Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion death. We hypothesize that TRALI requires 2 events: (1) the clinical condition of the patient and (2) the infusion of antibodies against MHC class I antigens or the plasma from stored blood. A 2-event rat model was developed with saline (NS) or endotoxin (LPS) as the first event and the infusion of plasma from packed red blood cells (PRBCs) or antibodies (OX18 and OX27) against MHC class I antigens as the second event. ALI was determined by Evans blue dye leak from the plasma to the bronchoalveolar lavage fluid (BALF), protein and CINC-1 concentrations in the BALF, and the lung histology. NS-treated rats did not evidence ALI with any second events, and LPS did not cause ALI. LPS-treated animals demonstrated ALI in response to plasma from stored PRBCs, both prestorage leukoreduced and unmodified, and to OX18 and OX27, all in a concentration-dependent fashion. ALI was neutrophil (PMN) dependent, and OX18/OX27 localized to the PMN surface in vivo and primed the oxidase of rat PMNs. We conclude that TRALI is the result of 2 events with the second events consisting of the plasma from stored blood and antibodies that prime PMNs. IntroductionTransfusion-related acute lung injury (TRALI) is the leading cause of transfusion mortality in the United States. 1,2 TRALI is the acute onset of noncardiogenic pulmonary edema as documented by chest radiograph and profound hypoxemia, in accordance with the definition of acute lung injury (ALI), that occurs within 6 hours of transfusion. 3,4 TRALI may occur with or without conditions that predispose the patient to ALI, and may be the worsening of pulmonary function in patients with preexisting ALI. 3,4 All blood products have been implicated in TRALI, but components that contain large amounts of plasma are mainly responsible. 5,6 The current incidence of TRALI has been estimated as 1/7900 to 1/1330 in the United Kingdom and the United States with lesser incidences in Europe. [5][6][7][8] Current mortality rates vary from 5% to 35% with the lesser mortality rates predominating. [5][6][7][8] The pathophysiology of TRALI has not been elucidated despite numerous studies. [9][10][11][12][13][14] The first mechanism proposed was the infusion of donor antibodies directed against the HLA class I or granulocyte-specific antigens on the recipient's leukocytes with animal models composed of an in vivo murine model and an isolated, perfused rabbit lung that provided physiologic relevance. [9][10][11][12]14 In addition, the neutrophil (PMN) was proposed to be the effector cell, identical to other forms of ALI and the acute respiratory distress syndrome (ARDS). [9][10][11][12]14 However, look-back studies of donors with specific antibodies directed against HLA or granulocyte antigens demonstrated that the infusion of donor antibodies into a recipient that expressed the cognate antigen resulted in TRALI in a minority of these patients, implying that the clinical condition of the recipient may be important for the d...

During T cell development, multipotent progenitors relinquish competence for other fates and commit to the T cell lineage by turning on the transcription factor Bcl11b. To clarify lineage commitment mechanisms, we followed developing T cells at single-cell level using Bcl11b knock-in fluorescent reporter mice. Notch signaling and Notch-activated transcription factors collaborate to activate Bcl11b expression, irrespective of Notch-dependent proliferation. These inputs work via three distinct, asynchronous mechanisms: an early locus poising function dependent on TCF-1 and GATA-3; a stochastic permissivity function dependent on Notch signaling; and a separate amplitude-control function dependent on Runx1, a factor already present in multipotent progenitors. Despite all being necessary for Bcl11b activation, these inputs act in a stage specific manner, providing a multi-tiered mechanism for developmental gene regulation.

High-mobility group box 1 (HMGB1) is a late mediator of the systemic inflammation associated with sepsis. Recently, HMGB1 has been shown in animals to be a mediator of hemorrhage-induced organ dysfunction. However, the time course of plasma HMGB1 elevations after trauma in humans remains to be elucidated. Consequently, we hypothesized that mechanical trauma in humans would result in early significant elevations of plasma HMGB1. Trauma patients at risk for multiple organ failure (ISS ≥15) were identified for inclusion (n = 23), and postinjury plasma samples were assayed for HMGB1 by enzyme-linked immunosorbent assay. Comparison of postinjury HMGB1 levels with markers for patient outcome (age, injury severity score, units of red blood cell (RBC) transfused per first 24 h, and base deficit) was performed. To investigate whether postinjury transfusion contributes to elevations of circulating HMGB1, levels were determined in both leuko-reduced and non–leuko-reduced packed RBCs. Plasma HMGB1 was elevated more than 30-fold above healthy controls within 1 h of injury (median, 57.76 vs. 1.77 ng/mL; P < 0.003), peaked from 2 to 6 h postinjury (median, 526.18 ng/mL; P < 0.01 vs. control), and remained elevated above control through 136 h. No clear relationship was evident between postinjury HMGB1 levels and markers for patient outcome. High-mobility group box 1 levels increase with duration of RBC storage, although concentrations did not account for postinjury plasma levels. Leuko-reduced attenuated HMGB1 levels in packed RBCs by approximately 55% (P < 0.01). Plasma HMGB1 is significantly increased within 1 h of trauma in humans with marked elevations occurring from 2 to 6 h postinjury. These results suggest that, in contrast to sepsis, HMGB1 release is an early event after traumatic injury in humans. Thus, HMGB1 may be integral to the early inflammatory response to trauma and is a potential target for future therapeutics.

