Chromosome 10q26-driven age-related macular degeneration is associated with reduced levels of HTRA1 in human retinal pigment epithelium.

Genome-wide association studies have identified the chromosome 10q26 (Chr10) locus, which contains the age-related maculopathy susceptibility 2 (ARMS2) and high temperature requirement A serine peptidase 1 (HTRA1) genes, as the strongest genetic risk factor for age-related macular degeneration (AMD) [L.G. Fritsche et al., Annu. Rev. Genomics Hum. Genet. 15, 151-171, (2014)]. To date, it has been difficult to assign causality to any specific single nucleotide polymorphism (SNP), haplotype, or gene within this region because of high linkage disequilibrium among the disease-associated variants [J. Jakobsdottir et al. Am. J. Hum. Genet. 77, 389-407 (2005); A. Rivera et al. Hum. Mol. Genet. 14, 3227-3236 (2005)]. Here, we show that HTRA1 messenger RNA (mRNA) is reduced in retinal pigment epithelium (RPE) but not in neural retina or choroid tissues derived from human donors with homozygous risk at the 10q26 locus. This tissue-specific decrease is mediated by the presence of a noncoding, cis-regulatory element overlapping the ARMS2 intron, which contains a potential Lhx2 transcription factor binding site that is disrupted by risk variant rs36212733. HtrA1 protein increases with age in the RPE-Bruch's membrane (BM) interface in Chr10 nonrisk donors but fails to increase in donors with homozygous risk at the 10q26 locus. We propose that HtrA1, an extracellular chaperone and serine protease, functions to maintain the optimal integrity of the RPE-BM interface during the aging process and that reduced expression of HTRA1 mRNA and protein in Chr10 risk donors impairs this protective function, leading to increased risk of AMD pathogenesis. HtrA1 augmentation, not inhibition, in high-risk patients should be considered as a potential therapy for AMD.

OMIP-095: 40-Color spectral flow cytometry delineates all major leukocyte populations in murine lymphoid tissues.

High-dimensional immunoprofiling is essential for studying host response to immunotherapy, infection, and disease in murine model systems. However, the difficulty of multiparameter panel design combined with a lack of existing murine tools has prevented the comprehensive study of all major leukocyte phenotypes in a single assay. Herein, we present a 40-color flow cytometry panel for deep immunophenotyping of murine lymphoid tissues, including the spleen, blood, Peyer's patches, inguinal lymph nodes, bone marrow, and thymus. This panel uses a robust set of surface markers capable of differentiating leukocyte subsets without the use of intracellular staining, thus allowing for the use of cells in downstream functional experiments or multiomic analyses. Our panel classifies T cells, B cells, natural killer cells, innate lymphoid cells, monocytes, macrophages, dendritic cells, basophils, neutrophils, eosinophils, progenitors, and their functional subsets by using a series of co-stimulatory, checkpoint, activation, migration, and maturation markers. This tool has a multitude of systems immunology applications ranging from serial monitoring of circulating blood signatures to complex endpoint analysis, especially in pre-clinical settings where treatments can modulate leukocyte abundance and/or function. Ultimately, this 40-color panel resolves a diverse array of immune cells on the axes of time, tissue, and treatment, filling the niche for a modern tool dedicated to murine immunophenotyping.

Perceptions Towards HCV Treatment with Direct Acting Antivirals (DAAs): A Qualitative Analysis with Persons with HIV/HCV Co-infection Who Delay or Refuse Treatment.

In the United States, approximately 25% of people with HIV (PWH) are co-infected with hepatitis C (HCV). Since 2014, highly effective and well-tolerated direct-acting antivirals (DAAs) have revolutionized HCV treatment. Uptake of DAAs by people with HIV/HCV co-infection has improved but remains suboptimal due to system, provider, and patient-level barriers. To explore patient-level issues by better understanding their attitudes towards DAA treatment, we conducted qualitative interviews with 21 persons with HIV/HCV co-infection who did not consent to DAA treatment or delayed treatment for at least 1 year after diagnosis. We found PWH perceived DAA treatment barriers and facilitators on multiple levels of the social-ecological environment: the individual (HCV disease and treatment literacy), interpersonal (peer influence), institutional (media and healthcare provider relationship), and structural levels (treatment cost and adherence support). Recommendations to improve DAA treatment uptake include HCV-treatment adherence support, HCV disease and treatment literacy training (particularly for substance use and DAA treatment interactions), and encouraging PWH who have successfully completed DAA treatment to speak with their peers.

