Constitutive Flt3 signaling impacts conventional dendritic cell function.

The development of dendritic cells (DCs) depends on signaling via the FMS-like tyrosine kinase 3 (Flt3) receptor. How Flt3 signaling impacts terminally differentiated DC function is unknown. This is important given the increasing interest in exploiting Flt3 for vaccination and tumor immunotherapy. Here, we examined DCs in mice harboring constitutively activated Flt3 (Flt3-ITD). Flt3ITD/ITD mice possessed expanded splenic DC subsets including plasmacytoid DC, conventional DC (cDC)1, cDC2, double positive (DP) cDC1 (CD11c+ CD8+ CD11b- CD103+ CD86+), noncanonical (NC) cDC1 (CD11c+ CD8+ CD11b- CD103- CD86-) and single positive (SP) cDC1 (CD11c+ CD8+ CD11b- CD103- CD86+). Outcomes of constitutive Flt3 signaling differed depending on the cDC subset examined. In comparison with wild type (WT) DCs, all Flt3ITD/ITD cDCs displayed an altered surface phenotype with changes in costimulatory molecules, major histocompatibility complex class I (MHC I) and II (MHC II). Cytokine secretion patterns, antigen uptake, antigen proteolysis and antigen presenting function differed between WT and Flt3ITD/ITD subsets, particularly cDC2. In summary, Flt3 signaling impacts the function of terminally differentiated cDCs with important consequences for antigen presentation.

GPR41 and GPR43 regulate CD8+ T cell priming during herpes simplex virus type 1 infection.

Naïve CD8+ T cells need to undergo a complex and coordinated differentiation program to gain the capacity to control virus infections. This not only involves the acquisition of effector functions, but also regulates the development of a subset of effector CD8+ T cells into long-lived and protective memory cells. Microbiota-derived metabolites have recently gained interest for their influence on T cells, but much remains unclear about their role in CD8+ T cell differentiation. In this study, we investigated the role of the G protein-coupled receptors (GPR)41 and GPR43 that can bind microbiota-derived short chain fatty acids (SCFAs) in CD8+ T cell priming following epicutaneous herpes simplex virus type 1 (HSV-1) infection. We found that HSV-specific CD8+ T cells in GPR41/43-deficient mice were impaired in the antigen-elicited production of interferon-gamma (IFN-γ), tumour necrosis factor-alpha (TNF-α), granzyme B and perforin, and failed to differentiate effectively into memory precursors. The defect in controlling HSV-1 at the site of infection could be restored when GPR41 and GPR43 were expressed exclusively by HSV-specific CD8+ T cells. Our findings therefore highlight roles for GPR41 and GPR43 in CD8+ T cell differentiation, emphasising the importance of metabolite sensing in fine-tuning anti-viral CD8+ T cell priming.

Asthma is associated with a lower incidence of metastatic colorectal cancer in a US patient cohort.

In previous pre-clinical studies, we examined the contribution of interleukin 4 receptor (IL4R) signaling in the progression and metastasis of colorectal cancer (CRC). Aberrant activation of this receptor can result in atopic diseases such as asthma. We hypothesized that further evidence for the contribution of excessive IL4R being associated with CRC progression could be seen in medical records, and specifically that chronic asthma patients were more likely to be diagnosed with metastatic CRC. To test this hypothesis, we took advantage of the Synthetic Derivative, a resource developed at Vanderbilt University Medical Center that hosts de-identified data taken from the electronic medical record. We developed search protocols that produced retrospective cohorts of invasive CRC patients and cancer-free equivalents. In comparing 787 metastatic CRC patients to 238 non-metastatic patients, we actually found significantly fewer asthmatics went on to develop metastatic CRC (P=0.0381). By comparing these groups together against 1197 cancer-free patients, even fewer asthmatic patients would develop invasive CRC (P<0.0001). While these results are clearly in opposition to our original hypothesis, they still support a link between chronic asthma and metastatic CRC development. One intriguing possibility, that will be examined in the future, is whether treatment for chronic asthma may be responsible for the reduction in metastatic cancer.

CD4+ T cell calibration of antigen-presenting cells optimizes antiviral CD8+ T cell immunity.

