Follicular helper- and peripheral helper-like T cells drive autoimmune disease in human immune system mice.

Human immune system (HIS) mice constructed in various ways are widely used for investigations of human immune responses to pathogens, transplants and immunotherapies. In HIS mice that generate T cells de novo from hematopoietic progenitors, T cell-dependent multisystem autoimmune disease occurs, most rapidly when the human T cells develop in the native NOD.Cg- Prkdc scid Il2rg tm1Wjl (NSG) mouse thymus, where negative selection is abnormal. Disease develops very late when human T cells develop in human fetal thymus grafts, where robust negative selection is observed. We demonstrate here that PD-1 + CD4 + peripheral (Tph) helper-like and follicular (Tfh) helper-like T cells developing in HIS mice can induce autoimmune disease. Tfh-like cells were more prominent in HIS mice with a mouse thymus, in which the highest levels of IgG were detected in plasma, compared to those with a human thymus. While circulating IgG and IgM antibodies were autoreactive to multiple mouse antigens, in vivo depletion of B cells and antibodies did not delay the development of autoimmune disease. Conversely, adoptive transfer of enriched Tfh- or Tph-like cells induced disease and autoimmunity-associated B cell phenotypes in recipient mice containing autologous human APCs without T cells. T cells from mice with a human thymus expanded and induced disease more rapidly than those originating in a murine thymus, implicating HLA-restricted T cell-APC interactions in this process. Since Tfh, Tph, autoantibodies and LIP have all been implicated in various forms of human autoimmune disease, the observations here provide a platform for the further dissection of human autoimmune disease mechanisms and therapies.

Population Pharmacokinetics of Single Bolus Dose Fentanyl in Obese Children.

BACKGROUND: Childhood obesity is a significant problem. Obesity may alter the pharmacokinetics (PKs) of medications. Fentanyl is commonly used for procedural sedation, but there is a paucity of bolus dose fentanyl PK data in obese children. Better understanding of fentanyl PK in obese children would facilitate dosing recommendations. We conducted a study involving children with and without obesity to assess the potential differences in bolus dose fentanyl PK between the 2 groups. METHODS: We enrolled children 2 to 12 years of age with and without obesity, defined as >95th percentile body mass index (BMI) for age and sex, undergoing elective tonsillectomy ± adenoidectomy. After induction, subjects had 2 intravenous (IV) lines placed in 2 different extremities: 1 for medications and IV fluids and 1 for obtaining blood aliquots for fentanyl concentration analysis. After administration of 1 mcg/kg of fentanyl based on total body weight (TBW), blood sample collections for fentanyl concentration analysis were attempted at 5, 15, 30, 60, 90, and 120 minutes. Five-minute fentanyl concentrations were compared between obese and nonobese cohorts. Population PK analysis to examine the differences between obese and nonobese children was performed and included various body size descriptors, such as TBW, BMI, and fat-free mass (FFM), to examine their influence on model parameters. RESULTS: Half of the 30 subjects were obese. Mean fentanyl concentrations at 5 minutes were 0.53 ng/mL for the nonobese group and 0.88 ng/mL for the obese group, difference 0.35 ng/mL (95% CI, 0.08-0.61 ng/mL; P = .01). Population PK analysis showed that FFM was a significant covariate for the central volume of distribution. The potential clinical effect of an IV bolus dose of fentanyl based on TBW versus FFM in an obese child was assessed in a simulation using our model. 1 mcg/kg fentanyl dose based on TBW resulted in an approximately 60% higher peak fentanyl effect site concentration than dosing based on FFM. CONCLUSIONS: Our data demonstrated higher peak plasma fentanyl concentrations in obese compared to nonobese subjects. Population PK analysis found that FFM was a significant covariate for the central volume of distribution. Model simulation showed dosing of fentanyl in obese children based on TBW resulted in significantly higher peak concentrations than dosing based on FFM. Based on this modeling and the known concentration-effect relationship between fentanyl and adverse effects, our results suggest that bolus dosing of fentanyl in obese children should be based on FFM rather than TBW, particularly for procedures of short duration.

Inpatient Care Utilization Following Mobile Crisis Response Encounters Among Racial/Ethnic Minoritized Youth.