Challenges to Achieving HCV Micro-Elimination in People With HIV in the United States: Provider Perspectives and the Role of Implicit Bias.

The prevalence of HIV/HCV (hepatitis C virus) co-infection is high particularly in persons who inject drugs (PWID) and is increasing because of the evolving opioid epidemic in the United States. The introduction of effective antiviral medications for HCV has raised the strategic goal of HCV micro-elimination, and efforts to understand the barriers to treatment are critical. In this study, we explored the provider perspective of factors that inhibit HCV micro-elimination efforts in people with HIV (PWH), including the role of implicit bias and related stigma in providers' health care decision making. We used the mixed-methods approach of nominal group technique (NGT) with 14 participants from 11 different clinics engaged in two virtual focus group sessions (n = 5 and n = 9). Responses from the NGTs were rank ordered during the sessions to identify providers' perspectives of major barriers and facilitators, then identified possible implicit bias after the NGTs concluded. There were 12 responses given for micro-elimination barriers with the three most prioritized being housing instability, medication nonadherence concerns, and inability to motivate patients. Of these, eight were categorized as potential implicit biases. Among the 14 responses given for facilitators of treatment, the three major solutions included distributive models of care, improved provider knowledge, and increased patient engagement. Although the solutions offered were insightful, there was consensus that the individual lives of patients were the root cause of most barriers to care. We recommend further research on behavioral design interventions that promote patients' involvement in decision making and focus on patients' eligibility criteria for HCV treatment as opposed to providers' perceived barriers to treatment.

Creating a Longitudinal HCV Care Cascade for Persons With HIV/HCV Coinfection in Selected HIV Clinics Using Data to Care Methods.

Highly effective direct-acting antiviral (DAA) treatments for hepatitis C have led to strategic goals promoting hepatitis C virus (HCV) cure particularly in focus populations including persons with HIV/HCV coinfection. Implementing treatment more broadly requires both clinic-level and public health approaches such as those inherent in Data to Care (D2C) originally developed to improve the treatment cascade for persons with HIV (PWH). We used D2C methods to characterize and improve HCV treatment for persons with HIV/HCV coinfection among 11 HIV clinics in Connecticut cities with high PWH prevalence. Providers who were local champions in HCV treatment were recruited to participate along with clinic data staff and were key to quality improvement via practice transformation. We developed a methodology whereby clinic-generated lists of PWH receiving care from 2009 to 2018 were matched by CT Department of Public Health (DPH) against the state-wide HCV surveillance system. The resultant coinfection list was reviewed by clinical staff who designated HCV treatment status, enabling creation of individual clinic-level HCV treatment cascades. Data from DPH, especially current residency and deaths, enabled better characterization and allowed for refinement of longitudinal cascades. There were 1,496 patients with HIV/HCV coinfection. Sustained virologic response (SVR) rates varied by clinic (range, 44%-100%) with an aggregate SVR rate of 71% in September 2020. SVR rates improved during the project through a combination of increased treatment initiation/completion as well as data clean-up including serial updates of patient treatment status. Lack of treatment initiation was associated with being female (odds ratio [OR] = 2.18) and not having HIV viral suppression (OR = 3.24).

Improving HIV and HCV Testing in Substance Use Disorder Programs (SUDs) That Provide Medications for Opiate Use Disorder (MOUD): Role of Addressing Barriers and Implementing Universal and Site-Specific Approaches.

