Journal: Frontiers in Immunology

Article Title: Immune and ionic mechanisms mediating the effect of dexamethasone in severe COVID-19

doi: 10.3389/fimmu.2023.1143350

Figure Lengend Snippet: Changes in ion channel expression with disease progression and dexamethasone treatment. Fold change in mRNA abundance of Kv1.3 ( KCNA3 , A ), KCa3.1 ( KCNN4 , B ), Orai ( ORAI1 , C ) and Stim1 ( STIM1 , D ) in PBMCs from healthy donors (n=4), and patients with mild COVID-19 (n=4), severe COVID-19 (Severe, n=3), and severe COVID-19 + dexamethasone (Dexa, n=4) was determined by RT-qPCR. Data are normalized to healthy donor PBMCs. Each sample was run in triplicate. 18S rRNA was used as the housekeeping gene. Bars represent means ± SD, and each symbol represents an individual patient. Data were analyzed by one-way analysis of variance (ANOVA) (P < 0.001 in A , P = 0.4270 in B , P=0.0003 in C, and P=0.0414 in D ), and post hoc testing was performed by the Holm-Sidak method.

Article Snippet: Cells were stained for surface guinea pig anti- Kv1.3 (Alomone Labs) and Orai1 (ATTO-633, Alomone Labs) primary antibodies overnight at 4°C in the dark.

Techniques: Expressing, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Immune and ionic mechanisms mediating the effect of dexamethasone in severe COVID-19

doi: 10.3389/fimmu.2023.1143350

Figure Lengend Snippet: Effect of dexamethasone on Kv1.3 channel abundance. (A) Kv1.3 ( KCNA3 ), Orai1 ( ORAI1 ), and Stim1 ( STIM1 ) mRNA expression in PBMCs from healthy donors (n=4) treated with 0.1 μM and 1μM dexamethasone or vehicle for 48 h was determined by RT-qPCR. Data were normalized to vehicle-treated control. Significance was determined by one-way analysis of variance (ANOVA) for KCNA3 (P < 0.001), ORAI1 (P=0.005), and by ANOVA on ranks for STIM1 (p=0.630). Post-hoc testing was performed by the Holm-Sidak method. (B) Kv1.3 and Orai1 abundance in immune cell subsets from healthy donor PBMCs treated with 1 μM dexamethasone for 48 h. (Left) Representative flow cytometry histograms showing Kv1.3 (top) and Orai1 (bottom) abundance in the presence or absence of dexamethasone treatment in CD3 + T cell subsets. (Right) Mean fluorescence intensity (MFI) of Kv1.3 and Orai1 in immune cell subsets from three healthy donors treated in vitro with 1 μM dexamethasone or vehicle for 48 h. Significance determined by paired t-test. means ± SD, and each symbol represents an individual donor. .

Article Snippet: Cells were stained for surface guinea pig anti- Kv1.3 (Alomone Labs) and Orai1 (ATTO-633, Alomone Labs) primary antibodies overnight at 4°C in the dark.

Techniques: Expressing, Quantitative RT-PCR, Control, Flow Cytometry, Fluorescence, In Vitro

Journal: Frontiers in Immunology

Article Title: Immune and ionic mechanisms mediating the effect of dexamethasone in severe COVID-19

doi: 10.3389/fimmu.2023.1143350

Figure Lengend Snippet: Effect of dexamethasone on Kv1.3 channel function, Ca 2+ signaling and cytokine production. (A) Inhibition of Kv1.3 currents in CD8 + T cells treated with 1 μM dexamethasone (Dexa) for 24 and 48 h. Representative Kv1.3 currents are shown on the left, and percentage inhibition of Kv1.3 currents by dexamethasone in n=4 donors is shown on the right. (B) Representative Ca 2+ response (shown as a ratio of Indo-1 fluorescence at 400 and 480 nm) recorded in activated healthy donor CD8 + T cells treated with either 1μM dexamethasone, 10 nM ShK-Dap22, or vehicle for 48 h are shown on the left. Cells were loaded with Indo-1 ratiometric dye, and the fluorescence was recorded by flow cytometry. Indo-1 loaded cells were first exposed to thapsigargin (arrow) in 0 mM Ca 2+ , followed by 2 mM Ca 2+ , which yields a rapid influx of Ca 2+ (see Materials and Methods). Data are representative of independent experiments performed in CD8 + T cells isolated from n=3 healthy donors. The average fold change in peak Ca 2+ levels in n = 3 healthy donors are shown on the right. Significance was determined by one-way analysis of variance (ANOVA, p=0.0309), and post hoc testing was performed by Tukey’s test. (C) IFN-γ release determined by ELISA in the supernatant of activated CD8 + T cells from n=4 healthy donors and n=3 severe COVID-19 patients. Significance was determined by unpaired t test (D) Percent inhibition of IFN-γ secretion as compared to vehicle treated cells after treatment of activated healthy donor (n=4) CD8 + T cells with either 0.1 μM and 1μM dexamethasone, 10 nM and 100 nM ShK-Dap22, or vehicle for 48 h. Significance was determined by one-way analysis of variance (ANOVA, p=0.9390). (E) Percent inhibition of IFN-γ secretion compared to vehicle treated cells after treatment of activated severe COVID-19 patient (n=3) CD8 + T cells with either 1μM dexamethasone, 10 nM ShK-Dap22, or vehicle for 48 h. Significance was determined by paired-t test. Activation of CD8 + T cells in panels (B–E) was done for 48 h with plate bound anti-CD3 and anti-CD28 antibodies. Bars represent means ± SD, and each symbol represents an individual.

