The Invariant NKT Cell Response Has Differential Signaling Requirements during Antigen-Dependent and Antigen-Independent Activation.

Invariant NKT (iNKT) cells are an innate-like population characterized by their recognition of glycolipid Ags and rapid cytokine production upon activation. Unlike conventional T cells, which require TCR ligation, iNKT cells can also be stimulated independently of their TCR. This feature allows iNKT cells to respond even in the absence of glycolipid Ags, for example, during viral infections. Although the TCR-dependent and -independent activation of iNKT cells have been relatively well established, the exact contributions of IL-12, IL-18, and TLRs remain unclear for these two activation pathways. To definitively investigate how these components affect the direct and indirect stimulation of iNKT cells, we used mice deficient for either MyD88 or the IL-12Rß2 in the T cell lineage. Using these tools, we demonstrate that IL-12, IL-18, and TLRs are completely dispensable for the TCR activation pathway when a strong agonist is used. In contrast, during murine CMV infection, when the TCR is not engaged, IL-12 signaling is essential, and TLR signaling is expendable. Importantly, to our knowledge, we discovered an intrinsic requirement for IL-18 signaling by splenic iNKT cells but not liver iNKT cells, suggesting that there might be diversity, even within the NKT1 population.

Cooperative activation of cardiac transcription through myocardin bridging of paired MEF2 sites.

Enhancers frequently contain multiple binding sites for the same transcription factor. These homotypic binding sites often exhibit synergy, whereby the transcriptional output from two or more binding sites is greater than the sum of the contributions of the individual binding sites alone. Although this phenomenon is frequently observed, the mechanistic basis for homotypic binding site synergy is poorly understood. Here, we identify a bona fide cardiac-specific Prkaa2 enhancer that is synergistically activated by homotypic MEF2 binding sites. We show that two MEF2 sites in the enhancer function cooperatively due to bridging of the MEF2C-bound sites by the SAP domain-containing co-activator protein myocardin, and we show that paired sites buffer the enhancer from integration site-dependent effects on transcription in vivo Paired MEF2 sites are prevalent in cardiac enhancers, suggesting that this might be a common mechanism underlying synergy in the control of cardiac gene expression in vivo.

Adults With Asthma Experience No Increase in Asthma-related Exacerbations When Digital Communication Technology Tools Are Employed to Offset Provider Workload: A Pragmatic Randomized Trial.

BACKGROUND: Challenges to health care efficiency are increasingly addressed with the help of digital communication technology tools (DCTs). OBJECTIVE: The objective of this study was to test whether DCT, compared with Usual Care, can reduce health care clinician burden without increasing asthma-related exacerbations among patients with asthma in a large integrated health care system. RESEARCH DESIGN: The (Breathewell) program was a pragmatic, randomized trial at (Kaiser Permanente Colorado), where asthma nurses screen patients for poor symptom control when beta2-agonist refill requests came within 60 days of previous fill or in the absence of a controller medication fill within 4 months (beta2-agonist overfill). A total of 14,978 adults with asthma were randomized to Usual Care or 1 of 2 DCT intervention groups (Text/Phone call or Email). SUBJECTS: Participants included adults 18 and older with an asthma diagnosis at the time of randomization and no history of chronic obstructive pulmonary disease. MEASURES: Primary outcome measures included asthma-related health care resource utilization (eg, asthma nurse contacts), medication use, and exacerbations. RESULTS: A total of 1933 patients had 4337 events which met beta2-agonist overfill criteria. Of the 2874 events in the intervention arm, 1188 (41%) were resolved by DCT contact and did not require additional clinician contact. Asthma medication use and exacerbations over 12 months did not differ among the 3 groups. CONCLUSIONS: DCT tools can successfully contact adult asthma patients to screen for symptoms and facilitate intervention. The absence of differences in medication fills and health care utilization indicates that the strategic replacement of nursing interventions by digital outreach did not reduce treatment adherence or compromise health care outcomes.

A standardized approach to bereavement risk-screening: a quality improvement project.

