An intergenic non-coding RNA promoter required for histone modifications in the human ß-globin chromatin domain.

Transcriptome analyses show a surprisingly large proportion of the mammalian genome is transcribed; much more than can be accounted for by genes and introns alone. Most of this transcription is non-coding in nature and arises from intergenic regions, often overlapping known protein-coding genes in sense or antisense orientation. The functional relevance of this widespread transcription is unknown. Here we characterize a promoter responsible for initiation of an intergenic transcript located approximately 3.3 kb and 10.7 kb upstream of the adult-specific human ß-globin genes. Mutational analyses in ß-YAC transgenic mice show that alteration of intergenic promoter activity results in ablation of H3K4 di- and tri-methylation and H3 hyperacetylation extending over a 30 kb region immediately downstream of the initiation site, containing the adult δ- and ß-globin genes. This results in dramatically decreased expression of the adult genes through position effect variegation in which the vast majority of definitive erythroid cells harbor inactive adult globin genes. In contrast, expression of the neighboring ε- and γ-globin genes is completely normal in embryonic erythroid cells, indicating a developmentally specific variegation of the adult domain. Our results demonstrate a role for intergenic non-coding RNA transcription in the propagation of histone modifications over chromatin domains and epigenetic control of ß-like globin gene transcription during development.

Systemic Angiopoietin-1/2 Dysregulation in Pediatric Sepsis and Septic Shock.

Background: Angiopoietin-1 and -2 are vascular growth factors that exert opposing effects on endothelial activation and dysfunction. The aim of this study was to assess the association of these biomarkers with outcomes in children with sepsis. Methods: Biomarkers were assayed from the blood collected in an emergency department prior to any intervention. Predictor variables were Ang-1 and Ang-2 levels and the Ang-2/Ang-1 ratio. Outcomes included mortality, length of time on vasopressors, and ICU and hospital lengths of stay. The Pediatric RISk of Mortality III Score was calculated. A vasoactive inotrope score was calculated every 12 hours. Results: Forty-five children with sepsis and 49 with septic shock were analyzed. The median Ang-2 was higher in septic shock. The Ang-2/Ang-1 ratio was approximately 2-fold greater in those with septic shock. The Ang-2/Ang-1 ratio was associated with higher doses of vasoactive agents at 12 hours and longer ICU length of stay. In septic shock, for every 0.35 unit increase in the Ang-2/Ang-1 ratio, the PRISM III score increased by 1. Conclusions: The Ang-2/Ang-1 ratio was higher in children with septic shock. Ang-2/Ang-1 was associated with higher vasoactive agents, longer ICU length of stay, and correlated with the severity of illness score.

Genetic Variation in MAP3K1 Associates with Ventilator-Free Days in Acute Respiratory Distress Syndrome.

Mitogen-activated protein kinase kinase kinase 1 (MAP3K1) regulates numerous intracellular signaling pathways involved in inflammation and apoptosis. We hypothesized that genetic variation in MAP3K1 might be associated with outcomes in patients with acute respiratory distress syndrome (ARDS), and that these variants would alter MAP3K1-mediated changes in inflammation and transcriptional regulation. To test this hypothesis, we genotyped single-nucleotide polymorphisms covering linkage disequilibrium bins in MAP3K1 in 306 subjects with ARDS from the ARDSNet FACTT (Fluid and Catheter Treatment Trial) study, and tested for associations between MAP3K1 single-nucleotide polymorphisms and ventilator-free days (VFDs) and mortality. We then validated these associations in a separate cohort of 241 patients with ARDS from Harborview Medical Center (Seattle, WA). We found the variant allele of rs832582 (MAP3K1906Val) was significantly associated with decreased VFDs using multivariate linear regression (-6.1 d, false discovery rate = 0.06) in the FACTT cohort. In the Harborview Medical Center cohort, subjects homozygous for MAP3K1906Val also had decreased VFDs (-15.1 d, false discovery rate < 0.01), and increased 28-day mortality (all subjects homozygous for the rare allele died). In whole blood stimulated with various innate immune agonists ex vivo, MAP3K1906Val was associated with increased IL-1ß, IL-6, IL-8, monocyte chemoattractant protein 1, and TNF-α production. Transcriptome analysis of whole blood stimulated with Toll-like receptor 4 agonist ex vivo demonstrated enrichment of inflammatory gene sets in subjects homozygous for MAP3K1906Val. Our findings show a robust association between the variant allele of rs832582 (MAP3K1906Val) and decreased VFDs in patients with ARDS and suggest that this variant may predispose individuals to a greater inflammatory response.

