Measuring the SPR Minimum Angle after analyte capture

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The biochemistry and
genetics of immune-related
disease
Meeting times: Wednesdays 4-6 pm BPB 201
michael.lynes@uconn.edu
486-4350
MCB 5255 intro meeting spring 2012
Today’s plans
•Course organization, grading, etc
•Course topic
•Overview
•my own research interests
•goals for the class
•Course organization, grading, etc
•Look over schedule (next slide)
•Grading:
•3 P’s
•Participation
•Presentation
•(grant) Proposal
Course schedule
January 18 no class; assigned reading for general discussion on 1/25 (come prepared on 1/25
to discuss the first pair of assigned papers!)
January 25 Introductory overview, group discussion of assigned papers (papers 1, 2)
Selection/Assignment of Student presentation topics
February 1 Grantsmanship, group discussion of assigned papers (papers 3-6)
February 8 Group discussion of assigned papers (papers 7,8)
February 15 Group discussion of assigned papers (papers 9,10)
Discussion of what a specific aim is designed to express in a grant proposal
February 22 Transcriptional regulation of CD4 and CD8 cells
Guest Presenters: Sara Colpitts, Ph.D. and Courtney Plumlee,
Ph.D. (Postdoctoral Fellows; Department of
Immunology, University of Connecticut
Health Center): Topic:
"Genetic control of CD4 and CD8 fate"
assigned papers 13,14
February 29 Guest Presenter: Maureen Sherry Lynes, Ph.D., Postdoctoral fellow, Stem Cell Center,
Harvard University. Topic: "Stat 3 and inflammatory disease"
assigned papers 11,12
March 7
Specific aims discussion (come with your grant specific aim thought out and written
down; email of this aim due to Dr Lynes by 5pm of March 6th
Student presenters #1,2
March 14 SPRING BREAK (3/11-3/17) Be working on your grant proposal
March 21 Student presenters #3
March 28 Student presenters #4
April 4
Student presenters #5
April 11
Student presenters #6
April 18
Student presenters #7
Student Grant Proposals are due today; reviewer assignments will be distributed
April 25
Grant review panel meeting
May 2-7
Final exam period
•
•Broad topic, so we will refine by selection of
topics (to be done next in class)
•Schedule for presentations will be emailed before
next week, schedule will be based on topics
selected
•ONE WEEK before you are to present, you must
forward either a link to the papers you will
present, or pdfs of the papers (which will be
assigned to all of the class)
•March 7: you must have specific aim for your
grant proposal defined and approved
•Course topics:
Epigenetic regulation of immune responses
Psychoneuroimmunology; the interaction between the nervous system
and the immune system
Obesity-induced activation of the NLrp3 inflammasome and insulin
resistance
Reactive oxygen and immune regulation
Ubiquitin as a regulator of chemotactic responses
Myeloid derived suppressor cells and their role in carcinogenesis
Myeloid derived suppressor cells and their role in autoimmune disease
pharmacologic manipulation of the immune response with biologic
agents; CTLA4Ig
Genome wide associational studies and the identification of
Rheumatoid arthritis susceptibility loci
 Metallothionein and immune regulation
Inflammasome regulation and microbial infections
mTOR as a central regulator of immunity
catastrophic dysregulation of the immune response; the cytokine
storm hemophagocytic syndrome and multi-organ failure in sepsis
Presentation Format
Overview
How do the two papers you have selected to present to the class tie
together? What is the general theme of your presentation?
Introduction
Answer the question:
“What does the audience need to know to appreciate the importance of
this topic?”
what was known before the work described in the two papers was done
what was the experimental approach/explain novel techniques
make sure to be familiar with the biological materials (e.g. reagents, cell
lines, animals)
Identify the most important experiments and discuss those with the controls
that help to understand the data
Summarize the conclusion of each experiment (what did the experiment tell us
and what did it open up as the next unknown)
Conclusion of paper:
what is the big picture of the paper, what do we know now that is most
important and what is left unknown
what would be the next interesting experiments
Practical aspects of presentation
One week before your presentation you must have emailed me links or pdf
files of the two papers that you will present and lead the discussion.
(papers should be related to each other and to the topic you have chosen,
and should have been published in the past 1-2 years.
Your presentation will be either 55 or 75 minutes (if presenting alone or with
another student). Earlier presentations will be doubled up and shorter
since there is less time to prepare
Plan on leading the discussion of the papers (remembering that each of you
will be graded on the quality and frequency of your participation in class)
•My own research interests
•The role of metallothionein in
immune modulation
•I’ll tell you a bit about the protein and its
biochemistry
•Examples of immunomodulation
•We have parallel interests in biotechnology
development
•Mab production (UC1MT)
•ECIS/taxis
•Grating coupled surface plasmon
resonance
Stressors initiate homeostatic responses,
including stress protein synthesis