OBJECTIVE: There has been an increase in youth psychiatric emergencies and psychiatric inpatient hospitalizations in recent years. Mobile crisis response (MCR) services offer an opportunity to meet acute youth mental health needs in the community and to provide linkage to care. However, an understanding of MCR encounters as a care pathway is needed, including how patterns of subsequent care may vary by youth race/ethnicity. The current study examines racial/ethnic differences in the rates of inpatient care use following MCR among youth. METHOD: Data included Los Angeles County Department of Mental Health (LACDMH) administrative claims for MCR in 2017 and psychiatric inpatient hospitalizations and outpatient services from 2017-2020 for youth aged 0 to 18 years. RESULTS: In this sample of 6,908 youth (70.4% racial/ethnic minoritized youth) who received an MCR, 3.2% received inpatient care within 30 days of their MCR, 18.6% received inpatient care beyond 30 days of their MCR, and 14.7% received repeated inpatient care episodes during the study period. Multivariate models revealed that Asian American/Pacific Islander (AAPI) youth were less likely to receive inpatient care, whereas American Indian/Alaska Native (AI/AN) youth were more likely to receive inpatient care following MCR. Youth age, primary language, primary diagnosis, and insurance status also predicted future inpatient episodes. CONCLUSION: Findings highlight differential rates of inpatient use following MCR among AAPI and AI/AN youth relative to youth from other groups. Alternative interpretations for the findings are offered related to differential levels of need and disparate penetration of community-based outpatient and prevention-focused services.

Post-COVID-19 Cardiopulmonary Symptoms: Predictors and Imaging Features in Patients after Hospital Discharge. / Sintomas Cardiopulmonares Pós-COVID-19: Preditores e Características de Imagem de Pacientes após a Alta Hospitalar.

BACKGROUND: Most of the evidence about the impact of the post-acute COVID-19 Syndrome (PACS) reports individual symptoms without correlations with related imaging. OBJECTIVES: To evaluate cardiopulmonary symptoms, their predictors and related images in COVID-19 patients discharged from hospital. METHODS: Consecutive patients who survived COVID-19 were contacted 90 days after discharge. The Clinic Outcome Team structured a questionnaire evaluating symptoms and clinical status (blinded for hospitalization data). A multivariate analysis was performed to address the course of COVID-19, comorbidities, anxiety, depression, and post-traumatic stress during hospitalization, and cardiac rehabilitation after discharge. The significance level was set at 5%. RESULTS: A total of 480 discharged patients with COVID-19 (age: 59±14 years, 67.5% males) were included; 22.3% required mechanical ventilation. The prevalence of patients with PACS-related cardiopulmonary symptoms (dyspnea, tiredness/fatigue, cough, and chest discomfort) was 16.3%. Several parameters of chest computed tomography and echocardiogram were similar in patients with and without cardiopulmonary symptoms. The multivariate analysis showed that PACS-related cardiopulmonary-symptoms were independently related to female sex (OR 3.023; 95% CI 1.319-6.929), in-hospital deep venous thrombosis (OR 13.689; 95% CI 1.069-175.304), elevated troponin I (OR 1.355; 95% CI 1.048-1.751) and C-reactive protein during hospitalization (OR 1.060; 95% CI 1.023-1.097) and depression (OR 6.110; 95% CI 2.254-16.558). CONCLUSION: PACS-related cardiopulmonary symptoms 90 days post-discharge are common and multifactorial. Beyond thrombotic and markers of inflammation/myocardial injury during hospitalization, female sex and depression were independently associated with cardiopulmonary-related PACS. These results highlighted the need for a multifaceted approach targeting susceptible patients.

