Nonperturbative Fluorogenic Labeling of Immunophilins Enables the Wash-free Detection of Immunosuppressants.

Immunosuppressants are clinically approved drugs to treat the potential rejection of transplanted organs and require frequent monitoring due to their narrow therapeutic window. Immunophilins are small proteins that bind immunosuppressants with high affinity, yet there are no examples of fluorogenic immunophilins and their potential application as optical biosensors for immunosuppressive drugs in clinical biosamples. In the present work, we designed novel diazonium BODIPY salts for the site-specific labeling of tyrosine residues in peptides via solid-phase synthesis as well as for late-stage functionalization of whole recombinant proteins. After the optimization of a straightforward one-step labeling procedure for immunophilins PPIA and FKBP12, we demonstrated the application of a fluorogenic analogue of FKBP12 for the selective detection of the immunosuppressant drug tacrolimus, including experiments in urine samples from patients with functioning renal transplants. This chemical methodology opens new avenues to rationally design wash-free immunophilin-based biosensors for rapid therapeutic drug monitoring.

Inserting "OFF-to-ON" BODIPY Tags into Cytokines: A Fluorogenic Interleukin IL-33 for Real-Time Imaging of Immune Cells.

The essential functions that cytokine/immune cell interactions play in tissue homeostasis and during disease have prompted the molecular design of targeted fluorophores to monitor their activity in real time. Whereas activatable probes for imaging immune-related enzymes are common, many immunological functions are mediated by binding events between cytokines and their cognate receptors that are hard to monitor by live-cell imaging. A prime example is interleukin-33 (IL-33), a key cytokine in innate and adaptive immunity, whose interaction with the ST2 cell-surface receptor results in downstream signaling and activation of NF-κB and AP-1 pathways. In the present work, we have designed a chemical platform to site-specifically introduce OFF-to-ON BODIPY fluorophores into full cytokine proteins and generate the first nativelike fluorescent analogues of IL-33. Among different incorporation strategies, chemical aminoacylation followed by bioorthogonal derivatization led to the best labeling results. Importantly, the BODIPY-labeled IL-33 derivatives-unlike IL-33-GFP constructs-exhibited ST2-specific binding and downstream bioactivity profiles comparable to those of the wild-type interleukin. Real-time fluorescence microscopy assays under no wash conditions confirmed the internalization of IL-33 through ST2 receptors and its intracellular trafficking through the endosomal pathway. We envision that the modularity and versatility of our BODIPY labeling platform will facilitate the synthesis of minimally tagged fluorogenic cytokines as the next generation of imaging reagents for real-time visualization of signaling events in live immune cells.

Outcomes of a Community-Based Participatory Research Partnership Self-Evaluation: The Rochester Healthy Community Partnership Experience.

BACKGROUND: Community-based participatory research (CBPR) can effectively address health disparities among groups that are historically difficult to reach, disadvantaged, of a minority status, or are otherwise underrepresented in research. Recent research has focused on the science of CBPR partnership constructs and on developing and testing tools for self-evaluation. Because CBPR requires substantial investment in human and material resources, specific factors that support successful and sustainable research partnerships must be identified. We sought to describe the evolution, implementation, and results of a self-evaluation of a CBPR partnership. METHODS: Academic and community members of the Rochester Healthy Community Partnership (RHCP) and researchers from the University of New Mexico-Center for Participatory Research collaborated to evaluate RHCP with qualitative and quantitative research methods and group analysis. RESULTS: The self-evaluation was used to provide an overall picture of the "health" of the partnership, in terms of sustainability and ability to effectively collaborate around community priorities. RHCP members revisited the partnership's mission and values; identified associations between partnership practices, dynamics, and outcomes; and elicited insight from community and academic partners to help guide decisions about future directions and the sustainability of the partnership. Positive partnership dynamics were associated with perceived improvements in health and equity outcomes. CONCLUSIONS: Although engaging in a comprehensive self-evaluation requires substantial investment from stakeholders, such assessments have significant value because they enable partners to reflect on the mission and values of the partnership, explore the history and context for its existence, identify factors that have contributed to outcomes, and plan strategically for the future.

An initiative to implement immediate postpartum long-acting reversible contraception in rural New Mexico.