Antiviral CD8+ T cell immunity depends on the integration of various contextual cues, but how antigen-presenting cells (APCs) consolidate these signals for decoding by T cells remains unclear. Here, we describe gradual interferon-α/interferon-ß (IFNα/ß)-induced transcriptional adaptations that endow APCs with the capacity to rapidly activate the transcriptional regulators p65, IRF1 and FOS after CD4+ T cell-mediated CD40 stimulation. While these responses operate through broadly used signaling components, they induce a unique set of co-stimulatory molecules and soluble mediators that cannot be elicited by IFNα/ß or CD40 alone. These responses are critical for the acquisition of antiviral CD8+ T cell effector function, and their activity in APCs from individuals infected with severe acute respiratory syndrome coronavirus 2 correlates with milder disease. These observations uncover a sequential integration process whereby APCs rely on CD4+ T cells to select the innate circuits that guide antiviral CD8+ T cell responses.

Implementation of a pharmacist toxicology service on treatment of paracetamol (acetaminophen) overdose.

INTRODUCTION: Paracetamol is a leading cause of fatality following a toxic ingestion. Individualized treatment is imperative in improving outcomes. Acetylcysteine is the standard of care for paracetamol overdose. Laboratory values and other clinical criteria can be used to guide treatment duration. Our hospital's protocol allows paracetamol overdose to be managed by the emergency department pharmacists. The purpose of this study was to evaluate the effect of a pharmacist toxicology service on the management of paracetamol overdose. METHODS: This was a single center, retrospective, cohort evaluation. All patients receiving acetylcysteine were divided into pre- and post-implementation groups with data obtained from August 1, 2013 to January 14, 2018 and January 15, 2018 to September 30, 2021, respectively. The primary outcome was the frequency of individualized acetylcysteine therapy. RESULTS: A total of 238 patients were screened for inclusion in the study with 120 patients included in the final analysis. There were 60 patients included in each cohort. The frequency of individualized acetylcysteine therapy was significantly higher in the post-implementation group versus the pre-implementation group (85% vs. 60% [95% CI 9.1-39.4; P = 0.002]). CONCLUSIONS: The implementation of a pharmacist toxicology service correlated with increased poison center consultation as well as increased frequency of individualized acetylcysteine therapy and decreased number of missed acetylcysteine doses.

Impact of the utilization of 500 mL IV bags on crystalloid resuscitation volumes administered to trauma patients.

INTRODUCTION: Administering large volumes of crystalloids to trauma patients has been shown to exacerbate metabolic complications of hemorrhage including dilutional coagulopathy and worsening acidosis The aim of this study was to evaluate crystalloid administration volumes in trauma patients after replacing 1 L IV containers with 500 mL IV containers in the emergency department trauma resuscitation bay. MATERIALS AND METHODS: This was a single-center, IRB-approved, retrospective cohort evaluation of adult trauma patients conducted at an 864-bed community tertiary referral center located in the southeastern United States. Patterns of crystalloid administration were examined before and after the trauma resuscitation bay began to exclusively stock 500 mL IV containers. The primary outcome was mean total crystalloid volume infused from time of injury to hospital admission. Secondary outcomes included mean total crystalloid volume infused prior to administration of blood products, proportion of patients who received less than 2 L total of crystalloids, time to initiation of blood products, and mortality in both the emergency department and in-hospital. RESULTS: Patient characteristics were largely similar between both groups including age, mechanism of injury, and Injury Severity Score. For the primary outcome, the mean total crystalloid volume infused from time of injury to hospital administration, patients in the 500 mL IV fluid container group were administered 555 mL less crystalloid when compared to the 1 L IV fluid container group, 1048 mL vs 1603 mL (p < 0.01; 95% CI 406 mL - 704 mL), respectively. After conversion to the 500 mL IV container bags, there was a 27.5% increase in the proportion of patients receiving less than 2 L of crystalloid, 90.5% vs 63.0% in the 500 mL IV fluid container and 1 L IV fluid container groups, respectively (p < 0.01). CONCLUSIONS: Due to reduced mortality, expanding literature and guidelines clearly support minimizing IV crystalloid resuscitation. Institutions must now work to minimize use of IV crystalloids to hemorrhaging trauma patients and a simple solution of using smaller IV fluid bags was shown to improve adherence to this practice.

MHC Class II Ubiquitination Regulates Dendritic Cell Function and Immunity.

