Dual α-globin and truncated EPO receptor knockin restores hemoglobin production in α-thalassemia-derived red blood cells.

Alpha-thalassemia is an autosomal recessive disease with increasing worldwide prevalence. The molecular basis is due to mutation or deletion of one or more duplicated α-globin genes, and disease severity is directly related to the number of allelic copies compromised. The most severe form, α-thalassemia major (αTM), results from loss of all four copies of α-globin and has historically resulted in fatality in utero. However, in utero transfusions now enable survival to birth. Postnatally, patients face challenges similar to ß-thalassemia, including severe anemia and erythrotoxicity due to imbalance of ß-globin and α-globin chains. While curative, hematopoietic stem cell transplantation (HSCT) is limited by donor availability and potential transplant-related complications. Despite progress in genome editing treatments for ß-thalassemia, there is no analogous curative option for patients suffering from α-thalassemia. To address this, we designed a novel Cas9/AAV6-mediated genome editing strategy that integrates a functional α-globin gene into the ß-globin locus in αTM patient-derived hematopoietic stem and progenitor cells (HSPCs). Incorporation of a truncated erythropoietin receptor transgene into the α-globin integration cassette dramatically increased erythropoietic output from edited HSPCs and led to the most robust production of α-globin, and consequently normal hemoglobin. By directing edited HSPCs toward increased production of clinically relevant RBCs instead of other divergent cell types, this approach has the potential to mitigate the limitations of traditional HSCT for the hemoglobinopathies, including low genome editing and low engraftment rates. These findings support development of a definitive ex vivo autologous genome editing strategy that may be curative for α-thalassemia.

Host and microbial characteristics associated with recurrent prosthetic joint infections.

Approximately 20% of patients after resection arthroplasty and antibiotic spacer placement for prosthetic joint infection develop repeat infections, requiring an additional antibiotic spacer before definitive reimplantation. The host and bacterial characteristics associated with the development of recurrent infection is poorly understood. A case-control study was conducted for 106 patients with intention to treat by two-stage revision arthroplasty for prosthetic joint infection at a single institution between 2009 and 2020. Infection was defined according to the 2018 Musculoskeletal Infection Society criteria. Thirty-nine cases ("recurrent-periprosthetic joint infection [PJI]") received at least two antibiotic spacers before clinical resolution of their infection, and 67 controls ("single-PJI") received a single antibiotic cement spacer before infection-free prosthesis reimplantation. Patient demographics, McPherson host grade, and culture results including antibiotic susceptibilities were compared. Fifty-two (78%) single-PJI and 32 (82%) recurrent-PJI patients had positive intraoperative cultures at the time of their initial spacer procedure. The odds of polymicrobial infections were 11-fold higher among recurrent-PJI patients, and the odds of significant systemic compromise (McPherson host-grade C) were more than double. Recurrent-PJI patients were significantly more likely to harbor Staphylococcus aureus. We found no differences between cases and controls in pathogen resistance to the six most tested antibiotics. Among recurrent-PJI patients, erythromycin-resistant infections were more prevalent at the final than initial spacer, despite no erythromycin exposure. Our findings suggest that McPherson host grade, polymicrobial infection, and S. aureus infection are key indicators of secondary or persistent joint infection following resection arthroplasty and antibiotic spacer placement, while bacterial resistance does not predict infection-related arthroplasty failure.

Demographic and socioeconomic disparities in receipt of ophthalmology consultation for facial trauma.

