Mast cells help organize the Peyer's patch niche for induction of IgA responses.

Peyer's patches (PPs) are lymphoid structures situated adjacent to the intestinal epithelium that support B cell responses that give rise to many intestinal IgA-secreting cells. Induction of isotype switching to IgA in PPs requires interactions between B cells and TGFß-activating conventional dendritic cells type 2 (cDC2s) in the subepithelial dome (SED). However, the mechanisms promoting cDC2 positioning in the SED are unclear. Here, we found that PP cDC2s express GPR35, a receptor that promotes cell migration in response to various metabolites, including 5-hydroxyindoleacetic acid (5-HIAA). In mice lacking GPR35, fewer cDC2s were found in the SED, and frequencies of IgA+ germinal center (GC) B cells were reduced. IgA plasma cells were reduced in both the PPs and lamina propria. These phenotypes were also observed in chimeric mice that lacked GPR35 selectively in cDCs. GPR35 deficiency led to reduced coating of commensal bacteria with IgA and reduced IgA responses to cholera toxin. Mast cells were present in the SED, and mast cell-deficient mice had reduced PP cDC2s and IgA+ cells. Ablation of tryptophan hydroxylase 1 (Tph1) in mast cells to prevent their production of 5-HIAA similarly led to reduced PP cDC2s and IgA responses. Thus, mast cell-guided positioning of GPR35+ cDC2s in the PP SED supports induction of intestinal IgA responses.

Outcomes of Distal Radius Fractures in Solid Organ Transplant Recipients.

PURPOSE: Distal radius fractures (DRFs) are one of the most common conditions that musculoskeletal providers treat. As the frequency of solid organ transplants (SOT) increases, these providers are often called upon to manage DRFs in these patients. These patients are at increased risk for osteopenia and osteoporosis, given the altered bone metabolism after SOT and frequent use of glucocorticoid and immunosuppressive medications. This study aimed to examine both surgical and nonsurgical treatment outcomes of DRFs in the SOT population and the prevalence of decreased bone mineral density. METHODS: A retrospective review of patients treated at a single institution who had previously undergone SOT and subsequently sustained DRF between 2013 and 2022 was completed. Patients were excluded for incomplete documentation and treatment initiation at an outside institution. Demographic variables, clinical outcomes, organ transplant, steroid use, and second metacarpal cortical percentage were collected for both groups. A telephone survey with the QuickDASH questionnaire was conducted for all available patients. RESULTS: A total of 34 DRFs in 33 patients were included in the analysis. Of these, 15 fractures in 14 patients underwent surgical intervention, and 19 fractures in 19 patients were managed nonsurgically. The following three adverse events were observed in the patients managed operatively: hardware failure, postoperative carpal tunnel syndrome, and tendon irritation. No reported treatment complications were recorded in the patients managed nonsurgically. Of the 33 included patients, 32 had radiographic evidence of decreased bone mineral density and five were receiving treatment for osteoporosis. CONCLUSIONS: Management of DRFs in SOT patients is challenging, given their increased medical complexity. Decreased bone mineral density was nearly universal and undertreated in this patient population. Most of these patients in both groups had good or excellent functional outcomes with both surgical and nonsurgical management. Additionally, surgery was well tolerated with no reported anesthesia complications, wound-healing issues, or infections. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.

Modifiable and non-modifiable risk factors for failure of non-operative treatment of pediatric forearm fractures: Where can we do better?

