Journal: Cell & Bioscience

Article Title: Enhancing striatal acetylcholine facilitates dopamine release and striatal output in parkinsonian mice

doi: 10.1186/s13578-024-01328-z

Figure Lengend Snippet: Activation of D1R and D2R regulated ACh dynamics in the dorsolateral striatum. A and C ACh3.0 sensor signals from DLS of 6-OHDA-lesioned mice aligned to the moment of the administration of D2R-selective agonist (Quinpirole, Qui), D2R-selective antagonist (Raclopride, RAC), L-DOPA, and saline. Mean values are shown as a blue line (Qui and saline-L-DOPA), a red line (RAC-L-DOPA), and a black line (saline), SEM, interval is shaded in red, blue, and gray. B Quantification of change in ACh3.0 sensor signals after administration of saline and Qui [n = 7, F(2,12) = 9.474, P = 0.0034, RM one-way ANOVA with Post hoc Bonferroni’s test]. D Quantification of the change in ACh3.0 sensor signals after the second administration of saline and L-DOPA [n = 4, F(2,6) = 12.32, P = 0.0075, RM one-way ANOVA with Post hoc Bonferroni’s test]. E and G ACh3.0 sensor signals from DLS of 6-OHDA-lesioned mice aligned to the moment of the administration of D1R-selective agonist (SKF-81297, SKF), D1R-selective antagonist (SCH23390, SCH), L-DOPA, and saline. Mean values are shown as a blue line (SKF and saline-L-DOPA), a red line (SCH-L-DOPA), and a black line (saline), SEM, interval is shaded in red, blue, and gray. F Quantification of change in ACh3.0 sensor signals after administration of saline and SKF [n = 7, F(2,12) = 33.98, P < 0.0001, RM one-way ANOVA with Post hoc Bonferroni’s test]. H Quantification of the change in ACh3.0 sensor signals after the second administration of saline and L-DOPA [n = 4, F(2,6) = 11.47, P = 0.0089, RM one-way ANOVA with Post hoc Bonferroni’s test]. I Diagram illustrating activation of D1R promoting GABA release from dSPNs acting on GABAARs on ChIs. J Left, schematic diagram of the fiber photometry recording system and local drugs infusion. Right, Image of ACh3.0 sensor, fiber channel, and cannula channel in the coronal DLS section. Scale bar: 500um. K ACh3.0 sensor signals from DLS aligned to the moment of the systemic administration of SKF and saline. Mean values are shown as a red line (PTX-SKF) a blue line (saline-SKF) and a black line (saline-saline), SEM, interval is shaded in red, blue, and gray. L Quantification of the change in ACh3.0 sensor signals after the second administration of saline and SKF [n = 3, F(2,4) = 59.58, P = 0.0011, RM one-way ANOVA with Post hoc Bonferroni’s test]

Article Snippet: We dissolved the D -like dopamine receptors (D1R)-selective agonist (SKF81297; 2.5 or 5 mg/kg; Sigma-Aldrich), D 2 -like dopamine receptors (D2R)-selective agonist (Quinpirole; 2.5 or 5 mg/kg; Sigma-Aldrich), D1R antagonist (SCH23390; 0.2 mg/kg; Sigma-Aldrich), D2R antagonist (Raclopride; 2 mg/kg; Sigma-Aldrich), L-DOPA (1, 6 or 10 mg/kg; Sigma-Aldrich) mixed with benserazide (15 mg/kg; Sigma-Aldrich), Donepezil (Den; 1 or 3 mg/kg; Selleck), Picrotoxin (PTX; 0.3 mg/mL; Tocris Bioscience), and Mecamylamine (Meca; 3 mg/kg and 50uM; MedChemExpress) in 0.9% saline solution.

Techniques: Activation Assay, Saline

Journal: Cell & Bioscience

Article Title: Enhancing striatal acetylcholine facilitates dopamine release and striatal output in parkinsonian mice

doi: 10.1186/s13578-024-01328-z

Figure Lengend Snippet: Activation of D1R and D2R regulated ACh dynamics in the dorsolateral striatum. A and C ACh3.0 sensor signals from DLS of 6-OHDA-lesioned mice aligned to the moment of the administration of D2R-selective agonist (Quinpirole, Qui), D2R-selective antagonist (Raclopride, RAC), L-DOPA, and saline. Mean values are shown as a blue line (Qui and saline-L-DOPA), a red line (RAC-L-DOPA), and a black line (saline), SEM, interval is shaded in red, blue, and gray. B Quantification of change in ACh3.0 sensor signals after administration of saline and Qui [n = 7, F(2,12) = 9.474, P = 0.0034, RM one-way ANOVA with Post hoc Bonferroni’s test]. D Quantification of the change in ACh3.0 sensor signals after the second administration of saline and L-DOPA [n = 4, F(2,6) = 12.32, P = 0.0075, RM one-way ANOVA with Post hoc Bonferroni’s test]. E and G ACh3.0 sensor signals from DLS of 6-OHDA-lesioned mice aligned to the moment of the administration of D1R-selective agonist (SKF-81297, SKF), D1R-selective antagonist (SCH23390, SCH), L-DOPA, and saline. Mean values are shown as a blue line (SKF and saline-L-DOPA), a red line (SCH-L-DOPA), and a black line (saline), SEM, interval is shaded in red, blue, and gray. F Quantification of change in ACh3.0 sensor signals after administration of saline and SKF [n = 7, F(2,12) = 33.98, P < 0.0001, RM one-way ANOVA with Post hoc Bonferroni’s test]. H Quantification of the change in ACh3.0 sensor signals after the second administration of saline and L-DOPA [n = 4, F(2,6) = 11.47, P = 0.0089, RM one-way ANOVA with Post hoc Bonferroni’s test]. I Diagram illustrating activation of D1R promoting GABA release from dSPNs acting on GABAARs on ChIs. J Left, schematic diagram of the fiber photometry recording system and local drugs infusion. Right, Image of ACh3.0 sensor, fiber channel, and cannula channel in the coronal DLS section. Scale bar: 500um. K ACh3.0 sensor signals from DLS aligned to the moment of the systemic administration of SKF and saline. Mean values are shown as a red line (PTX-SKF) a blue line (saline-SKF) and a black line (saline-saline), SEM, interval is shaded in red, blue, and gray. L Quantification of the change in ACh3.0 sensor signals after the second administration of saline and SKF [n = 3, F(2,4) = 59.58, P = 0.0011, RM one-way ANOVA with Post hoc Bonferroni’s test]

Article Snippet: We dissolved the D -like dopamine receptors (D1R)-selective agonist (SKF81297; 2.5 or 5 mg/kg; Sigma-Aldrich), D 2 -like dopamine receptors (D2R)-selective agonist (Quinpirole; 2.5 or 5 mg/kg; Sigma-Aldrich), D1R antagonist (SCH23390; 0.2 mg/kg; Sigma-Aldrich), D2R antagonist (Raclopride; 2 mg/kg; Sigma-Aldrich), L-DOPA (1, 6 or 10 mg/kg; Sigma-Aldrich) mixed with benserazide (15 mg/kg; Sigma-Aldrich), Donepezil (Den; 1 or 3 mg/kg; Selleck), Picrotoxin (PTX; 0.3 mg/mL; Tocris Bioscience), and Mecamylamine (Meca; 3 mg/kg and 50uM; MedChemExpress) in 0.9% saline solution.