Article Snippet: Cells were stained for surface guinea pig anti- Kv1.3 (Alomone Labs) and Orai1 (ATTO-633, Alomone Labs) primary antibodies overnight at 4°C in the dark.

Techniques: Inhibition, Fluorescence, Flow Cytometry, Isolation, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Expression of human Kv1.3 in Tetrahymena thermophila . A. Expression construct design. KCNA3 , the gene encoding human Kv1.3, was modified with a C-terminal FLAG/10Xhis tag and placed under the control of the MTT5 and MTT1 promoter and terminator, respectively. The entire expression cassette was cloned as a NotI fragment into an rDNA vector, pTRAS1. The relative positions of chromosome breakage sites (CBS) and ribosomal genes (17s, 5.8s, and 26s) are shown. B. Single cell isolates maintain expression of recombinant Kv1.3. Anti-Kv1.3 Western analysis of single cells isolated from pooled Tetrahymena transformants and tested for their ability to express Kv1.3. Eight of nine single cell isolates expressed Kv1.3 at similar levels to the original pool (T1) with one clone (#117) expressing higher-levels of Kv1.3. A lysate from wild-type cells (WT) was included as a negative control. C. Tetrahymena -expressed Kv1.3 is phosphorylated. Purified Kv1.3 was incubated in the absence (−) and presence (+) of calf-intestinal alkaline phosphatase (CIP) and subsequently detected by anti-Kv1.3 Western analysis as described above. D. Comparison of Kv1.3 expression levels in Tetrahymena and CHO cells. Cell lysates generated from 25,000 Kv1.3 expressing Tetrahymena (Tth) or CHO cells were resolved by SDS-PAGE. Kv1.3 was detected by Western analysis using an anti-Kv1.3 antibody and an anti-guinea pig HRP conjugated antibody. E. Tetrahymena -expressed Kv1.3 binds both Agitoxin-2 (AgTX-2) and ShK. Mock-induced wild-type cells (WT) and Kv1.3-expressing Tetrahymena cells were fixed and labeled with either 10 nM Agitoxin-2-TAMRA (AgTX-2-TAMRA) or ShK-TAMRA and visualized by fluorescence confocal microscopy. Inset shows a close-image of a single Tetrahymena cell. White arrows highlight the Tetrahymena plasma (surface) membrane. F. Binding of ShK to Tetrahymena Kv1.3 is specific. Fixed Tetrahymena cells expressing Kv1.3 were incubated with 10 nM ShK-TAMRA in the presence of saturating (10X) amounts of Margatoxin (MgTx) or Iberiotoxin (IbTx) and examined by fluorescence confocal microscopy.

Article Snippet: As a positive control, a polyclonal anti-Kv1.3-biotin antibody was also assessed (Alomone, #APC-101-B).