Objectives: Identifying family members at-risk of poor bereavement outcomes poses a challenge for clinicians, resulting in inconsistent bereavement follow-up. The current quality improvement study tests a method for identification of at-risk family members, and describes follow-up they received from the bereavement service at Dana-Farber Cancer Institute.Design: A standardized bereavement risk assessment, referral and follow-up process was piloted as part of a quality improvement project using a plan-do-study-act approach (PDSA).Methods: A convenience sample of eleven clinical social workers completed paper and pencil bereavement risk-screening assessments using the Bereavement Risk-Screening Tool (BRST) on a sample of bereaved family members known to them. The results of the BRST were passed onto the bereavement program for follow-up.Findings: Eleven out of a total of 17 social workers participated in the study. Social workers screened 100% (52/52) of identified bereaved family members, corresponding to 52 patient deaths. Approximately half (28/52) were identified as being 'at-risk' of a poor bereavement outcome based on the social worker's consideration of the presence of potential risk-factors and their response to a prediction-type question about the bereaved individual's future coping. 'Lack of preparation for the death', 'unexpected death within the context of an illness' and 'witnessing a difficult death' were the most commonly identified risk factors. Of those individuals who were identified to be 'at-risk', 89% received an outreach attempt by telephone from the director of bereavement services, surpassing our project target of 80%. Conclusions: The BRST has the potential to help clinicians in health care settings identify those family members who might be considered at heightened risk of a poor bereavement outcome, facilitating early outreach and recommendations for support. The tool was easy to complete and helped streamline the referral process to the bereavement program.

Ultrasensitive automated RNA in situ hybridization for kappa and lambda light chain mRNA detects B-cell clonality in tissue biopsies with performance comparable or superior to flow cytometry.

The assessment of B-cell clonality is a critical component of the evaluation of suspected lymphoproliferative disorders, but analysis from formalin-fixed, paraffin-embedded tissues can be challenging if fresh tissue is not available for flow cytometry. Immunohistochemical and conventional bright field in situ hybridization stains for kappa and lambda are effective for evaluation of plasma cells but are often insufficiently sensitive to detect the much lower abundance of light chains present in B-cells. We describe an ultrasensitive RNA in situ hybridization assay that has been adapted for use on an automated immunohistochemistry platform and compare results with flow cytometry in 203 consecutive tissues and 104 consecutive bone marrows. Overall, in 203 tissue biopsies, RNA in situ hybridization identified light chain-restricted B-cells in 85 (42%) vs 58 (29%) by flow cytometry. Within 83 B-cell non-Hodgkin lymphomas, RNA in situ hybridization identified restricted B-cells in 74 (89%) vs 56 (67%) by flow cytometry. B-cell clonality could be evaluated in only 23/104 (22%) bone marrow cases owing to poor RNA preservation, but evaluable cases showed 91% concordance with flow cytometry. RNA in situ hybridization allowed for recognition of biclonal/composite lymphomas not identified by flow cytometry and highlighted unexpected findings, such as coexpression of kappa and lambda RNA in 2 cases and the presence of lambda light chain RNA in a T lymphoblastic lymphoma. Automated RNA in situ hybridization showed excellent interobserver reproducibility for manual evaluation (average K=0.92), and an automated image analysis system showed high concordance (97%) with manual evaluation. Automated RNA in situ hybridization staining, which can be adopted on commonly utilized immunohistochemistry instruments, allows for the interpretation of clonality in the context of the morphological features in formalin-fixed, paraffin-embedded tissues with a clinical sensitivity similar or superior to flow cytometry.

NFIL3 Expression Distinguishes Tissue-Resident NK Cells and Conventional NK-like Cells in the Mouse Submandibular Glands.