Endothelial Activation and Blood-Brain Barrier Disruption in Neurotoxicity after Adoptive Immunotherapy with CD19 CAR-T Cells.

Lymphodepletion chemotherapy followed by infusion of CD19-targeted chimeric antigen receptor-modified T (CAR-T) cells can be complicated by neurologic adverse events (AE) in patients with refractory B-cell malignancies. In 133 adults treated with CD19 CAR-T cells, we found that acute lymphoblastic leukemia, high CD19+ cells in bone marrow, high CAR-T cell dose, cytokine release syndrome, and preexisting neurologic comorbidities were associated with increased risk of neurologic AEs. Patients with severe neurotoxicity demonstrated evidence of endothelial activation, including disseminated intravascular coagulation, capillary leak, and increased blood-brain barrier (BBB) permeability. The permeable BBB failed to protect the cerebrospinal fluid from high concentrations of systemic cytokines, including IFNγ, which induced brain vascular pericyte stress and their secretion of endothelium-activating cytokines. Endothelial activation and multifocal vascular disruption were found in the brain of a patient with fatal neurotoxicity. Biomarkers of endothelial activation were higher before treatment in patients who subsequently developed grade ≥4 neurotoxicity.Significance: We provide a detailed clinical, radiologic, and pathologic characterization of neurotoxicity after CD19 CAR-T cells, and identify risk factors for neurotoxicity. We show endothelial dysfunction and increased BBB permeability in neurotoxicity and find that patients with evidence of endothelial activation before lymphodepletion may be at increased risk of neurotoxicity. Cancer Discov; 7(12); 1404-19. ©2017 AACR.See related commentary by Mackall and Miklos, p. 1371This article is highlighted in the In This Issue feature, p. 1355.

Kinetics and biomarkers of severe cytokine release syndrome after CD19 chimeric antigen receptor-modified T-cell therapy.

Lymphodepletion chemotherapy followed by infusion of CD19-specific chimeric antigen receptor-modified (CAR) T cells has produced impressive antitumor responses in patients with refractory CD19+ B-cell malignancies but is often associated with cytokine release syndrome (CRS). Our understanding of CRS continues to evolve, and identification of the kinetics of CRS and predictive clinical and laboratory biomarkers of severity are needed to evaluate strategies to mitigate toxicity. We report the clinical presentation of and identify biomarkers of severe CRS in 133 adult patients who received CD19 CAR T cells. CRS developed in 70% of patients, including 62.5% with grade 1 to 3 CRS (grade 1, 26%; grade 2, 32%; grade 3, 4.5%), 3.8% with grade 4, and 3.8% with grade 5. A majority of cases of grade ≥4 CRS occurred during CAR T-cell dose finding. Multivariable analysis of baseline characteristics identified high marrow tumor burden, lymphodepletion using cyclophosphamide and fludarabine, higher CAR T-cell dose, thrombocytopenia before lymphodepletion, and manufacturing of CAR T cells without selection of CD8+ central memory T cells as independent predictors of CRS. Severe CRS was characterized by hemodynamic instability, capillary leak, and consumptive coagulopathy. Angiopoietin-2 and von Willebrand factor, which are biomarkers of endothelial activation, were increased during severe CRS and also before lymphodepletion in patients who subsequently developed CRS. We describe a classification-tree algorithm to guide studies of early intervention after CAR T-cell infusion for patients at high risk of severe CRS. These data provide a framework for early intervention studies to facilitate safer application of effective CD19 CAR T-cell therapy.