MHC class II (MHC II) Ag presentation by dendritic cells (DCs) is critical for CD4+ T cell immunity. Cell surface levels of MHC II loaded with peptide is controlled by ubiquitination. In this study, we have examined how MHC II ubiquitination impacts immunity using MHC IIKRKI/KI mice expressing mutant MHC II molecules that are unable to be ubiquitinated. Numbers of conventional DC (cDC) 1, cDC2 and plasmacytoid DCs were significantly reduced in MHC IIKRKI/KI spleen, with the remaining MHC IIKRKI/KI DCs expressing an altered surface phenotype. Whereas Ag uptake, endosomal pH, and cathepsin protease activity were unaltered, MHC IIKRKI/KI cDC1 produced increased inflammatory cytokines and possessed defects in Ag proteolysis. Immunization of MHC IIKRKI/KI mice identified impairments in MHC II and MHC class I presentation of soluble, cell-associated and/or DC-targeted OVA via mAb specific for DC surface receptor Clec9A (anti-Clec9A-OVA mAb). Reduced T cell responses and impaired CTL killing was observed in MHC IIKRKI/KI mice following immunization with cell-associated and anti-Clec9A-OVA. Immunization of MHC IIKRKI/KI mice failed to elicit follicular Th cell responses and generated barely detectable Ab to anti-Clec9A mAb-targeted Ag. In summary, MHC II ubiquitination in DCs impacts the homeostasis, phenotype, cytokine production, and Ag proteolysis by DCs with consequences for Ag presentation and T cell and Ab-mediated immunity.

Dendritic cell Flt3 - regulation, roles and repercussions for immunotherapy.

Dendritic cells (DCs) are essential for initiating immune responses. Depending on the environment, the type of DC and the way in which they interact with T cells, these immune responses can be beneficial or detrimental. DCs can be exploited as cellular vectors for vaccines against infection and cancer. The development and maintenance of DCs is dependent on the FMS-like tyrosine kinase 3 (Flt3)/Flt3 ligand (Flt3L) signaling cascade. Flt3 is also one of the most commonly mutated genes in acute myeloid leukemia and as such represents an attractive drug target. In this review, Flt3 is discussed with a particular focus on DCs. We detail the lifecycle of Flt3, from transcription to degradation, and interrogate recent studies as to how this pathway can be manipulated for immunotherapy, vaccination and treatment of autoimmune disease.

Multisystem Inflammatory Syndrome Presenting as Early Acute Appendicitis.

An 11-year-old male presented to the pediatric emergency department with a one-day history of peri-umbilical pain with nausea, anorexia, and scant vomiting. On examination, he had moderate tenderness in the right upper quadrant with moderate guarding and rebound tenderness. Imaging showed concern for early acute appendicitis. The patient was admitted and underwent laparoscopic appendectomy. Despite the appendectomy, the patient continued to have fevers and abdominal pain. Four days after the initial presentation, the patient decompensated and was diagnosed with multisystem inflammatory syndrome. This case is interesting because the patient never met diagnostic criteria for multisystem inflammatory syndrome in children (MIS-C) prior to his decompensation. If a patient's symptoms continue or worsen despite seemingly appropriate management, the patient must be reassessed for other causes of pathology. Surgeons must have a high index of suspicion for MIS-C in patients with recent COVID-19 diagnoses, and this case demonstrates that MIS-C can present in phases and not all at once.

Physiological substrates and ontogeny-specific expression of the ubiquitin ligases MARCH1 and MARCH8.

MARCH1 and MARCH8 are ubiquitin ligases that control the expression and trafficking of critical immunoreceptors. Understanding of their function is hampered by three major knowledge gaps: (i) it is unclear which cell types utilize these ligases; (ii) their level of redundancy is unknown; and (iii) most of their putative substrates have been described in cell lines, often overexpressing MARCH1 or MARCH8, and it is unclear which substrates are regulated by either ligase in vivo. Here we address these questions by systematically analyzing the immune cell repertoire of MARCH1- or MARCH8-deficient mice, and applying unbiased proteomic profiling of the plasma membrane of primary cells to identify MARCH1 and MARCH8 substrates. Only CD86 and MHC II were unequivocally identified as immunoreceptors regulated by MARCH1 and MARCH8, but each ligase carried out its function in different tissues. MARCH1 regulated MHC II and CD86 in professional and "atypical" antigen presenting cells of hematopoietic origin, including neutrophils, eosinophils and monocytes. MARCH8 only operated in non-hematopoietic cells, such as thymic and alveolar epithelial cells. Our results establish the tissue-specific functions of MARCH1 and MARCH8 in regulation of immune receptor expression and reveal that the range of cells constitutively endowed with antigen-presentation capacity is wider than generally appreciated.

Ubiquitination of MHC Class II Is Required for Development of Regulatory but Not Conventional CD4+ T Cells.