The submandibular salivary gland (SMG), a major site of persistent infection for many viruses, contains a large NK cell population. Using NFIL3-deficient mice, PLZF reporter/fate mapping mice, and mixed bone marrow chimeras, we identified two distinct populations of NK cells in the SMG. Although phenotypically unique, the main population relies on NFIL3, but not PLZF, for development and, therefore, is developmentally similar to the conventional NK cell subset. In contrast, we found that approximately one quarter of the SMG NK cells develop independently of NFIL3. Interestingly, NFIL3-independent SMG tissue-resident NK (trNK) cells are developmentally distinct from liver trNK cells. We also demonstrated that the SMG NK cell hyporesponsive phenotype during murine CMV infection is tissue specific and not cell intrinsic. In contrast, NFIL3-independent SMG trNK cells are intrinsically hyporesponsive. Altogether, our data show that the SMG tissue environment shapes a unique repertoire of NK-like cells with distinct phenotypes.

Role of SHIP1 in Invariant NKT Cell Development and Functions.

SHIP1 is a 5'-inositol phosphatase known to negatively regulate the signaling product of the PI3K pathway, phosphatidylinositol (3-5)-trisphosphate. SHIP1 is recruited to a large number of inhibitory receptors expressed on invariant NK (iNKT) cells. We hypothesized that SHIP1 deletion would have major effects on iNKT cell development by altering the thresholds for positive and negative selection. Germline SHIP1 deletion has been shown to affect T cells as well as other immune cell populations. However, the role of SHIP1 on T cell function has been controversial, and its participation on iNKT cell development and function has not been examined. We evaluated the consequences of SHIP1 deletion on iNKT cells using germline-deficient mice, chimeric mice, and conditionally deficient mice. We found that T cell and iNKT cell development are impaired in germline-deficient animals. However, this phenotype can be rescued by extrinsic expression of SHIP1. In contrast, SHIP1 is required cell autonomously for optimal iNKT cell cytokine secretion. This suggests that SHIP1 calibrates the threshold of iNKT cell reactivity. These data further our understanding of how iNKT cell activation is regulated and provide insights into the biology of this unique cell lineage.

Fully Automated RNAscope In Situ Hybridization Assays for Formalin-Fixed Paraffin-Embedded Cells and Tissues.

Biomarkers such as DNA, RNA, and protein are powerful tools in clinical diagnostics and therapeutic development for many diseases. Identifying RNA expression at the single cell level within the morphological context by RNA in situ hybridization provides a great deal of information on gene expression changes over conventional techniques that analyze bulk tissue, yet widespread use of this technique in the clinical setting has been hampered by the dearth of automated RNA ISH assays. Here we present an automated version of the RNA ISH technology RNAscope that is adaptable to multiple automation platforms. The automated RNAscope assay yields a high signal-to-noise ratio with little to no background staining and results comparable to the manual assay. In addition, the automated duplex RNAscope assay was able to detect two biomarkers simultaneously. Lastly, assay consistency and reproducibility were confirmed by quantification of TATA-box binding protein (TBP) mRNA signals across multiple lots and multiple experiments. Taken together, the data presented in this study demonstrate that the automated RNAscope technology is a high performance RNA ISH assay with broad applicability in biomarker research and diagnostic assay development. J. Cell. Biochem. 117: 2201-2208, 2016. © 2016 Wiley Periodicals, Inc.

The role of MHC class Ib-restricted T cells during infection.

Even though major histocompatibility complex (MHC) class Ia and many Ib molecules have similarities in structure, MHC class Ib molecules tend to have more specialized functions, which include the presentation of non-peptidic antigens to non-classical T cells. Likewise, non-classical T cells also have unique characteristics, including an innate-like phenotype in naïve animals and rapid effector functions. In this review, we discuss the role of MAIT and NKT cells during infection but also the contribution of less studied MHC class Ib-restricted T cells such as Qa-1-, Qa-2-, and M3-restricted T cells. We focus on describing the types of antigens presented to non-classical T cells, their response and cytokine profile following infection, as well as the overall impact of these T cells to the immune system.

SLI-1 Cbl inhibits the engulfment of apoptotic cells in C. elegans through a ligase-independent function.