Circulating levels of soluble Fas (sCD95) are associated with risk for development of a nonresolving acute kidney injury subphenotype.

BACKGROUND: Critically ill patients with acute kidney injury (AKI) can be divided into two subphenotypes, resolving or nonresolving, on the basis of the trajectory of serum creatinine. It is unknown if the biology underlying these two AKI recovery patterns is different. METHODS: We measured eight circulating biomarkers in plasma obtained from a cohort of patients admitted to an intensive care unit (ICU) (n = 1241) with systemic inflammatory response syndrome. The biomarkers were representative of several biologic processes: apoptosis (soluble Fas), inflammation (soluble tumor necrosis factor receptor 1, interleukin 6, interleukin 8) and endothelial dysfunction, (angiopoietin 1, angiopoietin 2, and soluble vascular cell adhesion molecule 1). We tested for associations between biomarker levels and AKI subphenotypes using relative risk regression accounting for multiple hypotheses with the Bonferroni correction. RESULTS: During the first 3 days of ICU admission, 868 (70%) subjects developed AKI; 502 (40%) had a resolving subphenotype, and 366 (29%) had a nonresolving subphenotype. Hospital mortality was 12% in the resolving subphenotype and 21% in the nonresolving subphenotype. Soluble Fas was the only biomarker associated with a nonresolving subphenotype after adjustment for age, body mass index, diabetes, and Acute Physiology and Chronic Health Evaluation III score (p = 0.005). CONCLUSIONS: Identifying modifiable targets in the Fas-mediated pathway may lead to strategies for prevention and treatment of a clinically important form of AKI.

A Two-Biomarker Model Predicts Mortality in the Critically Ill with Sepsis.

RATIONALE: Improving the prospective identification of patients with systemic inflammatory response syndrome (SIRS) and sepsis at low risk for organ dysfunction and death is a major clinical challenge. OBJECTIVES: To develop and validate a multibiomarker-based prediction model for 28-day mortality in critically ill patients with SIRS and sepsis. METHODS: A derivation cohort (n = 888) and internal test cohort (n = 278) were taken from a prospective study of critically ill intensive care unit (ICU) patients meeting two of four SIRS criteria at an academic medical center for whom plasma was obtained within 24 hours. The validation cohort (n = 759) was taken from a prospective cohort enrolled at another academic medical center ICU for whom plasma was obtained within 48 hours. We measured concentrations of angiopoietin-1, angiopoietin-2, IL-6, IL-8, soluble tumor necrosis factor receptor-1, soluble vascular cell adhesion molecule-1, granulocyte colony-stimulating factor, and soluble Fas. MEASUREMENTS AND MAIN RESULTS: We identified a two-biomarker model in the derivation cohort that predicted mortality (area under the receiver operator characteristic curve [AUC], 0.79; 95% confidence interval [CI], 0.74-0.83). It performed well in the internal test cohort (AUC, 0.75; 95% CI, 0.65-0.85) and the external validation cohort (AUC, 0.77; 95% CI, 0.72-0.83). We determined a model score threshold demonstrating high negative predictive value (0.95) for death. In addition to a low risk of death, patients below this threshold had shorter ICU length of stay, lower incidence of acute kidney injury, acute respiratory distress syndrome, and need for vasopressors. CONCLUSIONS: We have developed a simple, robust biomarker-based model that identifies patients with SIRS/sepsis at low risk for death and organ dysfunction.

Association of markers of endothelial dysregulation Ang1 and Ang2 with acute kidney injury in critically ill patients.