OBJECTIVE: Functional outcomes following facial and ocular trauma are time-sensitive and require prompt evaluation to minimise long-term vision loss, yet few studies have systematically evaluated disparities in the management of these cases. This study investigates whether a patient's race/ethnicity, primary language, insurance status, gender or age affects receipt of ophthalmology consultation for facial trauma. METHODS AND ANALYSIS: This study was a retrospective cohort analysis of patients from the Elmhurst City Hospital Trauma Registry in Queens, New York who were seen for facial trauma including open globe injuries and orbital fractures between January 2014 and May 2016. RESULTS: Of the 264 patients included, 43% reported as Hispanic, 23% white, 11% Asian, 8% black and 15% other/unknown. After controlling for confounding variables by multivariable logistic regression, neither race/ethnicity, gender, nor primary language were significantly associated with the likelihood of receiving an ophthalmology consult. However, patients with private insurance had 2.57 times greater odds of receiving an ophthalmology consultation than those with Medicaid or state corrections insurance (95% CI 1.37 to 4.95). As age increased, the likelihood of receiving an ophthalmology consultation decreased (p=0.009); patients 60 years of age and older had one-third the odds of ophthalmology consultation as younger patients (OR 0.33; 95% CI 0.16 to 0.68). CONCLUSIONS: This study highlights that lack of ophthalmology consultation in patients with facial trauma is linked to age and underinsurance. Extra attention must be paid during primary assessments to ensure elderly patients and those with public insurance have equitable access to timely and appropriate care for facial trauma.

STING inhibits the reactivation of dormant metastasis in lung adenocarcinoma.

Metastasis frequently develops from disseminated cancer cells that remain dormant after the apparently successful treatment of a primary tumour. These cells fluctuate between an immune-evasive quiescent state and a proliferative state liable to immune-mediated elimination1-6. Little is known about the clearing of reawakened metastatic cells and how this process could be therapeutically activated to eliminate residual disease in patients. Here we use models of indolent lung adenocarcinoma metastasis to identify cancer cell-intrinsic determinants of immune reactivity during exit from dormancy. Genetic screens of tumour-intrinsic immune regulators identified the stimulator of interferon genes (STING) pathway as a suppressor of metastatic outbreak. STING activity increases in metastatic progenitors that re-enter the cell cycle and is dampened by hypermethylation of the STING promoter and enhancer in breakthrough metastases or by chromatin repression in cells re-entering dormancy in response to TGFß. STING expression in cancer cells derived from spontaneous metastases suppresses their outgrowth. Systemic treatment of mice with STING agonists eliminates dormant metastasis and prevents spontaneous outbreaks in a T cell- and natural killer cell-dependent manner-these effects require cancer cell STING function. Thus, STING provides a checkpoint against the progression of dormant metastasis and a therapeutically actionable strategy for the prevention of disease relapse.

Comparative analysis of CRISPR off-target discovery tools following ex vivo editing of CD34+ hematopoietic stem and progenitor cells.

While a number of methods exist to investigate CRISPR off-target (OT) editing, few have been compared head-to-head in primary cells after clinically relevant editing processes. Therefore, we compared in silico tools (COSMID, CCTop, and Cas-OFFinder) and empirical methods (CHANGE-Seq, CIRCLE-Seq, DISCOVER-Seq, GUIDE-Seq, and SITE-Seq) after ex vivo hematopoietic stem and progenitor cell (HSPC) editing. We performed editing using 11 different gRNAs complexed with Cas9 protein (high-fidelity [HiFi] or wild-type versions), then performed targeted next-generation sequencing of nominated OT sites identified by in silico and empirical methods. We identified an average of less than one OT site per guide RNA (gRNA) and all OT sites generated using HiFi Cas9 and a 20-nt gRNA were identified by all OT detection methods with the exception of SITE-seq. This resulted in high sensitivity for the majority of OT nomination tools and COSMID, DISCOVER-Seq, and GUIDE-Seq attained the highest positive predictive value (PPV). We found that empirical methods did not identify OT sites that were not also identified by bioinformatic methods. This study supports that refined bioinformatic algorithms could be developed that maintain both high sensitivity and PPV, thereby enabling more efficient identification of potential OT sites without compromising a thorough examination for any given gRNA.

Ultra-deep sequencing validates safety of CRISPR/Cas9 genome editing in human hematopoietic stem and progenitor cells.