Introduction: Distal third forearm fractures are common fractures in children. While outcomes are generally excellent, some patients fail initial non-operative management and require intervention. The purpose of this study is to identify independent risk factors associated with failure of closed reduction. Methods: We conducted a retrospective review of distal third forearm fractures in children treated with closed reduction and casting. Patients were divided into two cohorts-those who were successfully closed reduced and those who failed initial non-operative management. Demographic characteristics, cast type, cast index, radiographic fracture, soft tissue characteristics, and quality of reduction were analyzed between groups. Results: A total of 207 children treated for distal third forearm fractures were included for analysis. A total of 190 (91.8%) children maintained their reduction while 17 (8.2%) failed initial non-operative management. Modifiable risk factors associated with loss of reduction on univariate analysis included the use of a long arm cast (p = 0.003), increased post-reduction displacement (p = 0.02), and increased post-reduction angular deformity (p = 0.01). Non-modifiable risk factors included increased body mass index (p = 0.02), increased presenting fracture displacement (p = 0.002), and increased width of the soft tissue envelope at the fracture site (p = 0.0001). The use of long arm casts (13% vs 2%, odds ratio = 6.44) and soft tissue width (60.6 vs 50.4 mm, odds ratio = 1.1) remained significant risk factors for loss of reduction after multivariate analysis. Conclusion: Both larger soft tissue envelope at the site of the fracture and long arm cast immobilization are independently associated with an increased risk of failing initial closed reduction in distal third forearm fractures in the pediatric population. Level of evidence: level III Case Control Study.

Platelets and mast cells promote pathogenic eosinophil recruitment during invasive fungal infection via the 5-HIAA-GPR35 ligand-receptor system.

Cryptococcus neoformans is the leading cause of fungal meningitis and is characterized by pathogenic eosinophil accumulation in the context of type-2 inflammation. The chemoattractant receptor GPR35 is expressed by granulocytes and promotes their migration to the inflammatory mediator 5-hydroxyindoleacetic acid (5-HIAA), a serotonin metabolite. Given the inflammatory nature of cryptococcal infection, we examined the role of GPR35 in the circuitry underlying cell recruitment to the lung. GPR35 deficiency dampened eosinophil recruitment and fungal growth, whereas overexpression promoted eosinophil homing to airways and fungal replication. Activated platelets and mast cells were the sources of GPR35 ligand activity and pharmacological inhibition of serotonin conversion to 5-HIAA, or genetic deficiency in 5-HIAA production by platelets and mast cells resulted in more efficient clearance of Cryptococcus. Thus, the 5-HIAA-GPR35 axis is an eosinophil chemoattractant receptor system that modulates the clearance of a lethal fungal pathogen, with implications for the use of serotonin metabolism inhibitors in the treatment of fungal infections.

Oncogenic CDK13 mutations impede nuclear RNA surveillance.

RNA surveillance pathways detect and degrade defective transcripts to ensure RNA fidelity. We found that disrupted nuclear RNA surveillance is oncogenic. Cyclin-dependent kinase 13 (CDK13) is mutated in melanoma, and patient-mutated CDK13 accelerates zebrafish melanoma. CDK13 mutation causes aberrant RNA stabilization. CDK13 is required for ZC3H14 phosphorylation, which is necessary and sufficient to promote nuclear RNA degradation. Mutant CDK13 fails to activate nuclear RNA surveillance, causing aberrant protein-coding transcripts to be stabilized and translated. Forced aberrant RNA expression accelerates melanoma in zebrafish. We found recurrent mutations in genes encoding nuclear RNA surveillance components in many malignancies, establishing nuclear RNA surveillance as a tumor-suppressive pathway. Activating nuclear RNA surveillance is crucial to avoid accumulation of aberrant RNAs and their ensuing consequences in development and disease.

Biomarkers in the early prediction of rescue therapy in acute severe colitis-a single-centre retrospective study.

Nil.

Antigen discrimination by T cells relies on size-constrained microvillar contact.

T cells use finger-like protrusions called 'microvilli' to interrogate their targets, but why they do so is unknown. To form contacts, T cells must overcome the highly charged, barrier-like layer of large molecules forming a target cell's glycocalyx. Here, T cells are observed to use microvilli to breach a model glycocalyx barrier, forming numerous small (<0.5 µm diameter) contacts each of which is stabilized by the small adhesive protein CD2 expressed by the T cell, and excludes large proteins including CD45, allowing sensitive, antigen dependent TCR signaling. In the absence of the glycocalyx or when microvillar contact-size is increased by enhancing CD2 expression, strong signaling occurs that is no longer antigen dependent. Our observations suggest that, modulated by the opposing effects of the target cell glycocalyx and small adhesive proteins, the use of microvilli equips T cells with the ability to effect discriminatory receptor signaling.

Impact of Baseline Clinical Biomarkers on Treatment Outcomes in Patients With Advanced NSCLC Receiving First-line Pembrolizumab-Based Therapy.