Heat shock proteins
glucose regulated
proteins
FKBP
cyclophilins





acute phase proteins
some cytokines
histone 2B
ubiquitin
glucocorticoids
metallothionein
Stressors
Immune changes
Metallothionein:
unusual molecular characteristics





small, heat stable molecule
about 61 amino acids
20/61 are cysteines (cys-cys, cys-X-cys and cys-X3cys motifs)
4-11 molecules of heavy metal divalent cation per
molecule of MT
no aromatic or histidine residues, no disulfide
linkages
MDPNCSCATGGSCTCAGSCKCKECKCTSCKKSCCSCCPVGCAKCAQGCV
CKGASDKCSCCA
alpha
subunit
Lys-Lys Dimer Bridge
Accessible Thiol-groups
beta
subunit
after Robbins et al.
Induction of MT Gene Transcription
inflammatory agents
Ca
ionophore
GC
TNF
IL-6
IL-1
H2O2
IFN
[Ca]
cAMP
CalmodulinPK
GC-R
ISRE
-800
GRE
-300
AP2
BLE
DAG
ROS
PKA
PKC
AP1
MRE
phorbal
metal
ester
cations
SP1
MBP
TRE GC MRE TATA
+1
GRE
1000
Structural MT gene: three exons interrupted by two introns
Chromosome 8 (mouse) and Chromosome 16 (human)
Metallothionein Functions

Intracellular functions:
• decreases toxic effects of heavy metals
• acts as a free radical scavenger, regulates cellular redox
potential
• serves as a reservoir for essential heavy metals
• regulates NF-kB, Sp-1 transcription factor activity