As CRISPR-based therapies enter the clinic, evaluation of safety remains a critical and active area of study. Here, we employ a clinical next generation sequencing (NGS) workflow to achieve high sequencing depth and detect ultra-low frequency variants across exons of genes associated with cancer, all exons, and genome wide. In three separate primary human hematopoietic stem and progenitor cell (HSPC) donors assessed in technical triplicates, we electroporated high-fidelity Cas9 protein targeted to three loci (AAVS1, HBB, and ZFPM2) and harvested genomic DNA at days 4 and 10. Our results demonstrate that clinically relevant delivery of high-fidelity Cas9 to primary HSPCs and ex vivo culture up to 10 days does not introduce or enrich for tumorigenic variants and that even a single SNP in a gRNA spacer sequence is sufficient to eliminate Cas9 off-target activity in primary, repair-competent human HSPCs.

Compliance to an enhanced recovery pathway among patients with a high frailty index after major gastrointestinal surgery results in improved 30-day outcomes.

BACKGROUND: Enhanced recovery pathways have been shown to improve clinical outcomes after surgery. Concerns exist about the feasibility of implementing enhanced recovery pathways in frail patients, who are at a greater risk for adverse postoperative outcomes. This study evaluated compliance and outcomes after enhanced recovery pathway implementation in high-risk, abdominal surgery patients. METHODS: Patients entered into the American College of Surgeons National Surgical Quality Improvement Program database who underwent abdominal surgery after enhanced recovery pathway implementation at two Johns Hopkins Medical Institutions were included. Risk was assessed using the American College of Surgeons National Surgical Quality Improvement Program validated, modified 5-item frailty index. Primary outcomes included compliance with 14 enhanced recovery pathway standards and postoperative length of stay, major complications (Clavien-Dindo score II-IV), and 30-day readmission. RESULTS: This study included 646 patients who participated in our enhanced recovery pathway program and 65 patients with modified 5-item frailty index ≥ 2 before enhanced recovery pathway implementation. Overall, 325 patients (50.3%) were high compliers (>75% compliance) with enhanced recovery pathway standards, with similar proportions of patients with a modified 5-item frailty index ≥ 2 or < 2 achieving high compliance (51.6% vs 50.2%, P = .89, respectively). Examining causality for "low compliers" among patients with a high frailty score (modified 5-item frailty index ≥2) demonstrated significant less use of goal-directed therapy when compared with "high compliers" (43% vs 75%, P = .01). Low compliers were also less likely than high compliers to experience mobilization the day of surgery (43% vs 78%, P = .01), postoperative day 1 (43% vs 88%, P < .01), and postoperative day 2 (60% vs 100%, P < .01). In addition, low compliers were less likely than high compliers to have their diet advanced to solids on postoperative day 1 (17% vs 59%, P < .01) and have their Foley catheter removed on postoperative day 1 (45% vs 97%, P < .01). Comparing our pre-enhanced recovery pathway patients with our enhanced recovery pathway cohort with a high frailty score, enhanced recovery pathway patients had a significantly shorter length of stay (4.5 vs 6 days, P = .04). However, adjusted analysis demonstrated that high compliance, and not just the enhanced recovery pathway intervention among patients with a high frailty score, was independently associated with a decrease in length of stay (odds ratio 0.72, 95% confidence interval 0.63-0.82, P < .01) and a significant reduction in major complications (odds ratio 0.30, 95% confidence interval 0.14-0.65, P < .01. CONCLUSION: This study demonstrates that frail patients comply well with a robust enhanced recovery pathway protocol and subsequently experience improved outcomes. Targeted interventions that seek to maximize compliance with specific enhanced recovery pathway standards may further improve outcomes in this population.

High Compliance to an Enhanced Recovery Pathway for Patients ≥65 Years Undergoing Major Small and Large Intestinal Surgery Is Associated With Improved Postoperative Outcomes.