BACKGROUND: The role of endothelial dysregulation with acute kidney injury (AKI) in critically ill patients is unclear. METHODS: We retrospectively assessed the associations of AKI with biomarkers of endothelial function and inflammation among 948 subjects admitted to the intensive care unit (ICU) at Harborview Medical Center (Seattle, WA, USA). From plasma obtained within 24 h of enrollment, we measured angiopoietin (Ang)-1 and Ang-2 alongside biomarkers of inflammation, including interleukin (IL)-6, IL-17 and granulocyte colony-stimulating factor. We tested for associations between standardized concentrations of biomarkers and AKI, defined by serum creatinine, from ICU admission to up to 7 days later. RESULTS: All biomarkers of inflammation and endothelial dysfunction were associated with AKI. After adjustment for demographics, comorbidities, and IL-6 concentration, every standard deviation of Ang-1 concentration was associated with a 19 % lower risk of AKI (relative risk (RR) = 0.85, 95 % confidence interval (CI) 0.77-0.93, p < 0.001). Conversely, higher Ang-2 concentration was associated with higher risk of AKI (RR per standard deviation = 1.17, 95 % CI 1.13-1.22, p < 0.001). CONCLUSIONS: In critically ill patients, plasma concentration of the endothelial growth factors Ang-1 and Ang-2 are associated with AKI, independently of inflammation.

A common TLR1 polymorphism is associated with higher parasitaemia in a Southeast Asian population with Plasmodium falciparum malaria.

BACKGROUND: The factors leading to poor outcomes in malaria infection are incompletely understood. Common genetic variation exists in the human genes for Toll like receptors (TLRs) that alter host responses to pathogen-associated molecular patterns. Genetic variation in TLR1 and TLR6 could alter the risk of development of complicated malaria and ability of the host to control the parasite burden during acute Plasmodium falciparum infection. METHODS: Five single nucleotide polymorphisms in TLR1 and TLR6 in 432 patients with clinical P. falciparum monoinfection acquired on the Thai-Myanmar border were genotyped. Using logistic regression, associations with the development of complicated malaria and the percentage of infected erythrocytes (parasitaemia) on the day of presentation to clinical care (day zero) were tested. RESULTS: Genotypes carrying the T (major) allele of TLR1 rs5743551--an allele associated with improved outcomes in sepsis--were associated with higher parasitaemia measured on day zero (p = 0.03). DISCUSSION: Since malaria exerts strong genetic pressure on the human genome, protection from parasitaemia associated with TLR1 rs5743551 may account for the maintenance of an allele associated with poor outcomes in Caucasians with sepsis. CONCLUSION: These data suggest that genetic variation in TLR1 has effects on the host response to Plasmodium falciparum malaria in Asian populations. Genotypes from TLR6 showed no evidence of association with either complicated malaria or parasite burden.

Biomarkers of Endothelial Activation Are Associated with Poor Outcome in Critical Illness.

BACKGROUND: Endothelial activation plays a role in organ dysfunction in the systemic inflammatory response syndrome (SIRS). Angiopoietin-1 (Ang-1) promotes vascular quiescence while angiopoietin-2 (Ang-2) mediates microvascular leak. Circulating levels of Ang-1 and Ang-2 in patients with SIRS could provide insight on risks for organ dysfunction and death distinct from inflammatory proteins. In this study, we determined if biomarkers of endothelial activation and inflammation exhibit independent associations with poor outcomes in SIRS. METHODS: We studied 943 critically ill patients with SIRS admitted to an Intensive Care Unit (ICU) of an academic medical center. We measured plasma levels of endothelial markers (Ang-1, Ang-2, soluble vascular cell adhesion molecule-1 (sVCAM-1)) and inflammatory markers (interleukin-6 (IL-6), interleukin-8 (IL-8), granulocyte-colony stimulating factor (G-CSF), soluble tumor necrosis factor receptor-1 (sTNFR-1)) within 24 hours of enrollment. We tested for associations between each marker and 28 day mortality, shock, and day 3 sequential organ failure assessment (SOFA) score. For 28 day mortality, we performed sensitivity analysis for those subjects with sepsis and those with sterile inflammation. We used multivariate models to adjust for clinical covariates and determine if associations identified with endothelial activation markers were independent of those observed with inflammatory markers. RESULTS: Higher levels of all biomarkers were associated with increased 28 day mortality except levels of Ang-1 which were associated with lower mortality. After adjustment for comorbidities and sTNFR-1 concentration, a doubling of Ang-1 concentration was associated with lower 28 day mortality (Odds ratio (OR) = 0.81; p<0.01), shock (OR = 0.82; p<0.001), and SOFA score (ß = -0.50; p<0.001), while Ang-2 concentration was associated with increased mortality (OR = 1.55; p<0.001), shock (OR = 1.51; p<0.001), and SOFA score (ß = +0.63; p<0.001). sVCAM-1 was not independently associated with SIRS outcomes. CONCLUSIONS: In critically ill patients with SIRS, early measurements of Ang-1 and Ang-2 are associated with death and organ dysfunction independently of simultaneously-measured markers of inflammation.