BACKGROUND: Over the past decade, many states have developed approaches to reimburse for immediate postpartum long-acting reversible contraception. Despite expanded coverage, few hospitals offer immediate postpartum long-acting reversible contraception. OBJECTIVES: Immediate postpartum long-acting reversible contraception implementation is complex and requires a committed multidisciplinary team. After New Mexico Medicaid approved reimbursement for this service, the New Mexico Perinatal Collaborative developed and initiated an evidence-based implementation program containing several components. We sought to evaluate timing of the implementation process and facilitators and barriers to immediate postpartum long-acting reversible contraception in several New Mexico rural hospitals. The primary study outcome was time from New Mexico Perinatal Collaborative program component introduction in each hospital to the hospital's completion of the corresponding implementation step. Secondary outcomes included barriers and facilitators to immediate postpartum contraception implementation. STUDY DESIGN: In this mixed-methods study, conducted from April 2017 to May 2018, we completed semistructured questionnaires and interviews with 20 key personnel from 7 New Mexico hospitals that planned to implement immediate postpartum long-acting reversible contraception. The New Mexico Perinatal Collaborative introduced program components to hospitals in a stepped-wedge design. Participants contributed baseline and follow-up data at 4 time periods detailing the steps taken towards program implementation and the timing of step completion at their hospital. Qualitative data were analyzed using directed qualitative content analysis principles based on the Consolidated Framework for Implementation Research. RESULTS: Investigators conducted 43 interviews during the 14-month study period. Median time to complete steps toward implementation-patient education, clinician training, nursing education, charge capture, available supplies, and protocols or guidelines-ranged from 7 days for clinician training to 357 days to develop patient education materials. Facilitators of immediate postpartum contraception readiness were local hospital clinical champions and institutional administrative and financial stability. Of the 7 hospitals, 4 completed all Perinatal Collaborative implementation program components and 3 of those piloted immediate postpartum long-acting reversible contraception services. Two publicly funded hospitals currently offer immediate postpartum long-acting reversible contraception without verification of payment for the device or insertion. The third hospital piloted the program with 8 contraceptive devices, did not receive reimbursement due to identified flaws in Medicaid billing guidance and does not currently offer the service. The remaining 3 of the 7 hospitals declined to complete the NMPC program; the hospital that completed the program but did not pilot immediate postpartum long-acting reversible contraception did so because Medicaid billing mechanisms were incompatible with their automated billing systems. Participants consistently reported that lack of reimbursement was the major barrier to immediate postpartum long-acting reversible contraception implementation. CONCLUSION: Despite the New Mexico Perinatal Collaborative's robust implementation process and hospital engagement, most hospitals did not offer immediate postpartum long-acting reversible contraception over the study period. Reimbursement obstacles prevented full service implementation. Interventions to improve immediate postpartum long-acting reversible contraception access must begin with implementation of seamless billing and reimbursement mechanisms to ensure adequate hospital payments.

Inhibition of ErbB kinase signalling promotes resolution of neutrophilic inflammation.

Neutrophilic inflammation with prolonged neutrophil survival is common to many inflammatory conditions, including chronic obstructive pulmonary disease (COPD). There are few specific therapies that reverse neutrophilic inflammation, but uncovering mechanisms regulating neutrophil survival is likely to identify novel therapeutic targets. Screening of 367 kinase inhibitors in human neutrophils and a zebrafish tail fin injury model identified ErbBs as common targets of compounds that accelerated inflammation resolution. The ErbB inhibitors gefitinib, CP-724714, erbstatin and tyrphostin AG825 significantly accelerated apoptosis of human neutrophils, including neutrophils from people with COPD. Neutrophil apoptosis was also increased in Tyrphostin AG825 treated-zebrafish in vivo. Tyrphostin AG825 decreased peritoneal inflammation in zymosan-treated mice, and increased lung neutrophil apoptosis and macrophage efferocytosis in a murine acute lung injury model. Tyrphostin AG825 and knockdown of egfra and erbb2 by CRISPR/Cas9 reduced inflammation in zebrafish. Our work shows that inhibitors of ErbB kinases have therapeutic potential in neutrophilic inflammatory disease.

The Development of a Collaborative Self-Evaluation Process for Community-Based Participatory Research Partnerships Using the Community-Based Participatory Research Conceptual Model and Other Adaptable Tools.