Techniques: Activation Assay, Saline

Journal: bioRxiv

Article Title: Microglial engulfing of glutamatergic inputs and diminished excitability of D1 medium spiny neurons of the nucleus accumbens by peripheral inflammatory insult

doi: 10.1101/2024.12.02.625901

Figure Lengend Snippet: (A) The schematic illustration for the test during systemic inflammation (n=4-8 for each treatment). (B) Open arm % and (C) total distance travel in EPM. (D) Center % and (E) total distance travel in OFT. (F) Light zone time % and (G) Total distance travel in light zone in LDB. (H) Total distance travel, (I) time spent in mouse area %, (J) number of contacts, and (K) average contact time in 3CT. (L) Representative images of cFos (red), cre (green) staining in the NAc. (M, N) Plots show % cFOS+ that are D1R positive, % D1R+ that are cfos positive and (O, P) % cfos+ that are D1R negative. All data are expressed as mean ± SEM and analyzed using unpaired t -test or non-parametric Mann Whitney test; * p<0.05, **p<0.01, and ***p<0.001.

Article Snippet: Male C57Bl/6 mice (Jackson Laboratory, Bar Harbor, ME, USA) and D1R Cre mice (C57Bl/6 background; in-house colony) were housed in a reverse 12h light-dark cycle (lights off at 10h) with ad libitum access to water and food.

Techniques: Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Microglial engulfing of glutamatergic inputs and diminished excitability of D1 medium spiny neurons of the nucleus accumbens by peripheral inflammatory insult

doi: 10.1101/2024.12.02.625901

Figure Lengend Snippet: (A) Whole-cell patch clamp electrophysiology was performed on LPS-injected (0.83 mg/kg, i.p.) D1R-cre mice with viral YFP expression (B) Representative images of patched cells in bright field and YFP. (C) Example EPSC traces in the NAc core. (D) Event frequency, (E) amplitude, and (F) resting membrane potentials of EPSCs of D1R neurons in the NAc core (n = 10-13 from mice for sal, n = 6-7 from mice for LPS). (G) Example EPSC traces in the NAc shell. (H) Event frequency, (I) peak amplitude, and (J) resting membrane potentials of D1R neurons of the NAc shell (n = 9-11 for sal from mice, n = 10-11 from mice for LPS. (K) Event frequency and (L) peak amplitude of IPSCs of D1R neurons in the NAc core. (M) Event frequency and (N) peak amplitude of IPSCs of D1R neurons in the NAc shell. (Data are expressed as mean ± SEM and analyzed using unpaired t -test or using non-parametric Mann Whitney test, *p<0.05.

Article Snippet: Male C57Bl/6 mice (Jackson Laboratory, Bar Harbor, ME, USA) and D1R Cre mice (C57Bl/6 background; in-house colony) were housed in a reverse 12h light-dark cycle (lights off at 10h) with ad libitum access to water and food.

Techniques: Patch Clamp, Injection, Expressing, Membrane, MANN-WHITNEY

Journal: bioRxiv

Article Title: The impact of systemic blockade of dopamine receptors on the acquisition of two-way active avoidance in rats

doi: 10.1101/2024.11.29.626069

Figure Lengend Snippet: Overview of the behavioral procedures of Experiment 1 and Experiment 2. SCH = D1R antagonist SCH 23390, SUL = D2R antagonist sulpiride, vehicle = 0.9% physiological saline + 17% DMSO.

Article Snippet: Rats were intraperitoneally injected with either D1R antagonist SCH 23390 (0.05 mg/kg or 0.025 mg/kg, Tocris Bioscience), D2R antagonist sulpiride (20 mg/kg, Sigma Aldrich) or vehicle (0.9% physiological saline + 17% DMSO).

Techniques: Saline

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A – J ) Violin plots depict AAb reactivity titrated from human sera against human D1R and D2R by ELISA from 4 cohorts. ( A and B ) In Cohort 1, significant differences in anti-D2R IgG levels were observed between SC patients ( n = 6) and age-matched healthy controls ( n = 18). ( C and D ) PANDAS patients with choreiform movements ( n = 11) showed significantly higher D1R and D2R IgG AAb levels compared with controls ( n = 18). ( E and F ) Cohort 2: PANDAS without choreiform movements ( n = 35) exhibited significantly elevated anti-D1R IgG levels compared with controls ( n = 18). ( G and H ) Cohort 3 with PANDAS/PANS ( n = 858) demonstrated elevated anti-D1R AAb titer versus controls ( n = 18). ( I and J ) Cohort 4: A second SC group (with acute SC) ( n = 14) showed elevated D1R and D2R titers compared with healthy controls ( n = 31). All groups were compared by the Mann-Whitney U test ( P < 0.05 considered significant).

Article Snippet: Briefly, Immunolon 4 microtiter plates (VWR) were coated overnight at 4°C with 10 μg/mL of antigens: D1R (Perkin-Elmer), D2R (Perkin-Elmer or in-house), GlcNAc conjugated to BSA (Vector Labs), HIB PRP (National Institute for Biological Standards and Controls, London, United Kingdom), pneumococcal capsular antigens ( Streptococcus pneumoniae type 23F, ATCC), or D1R and D2R synthesized peptides (for peptide epitope mapping) ( ) in carbonate/bicarbonate buffer pH 9.6.

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A and B ) In Cohort 1, ROC curves yield no predictive value for D1R titers and significance ( P < 0.05 considered significant) for D2R titers in SC. ( C and D ) PANDAS patients with choreiform movements in Cohort 1 exhibit significant values for both D1R and D2R titers. SEM is incorporated. ( E and F ) Cohort 2, PANDAS without choreiform movements, demonstrates highly significant for D1R titers, with no predictive value for D2R titers. ( G and H ) Cohort 3 (PANDAS/PANS) also demonstrated significant AUC for D1 titers, with no predictive AUC for D2 titers. ( I and J ) Cohort 4/acute SC for both D1R and D2R titers separating disease from controls. ROC curve analysis with 95% confidence intervals was calculated in Prism using the Wilson-Brown method.

Article Snippet: Briefly, Immunolon 4 microtiter plates (VWR) were coated overnight at 4°C with 10 μg/mL of antigens: D1R (Perkin-Elmer), D2R (Perkin-Elmer or in-house), GlcNAc conjugated to BSA (Vector Labs), HIB PRP (National Institute for Biological Standards and Controls, London, United Kingdom), pneumococcal capsular antigens ( Streptococcus pneumoniae type 23F, ATCC), or D1R and D2R synthesized peptides (for peptide epitope mapping) ( ) in carbonate/bicarbonate buffer pH 9.6.

Techniques:

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A ) Diagram illustrates the D1R primary sequence for synthetic peptides that reacted with PANDAS serum IgG versus control sera. ( B ) D1R peptide reactivity in SC ( n = 7, 8) and PANDAS ( n = 29–36, Cohorts 1 and 2) or control sera ( n = 8–9). One-way ANOVA with Tukey’s multiple-comparison test reveals * P < 0.05 for PANDAS (EL1a, EL2b, and EL3 epitopes) compared with control serum, and ** P < 0.01, *** P < 0.001 for PANDAS (NT, TM1, or EL1a epitopes) versus controls. ( C ) Linear map of D1R and corresponding amino acid residues. ( D ) Significant D2R immunoreactive epitopes mapped by SC sera. (Overlapping peptides in purple). ( E ) D2R peptide reactivity with control ( n = 11), SC ( n = 8), and PANDAS sera ( n = 34) from cohorts 1 and 2. One-way ANOVA with Tukey’s multiple-comparison test reveals greater reactivity for SC (* P < 0.05 NTa and NT1b, ** P < 0.01 for EL1) versus healthy controls and PANDAS NTb (* P < 0.005) versus controls. All graphs shown as mean ± SEM. ( F ) D2R mapped linear peptides with their corresponding amino acid residues.

