Susanna Harju-Baker

Silencing of Aγ-Globin Gene Expression during Adult Definitive Erythropoiesis Mediated by GATA-1-FOG-1-Mi2 Complex Binding at the −566 GATA Site

ABSTRACT Autonomous silencing of γ-globin transcription is an important developmental regulatory mechanism controlling globin gene switching. An adult stage-specific silencer of the Aγ-globin gene was identified between −730 and −378 relative to the mRNA start site. A marked copy of the Aγ-globin gene inserted between locus control region 5′ DNase I-hypersensitive site 1 and the ε-globin gene was transcriptionally silenced in adult β-globin locus yeast artificial chromosome (β-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 γ-globin levels, whereas only a weak signal was detected when γ-globin was expressed. Chromatin immunoprecipitation of fetal liver tissue from β-YAC transgenic mice demonstrated that GATA-1, FOG-1, and Mi2 were recruited to the Aγ-globin −566 or Gγ-globin −567 GATA site when γ-globin expression was low (day 18) but not when γ-globin was expressed (day 12). These data suggest that during definitive erythropoiesis, γ-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 γ-globin promoters.

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.

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-α-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 (KD, 400 nm) and CS-E (KD, 630 nm). Active matrilysin bound heparin (KD, 150 nm) but less so to CS-E (KD, 60 μm). Neither form bound heparan sulfate. These observations demonstrate that sulfated GAGs regulate matrilysin activation and its activity against specific substrates.

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.

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 (β = -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 (β = +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.

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.

Host derived biomarkers of inflammation, apoptosis, and endothelial activation are associated with clinical outcomes in patients with bacteremia and sepsis regardless of microbial etiology

William O. Hahn, Carmen Mikacenic, Brenda L. Price, Susanna Harju-Baker, Ronit Katz, Jonathan Himmelfarb, Mark M. Wurfelb,y, and W. Conrad Lilesa,y Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, WA, USA; Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Washington, Seattle, WA, USA; Department of Biostatistics, University of Washington, Seattle, WA, USA; Kidney Research Institute, University of Washington, Seattle, WA, USA

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.

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.

Alveolar levels of immuno-inflammatory mediators in diffuse alveolar hemorrhage after allogeneic transplant

Idiopathic pneumonia syndrome (IPS) is a severe and morbid complication of hematopoietic cell transplantation (HCT) that largely lacks effective treatments. The syndrome is defined as abnormal pulmonary physiology with multilobar opacities on chest imaging that are not attributable to lower respiratory tract infection (LRTI) or other organ failures [1]. Many pathologies meet IPS criteria, including organizing pneumonia, interstitial pneumonitis, and diffuse alveolar damage, among others. Biologic heterogeneity within this broadly defined syndrome may diminish the ability to detect the effect of treatments. An improved understanding of IPS pathobiology may catalyze the development of effective preventive and therapeutic strategies [2]. Several pro-inflammatory cytokines/chemokines are elevated in IPS [3, 4]. These biomarkers may be useful to distinguish IPS subphenotypes with unique biologic features. As a preliminary examination of this concept, we tested the hypothesis that immuno-inflammatory mediators measured in the blood and alveolar compartments of people with IPS differ according to the presence or absence of diffuse alveolar hemorrhage (DAH). DAH is a variably present manifestation of IPS that is proposed to have distinct biology [5]. We retrospectively studied 17 adults who underwent clinical bronchoscopy for diffusely abnormal chest imaging within 120 days of allogeneic HCT at Fred Hutchinson Cancer Research Center (FHCRC) between 2008 and 2010. None received specific tumor necrosis factor (TNF)-α inhibitors prior to bronchoscopy. Data and samples were banked prospectively with subjects’ consent under FHCRC protocol 999.209 and were approved for use in this research (FHCRC protocols 808 and 1829/substudy 050). We reviewed pertinent progress notes, microbiology reports, pathology reports, bronchoscopy reports, and radiographic images to identify IPS cases according to the American Thoracic Society definition [1]. We defined abnormal respiratory physiology as oxygen saturation ≤92% by pulse oximetry or arterial blood gas while breathing ambient air or supplemental oxygen. We defined LRTI as bronchoalveolar lavage fluid (BALF) growing any amount of Gram-negative rods, Mycoplasma spp., Chlamydophila spp., fungi/mold, Legionella, Nocardia, Mycobacteria, or >10 colony-forming units per mL of a single pathogenic Gram-positive coccus. Cytomegalovirus (CMV) infection required both culture and PCR positivity. We also classified as LRTI: any positive direct fluorescence antibody stain or >40 PCR copies of respiratory viruses (such as adenovirus, coronavirus, parainfluenza, influenza A/B, respiratory syncytial virus, human metapneumovirus, herpes simplex, and varicella zoster); positive stain for Pneumocystis jirovecii; and BALF or serum galactomannan >0.5 ng/L. DAH required documentation of increasingly bloody return from serial bronchoalveolar lavages, or report of “bloody” BALF followed by treatment with systemic corticosteroids for clinically diagnosed DAH. According to local protocols, blood samples were drawn at weekly intervals to survey for CMV and processed and cryopreserved immediately using local standard operating procedures. When available, we selected blood samples collected prior to bronchoscopy or within 2 days of bronchoscopy to best reflect biologic activity at the time of bronchoscopy. BALF samples were obtained using standard clinical techniques and cryopresevered without processing. We thawed samples on an ice batch and centrifuged them at * Lisa K. Vande Vusse lkvandev@u.washington.edu