The engulfment of apoptotic cells is required for normal metazoan development and tissue remodeling. In Caenorhabditis elegans, two parallel and partially redundant conserved pathways act in cell-corpse engulfment. One pathway, which includes the small GTPase CED-10 Rac and the cytoskeletal regulator ABI-1, acts to rearrange the cytoskeleton of the engulfing cell. The CED-10 Rac pathway is also required for proper migration of the distal tip cells (DTCs) during the development of the C. elegans gonad. The second pathway includes the receptor tyrosine kinase CED-1 and might recruit membranes to extend the surface of the engulfing cell. Cbl, the mammalian homolog of the C. elegans E3 ubiquitin ligase and adaptor protein SLI-1, interacts with Rac and Abi2 and modulates the actin cytoskeleton, suggesting it might act in engulfment. Our genetic studies indicate that SLI-1 inhibits apoptotic cell engulfment and DTC migration independently of the CED-10 Rac and CED-1 pathways. We found that the RING finger domain of SLI-1 is not essential to rescue the effects of SLI-1 deletion on cell migration, suggesting that its role in this process is ubiquitin ligase-independent. We propose that SLI-1 opposes the engulfment of apoptotic cells via a previously unidentified pathway.

Residential mobility during adolescence: Do even "upward" moves predict dropout risk?

This paper uses the National Longitudinal Study of Adolescent Health to investigate the impact of housing instability in adolescence on the likelihood of subsequent graduation from high school. Combining census data, self-reports, and information about respondents' residential changes, we use the variation in households' number of moves and neighborhood quality to predict whether participants obtain a high school diploma. Controlling for major predictors of housing mobility, students experiencing at least one move over a 12-month period have a roughly 50% decreased likelihood of obtaining a high school diploma by the age of 25. These associations are identified regardless of whether students move to a poorer or less-poor neighborhood. Our results carry implications for the development of housing policies and interventions designed for disadvantaged populations.

Small Felids Coexist in Mixed-Use Landscape in the Bolivian Amazon.

In the face of global species loss, it is paramount to understand the effects of human activity on vulnerable species, particularly in highly diverse, complex systems. The Greater Madidi Landscape in the Bolivian Amazon includes several biodiverse protected areas that were created with the goal of sustaining healthy and diverse ecosystems while not impeding the livelihoods of local indigenous peoples. In this study, we sought to use camera trap data and single-species occupancy analysis to assess the impacts of different forms of human activity on four species of small felids: ocelots (Leopardus pardalis), margays (Leopardus wiedii), jaguarundi (Herpailurus yagouaroundi), and oncilla (Leopardus tigrinus). We modeled both human variables (proximity to indigenous communities, roads, and tourist camps) and non-human variables (terrain ruggedness, proximity to rivers, canopy height, prey availability, and large cat abundance). Margay occupancy was unaffected by any of these human variables and ocelots showed only weak evidence of being affected by tourism. Ocelots were particularly pervasive throughout the study area and were consistently estimated to have high occupancy probability. We did not obtain sufficient data on jaguarundi or oncilla to reliably model these effects. Our results indicate that small cats successfully coexist both with each other and with the surrounding human activity in this unique landscape, which serves as a model for global protected area management.

Genetic hemoglobin disorders, infection, and deficiencies of iron and vitamin A determine anemia in young Cambodian children.

In Cambodia, many factors may complicate the detection of iron deficiency. In a cross-sectional survey, we assessed the role of genetic hemoglobin (Hb) disorders, iron deficiency, vitamin A deficiency, infections, and other factors on Hb in young Cambodian children. Data on sociodemographic status, morbidity, and growth were collected from children (n = 3124) aged 6 to 59 mo selected from 3 rural provinces and Phnom Penh municipality. Blood samples were collected (n = 2695) for complete blood count, Hb type (by DNA analysis), ferritin, soluble transferrin receptor (sTfR), retinol-binding protein (RBP), C-reactive protein, and α(1)-acid glycoprotein (AGP). Genetic Hb disorders, anemia, and vitamin A deficiency were more common in rural than in urban provinces (P < 0.001): 60.0 vs. 40.0%, 58.2 vs. 32.7%, and 7.4 vs. 3.1%, respectively. Major determinants of Hb were age group, Hb type, ferritin, sTfR, RBP, AGP >1.0 g/L (P < 0.001), and rural setting (P < 0.05). Age group, Hb type, RBP, elevated AGP, and rural setting also influenced ferritin and sTfR (P < 0.02). Multiple factors affected anemia status, including the following: age groups 6-11.99 mo (OR: 6.1; 95% CI: 4.3, 8.7) and 12-23.99 mo (OR: 2.7; 95% CI: 2.1, 3.6); Hb type, notably Hb EE (OR: 18.5; 95% CI: 8.5, 40.4); low ferritin (OR: 3.2; 95% CI: 2.2, 4.7); elevated AGP (OR: 1.4; 95% CI: 1.2,1.7); rural setting (OR: 2.3; 95% CI: 1.7, 3.1); low RBP (OR: 3.6; 95% CI: 2.2, 5.9); and elevated sTfR (OR: 2.1; 95% CI: 1.7, 2.7). In Cambodia, where a high prevalence of genetic Hb disorders exists, ferritin and sTfR are of limited use for assessing the prevalence of iron deficiency. New low-cost methods for detecting genetic Hb disorders are urgently required.

