCLUSIONS: BOS 2.0 mg b.i.d. significantly improved most efficacy outcomes in adolescents with EoE versus placebo.

A Placebo-Controlled Randomized Trial of Vigabatrin in the Management of Acute Alcohol Withdrawal.

OBJECTIVE: To undertake a double blinded randomised placebo-controlled trial to assess the efficacy of vigabatrin, a GABA-transaminase inhibitor, as a benzodiazepine sparing agent in the management of acute alcohol withdrawal syndrome in a residential setting. METHODS: We enrolled 120 patients with alcohol use disorder who were randomly assigned to either treatment with vigabatrin (2g/day for 4 days) or placebo. The primary outcome was defined as the number of participants in each treatment arm needing diazepam for withdrawal management. A secondary outcome prespecified was the total dose of diazepam received by participants in each treatment arm. Participants were recruited on admission to a residential withdrawal unit at St Vincent's Hospital Melbourne from December 2014 to April 2019. RESULTS: No significant difference was observed in the number of participants requiring benzodiazepines during their residential withdrawal stay with 44 participants (78.6%) in placebo arm requiring at least one dose of diazepam compared to 38 (66.7%) in vigabatrin arm (p = .156). An 18.1% difference was observed between the proportion of participants who received a total dose of >100mg of diazepam during their residential withdrawal stay in placebo arm (32.1%), compared to vigabatrin arm (14.0%, p = .022). There were higher rates of reported adverse events in placebo arm with nine (15.0%) participants reporting adverse events compared with two (3.3%) participants in vigabatrin arm (p = .027). CONCLUSION: Vigabatrin significantly reduced the number of participants requiring >100mg diazepam over the course of their alcohol withdrawal and was associated with a reduction in adverse effects when compared to placebo.

"Let's Not Have the Perfect Be the Enemy of the Good": Social Impact Bonds, Randomized Controlled Trials, and the Valuation of Social Programs.

This article uses the case of "social impact bonds" (SIBs) to explore the role of social science methods in new markets in "social investment." Pioneered in the UK in 2010, SIBs use private capital to fund social programs with governments paying returns for successful outcomes. Central to the SIB model is the question of evaluation and the method to be used in determining program outcomes and investor returns. In the United States, the randomized controlled trial (RCT) has been the dominant method. However, this has not been without controversy. Some SIB practitioners and investors have argued that, while this may be the perfect tool, the need to grow the SIB market demands a more pragmatic approach. Drawing from a three-year study of SIBs, and informed by Science and Technology Studies (STS)-inspired work on valuation and the social life of methods, the article explores RCTs as both a valuation technology central to SIB design and the object of a micropolitics of valuation which has impeded market growth. It is the relationship between, and the politics of, evaluation and valuation that is a key lesson of the SIB experiment and an important insight for future research on "social investment" and other settings where methods are constitutive of financial value.

Engineering Human Cells Expressing CRISPR/Cas9-Synergistic Activation Mediators for Recombinant Protein Production.

Recombinant engineering for protein production commonly employs plasmid-based gene templates for introduction and expression of genes in a candidate cell system in vitro. Challenges to this approach include identifying cell types that can facilitate proper post-translational modifications and difficulty expressing large multimeric proteins. We hypothesized that integration of the CRISPR/Cas9-synergistic activator mediator (SAM) system into the human genome would be a powerful tool capable of robust gene expression and protein production. SAMs are comprised of a "dead" Cas9 (dCas9) linked to transcriptional activators viral particle 64 (VP64), nuclear factor-kappa-B p65 subunit (p65), and heat shock factor 1 (HSF1) and are programmable to single or multiple gene targets. We integrated the components of the SAM system into human HEK293, HKB11, SK-HEP1, and HEP-g2 cells using coagulation factor X (FX) and fibrinogen (FBN) as proof of concept. We observed upregulation of mRNA in each cell type with concomitant protein expression. Our findings demonstrate the capability of human cells stably expressing SAM for user-defined singleplex and multiplex gene targeting and highlight their broad potential utility for recombinant engineering as well as transcriptional modulation across networks for basic, translational, and clinical modeling and applications.

Kidney stone formation in a novel murine model of polycystic kidney disease.

Individuals with autosomal dominant polycystic kidney disease have a higher incidence of stone formation than the general population. However, there are no cystic animal models known to develop stones. Cystic mice compound heterozygous for hypomorphic Pkd1V and Pkd1RC alleles develop cystic kidneys within a few weeks of birth but live beyond 20 wk of age, allowing for the study of cystic comorbidities including stone formation. Cystic Pkd1V/RC mice were euthanized at 3, 13, or 26 wk of age, and their kidneys were analyzed by microcomputed tomography (µCT) for stone formation. Mice had occasional mineral aggregates that could be detected by µCT analysis at 3 wk of age. At 13 or 26 wk of age, numerous white masses were visible beneath the kidney surface. µCT analysis confirmed the masses to be large mineral stone deposits throughout the renal cortex, with mineral content increasing with age. Staining of histological sections with alizarin red and von Kossa suggested that the stone deposits were composed primarily of calcium and phosphate. Microdissection confirmed stones localized within cyst lumens. Analysis of individual stones by µCT and infrared spectroscopy confirmed apatite mineral composition. Urinalysis revealed elevated levels of phosphate and citrate at 3 wk of age and lower pH and elevated levels of calcium and citrate at 13 wk of age, suggesting altered phosphate and calcium homeostasis as a potential cause of mineralization and renal stone formation. This is the first animal model exhibiting overt kidney stone formation in the context of cystic kidney disease.NEW & NOTEWORTHY Compound heterozygous Pkd1V/RC mice were found to form calcium phosphate-containing stones within cysts of the renal cortex by 13 wk of age. This is the first polycystic kidney disease animal model exhibiting spontaneous stone formation. A growing body of evidence suggests a link between renal stone formation and cystic kidney disease. This mouse model may be useful for studying the interplay between stone and cyst formation and the functional role of polycystins in mineral homeostasis.

