Simultaneous Monitoring of Protein Adsorption Kinetics Using a Quartz Crystal Microbalance and Field-Effect Transistor Integrated Device

We developed an integrated device comprising a quartz crystal microbalance (QCM) and a field-effect transistor (FET) with a single common gold electrode in a flow chamber. An alternating current inducing oscillations in the piezoelectric quartz of the QCM sensor is electrically independent of the circuit for the FET output so that the two sensors in different detection mechanisms simultaneously record binding kinetics from a single protein solution on the same electrode. A conjunction of adsorbed mass from QCM with electric nature of bound protein from FET provided deeper understanding on a complex process of nonspecific protein adsorption and subsequent conformational changes at a solid/liquid interface. Lower apparent <i>k</i>_on values obtained by FET than those obtained by QCM on hydrophobic surfaces are interpreted as preferred binding of protein molecules facing uncharged domains to the electrode surface, whereas higher <i>k</i>_off values by FET than those by QCM imply active macromolecular rearrangements on the surfaces mainly driven by hydrophobic association in an aqueous medium. The advanced features of the combined sensor including in situ, label-free, and real-time monitoring provide information on structural dynamics, beyond measurements of affinities and kinetics in biological binding reactions

EPO reduces total number of Mononuclear and T cells in EAE lymph nodes

a, b<p>EPO or sham PBS daily treatment for 3 days was initiated 7 days post MOG-immunization. Mice (n = 6/group) were sacrificed on day 11 (^a) or day 17 (^b). DILNs were harvested for cell quantification. Normal mice (n = 6) were used as controls.</p>c<p>mean±SEM</p>d<p>p<0.001 (sham PBS treated EAE vs NL or EPO-treated EAE mice)</p>e<p>p<0.05 (sham PBS treated EAE vs NL mice)</p

EPO induced sustained immuno/inflammatory modulation by expanding peripheral Treg cell numbers and reducing Th17 positive cells.

<p>Mice were immunized with MOG and received daily EPO treatment for 6 days (day1–6). a). EPO therapy for 6 days delayed onset of clinical neurologic signs in animals and reduced the magnitude of clinical deficit in EAE mice (•) compared to sham treated control (○) EAE mice (*, p<0.05). The clinical score was determined as the mean±SEM of 8 mice per group. Data represents the mean±SEM of 8 individual mice. b–c) DILNs were obtained from mice after 6 days treatment with either EPO or PBS. Cells were quantified for number of Tregs (CD4⁺Foxp3⁺) and Th17 by flow cytometry. Panel b shows that about 3.4% Treg cells were detectable in normal mouse inguinal lymph node (left). A reduced number (2.4%) of Treg cells was detected in sham treated EAE control mice nodes (middle), whereas EPO therapy induced a 2-fold increase in Foxp3+ Treg cells on day 16 compared to sham treatment (right). Panel c shows <0.2% of normal healthy lymph node cells stained positive for Th17 (left). Numbers of peripheral Th17 cells greatly increased on day 16 (15-fold) in EAE mice treated with PBS (middle, 3.2%). In marked contrast, EPO therapy (right) sharply reduced the number of peripheral Th17 cells in MOG-immunized animals.</p

In vivo effect of EPO treatment on MHC expression and number of MOG antigen specific T cells in inguinal lymph nodes.

<p>EPO treatment was started 7 days after mice received MOG_35–55 immunization. Bilateral draining inguinal lymph nodes (DILNs) were obtained from mice on day 11 (after 3 day treatment with either EPO at 5000 U/kg/day or sham treatment with PBS) and single cell suspensions were prepared. a) EPO treatment down-regulated mononuclear cell MHC class I (left) and class II (right) expression in lymph nodes from EAE mice. b) Less than 0.7% of single cells from normal mouse inguinal lymph nodes were double-positive for MOG_44–54 dimer and anti-CD3 antibody. In contrast, about 4% of single cell suspensions from sham treated EAE mice were positive for FITC-CD3 and PE-MOG_40–54/H-2Db dimer double staining (middle), whereas EPO treatment reduced in vivo proliferation of MOG-specific T cells back to 1.5% (right).</p

EPO treatment increased numbers of Treg cells and reduced numbers of Th17 cells in MOG-EAE mouse spinal cord.

<p>MOG-immunized C57 mice received daily EPO or PBS treatment for 6 days (day 1–6). Spinal cords were obtained at two different time points (day 16 and day 45) from mice treated with EPO or sham treated with PBS. Cells were quantified for number of Tregs (CD4⁺Foxp3⁺), Th17 and MOG-antigen specific T cells by flow cytometry. a) EPO therapy induced a substantial increase in Foxp3+ Treg cells in EAE spinal cords. The EPO induced expansion of Tregs in the CNS became even more evident in late stages of the disease and correlated with less severe neurologic deficit. b–c) Foxp3⁺ labeled Treg cells in PBS or EPO treated MOG-EAE spinal cord by IHC. Panel b shows a sham treated EAE spinal cord section reacted with Foxp3 antibody containing few labeled cells. By contrast, many more Foxp3+ cells were present in the infiltrates of EPO treated EAE spinal cord (panel c). d) Significantly increased numbers of Th17 cells occurred in sham treated EAE mouse spinal cord and this correlated with more severe clinical neurologic deficits whereas EPO therapy suppressed the number of spinal cord Th17 cells. e) Increased MOG antigen-specific T cells occurred within untreated EAE spinal cord, and EPO induced large reductions in MOG-specific T effector cells within the CNS. Data represents mean±SEM for 6 individual mice. *, p<0.05; **, P<0.001.</p

EPO treatment induced peripheral Treg cell expansion while reducing MOG-specific T cells and Th17 positive cells in peripheral lymphoid tissues.

