Leukotriene signaling via ALOX5 and cysteinyl leukotriene receptor 1 is dispensable for in vitro growth of CD34 (+)CD38(-) stem and progenitor cells in chronic myeloid leukemia

Tyrosine kinase inhibitors targeting the BCR-ABL oncoprotein in chronic myeloid leukemia (CML) are remarkably effective inducing deep molecular remission in most patients. However, they are less effective to eradicate the leukemic stem cells (LSC), resulting in disease persistence. Therefore, there is great need to develop novel therapeutic strategies to specifically target the LSC. In an experimental mouse CML model system, the leukotriene pathway, and specifically, the expression ALOX5, encoding 5-lipoxygenase (5-LO), has been reported as a critical regulator of the LSC. Based on these results, the 5-LO inhibitor zileuton has been introduced in clinical trials as a therapeutic option to target the LSC although its effect on primary human CML LSC has not been studied. We have here by using multiplex single cell PCR analyzed the expression of the mediators of the leukotriene pathway in bone marrow (BM) BCR-ABL(+)CD34(+)CD38(-) cells at diagnosis, and found low or undetectable expression of ALOX5. In line with this, zileuton did not exert significant overall growth inhibition in the long-term culture-initiating cell (LTC-IC) and colony (CFU-C) assays of BM CD34(+)CD38(-) cells from 7 CML patients. The majority of the single leukemic BCR-ABL(+)CD34(+)CD38(-) cells expressed cysteinyl leukotriene receptors CYSLTI and CYSLT2. However, montelukast, an inhibitor of CYSLTI, also failed to significantly suppress CFU-C and LTC-IC growth. These findings indicate that targeting ALOX5 or CYSLTI signaling with leukotriene antagonists, introduced into the clinical practice primarily as prophylaxis and treatment for asthma, may not be a promising pharmacological strategy to eradicate persisting LSC in CML patients. (C) 2017 The Author(s). Published by Elsevier Inc.Peer reviewe

Acute Histologic Chorioamnionitis at Term: Nearly Always Noninfectious

Background: The link between histologic acute chorioamnionitis and infection is well established in preterm deliveries, but less well-studied in term pregnancies, where infection is much less common. Methodology/Principal Findings We conducted a secondary analysis among 195 low-risk women with term pregnancies enrolled in a randomized trial. Histologic and microbiologic evaluation of placentas included anaerobic and aerobic cultures (including mycoplasma/ureaplasma species) as well as PCR. Infection was defined as ≥1,000 cfu of a single known pathogen or a ≥2 log difference in counts for a known pathogen versus other organisms in a mixed culture. Placental membranes were scored and categorized as: no chorioamnionitis, Grade 1 (subchorionitis and patchy acute chorioamnionitis), or Grade 2 (severe, confluent chorioamnionitis). Grade 1 or grade 2 histologic chorioamnionitis was present in 34% of placentas (67/195), but infection was present in only 4% (8/195). Histologic chorioamnionitis was strongly associated with intrapartum fever >38°C [69% (25/36) fever, 26% (42/159) afebrile, P<.0001]. Fever occurred in 18% (n = 36) of women. Most febrile women [92% (33/36)] had received epidural for pain relief, though the association with fever was present with and without epidural. The association remained significant in a logistic regression controlling for potential confounders (OR = 5.8, 95% CI = 2.2,15.0). Histologic chorioamnionitis was also associated with elevated serum levels of interleukin-8 (median = 1.3 pg/mL no histologic chorioamnionitis, 1.5 pg/mL Grade 1, 2.1 pg/mL Grade 2, P = 0.05) and interleukin-6 (median levels = 2.2 pg/mL no chorioamnionitis, 5.3 pg/mL Grade 1, 24.5 pg/mL Grade 2, P = 0.02) at admission for delivery as well as higher admission WBC counts (mean = 12,000cells/mm$^3$ no chorioamnionitis, 13,400cells/mm$^3$ Grade 1, 15,700cells/mm$^3$ Grade 2, P = 0.0005). Conclusion/Significance: Our results suggest histologic chorioamnionitis at term most often results from a noninfectious inflammatory process. It was strongly associated with fever, most of which was related to epidural used for pain relief. A more ‘activated’ maternal immune system at admission was also associated with histologic chorioamnionitis

Peptidylarginine deiminases as drug targets in neonatal hypoxic-ischemic encephalopathy

Oxygen deprivation and infection are major causes of perinatal brain injury leading to cerebral palsy and other neurological disabilities. The identification of novel key factors mediating white and grey matter damage are crucial to allow better understanding of the specific contribution of different cell types to the injury processes and pathways for clinical intervention. Recent studies in the Rice-Vannucci mouse model of neonatal hypoxic ischaemia (HI) have highlighted novel roles for calcium-regulated peptidylarginine deiminases (PADs) and demonstrated neuro-protective effects of pharmacological PAD inhibition following HI and synergistic infection mimicked by LPS stimulation

