Implementing a Family-Centered Rounds Intervention Using Novel Mentor-Trios.

BACKGROUND AND OBJECTIVES: Patient and Family Centered I-PASS (PFC I-PASS) emphasizes family and nurse engagement, health literacy, and structured communication on family-centered rounds organized around the I-PASS framework (Illness severity-Patient summary-Action items-Situational awareness-Synthesis by receiver). We assessed adherence, safety, and experience after implementing PFC I-PASS using a novel "Mentor-Trio" implementation approach with multidisciplinary parent-nurse-physician teams coaching sites. METHODS: Hybrid Type II effectiveness-implementation study from 2/29/19-3/13/22 with ≥3 months of baseline and 12 months of postimplementation data collection/site across 21 US community and tertiary pediatric teaching hospitals. We conducted rounds observations and surveyed nurses, physicians, and Arabic/Chinese/English/Spanish-speaking patients/parents. RESULTS: We conducted 4557 rounds observations and received 2285 patient/family, 1240 resident, 819 nurse, and 378 attending surveys. Adherence to all I-PASS components, bedside rounding, written rounds summaries, family and nurse engagement, and plain language improved post-implementation (13.0%-60.8% absolute increase by item), all P < .05. Except for written summary, improvements sustained 12 months post-implementation. Resident-reported harms/1000-resident-days were unchanged overall but decreased in larger hospitals (116.9 to 86.3 to 72.3 pre versus early- versus late-implementation, P = .006), hospitals with greater nurse engagement on rounds (110.6 to 73.3 to 65.3, P < .001), and greater adherence to I-PASS structure (95.3 to 73.6 to 72.3, P < .05). Twelve of 12 measures of staff safety climate improved (eg, "excellent"/"very good" safety grade improved from 80.4% to 86.3% to 88.0%), all P < .05. Patient/family experience and teaching were unchanged. CONCLUSIONS: Hospitals successfully used Mentor-Trios to implement PFC I-PASS. Family/nurse engagement, safety climate, and harms improved in larger hospitals and hospitals with better nurse engagement and intervention adherence. Patient/family experience and teaching were not affected.

Family, nurse, and physician beliefs on family-centered rounds: A 21-site study.

BACKGROUND: Variation exists in family-centered rounds (FCR). OBJECTIVE: We sought to understand patient/family and clinician FCR beliefs/attitudes and practices to support implementation efforts. DESIGNS, SETTINGS AND PARTICIPANTS: Patients/families and clinicians at 21 geographically diverse US community/academic pediatric teaching hospitals participated in a prospective cohort dissemination and implementation study. INTERVENTION: We inquired about rounding beliefs/attitudes, practices, and demographics using a 26-question survey coproduced with family/nurse/attending-physician collaborators, informed by prior research and the Consolidated Framework for Implementation Research. MAIN OUTCOME AND MEASURES: Out of 2578 individuals, 1647 (64%) responded to the survey; of these, 1313 respondents participated in FCR and were included in analyses (616 patients/families, 243 nurses, 285 resident physicians, and 169 attending physicians). Beliefs/attitudes regarding the importance of FCR elements varied by role, with resident physicians rating the importance of several FCR elements lower than others. For example, on adjusted multivariable analysis, attending physicians (odds ratio [OR] 3.0, 95% confidence interval [95% CI] 1.2-7.8) and nurses (OR 3.1, 95% CI 1.3-7.4) were much more likely than resident physicians to report family participation on rounds as very/extremely important. Clinician support for key FCR elements was higher than self-reported practice (e.g., 88% believed family participation was important on rounds; 68% reported it often/always occurred). In practice, key elements of FCR were reported to often/always occur only 23%-70% of the time. RESULT: Support for nurse and family participation in FCR is high among clinicians but varies by role. Physicians, particularly resident physicians, endorse several FCR elements as less important than nurses and patients/families. The gap between attitudes and practice and between clinician types suggests that attitudinal, structural, and cultural barriers impede FCR.