Extracellular functions:
• may redistribute metal cations within body
• may bind membrane bound receptors (astrocyte receptor?)
Hypothesis: Metallothionein that is synthesized
as a result of stress can alter the capacity of the
immune system
Adaptive immune
mechanisms
From Kuby, Immunology, 2001 Freeman Press
MT Suppresses In Vitro CTL Induction
50
CTL induction
phase
40
Cytotoxicity
(%)
30
PBS
20
MT
10
0
30:1
10:1
1:1
Effector:Target
Splenic T lymphocytes of BALB/cByJ (H-2d) mice were co-cultured with mitomycin Ctreated splenocytes of C57BL/6J (H-2b) in the presence or absence of 20 uM MT for 5
days. Effector cells recovered from this mixed culture were assayed for CTL activity as
measured by LDH release from EL4 (H-2b) target cells. Results are reported as the
average of triplicates + s.d. and are representative of three independent experiments.
Metallothionein-mediated in vivo
humoral immunosuppression
mOD/min
80
60
40
ova
ova/mt
20
0
10
12
14
16
18
20
22
days
Mice were injected with 200 ug OVA with or without the addition of 120 ug MT on day 0
and day 10. Samples obtained on the days indicated were used in ELISA to determine
the anti-OVA activity. Results are representative of three independent experiments and
are reported as the average of triplicates + s.d.
anti OVA response (average mOD/min)
UC1MT enhances the humoral response
to
OVA immunization
300
OVA
OVA w/ UC1MT
OVA w/ Ig Control
250
200
150
100
50
0
0
14
18
21
25
32
35
43
Time (days)
BALB/cByJ mice were challenged with 200 ug OVA in the presence or absence of
UC1MT or isotype control on day 0 and day 10.
Targeted disruption of metallothionein
enhances the humoral response
180
average mOD/min
160
140
120
100
80
60
40
20
129 WT
129 KO
0
Day 0
Day 11
Day 13
Day 15
Day 17
Day 19
Time (days)
Day 25
Day 27
Serum MT (% of control)
SERUM METALLOTHIONEIN IN
B6-Hcph-1me-v (“viable motheaten”)
MICE
600
400
200
0
0
10
20
30
40
Age (days)
50
60
Metallothionein is also found on the surface of leukocytes
from mice with a congenital chronic inflammatory disease
_____
_____
_____
_____
+/mev
mev/mev
+/mev
mev/mev
(unstained)
(unstained)
(UC1MT-FITC)
(UC1MT-FITC)
UC1MT-FITC binding to splenocytes from B6-Hcph-1mev/mev and B6Hcph-1+/mev mice
MT suppresses the severity of collagen-induced arthritis
6
5
Arthritic index
Metallothionein also influences the onset
and severity of inflammatory bowel disease
vehicle group
MT group
(collaboration with University of Gent,
ZnCl group
Belgium); with inflammation associated
with optic wound healing (collaboration with
University of Tasmania, Australia), and with
23 24 25 26 27 28 29 30 31 32 33 post immunization
experimental epidermolysis bullosaMT injection
ZnCl 2 injection
acquisita (Universitätsklinikum SchleswigHolstein , Germany)
4
3
2
2
1
0
To induce CIA, DBA/1J mice were initially immunized by injection with CII/CFA into the base of the tail. 2 weeks later, mice received a
booster injection with CII/IFA in the footpad. After 7 days, each group of mice were injected i.p. with MT (), ZnCl2 () or PBS(vehicle
control) and were assessed for indications of arthritis. The clinical severity of CIA, determined by arthritis index, was significantly suppressed
in MT and ZnCl2 treatment groups.
-Youn et al. 2002
Metallothionein and Chemotactic
Factor sequence comparison
(amino acid residues enclosed in boxes
are identical, shaded amino acids are
homologous at 85% by clustal analysis)
Metallothionein
gene cluster
synteny: human
16 and mouse 8
chromosomes
CELL MOVEMENT: BIOLOGICAL REGULATION
The directed movement of cells (chemotaxis) from one location to
another can be controlled by molecular signals that serve as maps to
guide the cells to their destinations. These molecular maps can be
gradients of soluble molecules or gradients of molecules attached to cells
or other surfaces that the cell can encounter (haptotaxis).
Cells can also move randomly (chemokinesis), with their speed of
movement governed by external molecular signals.
CELL MOVEMENT: THE BASICS
Immune cells migrate from place to place through tissues and organs in
the body for many reasons:
- to rebuild wounded or damaged tissues
- to respond to necrotic or apoptotic cell
death
- to fight infection, attack transplanted
tissues
- to contribute to autoimmune disease
processes, hypersensitivities
Neutrophil chemotaxis is governed by very small gradients
This movie was made with a 16mm camera in the 1950s by David Rogers at
Vanderbilt University. The neutrophil is "chasing” a Staphylococcus aureus
bacterium, added to the blood film (the non-motile cells are erythrocytes)
http://expmed.bwh.harvard.edu/projects/motility/neutrophil.html.
8 Well ECIS Chamber
Collaboration with David
Knecht’s laboratory and with
Applied Biophysics, Inc.
Courtesy of Applied Biophysics Inc.
ECIS/taxis Side View
Agarose
Che m oa tt r a c ta nt
Cell Well
La r ge
E l e c tr ode
Ta r ge t
E l e c tr ode
To ECIS
Instrumentation
ECIS/taxis- automated measurement of
dictyostelium folate chemotaxis
(e-)
Surface Plasmon Resonance; the basics
Based on the physical phenomenon of energy transfer at a metal-dielectric
interface (e.g., a gold and water interface ).
Under specific optical conditions (e.g. wave vector, incident angle and frequency of the incident
light), the energy of the light excites electron movement (the plasmon) within the metal. This
energy transfer reduces the intensity of the reflected light.
SPR technologies can use different ways to match the
wave vector of the illuminating beam of light with the
plasmon wave vector
Using a prism (Kretschman configuration)