Leukocyte activation in newborns in relation to prenatal stress

The aim of this project is to describe the pattern of innate immunity responses at birth with focus on the recruitment of leukocytes such as eosinophils and neutrophils and their content of cathelicidin antimicrobial peptide LL-37 associated to different degrees of fetal stress and delivery modes. We know that delivery is associated to fetal stress including release of cortisol and cathecolamines. Stress has profound effects on the immune system, also by acting on the trafficking of leukocytes, a process in which adhesion and chemotaxis are primordial and critical events for the development of effective antimicrobial defences. Birth and postnatal adaptation are associated with leukocyte recruitment and activation, an acute phase response and inflammatory skin reactions in the human newborn, all together indicating an enhanced innate immunity at that time-point in life. We found that with a progressive increase in fetal stress, there are significant elevations in total white blood count, in particular neutrophils and monocytes, as well as an enhanced IL-8 and soluble ESelectin level. Assisted delivery, associated with the highest degree of fetal stress in addition has an increased Interferon-gamma level. The direct correlation between specific leukocytes and Interferon-gamma respectively, with stress markers such as cortisol, beta-endorphin and cathecolamines reflects the impact of stress on fetal immunity. Also, after normal delivery cord plasma LL-37 levels were 3 times higher compared to C-section indicating fetal peptide release. Neutrophils from cord blood after normal delivery contain 10 times more cytoplasmatic cathelicidin peptide compared to corresponding cells after C-section where a strict granular localization is found, a possible sign of cell activation. A highly significant correlation was observed between maternal and cord plasma LL-37 levels, most probably reflects maternal cathelicidin placental transfer. We also could show that cord eosinophils contain and to some degree release LL-37. Interesting, cord eosinophils contain 3 times more LL-37 compared to adults. Thus, it seems reasonable to deduce that these cells may exert important regulatory functions during the neonatal period, also including antimicrobial activity and immunomodulatory effects by peptide LL-37. Our data analysis shows gene expression differences in eosinophils between the two delivery modes. We have identified the genes with most significant expression difference in C-section eosinophils compared to normal delivery eosinophils, here, being IFN-gamma receptor and STAT1. Our RT-PCR data confirms the upregulation of these genes in eosinophils from cord blood compared to normal delivery. This indicates a more enhanced immunity in eosinophils after normal delivery. This study assembles a picture of newborn infant s ability to mount a powerful inflammatory immune response at birth and that the stress of normal labor and terrestrial adaptation induces profound structural and functional changes in fetal leukocytes

Maternal plasma level of antimicrobial peptide LL37 is a major determinant factor of neonatal plasma LL37 level

Aim: To determine cathelicidin antimicrobial peptide LL37subcellular distribution in cord neutrophils and normal plasma LL37 levels in mothers and neonates, relate them to delivery mode and relevant biochemical markers, including 25-OHvitamin D [25(OH)D] as this molecules increases cathelicidin gene expression. Methods: A total of 115 infants were included, n = 68 with normal delivery and n = 47 with elective Caesarean section (C-section), a subset of these being 50 mother-infant pairs. Biomarkers were determined in maternal and cord blood. Subcellular peptide LL37 distribution was analysed with immunoelectron microscopy. Results: Cord plasma LL37 levels were three-times higher after normal delivery compared with C-section. A highly significant correlation was observed between maternal and cord plasma LL37 levels, regardless of delivery mode. No relationship was found between LL37 and 25(OH)D levels. Neutrophils from cord blood after normal delivery contained 10-times more cytoplasmatic cathelicidin peptide compared with corresponding cells after C-section where a strict granular localization was found. Conclusion: These data are consistent with a placental transfer of LL37 and identifies maternal stores as the critical factor determining neonatal plasma LL37 level. An additional enhancement of neonatal cathelicidin mobilization and release is connected to normal delivery stress

IFN gamma directly counteracts imatinib-induced apoptosis of primary human CD34+CML stem/progenitor cells potentially through the upregulation of multiple key survival factors

Tyrosine kinase inhibitors (TKIs) have dramatically improved the survival in chronic myeloid leukemia (CML), but residual disease typically persists even after prolonged treatment. Several lines of evidence suggest that TKIs administered to CML patients upregulate interferon gamma (IFN gamma) production, which may counteract the anti-tumorigenic effects of the therapy. We now show that activated T cell-conditioned medium (TCM) enhanced proliferation and counteracted imatinib-induced apoptosis of CML cells, and addition of a neutralizing anti-IFN gamma antibody at least partially inhibited the anti-apoptotic effect. Likewise, recombinant IFN gamma also reduced imatinib-induced apoptosis of CML cells. This anti-apoptotic effect of IFN gamma was independent of alternative IFN gamma signaling pathways, but could be notably diminished by STAT1-knockdown. Furthermore, IFN gamma upregulated the expression of several anti-apoptotic proteins, including MCL1, PARP9, and PARP14, both in untreated and imatinib-treated primary human CD34+ CML stem/progenitor cells. Our results suggest that activated T cells in imatinib-treated CML patients can directly rescue CML cells from imatinib-induced apoptosis at least partially through the secretion of IFN gamma, which exerts a rapid, STAT1-dependent anti-apoptotic effect potentially through the simultaneous upregulation of several key hematopoietic survival factors. These mechanisms may have a major clinical impact, when targeting residual leukemic stem/progenitor cells in CML.Funding Agencies|Cancer Research Funds of Radiumhemmet [174283]; Thorsmans stiftelse for preleukemiforskning; Cathrine Everts Research Foundation; Lars Hierta Memorial Foundation; Swedish Research Council [2013-08807]; Karolinska Institute Foundation and Funds; Gunnar Grimfors Gavofond for Hematologisk Forskning; Emil Andersson Fund for Medical Research</p