BACKGROUND: While the introduction of immune checkpoint inhibitors (ICI) such as pembrolizumab has significantly improved survival for patients with metastatic non-small cell lung cancer (NSCLC), there remains a need for improved predictive and prognostic biomarkers. PATIENTS AND METHODS: We conducted a retrospective, 3-center study using electronic medical record data for patients with stage IV NSCLC treated with first-line pembrolizumab, either as monotherapy or in combination with chemotherapy, between 2014 and 2019. We categorized variables as covariates or confounders. Covariates, which were the focus of analysis due to their emerging prognostic value, included pretreatment body mass index (BMI), neutrophil-to-lymphocyte ratio (NLR), albumin, and antibiotic exposure. Confounders, which highlighted characteristics for each patient and their cancer, included sex, age at start of immunotherapy, Programmed death-ligand 1 (PD-L1) expression, performance status (PS), tumor mutational burden and whether pembrolizumab was given as monotherapy or in combination with chemotherapy. The association between these variables with time to treatment failure (TTF) and overall survival (OS) was assessed using Kaplan-Meier method and Cox proportional hazards models. RESULTS: One hundred thirty-six patients were included in our study. Antibiotics usage, serum albumin, and NLR have univariate relationships with TTF. Serum albumin, NLR, and BMI were associated with OS in univariate analyses. In our multivariate analysis, antibiotic usage had a strong negative association with TTF when adjusting for all 6 confounders. CONCLUSION: Pretreatment usage of antibiotics, as well as albumin, NLR, and BMI have potential to predict treatment outcomes in patients with advanced NSCLC receiving first-line immunotherapy.

Plerixafor strategies for autologous hematopoietic cell transplant mobilization: A comparison of efficacy and cost.

Addition of plerixafor (P) to granulocyte colony stimulating factor (G-CSF) during peripheral blood mobilization of hematopoietic stem cells (HSC) increases the number of patients meeting collection goals prior to autologous stem cell transplant (aSCT). However, use of P is not universal among transplant centers due to cost. This study aims to compare clinical and financial impacts of using an algorithm-based P mobilization strategy versus use in all patients. This was a single center, retrospective analysis of adult patients with myeloma or amyloidosis receiving aSCT who received apheresis of their HSC between 3/1/2017 and 3/1/2019. Patients prior to 3/1/2018 were classified as receiving P "per algorithm" and those after this date were classified as "up-front" P. For the per-algorithm group, P was given for a pre-apheresis CD34+ cell count of <20 cells/µL on mobilization day 5 and patients returned on day 6 for apheresis. Of the 129 patients included, 55 received P per-algorithm and 74 received up-front P. There was a reduction in median number of apheresis days (1.5 vs 1 day, p < 0.001) and an increase in median number of CD34+ cells collected (6.6 vs 8.5 × 106 cells/kg, p < 0.001) with up-front P. Up-front P increased drug cost but reduced apheresis costs, which resulted in a net savings of $121 per patient in total mobilization costs. These findings suggest that use of up-front P for mobilization significantly reduces apheresis days and increases HSC collection yield without increasing overall cost per patient.

Trapping or slowing the diffusion of T cell receptors at close contacts initiates T cell signaling.

T cell activation is initiated by T cell receptor (TCR) phosphorylation. This requires the local depletion of large receptor-type phosphatases from "close contacts" formed when T cells interact with surfaces presenting agonistic TCR ligands, but exactly how the ligands potentiate signaling is unclear. It has been proposed that TCR ligands could enhance receptor phosphorylation and signaling just by holding TCRs in phosphatase-depleted close contacts, but this has not been directly tested. We devised simple methods to move the TCR in and out of close contacts formed by T cells interacting with supported lipid bilayers (SLBs) and to slow the receptor's diffusion in the contacts, using a series of anti-CD3ε Fab- and ligand-based adducts of the receptor. TCRs engaging a Fab extended with the large extracellular region of CD45 were excluded from contacts and produced no signaling. Conversely, allowing the extended Fab to become tethered to the SLB trapped the TCR in the close contacts, leading to very strong signaling. Importantly, attaching untethered anti-CD3ε Fab or peptide/MHC ligands, each of which were largely inactive in solution but both of which reduced TCR diffusion in close contacts approximately fivefold, also initiated signaling during cell/SLB contact. Our findings indicate that holding TCRs in close contacts or simply slowing their diffusion in phosphatase-depleted regions of the cell surface suffices to initiate signaling, effects we could reproduce in single-particle stochastic simulations. Our study shows that the TCR is preconfigured for signaling in a way that allows it to be triggered by ligands acting simply as receptor "traps."