Introduction. National strategies to end the HIV epidemic and eliminate hepatitis c (HCV) through a syndemic approach require improvements in testing for HIV and HCV. Given the intersection of the opioid crisis with HIV and HCV acquisition, substance use disorder (SUD) treatment centers providing medications for opiate use disorder (MOUD) provide a critical opportunity to expand testing. Rates of testing in MOUD clinics have been suboptimal. Method. We employed the Nominal Group Technique (NGT), Ishikawa cause and effect diagrams, and individualized Quality Improvement (QI) efforts at two SUD clinics (SUD A and B) in Connecticut (CT) as part of an HRSA-funded grant focused on improving HCV cure in persons with HIV/HCV coinfection. Baseline and longitudinal data were collected on rates of HIV and HCV testing and positivity as well as linkage to treatment. Results. Between April 1, 2019, and May 31, 2021, for SUD A and B respectively, HIV testing increased from 13% to 90% and 33% to 83%; HCV testing increased from 4% to 90% and 30% to 82%, with few reported cases of HIV/HCV coinfection. HCV testing revealed new and prior diagnoses at both sites, with subsequent referrals for treatment. Qualitative assessments identified best practices which included the institution of formal policies and procedures, streamlining of testing logistics, designation of a site champion, and broadening relevant education to staff and clients. Conclusion. Strategic assessment of barriers and facilitators to HIV and HCV testing at MOUD clinics can lead to improved testing and referral rates that are key to improving the cascade of care for both diseases.

National Needs Assessment for a Pediatric Trauma Toolkit: General Requirements for Moderate- and High-Volume Hospitals.

BACKGROUND: Optimal outcomes have been reported for children treated at pediatric trauma centers; however, most children are treated at nonpediatric trauma centers or nonpediatric general hospitals. Hospitals that are not verified or designated pediatric trauma centers may lack the training and level of comfort and skill when treating severely injured children. OBJECTIVE: This study focused on identifying common pediatric guidelines for standardization across all trauma centers to inform a pediatric trauma toolkit. METHODS: A needs assessment survey was developed highlighting the guidelines from an expert committee review. The purpose of the survey was to prioritize needed items for the development of a pediatric trauma toolkit. Professional trauma organizations distributed the survey to their respective memberships to ensure good representation of people who care for traumatically injured children and work in trauma centers. Deidentified survey results were analyzed with frequencies and descriptive statistics provided. Data were compared by hospital trauma verification level using a chi-square test. The value of p < .05 was considered statistically significant. RESULTS: A total of 303 people responded to the survey. The majority of respondents reported a high value in the creation of a pediatric trauma toolkit for the guidelines that were included. There was variability in the reported access to the guidelines, indicating a significant need for the toolkit development and dissemination. CONCLUSION: As expected, Level III centers reported the largest gaps in access to standardized pediatric guidelines and demonstrated high levels of interest and need.

Tracking expression and subcellular localization of RNA and protein species using high-throughput single cell imaging flow cytometry.

We report a high-throughput application of multispectral imaging flow cytometry (MIFC) for analyzing the expression and localization of both RNA and protein molecules in a heterogeneous population of cells. The approach was developed using polyadenylated nuclear (PAN) RNA, an abundant, noncoding RNA expressed by Kaposi's sarcoma-associated herpesvirus (KSHV) during the lytic phase of infection. High levels of PAN RNA are, in part, dependent on its interaction with poly(A)-binding protein C1 (PABPC1), which relocalizes from the cytoplasm to the nucleus of lytically infected cells. We quantitatively tracked the cytoplasmic to nuclear translocation of PABPC1 and examined how this translocation relates to the expression and localization of viral RNA and protein molecules in KSHV-infected cells. This high-throughput approach will be useful for other systems in which changes in subcellular localization of RNA and protein molecules need to be monitored simultaneously.

Associations between injection risk and community disadvantage among suburban injection drug users in southwestern Connecticut, USA.

Increases in drug abuse, injection, and opioid overdoses in suburban communities led us to study injectors residing in suburban communities in southwestern Connecticut, US. We sought to understand the influence of residence on risk and injection-associated diseases. Injectors were recruited by respondent-driven sampling and interviewed about sociodemographics, somatic and mental health, injection risk, and interactions with healthcare, harm reduction, substance abuse treatment, and criminal justice systems. HIV, hepatitis B and C (HBV and HCV) serological testing was also conducted. Our sample was consistent in geographic distribution and age to the general population and to the patterns of heroin-associated overdose deaths in the suburban towns. High rates of interaction with drug abuse treatment and criminal justice systems contrasted with scant use of harm reduction services. The only factors associated with both dependent variables-residence in less disadvantaged census tracts and more injection risk-were younger age and injecting in one's own residence. This contrasts with the common association among urban injectors of injection-associated risk behaviors and residence in disadvantaged communities. Poor social support and moderate/severe depression were associated with risky injection practices (but not residence in specific classes of census tracts), suggesting that a region-wide dual diagnosis approach to the expansion of harm reduction services could be effective at reducing the negative consequences of injection drug use.