Techniques: Expressing, Construct, Modification, Clone Assay, Plasmid Preparation, Recombinant, Western Blot, Isolation, Negative Control, Purification, Incubation, Generated, SDS Page, Labeling, Fluorescence, Confocal Microscopy, Binding Assay

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Purification of recombinant Kv1.3. a. SDS-PAGE analysis of purified and reconstituted Kv1.3. Kv1.3 was purified as described in Materials, resolved by SDS-PAGE before and after reconstitution into liposomes and stained with SimplyBlue™ SafeStain™. B. Ligand Binding Analysis. Kv1.3-containing liposomes were incubated with FAM-ShK (3 nM) in the presence or absence of a 50-fold excess of either MgTx or IbTx. Top Panel is a representative experiment showing total binding expressed as Anisotropy measured by fluorescence polarization. Bottom Panel represents specific binding to FAM-ShK. Kd was estimated as 11.5 nM +/− 3.4 nM based on specific binding curves generated in three separate experiments c. Fluorescence microscopy analysis of Kv1.3 magnetic beads. Magnetic beads containing tethered Kv1.3 reconstituted into a lipid bilayer consisting of rhodamine-labeled PE and non-labeled PC were examined by fluorescence microscopy. Beads were examined under a rhodamine filter to detect labeled PE incorporation (Left Panel) and with a FITC-filter following labelling with an anti-Kv1.3 antibody that recognizes an epitope on the first extracellular loop and anti-guinea pig conjugated FITC (Middle Panel). Rhodamine and FITC- images were merged (Right Panel) to confirm co-localization of Kv1.3 and the lipid bilayer (yellow fluorescence). D. Schematic illustration of Kv1.3 magnetic beads. Shown is the magnetic bead surface; the lipid bilayer consisting of PC (yellow lipids) and Rhodamine-labeled PE (red lipids); the six transmembrane domains of the Kv1.3 monomer (S1-S6); the C-terminal engineered FLAG (orange Triangle) and 10Xhis (Green Box) tags; a star indicates the position of the epitope on the first extracellular loop that is recognized by the Kv1.3 antibody utilized in c and e. e. Kv1.3 magnetic beads preferentially precipitate an antibody that recognizes an extracellular epitope. Kv1.3 magnetic beads or control beads were incubated with antibodies (6.7 nM) that recognize either internal (FLAG and 10Xhis) or external (Kv1.3) epitopes. Beads were washed and bound IgG eluted directly in SDS-PAGE loading buffer. IgG was detected by Western analysis using either anti-mouse-HRP (anti-FLAG and -His) or anti-guinea pig-HRP (anti-Kv1.3).

Article Snippet: As a positive control, a polyclonal anti-Kv1.3-biotin antibody was also assessed (Alomone, #APC-101-B).

Techniques: Purification, Recombinant, SDS Page, Staining, Ligand Binding Assay, Incubation, Binding Assay, Fluorescence, Generated, Microscopy, Magnetic Beads, Labeling, Western Blot

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Summary of anti-Kv1.3 antibody identification.

Article Snippet: As a positive control, a polyclonal anti-Kv1.3-biotin antibody was also assessed (Alomone, #APC-101-B).

Techniques: Enzyme-linked Immunosorbent Assay, Functional Assay, Magnetic Beads

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Identification of anti-Kv1.3 antibodies. A. Chicken anti-Kv1.3 antibodies. ScFv-Fc screening was carried out by ELISA using three-fold serial dilutions of antibody against proteoliposomes containing Kv1.3 or an irrelevant human VGIC also expressed in Tetrahymena . An isotype control (IC) for generated antibodies was also included. Shown are results for antibodies that inhibit Kv1.3 activity. Note the relative lack of reactivity of clone ch_p1E6 against Kv1.3 and clone p1F8 reactivity against the irrelevant proteoliposome control. B. Llama anti-Kv1.3 antibodies. ScFv-Fc screening was carried out using ten-fold serial dilutions of antibody on mesoscale against proteoliposomes containing Kv1.3 or an irrelevant human VGIC also expressed in Tetrahymena . IC1, isotype control for generated antibodies; IC2, isotype control for anti-Kv1.3 polyclonal antibody. Data is shown for 6 of 19 specific Kv1.3 binding scFv-Fc antibodies.

Article Snippet: As a positive control, a polyclonal anti-Kv1.3-biotin antibody was also assessed (Alomone, #APC-101-B).