Article Snippet: Briefly, Immunolon 4 microtiter plates (VWR) were coated overnight at 4°C with 10 μg/mL of antigens: D1R (Perkin-Elmer), D2R (Perkin-Elmer or in-house), GlcNAc conjugated to BSA (Vector Labs), HIB PRP (National Institute for Biological Standards and Controls, London, United Kingdom), pneumococcal capsular antigens ( Streptococcus pneumoniae type 23F, ATCC), or D1R and D2R synthesized peptides (for peptide epitope mapping) ( ) in carbonate/bicarbonate buffer pH 9.6.

Techniques: Sequencing, Control, Comparison

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A – C ) ELISA titers of anti–microbial polysaccharide antigen IgG antibodies in PANDA/PANS and control serum. ( A ) Elevated anti-GlcNAc titers in PANDAS/PANS ( n = 22) versus controls ( n = 14). Mann-Whitney U test. ( B and C ) AAb titers against pneumococcal polysaccharide 23F (PP-23F) or Haemophilus influenzae type b capsular polyribitol phosphate (HIB PRP) were not significantly elevated (NS). ( D and E ) Competitive inhibition ELISA of D1R ( D ) or D2R ( E ) with PANDAS serum IgG ( n = 7) preabsorbed with HIB-PRP, PP-23F, or GlcNAc at 500 μg/mL. GlcNAc and HIB PRP significantly inhibit PANDAS AAb IgG reactivity of D1R (** P < 0.005) and D2R (** P < 0.05). In D and E , analysis was by nonparametric Kruskal-Wallis test followed by Dunn’s multiple-comparison test.

Article Snippet: Briefly, Immunolon 4 microtiter plates (VWR) were coated overnight at 4°C with 10 μg/mL of antigens: D1R (Perkin-Elmer), D2R (Perkin-Elmer or in-house), GlcNAc conjugated to BSA (Vector Labs), HIB PRP (National Institute for Biological Standards and Controls, London, United Kingdom), pneumococcal capsular antigens ( Streptococcus pneumoniae type 23F, ATCC), or D1R and D2R synthesized peptides (for peptide epitope mapping) ( ) in carbonate/bicarbonate buffer pH 9.6.

Techniques: Enzyme-linked Immunosorbent Assay, Control, MANN-WHITNEY, Inhibition, Comparison

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A ) PANDAS IgG-secreting hybridoma of B4C immunoreactivity with the synthesized human D1R and the D1R extracellular loop 1 peptide (EL1a), but not ( B ) D2R or D2R peptides. ( C ) Cell-based assay of PANDAS mAb B4C immunostaining (in red) of CHO-K1 cells transfected with D1R or the empty vector control. B4C preadsorption with the D1R EL1a peptide abolished B4C immunoreactivity with D1R. Original magnification, ×20. ( D ) mAb B4C mediates D1R dose-dependent signaling. CHO-K1 cells were transfected with full-length human D1R or the empty vector (EV) control and were treated with serial dilutions of mAb B4C starting with 1 ng/mL. * P < 0.05 by paired, 2-tailed t test between the D1R-transfected cells and vehicle. ( E ) CHO-K1 cells transfected with either D1R or EV were treated with mAb B4C alongside serially diluted dopamine (red) or dopamine alone (black). Dopamine dose response of D1R-transfected cells treated with dopamine versus B4C and dopamine was analyzed by nonlinear regression, showing significance. *** P = 0.0001 by extra sum-of-squares F test ( F test). ( F ) CHO-K1 cells, transfected with D1R or EV, underwent treatment with serially diluted dopamine (black diamonds), mAb B4C alongside dopamine (red circles), or D1R-transfected cells with preadsorbed mAb B4C peptide EL1a (1 μg/mL) and dopamine (blue squares). Dose-response curves from the cAMP assay in D1R-transfected cells treated with mAb B4C and dopamine versus B4C and dopamine preadsorbed with D1R EL1a peptide. * P = 0.036, nonlinear regression, logEC 50 , F test.

Article Snippet: Briefly, Immunolon 4 microtiter plates (VWR) were coated overnight at 4°C with 10 μg/mL of antigens: D1R (Perkin-Elmer), D2R (Perkin-Elmer or in-house), GlcNAc conjugated to BSA (Vector Labs), HIB PRP (National Institute for Biological Standards and Controls, London, United Kingdom), pneumococcal capsular antigens ( Streptococcus pneumoniae type 23F, ATCC), or D1R and D2R synthesized peptides (for peptide epitope mapping) ( ) in carbonate/bicarbonate buffer pH 9.6.

Techniques: Synthesized, Cell Based Assay, Immunostaining, Transfection, Plasmid Preparation, Control, cAMP Assay

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A ) PANDAS serum–induced cellular cAMP concentrations from CHO-K1 cells transfected with either D1R cDNA or the empty vector (sera diluted 1:40). ** P < 0.01 by Mann-Whitney U test. ( B ) PANDAS sera stimulated cAMP levels in stably expressed D1R (Tango D1- bla U2OS cells). *** P < 0.001 by Mann-Whitney U test. ( C ) D1R percentage increase in β-arrestin recruitment to D1R produced by SC ( n = 6), PANDAS ( n = 9), and control ( n = 6) serum from Cohort 1. ** P < 0.005 controls vs. PANDAS by 1-way ANOVA with Dunn’s post hoc test. ( D ) β-Arrestin recruitment to D1R stimulated by diluted PANDAS ( n = 19) vs. controls ( n = 7) from Cohort 2. ** P < 0.005 by Mann-Whitney U test. β-Arrestin recruitment to D1R was assayed using the FRET-based TANGO GeneBLAzer GPCR assay system. Values are expressed as a percentage of the FRET signal in cells relative to vehicle. ( E ) Serum-mediated D1R signaling dose response (Tango GeneBLAzer GPCR Assay) showed a significant difference between PANDAS and healthy controls (mean nonlinear regression analysis, sum-of-squares F test). ( F ) D1R activation is shown by the dose-response curves of PANDAS sera added in combination with dopamine (red square or blue squares) compared to dopamine alone (depicted by black circles) in Tango D1- bla U2OS cells.

Article Snippet: Briefly, Immunolon 4 microtiter plates (VWR) were coated overnight at 4°C with 10 μg/mL of antigens: D1R (Perkin-Elmer), D2R (Perkin-Elmer or in-house), GlcNAc conjugated to BSA (Vector Labs), HIB PRP (National Institute for Biological Standards and Controls, London, United Kingdom), pneumococcal capsular antigens ( Streptococcus pneumoniae type 23F, ATCC), or D1R and D2R synthesized peptides (for peptide epitope mapping) ( ) in carbonate/bicarbonate buffer pH 9.6.

