Journal: Oncology Reports

Article Title: Kevetrin induces apoptosis in TP53 wild-type and mutant acute myeloid leukemia cells

doi: 10.3892/or.2020.7730

Figure Lengend Snippet: Effect of continuous kevetrin treatment on viability and apoptosis induction in AML cell lines and primary samples. (A and B) Viability of TP53 -wt OCI-AML3 and MOLM-13 and TP53 -mut KASUMI-1 and NOMO-1 cell lines treated with different concentrations of kevetrin (85, 170 and 340 µM) for 24 and 48 h. (C and D) Quantification of Annexin V + cells in AML cell lines treated with kevetrin at different concentrations (85, 170 and 340 µM) for 24 and 48 h. (E and F) Cell cycle analysis of cell lines treated with 340 µM kevetrin for 24 and 48 h. Values represent the mean ± standard deviation of three biological replicates. (G) Cell viability and (H) percentage of Annexin V + cells in bone marrow mononuclear cells from AML patients exposed to increasing concentrations of kevetrin. (I) Percentage of AML blasts relative to control. Values represent the mean ± standard deviation. Symbols indicate samples from AML patients (no. 01: TP53 -mut AML; nos. 02, 03 and 04: TP53 -wt AML). (J) Percentage of Annexin V − cells in AML blasts, monocytes and lymphocytes. Blasts were defined as CD33 + CD14 − cells in the CD45/side scatter ‘blast gate’, monocytes as CD33 + CD14 + , B lymphocytes as CD19 + and T lymphocytes as CD3 + . *P<0.05, **P<0.01, ***P<0.001. AML, acute myeloid leukemia; KEV, kevetrin; CTRL, control; wt, wild-type.

Article Snippet: In primary samples, Annexin V staining was combined with surface markers using the following antibodies: CD45-APC Vio770 (cat. no. 130-110-635), CD33-APC (cat. no. 130-111-020), CD14-PerCP Vio 700 (cat. no. 130-110-523), CD3-PE (cat. no. 130-113-139) (all from Miltenyi Biotec GmbH, dilution 1:50) and CD19-PE Cy7 (cat. no. 302216, 1:10, BioLegend, Inc.).

Techniques: Cell Cycle Assay, Standard Deviation, Control

Journal: The Journal of Clinical Investigation

Article Title: CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen

doi: 10.1172/JCI125456

Figure Lengend Snippet: (A) Schematic representation of an antigenic liposome (TNP-LP, left) or an antigenic liposome displaying human CD33 ligands (TNP-LP-CD33L, right). (B) Antibody staining of various Siglecs (Sig-) on LAD2 cells analyzed by flow cytometry. (C) Flow cytometric analysis of binding of fluorescent liposomes with or without CD33L (20 μM) to LAD2 cells pretreated with isotype control or anti-CD33 (clone WM53). (D) Calcium flux of LAD2 cells induced by addition (arrow) of TNP-LP or TNP-LP-CD33 (2.5 μM) or PBS (1 μl). Graph shows quantification of the AUC of calcium flux induced by 2.5 μM TNP-LP or TNP-LP-CD33L. Results were combined from 2 independent experiments. (E) Degranulation induced by TNP-LP or TNP-LP-CD33L as measured by the percentage of β-hex release (n = 3 per condition; values are plotted as the mean ± SD). (F) Degranulation induced by TNP-LP (30 μM), TNP-LP-CD33L (30 μM), or a mixture of TNP-LP and LP-CD33L (30 μM each). (G) Degranulation induced by TNP-LP or TNP-LP-CD33L (30 μM) in the presence of LP-CD33L (10 μM). Control cells received buffer only. (H) Degranulation induced by TNP-LP or TNP-LP-CD33L (30 μM) in the presence of isotype or anti-CD33 (clone WM53, 1 μg/ml). (I) Degranulation induced by Ah2-LP or Ah2-LP-CD33L (30 μM), with final Ah2 at 750 ng/ml using LAD2 cells sensitized with atopic plasma reactive to peanut (PlasmaLab). (J) Degranulation induced by OVA-LP or OVA-LP-CD33L (30 μM), with the final OVA dose at 1.5 μg/ml using LAD2 cells sensitized with human anti–OVA-IgE. Results in E–J are representative of 3 independent experiments. ***P < 0.001 and ****P < 0.0001, by 2-tailed Student’s t test (D and E) and 1-way ANOVA followed by Tukey’s test (F–J). α, anti; Max, maximum.