MHC class II (MHC II) displays peptides at the cell surface, a process critical for CD4+ T cell development and priming. Ubiquitination is a mechanism that dictates surface MHC II with the attachment of a polyubiquitin chain to peptide-loaded MHC II, promoting its traffic away from the plasma membrane. In this study, we have examined how MHC II ubiquitination impacts the composition and function of both conventional CD4+ T cell and regulatory T cell (Treg) compartments. Responses were examined in two models of altered MHC II ubiquitination: MHCIIKRKI /KI mice that express a mutant MHC II unable to be ubiquitinated or mice that lack membrane-associated RING-CH 8 (MARCH8), the E3 ubiquitin ligase responsible for MHC II ubiquitination specifically in thymic epithelial cells. Conventional CD4+ T cell populations in thymus, blood, and spleen of MHCIIKRKI/KI and March8 -/- mice were largely unaltered. In MLRs, March8 -/-, but not MHCIIKRKI/KI, CD4+ T cells had reduced reactivity to both self- and allogeneic MHC II. Thymic Treg were significantly reduced in MHCIIKRKI/KI mice, but not March8 -/- mice, whereas splenic Treg were unaffected. Neither scenario provoked autoimmunity, with no evidence of immunohistopathology and normal levels of autoantibody. In summary, MHC II ubiquitination in specific APC types does not have a major impact on the conventional CD4+ T cell compartment but is important for Treg development.

Blocked transcription through KvDMR1 results in absence of methylation and gene silencing resembling Beckwith-Wiedemann syndrome.

The maternally methylated KvDMR1 ICR regulates imprinted expression of a cluster of maternally expressed genes on human chromosome 11p15.5. Disruption of imprinting leads to Beckwith-Wiedemann syndrome (BWS), an overgrowth and cancer predisposition condition. In the majority of individuals with BWS, maternal-specific methylation at KvDMR1 is absent and genes under its control are repressed. We analyzed a mouse model carrying a poly(A) truncation cassette inserted to prevent RNA transcripts from elongation through KvDMR1. Maternal inheritance of this mutation resulted in absence of DNA methylation at KvDMR1, which led to biallelic expression of Kcnq1ot1 and suppression of maternally expressed genes. This study provides further evidence that transcription is required for establishment of methylation at maternal gametic DMRs. More importantly, this mouse model recapitulates the molecular phenotypic characteristics of the most common form of BWS, including loss of methylation at KvDMR1 and biallelic repression of Cdkn1c, suggesting that deficiency of maternal transcription through KvDMR1 may be an underlying cause of some BWS cases.

Phosphorylation of Tyr188 in the WW domain of YAP1 plays an essential role in YAP1-induced cellular transformation.

The Hippo signaling pathway regulates cellular proliferation and survival, thus exerting profound effects on normal cell fate and tumorigenesis. The pivotal effector of this pathway is YAP1, a transcriptional co-activator amplified in mouse and human cancers where it promotes epithelial-to-mesenchymal transition (EMT) and malignant transformation. The Hippo tumor suppressor pathway has been suggested to inhibit the YAP1 function through serine phosphorylation-induced cytoplasmic retention and degradation. Here we report that the tyrosine188 (Y188) site of YAP1 isoform with 2 WW domains (known as YAP1-2) plays an important role in YAP1-induced cellular transformation. IP-Mass Spectrometry analysis of YAP1 identified the phosphorylation of Y188 but not other tyrosine residues. In contrast to the aberrant 3D acinus formation observed in YAP1-WT transduced cells, overexpression of YAP1-Y188F (non-phosphorylated mimic) displayed normal 3D structures. In addition, knockdown of the endogenous YAP1 in MDA-MB231 breast cancer cells inhibited cell proliferation and migration, which were then successfully rescued by the exogenous YAP1-WT and YAP1-Y188E but not Y188F. Mechanistically, we also demonstrated that YAP1-Y188F had a higher affinity to the upstream negative regulator PTPN14 and was extensively localized in the cytoplasm. Since the Y188 is located in the conserved aromatic core of the WW domain of YAP1, our finding has a wide implication for WW domain signaling in general, where Y phosphorylation may act as a common positive regulator of the complex formation via WW domains. In summary, our results indicate that tyrosine 188 plays an important role in the YAP1-induced cellular transformation and its phosphorylation may intriguingly serve as a positive indicator of YAP1 activation.

The Regulatory Role of KIBRA and PTPN14 in Hippo Signaling and Beyond.