FUNDAMENTO: A maioria da evidência sobre o impacto da síndrome COVID pós-aguda (PACS, do inglês, post-acute COVID-19 syndrome) descreve sintomas individuais sem correlacioná-los com exames de imagens. OBJETIVOS: Avaliar sintomas cardiopulmonares, seus preditores e imagens relacionadas em pacientes com COVID-19 após alta hospitalar. MÉTODOS: Pacientes consecutivos, que sobreviveram à COVID-19, foram contatados 90 dias após a alta hospitalar. A equipe de desfechos clínicos (cega quanto aos dados durante a internação) elaborou um questionário estruturado avaliando sintomas e estado clínico. Uma análise multivariada foi realizada abordando a evolução da COVID-19, comorbidades, ansiedade, depressão, e estresse pós-traumático durante a internação, e reabilitação cardíaca após a alta. O nível de significância usado nas análises foi de 5%. RESULTADOS: Foram incluídos 480 pacientes (idade 59±14 anos, 67,5% do sexo masculino) que receberam alta hospitalar por COVID-19; 22,3% necessitaram de ventilação mecânica. A prevalência de pacientes com sintomas cardiopulmonares relacionados à PACS (dispneia, cansaço/fadiga, tosse e desconforto no peito) foi de 16,3%. Vários parâmetros de tomografia computadorizada do tórax e de ecocardiograma foram similares entre os pacientes com e sem sintomas cardiopulmonares. A análise multivariada mostrou que sintomas cardiopulmonares foram relacionados de maneira independente com sexo feminino (OR 3,023; IC95% 1,319-6,929), trombose venosa profunda durante a internação (OR 13,689; IC95% 1,069-175,304), nível elevado de troponina (OR 1,355; IC95% 1,048-1,751) e de proteína C reativa durante a internação (OR 1,060; IC95% 1,023-1,097) e depressão (OR 6,110; IC95% 2,254-16,558). CONCLUSÃO: Os sintomas cardiopulmonares relacionados à PACS 90 dias após a alta hospitalar são comuns e multifatoriais. Além dos marcadores trombóticos, inflamatórios e de lesão miocárdica durante a internação, sexo feminino e depressão foram associados independentemente com sintomas cardiopulmonares relacionados à PACS. Esses resultados destacaram a necessidade de uma abordagem multifacetada direcionada a pacientes susceptíveis.

Vaccination status and long COVID symptoms in patients discharged from hospital.

Effective vaccination against coronavirus mitigates the risk of hospitalisation and mortality; however, it is unclear whether vaccination status influences long COVID symptoms in patients who require hospitalisation. The available evidence is limited to outpatients with mild disease. Here, we evaluated 412 patients (age: 60 ± 16 years, 65% males) consecutively admitted to two Hospitals in Brazil due to confirmed coronavirus disease 2019 (COVID-19). Compared with patients with complete vaccination (n = 185) before infection or hospitalisation, those with no or incomplete vaccination (n = 227) were younger and had a lower frequency of several comorbidities. Data during hospitalisation revealed that the no or incomplete vaccination group required more admissions to the intensive care unit (ICU), used more corticosteroids, and had higher rates of pulmonary embolism or deep venous thrombosis than the complete vaccination group. Ninety days after hospital discharge, patients with no or incomplete vaccination presented a higher frequency of symptoms (≥ 1) than patients with complete vaccination (40 vs. 27%; p = 0.013). After adjusting for confounders, no or incomplete vaccination (odds ratio [OR] 1.819; 95% confidence interval [CI] 1.175-2.815), female sex (OR 2.435; 95% CI 1.575-3.764) and ICU admission during hospitalisation (OR 1.697; 95% CI 1.062-2.712) were independently associated with ≥ 1 symptom 90 days after hospital discharge. In conclusion, even in patients with severe COVID-19, vaccination mitigates the probability of long COVID symptoms.

Feeding and Swallowing Characteristics of Children With Esophageal Atresia and Tracheoesophageal Fistula.

