The rockefeller university

Cancer is a systemic disease with complications beyond the primary tumor site. Among them, thrombosis is the second leading cause of death in patients with certain cancers (e.g., pancreatic ductal adenocarcinoma [PDAC]) and advanced-stage disease. Here, we demonstrate that pro-thrombotic small extracellular vesicles (sEVs) are secreted by C-X-C motif chemokine 13 (CXCL13)-reprogrammed interstitial macrophages in the non-metastatic lung microenvironment of multiple cancers, a niche that we define as the pro-thrombotic niche (PTN). These sEVs package clustered integrin beta(2) that dimerizes with integrin alpha(X) and interacts with platelet-bound glycoprotein (GP)Ib to induce platelet aggregation. Blocking integrin beta(2) decreases both sEV-induced thrombosis and lung metastasis. Importantly, sEV-beta(2) levels are elevated in the plasma of PDAC patients prior to thrombotic events compared with patients with no history of thrombosis. We show that lung PTN establishment is a systemic consequence of cancer progression and identify sEV-beta(2) as a prognostic biomarker of thrombosis risk as well as a target to prevent thrombosis and metastasis.

Embryo selection (ES) during IVF is expected to select the 'best' embryo(s) from among a cycle's embryo cohort and has been a core concept of IVF for over 40 years. However, among 36 492 articles on ES in a recent PubMed search, we were unable to locate even a single one questioning the concept that, beyond standard oocyte and embryo morphology, ES has remained an unproven hypothesis. In unselected patient populations, attempts at ES have universally, indeed, failed to improve cumulative pregnancy and live birth rates. The only benefit ES appears to offer is a marginal shortening in time to pregnancy, and even this benefit manifests only in best-prognosis patients with large oocyte and embryo numbers. Excluding in vitro maturation efforts, oocytes, once retrieved, and their resulting embryos have predetermined finite cumulative pregnancy and live birth chances that cannot be further improved. The hypothesis of ES has, however, remained a driving force for research and the introduction of a multitude of 'add-ons' to IVF. Enormous investments over decades in ES, therefore, should be better redirected from post- to pre-retrieval efforts.

The soles of staff shoes accessing vivaria can become contaminated on urban streets, potentially serving as a source of fomite-mediated transmission of adventitious agents to laboratory rodents. While shoe covers may mitigate this risk, donning them can lead to hand contamination. Staff accessing our vivaria use motor-driven shoe cleaners hundreds of times daily to remove and collect particulates via a vacuum collection system from the top, sole, and sides of shoes instead of shoe covers. Shoe cleaner debris (SCD) and contact media (CM) exposed to SCD from shoe cleaners in 5 vivaria were assessed by PCR for 84 adventitious agents. SCD and CM samples tested positive for 33 and 37 agents, respectively, and a combined 39 agents total. To assess SCD infectivity, NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) and Swiss outbred mice were housed for 7 d in direct contact with SCD and oronasally inoculated with a suspension created from SCD collected from each of the 5 vivaria. Mice were tested by PCR and serology at 3, 7, 14, and 63 d postinoculation. All mice remained healthy until the study's end and tested negative for all agents found in SCD/CM except murine astrovirus 1, Staphylococcus xylosus, and Candidatus Savagella, agents known to be enzootic in the experimental mouse source colony. In a follow-up study, the soles of 27 staff street shoes were directly sampled using CM. Half of CM was used for PCR, while the other half was added as bedding material to a cage containing NSG and Swiss outbred mice. While CM tested positive for 11 agents, all mice were healthy at 63 d postexposure and again positive for only enzootic agents. These results suggest that shoe debris might not be a significant biosecurity risk to laboratory mice, questioning the need for shoe covers or cleaners when entering experimental barrier vivaria.