Budesonide Oral Suspension Improves Outcomes in Patients With Eosinophilic Esophagitis: Results from a Phase 3 Trial.

BACKGROUND & AIMS: Eosinophilic esophagitis (EoE) is a chronic, immune-mediated disease for which there is currently no pharmacologic therapy approved by the US Food and Drug Administration. METHODS: In this double-blind, placebo-controlled, phase 3 trial, patients 11-55 years of age with EoE and dysphagia were randomized 2:1 to receive budesonide oral suspension (BOS) 2.0 mg twice daily or placebo for 12 weeks at academic or community care practices. Co-primary endpoints were the proportion of stringent histologic responders (≤6 eosinophils/high-power field) or dysphagia symptom responders (≥30% reduction in Dysphagia Symptom Questionnaire [DSQ] score) over 12 weeks. Changes in DSQ score (key secondary endpoint) and EoE Endoscopic Reference Score (EREFS) (secondary endpoint) from baseline to week 12, and safety parameters were examined. RESULTS: Overall, 318 patients (BOS, n = 213; placebo, n = 105) were randomized and received ≥1 dose of study treatment. More BOS-treated than placebo-treated patients achieved a stringent histologic response (53.1% vs 1.0%; Δ52% [95% confidence interval (CI), 43.3%-59.1%]; P < .001) or symptom response (52.6% vs 39.1%; Δ13% [95% CI, 1.6%-24.3%]; P = .024) over 12 weeks. BOS-treated patients also had greater improvements in least-squares mean DSQ scores and EREFS over 12 weeks than placebo-treated patients: DSQ, -13.0 (SEM 1.2) vs -9.1 (SEM 1.5) (Δ-3.9 [95% CI, -7.1 to -0.8]; P = .015); EREFS, -4.0 (SEM 0.3) vs -2.2 (SEM 0.4) (Δ-1.8 [95% CI, -2.6 to -1.1]; P < .001). BOS was well tolerated; most adverse events were mild or moderate in severity. CONCLUSIONS: In patients with EoE, BOS 2.0 mg twice daily was superior to placebo in improving histologic, symptomatic, and endoscopic outcomes over 12 weeks. BOS 2.0 mg twice daily was well tolerated. ClinicalTrials.gov number: NCT02605837.

Long-Term Treatment of Eosinophilic Esophagitis With Budesonide Oral Suspension.

BACKGROUND & AIMS: We evaluated treatment withdrawal, long-term outcomes, and safety of budesonide oral suspension (BOS) 2.0 mg twice daily in patients with eosinophilic esophagitis who completed a 12-week induction study. METHODS: Induction full responders (≤6 eosinophils per high-power field [eos/hpf] and ≥30% reduction in the Dysphagia Symptom Questionnaire score) to BOS 2.0 mg twice daily (ORBIT1/SHP621-301/NCT02605837) were randomized to continue BOS (BOS-BOS) or withdraw to placebo (BOS-PBO) for 36 weeks (ORBIT2/SHP621-302/NCT02736409). Induction partial responders and nonresponders, and patients who received induction placebo, received BOS for 36 weeks. The primary end point was the proportion of BOS-BOS and BOS-PBO patients who relapsed (≥15 eos/hpf and ≥4 days of dysphagia [Dysphagia Symptom Questionnaire] over 2 weeks) by week 36. The key secondary end point was the proportion of induction partial responders and nonresponders who fully responded after 52 weeks of total BOS therapy. Other secondary end points included the proportion of induction full responders with histologic responses (≤1, ≤6, <15 eos/hpf) at week 12 of the extension study, and safety outcomes. RESULTS: The randomized withdrawal period enrolled 48 patients (BOS-BOS, n = 25; BOS-PBO, n = 23); 106 induction partial responders and nonresponders, and 65 induction placebo patients received BOS. More BOS-PBO than BOS-BOS patients relapsed over 36 weeks (43.5% vs 24.0%; P = .131) and had histologic responses at week 12 of therapy (P < .001). Overall, 13.2% of induction partial responders and nonresponders fully responded at week 36. BOS was well tolerated; therapy duration was not associated with new safety concerns. CONCLUSIONS: For induction full responders, continuing BOS numerically improved maintenance of efficacy vs withdrawal. A longer therapy duration did not raise safety concerns. (ClinicalTrials.gov: NCT02736409.).