Article Snippet: Rosa26-Stop fl/fl -CD33 mice were generated by subcloning cDNA encoding full-length CD33 (OriGene, catalog SC122608, sequence identical to that of GenBank {"type":"entrez-nucleotide","attrs":{"text":"BC028152.1","term_id":"20381381"}} BC028152.1 ) into the AscI site of a CTV targeting vector (Addgene, plasmid 15912).

Techniques: Staining, Flow Cytometry, Binding Assay, Liposomes, Control, Clinical Proteomics

Journal: The Journal of Clinical Investigation

Article Title: CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen

doi: 10.1172/JCI125456

Figure Lengend Snippet: (A) Flow cytometric analysis of GFP expression on representative peritoneal mast cells harvested from C57BL/6J, control-Tg (Mcpt5-Cre–Rosa26-Stopfl/fl-CD33+), and CD33-Tg (Mcpt5-Cre+/–Rosa26-Stopfl/fl-CD33+) mice. Mast cells were defined as PI–CD45+c-Kit+. Baseline GFP signal was determined by mast cells from C57BL/6J mice. (B) Quantification of the percentage of GFP+ peritoneal mast cells from mice of the 3 genotypes. Tg mice bearing 1 or 2 copies of CD33 were used. Both male and female mice 8 weeks or older were analyzed, with no difference observed. (C) Numbers of peritoneal mast cells from the same mice of the 3 genotypes as in B. (A–C) Results were compiled from 6 experiments. (D) Staining of peritoneal cells harvested from control-Tg or CD33-Tg mice with anti-CD33 (clone WM53) or isotype control, as analyzed by flow cytometry. (E) Binding of fluorescent liposome, with or without CD33L (20 μM), to peritoneal mast cells (c-Kit+FcεRI+CD45+). (F) Degranulation of CD33+ BMMCs induced by TNP-LP or TNP-LP-CD33L. (G) Cytokine induction of CD33+ BMMCs following treatment with TNP-LP (40 μM), TNP-LP-CD33L (40 μM), LP-CD33L (40 μM), or a mixture of TNP-LP and LP-CD33L (40 μM each). Supernatant from the unstimulated cells was subtracted as a background. (H) Degranulation of CD33+ BMMCs induced by TNP-LP or TNP-LP-CD33L (30 μM) in the presence of anti-CD33 (2 μg/ml). (I and J) Cytokine production of CD33+ BMMCs induced by TNP-LP or TNP-LP-CD33L (40 μM) in the presence of anti-CD33 (10 μg/ml). Supernatant from untreated cells was subtracted as a background. Results shown are representative of 3 (D–G) or 2 (H–J) independent experiments. (F–J) Values are plotted as the mean ± SD (n = 3 per condition). ***P < 0.001 and ****P < 0.0001, by 2-tailed Student’s t test (F) and 1-way ANOVA followed by Tukey’s test (C and G–J).

Article Snippet: Rosa26-Stop fl/fl -CD33 mice were generated by subcloning cDNA encoding full-length CD33 (OriGene, catalog SC122608, sequence identical to that of GenBank {"type":"entrez-nucleotide","attrs":{"text":"BC028152.1","term_id":"20381381"}} BC028152.1 ) into the AscI site of a CTV targeting vector (Addgene, plasmid 15912).