Shifting gears: Id3 enables recruitment of E proteins to new targets during T cell development and differentiation.

Shifting levels of E proteins and Id factors are pivotal in T cell commitment and differentiation, both in the thymus and in the periphery. Id2 and Id3 are two different factors that prevent E proteins from binding to their target gene cis-regulatory sequences and inducing gene expression. Although they use the same mechanism to suppress E protein activity, Id2 and Id3 play very different roles in T cell development and CD4 T cell differentiation. Id2 imposes an irreversible choice in early T cell precursors between innate and adaptive lineages, which can be thought of as a railway switch that directs T cells down one path or another. By contrast, Id3 acts in a transient fashion downstream of extracellular signals such as T cell receptor (TCR) signaling. TCR-dependent Id3 upregulation results in the dislodging of E proteins from their target sites while chromatin remodeling occurs. After the cessation of Id3 expression, E proteins can reassemble in the context of a new genomic landscape and molecular context that allows induction of different E protein target genes. To describe this mode of action, we have developed the "Clutch" model of differentiation. In this model, Id3 upregulation in response to TCR signaling acts as a clutch that stops E protein activity ("clutch in") long enough to allow shifting of the genomic landscape into a different "gear", resulting in accessibility to different E protein target genes once Id3 decreases ("clutch out") and E proteins can form new complexes on the DNA. While TCR signal strength and cytokine signaling play a role in both peripheral and thymic lineage decisions, the remodeling of chromatin and E protein target genes appears to be more heavily influenced by the cytokine milieu in the periphery, whereas the outcome of Id3 activity during T cell development in the thymus appears to depend more on the TCR signal strength. Thus, while the Clutch model applies to both CD4 T cell differentiation and T cell developmental transitions within the thymus, changes in chromatin accessibility are modulated by biased inputs in these different environments. New emerging technologies should enable a better understanding of the molecular events that happen during these transitions, and how they fit into the gene regulatory networks that drive T cell development and differentiation.

Patient and Family-Centered I-PASS SCORE Program: Resident and Advanced Care Provider Training Materials.

Introduction: Patient and family-centered rounds (PFCRs) are an important element of family-centered care often used in the inpatient pediatric setting. However, techniques and best practices vary, and faculty, trainees, nurses, and advanced care providers may not receive formal education in strategies that specifically enhance communication on PFCRs. Methods: Harnessing the use of structured communication, we developed the Patient and Family-Centered I-PASS Safer Communication on Rounds Every Time (SCORE) Program. The program uses a standardized framework for rounds communication via the I-PASS mnemonic, principles of health literacy, and techniques for patient/family engagement and bidirectional communication. The resident and advanced care provider training materials, a component of the larger SCORE Program, incorporate a flipped classroom approach as well as interactive exercises, simulations, and virtual learning options to optimize learning and retention via a 90-minute workshop. Results: Two hundred forty-six residents completed the training and were evaluated on their knowledge and confidence regarding key elements of the curriculum. Eighty-eight percent of residents agreed/strongly agreed that after training they could activate and engage families and all members of the interprofessional team to create a shared mental model; 90% agreed/strongly agreed that they could discuss the roles/responsibilities of various team members during PFCRs. Discussion: The Patient and Family-Centered I-PASS SCORE Program provides a structured framework for teaching advanced communication techniques that can improve provider knowledge of and confidence with engaging and communicating with patients/families and other members of the interprofessional team during PFCRs.

Regulation of the Signal-Dependent E Protein HEBAlt Through a YYY Motif Is Required for Progression Through T Cell Development.