BACKGROUND: Established community-based participatory research (CBPR) partnerships need tools to assist with self-evaluation of the effectiveness and engagement with CBPR principles and to inform ongoing work. A growing part of the CBPR field is focused on the evaluation of partnering processes and outcomes. OBJECTIVES: The Rochester Healthy Community Partnership (RHCP), a partnership with more than a decade of engagement in health promotion research, performed a self-evaluation in collaboration with the University of New Mexico Center for Participatory Research (UNM-CPR). METHODS: We collaboratively developed and implemented a facilitated self-evaluation using adaptations of existing tools and the CBPR conceptual model. Partners contributed through surveys and qualitative interviews. Initially, data were analyzed collaboratively by members of RHCP and UNM-CPR, but RHCP partners further processed and consolidated findings, leading to the development of key questions that guided a full partnership discussion of action steps. RESULTS: Our process confirmed the adaptability of existing tools and the CBPR conceptual model for the purpose of partnership reflection and self-evaluation. We offer the key findings of our assessment of partnering practices and directions for the future, and share our approach to collaborative analysis and dissemination. Our discussion includes lessons learned, with applicability to other established partnerships. CONCLUSIONS: Our experience indicates that collective reflection is empowering for members of established partnerships, which can be facilitated by engagement in self-evaluation through the use of adapted, available tools. The incorporation of participatory processes adds complexity, but leads to a level of resonance and usefulness that would not have been obtained from a traditional evaluation.

Pellino-1 Regulates Immune Responses to Haemophilus influenzae in Models of Inflammatory Lung Disease.

Non-typeable Haemophilus influenzae (NTHi) is a frequent cause of lower respiratory tract infection in people with chronic obstructive pulmonary disease (COPD). Pellino proteins are a family of E3 ubiquitin ligases that are critical regulators of TLR signaling and inflammation. The aim of this study was to identify a role for Pellino-1 in airway defense against NTHi in the context of COPD. Pellino-1 is rapidly upregulated by LPS and NTHi in monocyte-derived macrophages (MDMs) isolated from individuals with COPD and healthy control subjects, in a TLR4 dependent manner. C57BL/6 Peli1-/- and wild-type (WT) mice were subjected to acute (single LPS challenge) or chronic (repeated LPS and elastase challenge) airway inflammation followed by NTHi infection. Both WT and Peli1-/- mice develop airway inflammation in acute and chronic airway inflammation models. Peli1-/- animals recruit significantly more neutrophils to the airway following NTHi infection which is associated with an increase in the neutrophil chemokine, KC, in bronchoalveolar lavage fluid as well as enhanced clearance of NTHi from the lung. These data suggest that therapeutic inhibition of Pellino-1 may augment immune responses in the airway and enhance bacterial clearance in individuals with COPD.

Expression analysis of the osteoarthritis genetic susceptibility mapping to the matrix Gla protein gene MGP.

BACKGROUND: Osteoarthritis (OA) is a common disease of older individuals that impacts detrimentally on the quality and the length of life. It is characterised by the painful loss of articular cartilage and is polygenic and multifactorial. Genome-wide association scans have highlighted over 90 osteoarthritis genetic signals, some of which reside within or close to highly plausible candidate genes. An example is an association to polymorphisms within and adjacent to the matrix Gla protein gene MGP. We set out to undertake a functional study of this gene. METHODS: Nucleic acid was extracted from cartilage, infrapatellar fat pad, synovium, trabecular bone, trapezium and peripheral whole blood from OA patients and also from mesenchymal stem cells (MSCs) subjected to chondrogenesis. Expression of MGP was measured by quantitative PCR (qPCR), RNA-sequencing and allelic expression imbalance (AEI) analysis. Matrix Gla protein was depleted in chondrocytes by knocking down MGP expression using RNA interference (RNAi) and the effect on a range of genes assessed by qPCR. RESULTS: MGP is expressed in joint tissues, blood and chondrocytes cultured from MSCs. There is a higher expression in diseased versus non-diseased cartilage. Polymorphisms that are associated with OA also correlate with the expression of MGP, with the OA risk-conferring allele showing significantly reduced expression in cartilage, fat pad and synovium but increased expression in blood. Depletion of Matrix Gla protein had a significant effect on the majority of genes tested, with an increased expression of catabolic genes that encode enzymes that degrade cartilage. CONCLUSIONS: MGP expression is subject to cis-acting regulators that correlate with the OA association signal. These are active in a range of joint tissues but have effects which are particularly strong in cartilage. An opposite effect is observed in blood, highlighting the context-specific nature of the regulation of this gene's expression. Recapitulation of the genetic deficit in cartilage chondrocytes is pro-catabolic.