Techniques: Expressing, Control, Staining, Flow Cytometry, Binding Assay

Journal: The Journal of Clinical Investigation

Article Title: CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen

doi: 10.1172/JCI125456

Figure Lengend Snippet: (A) Phosphorylation of Syk, PLCγ1, MEK, and ERK in LAD2 cells after a 3-, 10-, or 30-minute stimulation using TNP-LP or TNP-LP-CD33L (2 μM), as evaluated by Western blotting. (B) Phosphorylation of Syk and ERK in LAD2 cells after a 10-minute stimulation using TNP-LP, TNP-LP-CD33L, or LP-CD33L (2 μM), as evaluated by Western blotting. (C) Phosphorylation of Syk, PLCγ1, PLCγ2, JNK, AKT, and ERK in CD33+ BMMCs cells after a 10-minute stimulation with TNP-LP, TNP-LP-CD33L, or LP-CD33L (2 μM), as evaluated by Western blotting. (A–C) Total Syk and ERK were used as loading controls. (D–F) Proposed mechanisms of IgE/FcεRI signaling induced by antigenic liposomes and recruitment of CD33 by CD33L. (D) TNP-LP stabilizes the anti–TNP-IgE–FcεRI complex in lipid rafts with Src kinases that initiate the FcεRI signaling cascade. We propose that CD33 has no basal impact on signaling, because it is not constitutively localized in the same microdomain with FcεRI. (E) TNP-LP-CD33L recruits CD33 to the anti–TNP-IgE–FcεRI immunological synapse. Our results suggest that the cytoplasmic ITIMs of CD33 were phosphorylated by Src kinases and then recruited tyrosine phosphatases such as Shp-1, which dephosphorylated Syk, and other kinases. (F) Proposed model showing that monoclonal anti-CD33 antibodies (or LP-CD33L) block recruitment of CD33 to the IgE-FcεRI complex and enable mast cell degranulation induced by TNP-LP-CD33L.

Article Snippet: Rosa26-Stop fl/fl -CD33 mice were generated by subcloning cDNA encoding full-length CD33 (OriGene, catalog SC122608, sequence identical to that of GenBank {"type":"entrez-nucleotide","attrs":{"text":"BC028152.1","term_id":"20381381"}} BC028152.1 ) into the AscI site of a CTV targeting vector (Addgene, plasmid 15912).

Techniques: Phospho-proteomics, Western Blot, Liposomes, Blocking Assay

Journal: The Journal of Clinical Investigation

Article Title: CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen

doi: 10.1172/JCI125456

Figure Lengend Snippet: Display of CD33L on antigenic liposomes suppresses PCA and PSA in CD33-Tg mice (Mcpt5-Cre+/–Rosa26-Stopfl/fl-CD33+), but not in control-Tg mice (Mcpt5-Cre– Rosa26-Stopfl/fl-CD33+). Mice bearing 1 or 2 copies of the CD33 transgene were used. In I, Mcpt5-Cre+/– mice expressing human CD33 (CD33-Tg) were crossed with Ptpn6fl/fl mice to yield mice with mast cells expressing CD33 and no Shp-1 (CD33-Tg/Shp-1–KO). (A) Injection scheme for the PCA model. The genotypes of the mice were determined by PCR after the experiments. (B) Representative images of vascular leakage induced by TNP-LP or TNP-LP-CD33L (50 μg) in control-Tg mice. (C) Quantification of local mast cell activation (absorbance at 650 nm) induced by TNP-LP (50 μg, n = 14) or TNP-LP-CD33L (50 μg, n = 28) in control-Tg mice. (D) Representative images of vascular leakage induced by TNP-LP or TNP-LP-CD33L (50 μg) in CD33-Tg mice. (E) Quantification of local mast cell activation (absorbance at 650 nm) induced by TNP-LP (50 μg, n = 21) or TNP-LP-CD33L (50 μg, n =27) in CD33-Tg mice. (F) Injection scheme for the PSA model. (G–I) Decrease in rectal temperature induced by TNP-LP or TNP-LP-CD33L (150 μg) in control-Tg mice (G), CD33-Tg mice (H), and CD33-Tg mice lacking Shp-1 (I) that were sensitized with 10 μg anti–TNP-IgE. (G–I) Values are plotted as the mean ± SEM at the indicated time points. Data are from 1 experiment (G and I) or were compiled from 3 (H) or 9 sets of experiments (C and E). ***P < 0.001 and ****P < 0.0001, by 1-way ANOVA followed by Tukey’s test (C and E), repeated-measures (RM) 2-way ANOVA (G and H), and RM 2-way ANOVA followed by Tukey’s test (I).