The Hippo signaling pathway regulates cellular proliferation and survival, thus exerting profound effects on normal cell fate and tumorigenesis. Pivotal effectors of this pathway are YAP/TAZ, transcriptional co-activators whose dysfunction contributes to the development of cancer. Complex networks of intracellular and extracellular signaling pathways that modulate YAP and TAZ activities have recently been identified. Among them, KIBRA and PTPN14 are two evolutionarily-conserved and important YAP/TAZ upstream regulators. They can negatively regulate YAP/TAZ functions separately or in concert. In this review, we summarize the current and emerging regulatory roles of KIBRA and PTPN14 in the Hippo pathway and their functions in cancer.

Sox4 Expression Confers Bladder Cancer Stem Cell Properties and Predicts for Poor Patient Outcome.

Genetic and epigenetic alterations have been identified as to contribute directly or indirectly to the generation of transitional cell carcinoma of the urinary bladder (TCC-UB). We have previously found that amplification of chromosome 6p22 is significantly associated with the muscle-invasive rather than superficial TCC-UB. Here, we demonstrated that Sox4, one of the candidate oncogenes located within the chromosome 6p22 amplicon, confers bladder cancer stem cell (CSC) properties. Down-regulation of Sox4 led to the inhibition of cell migration, colony formation as well as mesenchymal-to-epithelial transition (MET). Interestingly, knockdown of Sox4 also reduced the sphere formation, enriched cell population with high levels of aldehyde dehydrogenase (ALDH (high)) and tumor formation potential. Using gene expression profiling, we further identified novel Sox4 target genes. Last, immunohistochemistry analysis of human bladder tumor tissue microarrays (TMAs) indicated that high Sox4 expression was correlated with advanced cancer stages and poor survival rate. In summary, our data show that Sox4 is an important regulator of the bladder CSC properties and it may serve as a biomarker of the aggressive phenotype in bladder cancer.

Molecular profiling and computational network analysis of TAZ-mediated mammary tumorigenesis identifies actionable therapeutic targets.

Triple-negative breast cancer (TNBC) accounts for approximately 15-20% of all breast cancer (BC) cases and contributes disproportionately to BC mortality. TAZ, a key transducer of the Hippo pathway, has recently been demonstrated to confer breast cancer stem cell (CSC) traits. However, TAZ target genes and the underlying transcriptional regulatory pathways responsible for the CSC phenomenon remain unknown. Here, we demonstrate that the oncogenic activity of TAZ is essential for propagation of the malignant phenotype. We further show that constitutively active TAZ tumor-derived cells exhibit unique tumor-initiating properties, including increased self-renewal and metastatic seeding potential, acquired chemotherapy resistance and the ability to efficiently regenerate tumor formation in vivo. Combined digital RNA expression analysis and computational network approaches identify several signaling pathways that distinguish breast cancer tumor-initiating cells (T-ICs) from bulk tumor cells. We demonstrate the utility of this approach by repositioning the small molecule tyrosine kinase inhibitor, Dasatinib, which selectively targets T-ICs and inhibits TNBC growth in vivo.

PTPN14 forms a complex with Kibra and LATS1 proteins and negatively regulates the YAP oncogenic function.

The Hippo signaling pathway regulates cellular proliferation and survival, thus exerting profound effects on normal cell fate and tumorigenesis. Pivotal effectors of this pathway are YAP/TAZ, transcriptional co-activators whose dysfunction contributes to epithelial-to-mesenchymal transition and malignant transformation. Therefore, it is of great importance to decipher the mechanisms underlying the regulations of YAP/TAZ at various levels. Here we report that non-receptor tyrosine phosphatase 14 (PTPN14) interacts with the Kibra protein. The interaction between PTPN14 and Kibra is through the PPXY domain of PTPN14 and WW domain of Kibra. PTPN14 and Kibra can induce the LATS1 activation independently and cooperatively. Interestingly, activation of LATS1 by PTPN14 is dependent on the C terminus of PTPN14 and independent of the upstream mammalian STE20-like kinase (MST) proteins. Furthermore, we demonstrate that PTPN14 increases the LAST1 protein stability. Last, overexpression of Kibra rescues the increased cell migration and aberrant three-dimensional morphogenesis induced by knockdown of PTPN14, and this rescue is mediated through the activation of the upstream LATS1 kinase and subsequent cytoplasmic sequestration of YAP. In summary, our results indicate a potential regulatory role of PTPN14 in the Hippo pathway and demonstrate another layer of regulation in the YAP oncogenic function.