OBJECTIVES: This study aimed to characterize feeding/swallowing difficulties in children with esophageal atresia and/or tracheoesophageal fistula (EA/TEF) and evaluate associations among feeding difficulties, pharyngeal dysphagia (PD), and other aerodigestive evaluation findings. METHODS: This was a retrospective cohort study of feeding/swallowing characteristics of 44 patients with EA/TEF treated in the aerodigestive program of a single academic medical institution from 2010 to 2015. Demographics, comorbidities, presence and characteristics of feeding/swallowing difficulties, and results of relevant diagnostic tests [videofluoroscopic swallow studies (VFSS), clinical feeding evaluations (CFEs), chest computerized tomography (CT) scans, pulmonary bronchoscopies, and upper GI (UGI)/esophagrams] were reviewed. RESULTS: Fifty percent of the cohort had PD and 88.6% had feeding difficulties. Across 118 encounters (87 VFSS and 31 CFEs), feeding difficulties suggestive of esophageal dysphagia were most frequently seen in children over 48 months and feeding difficulties suggestive of developmental feeding problems were most frequently seen in children from 24 to 48 months. Abnormal findings were present in 59.8% of VFSS, with aspiration (34.5%) and pharyngeal residue (26.4%) the most frequently observed signs of dysphagia. Abnormal UGI/esophagram findings were not associated with significantly increased risk of feeding difficulties during visits within 3 months (risk ratio, RR = 1.33). Presence of dysphagia was associated with increased risk for some abnormal CT findings (RR= 3.0 for airspace and 3.0 for bronchiectasis). CONCLUSIONS: Feeding/swallowing difficulties are common in EA/TEF, and types of feeding difficulties vary by patient age. The presence of abnormal findings on UGI/esophagram did not increase the risk of feeding complaints; however, the presence of dysphagia increased the risk of abnormal chest CT.

Sintomas Cardiopulmonares Pós-COVID-19: Preditores e Características de Imagem de Pacientes após a Alta Hospitalar / Post-COVID-19 Cardiopulmonary Symptoms: Predictors and Imaging Features in Patients after Hospital Discharge

Resumo Fundamento A maioria da evidência sobre o impacto da síndrome COVID pós-aguda (PACS, do inglês, post-acute COVID-19 syndrome) descreve sintomas individuais sem correlacioná-los com exames de imagens. Objetivos Avaliar sintomas cardiopulmonares, seus preditores e imagens relacionadas em pacientes com COVID-19 após alta hospitalar. Métodos Pacientes consecutivos, que sobreviveram à COVID-19, foram contatados 90 dias após a alta hospitalar. A equipe de desfechos clínicos (cega quanto aos dados durante a internação) elaborou um questionário estruturado avaliando sintomas e estado clínico. Uma análise multivariada foi realizada abordando a evolução da COVID-19, comorbidades, ansiedade, depressão, e estresse pós-traumático durante a internação, e reabilitação cardíaca após a alta. O nível de significância usado nas análises foi de 5%. Resultados Foram incluídos 480 pacientes (idade 59±14 anos, 67,5% do sexo masculino) que receberam alta hospitalar por COVID-19; 22,3% necessitaram de ventilação mecânica. A prevalência de pacientes com sintomas cardiopulmonares relacionados à PACS (dispneia, cansaço/fadiga, tosse e desconforto no peito) foi de 16,3%. Vários parâmetros de tomografia computadorizada do tórax e de ecocardiograma foram similares entre os pacientes com e sem sintomas cardiopulmonares. A análise multivariada mostrou que sintomas cardiopulmonares foram relacionados de maneira independente com sexo feminino (OR 3,023; IC95% 1,319-6,929), trombose venosa profunda durante a internação (OR 13,689; IC95% 1,069-175,304), nível elevado de troponina (OR 1,355; IC95% 1,048-1,751) e de proteína C reativa durante a internação (OR 1,060; IC95% 1,023-1,097) e depressão (OR 6,110; IC95% 2,254-16,558). Conclusão Os sintomas cardiopulmonares relacionados à PACS 90 dias após a alta hospitalar são comuns e multifatoriais. Além dos marcadores trombóticos, inflamatórios e de lesão miocárdica durante a internação, sexo feminino e depressão foram associados independentemente com sintomas cardiopulmonares relacionados à PACS. Esses resultados destacaram a necessidade de uma abordagem multifacetada direcionada a pacientes susceptíveis.