The E protein transcription factors E2A and HEB are critical for many developmental processes, including T cell development. We have shown that the Tcf12 locus gives rise to two distinct HEB proteins, with alternative (HEBAlt) and canonical (HEBCan) N-terminal domains, which are co-expressed during early T cell development. While the functional domains of HEBCan have been well studied, the nature of the HEBAlt-specific (Alt) domain has been obscure. Here we provide compelling evidence that the Alt domain provides a site for the molecular integration of cytokine signaling and E protein activity. Our results indicate that phosphorylation of a unique YYY motif in the Alt domain increases HEBAlt activity by 10-fold, and that this increase is dependent on Janus kinase activity. To enable in vivo studies of HEBAlt in the T cell context, we generated ALT-Tg mice, which can be induced to express a HA-tagged HEBAlt coding cassette in the presence of Cre recombinases. Analysis of ALT-Tg mice on the Vav-iCre background revealed a minor change in the ratio of ISP cells to CD8+ SP cells, and a mild shift in the ratio of T cells to B cells in the spleen, but otherwise the thymus, spleen, and bone marrow lymphocyte subsets were comparable at steady state. However, kinetic analysis of T cell development in OP9-DL4 co-cultures revealed a delay in early T cell development and a partial block at the DN to DP transition when HEBAlt levels or activity were increased. We also observed that HEBCan and HEBAlt displayed significant differences in protein stability that were resolved in the thymocyte context. Finally, a proteomic screen identified STAT1 and Xpo1 as potential members of HEBAlt-containing complexes in thymocytes, consistent with JAK-induced activation of HEBAlt accompanied by translocation to the nucleus. Thus, our results show that the Alt domain confers access to multiple layers of post-translational control to HEBAlt that are not available to HEBCan, and thus may serve as a rheostat to tune E protein activity levels as cells move through different thymic signaling environments during T cell development.

Direct regulation of TCR rearrangement and expression by E proteins during early T cell development.

γδ T cells are widely distributed throughout mucosal and epithelial cell-rich tissues and are an important early source of IL-17 in response to several pathogens. Like αß T cells, γδ T cells undergo a stepwise process of development in the thymus that requires recombination of genome-encoded segments to assemble mature T cell receptor (TCR) genes. This process is tightly controlled on multiple levels to enable TCR segment assembly while preventing the genomic instability inherent in the double-stranded DNA breaks that occur during this process. Each TCR locus has unique aspects in its structure and requirements, with different types of regulation before and after the αß/γδ T cell fate choice. It has been known that Runx and Myb are critical transcriptional regulators of TCRγ and TCRδ expression, but the roles of E proteins in TCRγ and TCRδ regulation have been less well explored. Multiple lines of evidence show that E proteins are involved in TCR expression at many different levels, including the regulation of Rag recombinase gene expression and protein stability, induction of germline V segment expression, chromatin remodeling, and restriction of the fetal and adult γδTCR repertoires. Importantly, E proteins interact directly with the cis-regulatory elements of the TCRγ and TCRδ loci, controlling the predisposition of a cell to become an αß T cell or a γδ T cell, even before the lineage-dictating TCR signaling events. This article is categorized under: Immune System Diseases > Stem Cells and Development Immune System Diseases > Genetics/Genomics/Epigenetics.

Intervention, individual, and contextual determinants to high adherence to structured family-centered rounds: a national multi-site mixed methods study.

BACKGROUND: Effective communication in transitions between healthcare team members is associated with improved patient safety and experience through a clinically meaningful reduction in serious safety events. Family-centered rounds (FCR) can serve a critical role in interprofessional and patient-family communication. Despite widespread support, FCRs are not utilized consistently in many institutions. Structured FCR approaches may prove beneficial in increasing FCR use but should address organizational challenges. The purpose of this study was to identify intervention, individual, and contextual determinants of high adherence to common elements of structured FCR in pediatric inpatient units during the implementation phase of a large multi-site study implementing a structured FCR approach. METHODS: We performed an explanatory sequential mixed methods study from September 2019 to October 2020 to evaluate the variation in structured FCR adherence across 21 pediatric inpatient units. We analyzed 24 key informant interviews of supervising physician faculty, physician learners, nurses, site administrators, and project leaders at 3 sites using a qualitative content analysis paradigm to investigate site variation in FCR use. We classified implementation determinants based on the Consolidated Framework for Implementation Research. RESULTS: Provisional measurements of adherence demonstrated considerable variation in structured FCR use across sites at a median time of 5 months into the implementation. Consistent findings across all three sites included generally positive clinician beliefs regarding the use of FCR and structured rounding approaches, benefits to learner self-efficacy, and potential efficiency gains derived through greater rounds standardization, as well as persistent challenges with nurse engagement and interaction on rounds and coordination and use of resources for families with limited English proficiency. CONCLUSIONS: Studies during implementation to identify determinants to high adherence can provide generalizable knowledge regarding implementation determinants that may be difficult to predict prior to implementation, guide adaptation during the implementation, and inform sustainment strategies.

