Penn Mental Health AIDS Research Center
Core E: Laboratory and Biobehavioral Marker Core
May 9, 2019
Core Director: Steven D. Douglas, M.D.
Co-Director: Ruben C. Gur, Ph.D.
Core E: Specific Aims
Aim 1 The Core E Laboratory performs assays of body fluids including serum, plasma and CSF markers related to HIV/AIDS, psychiatric disease, and comorbidities.
Aim 2 Core E utilizes novel assays generating “big data” output such as RNAseq, SOMAscan and CyTOF available at UPenn and CHOP to identify new biomarkers related to HIV/AIDS, psychiatric disease, and comorbidities.
Aim 3 Core E, in collaboration with the Center for Neuroimaging in Psychiatry and the Brain Behavior Lab, perform state of the art assessments of brain-behavior function in normative and clinical populations.
Aim 4 The Core E maintains a specimen BioBank, which includes plasma, serum, cells, and genomic DNA.
Core E: Services
Routine Pilot Panel
Immune/Inflamm Immune Inflamm Stress Neurodamage Cardio Psychiatric Fractalkine MIP-1α IFNγ hsCRP Cortisol NFH Fractalkine Cortisol
IFN-γ sCD14 CD4/CD8 LPS NFL sICAM IL-6
IL-1β sCD163 Neopterin sVCAM TNFαIL-6 sICAM sCD163 hsCRPIL-8 sVCAM IFN-γ
IL-10 TNFα CD4/CD8MCP-1 TNFα-R1
Custom panels available upon request
Analysis of Viral Reservoirs
• QVOA - Quantitative Viral outgrowth assay
• Single copy assay for residual viremia
• Total HIV-1 DNA in PBMC
• Total HIV-1 DNA in purified resting CD4+ T cells
• Integrated HIV-1 DNA
• HIV-1 DNA and RNA in biopsy samples (rectal, lymph nodes)
• Total HIV-1 RNA in PBMC or CD4+ T cells
New Aim 2:
• SOMAscan• RNAseq• CyTOF• ELLA• SiMOA• Bioinformatics (CHRI informatics)
Assays generating “big data” output to identify new biomarkers related to
HIV/AIDS, psychiatric disease, and comorbidities
SOMAscan: Pathway Map
Pilot study with SOMAscan, unique protein biomarker discovery tools, which measure 1,300 plasma proteins including biomarkers of inflammation and other indicators of
monocyte/macrophage polarization, as part of U01MH090325 - Anti-HIV Neuroimmunomodulatory Therapy with Neurokinin-1 Antagonists
Principles of RNA sequencing
Bauer JW et al. (2009) Gene-expression profiling in rheumatic disease: tools and therapeutic potential
Nat Rev Rheumatol doi:10.1038/nrrheum.2009.503
http://tucf-genomics.tufts.edu/home/faq
Effect of SP treatment on gene expression of non-classical compared to classical monocytes
DifferentialGene Expression
Shiroguchi et al. PNAS 2012
SP treatment FACS sorting RNA sequencing
Differentially Expressed Genes in non-classical
monocytes compared to classical monocytes
Differentially Expressed Genes in SP treated
monocytes compared to control monocytes
Effect of SP treatment on gene expression of monocytes compared to controls
CyTOF
Citrus analysis of NK cell abundance reveals phenotypic differences after cytokine treatment.Cytometry Part B: Clinical CytometryJulia Fukuyama, Dara M. Strauss-Albee, Susan Holmes, Catherine A. BlishVolume 92, Issue 1, pages 57-67, 20 JAN 2017
Mass Cytometry Analytical Approaches Reveal Cytokine‐Induced Changes in
Natural Killer Cells
Ella Assay: Advantages
• High reproducibility across multiple labs
• Less hands-on, more automated
• Ability to measure endogenous levels at high and low concentrations
• Small sample volume
• Multiplexing without cross-reactivity
SiMOA Assay: Advantages
• Utilizes Single Molecule Array technology to deliver femtogram per milliliter (fg/ml) level sensitivity for measurement of low-abundance biomarkers in serum, plasma and other matrices
• Unique immunoassay technology provides robust detection for various single analytes and select multiplex panel options
• High precision service offers accurate and reproducible data with < 10% inter- and intra-assay CVs
• Over 50 analytes available including IL-17A, TNF-alpha, IL-23, IP-10, TRAIL, IL-6, IL-10 and IL-12p70
SiMOA
0 50 100 150 200 2500
2
4
6
NHP
CSF
plas
ma
Pearson r 0.9362R square 0.8764
0 2000 4000 6000 8000 100000
5
10
15
20
25
Human
CSF
plas
ma
Pearson r 0.7912R square 0.6259
Comparison of plasma and CSF levels of neurofilamentsmeasured by conventional ELISA (CSF) and plasma (Simoa) in humans and NHPs
Core E: Pilot Support
• Year 1-Core E supported 4 of the 6 Pilot Projects-Epperson, Kayser, Langleben,
McGuire
• Year 2-Core E supported 5 Pilot Projects-Arnold, Coviello, Dowshen, McLean, Thase
• Year 3-Core E supported 5 Pilot Projects-Gibbons, Greeson, Kolson, Scott, Weljie
• Year 4-Core E supported 4 Pilot Projects-Ashare, Berrettini, DeBiasi, Gross
• Year 5-Core E supported 3 Pilot Projects-Anderson, Hill/Lazar, Wood
• Year 6-Core E supported 3 Pilot Projects-Brooks, Colon-Rivera/Montaner, Deo
• Year 7-Core E is planning to support 3 Pilot Projects-Spitsin/Benton, Bien-
Gund/Ashare/Schnoll/Gross, Kornfield
Core E: Goals• Measure novel biomarkers
• Develop assays to detect HIV DNA and RNA for reservoir
quantitation
• Assess non-cytolytic NK function interferon gamma production (UO1
HIV SSRI Depression)
• Assess the reliability and validity of the Penn Computerized
Neurocognitive Battery (CNB) for the diagnosis of HAND
• Proteomics (SOMAscan), RNASeq, CyTOF, ELLA, SIMOA
Future Directions
• Testing new bioanalytic platforms • Single cell RNAseq• Alternative splicing • Supporting new collaborations and pilot
projects – IFI – Microbiome
Reverse Transcription and Nucleocapsid
David S. Goodsell 2015 (http://hive.scripps.edu/resources.html)
HIV Life Cycle
Core E: ServicesPeripheral Blood Leukocyte Cell Phenotypes
T cell phenotypes:
Naïve T cells
Central memory T cells
Effector memory T cells
Effector T cells
Stem cell-like T cells
Core E: ServicesPeripheral Blood Leukocyte Cell Phenotypes
Monocyte and NK Cell phenotypes
Classical monocytes
Intermediate monocytes
Non-classical monocytes
Typical NK cells
NK cells precursor-like