Camille Apple, Elizabeth Miller, Julie A Stortz, Juan C Mira, McKenzie K Hollen, Tyler J Loftus, Maria Cecilia Lopez, Zhongkai Wang, Kolenkode B Kannan, Dina C Nacionales, Christopher R Cogle, Hari K Parvataneni, Kalia K Sadasivan, Matthew Patrick, Jennifer E Hagen, Scott Brakenridge, Frederick A Moore, Henry V Baker, Lyle L Moldawer, Philip A Efron, Alicia M Mohr
AMM was supported by R01 GM105893‐01A1 I do not have any relevant financial relationships with any commercial interest that pertains to the content of my presentation
Our patients: PIAA BM suppression of erythroid Acute trauma progenitor colony growth Hemorrhagic shock Hematopoietic progenitor cell (HPC) sequestration at site of injury Critically ill (ICU) Persistent Anemia HPC mobilization to peripheral (up to 6mo.) blood Supraphysiologic Impaired iron homeostasis catecholamine levels Bone marrow (BM) Myelo‐erythroid reprioritization dysfunction
Regulatory RNAs that target mRNA Significant as a distinct class of biological regulators in many organisms Dickinson B, et al. Nat Biotechnol. 2013;31:965‐967.
Hong SH et al. Stem Cells. 2015;33(1):1‐7.
Hattangadi SM et al. Blood. 2011;118(24):6258‐68.
The expression of erythropoiesis‐related miRNAs is altered in trauma patients miRNAs play a role in persistent injury‐associated anemia
Blood and bone marrow (BM) collected intra‐ operatively from: Severely injured trauma patients who underwent fracture fixation (n=27) Controls ‐ elective hip replacement patients (n=10)
Bone marrow to assess GEMM, BFU‐E, CFU‐E colony growth formation Total RNA and miRNA were isolated from HSCs Genome‐wide gene and miRNA expression was assayed Threshold of significance *p < 0.01 For visualization, expression differences were clustered using a heat map
CONTROLS TRAUMA (n = 10) (n =27) P‐value Male (n) 4 (40%) 16 (59%) NS Age (years) 66* 43* 0.0004 Admission heart rate (bpm) 72* 99* 0.0005 Admission SBP (mmHg) 128* 110* 0.0318 Admission MAP (mmHg) 86* 73* 0.0199 Admission Lactate (mg/dL) NA 2.9 NA Pre‐operative Hemoglobin (g/dL) 13.7* 9.4* 0.0001 Discharge Hemoglobin (g/dL) 10.3* 8.9* 0.0012
miRNA Trauma/Control Function Fold Change hsa‐miR‐150‐3p 1.7* Regulates genes whose downstream products encourage megakaryocytic differentiation rather than erythrocytic differentiation Promotes granulocytic differentiation and hsa‐miR‐223 1.8* suppresses erythrocytic differentiation hsa‐miR‐15a 1.2* Negatively regulates normal erythropoiesis by directly targeting the human activin type I receptors c‐Kit Negatively regulates normal erythropoiesis by hsa‐miR‐24‐1 1.2* directly targeting the human activin type I receptors ALK4 hsa‐miR‐23a‐5p 4.5* Acts as key regulator for erythroid differentiation of CD34+ HPCs by targeting SHP2 * p<0.01
Following trauma there is decreased growth of bone marrow erythroid progenitor cells and increased expression of five miRNAs that negatively regulate erythropoiesis Despite the presence of anemia following hip replacement and trauma, microRNA regulation within the bone marrow is not similar
Acknowledgements Alicia Mohr, MD Elizabeth Miller, MD Kolenkode Kannan, PhD Moldawer Lab Henry Baker, PhD Maria Cecilia Lopez, BS
miRNAs as novel targets for therapeutic intervention Microvesicle‐mediated transfer of miRNAs between HSCs and the bone marrow niche could help us regulate HSCs miRNA replacement therapy using miRNA mimics Inhibition of miRNA function by anti‐miRNAs
Preterm Neonates Have Persistent Neutrophil Preterm Neonates Have Persistent Neutrophil Velocity and Transcriptomic Changes that Fail Velocity and Transcriptomic Changes that Fail to Resolve Despite Reaching Term Corrected to Resolve Despite Reaching Term Corrected Gestational Age Gestational Age RB Hawkins, SL Raymond, JC Rincon, R Ungaro, MC Lopez, HV Baker, JL Wynn, LL Moldawer, SD Larson
Disclosures • Nothing to disclose • Work Supported by: – NIH R01 GM097531 (SDL, LLM) – NIH R01 HD089939 (JLW, LLM) – NIH T32 GM008721 (RBH)
Introduction • Prematurity and its associated complications are leading causes of death in the neonatal period • Preterm neonates have quantitative and functional impairments in immunity that are poorly understood • To date, assessment of human neonatal immune function has been limited by high blood volume requirements
Microfluidics • Microfluidic technologies have emerged, allowing analysis with only a drop of blood • Microfluidic assays developed for: – Neutrophil migration – Phagocytosis – Chemotaxis – Sepsis diagnostics