<p>Mice were immunized with MOG and received daily EPO treatment for 6 days (day1–6). DILNs and spinal cords were obtained at three different time points (day 7, day 16 and day 45) from mice treated with EPO or sham treated with PBS. Cells were quantified for number of Tregs (CD4⁺Foxp3⁺), Th17 and MOG-antigen specific T cells by flow cytometry. a) There were reduced numbers of Treg cells in DILNs from sham treated EAE mice at early stages (day 7) as well as at the peak of clinical signs (d16), and during recovery (day 45) compared to normal healthy control animals. b) Numbers of peripheral Th17 cells increased in sham treated EAE mice, whereas EPO therapy sharply reduced the number of peripheral Th17 cells in MOG-immunized animals. c) EPO treatment significantly reduced peripheral MOG_40–54/H-2Db specific T effector cell population compared to sham treated EAE mice. Data represents mean±SEM for 6 individual mice. *, p<0.05; **, p<0.001.</p

EPO down-regulates proliferation of MOG-specific T cells and suppresses T cell cytokine production.

<p>a) Single cell suspensions of hyperimmune EAE spleen were prepared 7 days post MOG peptide immunization and quantified by flow cytometry for CD8 and MOG_44–54/H-2Db dimer double positive binding. In normal mice, 0.42% of splenic CD8 cells bound to the MOG_44–54/H-2Db dimer (left side), whereas an 8-fold increase in MOG_44–54/H-2Db dimer positive staining occurred in CD8 T cells obtained from MOG_35–55 immunized EAE animals (middle). In contrast, when a non-specific control peptide was tested, only 0.6% of CD8 cells exhibited non-specific binding to the NP_366–374 dimer (right). b & c) MOG-EAE antigen specific T cells were enriched by exposure to antigen in long term tissue culture, and then exposed to EPO (1 or 10 U/ml) for 72 hrs. Supernatants were collected from the cultured MOG-T cells for cytokine profile analysis and the remaining cells were quantified for number of CD8 and MOG_44–54/H-2Db dimer double binding cells after exposure to EPO or control treatment. EPO induced a marked decrease in pro-inflammatory cytokine production (black bars) when compared to controls (white bars). EPO at 1 U/ml induced a substantial decline in the MOG-specific CD8 T cell population (c-middle panel). Some of the MOG_44–54-peptide/H-2Db dimer positive, non-CD8 T cell (including portion of CD11c positive cells) populations were also reduced by EPO treatment (c-middle panel). Each data point is the mean±SEM of three experiments performed in triplicate. *, p<0.005.</p

Early EPO treatment sharply reduced total numbers of inflammatory cells and limited expansion of the dendritic cell population in DILNs during the EAE induction phase.

<p>C57 mice received EPO or PBS sham treatment starting on the day of MOG-immunization for 6 days and lymph nodes were obtained on day 7. a) Significantly enlarged inguinal lymph nodes were observed in control sham treated MOG-EAE mice (middle), whereas much smaller nodes were found in EPO treated EAE mice (right) similar in size to nodes from normal animals (left). b) Total MNCs were quantified from single cell suspensions of DILNs after lysis of RBCs. Greatly increased inflammatory mononuclear cell numbers were observed in sham treated EAE lymph nodes compared to DILNs from EPO treated EAE mice or normal mice. c) Cells were reacted with fluorophore labeled mAbs specific for surface markers (MHC class II and CD11c) and analyzed by flow cytometry. Note that the large expansion in dendritic cell population found in sham treated EAE mice was dramatically suppressed by EPO therapy. Data represents mean±SEM for 6 individual mice. **, p<0.0001.</p

LIPS for gAChR in the sera from patients with autoimmune autonomic ganglionopathy (AAG) and controls.

<p>We tested the sera from patients with AAG, disease controls (DC), and healthy controls (HC). a) Anti-gAChRα3 antibodies were detected in 23 samples. The mean anti-gAChRα3 antibody level in the HC was 0.305 antibody index (A.I.), which was significantly lower than in the AAG samples with a mean level of 1.210 A.I. (<i>p</i> < 0.001). b) Anti-gAChRβ4 antibodies were also detected in seven samples, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118312#pone.0118312.g004" target="_blank">Fig. 4B</a> (<i>p</i> = 0.005). The mean anti-gAChRβ4 antibody level in the HC was 0.367 A.I., which was significantly lower than the measn level of 0.618 A.I. in the AAG samples (<i>p</i> < 0.001).</p

Autonomic function tests at baseline of anti-gAChR Ab positive AAG patients.

<p>Anti-gAChRα3 Ab = ganglionic acetylcholine receptor α3 antibody; Anti-gAChRβ4 Ab = ganglionic acetylcholine receptor β4 antibody; A.I. = Antibody Index; OH = orthostatic hypotension; HUTT = head-up tilt test; CV R-R = CV = coefficient of variation R-R interval; H/M ratio = heart-to-mediastinum ratio in ¹²³I-MIBG myocardial scintigraphy; TST = thermoregulatory sweat test; NE = norepinephrine; SSR = sympathetic skin response; QSART = quantitative sudomotor axon reflex test; CSF = cerebrospinal fluid</p><p>Autonomic function tests at baseline of anti-gAChR Ab positive AAG patients.</p