Implementing a Surveillance-Based Approach to Create a Statewide Viral Clearance Cascade for Hepatitis C Among People With HIV and HCV Coinfection in Connecticut.

OBJECTIVES: Highly effective direct-acting antiviral medications have made it feasible to achieve elimination of hepatis C virus (HCV), including for people with HIV and HCV coinfection. The Centers for Disease Control and Prevention offers guidance for a laboratory surveillance-based HCV viral clearance cascade, which allows public health departments to track the outcomes of people with HCV based on the following steps: ever infected, virally tested, initial infection, and cured or cleared. We examined the feasibility of this approach among people with HIV and HCV coinfection in Connecticut. METHODS: We matched an HIV surveillance database, which included cases from the enhanced HIV/AIDS Reporting System as of December 31, 2019, and the HCV surveillance database, the Connecticut Electronic Disease Surveillance System, to define a cohort of coinfected people. We used HCV laboratory results obtained from January 1, 2016, through August 3, 2020, to determine HCV status. RESULTS: Of 1361 people who were ever infected with HCV as of December 31, 2019, 1256 (92.3%) received HCV viral testing, 865 of 1256 people tested (68.9%) were HCV infected, and 336 of 865 infected people (38.8%) were cleared or cured. People who had undetectable HIV viral loads at most recent HIV test (<200 copies/mL) were more likely than those with detectable HIV viral loads to achieve HCV cure (P = .02). CONCLUSIONS: A surveillance-based approach that includes data based on the Centers for Disease Control and Prevention HCV viral clearance cascade is feasible to implement, can help track population-level outcomes longitudinally, and can help identify gaps to inform HCV elimination strategies.

Using Data-To-Care Strategies to Optimize the HIV Care Continuum in Connecticut: Results From a Randomized Controlled Trial.

BACKGROUND: Re-engaging people with HIV who are newly out-of-care remains challenging. Data-to-care (D2C) is a potential strategy to re-engage such individuals. METHODS: A prospective randomized controlled trial compared a D2C strategy using a disease intervention specialist (DIS) vs standard of care where 23 HIV clinics in 3 counties in Connecticut could re-engage clients using existing methods. Using a data reconciliation process to confirm being newly out-of-care, 655 participants were randomized to DIS (N = 333) or standard of care (N = 322). HIV care continuum outcomes included re-engagement at 90 days, retention in care, and viral suppression by 12 months. Multivariable regression models were used to assess factors predictive of attaining HIV care continuum outcomes. RESULTS: Participants randomized to DIS were more likely to be re-engaged at 90 days (adjusted odds ratios [aOR] = 1.42, P = 0.045). Independent predictors of re-engagement at 90 days were age older than 40 years (aOR = 1.84, P = 0.012) and perinatal HIV risk category (aOR = 3.19, P = 0.030). Predictors of retention at 12 months included re-engagement at 90 days (aOR = 10.31, P < 0.001), drug injection HIV risk category (aOR = 1.83, P = 0.032), detectable HIV-1 RNA before randomization (aOR = 0.40, P = 0.003), and county (Hartford aOR = 1.74, P = 0.049; New Haven aOR = 1.80, P = 0.030). Predictors of viral suppression included re-engagement at 90 days (aOR = 2.85, P < 0.001), retention in HIV care (aOR = 7.07, P < 0.001), and detectable HIV-1 RNA prerandomization (aOR = 0.23, P < 0.001). CONCLUSIONS: A D2C strategy significantly improved re-engagement at 90 days. Early re-engagement improved downstream benefits along the HIV care continuum like retention in care and viral suppression at 12 months. Moreover, other factors predictive of care continuum outcomes can be used to improve D2C strategies.

Connections Network: Harnessing the Collective Influence of Grassroots Leaders to Address Health-Related Problems in Hawkins and Hancock County, TN.