Abstract Background Most of the evidence about the impact of the post-acute COVID-19 Syndrome (PACS) reports individual symptoms without correlations with related imaging. Objectives To evaluate cardiopulmonary symptoms, their predictors and related images in COVID-19 patients discharged from hospital. Methods Consecutive patients who survived COVID-19 were contacted 90 days after discharge. The Clinic Outcome Team structured a questionnaire evaluating symptoms and clinical status (blinded for hospitalization data). A multivariate analysis was performed to address the course of COVID-19, comorbidities, anxiety, depression, and post-traumatic stress during hospitalization, and cardiac rehabilitation after discharge. The significance level was set at 5%. Results A total of 480 discharged patients with COVID-19 (age: 59±14 years, 67.5% males) were included; 22.3% required mechanical ventilation. The prevalence of patients with PACS-related cardiopulmonary symptoms (dyspnea, tiredness/fatigue, cough, and chest discomfort) was 16.3%. Several parameters of chest computed tomography and echocardiogram were similar in patients with and without cardiopulmonary symptoms. The multivariate analysis showed that PACS-related cardiopulmonary-symptoms were independently related to female sex (OR 3.023; 95% CI 1.319-6.929), in-hospital deep venous thrombosis (OR 13.689; 95% CI 1.069-175.304), elevated troponin I (OR 1.355; 95% CI 1.048-1.751) and C-reactive protein during hospitalization (OR 1.060; 95% CI 1.023-1.097) and depression (OR 6.110; 95% CI 2.254-16.558). Conclusion PACS-related cardiopulmonary symptoms 90 days post-discharge are common and multifactorial. Beyond thrombotic and markers of inflammation/myocardial injury during hospitalization, female sex and depression were independently associated with cardiopulmonary-related PACS. These results highlighted the need for a multifaceted approach targeting susceptible patients.

Creating a positive feedback loop in leadership to accelerate cultural change.

Leadership skills are highly valued in institutional environments, but in the context of persons excluded because of ethnicity or race (PEERs) in science, support for developing those skills often is lacking. To increase leadership diversity, institutions and programs should practice and teach diverse leadership strategies.

Control of Gαq signaling dynamics and GPCR cross-talk by GRKs.

Numerous processes contribute to the regulation of G protein-coupled receptors (GPCRs), but relatively little is known about rapid mechanisms that control signaling on the seconds time scale or regulate cross-talk between receptors. Here, we reveal that the ability of some GPCR kinases (GRKs) to bind Gαq both drives acute signaling desensitization and regulates functional interactions between GPCRs. GRK2/3-mediated acute desensitization occurs within seconds, is rapidly reversible, and can occur upon local, subcellular activation. This rapid desensitization is kinase independent, insensitive to pharmacological inhibition, and generalizable across receptor families and effectors. We also find that the ability of GRK2 to bind G proteins also enables it to regulate the extent and timing of Gαq-dependent signaling cross-talk between GPCRs. Last, we find that G protein/GRK2 interactions enable a novel form of GPCR trafficking cross-talk. Together, this work reveals potent forms of Gαq-dependent GPCR regulation with wide-ranging pharmacological and physiological implications.

Human stem cell-derived thymic epithelial cells enhance human T-cell development in a xenogeneic thymus.

BACKGROUND: Generation of thymic tissue from pluripotent stem cells would provide therapies for acquired and congenital thymic insufficiency states. OBJECTIVES: This study aimed to generate human thymic epithelial progenitors from human embryonic stem cells (hES-TEPs) and to assess their thymopoietic function in vivo. METHODS: This study differentiated hES-TEPs by mimicking developmental queues with FGF8, retinoic acid, SHH, Noggin, and BMP4. Their function was assessed in reaggregate cellular grafts under the kidney capsule and in hybrid thymi by incorporating them into swine thymus (SwTHY) grafts implanted under the kidney capsules of immunodeficient mice that received human hematopoietic stem and progenitor cells (hHSPCs) intravenously. RESULTS: Cultured hES-TEPs expressed FOXN1 and formed colonies expressing EPCAM and both cortical and medullary thymic epithelial cell markers. In thymectomized immunodeficient mice receiving hHSPCs, hES-TEPs mixed with human thymic mesenchymal cells supported human T-cell development. Hypothesizing that support from non-epithelial thymic cells might allow long-term function of hES-TEPs, the investigators injected them into SwTHY tissue, which supports human thymopoiesis in NOD severe combined immunodeficiency IL2Rγnull mice receiving hHSPCs. hES-TEPs integrated into SwTHY grafts, enhanced human thymopoiesis, and increased peripheral CD4+ naive T-cell reconstitution. CONCLUSIONS: This study has developed and demonstrated in vivo thymopoietic function of hES-TEPs generated with a novel differentiation protocol. The SwTHY hybrid thymus model demonstrates beneficial effects on human thymocyte development of hES-TEPs maturing in the context of a supportive thymic structure.