OBJECTIVE: This study was performed to evaluate compliance to an Enhanced Recovery Pathway (ERP) among patients ≥65 years and determine the effect of compliance on postoperative outcomes. SUMMARY BACKGROUND DATA: ERPs improve postoperative outcomes in patients undergoing major surgery. Given the inherent decline of the older surgical patient, the benefit of an ERP in this population has been questioned. METHODS: Patients undergoing major small and large intestinal surgery prior to and following ERP implementation at the Johns Hopkins Medical Institutions were entered into the ACS-NSQIP database. Outcomes included ERP compliance rates, complications, length of stay (LOS), and 30-day readmission rates were determined for older patients. RESULTS: Nine hundred seventy-four patients (693 < 65 yrs and 281 ≥ 65 yrs) were included. Of those ≥ 65 years, 142 (51%) were entered prior to and 139 (49%) were entered following ERP implementation. More ERP than pre-ERP patients underwent laparoscopic procedures (45.3% vs. 32.4%, P = 0.02), had disseminated malignancies (9.4% vs. 2.8%, P = 0.03), and smoked (14.4% vs. 4.9%, P = 0.01). Overall compliance was 74.5%, and 47% of older ERP patients achieved high compliance (≥75% compliance with ERP variables). High compliance was associated with a 30% decrease LOS (IRR: 0.7 P = 0.001) and 60% decrease in major (CD ≥ II) complications (OR: 0.4 P = 0.05). CONCLUSION: LOS and complication rates following implementation of an ERP were significantly improved in highly compliant elderly patients. Interventions to further improve outcomes should target decreasing variability by increasing individual compliance with an effective clinical pathway.

A Nigro-Vagal Pathway Controls Gastric Motility and Is Affected in a Rat Model of Parkinsonism.

BACKGROUND & AIMS: In most patients with Parkinson's disease, gastrointestinal (GI) dysfunctions, such as gastroparesis and constipation, are prodromal to the cardinal motor symptoms of the disease. Sporadic Parkinson's disease has been proposed to develop after ingestion of neurotoxicants that affect the brain-gut axis via the vagus nerve, and then travel to higher centers, compromising the substantia nigra pars compacta (SNpc) and, later, the cerebral cortex. We aimed to identify the pathway that connects the brainstem vagal nuclei and the SNpc, and to determine whether this pathway is compromised in a rat model of Parkinsonism. METHODS: To study this neural pathway in rats, we placed tracers in the dorsal vagal complex or SNpc; brainstem and midbrain were examined for tracer distribution and neuronal neurochemical phenotype. Rats were given injections of paraquat once weekly for 3 weeks to induce features of Parkinsonism, or vehicle (control). Gastric tone and motility were recorded after N-methyl-d-aspartate microinjection in the SNpc and/or optogenetic stimulation of nigro-vagal terminals in the dorsal vagal complex. RESULTS: Stimulation of the SNpc increased gastric tone and motility via activation of dopamine 1 receptors in the dorsal vagal complex. In the paraquat-induced model of Parkinsonism, this nigro-vagal pathway was compromised during the early stages of motor deficit development. CONCLUSIONS: We identified and characterized a nigro-vagal monosynaptic pathway in rats that controls gastric tone and motility. This pathway might be involved in the prodromal gastric dysmotility observed in patients with early-stage Parkinson's disease.

Thyroid gland disorder emergencies: thyroid storm and myxedema coma.

Although thyroid dysfunction will develop in more than 12% of the US population during their lifetimes, true thyroid emergencies are rare. Thyroid storm and myxedema coma are endocrine emergencies resulting from thyroid hormone dysregulation, usually coupled with an acute illness as a precipitant. Careful assessment of risk and rapid action, once danger is identified, are essential for limiting morbidity and mortality related to thyroid storm and myxedema coma. This article reviews which patients are at risk, explains thyroid storm and myxedema coma, and describes pharmacological treatment and supportive cares.

Apelin protects against angiotensin II-induced cardiovascular fibrosis and decreases plasminogen activator inhibitor type-1 production.