The Dimensionality of Language and Literacy in the School-Age Years.

PURPOSE: This was an investigation of the dimensionality of oral and written language to test the hypothesis that a two-factor model with sound/word and sentence/discourse language levels would best fit language and literacy data for a population-based sample in the school-age years. METHOD: A stratified secondary data set of 1,500 participants was drawn randomly from a larger nationally representative U.S. data set (N = 1,853) gathered during standardization of the Test of Integrated Language and Literacy Skills. A sample of 254 students with prior diagnoses of language and literacy disorders (LLD) was drawn from the full data set. Confirmatory factor analysis was used to compare the hypothesized two-factor model with other theoretically possible models. RESULTS: The hypothesized two-factor language-levels model had an acceptable-to-good fit to the full data set, as did the three-factor model, with verbal memory added. High interfactor correlation between verbal memory and sentence/discourse constructs, as well as a preference for parsimony, led to the acceptance of the two-factor model as best. This language-levels model had a good fit to the data at ages 8-11 years, and an excellent fit at ages 12-18 years, but only a poor fit for ages 6-7 years (yet still better than other two-factor or unitary models). It had a reasonable fit for students with LLD, although the three-factor model fit their data slightly better. CONCLUSIONS: Oral and written language abilities during the school-age years are best explained by a two-factor model with sound/word and sentence/discourse language levels and memory as a contributing factor. Implications for identifying and treating language and literacy disorders as multidimensional rather than categorical are discussed.

Realization of the T Lineage Program Involves GATA-3 Induction of Bcl11b and Repression of Cdkn2b Expression.

The zinc-finger transcription factor GATA-3 plays a crucial role during early T cell development and also dictates later T cell differentiation outcomes. However, its role and collaboration with the Notch signaling pathway in the induction of T lineage specification and commitment have not been fully elucidated. We show that GATA-3 deficiency in mouse hematopoietic progenitors results in an early block in T cell development despite the presence of Notch signals, with a failure to upregulate Bcl11b expression, leading to a diversion along a myeloid, but not a B cell, lineage fate. GATA-3 deficiency in the presence of Notch signaling results in the apoptosis of early T lineage cells, as seen with inhibition of CDK4/6 (cyclin-dependent kinases 4 and 6) function, and dysregulated cyclin-dependent kinase inhibitor 2b (Cdkn2b) expression. We also show that GATA-3 induces Bcl11b, and together with Bcl11b represses Cdkn2b expression; however, loss of Cdkn2b failed to rescue the developmental block of GATA-3-deficient T cell progenitor. Our findings provide a signaling and transcriptional network by which the T lineage program in response to Notch signals is realized.

Fetal Thymic Organ Culture (FTOC) Optimized for Gamma-Delta T Cell Studies.