Approximately 17% of our genome consists of copies of the retrotransposon "long interspersed element-1" (LINE-1 or L1). Patients with systemic lupus erythematosus (SLE) frequently have autoantibodies against the L1-encoded ORF1 protein (ORF1p), which correlate with disease activity and interferon gene signature. ORF1p is present in neutrophils from patients with active disease in perinuclear ribonucleoprotein particles that also contain Ro60 and nucleic acid sensors. Here, we report that treatment of neutrophils or monocytes with the demethylating agent 5-aza-deoxycytidine, interferon-alpha, tumor necrosis factor-alpha, and other cytokines or toll-like receptor agonists, induce a rapid increase in L1 transcripts. This increase was greater in cells from patients with SLE or rheumatoid arthritis (RA) than in cells from healthy donors, except that cells from SLE did not respond to interferon-alpha, presumably because most SLE patients have elevated type I interferons in vivo. Interferon-alpha also induced ORF1p in RA neutrophils with a subcellular distribution like that of ORF1p in freshly isolated SLE neutrophils. A luciferase reporter gene driven by the 5' untranslated region of L1, which controls its transcription, was also stimulated by interferon-alpha. These new insights into L1 transcriptional regulation indicate that it may play a more active role in antiviral immune responses.

IL-23 is implicated in the pathogenesis of immune-mediated inflammatory diseases, and myeloid cells that express Fc gamma receptor 1 (Fc gamma RI or CD64) on their surface have been recently identified as a primary source of IL-23 in inflamed tissue. Our complementary analyses of transcriptomic datasets from psoriasis and IBD showed increased expression of CD64 and IL-23 transcripts in inflamed tissue, and greater abundance of cell types with co-expression of CD64 and IL-23. These findings led us to explore potential implications of CD64 binding on the function of IL-23-targeting monoclonal antibodies (mAbs). Guselkumab and risankizumab are mAbs that target the IL-23p19 subunit. Guselkumab has a native Fc domain while risankizumab contains mutations that diminish binding to Fc gamma Rs. In flow cytometry assays, guselkumab, but not risankizumab, showed Fc-mediated binding to CD64 on IFN gamma-primed monocytes. Guselkumab bound CD64 on IL-23-producing inflammatory monocytes and simultaneously captured IL-23 secreted from these cells. Guselkumab binding to CD64 did not induce cytokine production. In live-cell confocal imaging of CD64+ macrophages, guselkumab, but not risankizumab, mediated IL-23 internalization to low-pH intracellular compartments. Guselkumab and risankizumab demonstrated similar potency for inhibition of IL-23 signaling in cellular assays with exogenous addition of IL-23. However, in a co-culture of IL-23-producing CD64+ THP-1 cells with an IL-23-responsive reporter cell line, guselkumab demonstrated Fc-dependent enhanced potency compared to risankizumab for inhibiting IL-23 signaling. These in vitro data highlight the potential for guselkumab binding to CD64 in inflamed tissue to contribute to the potent neutralization of IL-23 at its cellular source.

The high resource intensity of industrial beef in high- income economies has prompted growing interest in alternative, potentially lower environmental impact beef production pathways. Of those, grass feeding is promoted by some as one such alternative, but rigorous quantification of this claim is required. Motivated to bridge this knowledge gap, we integrate empirical evidence with a model based on authoritative equations governing beef cattle performance to quantify the greenhouse gas emissions of producing grass- fed beef. Because geographical specificity and dependence on agricultural intensity are key, we model widely varied herds, from extensive operations on semiarid, marginal rangelands to partially industrial, intensive ones in lusher, more accommodating settings. We find that emissions per kg protein of even the most efficient grass- fed beef are 10 to 25% higher than those of industrial US beef and 3- to over 40- fold higher than a wide range of plant and animal alternatives. Soil sequestration enhancement by rangeland grazing reduces these emissions from 280-390 to 180-290 kg CO2eq (kg protein)-1, still somewhat above industrial beef's 180 to 220 kg CO2eq (kg protein)-1, and well above nonbeef alternatives' 10 to 70 kg CO2eq (kg protein)-1. These differences prove robust across a broad set of combinations of grass- fed beef operation types, management practices, and ration qualities. Consequently, even with maximal credit for putative sequestration enhancement, grass- fed beef is still no less carbon intensive than industrial beef, and severalfold more intensive than nonbeef alternatives.

The spectrum of known monogenic inborn errors of immunity is growing, with certain disorders underlying a specific and narrow range of infectious diseases. These disorders reveal the core mechanisms by which these infections occur in various settings, including inherited and acquired immunodeficiencies, thereby delineating the essential mechanisms of protective immunity to the corresponding pathogens. These findings also have medical implications, facilitating diagnosis and improving the management of individuals at risk of disease. (J Allergy Clin Immunol 2025;155:768-83.)