Palmitate-Induced IRE1-XBP1-ZEB Signaling Represses Desmoplakin Expression and Promotes Cancer Cell Migration.

Elevated uptake of saturated fatty acid palmitate is associated with metastatic progression of cancer cells; however, the precise signaling mechanism behind the phenomenon is unclear. The loss of cell adhesion proteins, such as desmoplakin (DSP), is a key driving event in the transformation of cancer cells to more aggressive phenotypes. Here, we investigated the mechanism by which palmitate induces the loss of DSP in liver and breast cancer cells. We propose that palmitate activates the IRE1-XBP1 branch of the endoplasmic reticulum (ER) stress pathway to upregulate the ZEB transcription factor, leading to transcriptional repression of DSP. Using liver and breast cancer cells treated with palmitate, we found loss of DSP leads to increased cell migration independent of E-cadherin. We report that the ZEB family of transcription factors function as direct transcriptional repressors of DSP. CRISPR-mediated knockdown of IRE1 confirmed that the transcription of ZEB, loss of DSP, and enhanced migration in the presence of palmitate is dependent on the IRE1-XBP1 pathway. In addition, by analyzing the somatic expression and copy number variation profiles of over 11,000 tumor samples, we corroborate our hypothesis and establish the clinical relevance of DSP loss via ZEB in human cancers. IMPLICATIONS: Provides mechanistic link on palmitate-induced activation of IRE1α to cancer cell migration.

Soft Polydimethylsiloxane-Supported Lipid Bilayers for Studying T Cell Interactions.

Much of what we know about the early stages of T cell activation has been obtained from studies of T cells interacting with glass-supported lipid bilayers that favor imaging but are orders of magnitude stiffer than typical cells. We developed a method for attaching lipid bilayers to polydimethylsiloxane polymer supports, producing "soft bilayers" with physiological levels of mechanical resistance (Young's modulus of 4 kPa). Comparisons of T cell behavior on soft and glass-supported bilayers revealed that whereas late stages of T cell activation are thought to be substrate-stiffness dependent, early calcium signaling was unaffected by substrate rigidity, implying that early steps in T cell receptor triggering are not mechanosensitive. The exclusion of large receptor-type phosphatases was observed on the soft bilayers, however, even though it is yet to be demonstrated at authentic cell-cell contacts. This work sets the stage for an imaging-based exploration of receptor signaling under conditions closely mimicking physiological cell-cell contact.

Testing of a Program to Automatically Analyze Students' Concept Maps.

Concept maps are graphical representations of how various concepts relate to one another. Assessment of concept maps developed by students in the pharmacy curriculum helps to evaluate student understanding of course material. However, providing feedback on concept maps can be time-consuming and often requires the grader to be a content expert. The purpose of this study was to develop and validate a software program to provide students with feedback on their concept map performance. Student maps for four different disease states were compared against expert concept maps. The analysis of the program compared favorably to a manual assessment of student maps for the maps' complexity and content but did not correlate for their organization. The value of using a software program to quickly and efficiently analyze concept maps is discussed.

Fabrication of Zero Mode Waveguides for High Concentration Single Molecule Microscopy.

In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to nanomolar ranges, several orders of magnitude less than many physiological ligand concentrations. Optical nanostructures called zero mode waveguides (ZMWs), which are 100-200 nm in diameter apertures fabricated in a thin conducting metal such as aluminum or gold, allow imaging of individual molecules at micromolar concentrations of fluorophores by confining visible light excitation to zeptoliter effective volumes. However, the need for expensive and specialized nanofabrication equipment has precluded the widespread use of ZMWs. Typically, nanostructures such as ZMWs are obtained by direct writing using electron beam lithography, which is sequential and slow. Here, colloidal, or nanosphere, lithography is used as an alternative strategy to create nanometer-scale masks for waveguide fabrication. This report describes the approach in detail, with practical considerations for each phase. The method allows thousands of aluminum or gold ZMWs to be made in parallel, with final waveguide diameters and depths of 100-200 nm. Only common lab equipment and a thermal evaporator for metal deposition are required. By making ZMWs more accessible to the biochemical community, this method can facilitate the study of molecular processes at cellular concentrations and rates.