Techniques: Enzyme-linked Immunosorbent Assay, Generated, Activity Assay, Binding Assay

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Identification of anti-Kv1.3 antibodies that functionally inhibit Kv1.3 channel activity. Purified scFv-Fc anti-Kv1.3 antibodies from either chickens or llamas were tested at a concentration of 400nM via electrophysiology for their ability to block current from human Kv1.3 expressed in L929 fibroblast cells. Shown are representative traces for each of the antibodies that blocked Kv1.3 current. Black lines represent control currents, red lines represent currents following addition of antibody. Inhibiting anti-Kv1.3 antibody clones derived from chickens are shown in a, the functional llama anti-Kv1.3 antibody is shown in b. An example of an antibody that was tested and shown not to modulate Kv1.3 activity is shown in c. d. Time-dependent development of current inhibition by monoclonal antibodies targeting Kv1.3. (Left Panel) Time-current plots showing current inhibition of three (3) individual cells expressing hKv1.3 channels by the monoclonal antibody ScFv-Fc L1A3. Antibodies were added after current stabilization at 0 second. Currents were elicited by pulsing to +40 mV for 200 ms from a holding potential of −80 mV every 30 seconds. (Right Panel) Means ± SD plot of the current inhibition of three individual cells in the left panel. Similar time-dependent profiles were observed for each of the blocking antibodies.

Article Snippet: As a positive control, a polyclonal anti-Kv1.3-biotin antibody was also assessed (Alomone, #APC-101-B).

Techniques: Activity Assay, Purification, Concentration Assay, Blocking Assay, Clone Assay, Derivative Assay, Functional Assay, Inhibition, Expressing

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Summary of anti-Kv1.3 antibody functional activity.

Article Snippet: As a positive control, a polyclonal anti-Kv1.3-biotin antibody was also assessed (Alomone, #APC-101-B).

Techniques: Functional Assay, Activity Assay, Blocking Assay

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Anti-Kv1.3 antibody potency analysis. A. Dose response curves. Ten-fold serial dilutions of chicken antibody p1E6 (red traces) and llama antibody L1A3 (green traces) were used to generate dose-response curves. Black lines represent control currents. B. IC 50 Determinations. IC 50 s for p1E6 (red curve) and L1A3 (green curve) were determined by fitting the calculated percentage of current block to a Hill equation. IC 50 s for p1E6 and L1A3 were estimated as 6 and 109 nM, respectively c. Analysis of p1E6 and L1A3 selectivity. ScFv-Fc clones p1E6 and L1A3 were tested for their ability to block the activity of related Kv1.x family members (Kv1.1, Kv1.2, Kv1.5), hERG and Nav1.5 at a concentration of 1 μM. Shown are representative traces from each experiment. Black lines, control current; Red lines, currents following addition of antibody.

Article Snippet: As a positive control, a polyclonal anti-Kv1.3-biotin antibody was also assessed (Alomone, #APC-101-B).

Techniques: Blocking Assay, Clone Assay, Activity Assay, Concentration Assay

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: L1A3 and p1E6 selectivity profile.

Article Snippet: As a positive control, a polyclonal anti-Kv1.3-biotin antibody was also assessed (Alomone, #APC-101-B).

Techniques:

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Kv1.3 epitope binning analysis. A. Binning analysis heat map. Shown are antibodies colored by bin (1–5) and by functional data. Antibodies that block Kv1.3 current are highlighted in blue (Ephys only), those that additionally bind Jurkat cells are highlighted in red (Ephys + Jurkat) and those that do neither are highlighted in grey. Relative competition activity of each antibody is color coded and indicates strong competition (red boxes), intermediate/weak competition (yellow boxes) or no competition (green boxes). Dark red boxes indicate competition from the same antibody pair. B. Binning network plot. Antibodies are colored to identify those that inhibit channel activity (blue), additionally bind Jurkat cells (red) or do neither (grey). Note antibody L1A3 was not tested for its ability to bind Jurkat cells, however, for simplicity was denoted as inhibiting ion channel only (blue) in a & b.

Article Snippet: As a positive control, a polyclonal anti-Kv1.3-biotin antibody was also assessed (Alomone, #APC-101-B).

Techniques: Functional Assay, Blocking Assay, Activity Assay

Journal: mAbs

Article Title: A multiplatform strategy for the discovery of conventional monoclonal antibodies that inhibit the voltage-gated potassium channel Kv1.3

doi: 10.1080/19420862.2018.1445451

Figure Lengend Snippet: Kv1.3 ortholog sequence alignment analysis.

Article Snippet: As a positive control, a polyclonal anti-Kv1.3-biotin antibody was also assessed (Alomone, #APC-101-B).

Techniques: Sequencing

Journal: Cell Reports Medicine

Article Title: A target discovery pipeline identified ILT3 as a target for immunotherapy of multiple myeloma

doi: 10.1016/j.xcrm.2023.101110

Figure Lengend Snippet:

Article Snippet: KCNA3 , Prosci , 57–918.

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Recombinant, Magnetic Beads, Software, CRISPR