Article Snippet: Rosa26-Stop fl/fl -CD33 mice were generated by subcloning cDNA encoding full-length CD33 (OriGene, catalog SC122608, sequence identical to that of GenBank {"type":"entrez-nucleotide","attrs":{"text":"BC028152.1","term_id":"20381381"}} BC028152.1 ) into the AscI site of a CTV targeting vector (Addgene, plasmid 15912).

Techniques: Liposomes, Control, Expressing, Injection, Activation Assay

Journal: The Journal of Clinical Investigation

Article Title: CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen

doi: 10.1172/JCI125456

Figure Lengend Snippet: (A) Injection scheme for desensitization to TNP. CD33-Tg mice were used in the TNP-LP-CD33L–treated group (red). Both CD33-Tg and control-Tg mice were used in the 2 untreated groups (black, gray). (B) Changes in rectal temperature induced by treatment or the challenges indicated in A. (C) Injection scheme to determine antigen specificity of desensitization. CD33-Tg mice were used in the OVA-LP-CD33L–treated group (red circles and squares). Both CD33-Tg and control-Tg mice were used in the untreated group (gray circles and squares). (D) Rectal temperature induced by the treatment or challenge illustrated in C. (B and D) Values are plotted as the mean ± SEM. (E) Injection scheme used to evaluate the impact of TNP-LP-CD33L on mast cell frequency and anti–TNP-IgE on mast cells. Control mice received 200 μl PBS. (F) Frequencies of mast cells from peritoneal fluid from mice treated in E. Mast cell frequencies were determined by c-Kit+CD45+PI– cells. (G) In vitro binding of fluorescent TNP-LP (20 μM) to peritoneal mast cells harvested from mice treated as illustrated in C. (H) MFI of fluorescent TNP-LP binding to peritoneal mast cells quantified in G. The background was determined using untreated cells from a naive mouse. (I) Serum anti–TNP-IgE quantified prior to and 6 hours and 24 hours after treatment with TNP-LP-CD33L (450 μg) using CD33-Tg mice sensitized with 10 μg anti–TNP-IgE. Control mice received 200 μl PBS. Data in B were compiled from 2 experiments. Data are representative of 2 (F–H) or 3 (I) independent experiments. **P < 0.01, ***P < 0.001, and ****P < 0.0001, by RM 2-way ANOVA (B), RM 2-way ANOVA followed by Tukey’s test (D), and unpaired, 2-tailed Student’s t test (F–I).

Article Snippet: Rosa26-Stop fl/fl -CD33 mice were generated by subcloning cDNA encoding full-length CD33 (OriGene, catalog SC122608, sequence identical to that of GenBank {"type":"entrez-nucleotide","attrs":{"text":"BC028152.1","term_id":"20381381"}} BC028152.1 ) into the AscI site of a CTV targeting vector (Addgene, plasmid 15912).

Techniques: Injection, Control, In Vitro, Binding Assay

Journal: The Journal of Clinical Investigation

Article Title: CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen

doi: 10.1172/JCI125456

Figure Lengend Snippet: (A) Flow cytometric analysis of mast cells isolated from discarded human skin (c-KithiFcεRI+ gated on PI–CD45+CD3–CD19–CD56– cells) (left) and overlay of isotype control and anti-CD33 staining of gated mast cells (right). (B) MFI of antibody staining of Siglecs on mast cells isolated from skin that was discarded following surgical procedures (n = 1 to 10 donors). (C) Time course of the percentage of bronchoconstriction of hPCLSs. Lung slices were sensitized with human IgE (4 mg/ml, gray) or anti–TNP-IgE (10 μg/ml, black and red) with recombinant human SCF (200 ng/ml, R&D Systems) overnight. Slices were challenged with anti–human IgE (20 μg/ml, gray), TNP-LP (50 μM, black), or TNP-LP-CD33L (50 μM, red) over a 10-minute period. The airway luminal area over time was compared with the baseline luminal area and expressed as the percentage of bronchoconstriction. Values represent the mean ± SEM. (D) AUC induced by the indicated treatments. AUC values below 0 are plotted as 0. (E) Percentage of bronchoconstriction induced by the indicated agents at 10 minutes. (F) Following stimulation with the indicated reagents, the percentage of bronchoconstriction induced by CCh (0.1 mM) was measured. *P < 0.05 and **P < 0.01, by 1-way ANOVA followed by Tukey’s test (D–F). Gating of skin mast cells in A is representative of the 10 donors. Data in C–F were compiled from 5 or 9 lung slices from 2 donors.