Using a diffraction grating (Grating-coupled surface plasmon resonance ; GCSPR) -- the
first order diffracted light from the grating matches the plasmon requirements
There is a critical angle of illumination at which this vector
matching is best, and at which the energy transfer into the
metal is largest.
The angle at which maximum energy transfer occurs is the “SPR angle ” or “resonance angle”.
Energy transfer is sensitive to the refractive index of the dielectric/metal interface
The addition of proteins to the sensor chip surface (which will then have a higher index of
refraction than water) will increase the angle at which maximum coupling occurs.
Measuring the SPR Minimum Angle baseline
CCD Camera and
Detection Optics
q
Data capture and real-time display
Intensity
Collimated
light source
Interrogation angle
Measuring the SPR Minimum Angle baseline
CCD Camera and
Detection Optics
q
Data capture and real-time display
Intensity
Collimated
light source
Measuring the SPR Minimum Angle baseline
CCD Camera and
Detection Optics
q
Data capture and real-time display
Intensity
Collimated
light source
Measuring the SPR Minimum Angle baseline
CCD Camera and
Detection Optics
q
Data capture and real-time display
Intensity
Collimated
light source
SPR
angle
Interrogation angle
Measuring the SPR Minimum Angle after
analyte capture
CCD Camera and
Detection Optics
Collimated
light source
q
Data capture and real-time display
Intensity
Antigen flow
Interrogation angle
Measuring the SPR Minimum Angle after
analyte capture
CCD Camera and
Detection Optics
Collimated
light source
q
Data capture and real-time display
Intensity
Antigen flow
Interrogation angle
Measuring the SPR Minimum Angle after analyte capture
CCD Camera and
Detection Optics
q
Data capture and real-time display
Intensity
Collimated
light source
Measuring the SPR Minimum Angle after analyte capture
CCD Camera and
Detection Optics
q
Data capture and real-time display
Intensity
Collimated
light source
Measuring the SPR Minimum Angle after analyte capture
CCD Camera and
Detection Optics
q
Data capture and real-time display
Intensity
Collimated
light source
Measuring the SPR Minimum Angle after analyte capture
CCD Camera and
Detection Optics
Collimated
light source
Data capture and real-time display
Intensity
q
SPR
angle
Change in SPR angle is proportional to
mass adsorbed at sensor chip surface
Interrogation angle
High Content assay results
32 x 32 array
High signal:noise
ratio
> 1000 independent measurements/cm2
with parallel measurements at regional
reference spots
Combined GCSPR/GCSPCE detection enlarges total
dynamic range
pg/ml
µg/ml
Potential diagnostic applications in a heterogeneous world-single
biomarkers are often not enough
TNF-a
SPEF Microarray analysis of
Rheumatoid arthritis patient
and normal control sera.
Serum from 2 RA patients and
one control were evaluated
for the presence of 17
biomarkers by SPEF
microarray. Bars indicate
average fluorescent intensity
values from 5 ROIs on the
sensor chip surface.
Single biomarkers can differ between individuals
with the same disease
Comparison of GCSPRI values with scanning electron micrograph
enumeration of captured Bacillus globigii
Limit of detection: ~100 bacteria
B. globigii (BG) cells were grown overnight in TSB at 37C.
GCSPR sensor chips were spotted with various antibodies at
concentrations ranging from 0-500 ug/ml. The chip was
blocked with 2% BSA for 30 min, and equilibrated with PBST
prior to flowing the bacterial cell sample. Immediately after
the GCSPR capture assay the chip was gently dipped in ddH20,
air dried, and stored in a dessication chamber until SEM
imaging (48 hrs later). Bacterial cell counts were done on
SEM images of the ROIs that generated different SPR angle
shifts. The lower horizontal dotted line represents the average
of 22 background spots (bare gold regions on the chip). The
upper horizontal dotted line represents the background value
plus 2 Standard deviations. INSET: Two linked BG cells
captured on the GCSPRI grating and imaged with SEM.
Magnification: 17,000x. Cells measure approximately 3.3 x
0.86 M.
Capture of antigen-specific T cells by sensor-chip
A MHC class II monomers associated with the
immobilized
cognate antigen: can detect 1/1000 targets
Influenza Peptide Reactive CD4
+
T-Cell Capture and Detection on a GCSPRI
300
² SPR Angle (mdeg)
250
200
MHC II / HA peptide
MHC II / Tet peptide
anti- CD3 antibody
150
*
100
*
*
50
*
*
0
100
B
50
10
1
0.1
0.01
Percent HA Reactive T cells
MHC II / HA peptide
MHC II / Tet peptide
(negative control)
anti-CD3 Antibody
(positive control)
Limits of Sensitivity for Influenza Reactive T Cell Detection. CD4+ HA peptide reactive T cells were mixed with CD4+ Jurkat T cells in 6 ratios ranging from 100% 0.01% HA peptide specific T cells in 6 million totals cells. Cells were resuspended in RPMI + FBS to a final volume of 3ml and passed over a gold GCSPR sensor chip
with a PDEC Dextran surface and covalently attached MHC/peptide constructs or antibodies. A. Shifts in SPR angle were determined as average of 3 capture spots
consisting of MHC II / HA peptide, MHC / Tet peptide (negative control), or anti-CD3 antibody (positive control) *signal is more than 2 std above negative control B.
Pictures correspond to one representative cell capture spot on sensor surface.
JMR021208
D3-SPR Dual-Channel Technology
 Permits matched-pair analyses between patients and
controls
 Facilitates simultaneous measure of macromolecular
constituents and cellular phenotypes in sample
 Integrates with Dual-Fluor technology to increase
statistical power of diagnostic models
Conclusions