Fetal thymic organ culture (FTOC) provides a method for analyzing T cell development in a physiological context outside the animal. This technique enables studies of genetically altered mice that are embryonic or neonatal lethal, in addition to bypassing the complication of migration of successive waves of T cells out of the thymus. The hanging drop method involves depletion of thymocytes from host lobes using deoxyguanosine, followed by reconstitution with hematopoietic progenitors. This method has become standard for analysis of fetal liver precursors, bone marrow precursors, and early thymocytes. However, difficulties are encountered in the analysis of γδ T cell precursors using this method. We have developed a modification of FTOC in which partial depletion of hematopoietic precursors by shortened deoxyguanosine treatment, coupled with the use of TCRδ-deficient host lobes, enables engraftment and development of fetal γδTCR+ thymocytes. This method allows comparisons of development and functional differentiation of γδ T cell precursors between cells of different genotypes or treatments, in the context of a permissive thymic microenvironment.

DL4-µbeads induce T cell lineage differentiation from stem cells in a stromal cell-free system.

T cells are pivotal effectors of the immune system and can be harnessed as therapeutics for regenerative medicine and cancer immunotherapy. An unmet challenge in the field is the development of a clinically relevant system that is readily scalable to generate large numbers of T-lineage cells from hematopoietic stem/progenitor cells (HSPCs). Here, we report a stromal cell-free, microbead-based approach that supports the efficient in vitro development of both human progenitor T (proT) cells and T-lineage cells from CD34+cells sourced from cord blood, GCSF-mobilized peripheral blood, and pluripotent stem cells (PSCs). DL4-µbeads, along with lymphopoietic cytokines, induce an ordered sequence of differentiation from CD34+ cells to CD34+CD7+CD5+ proT cells to CD3+αß T cells. Single-cell RNA sequencing of human PSC-derived proT cells reveals a transcriptional profile similar to the earliest thymocytes found in the embryonic and fetal thymus. Furthermore, the adoptive transfer of CD34+CD7+ proT cells into immunodeficient mice demonstrates efficient thymic engraftment and functional maturation of peripheral T cells. DL4-µbeads provide a simple and robust platform to both study human T cell development and facilitate the development of engineered T cell therapies from renewable sources.

Ontogenic timing, T cell receptor signal strength, and Notch signaling direct γδ T cell functional differentiation in vivo.

γδ T cells form an integral arm of the immune system and are critical during protective and destructive immunity. However, how γδ T cells are functionally programmed in vivo remains unclear. Here, we employ RBPJ-inducible and KN6-transgenic mice to assess the roles of ontogenic timing, T cell receptor (TCR) signal strength, and Notch signaling. We find skewing of Vγ1+ cells toward the PLZF+Lin28b+ lineage at the fetal stage. Generation of interleukin-17 (IL-17)-producing γδ T cells is favored during, although not exclusive to, the fetal stage. Surprisingly, Notch signaling is dispensable for peripheral γδ T cell IL-17 production. Strong TCR signals, together with Notch, promote IL-4 differentiation. Conversely, less strong TCR signals promote Notch-independent IL-17 differentiation. Single-cell transcriptomic analysis reveals differential programming instilled by TCR signal strength and Notch for specific subsets. Thus, our results precisely define the roles of ontogenic timing, TCR signal strength, and Notch signaling in γδ T cell functional programming in vivo.

Cutting Edge: TCR-ß Selection Is Required at the CD4+CD8+ Stage of Human T Cell Development.

T cell development is predicated on the successful rearrangement of the TCR gene loci, which encode for Ag-specific receptors. Recombination-activating gene (RAG) 2 is required for TCR gene rearrangements, which occur during specific stages of T cell development. In this study, we differentiated human pluripotent stem cells with a CRISPR/Cas9-directed deletion of the RAG2 gene (RAG2-KO) to elucidate the requirement for the TCR ß-chain in mediating ß-selection during human T cell development. In stark contrast to mice, human RAG2-KO T lineage progenitors progressed to the CD4+CD8+ double-positive (DP) stage in the absence of TCRß rearrangements. Nonetheless, RAG2-KO DPs retrovirally transduced to express a rearranged TCR ß-chain showed increased survival and proliferation as compared with control-transduced RAG2-KO DPs. Furthermore, transcriptomic analysis showed that TCRß- and control-transduced RAG2-KO DPs differed in gene pathways related to survival and proliferation. Our results provide important insights as to the distinct requirement for the TCR ß-chain during human T cell development.