Techniques: Transfection, Plasmid Preparation, MANN-WHITNEY, Stable Transfection, Produced, Control, Activation Assay

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A ) Human mAb 42.4.1 and mAb 3C3.1 reactivity with the human D1R by ELISA . ( B ) Human mAb 42.4.1 and mAb 3C3.1 stimulated cAMP induction in Tango D1- bla U2OS cells; dopamine was used at (0.001 M) as reference. Nonspecific mAb and mAb media controls were not quantifiable (NQ), as the ELISA values were below the standard curve. ( C ) Human mAbs 42.4.1 (red, ** P = 0.0067) and 3C3.1 (blue, **** P < 0.0001) induced dose-dependent D1R agonistic signaling in Tango D1- bla U2OS cells versus media control (nonlinear regression, F test). ( D ) Human mAb 3C3.1 treatment with dopamine enhanced Tango D1- bla U2OS cells’ dopamine dose-response curve significantly, compared with dopamine alone, as per nonlinear regression, F test. ( E ) PANDAS sera–mediated ( n = 5) D1R signaling of NS (nonsilencing control siRNA) or D1R siRNA–transfected D1R- bla U2OS. ( F ) PANDAS mAb–mediated signaling following D1R or NS siRNA transfection. In E and F , data represent the average of 3 technical replicates. * P < 0.05, ** P < 0.01 by Mann-Whitney U test.

Article Snippet: Briefly, Immunolon 4 microtiter plates (VWR) were coated overnight at 4°C with 10 μg/mL of antigens: D1R (Perkin-Elmer), D2R (Perkin-Elmer or in-house), GlcNAc conjugated to BSA (Vector Labs), HIB PRP (National Institute for Biological Standards and Controls, London, United Kingdom), pneumococcal capsular antigens ( Streptococcus pneumoniae type 23F, ATCC), or D1R and D2R synthesized peptides (for peptide epitope mapping) ( ) in carbonate/bicarbonate buffer pH 9.6.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Transfection, MANN-WHITNEY

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: Sequence alignment between the human dopamine D1R and D2R consensus sequences of similar epitopes. Identical residues are highlighted (gray). Residues with similar properties are in bold. Asterisks and/or lines indicate a single, fully conserved residue. A colon indicates conservation of similar properties. A period indicates conservation of weakly similar properties. Emboss pairwise alignments ( https://www.ebi.ac.uk/jdispatcher/psa/emboss_needle ) were used for 2 sequences and Clustal Omega ( https://www.ebi.ac.uk/jdispatcher/msa/clustalo ) for multiple sequence alignments.

Article Snippet: Briefly, Immunolon 4 microtiter plates (VWR) were coated overnight at 4°C with 10 μg/mL of antigens: D1R (Perkin-Elmer), D2R (Perkin-Elmer or in-house), GlcNAc conjugated to BSA (Vector Labs), HIB PRP (National Institute for Biological Standards and Controls, London, United Kingdom), pneumococcal capsular antigens ( Streptococcus pneumoniae type 23F, ATCC), or D1R and D2R synthesized peptides (for peptide epitope mapping) ( ) in carbonate/bicarbonate buffer pH 9.6.

Techniques: Sequencing, Residue

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A – J ) Violin plots depict AAb reactivity titrated from human sera against human D1R and D2R by ELISA from 4 cohorts. ( A and B ) In Cohort 1, significant differences in anti-D2R IgG levels were observed between SC patients ( n = 6) and age-matched healthy controls ( n = 18). ( C and D ) PANDAS patients with choreiform movements ( n = 11) showed significantly higher D1R and D2R IgG AAb levels compared with controls ( n = 18). ( E and F ) Cohort 2: PANDAS without choreiform movements ( n = 35) exhibited significantly elevated anti-D1R IgG levels compared with controls ( n = 18). ( G and H ) Cohort 3 with PANDAS/PANS ( n = 858) demonstrated elevated anti-D1R AAb titer versus controls ( n = 18). ( I and J ) Cohort 4: A second SC group (with acute SC) ( n = 14) showed elevated D1R and D2R titers compared with healthy controls ( n = 31). All groups were compared by the Mann-Whitney U test ( P < 0.05 considered significant).

Article Snippet: D1R-specific primers were generated against the D1R CDS sequence provided by Thermo Fisher Scientific and ordered from IDT. cDNA (10 ng) was amplified with D1R primers (0.5 μM) using the QuantiTect Transcription Kit (Qiagen).

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A and B ) In Cohort 1, ROC curves yield no predictive value for D1R titers and significance ( P < 0.05 considered significant) for D2R titers in SC. ( C and D ) PANDAS patients with choreiform movements in Cohort 1 exhibit significant values for both D1R and D2R titers. SEM is incorporated. ( E and F ) Cohort 2, PANDAS without choreiform movements, demonstrates highly significant for D1R titers, with no predictive value for D2R titers. ( G and H ) Cohort 3 (PANDAS/PANS) also demonstrated significant AUC for D1 titers, with no predictive AUC for D2 titers. ( I and J ) Cohort 4/acute SC for both D1R and D2R titers separating disease from controls. ROC curve analysis with 95% confidence intervals was calculated in Prism using the Wilson-Brown method.

Article Snippet: D1R-specific primers were generated against the D1R CDS sequence provided by Thermo Fisher Scientific and ordered from IDT. cDNA (10 ng) was amplified with D1R primers (0.5 μM) using the QuantiTect Transcription Kit (Qiagen).

Techniques:

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A ) Diagram illustrates the D1R primary sequence for synthetic peptides that reacted with PANDAS serum IgG versus control sera. ( B ) D1R peptide reactivity in SC ( n = 7, 8) and PANDAS ( n = 29–36, Cohorts 1 and 2) or control sera ( n = 8–9). One-way ANOVA with Tukey’s multiple-comparison test reveals * P < 0.05 for PANDAS (EL1a, EL2b, and EL3 epitopes) compared with control serum, and ** P < 0.01, *** P < 0.001 for PANDAS (NT, TM1, or EL1a epitopes) versus controls. ( C ) Linear map of D1R and corresponding amino acid residues. ( D ) Significant D2R immunoreactive epitopes mapped by SC sera. (Overlapping peptides in purple). ( E ) D2R peptide reactivity with control ( n = 11), SC ( n = 8), and PANDAS sera ( n = 34) from cohorts 1 and 2. One-way ANOVA with Tukey’s multiple-comparison test reveals greater reactivity for SC (* P < 0.05 NTa and NT1b, ** P < 0.01 for EL1) versus healthy controls and PANDAS NTb (* P < 0.005) versus controls. All graphs shown as mean ± SEM. ( F ) D2R mapped linear peptides with their corresponding amino acid residues.

Article Snippet: D1R-specific primers were generated against the D1R CDS sequence provided by Thermo Fisher Scientific and ordered from IDT. cDNA (10 ng) was amplified with D1R primers (0.5 μM) using the QuantiTect Transcription Kit (Qiagen).