In March 2021, grassroots leaders in two counties in northeast Tennessee formed a new network called Connections. Leaders are working to strengthen the capacity of the network and member organizations by promoting partnerships as vital to address effectively rural social determinants of health. Connections provides network members with capacity-building tools and resources, including two funding opportunities, to achieve their missions and sustain impact. Network members are also aligning around common goals to address the socioeconomic conditions affecting health outcomes. Connections will utilize findings from network activities and collaborations to identify synergies that can accelerate improvements in community health and well-being.

Costs and cost-effectiveness of a collaborative data-to-care intervention for HIV treatment and care in the United States.

INTRODUCTION: Data-to-care programmes utilize surveillance data to identify persons who are out of HIV care, re-engage them in care and improve HIV care outcomes. We assess the costs and cost-effectiveness of re-engagement in an HIV care intervention in the United States. METHODS: The Cooperative Re-engagement Control Trial (CoRECT) employed a data-to-care collaborative model between health departments and HIV care providers, August 2016-July 2018. The health departments in Connecticut (CT), Massachusetts (MA) and Philadelphia (PHL) collaborated with HIV clinics to identify newly out-of-care patients and randomize them to receive usual linkage and engagement in care services (standard-of-care control arm) or health department-initiated active re-engagement services (intervention arm). We used a microcosting approach to identify the activities and resources involved in the CoRECT intervention, separate from the standard-of-care, and quantified the costs. The cost data were collected at the start-up and recurrent phases of the trial to incorporate potential variation in the intervention costs. The costs were estimated from the healthcare provider perspective. RESULTS: The CoRECT trial in CT, MA and PHL randomly assigned on average 327, 316 and 305 participants per year either to the intervention arm (n = 166, 159 and 155) or the standard-of-care arm (n = 161, 157 and 150), respectively. Of those randomized, the number of participants re-engaged in care within 90 days in the intervention and standard-of-care arms was 85 and 70 in CT, 84 and 70 in MA, and 98 and 67 in PHL. The additional number of participants re-engaged in care in the intervention arm compared with those in the standard-of-care arm was 15 (CT), 14 (MA) and 31 (PHL). We estimated the annual total cost of the CoRECT intervention at $490,040 in CT, $473,297 in MA and $439,237 in PHL. The average cost per participant enrolled was $2952, $2977 and $2834 and the average cost per participant re-engaged in care was $5765, $5634 and $4482. We estimated an incremental cost per participant re-engaged in care at $32,669 (CT), $33,807 (MA) and $14,169 (PHL). CONCLUSIONS: The costs of the CoRECT intervention that identified newly out-of-care patients and re-engaged them in HIV care are comparable with other similar interventions, suggesting a potential for its cost-effectiveness in the US context.

Mechanisms of spatial and temporal development of autoimmune vitiligo in tyrosinase-specific TCR transgenic mice.

Generalized vitiligo is thought to have an autoimmune etiology and has been correlated with the presence of CD8 T cells specific for melanocyte differentiation Ag. However, limited animal models for the disease have hampered its understanding. Thus, we generated TCR transgenic mice that recognize an epitope of the melanocyte protein, tyrosinase. These animals develop vitiligo with strikingly similar characteristics to the human disease. Vitiligo develops temporally and spatially, with juvenile lesions forming bilaterally in head and facial areas, and only arising later in the body of adult animals. Vitiligo is entirely dependent on CD8 T cells, whereas CD4 T cells exert a negative regulatory effect. Importantly, CD8 T cells can be pervasively present in the skin in the steady state without inducing vitiligo in most areas. This points to developmental differences in melanocyte susceptibility and/or immunological effector mechanisms over time, or in different body locations. Disease is strongly dependent on both IFN-gamma and CXCR3, whereas dependence on CCR5 is more limited, and both CCR4 and perforin are dispensable. Genetic ablation of CXCR3 or IFN-gamma also resulted in scarce CD8 T cell infiltration into the skin. Our results identify unexpected complexity in vitiligo development and point toward possible therapeutic interventions.

Strategies and challenges in eliciting immunity to melanoma.