Mandibular muscle troponin of the Florida carpenter ant Camponotus floridanus: extending our insights into invertebrate Ca2+ regulation.

Ants use their mandibles for a variety of functions and behaviors. We investigated mandibular muscle structure and function from major workers of the Florida carpenter ant Camponotus floridanus: force-pCa relation and velocity of unloaded shortening of single, permeabilized fibres, primary sequences of troponin subunits (TnC, TnI and TnT) from a mandibular muscle cDNA library, and muscle fibre ultrastructure. From the mechanical measurements, we found Ca2+-sensitivity of isometric force was markedly shifted rightward compared with vertebrate striated muscle. From the troponin sequence results, we identified features that could explain the rightward shift of Ca2+-activation: the N-helix of TnC is effectively absent and three of the four EF-hands of TnC (sites I, II and III) do not adhere to canonical sequence rules for divalent cation binding; two alternatively spliced isoforms of TnI were identified with the alternatively spliced exon occurring in the region of the IT-arm α-helical coiled-coil, and the N-terminal extension of TnI may be involved in modulation of regulation, as in mammalian cardiac muscle; and TnT has a Glu-rich C-terminus. In addition, a structural homology model was built of C. floridanus troponin on the thin filament. From analysis of electron micrographs, we found thick filaments are almost as long as the 6.8 µm sarcomeres, have diameter of ~ 16 nm, and typical center-to-center spacing of ~ 46 nm. These results have implications for the mechanisms by which mandibular muscle fibres perform such a variety of functions, and how the structure of the troponin complex aids in these tasks.

The Trypanosoma brucei subpellicular microtubule array is organized into functionally discrete subdomains defined by microtubule associated proteins.

Microtubules are inherently dynamic cytoskeletal polymers whose length and organization can be altered to perform essential functions in eukaryotic cells, such as providing tracks for intracellular trafficking and forming the mitotic spindle. Microtubules can be bundled to create more stable structures that collectively propagate force, such as in the flagellar axoneme, which provides motility. The subpellicular microtubule array of the protist parasite Trypanosoma brucei, the causative agent of African sleeping sickness, is a remarkable example of a highly specialized microtubule bundle. It is comprised of a single layer of microtubules that are crosslinked to each other and to the overlying plasma membrane. The array microtubules appear to be highly stable and remain intact throughout the cell cycle, but very little is known about the pathways that tune microtubule properties in trypanosomatids. Here, we show that the subpellicular microtubule array is organized into subdomains that consist of differentially localized array-associated proteins at the array posterior, middle, and anterior. The array-associated protein PAVE1 stabilizes array microtubules at the cell posterior and is essential for maintaining its tapered shape. PAVE1 and the newly identified protein PAVE2 form a complex that binds directly to the microtubule lattice, demonstrating that they are a true kinetoplastid-specific MAP. TbAIR9, which localizes to the entirety of the subpellicular array, is necessary for maintaining the localization of array-associated proteins within their respective subdomains of the array. The arrangement of proteins within the array likely tunes the local properties of array microtubules and creates the asymmetric shape of the cell, which is essential for parasite viability.

Mechanisms of differential desensitization of metabotropic glutamate receptors.

G protein-coupled receptors (GPCRs) interact with intracellular transducers to control both signal initiation and desensitization, but the distinct mechanisms that control the regulation of different GPCR subtypes are unclear. Here we use fluorescence imaging and electrophysiology to examine the metabotropic glutamate receptor (mGluR) family. We find distinct properties across subtypes in both rapid desensitization and internalization, with striking differences between the group II mGluRs. mGluR3, but not mGluR2, undergoes glutamate-dependent rapid desensitization, internalization, trafficking, and recycling. We map differences between mGluRs to variable Ser/Thr-rich sequences in the C-terminal domain (CTD) that control interaction with both GPCR kinases and ß-arrestins. Finally, we identify a cancer-associated mutation, G848E, within the mGluR3 CTD that enhances ß-arrestin coupling and internalization, enabling an analysis of mGluR3 ß-arrestin-coupling properties and revealing biased variants. Together, this work provides a framework for understanding the distinct regulation and functional roles of mGluR subtypes.