Data Sharing to Combat Segregation.

Data sharing between housing and education agencies will provide housing agencies with resources to assist them with efforts to decrease segregation and mitigate the adverse health outcomes experienced by people of color. The Fair Housing Act has the potential to fulfill its original integrationist purpose if housing and education agencies combine resources and data to create and implement fair housing plans. The Biden Administration's restored rule to affirmatively further fair housing pursuant to the Fair Housing Act of 1968 which seeks to reduce segregation and increase housing equity. However, it omits most of the processes set forth by the Obama Administration whereby federal agencies provide the proper tools to housing agencies so that they are able to make data-based decisions about housing policies. This article advocates for the sharing of data between housing and education agencies to optimize the positive impact of fair housing not only within housing, but also on the education, employment, and health opportunities for communities of color.

Simultaneous Visualization of RNA and Protein Expression in Tissue Using a Combined RNAscope™ In Situ Hybridization and Immunofluorescence Protocol.

Gene expression analysis is critical to precisely characterize complex tissues and provide insight into a disease condition. Techniques like PCR, sequencing, and northern blotting are highly sensitive and specific but are unable to provide information about spatial positioning of target genes. Visualization of gene expression with a spatial context can be critical in identifying complex milieus in heterogenous tissues like tumors. The RNAscope in situ hybridization (ISH) technology detects target RNA expression with high sensitivity and specificity at single-cell resolution. To understand the cellular cross talk between different cell populations, it is essential to simultaneously study gene and protein expression within a complex tissue. This chapter details combining the RNAscope ISH assay with immunofluorescence (IF) in one protocol to simultaneously visualize gene expression and protein expression in human tumor tissue and mouse brain tissue.

Planning for Implementation Success Using RE-AIM and CFIR Frameworks: A Qualitative Study.

Background: RE-AIM (Reach, Effectiveness, Adoption, Implementation, Maintenance) and CFIR (Consolidated Framework for Implementation Research) dissemination and implementation frameworks define theory-based domains associated with the adoption, implementation and maintenance of evidence-based interventions. Used together, the two frameworks identify metrics for evaluating implementation success, i.e., high reach and effectiveness resulting in sustained practice change (RE-AIM), and modifiable factors that explain and enhance implementation outcomes (CFIR). We applied both frameworks to study the implementation planning process for a technology-delivered asthma care intervention called Breathewell within an integrated care organization. The goal of the Breathewell intervention is to increase the efficiency of delivering resource-intensive asthma care services. Methods: We reviewed historical documents (i.e., meeting agendas; minutes) from 14 months of planning to evaluate alignment of implementation team priorities with RE-AIM domains. Key content was extracted and analyzed on topics, frequency and amount of discussion within each RE-AIM domain. Implementation team members were interviewed using questions adapted from the CFIR Interview Guide Tool to focus their reflection on the process and contextual factors considered during pre-implementation planning. Documents and transcripts were initially coded using RE-AIM domain definitions, and recoded using CFIR constructs, with intent to help explain how team decisions and actions can contribute to adoption, implementation and maintenance outcomes. Results: Qualitative analysis of team documents and interviews demonstrated strong alignment with the RE-AIM domains: Reach, Effectiveness, and Implementation; and with the CFIR constructs: formal inclusion of provider and staff stakeholders in implementation planning, compatibility of the intervention with workflows and systems, and alignment of the intervention with organizational culture. Focus on these factors likely contributed to RE-AIM outcomes of high implementation fidelity. However, team members expressed low confidence that Breathewell would be adopted and maintained post-trial. A potential explanation was weak alignment with several CFIR constructs, including tension for change, relative priority, and leadership engagement that contribute to organizational receptivity and motivation to sustain change. Conclusions: While RE-AIM provides a practical framework for planning and evaluating practice change interventions to assure their external validity, CFIR explains why implementation succeeded or failed, and when used proactively, identifies relevant modifiable factors that can promote or undermine adoption, implementation, and maintenance.