Metallothionein synthetic capacity represents a risk
factor for toxicant-induced immunomodulation
Induction of metallothionein synthesis may have
therapeutic effects in some forms of autoimmunity
Metallothionein analogs may represent novel
immunotherapeutics
The propensity to synthesize metallothionein may
influence disease susceptibility
Monoclonal antibody-mediated manipulation of
metallothionein or other metallothionein antagonists in
vivo can enhance vaccination efficacy
Technologies on our horizon


Grating-coupled surface plasmon resonance imaging as
diagnostic for molecular signatures of agricultural diseases
Grating coupled fluorescence plasmonics for the identification of
CD4+ and CD8+ T cells that are diabetogenic
• Capture of cells with immobilized MHC Class I and Class II
monomers folded with diabetogenic peptides
• Functional analysis of captured cells

Design and validate chips for specific
diagnostic/therapeutic/monitoring needs
•
•
•
•
Stress responses
Protein synthesis and secretion
Cellular differentiation
Vaccine responses (simultaneous assessments of vaccine
efficacy, immune capacity, and pathogen presence using the
same unfractionated blood sample).
• Immune assessment (infection, autoimmune disease and
cancer)
• Environmental assessment
Regulatory T cells: central players
differentiated by cytokine profile and
transcription factor expression
http://www.uke.de/institute/immunologie/index_ENG_72302.php
IL-23/IL-17 axis in inflammation
The IL-23/IL-17 axis plays an important role in the
development of chronic inflammation and in host defenses
against bacterial infection. (A) In chronic inflammation,
antigen-stimulated dendritic cells and macrophages
produce IL-23, which promotes the development of
Th17/ThIL-17 cells. Th17/ThIL-17 cells produce IL-17,
which enhances T cell priming and triggers potent
inflammatory responses by inducing the production of a
variety of inflammatory mediators. IL-23 also acts on
dendritic cells and macrophages in an
autocrine/paracrine manner to stimulate the generation
of proinflammatory cytokines, such as IL-1, IL-6, and
TNF-α. IL-12–stimulated Th1 cells produce IFN-γ and
suppress the differentiation of Th17/ThIL-17 cells. Th1
cells may play an immunoregulatory, not a pathogenic,
role in the development of chronic inflammation. (B)
Upon bacterial infection, IL-23 is rapidly produced by
activated macrophages and dendritic cells at the site of
infection. IL-23 then activates local resident Th17/ThIL17 cells and other IL-17–producing cells, such as CD8+
T cells and γδ T cells. Production of IL-17 by these cells
induces G-CSF production from stromal cells. The IL23/IL-17/G-CSF pathway augments neutrophil
recruitment to the infection site, contributing to
extracellular bacterial clearance. IL-23 also increases
the production of IL-1, IL-6, and TNF-α in an
autocrine/paracrine manner. In contrast, Th1 cells
produce IFN-γ and stimulate CD8+ cytotoxic T
lympocytes, NK cells, and macrophages. IFN-γ enhances
antigen presentation by inducing expression of MHC
molecules and activates cells to produce cytolytic
molecules, including perforin and granzyme, which
promote the elimination of intracellular bacteria.
J Clin Invest. 2006; 116(5):1218–1222 doi:10.1172/JCI28508
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