Article Snippet: Rosa26-Stop fl/fl -CD33 mice were generated by subcloning cDNA encoding full-length CD33 (OriGene, catalog SC122608, sequence identical to that of GenBank {"type":"entrez-nucleotide","attrs":{"text":"BC028152.1","term_id":"20381381"}} BC028152.1 ) into the AscI site of a CTV targeting vector (Addgene, plasmid 15912).

Techniques: Isolation, Control, Staining, Recombinant

Journal: Microbiology Spectrum

Article Title: O -Acetylation of Capsular Polysialic Acid Enables Escherichia coli K1 Escaping from Siglec-Mediated Innate Immunity and Lysosomal Degradation of E. coli -Containing Vacuoles in Macrophage-Like Cells

doi: 10.1128/spectrum.00399-21

Figure Lengend Snippet: The binding of E. coli K1 strains to Siglecs. The human CD33 rSiglec-Fc chimeras (i.e., rhSiglec-5/7/11/14-Fc) were separately plated on a protein A-coated well and incubated with FITC-labeled E. coli strains WT, Δ neuD , and NeuO + , respectively. Bindings between bacteria and Siglec were examined and quantified by fluorescence intensity. Each experiment was carried out at three separate times, in triplicate. Data are shown as mean ± SD and were analyzed by a two-tailed unpaired Student's t test. * , P < 0.05; * * , P < 0.005; ** * , P < 0.0005.

Article Snippet: Human CD33 rSiglec-Fc chimeras (R & D Systems) were added to wells at 0.5 mg/mL and adhered at 4°C overnight.

Techniques: Binding Assay, Incubation, Labeling, Bacteria, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Rational design of oxidation-resistant antibodies through local electrostatic modulation

doi: 10.1101/2025.06.29.662139

Figure Lengend Snippet: a) Cryo-EM structure of 15G15.3 Fab (gray) bound to CD33 (violet), showing Trp96 forming key interactions with Lys52 (CD33) and Asp101 (Fab). Trp96 oxidizes at 97% under AAPH stress; W96F mutation abolishes binding ( > 1000-fold loss). b) Electrostatic potential of the lead candidate, with 12 mutated residues shown as pink spheres. c) Scatter plot of Trp oxidation vs. Epot for the lead and 13 variants; point color reflects relative KD. d) Summary of 13 engineered variants. S11 and S13 show improved oxidation resistance with preserved binding.

Article Snippet: Briefly, each antibody variant was captured by Protein A sensor chip (Series S) on the different flow cell to achieve approximately 150 response units (RU), followed by the injection of fivefold serial dilutions of human CD33 protein (R&D Systems; 0.16 nM to 100 nM) in HBS-EP buffer.

Techniques: Cryo-EM Sample Prep, Mutagenesis, Binding Assay

Journal: bioRxiv

Article Title: CD33-CD45 Interaction Reveals a Mechanistic Link to Alzheimer’s Disease Susceptibility

doi: 10.1101/2025.07.28.667311

Figure Lengend Snippet: a. Proteins that were significantly enriched as CD33 binding proteins, defined as associated with CD33 after THP-1 cells were treated with Ac 4 ManNDAz and treated with UV light compared to THP-1 cells treated with DMSO alone and UV light. b. Proteins that were not enriched in the CD33 immunoprecipitation without Ac 4 ManNDAz compared to IgG control immunoprecipitation, demonstrating their specificity for the sialic acid binding domain. These proteins were enriched after the Ac 4 ManNDAz plus UV light treatment compared to the Ac 4 ManNDAz treatment without UV light crosslinking. Error bars denote the standard deviation between technical replicates.

Article Snippet: A CD33 encoding lentivirus was obtained from Origene (RC207023L1) and various CD33 mutants were made (R119K, Y340F, Y358F, and Y340F/Y358F) using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs).