OBJECTIVE: To test the hypothesis that apelin protects against angiotensin II (Ang II)-induced cardiovascular fibrosis and vascular remodeling. METHODS AND RESULTS: Wild-type mice administered apelin or apelin along with Ang II exhibited less cardiovascular fibrosis and decreased plasminogen activator inhibitor type-1 (PAI-1) gene expression than mice receiving Ang II, N-nitro-L-arginine methyl ester (L-NAME), apelin plus L-NAME or apelin plus Ang II plus L-NAME. In-vitro analysis using a luciferase construct driven by 3.1 kb of the human PAI-1 promoter revealed that apelin blocked Ang II-mediated PAI-1 gene expression. Immunoblotting for phosphorylated myosin phosphatase subunit and myosin light chain revealed that apelin blocked Ang II activation of the Rho kinase pathway, which is associated with induction of PAI-1 gene expression by Ang II. In addition, treatment of human aortic smooth muscle cells with apelin reduced PAI-1 mRNA and protein production in the presence and absence of Ang II. Conversely, L-NAME treatment attenuated the downregulation of PAI-1 by apelin in cells. CONCLUSION: Apelin protects against cardiac fibrosis and vascular remodeling through direct regulation of PAI-1 gene expression. This protective effect is mediated through the synergistic inhibition of Ang II signaling and increased production of nitric oxide by apelin. Our data extend previous findings and provide new insight into the molecular mechanisms by which apelin elicits a cardioprotective effect.

Attribution of vascular phenotypes of the murine Egfl7 locus to the microRNA miR-126.

Intronic microRNAs have been proposed to complicate the design and interpretation of mouse knockout studies. The endothelial-expressed Egfl7/miR-126 locus contains miR-126 within Egfl7 intron 7, and angiogenesis deficits have been previously ascribed to Egfl7 gene-trap and lacZ knock-in mice. Surprisingly, selectively floxed Egfl7(Delta) and miR-126(Delta) alleles revealed that Egfl7(Delta/Delta) mice were phenotypically normal, whereas miR-126(Delta/Delta) mice bearing a 289-nt microdeletion recapitulated previously described Egfl7 embryonic and postnatal retinal vascular phenotypes. Regulation of angiogenesis by miR-126 was confirmed by endothelial-specific deletion and in the adult cornea micropocket assay. Furthermore, miR-126 deletion inhibited VEGF-dependent Akt and Erk signaling by derepression of the p85beta subunit of PI3 kinase and of Spred1, respectively. These studies demonstrate the regulation of angiogenesis by an endothelial miRNA, attribute previously described Egfl7 vascular phenotypes to miR-126, and document inadvertent miRNA dysregulation as a complication of mouse knockout strategies.

Plasmalemmal vesicle associated protein-1 is a novel marker implicated in brain tumor angiogenesis.

PURPOSE: Plasmalemmal vesicle associated protein-1 (PV-1) is up-regulated in the endothelium of human glioblastoma. We sought to further characterize the expression pattern of PV-1 in human brain tumors and interrogate its role in brain tumor angiogenesis. EXPERIMENTAL DESIGN: Quantitative reverse transcription-PCR and in situ hybridization were used to measure PV-1 expression in a panel of 46 human brain tumors and related pathologic states. Matrigel tubulogenesis assays and cell migration assays were used to show function of PV-1 in primary human endothelial cells (HMVEC) under gene knockdown conditions. RESULTS: PV-1 is selectively up-regulated in a variety of high-grade human brain tumors, including glioblastoma and metastatic carcinoma, as well as other cerebral disorders associated with blood-brain barrier disruption, such as acute ischemia. Expression levels were reduced in low-grade neoplasia; however, tumors associated with the ependyma and choroid plexus, known sites of PV-1 expression, also exhibited robust expression. Cerebral expression of PV-1 mRNA was confined to endothelial cells in all cases. PV-1 expression was induced in HMVEC cells in vitro by exposure to medium conditioned by U87MG and U251MG human brain tumor cell lines and by medium supplemented with exogenous vascular endothelial growth factor or scatter factor/hepatocyte growth factor. RNA interference-mediated inhibition of PV-1 induction in HMVEC cells blocked Matrigel-induced tubulogenesis and inhibited cell migration induced by conditioned medium or angiogenic growth factors. CONCLUSIONS: Our results confirm that PV-1 is preferentially induced in the endothelium of high-grade human brain tumors. Inhibition of PV-1 expression is associated with failure of endothelial differentiation in vitro. PV-1 represents a novel marker of brain tumor angiogenesis and integrity of the blood-brain barrier and is a potential therapeutic target.