E2A regulates neural ectoderm fate specification in human embryonic stem cells.

E protein transcription factors are crucial for many cell fate decisions. However, the roles of E proteins in the germ-layer specification of human embryonic stem cells (hESCs) are poorly understood. We disrupted the TCF3 gene locus to delete the E protein E2A in hESCs. E2A knockout (KO) hESCs retained key features of pluripotency, but displayed decreased neural ectoderm coupled with enhanced mesoendoderm outcomes. Genome-wide analyses showed that E2A directly regulates neural ectoderm and Nodal pathway genes. Accordingly, inhibition of Nodal or E2A overexpression partially rescued the neural ectoderm defect in E2A KO hESCs. Loss of E2A had little impact on the epigenetic landscape of hESCs, whereas E2A KO neural precursors displayed increased accessibility of the gene locus encoding the Nodal agonist CRIPTO. Double-deletion of both E2A and HEB (TCF12) resulted in a more severe neural ectoderm defect. Therefore, this study reveals critical context-dependent functions for E2A in human neural ectoderm fate specification.

Interaction between γδTCR signaling and the E protein-Id axis in γδ T cell development.

γδ T cells acquire their functional properties in the thymus, enabling them to exert rapid innate-like responses. To understand how distinct γδ T cell subsets are generated, we have developed a Two-Stage model for γδ T cell development. This model is predicated on the finding that γδTCR signal strength impacts E protein activity through graded upregulation of Id3. Our model proposes that cells enter Stage 1 in response to a γδTCR signaling event in the cortex that activates a γδ T cell-specific gene network. Part of this program includes the upregulation of chemokine receptors that guide them to the medulla. In the medulla, Stage 1 cells receive distinct combinations of γδTCR, cytokine, and/co-stimulatory signals that induce their transit into Stage 2, either toward the γδT1 or the γδT17 lineage. The intersection between γδTCR and cytokine signals can tune Id3 expression, leading to different outcomes even in the presence of strong γδTCR signals. The thymic signaling niches required for γδT17 development are segregated in time and space, providing transient windows of opportunity during ontogeny. Understanding the regulatory context in which E proteins operate at different stages will be key in defining how their activity levels impose functional outcomes.

Squamous Cell Carcinoma of the Bladder Is Not Associated With High-risk HPV.

OBJECTIVE: To evaluate the clinical features, pathologic features, and prevalence of human papilloma virus (HPV) in squamous cell carcinoma (SCC) of the bladder. SCC of the bladder is known to be associated with conditions that cause chronic inflammation/irritation. The literature is inconsistent regarding the association of HPV with pure SCC of the bladder. METHODS: A multi-institutional study identified cases of SCC of the bladder. Pure squamous histology and the absence of urothelial carcinoma in situ were required for inclusion. Clinical and pathologic features were collected, and tissues were evaluated for high-risk HPV using p16 immunohistochemistry and in situ hybridization. RESULTS: We identified 207 cases of SCC of the bladder. Risk factors for bladder cancer included smoking (133/207, 64%) and chronic bladder irritation (83/207, 40%). The majority (155/207, 75%) of patients had > pT2 disease. Mean tumor size was 5.6 ± 3.0 cm and 36/207 (17%) patients had lymph node positive disease. p16 immunohistochemistry was positive in 52/204 (25%) cases but high-risk HPV was identified with in situ hybridization in only 1 (0.5%) case. Tumor size, stage, number of lymph nodes removed, number of positive lymph nodes, lymphovascular invasion, perineural invasion, and positive margins each were associated with cancer-specific mortality when adjusted for demographic factors. A multivariate analysis of variable importance further revealed sex and race as important factors in predicting cancer-specific mortality. CONCLUSION: SCC of the bladder is an aggressive histologic subtype. Although bladder SCC can express p16, it is not typically associated with high-risk HPV, although rare cases can occur.