Hypothesis Preterm neonates have distinct neutrophil motility and transcriptomic phenotypes at birth that normalize over time
Study Design • Human preterm and term neonates enrolled • Inclusion criteria: – Preterm cohort: <32 weeks gestational age – Term cohort: >36 weeks gestational age – Ability to obtain consent from parent/guardian • Exclusion criteria: – Congenital defects, suspected genetic disorders, 32‐36 weeks completed gestation, lack of consent
Study Design • Term neonates: single 250 µL blood sample DOL1 • Preterm neonates: serial 250 µL blood samples during time period at highest risk of sepsis/complications – DOL 4, twice weekly x 3 weeks, weekly until discharge
Microfluidic Analysis 200 µL Leukocyte Transcriptomics (GeneChip™) 250 µL 50 µL Whole Spontaneous Blood Migration
Enrollment 25 Neonates 114 Total Blood Samples Processed 14 Preterm 11 Term • Preterm cohort: 50% male, mean gestational age 29.5 weeks, mean birth weight 1254 grams
Neutrophil Velocity Increases After Birth for Preterm Neonates, Approaches Term Levels Control: 21 µm/min R 2 = 0.36 p = <0.0001
Preterm neonates have distinct transcriptomic pattern at birth, does not normalize by 37 corrected gestational age
Preterm neonates have differentially expressed gene pathways at birth and at 2 months of age • Time ANOVA performed comparing preterm neonatal samples over time to full term cohort • 618 genes differentially expressed over time between preterm and term groups • Ingenuity Pathway Analysis™ revealed: – At DOL4, upregulation DNA synthesis and repair, downregulation of apoptosis – At 2 months of age, increased activation of phagocyte degranulation, inhibition of transcription regulation
Conclusions • Preterm neonates have decreased neutrophil velocity at birth, which corrects to term levels by ~1 month • Leukocyte transcriptomic expression follows a distinct pattern after preterm birth, and does not reach term levels even by hospital discharge • Transcriptomics suggest ongoing inflammatory response • These results may partially explain persistent long‐term immune dysfunction in preterm neonates
Acknowledgements • University of Florida Department of Surgery – Dr. Gib Upchurch, Dr. Saleem Islam, Dr. Lyle Moldawer, Dr. Shawn Larson
Immune Modulation by Bacterial Endotoxin Suppresses Pancreatic Cancer Progression Anthony Ferrantella MD, Prateek Sharma MD, Saba Kurtom MD, Vrishketan Sethi MD, Bhuwan Giri MD, Mohammad Tarique PhD, Harrys KC Jacob PhD, Pooja Roy MD, Shweta Lavania MS, Ashok Saluja PhD, Vikas Dudeja MD DeWitt Daughtry Family Department of Surgery Leonard M. Miller School of Medicine University of Miami Miami, FL
Disclosure Statement Anthony Ferrantella, M.D. ‐Nothing to disclose
Background • Pancreatic ductal adenocarcinoma (PDAC) is an immunologically “cold” tumor. Donahue et al. (2016) • Immunotherapy has not been effective in PDAC. • Toll‐like receptor 4 (TLR4) signaling promotes inflammatory pathways. Akira et al. (2004)
Resection model of pancreatic cancer KPC Pancreatic Cancer Cells Wild‐type (C57BL6) Mice Resection Resection Sham Surgery + LPS (5mg/kg + Saline twice weekly)
TLR4 activation reduces cancer recurrence 100 Sham Surgery Resection Only 75 Resection + LPS 50 25 0 0 10 20 30 40 50 60 70 80 90 Days Following Resection Sham Surgery Resection Only Resection + LPS n = 16-19 per group Median Survival 34 days 41 days not reached log-rank p < 0.001
Liver metastasis model of pancreatic cancer KPC Pancreatic Cancer Cells Wild‐type (C57BL/6) and Rag1 ‐/‐ Mice LPS (1mg/kg Saline weekly)
TLR4 activation suppresses liver metastasis Rag1 -/- Mice Wild-type Mice n.s. Liver metastases weight (g) Liver metastases weight (g) 2.0 n = 8-9 per group 1.5 ** p < 0.01 n.s. = not significant 1.0 0.5 0.0 Saline LPS
TLR4 activation promotes macrophage activation MHC-II SALINE % MHC-II+ of CD45/F4-80 cells % F4-80+ of CD45 cells LPS n = 5 per group * p < 0.05 F4/80 ** p < 0.01
TLR4 activation reduces pro-tumorigenic MDSCs CD11b SALINE % Gr-1/CD11b+ of CD45 cells LPS n = 5 per group *** p < 0.001 Ly6G/Ly6C
TLR4 activation reduces tumor growth Tumor volume (mm 3 ) *** 0 5 0 5 0 5 1 1 2 2 n = 10 per group *** p < 0.001
TLR4 activation reduces tumor growth Tumor volume (mm 3 ) *** 0 5 0 5 0 5 1 1 2 2 n = 10 per group *** p < 0.001
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