LCR 5' hypersensitive site specificity for globin gene activation within the active chromatin hub.

The DNaseI hypersensitive sites (HSs) of the human ß-globin locus control region (LCR) may function as part of an LCR holocomplex within a larger active chromatin hub (ACH). Differential activation of the globin genes during development may be controlled in part by preferential interaction of each gene with specific individual HSs during globin gene switching, a change in conformation of the LCR holocomplex, or both. To distinguish between these possibilities, human ß-globin locus yeast artificial chromosome (ß-YAC) lines were produced in which the ε-globin gene was replaced with a second marked ß-globin gene (ß(m)), coupled to an intact LCR, a 5'HS3 complete deletion (5'ΔHS3) or a 5'HS3 core deletion (5'ΔHS3c). The 5'ΔHS3c mice expressed ß(m)-globin throughout development; γ-globin was co-expressed in the embryonic yolk sac, but not in the fetal liver; and wild-type ß-globin was co-expressed in adult mice. Although the 5'HS3 core was not required for ß(m)-globin expression, previous work showed that the 5'HS3 core is necessary for ε-globin expression during embryonic erythropoiesis. A similar phenotype was observed in 5'HS complete deletion mice, except ß(m)-globin expression was higher during primitive erythropoiesis and γ-globin expression continued into fetal definitive erythropoiesis. These data support a site specificity model of LCR HS-globin gene interaction.

Matrilysin (MMP-7) inhibition of BMP-7 induced renal tubular branching morphogenesis suggests a role in the pathogenesis of human renal dysplasia.

Congenital renal dysplasia (RD) is a severe form of congenital renal malformation characterized by disruption of normal renal development with cyst formation, reduced or absent nephrons, and impaired renal growth. The authors previously identified that matrilysin (matrix metalloproteinase-7) was overexpressed in a microarray gene expression analysis of human RD compared to normal control kidneys. They now find that active matrilysin gene transcription and protein synthesis occur within dysplastic tubules and epithelial cells lining cysts in human RD by RT-PCR and immunohistochemistry. Similar staining patterns were seen in obstructed kidneys of pouch opossums that show histological features similar to that of human RD. In vitro, matrilysin inhibits formation of branching structures in mIMCD-3 cells stimulated by bone morphogenetic protein-7 (BMP-7) but does not inhibit hepatocyte growth factor-stimulated branching. BMP-7 signaling is essential for normal kidney development, and overexpression of catalytically active matrilysin in human embryonic kidney 293 cells reduces endogenous BMP-7 protein levels and inhibits phosphorylation of BMP-7 SMAD signaling intermediates. These findings suggest that matrilysin expression in RD may be an injury response that disrupts normal nephrogenesis by impairing BMP-7 signaling.

Epilysin (matrix metalloproteinase-28) contributes to airway epithelial cell survival.