Techniques: Sequencing, Control, Comparison

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A – C ) ELISA titers of anti–microbial polysaccharide antigen IgG antibodies in PANDA/PANS and control serum. ( A ) Elevated anti-GlcNAc titers in PANDAS/PANS ( n = 22) versus controls ( n = 14). Mann-Whitney U test. ( B and C ) AAb titers against pneumococcal polysaccharide 23F (PP-23F) or Haemophilus influenzae type b capsular polyribitol phosphate (HIB PRP) were not significantly elevated (NS). ( D and E ) Competitive inhibition ELISA of D1R ( D ) or D2R ( E ) with PANDAS serum IgG ( n = 7) preabsorbed with HIB-PRP, PP-23F, or GlcNAc at 500 μg/mL. GlcNAc and HIB PRP significantly inhibit PANDAS AAb IgG reactivity of D1R (** P < 0.005) and D2R (** P < 0.05). In D and E , analysis was by nonparametric Kruskal-Wallis test followed by Dunn’s multiple-comparison test.

Article Snippet: D1R-specific primers were generated against the D1R CDS sequence provided by Thermo Fisher Scientific and ordered from IDT. cDNA (10 ng) was amplified with D1R primers (0.5 μM) using the QuantiTect Transcription Kit (Qiagen).

Techniques: Enzyme-linked Immunosorbent Assay, Control, MANN-WHITNEY, Inhibition, Comparison

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A ) PANDAS IgG-secreting hybridoma of B4C immunoreactivity with the synthesized human D1R and the D1R extracellular loop 1 peptide (EL1a), but not ( B ) D2R or D2R peptides. ( C ) Cell-based assay of PANDAS mAb B4C immunostaining (in red) of CHO-K1 cells transfected with D1R or the empty vector control. B4C preadsorption with the D1R EL1a peptide abolished B4C immunoreactivity with D1R. Original magnification, ×20. ( D ) mAb B4C mediates D1R dose-dependent signaling. CHO-K1 cells were transfected with full-length human D1R or the empty vector (EV) control and were treated with serial dilutions of mAb B4C starting with 1 ng/mL. * P < 0.05 by paired, 2-tailed t test between the D1R-transfected cells and vehicle. ( E ) CHO-K1 cells transfected with either D1R or EV were treated with mAb B4C alongside serially diluted dopamine (red) or dopamine alone (black). Dopamine dose response of D1R-transfected cells treated with dopamine versus B4C and dopamine was analyzed by nonlinear regression, showing significance. *** P = 0.0001 by extra sum-of-squares F test ( F test). ( F ) CHO-K1 cells, transfected with D1R or EV, underwent treatment with serially diluted dopamine (black diamonds), mAb B4C alongside dopamine (red circles), or D1R-transfected cells with preadsorbed mAb B4C peptide EL1a (1 μg/mL) and dopamine (blue squares). Dose-response curves from the cAMP assay in D1R-transfected cells treated with mAb B4C and dopamine versus B4C and dopamine preadsorbed with D1R EL1a peptide. * P = 0.036, nonlinear regression, logEC 50 , F test.

Article Snippet: D1R-specific primers were generated against the D1R CDS sequence provided by Thermo Fisher Scientific and ordered from IDT. cDNA (10 ng) was amplified with D1R primers (0.5 μM) using the QuantiTect Transcription Kit (Qiagen).

Techniques: Synthesized, Cell Based Assay, Immunostaining, Transfection, Plasmid Preparation, Control, cAMP Assay

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A ) PANDAS serum–induced cellular cAMP concentrations from CHO-K1 cells transfected with either D1R cDNA or the empty vector (sera diluted 1:40). ** P < 0.01 by Mann-Whitney U test. ( B ) PANDAS sera stimulated cAMP levels in stably expressed D1R (Tango D1- bla U2OS cells). *** P < 0.001 by Mann-Whitney U test. ( C ) D1R percentage increase in β-arrestin recruitment to D1R produced by SC ( n = 6), PANDAS ( n = 9), and control ( n = 6) serum from Cohort 1. ** P < 0.005 controls vs. PANDAS by 1-way ANOVA with Dunn’s post hoc test. ( D ) β-Arrestin recruitment to D1R stimulated by diluted PANDAS ( n = 19) vs. controls ( n = 7) from Cohort 2. ** P < 0.005 by Mann-Whitney U test. β-Arrestin recruitment to D1R was assayed using the FRET-based TANGO GeneBLAzer GPCR assay system. Values are expressed as a percentage of the FRET signal in cells relative to vehicle. ( E ) Serum-mediated D1R signaling dose response (Tango GeneBLAzer GPCR Assay) showed a significant difference between PANDAS and healthy controls (mean nonlinear regression analysis, sum-of-squares F test). ( F ) D1R activation is shown by the dose-response curves of PANDAS sera added in combination with dopamine (red square or blue squares) compared to dopamine alone (depicted by black circles) in Tango D1- bla U2OS cells.

Article Snippet: D1R-specific primers were generated against the D1R CDS sequence provided by Thermo Fisher Scientific and ordered from IDT. cDNA (10 ng) was amplified with D1R primers (0.5 μM) using the QuantiTect Transcription Kit (Qiagen).

Techniques: Transfection, Plasmid Preparation, MANN-WHITNEY, Stable Transfection, Produced, Control, Activation Assay

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A ) Human mAb 42.4.1 and mAb 3C3.1 reactivity with the human D1R by ELISA . ( B ) Human mAb 42.4.1 and mAb 3C3.1 stimulated cAMP induction in Tango D1- bla U2OS cells; dopamine was used at (0.001 M) as reference. Nonspecific mAb and mAb media controls were not quantifiable (NQ), as the ELISA values were below the standard curve. ( C ) Human mAbs 42.4.1 (red, ** P = 0.0067) and 3C3.1 (blue, **** P < 0.0001) induced dose-dependent D1R agonistic signaling in Tango D1- bla U2OS cells versus media control (nonlinear regression, F test). ( D ) Human mAb 3C3.1 treatment with dopamine enhanced Tango D1- bla U2OS cells’ dopamine dose-response curve significantly, compared with dopamine alone, as per nonlinear regression, F test. ( E ) PANDAS sera–mediated ( n = 5) D1R signaling of NS (nonsilencing control siRNA) or D1R siRNA–transfected D1R- bla U2OS. ( F ) PANDAS mAb–mediated signaling following D1R or NS siRNA transfection. In E and F , data represent the average of 3 technical replicates. * P < 0.05, ** P < 0.01 by Mann-Whitney U test.

Article Snippet: D1R-specific primers were generated against the D1R CDS sequence provided by Thermo Fisher Scientific and ordered from IDT. cDNA (10 ng) was amplified with D1R primers (0.5 μM) using the QuantiTect Transcription Kit (Qiagen).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Transfection, MANN-WHITNEY

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: Sequence alignment between the human dopamine D1R and D2R consensus sequences of similar epitopes. Identical residues are highlighted (gray). Residues with similar properties are in bold. Asterisks and/or lines indicate a single, fully conserved residue. A colon indicates conservation of similar properties. A period indicates conservation of weakly similar properties. Emboss pairwise alignments ( https://www.ebi.ac.uk/jdispatcher/psa/emboss_needle ) were used for 2 sequences and Clustal Omega ( https://www.ebi.ac.uk/jdispatcher/msa/clustalo ) for multiple sequence alignments.

Article Snippet: D1R-specific primers were generated against the D1R CDS sequence provided by Thermo Fisher Scientific and ordered from IDT. cDNA (10 ng) was amplified with D1R primers (0.5 μM) using the QuantiTect Transcription Kit (Qiagen).