GATA-3 expression is crucial for T cell development and peaks during commitment to the T-cell lineage, midway through the CD4−CD8− (DN) 1-3 stages. We used RNA interference and conditional deletion to reduce GATA-3 protein acutely at specific points during T-cell differentiation in vitro. Even moderate GATA-3 reduction killed DN1 cells, delayed progression to DN2 stage, skewed DN2 gene regulation, and blocked appearance of DN3 phenotype. Although a Bcl-2 transgene rescued DN1 survival and improved DN2 cell generation, it did not restore DN3 differentiation. Gene expression analyses (qPCR, RNA-seq) showed that GATA-3-deficient DN2 cells quickly upregulated genes including Spi1 (PU.1) and Bcl11a and downregulated genes including Cpa3, Ets1, Zfpm1, Bcl11b, Il9r and Il17rb, with gene-specific kinetics and dose-dependencies. These targets could mediate two distinct roles played by GATA-3 in lineage commitment, as revealed by removing wildtype or GATA-3-deficient early T-lineage cells from environmental Notch signals. GATA-3 worked as a potent repressor of B-cell potential even at low expression levels, so that only full deletion of GATA-3 enabled pro-T cells to reveal B-cell potential. The ability of GATA-3 to block B-cell development did not require T-lineage commitment factor Bcl11b. In prethymic multipotent precursors, however, titration of GATA-3 activity using tamoxifen-inducible GATA-3 showed that GATA-3 inhibits B and myeloid developmental alternatives at different threshold doses. Furthermore, differential impacts of a GATA-3 obligate repressor construct imply that B and myeloid development are inhibited through distinct transcriptional mechanisms. Thus, the pattern of GATA-3 expression sequentially produces B-lineage exclusion, T-lineage progression, and myeloid-lineage exclusion for commitment.

The ETS family transcription factor PU.1 is essential for the development of several blood lineages, including T cells, but its function in intrathymic T-cell precursors has been poorly defined. In the thymus, high PU.1 expression persists through multiple cell divisions in early stages but then falls sharply during T-cell lineage commitment. PU.1 silencing is critical for T-cell commitment, but it has remained unknown how PU.1 activities could contribute positively to T-cell development. Here we employed conditional knockout and modified antagonist PU.1 constructs to perturb PU.1 function stage-specifically in early T cells. We show that PU.1 is needed for full proliferation, restricting access to some non-T fates, and controlling the timing of T-cell developmental progression such that removal or antagonism of endogenous PU.1 allows precocious access to T-cell differentiation. Dominant-negative effects reveal that this repression by PU.1 is mediated indirectly. Genome-wide transcriptome analysis identifies novel targets of PU.1 positive and negative regulation affecting progenitor cell signaling and cell biology and indicating distinct regulatory effects on different subsets of progenitor cell transcription factors. Thus, in addition to supporting early T-cell proliferation, PU.1 regulates the timing of activation of the core T-lineage developmental program.

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Viable constitutive and tamoxifen inducible liver-specific RNase H1 knockout mice that expressed no RNase H1 activity in hepatocytes showed increased R-loop levels and reduced mitochondrial encoded DNA and mRNA levels, suggesting impaired mitochondrial R-loop processing, transcription and mitochondrial DNA replication. These changes resulted in mitochondrial dysfunction with marked changes in mitochondrial fusion, fission, morphology and transcriptional changes reflective of mitochondrial damage and stress. Liver degeneration ensued, as indicated by apoptosis, fibrosis and increased transaminase levels. Antisense oligonucleotides (ASOs) designed to serve as substrates for RNase H1 were inactive in the hepatocytes from the RNase H1 knockout mice and in vivo, demonstrating that RNase H1 is necessary for the activity of DNA-like ASOs. During liver regeneration, a clone of hepatocytes that expressed RNase H1 developed and partially restored mitochondrial and liver function.

Highlights d RNase H1-dependent ASOs cause cleavage and degradation of nascent transcripts d ASO-directed degradation of transcripts results in RNA Pol II transcription termination d ASO-directed RNA Pol II transcription termination is mediated by the exonuclease XRN2 d Judicious design of ASOs allows for transcript degradation without causing termination