The ability of CD8+ T cells to recognize melanoma tumors has led to the development of immunotherapeutic approaches that use the antigens CD8+ T cells recognize. However, clinical response rates have been disappointing. Here we summarize our work to understand the mechanisms of self-tolerance that limit responses to currently utilized antigens and our approach to identify new antigens directly tied to malignancy. We also explore several aspects of the anti-tumor immune response induced by peptide-pulsed dendritic cells (DCs). DCs differentially augment the avidity of recall T cells specific for self-antigens and overcome a process of aberrant CD8+ T-cell differentiation that occurs in tumor-draining lymph nodes. DC migration is constrained by injection route, resulting in immune responses in localized lymphoid tissue, and differential control of tumors depending on their location in the body. We demonstrate that CD8+ T-cell differentiation in different lymphoid compartments alters the expression of homing receptor molecules and leads to the presence of systemic central memory cells. Our studies highlight several issues that must be addressed to improve the efficacy of tumor immunotherapy.

Rethinking pediatric trauma triage.

Despite advances in the delivery of trauma care, trauma remains the leading cause of death amongst the pediatric population within the United States and is one of the leading causes of death in children worldwide.  Accurately triaging pediatric trauma patients is essential to minimize preventable mortality without burdening the system by utilizing unnecessary resources.  This article will review the accuracy of current pediatric trauma triage practices and how it will evolve in the future including moving away from mechanism of injury towards physiologic scoring tools such as the pediatric age-adjust shock index, and intervention-based systems including. Need for Surgeon Presence and Need For Trauma Intervention. This paper will also present evidence regarding over-utilization of air transport for pediatric trauma patients and the associated unnecessary costs placed on the trauma system.

Advancing data to care strategies for persons with HIV using an innovative reconciliation process.

BACKGROUND: UN AIDS has set ambitious 95-95-95 HIV care continuum targets for global HIV elimination by 2030. The U.S. HIV Care Continuum in 2018 showed that 65% of persons with HIV(PWH) are virally suppressed and 58% retained in care. Incomplete care-engagement not only affects individual health but drives ongoing HIV transmission. Data to Care (D2C) is a strategy using public health surveillance data to identify and re-engage out-of-care (OOC) PWH. Optimization of this strategy is needed. SETTING: Statewide partnership with Connecticut Department of Public Health (CT DPH), 23 HIV clinics and Yale University School of Medicine (YSM). Our site was one of 3 participants in the CDC-sponsored RCT evaluating the efficacy of DPH-employed Disease Intervention Specialists (DIS) for re-engagement in care. METHODS: From 11/2016-7/2018, a data reconciliation process using public health surveillance and clinic visit data was used to identify patients eligible for randomization (defined as in-Care for 12 months and OOC for subsequent 6-months) to receive DIS intervention. Clinic staff further reviewed this list and designated those who would not be randomized based on established criteria. RESULTS: 2958 patients were eligible for randomization; 655 (22.1%) were randomized. Reasons for non-randomizing included: well patient [499 (16.9%)]; recent visit [946 (32.0%)]; upcoming visit [398 (13.5%)]. Compared to non-randomized patients, those who were randomized were likely to be younger (mean age 46.1 vs. 51.6, p < .001), Black (40% vs 35%)/Hispanic (37% vs 32.8%) [(p < .001)], have CD4<200 cells/ul (15.9% vs 8.5%, p < .001) and viral load >20 copies/ml (43.8% vs. 24.1%, 0<0.001). Extrapolating these estimates to a statewide HIV care continuum suggests that only 8.3% of prevalent PWH are truly OOC. CONCLUSIONS: A D2C process that integrated DPH surveillance and clinic data successfully refined the selection of newly OOC PWH eligible for DIS intervention. This approach more accurately reflects real world care engagement and can help prioritize DPH resources.

In situ T-cell transfection by anti-CD3-conjugated lipid nanoparticles leads to T-cell activation, migration, and phenotypic shift.