Pharmacists' Role in Managing Patients with Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a hematologic malignancy that has seen significant advances in care over the last 5 years with the approval of oral agents such as ibrutinib and venetoclax for the treatment of this disease. As such, there has been a substantial shift away from the traditional chemotherapy infusions which have allowed patients greater autonomy with oral cancer therapies. This paradigm shift poses new challenges for the medical team, including drug-drug interactions, adherence counseling, and financial toxicity. Pharmacists are uniquely trained and equipped to help to manage the changing landscape of CLL care. From identifying common medications which may impair ibrutinib clearance to ensuring patients are on the appropriate anti-infective prophylaxis while receiving obinutuzumab, pharmacists can play a vital role in ensuring the highest quality of patient care. Furthermore, additional credentialing of clinical pharmacists in select states allows for independent visits with the pharmacists, allowing for greater involvement, particularly for initiation of venetoclax and management of ibrutinib-induced toxicities. Pharmacists are essential to both expanding and enhancing the care of patients with CLL and should be leveraged to improve patient outcomes whenever possible.

Gain-of-Function Genetic Alterations of G9a Drive Oncogenesis.

Epigenetic regulators, when genomically altered, may become driver oncogenes that mediate otherwise unexplained pro-oncogenic changes lacking a clear genetic stimulus, such as activation of the WNT/ß-catenin pathway in melanoma. This study identifies previously unrecognized recurrent activating mutations in the G9a histone methyltransferase gene, as well as G9a genomic copy gains in approximately 26% of human melanomas, which collectively drive tumor growth and an immunologically sterile microenvironment beyond melanoma. Furthermore, the WNT pathway is identified as a key tumorigenic target of G9a gain-of-function, via suppression of the WNT antagonist DKK1. Importantly, genetic or pharmacologic suppression of mutated or amplified G9a using multiple in vitro and in vivo models demonstrates that G9a is a druggable target for therapeutic intervention in melanoma and other cancers harboring G9a genomic aberrations. SIGNIFICANCE: Oncogenic G9a abnormalities drive tumorigenesis and the "cold" immune microenvironment by activating WNT signaling through DKK1 repression. These results reveal a key druggable mechanism for tumor development and identify strategies to restore "hot" tumor immune microenvironments.This article is highlighted in the In This Issue feature, p. 890.

Nanoaperture fabrication via colloidal lithography for single molecule fluorescence analysis.

In single molecule fluorescence studies, background emission from labeled substrates often restricts their concentrations to non-physiological nanomolar values. One approach to address this challenge is the use of zero-mode waveguides (ZMWs), nanoscale holes in a thin metal film that physically and optically confine the observation volume allowing much higher concentrations of fluorescent substrates. Standard fabrication of ZMWs utilizes slow and costly E-beam nano-lithography. Herein, ZMWs are made using a self-assembled mask of polystyrene microspheres, enabling fabrication of thousands of ZMWs in parallel without sophisticated equipment. Polystyrene 1 µm dia. microbeads self-assemble on a glass slide into a hexagonal array, forming a mask for the deposition of metallic posts in the inter-bead interstices. The width of those interstices (and subsequent posts) is adjusted within 100-300 nm by partially fusing the beads at the polystyrene glass transition temperature. The beads are dissolved in toluene, aluminum or gold cladding is deposited around the posts, and those are dissolved, leaving behind an array ZMWs. Parameter optimization and the performance of the ZMWs are presented. By using colloidal self-assembly, typical laboratories can make use of sub-wavelength ZMW technology avoiding the availability and expense of sophisticated clean-room environments and equipment.

Activation of neural stem cells from quiescence drives reactive hippocampal neurogenesis after alcohol dependence.