Techniques: Sequencing, Residue

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A – J ) Violin plots depict AAb reactivity titrated from human sera against human D1R and D2R by ELISA from 4 cohorts. ( A and B ) In Cohort 1, significant differences in anti-D2R IgG levels were observed between SC patients ( n = 6) and age-matched healthy controls ( n = 18). ( C and D ) PANDAS patients with choreiform movements ( n = 11) showed significantly higher D1R and D2R IgG AAb levels compared with controls ( n = 18). ( E and F ) Cohort 2: PANDAS without choreiform movements ( n = 35) exhibited significantly elevated anti-D1R IgG levels compared with controls ( n = 18). ( G and H ) Cohort 3 with PANDAS/PANS ( n = 858) demonstrated elevated anti-D1R AAb titer versus controls ( n = 18). ( I and J ) Cohort 4: A second SC group (with acute SC) ( n = 14) showed elevated D1R and D2R titers compared with healthy controls ( n = 31). All groups were compared by the Mann-Whitney U test ( P < 0.05 considered significant).

Article Snippet: D1R-specific primers were generated against the D1R CDS sequence provided by Thermo Fisher Scientific and ordered from IDT. cDNA (10 ng) was amplified with D1R primers (0.5 μM) using the QuantiTect Transcription Kit (Qiagen).

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A and B ) In Cohort 1, ROC curves yield no predictive value for D1R titers and significance ( P < 0.05 considered significant) for D2R titers in SC. ( C and D ) PANDAS patients with choreiform movements in Cohort 1 exhibit significant values for both D1R and D2R titers. SEM is incorporated. ( E and F ) Cohort 2, PANDAS without choreiform movements, demonstrates highly significant for D1R titers, with no predictive value for D2R titers. ( G and H ) Cohort 3 (PANDAS/PANS) also demonstrated significant AUC for D1 titers, with no predictive AUC for D2 titers. ( I and J ) Cohort 4/acute SC for both D1R and D2R titers separating disease from controls. ROC curve analysis with 95% confidence intervals was calculated in Prism using the Wilson-Brown method.

Article Snippet: D1R-specific primers were generated against the D1R CDS sequence provided by Thermo Fisher Scientific and ordered from IDT. cDNA (10 ng) was amplified with D1R primers (0.5 μM) using the QuantiTect Transcription Kit (Qiagen).

Techniques:

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A ) Diagram illustrates the D1R primary sequence for synthetic peptides that reacted with PANDAS serum IgG versus control sera. ( B ) D1R peptide reactivity in SC ( n = 7, 8) and PANDAS ( n = 29–36, Cohorts 1 and 2) or control sera ( n = 8–9). One-way ANOVA with Tukey’s multiple-comparison test reveals * P < 0.05 for PANDAS (EL1a, EL2b, and EL3 epitopes) compared with control serum, and ** P < 0.01, *** P < 0.001 for PANDAS (NT, TM1, or EL1a epitopes) versus controls. ( C ) Linear map of D1R and corresponding amino acid residues. ( D ) Significant D2R immunoreactive epitopes mapped by SC sera. (Overlapping peptides in purple). ( E ) D2R peptide reactivity with control ( n = 11), SC ( n = 8), and PANDAS sera ( n = 34) from cohorts 1 and 2. One-way ANOVA with Tukey’s multiple-comparison test reveals greater reactivity for SC (* P < 0.05 NTa and NT1b, ** P < 0.01 for EL1) versus healthy controls and PANDAS NTb (* P < 0.005) versus controls. All graphs shown as mean ± SEM. ( F ) D2R mapped linear peptides with their corresponding amino acid residues.

Article Snippet: D1R-specific primers were generated against the D1R CDS sequence provided by Thermo Fisher Scientific and ordered from IDT. cDNA (10 ng) was amplified with D1R primers (0.5 μM) using the QuantiTect Transcription Kit (Qiagen).

Techniques: Sequencing, Control, Comparison

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A – C ) ELISA titers of anti–microbial polysaccharide antigen IgG antibodies in PANDA/PANS and control serum. ( A ) Elevated anti-GlcNAc titers in PANDAS/PANS ( n = 22) versus controls ( n = 14). Mann-Whitney U test. ( B and C ) AAb titers against pneumococcal polysaccharide 23F (PP-23F) or Haemophilus influenzae type b capsular polyribitol phosphate (HIB PRP) were not significantly elevated (NS). ( D and E ) Competitive inhibition ELISA of D1R ( D ) or D2R ( E ) with PANDAS serum IgG ( n = 7) preabsorbed with HIB-PRP, PP-23F, or GlcNAc at 500 μg/mL. GlcNAc and HIB PRP significantly inhibit PANDAS AAb IgG reactivity of D1R (** P < 0.005) and D2R (** P < 0.05). In D and E , analysis was by nonparametric Kruskal-Wallis test followed by Dunn’s multiple-comparison test.

Article Snippet: D1R-specific primers were generated against the D1R CDS sequence provided by Thermo Fisher Scientific and ordered from IDT. cDNA (10 ng) was amplified with D1R primers (0.5 μM) using the QuantiTect Transcription Kit (Qiagen).

Techniques: Enzyme-linked Immunosorbent Assay, Control, MANN-WHITNEY, Inhibition, Comparison

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A ) PANDAS IgG-secreting hybridoma of B4C immunoreactivity with the synthesized human D1R and the D1R extracellular loop 1 peptide (EL1a), but not ( B ) D2R or D2R peptides. ( C ) Cell-based assay of PANDAS mAb B4C immunostaining (in red) of CHO-K1 cells transfected with D1R or the empty vector control. B4C preadsorption with the D1R EL1a peptide abolished B4C immunoreactivity with D1R. Original magnification, ×20. ( D ) mAb B4C mediates D1R dose-dependent signaling. CHO-K1 cells were transfected with full-length human D1R or the empty vector (EV) control and were treated with serial dilutions of mAb B4C starting with 1 ng/mL. * P < 0.05 by paired, 2-tailed t test between the D1R-transfected cells and vehicle. ( E ) CHO-K1 cells transfected with either D1R or EV were treated with mAb B4C alongside serially diluted dopamine (red) or dopamine alone (black). Dopamine dose response of D1R-transfected cells treated with dopamine versus B4C and dopamine was analyzed by nonlinear regression, showing significance. *** P = 0.0001 by extra sum-of-squares F test ( F test). ( F ) CHO-K1 cells, transfected with D1R or EV, underwent treatment with serially diluted dopamine (black diamonds), mAb B4C alongside dopamine (red circles), or D1R-transfected cells with preadsorbed mAb B4C peptide EL1a (1 μg/mL) and dopamine (blue squares). Dose-response curves from the cAMP assay in D1R-transfected cells treated with mAb B4C and dopamine versus B4C and dopamine preadsorbed with D1R EL1a peptide. * P = 0.036, nonlinear regression, logEC 50 , F test.

Article Snippet: D1R-specific primers were generated against the D1R CDS sequence provided by Thermo Fisher Scientific and ordered from IDT. cDNA (10 ng) was amplified with D1R primers (0.5 μM) using the QuantiTect Transcription Kit (Qiagen).