Ischemic stroke is a major cause of disability in adults. Early treatment with thrombolytics and/or thrombectomy can significantly improve outcomes; however, following these acute interventions, treatment is limited to rehabilitation therapies. Thus, identification of therapeutic strategies that can help restore brain function in the post-acute phase remains a major challenge. Here we report that genetic or pharmacologic inhibition of the PDGF-CC/PDGFR alpha pathway, which has previously been implicated in stroke pathology, significantly reduced myofibroblast expansion in the border of the fibrotic scar and improved outcome in a sensory-motor integration test after experimental ischemic stroke. This was supported by gene expression analyses of cerebrovascular fragments showing upregulation of profibrotic/proinflammatory genes, including genes of the TGF pathway, after ischemic stroke or intracerebroventricular injection of active PDGF-CC. Further, longitudinal intravital 2-photon imaging revealed that inhibition of PDGFR alpha dampened the biphasic pattern of stroke-induced vascular leakage and enhanced vascular perfusion in the ischemic lesion. Importantly, we found PDGFR alpha inhibition to be effective in enhancing functional recovery when initiated 24 hours after ischemic stroke. Our data implicate the PDGF-CC/PDGFR alpha pathway as a crucial mediator modulating post-stroke pathology and suggest a post-acute treatment opportunity for patients with ischemic stroke targeting myofibroblast expansion to foster long-term CNS repair.

Platelet clot retraction, the ultimate phase of platelet thrombus formation, is critical for clot stabilization. It requires functional alpha IIb(33 receptors, fibrin, and the integrated actions of the actin-myosin contractile and cytoskeletal systems. Disturbances in clot retraction have been associated with both bleeding and thrombosis. We recently demonstrated that platelets treated with the alpha IIb(33 antagonist peptide Arg-Gly-Asp-Trp, which eliminates fibrinogen- mediated platelet aggregation, are still able to retract clots. We have exploited this observation to develop an unbiased, functional high-throughput assay to identify small- molecule inhibitors of fibrin-mediated clot retraction adapted for a 384-well plate format. We tested 9710 compounds from drug-repurposing libraries (DRLs). These libraries contain compounds that are either US Food and Drug Administration approved or have undergone preclinical/clinical development. We identified 27 compounds from the Library of Pharmacologically Active Compounds library as inhibitors of clot retraction, of which 14 are known inhibitors of platelet function. From the DRLs, we identified 135 compounds (1.6% hit rate). After extensive curation, these compounds were categorized based on the activity of their reported target. Multiple kinase and phosphodiesterase inhibitors with known antiplatelet effects were identified, along with multiple deubiquitination and receptor inhibitors, as well as compounds that have not previously been reported to have antiplatelet activity. Studies of 1 of the deubiquitination inhibitors (degrasyn) suggest that its effects are downstream of thrombin-induced platelet-fibrinogen interactions and thus may permit the separation of platelet thrombin-induced aggregation-mediated events from clot retraction. Additional studies of the identified compounds may lead to novel mechanisms of inhibiting thrombosis.

Vimentin intermediate filaments (VIFs) form complex, tightly packed networks; due to this density, traditional imaging approaches cannot discern single-filament behavior. To address this, we developed and validated a sparse vimentin-SunTag labeling strategy, enabling single-particle tracking of individual VIFs and providing a sensitive, unbiased, and quantitative method for measuring global VIF motility. Using this approach, we define the steady-state VIF motility rate, showing a constant similar to 8% of VIFs undergo directed microtubule-based motion irrespective of subcellular location or local filament density. Significantly, our single-particle tracking approach revealed uncorrelated motion of individual VIFs within bundles, an observation seemingly at odds with conventional models of tightly cross-linked bundles. To address this, we acquired high-resolution focused ion beam scanning electron microscopy volumes of vitreously frozen cells and reconstructed three-dimensional VIF bundles, finding that they form only loosely organized, semi-coherent structures from which single VIFs frequently emerge to locally engage neighboring microtubules. Overall, this work demonstrates single VIF dynamics and organization in the cellular milieu for the first time.