A fine-tuned azobenzene for enhanced photopharmacology in vivo.

Despite the power of photopharmacology for interrogating signaling proteins, many photopharmacological systems are limited by their efficiency, speed, or spectral properties. Here, we screen a library of azobenzene photoswitches and identify a urea-substituted "azobenzene-400" core that offers bistable switching between cis and trans with improved kinetics, light sensitivity, and a red-shift. We then focus on the metabotropic glutamate receptors (mGluRs), neuromodulatory receptors that are major pharmacological targets. Synthesis of "BGAG12,400," a photoswitchable orthogonal, remotely tethered ligand (PORTL), enables highly efficient, rapid optical agonism following conjugation to SNAP-tagged mGluR2 and permits robust optical control of mGluR1 and mGluR5 signaling. We then produce fluorophore-conjugated branched PORTLs to enable dual imaging and manipulation of mGluRs and highlight their power in ex vivo slice and in vivo behavioral experiments in the mouse prefrontal cortex. Finally, we demonstrate the generalizability of our strategy by developing an improved soluble, photoswitchable pore blocker for potassium channels.

Whole-Proteome Differential Screening Identifies Novel Vaccine Candidates for Schistosomiasis japonica.

Schistosomiasis remains a leading cause of chronic morbidity in endemic regions despite decades of widespread mass chemotherapy with praziquantel. Using our whole proteome differential screening approach, and plasma and epidemiologic data from a longitudinal cohort of individuals living in a Schistosoma japonicum-endemic region of the Philippines, we interrogated the parasite proteome to identify novel vaccine candidates for Schistosoma japonicum. We identified 16 parasite genes which encoded proteins that were recognized by immunoglobulin G or immunoglobulin E antibodies in the plasma of individuals who had developed resistance to reinfection, but were not recognized by antibodies in the plasma of individuals who remained susceptible to reinfection. Antibody levels to Sj6-8 and Sj4-1 measured in the entire cohort (N = 505) 1 month after praziquantel treatment were associated with significantly decreased risk of reinfection and lower intensity of reinfection over 18 months of follow-up.

Role of the thymus in spontaneous development of a multi-organ autoimmune disease in human immune system mice.

We evaluated the role of the thymus in development of multi-organ autoimmunity in human immune system (HIS) mice. T cells were essential for disease development and the same T cell clones with varying phenotypes infiltrated multiple tissues. De novo-generated hematopoietic stem cell (HSC)-derived T cells were the major disease drivers, though thymocytes pre-existing in grafted human thymi contributed if not first depleted. HIS mice with a native mouse thymus developed disease earlier than thymectomized mice with a thymocyte-depleted human thymus graft. Defective structure in the native mouse thymus was associated with impaired negative selection of thymocytes expressing a transgenic TCR recognizing a self-antigen. Disease developed without direct recognition of antigens on recipient mouse MHC. While human thymus grafts had normal structure and negative selection, failure to tolerize human T cells recognizing mouse antigens presented on HLA molecules may explain eventual disease development. These new insights have implications for human autoimmunity and suggest methods of avoiding autoimmunity in next-generation HIS mice.

Targeting dual signalling pathways in concert with immune checkpoints for the treatment of pancreatic cancer.

OBJECTIVE: This study exploits the intersection between molecular-targeted therapies and immune-checkpoint inhibition to define new means to treat pancreatic cancer. DESIGN: Patient-derived cell lines and xenograft models were used to define the response to CDK4/6 and MEK inhibition in the tumour compartment. Impacts relative to immunotherapy were performed using subcutaneous and orthotopic syngeneic models. Single-cell RNA sequencing and multispectral imaging were employed to delineate effects on the immunological milieu in the tumour microenvironment. RESULTS: We found that combination treatment with MEK and CDK4/6 inhibitors was effective across a broad range of PDX models in delaying tumour progression. These effects were associated with stable cell-cycle arrest, as well as the induction of multiple genes associated with interferon response and antigen presentation in an RB-dependent fashion. Using single-cell sequencing and complementary approaches, we found that the combination of CDK4/6 and MEK inhibition had a significant impact on increasing T-cell infiltration and altering myeloid populations, while potently cooperating with immune checkpoint inhibitors. CONCLUSIONS: Together, these data indicate that there are canonical and non-canonical features of CDK4/6 and MEK inhibition that impact on the tumour and immune microenvironment. This combination-targeted treatment can promote robust tumour control in combination with immune checkpoint inhibitor therapy.