Techniques: Synthesized, Cell Based Assay, Immunostaining, Transfection, Plasmid Preparation, Control, cAMP Assay

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A ) PANDAS serum–induced cellular cAMP concentrations from CHO-K1 cells transfected with either D1R cDNA or the empty vector (sera diluted 1:40). ** P < 0.01 by Mann-Whitney U test. ( B ) PANDAS sera stimulated cAMP levels in stably expressed D1R (Tango D1- bla U2OS cells). *** P < 0.001 by Mann-Whitney U test. ( C ) D1R percentage increase in β-arrestin recruitment to D1R produced by SC ( n = 6), PANDAS ( n = 9), and control ( n = 6) serum from Cohort 1. ** P < 0.005 controls vs. PANDAS by 1-way ANOVA with Dunn’s post hoc test. ( D ) β-Arrestin recruitment to D1R stimulated by diluted PANDAS ( n = 19) vs. controls ( n = 7) from Cohort 2. ** P < 0.005 by Mann-Whitney U test. β-Arrestin recruitment to D1R was assayed using the FRET-based TANGO GeneBLAzer GPCR assay system. Values are expressed as a percentage of the FRET signal in cells relative to vehicle. ( E ) Serum-mediated D1R signaling dose response (Tango GeneBLAzer GPCR Assay) showed a significant difference between PANDAS and healthy controls (mean nonlinear regression analysis, sum-of-squares F test). ( F ) D1R activation is shown by the dose-response curves of PANDAS sera added in combination with dopamine (red square or blue squares) compared to dopamine alone (depicted by black circles) in Tango D1- bla U2OS cells.

Article Snippet: D1R-specific primers were generated against the D1R CDS sequence provided by Thermo Fisher Scientific and ordered from IDT. cDNA (10 ng) was amplified with D1R primers (0.5 μM) using the QuantiTect Transcription Kit (Qiagen).

Techniques: Transfection, Plasmid Preparation, MANN-WHITNEY, Stable Transfection, Produced, Control, Activation Assay

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A ) Human mAb 42.4.1 and mAb 3C3.1 reactivity with the human D1R by ELISA . ( B ) Human mAb 42.4.1 and mAb 3C3.1 stimulated cAMP induction in Tango D1- bla U2OS cells; dopamine was used at (0.001 M) as reference. Nonspecific mAb and mAb media controls were not quantifiable (NQ), as the ELISA values were below the standard curve. ( C ) Human mAbs 42.4.1 (red, ** P = 0.0067) and 3C3.1 (blue, **** P < 0.0001) induced dose-dependent D1R agonistic signaling in Tango D1- bla U2OS cells versus media control (nonlinear regression, F test). ( D ) Human mAb 3C3.1 treatment with dopamine enhanced Tango D1- bla U2OS cells’ dopamine dose-response curve significantly, compared with dopamine alone, as per nonlinear regression, F test. ( E ) PANDAS sera–mediated ( n = 5) D1R signaling of NS (nonsilencing control siRNA) or D1R siRNA–transfected D1R- bla U2OS. ( F ) PANDAS mAb–mediated signaling following D1R or NS siRNA transfection. In E and F , data represent the average of 3 technical replicates. * P < 0.05, ** P < 0.01 by Mann-Whitney U test.

Article Snippet: D1R-specific primers were generated against the D1R CDS sequence provided by Thermo Fisher Scientific and ordered from IDT. cDNA (10 ng) was amplified with D1R primers (0.5 μM) using the QuantiTect Transcription Kit (Qiagen).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Transfection, MANN-WHITNEY

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: Sequence alignment between the human dopamine D1R and D2R consensus sequences of similar epitopes. Identical residues are highlighted (gray). Residues with similar properties are in bold. Asterisks and/or lines indicate a single, fully conserved residue. A colon indicates conservation of similar properties. A period indicates conservation of weakly similar properties. Emboss pairwise alignments ( https://www.ebi.ac.uk/jdispatcher/psa/emboss_needle ) were used for 2 sequences and Clustal Omega ( https://www.ebi.ac.uk/jdispatcher/msa/clustalo ) for multiple sequence alignments.

Article Snippet: D1R-specific primers were generated against the D1R CDS sequence provided by Thermo Fisher Scientific and ordered from IDT. cDNA (10 ng) was amplified with D1R primers (0.5 μM) using the QuantiTect Transcription Kit (Qiagen).

Techniques: Sequencing, Residue

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A – J ) Violin plots depict AAb reactivity titrated from human sera against human D1R and D2R by ELISA from 4 cohorts. ( A and B ) In Cohort 1, significant differences in anti-D2R IgG levels were observed between SC patients ( n = 6) and age-matched healthy controls ( n = 18). ( C and D ) PANDAS patients with choreiform movements ( n = 11) showed significantly higher D1R and D2R IgG AAb levels compared with controls ( n = 18). ( E and F ) Cohort 2: PANDAS without choreiform movements ( n = 35) exhibited significantly elevated anti-D1R IgG levels compared with controls ( n = 18). ( G and H ) Cohort 3 with PANDAS/PANS ( n = 858) demonstrated elevated anti-D1R AAb titer versus controls ( n = 18). ( I and J ) Cohort 4: A second SC group (with acute SC) ( n = 14) showed elevated D1R and D2R titers compared with healthy controls ( n = 31). All groups were compared by the Mann-Whitney U test ( P < 0.05 considered significant).

Article Snippet: D1R GPCR signaling assays were performed with Tango D1- bla USOS cells (Invitrogen).

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A and B ) In Cohort 1, ROC curves yield no predictive value for D1R titers and significance ( P < 0.05 considered significant) for D2R titers in SC. ( C and D ) PANDAS patients with choreiform movements in Cohort 1 exhibit significant values for both D1R and D2R titers. SEM is incorporated. ( E and F ) Cohort 2, PANDAS without choreiform movements, demonstrates highly significant for D1R titers, with no predictive value for D2R titers. ( G and H ) Cohort 3 (PANDAS/PANS) also demonstrated significant AUC for D1 titers, with no predictive AUC for D2 titers. ( I and J ) Cohort 4/acute SC for both D1R and D2R titers separating disease from controls. ROC curve analysis with 95% confidence intervals was calculated in Prism using the Wilson-Brown method.

Article Snippet: D1R GPCR signaling assays were performed with Tango D1- bla USOS cells (Invitrogen).

Techniques:

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A ) Diagram illustrates the D1R primary sequence for synthetic peptides that reacted with PANDAS serum IgG versus control sera. ( B ) D1R peptide reactivity in SC ( n = 7, 8) and PANDAS ( n = 29–36, Cohorts 1 and 2) or control sera ( n = 8–9). One-way ANOVA with Tukey’s multiple-comparison test reveals * P < 0.05 for PANDAS (EL1a, EL2b, and EL3 epitopes) compared with control serum, and ** P < 0.01, *** P < 0.001 for PANDAS (NT, TM1, or EL1a epitopes) versus controls. ( C ) Linear map of D1R and corresponding amino acid residues. ( D ) Significant D2R immunoreactive epitopes mapped by SC sera. (Overlapping peptides in purple). ( E ) D2R peptide reactivity with control ( n = 11), SC ( n = 8), and PANDAS sera ( n = 34) from cohorts 1 and 2. One-way ANOVA with Tukey’s multiple-comparison test reveals greater reactivity for SC (* P < 0.05 NTa and NT1b, ** P < 0.01 for EL1) versus healthy controls and PANDAS NTb (* P < 0.005) versus controls. All graphs shown as mean ± SEM. ( F ) D2R mapped linear peptides with their corresponding amino acid residues.