Refill Reminder Preference and Inhaled Corticosteroid Adherence Among Patients with Asthma.

CONTEXT: Refill reminders can help patients improve adherence to inhaled corticosteroid (ICS) therapy. However, little is known about patient preferences for reminder type or whether patients who express a preference differ from patients who do not. OBJECTIVES: To describe patient preferences for ICS prescription refill reminder type and to compare baseline ICS therapy adherence, measured as proportion of days covered (PDC) 1 year before initiating preference-based reminders, between patients who did and did not express a preference. DESIGN: This substudy within a randomized multi-intervention study was conducted at Kaiser Permanente Colorado. Adults with asthma randomized to intervention were offered the opportunity to choose text, telephone, or email reminders. Patients who did and did not provide a preference were compared by baseline characteristics using log-binomial models. MAIN OUTCOME MEASURE(S): The primary outcomes were reminder preference and type. RESULTS: A total of 1497 of 4545 patients (32.9%) expressed a preference; 789 (52.7%) chose text. The adjusted relative risk (aRR) of not providing a preference increased with decreasing PDC (PDC of 0.50 to < 0.80: aRR, 1.14; 95% confidence interval [CI], 1.04-1.25; PDC < 0.5: aRR, 1.76; 95% CI, 1.59-1.95) compared with patients with a PDC of 0.80 or greater. CONCLUSION: Among patients who expressed a preference, text reminders were preferred. Patients who expressed a preference had higher baseline adherence. Further research is needed to determine whether expressing a preference for a refill reminder type is itself associated with adherence. Given that offering the opportunity to choose a reminder type only engaged a subset of patients, further work is needed to understand how best to leverage technology-enabled communication outreach to help patients optimize adherence.

Complete Genome Sequences of Four Isolates of Vancomycin-Resistant Enterococcus faecium with the vanA Gene and Two Daptomycin Resistance Mutations, Obtained from Two Inpatients with Prolonged Bacteremia.

Here, we present complete genome sequences of four Enterococcus faecium isolates, obtained from two patients with apparent vancomycin-resistant Enterococcus faecium bacteremia; these isolates also carried two mutations known to be associated with daptomycin resistance. Sequences were obtained using de novo and hybrid assembly of Oxford Nanopore and Illumina sequence data.

Two Cases of Vancomycin-Resistant Enterococcus faecium Bacteremia With Development of Daptomycin-Resistant Phenotype and its Detection Using Oxford Nanopore Sequencing.

In this work, we report 2 cases of vancomycin-resistant Enterococcus faecium bacteremia with development of daptomycin resistance in 2 patients with acute myeloid leukemia and myelodysplastic syndrome. Mutations related to daptomycin-nonsusceptible phenotype in liaSR genes were found in all strains of the study, including those with a minimum inhibitory concentration <1 µg/mL collected before daptomycin therapy. Epidemiological investigation using core genome single nucleotide polymorphism and core genome multilocus sequence typing revealed clonality of all the isolates. In this study, we conclude that real-time genome sequencing of clinical isolates can provide rapid access to timely information on daptomycin-resistant genotypes that would help clinicians speed up and optimize the selection of the antibiotic for treatment.