3 Mechanistic Investigation of Pediatric Rheumatic Diseases

Question 1

T/F Separation of cells by their relative density (e.g. Fickle-based or elutriation) yields specific cell populations

Answer 1

F, does NOT yield specific cell populations

Question 2

Cell separation which is a bead-based removal of undesired cells, leaving desired cells untouched, but is generally of lower purity

Answer 2

Negative selection

Question 3

Cell separation which refers to enrichment for the cells attached to magnetic beads and generally leads to higher purity

Answer 3

Positive selection

Question 4

Refers to the process of using a fluid steam to align cells in a single file for analysis

Answer 4

Flow cytometry

Question 5

Substrates with which antibodies can be conjugated for detection

Answer 5

1) Fluorophores
2) Heavy metals then detected by mass spectrometry
3) DNA barcodes then detected by NGS
4) Magnetic beads

Question 6

MC method of rendering antibodies detectable by flow cytometry

Answer 6

Labeling to a fluorophore

Question 7

A fluorescent chemical compound that becomes excited at a specific wavelength spectrum, and then reemits light of a higher wavelength spectrum

Answer 7

Fluorophore

Question 8

In flow cytometry, the brightness of any given target is determined by

Answer 8

1) Abundance of the target on the cell
2) Affinity of the antibody
3) Brightness of the attached fluorophore

Question 9

Flow cytometry, forward scatter vs side scatter: Relative estimation of a cell’s size

Answer 9

Forward scatter

Question 10

Flow cytometry, forward scatter vs side scatter: Cell heterogeneity/intrinsic reflection of light

Answer 10

Side scatter

Question 11

Refers to the ability to sort cells to an extremely high-degree of of purity by using emitted light to tag cellular targets

Answer 11

Fluorescence-activated cell sorting (FACS)

Question 12

Great challenge inherent to FACS

Answer 12

“Compensation” which refers to overlapping light emission, thereby limiting simultaneous detection of fluorophores associated with the same cell

Question 13

Method that overcomes compensation in FACS where antibodies are conjugated to heavy metals and then abundance of heavy metals is analyzed by mass spectrometry

Answer 13

Cytometry by time-of-flight (CyTOF), aka mass cytometry

Question 14

Disadvantages of CyTOF

Answer 14

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

Question 15

Involves coupling an enzymatic reaction to an antibody bound to a fixed sample

Answer 15

Immunohistochemistry (IHC)

Question 16

Staining for ___ facilitates the identification of hemophagocytes on crowded bone marrow aspirates

Answer 16

CD163 (a plasma membrane protein present on most tissue resident macrophages)

Question 17

Challenge in using IHC overcome by immunofluorescence (IF)

Answer 17

Ability to detect more than one protein per slide by IHC; IF overcomes this limitation by conjugating target antibodies to different fluorophores

Question 18

IHC vs IF: generally provides better resolution

Answer 18

IF

Question 19

Preferred method for detecting the pattern of ANAs, ANCAs, and complement deposition in lupus kidney biopsies

Answer 19

IF

Question 20

IHC vs IF: Quantitative

Answer 20

Neither

Question 21

ELISA process

Answer 21

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

Question 22

T/F Distinct proteins can be quantitated simultaneously from the same biological sample using ELISA

Answer 22

T

Question 23

Important caveat of all ELISA-like assays

Answer 23

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)

Question 24

T/F Hook effect or prozone phenomenon results in falsely low quantitation

Answer 24

T

Question 25

Method of quantitating proteins in biological samples using carefully engineered DNA molecules

Answer 25

Aptamer Array

Question 26

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

Answer 26

Western blot

Question 27

Western blotting can be used to identify and roughly quantitate the presence of proteins within biological samples, with the added advantage of ___

Answer 27

Indicating their molecular weights

Question 28

Confirmatory testing of Lyme infection uses what protein quantitation technique

Answer 28

Western blot

Question 29

Relies on separation of different proteins as a result of differences in their isoelectric point and their molecular size

Answer 29

2D-PAGE

Question 30

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

Answer 30

Mass spectrometry

Question 31

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.

Answer 31

Antiinflammatory IL-10

Question 32

A specific biomarker found in aqueous humor for JIA-associated uveitis compared with other etiologies of uveitis

Answer 32

Transthyretin

Question 33

This IL has been shown to have a pivotal role during active systemic JIA

Answer 33

IL-1

Question 34

Parameters that have been shown to be useful for differentiation of both polyarticular sJIA and infections from sJIA patients with active systemic inflammation

Answer 34

Myeloid-related protein 8 (MRP8) and MRP14 (S100A8 and S100A9 [calprotectin])

Question 35

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

Answer 35

MRP8 and MRP14

Question 36

A predominant role of this cytokine has been confirmed in sJIA eventually resulting in the introduction of targeted therapies

Answer 36

IL-1β

Question 37

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

Answer 37

T

Question 38

A critical signaling molecule in the pathogenesis of hemophagocytosis and MAS

Answer 38

Hypoxia-inducible factor 1A (HIF1A)

Question 39

Gene signatures seen in JDM

Answer 39

Type I IFN and TLR7-pathway signatures

Question 40

Gene signature characteristic of SLE

Answer 40

Type 1 IFN

Question 41

Defective regulation of ___ is now accepted as a common mechanism of autoimmunity/autoinflammation

Answer 41

Type 1 IFN response