Article Snippet: D1R GPCR signaling assays were performed with Tango D1- bla USOS cells (Invitrogen).

Techniques: Sequencing, Control, Comparison

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A – C ) ELISA titers of anti–microbial polysaccharide antigen IgG antibodies in PANDA/PANS and control serum. ( A ) Elevated anti-GlcNAc titers in PANDAS/PANS ( n = 22) versus controls ( n = 14). Mann-Whitney U test. ( B and C ) AAb titers against pneumococcal polysaccharide 23F (PP-23F) or Haemophilus influenzae type b capsular polyribitol phosphate (HIB PRP) were not significantly elevated (NS). ( D and E ) Competitive inhibition ELISA of D1R ( D ) or D2R ( E ) with PANDAS serum IgG ( n = 7) preabsorbed with HIB-PRP, PP-23F, or GlcNAc at 500 μg/mL. GlcNAc and HIB PRP significantly inhibit PANDAS AAb IgG reactivity of D1R (** P < 0.005) and D2R (** P < 0.05). In D and E , analysis was by nonparametric Kruskal-Wallis test followed by Dunn’s multiple-comparison test.

Article Snippet: D1R GPCR signaling assays were performed with Tango D1- bla USOS cells (Invitrogen).

Techniques: Enzyme-linked Immunosorbent Assay, Control, MANN-WHITNEY, Inhibition, Comparison

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A ) PANDAS IgG-secreting hybridoma of B4C immunoreactivity with the synthesized human D1R and the D1R extracellular loop 1 peptide (EL1a), but not ( B ) D2R or D2R peptides. ( C ) Cell-based assay of PANDAS mAb B4C immunostaining (in red) of CHO-K1 cells transfected with D1R or the empty vector control. B4C preadsorption with the D1R EL1a peptide abolished B4C immunoreactivity with D1R. Original magnification, ×20. ( D ) mAb B4C mediates D1R dose-dependent signaling. CHO-K1 cells were transfected with full-length human D1R or the empty vector (EV) control and were treated with serial dilutions of mAb B4C starting with 1 ng/mL. * P < 0.05 by paired, 2-tailed t test between the D1R-transfected cells and vehicle. ( E ) CHO-K1 cells transfected with either D1R or EV were treated with mAb B4C alongside serially diluted dopamine (red) or dopamine alone (black). Dopamine dose response of D1R-transfected cells treated with dopamine versus B4C and dopamine was analyzed by nonlinear regression, showing significance. *** P = 0.0001 by extra sum-of-squares F test ( F test). ( F ) CHO-K1 cells, transfected with D1R or EV, underwent treatment with serially diluted dopamine (black diamonds), mAb B4C alongside dopamine (red circles), or D1R-transfected cells with preadsorbed mAb B4C peptide EL1a (1 μg/mL) and dopamine (blue squares). Dose-response curves from the cAMP assay in D1R-transfected cells treated with mAb B4C and dopamine versus B4C and dopamine preadsorbed with D1R EL1a peptide. * P = 0.036, nonlinear regression, logEC 50 , F test.

Article Snippet: D1R GPCR signaling assays were performed with Tango D1- bla USOS cells (Invitrogen).

Techniques: Synthesized, Cell Based Assay, Immunostaining, Transfection, Plasmid Preparation, Control, cAMP Assay

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A ) PANDAS serum–induced cellular cAMP concentrations from CHO-K1 cells transfected with either D1R cDNA or the empty vector (sera diluted 1:40). ** P < 0.01 by Mann-Whitney U test. ( B ) PANDAS sera stimulated cAMP levels in stably expressed D1R (Tango D1- bla U2OS cells). *** P < 0.001 by Mann-Whitney U test. ( C ) D1R percentage increase in β-arrestin recruitment to D1R produced by SC ( n = 6), PANDAS ( n = 9), and control ( n = 6) serum from Cohort 1. ** P < 0.005 controls vs. PANDAS by 1-way ANOVA with Dunn’s post hoc test. ( D ) β-Arrestin recruitment to D1R stimulated by diluted PANDAS ( n = 19) vs. controls ( n = 7) from Cohort 2. ** P < 0.005 by Mann-Whitney U test. β-Arrestin recruitment to D1R was assayed using the FRET-based TANGO GeneBLAzer GPCR assay system. Values are expressed as a percentage of the FRET signal in cells relative to vehicle. ( E ) Serum-mediated D1R signaling dose response (Tango GeneBLAzer GPCR Assay) showed a significant difference between PANDAS and healthy controls (mean nonlinear regression analysis, sum-of-squares F test). ( F ) D1R activation is shown by the dose-response curves of PANDAS sera added in combination with dopamine (red square or blue squares) compared to dopamine alone (depicted by black circles) in Tango D1- bla U2OS cells.

Article Snippet: D1R GPCR signaling assays were performed with Tango D1- bla USOS cells (Invitrogen).

Techniques: Transfection, Plasmid Preparation, MANN-WHITNEY, Stable Transfection, Produced, Control, Activation Assay

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: ( A ) Human mAb 42.4.1 and mAb 3C3.1 reactivity with the human D1R by ELISA . ( B ) Human mAb 42.4.1 and mAb 3C3.1 stimulated cAMP induction in Tango D1- bla U2OS cells; dopamine was used at (0.001 M) as reference. Nonspecific mAb and mAb media controls were not quantifiable (NQ), as the ELISA values were below the standard curve. ( C ) Human mAbs 42.4.1 (red, ** P = 0.0067) and 3C3.1 (blue, **** P < 0.0001) induced dose-dependent D1R agonistic signaling in Tango D1- bla U2OS cells versus media control (nonlinear regression, F test). ( D ) Human mAb 3C3.1 treatment with dopamine enhanced Tango D1- bla U2OS cells’ dopamine dose-response curve significantly, compared with dopamine alone, as per nonlinear regression, F test. ( E ) PANDAS sera–mediated ( n = 5) D1R signaling of NS (nonsilencing control siRNA) or D1R siRNA–transfected D1R- bla U2OS. ( F ) PANDAS mAb–mediated signaling following D1R or NS siRNA transfection. In E and F , data represent the average of 3 technical replicates. * P < 0.05, ** P < 0.01 by Mann-Whitney U test.

Article Snippet: D1R GPCR signaling assays were performed with Tango D1- bla USOS cells (Invitrogen).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Transfection, MANN-WHITNEY

Journal: JCI Insight

Article Title: Dopamine receptor autoantibody signaling in infectious sequelae differentiates movement versus neuropsychiatric disorders

doi: 10.1172/jci.insight.164762

Figure Lengend Snippet: Sequence alignment between the human dopamine D1R and D2R consensus sequences of similar epitopes. Identical residues are highlighted (gray). Residues with similar properties are in bold. Asterisks and/or lines indicate a single, fully conserved residue. A colon indicates conservation of similar properties. A period indicates conservation of weakly similar properties. Emboss pairwise alignments ( https://www.ebi.ac.uk/jdispatcher/psa/emboss_needle ) were used for 2 sequences and Clustal Omega ( https://www.ebi.ac.uk/jdispatcher/msa/clustalo ) for multiple sequence alignments.

Article Snippet: D1R GPCR signaling assays were performed with Tango D1- bla USOS cells (Invitrogen).

Techniques: Sequencing, Residue