MMP28 is constitutively expressed by epithelial cells in many tissues, including the respiratory epithelium in the lung and keratinocytes in the skin. This constitutive expression suggests that MMP28 may serve a role in epithelial cell homeostasis. In an effort to determine its function in epithelial cell biology, we generated cell lines expressing wild-type or catalytically-inactive mutant MMP28 in two pulmonary epithelial cell lines, A549 and BEAS-2B. We observed that over-expression of MMP28 provided protection against apoptosis induced by either serum-deprivation or treatment with a protein kinase inhibitor, staurosporine. Furthermore, we observed increased caspase-3/7 activity in influenza-infected lungs from Mmp28-/- mice compared to wild-type mice, and this activity localized to the airway epithelium but was not associated with a change in viral load. Thus, we have identified a novel role of MMP28 in promoting epithelial cell survival in the lung.

Control of promatrilysin (MMP7) activation and substrate-specific activity by sulfated glycosaminoglycans.

Matrix metalloproteinases are maintained in an inactive state by a bond between the thiol of a conserved cysteine in the prodomain and a zinc atom in the catalytic domain. Once this bond is disrupted, MMPs become active proteinases and can act on a variety of extracellular protein substrates. In vivo, matrilysin (MMP7) activates pro-alpha-defensins (procryptdins), but in vitro, processing of these peptides is slow, with about 50% conversion in 8-12 h. Similarly, autolytic activation of promatrilysin in vitro can take up to 12-24 h for 50% conversion. These inefficient reactions suggest that natural cofactors enhance the activation and activity of matrilysin. We determined that highly sulfated glycosaminoglycans (GAG), such as heparin, chondroitin-4,6-sulfate (CS-E), and dermatan sulfate, markedly enhanced (>50-fold) the intermolecular autolytic activation of promatrilysin and the activity of fully active matrilysin to cleave specific physiologic substrates. In contrast, heparan sulfate and less sulfated forms of chondroitin sulfate did not augment matrilysin activation or activity. Chondroitin-2,6-sulfate (CS-D) also did not enhance matrilysin activity, suggesting that the presentation of sulfates is more important than the overall degree of sulfation. Surface plasmon resonance demonstrated that promatrilysin bound heparin (K(D), 400 nm) and CS-E (K(D), 630 nm). Active matrilysin bound heparin (K(D), 150 nm) but less so to CS-E (K(D), 60 microm). Neither form bound heparan sulfate. These observations demonstrate that sulfated GAGs regulate matrilysin activation and its activity against specific substrates.

Silencing of Agamma-globin gene expression during adult definitive erythropoiesis mediated by GATA-1-FOG-1-Mi2 complex binding at the -566 GATA site.

Autonomous silencing of gamma-globin transcription is an important developmental regulatory mechanism controlling globin gene switching. An adult stage-specific silencer of the (A)gamma-globin gene was identified between -730 and -378 relative to the mRNA start site. A marked copy of the (A)gamma-globin gene inserted between locus control region 5' DNase I-hypersensitive site 1 and the epsilon-globin gene was transcriptionally silenced in adult beta-globin locus yeast artificial chromosome (beta-YAC) transgenic mice, but deletion of the 352-bp region restored expression. This fragment reduced reporter gene expression in K562 cells, and GATA-1 was shown to bind within this sequence at the -566 GATA site. Further, the Mi2 protein, a component of the NuRD complex, was observed in erythroid cells with low gamma-globin levels, whereas only a weak signal was detected when gamma-globin was expressed. Chromatin immunoprecipitation of fetal liver tissue from beta-YAC transgenic mice demonstrated that GATA-1, FOG-1, and Mi2 were recruited to the (A)gamma-globin -566 or (G)gamma-globin -567 GATA site when gamma-globin expression was low (day 18) but not when gamma-globin was expressed (day 12). These data suggest that during definitive erythropoiesis, gamma-globin gene expression is silenced, in part, by binding a protein complex containing GATA-1, FOG-1, and Mi2 at the -566/-567 GATA sites of the proximal gamma-globin promoters.