Neural stem cell-driven adult neurogenesis contributes to the integrity of the hippocampus. Excessive alcohol consumption in alcoholism results in hippocampal degeneration that may recover with abstinence. Reactive, increased adult neurogenesis during abstinence following alcohol dependence may contribute to recovery, but the mechanism driving reactive neurogenesis is not known. Therefore, adult, male rats were exposed to alcohol for four days and various markers were used to examine cell cycle dynamics, the percentage and number of neural progenitor cell subtypes, and the percentage of quiescent versus activated progenitors. Using a screen for cell cycle perturbation, we showed that the cell cycle is not likely altered at 7 days in abstinence. As the vast majority of Bromodeoxyuridine-positive (+) cells were co-labeled with progenitor cell marker, Sox2, we then developed a quadruple fluorescent labeling scheme to examine Type-1, -2a, -2b and -3 progenitor cells simultaneously. Prior alcohol dependence indiscriminately increased all subtypes at 7 days, the peak of the reactive proliferation. An evaluation of the time course of reactive cell proliferation revealed that cells begin proliferating at 5 days post alcohol, where only actively dividing Type 2 progenitors were increased by alcohol. Furthermore, prior alcohol increased the percentage of actively dividing Sox2+ progenitors, which supported that reactive neurogenesis is likely due to the activation of progenitors out of quiescence. These observations were associated with granule cell number returning to normal at 28 days. Therefore, activating stem and progenitor cells out of quiescence may be the mechanism underlying hippocampal recovery in abstinence following alcohol dependence.

Increased expression of M1 and M2 phenotypic markers in isolated microglia after four-day binge alcohol exposure in male rats.

Microglia activation and neuroinflammation are common features of neurodegenerative conditions, including alcohol use disorders (AUDs). When activated, microglia span a continuum of diverse phenotypes ranging from classically activated, pro-inflammatory (M1) microglia/macrophages to alternatively activated, growth-promoting (M2) microglia/macrophages. Identifying microglia phenotypes is critical for understanding the role of microglia in the pathogenesis of AUDs. Therefore, male rats were gavaged with 25% (w/v) ethanol or isocaloric control diet every 8 h for 4 days and sacrificed at 0, 2, 4, and 7 days after alcohol exposure (e.g., T0, T2, etc.). Microglia were isolated from hippocampus and entorhinal cortices by Percoll density gradient centrifugation. Cells were labeled with microglia surface antigens and analyzed by flow cytometry. Consistent with prior studies, isolated cells yielded a highly enriched population of brain macrophages/microglia (>95% pure), evidenced by staining for the macrophage/microglia antigen CD11b. Polarization states of CD11b+CD45low microglia were evaluated by expression of M1 surface markers, major histocompatibility complex (MHC) II, CD32, CD86, and M2 surface marker, CD206 (mannose receptor). Ethanol-treated animals begin to show increased expression of M1 and M2 markers at T0 (p = n.s.), with significant changes at the T2 time point. At T2, expression of M1 markers, MHC-II, CD86, and CD32 were increased (p < 0.05) in hippocampus and entorhinal cortices, while M2 marker, CD206, was increased significantly only in entorhinal cortices (p < 0.05). All effects resolved to control levels by T4. In summary, four-day binge alcohol exposure produces a transient increase in both M1 (MHC-II, CD32, and CD86) and M2 (CD206) populations of microglia isolated from the entorhinal cortex and hippocampus. Thus, these findings that both pro-inflammatory and potentially beneficial, recovery-promoting microglia phenotypes can be observed after a damaging exposure of alcohol are critically important to our understanding of the role of microglia in the pathogenesis of AUDs.

Millstone: software for multiplex microbial genome analysis and engineering.

Inexpensive DNA sequencing and advances in genome editing have made computational analysis a major rate-limiting step in adaptive laboratory evolution and microbial genome engineering. We describe Millstone, a web-based platform that automates genotype comparison and visualization for projects with up to hundreds of genomic samples. To enable iterative genome engineering, Millstone allows users to design oligonucleotide libraries and create successive versions of reference genomes. Millstone is open source and easily deployable to a cloud platform, local cluster, or desktop, making it a scalable solution for any lab.