3 Mechanistic Investigation of Pediatric Rheumatic Diseases Flashcards
T/F Separation of cells by their relative density (e.g. Fickle-based or elutriation) yields specific cell populations
F, does NOT yield specific cell populations
Cell separation which is a bead-based removal of undesired cells, leaving desired cells untouched, but is generally of lower purity
Negative selection
Cell separation which refers to enrichment for the cells attached to magnetic beads and generally leads to higher purity
Positive selection
Refers to the process of using a fluid steam to align cells in a single file for analysis
Flow cytometry
Substrates with which antibodies can be conjugated for detection
1) Fluorophores
2) Heavy metals then detected by mass spectrometry
3) DNA barcodes then detected by NGS
4) Magnetic beads
MC method of rendering antibodies detectable by flow cytometry
Labeling to a fluorophore
A fluorescent chemical compound that becomes excited at a specific wavelength spectrum, and then reemits light of a higher wavelength spectrum
Fluorophore
In flow cytometry, the brightness of any given target is determined by
1) Abundance of the target on the cell
2) Affinity of the antibody
3) Brightness of the attached fluorophore
Flow cytometry, forward scatter vs side scatter: Relative estimation of a cell’s size
Forward scatter
Flow cytometry, forward scatter vs side scatter: Cell heterogeneity/intrinsic reflection of light
Side scatter
Refers to the ability to sort cells to an extremely high-degree of of purity by using emitted light to tag cellular targets
Fluorescence-activated cell sorting (FACS)
Great challenge inherent to FACS
“Compensation” which refers to overlapping light emission, thereby limiting simultaneous detection of fluorophores associated with the same cell
Method that overcomes compensation in FACS where antibodies are conjugated to heavy metals and then abundance of heavy metals is analyzed by mass spectrometry
Cytometry by time-of-flight (CyTOF), aka mass cytometry
Disadvantages of CyTOF
1) More costs the flow cytometry
2) Low efficiency (only a small fraction of the cells are able to be analyzed)
3) Cells are incinerated and thus are unable to be sorted or purified
Involves coupling an enzymatic reaction to an antibody bound to a fixed sample
Immunohistochemistry (IHC)
Staining for ___ facilitates the identification of hemophagocytes on crowded bone marrow aspirates
CD163 (a plasma membrane protein present on most tissue resident macrophages)
Challenge in using IHC overcome by immunofluorescence (IF)
Ability to detect more than one protein per slide by IHC; IF overcomes this limitation by conjugating target antibodies to different fluorophores
IHC vs IF: generally provides better resolution
IF
Preferred method for detecting the pattern of ANAs, ANCAs, and complement deposition in lupus kidney biopsies
IF
IHC vs IF: Quantitative
Neither
ELISA process
1) “Capture” antibody specific to protein of interest bound to a protein substrate (plastic plate or magnetic bead)
2) Liquid biological sample; target protein will be bound
3) Unbound proteins are washed out from substrate
4) Substrate-capture antibody-protein complex is exposed to a “detection antibody” also specific for the protein of interest
5) Unbound detection antibodies removed
6) Amount of detection antibody remaining (indirectly) attached to the substrate is quantified via chemical reaction or fluorescence
T/F Distinct proteins can be quantitated simultaneously from the same biological sample using ELISA
T
Important caveat of all ELISA-like assays
Hook effect or prozone phenomenon (occurs when concentrations of the target protein are extremely high, saturating the involved antibodies and preventing the capture and detection antibodies from binding to the same protein)
T/F Hook effect or prozone phenomenon results in falsely low quantitation
T
Method of quantitating proteins in biological samples using carefully engineered DNA molecules
Aptamer Array
Involves the use chemicals like SDS to both denature proteins and coat them with negative ionic charges, then separated by molecular weight through polyacrylamide gel electrophoresis; substrate is exposed to antibodies specific to a protein of interest, followed by reagents that detect the antibody bound to the immobilized protein
Western blot
Western blotting can be used to identify and roughly quantitate the presence of proteins within biological samples, with the added advantage of ___
Indicating their molecular weights
Confirmatory testing of Lyme infection uses what protein quantitation technique
Western blot
Relies on separation of different proteins as a result of differences in their isoelectric point and their molecular size
2D-PAGE
Simultaneous identification and quantification of thousands of proteins in complex biological samples by measuring the mass-to-charge (m/z) ratio of ionized protein fragments
Mass spectrometry
Antibody array studies revealed that fibroblast-like cells obtained from JIA patients secrete significantly higher amounts of proinflammatory molecules like IL-4, IL-6, IL-17, CXCL1, and CXCL6 but downregulate ___ compared with control cells.
Antiinflammatory IL-10
A specific biomarker found in aqueous humor for JIA-associated uveitis compared with other etiologies of uveitis
Transthyretin
This IL has been shown to have a pivotal role during active systemic JIA
IL-1
Parameters that have been shown to be useful for differentiation of both polyarticular sJIA and infections from sJIA patients with active systemic inflammation
Myeloid-related protein 8 (MRP8) and MRP14 (S100A8 and S100A9 [calprotectin])
Inflammatory molecules of the S100-alarmin family that play a role in the pathogenesis of sJIA and have been used as reliable markers for diagnosis and monitoring of sJIA for more than 10 years
MRP8 and MRP14
A predominant role of this cytokine has been confirmed in sJIA eventually resulting in the introduction of targeted therapies
IL-1β
T/F The best response to therapy for sJIA is indicated by higher baseline expression of dysregulated genes followed by an early transcriptional normalization after initiation of therapy
T
A critical signaling molecule in the pathogenesis of hemophagocytosis and MAS
Hypoxia-inducible factor 1A (HIF1A)
Gene signatures seen in JDM
Type I IFN and TLR7-pathway signatures
Gene signature characteristic of SLE
Type 1 IFN
Defective regulation of ___ is now accepted as a common mechanism of autoimmunity/autoinflammation
Type 1 IFN response