Cost of closure: Comparing success and spending of fat graft myringoplasties with and without hyaluronic acid.

INTRODUCTION: Myringoplasties are common pediatric procedures used to surgically close a perforated tympanic membrane. While a wide variety of graft materials are available to surgeons, the cost effectiveness of these different techniques is not well studied. OBJECTIVES: To compare the cost effectiveness of the fat graft myringoplasty (FGM) with the hyaluronic acid fat graft myringoplasty (HAFGM). METHODS: Retrospective chart review of patients ages 31 days to 18 years who had undergone either FGM or HAFGM from 2006 to 2016. RESULTS: We identified 85 patients who had undergone FGM and 51 patients who had undergone HAFGM. The two groups were statistically similar in age (CI -0.51, 1.9; p = 0.23), sex (CI 0.3, 1.4; p = 0.27), and history of prior tympanostomy tube placement (CI -0.07, 0.07; p = 0.69). Both groups had a similar number of total comorbidities (40.0% of patients in the FGM group and 27.5% of patients in the HAFGM; CI -0.04, 0.29; p = 0.19). The FGM and the HAFGM procedure did not have statistically significant differences in perforation closure rates, 82.4% and 92.2% respectively (CI 0.81, 7.3; p = 0.13). In comparing the total surgeons' cost of closing a tympanic membrane deficit, the FGM incurred a greater cost per perforation. The cost/tympanic membrane perforation closure for the FGM totaled $3011.88 per deficit, whereas the HAFGM totaled $2742.98. CONCLUSION: As financial stewardship becomes more important for medical decision making, it is imperative that providers consider cost and outcomes data together when comparing similar treatment options. The FGM and the HAFGM have statistically similar rates of success in closing tympanic membrane perforations. In this study, the FGM cohort consumed more health care dollars per perforation secondary to the need for revision surgeries. As such, this study offers that the additional use of a hyaluronic disc does not increase overall cost to the healthcare system when performing a fat graft myringoplasty over a large cohort of patients.

Demonstrating the benefits of a multidisciplinary aerodigestive program.

OBJECTIVES/HYPOTHESIS: The Aerodigestive Program (the Aero Program) at Children's Hospital Colorado is a multidisciplinary program focused on airway, digestive, and lung disorders in complex children, involving collaboration between gastroenterology, pulmonology, anesthesiology, and otolaryngology in clinic and operating room. These programs have proliferated as institutions focus on providing greater care coordination and family satisfaction. However, few cost, charge, and satisfaction data exist to support these resource-intensive programs. The goal of this study was to investigate the value of combined triple endoscopy delivered by the Aero Program through analysis of institutional charges, direct costs, operating room efficiency metrics, and parent satisfaction. STUDY DESIGN: Program evaluation. METHODS: Finance, satisfaction, efficiency, and quality-of-care metrics were evaluated within and outside of the Aero Program through retrospective queries of electronic health records, administrative databases, and parent surveys at our institution. RESULTS: Mean anesthesia time in the Aero Program was 54 minutes (49-60; 95% confidence interval), which was significantly less (P < .0001) than the estimated 89 minutes of having the three procedures done separately. Average charges and average direct costs for triple endoscopy were 38.8% and 41.9% less than the sum of the averages for separate procedures, respectively. Parent satisfaction was high for the Aero Program care. CONCLUSIONS: As organizations move toward greater coordination of care for complex patients, multidisciplinary programs must demonstrate their value by delivering cost-effective care. Aerodigestive programs have the potential to provide satisfying care that is less costly to the organization, insurer, and family. These programs represent a step in the evolution toward higher value care and value-based payment methodology. LEVEL OF EVIDENCE: 4 Laryngoscope, 130:521-525, 2020.