Ex vivo programming of T cells can be efficacious but is complex and expensive; therefore, the development of methods to transfect T cells in situ is important. We developed and optimized anti-CD3-targeted lipid nanoparticles (aCD3-LNPs) to deliver tightly packed, reporter gene mRNA specifically to T cells. In vitro, targeted LNPs efficiently delivered mCherry mRNA to Jurkat T cells, and T-cell activation and depletion were associated with aCD3 antibody coating on the surface of LNPs. aCD3-LNPs, but not non-targeted LNPs, accumulated within the spleen following systemic injection, with mCherry and Fluc signals visible within 30 min after injection. At 24 h after aCD3-LNP injection, 2-4% of all splenic T cells and 2-7% of all circulating T cells expressed mCherry, and this was dependent on aCD3 coating density. Targeting and transfection were accompanied by systemic CD25+, OX40+, and CD69+ T-cell activation with temporary CD3e ligand loss and depletion of splenic and circulating subsets. Migration of splenic CD8a+ T cells from the white-pulp to red-pulp, and differentiation from naïve to memory and effector phenotypes, followed upon aCD3-LNP delivery. Additionally, aCD3-LNP injection stimulated the secretion of myeloid-derived chemokines and T-helper cytokines into plasma. Lastly, we administered aCD3-LNPs to tumor bearing mice and found that transfected T cells localized within tumors and tumor-draining lymph nodes following immunotherapy treatment. In summary, we show that CD3-targeted transfection is feasible, yet associated with complex immunological consequences that must be further studied for potential therapeutic applications.

Double emulsion flow cytometry with high-throughput single droplet isolation and nucleic acid recovery.

Droplet microfluidics has made large impacts in diverse areas such as enzyme evolution, chemical product screening, polymer engineering, and single-cell analysis. However, while droplet reactions have become increasingly sophisticated, phenotyping droplets by a fluorescent signal and sorting them to isolate individual variants-of-interest at high-throughput remains challenging. Here, we present sdDE-FACS (s[combining low line]ingle d[combining low line]roplet D[combining low line]ouble E[combining low line]mulsion-FACS), a new method that uses a standard flow cytometer to phenotype, select, and isolate individual double emulsion droplets of interest. Using a 130 µm nozzle at high sort frequency (12-14 kHz), we demonstrate detection of droplet fluorescence signals with a dynamic range spanning 5 orders of magnitude and robust post-sort recovery of intact double emulsion (DE) droplets using 2 commercially-available FACS instruments. We report the first demonstration of single double emulsion droplet isolation with post-sort recovery efficiencies >70%, equivalent to the capabilities of single-cell FACS. Finally, we establish complete downstream recovery of nucleic acids from single, sorted double emulsion droplets via qPCR with little to no cross-contamination. sdDE-FACS marries the full power of droplet microfluidics with flow cytometry to enable a variety of new droplet assays, including rare variant isolation and multiparameter single-cell analysis.

Trends in Abstinence and Retention Associated with a Medication-Assisted Treatment Program for People with Opioid Use Disorders.

BACKGROUND: Medication-assisted treatment (MAT) is an evidence-based program for patients with opioid use disorders. Yet, within the state of Utah, MAT had not been widely available, promoted, or adopted within the public sector. Recognizing the potential benefit, a collective impact approach was used to promote social change and increase the use of MAT in the community for treatment of opioid use disorders. OBJECTIVE: Conduct a retrospective, observational case series study to measure the effect of a community-based, collective impact approach implementing the MAT program to improve the rate of abstinence and retention among individuals identified with an opioid use disorder in three Utah counties. METHODS: The study was designed and implemented by the Utah Opioid Community Collaborative (OCC) using a collective impact approach, which included broad sector coordination (public-private collaboration), a common agenda, participation in mutually reinforcing activities, continuous communication, consistent measurement of results, and identification of a backbone organization. The MAT intervention program includes use of medications approved by the U.S. Food and Drug Administration in combination with counseling and behavioral therapies delivered within two community sites. Analysis was performed over time to describe the rate of abstinence and retention associated with participation in the MAT program during 2015 through 2017. RESULTS: Of the 339 identified with risk of an opioid use disorders, 228 enrolled in the MAT Program. At MAT enrollment, average age was 32.6 ± 8.2 years old and 58.0% were female. At 365 days after MAT enrollment, 84% of participants were abstinent from opioid substances and 62% from all illicit substances. CONCLUSIONS: Use of a collective impact approach provides a successful mobilization framework in Utah for increasing community engagement and expanding patient access to underresourced MAT programs while suggesting a high rate of abstinence from illicit substances at 12 months.