Techniques: Binding Assay, Immunoprecipitation, Control, Standard Deviation

Journal: bioRxiv

Article Title: CD33-CD45 Interaction Reveals a Mechanistic Link to Alzheimer’s Disease Susceptibility

doi: 10.1101/2025.07.28.667311

Figure Lengend Snippet: a. The mass spectrometry findings of the CD33 and CD45 interaction were validated in THP-1 cells using the proximity ligation assay (PLA). Knock-out (KO) of CD33 from the THP-1 cell line led to the elimination of the interaction between CD33 and CD45, as detected by PLA. b. Combining THP-1 cells with either CD33 KO or CD45 KO via CRISPR/Cas9 leads to a reduction in the interaction between CD33 and CD45, suggesting that the interaction is occurring in cis. c. In a solid phase binding assay, recombinant human CD33 bound to immobilized recombinant human CD45 in a dose dependent manner. Increasing concentrations of recombinant hCD33-Fc (0.005µg/mL-5µg/mL) were incubated overnight at 4°C to recombinant hCD45 coated plates and an anti-human Fc antibody was used to detect recombinant bound hCD33. Each dot represents the average of three independent experiments. Two-way ANOVA with Tukey’s multiple comparison test, p*<0.05, p**<0.01, p***<0.001. Asterisks denote comparison of 0.5 μg/mL (blue asterisks) or 1 μg/mL (black asterisk) to 0 μg/mL CD45 concentration treatment. d. Representative images of CD33-CD45 PLA signal in CD33 mutant THP-1 cell lines. Mutations in the sialic acid binding domain (R119K) and the ITIM (Y340F) disrupt the interaction between CD33 and CD45 in THP-1 monocytes, as demonstrated by reduced PLA signal. Mutation in the ITIM-like domain (Y358F) leads to a reduction in CD33-CD45 interaction, as does the double mutant (Y358F/Y340F). e. Quantification of the CD33-CD45 PLA signal in each mutant THP-1 line, as PLA intensity per DAPI positive cell. Each dot represents a technical replicate. Unpaired t-test. *p<0.05, **p<0.01. f. Median fluorescence intensity of CD33-CD45 Flow Cytometry PLA in primary monocytes from 10 individuals, with and without pretreatment with 60nM clusterin (CLU) for 15 minutes. Error bars = mean + SEM. ***p<0.001, paired t-test. g. Representative overlay flow cytometry histogram of CD33-CD45 PLA fluorescent signal from monocytes with and without pretreatment with 60nM clusterin for 15 minutes. h. Median fluorescence intensity of CD33-CD45 PLA in primary monocytes from individuals genotyped for CD33 rs3865444 . N = 16 rs3865444 AA and 18 rs3865444 CC . Error bars = mean + SEM. *p<0.05, unpaired t-test. i. Representative overlayed flow cytometry histogram of PLA fluorescent signal from one CD33 rs3865444 AA and one CD33 rs3865444 CC individual.

Article Snippet: A CD33 encoding lentivirus was obtained from Origene (RC207023L1) and various CD33 mutants were made (R119K, Y340F, Y358F, and Y340F/Y358F) using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs).

Techniques: Mass Spectrometry, Proximity Ligation Assay, Knock-Out, CRISPR, Binding Assay, Recombinant, Incubation, Comparison, Concentration Assay, Mutagenesis, Fluorescence, Flow Cytometry

Journal: bioRxiv

Article Title: CD33-CD45 Interaction Reveals a Mechanistic Link to Alzheimer’s Disease Susceptibility

doi: 10.1101/2025.07.28.667311

Figure Lengend Snippet: a. CD33KO THP-1 cells have higher phosphatase activity compared to WT THP-1 cells, both at baseline and after stimulation with LPS. Phosphatase activity was measured with a colorimetric assay, reported with absorbance units. Unpaired t-test. ***p<0.001. b. Mutant CD33KO THP-1 monocytes with CD33 R119K re-expressed have higher phosphatase activity compared to CD33KO THP-1 cells with WT CD33 re-expressed, both at baseline and after stimulation with LPS. Unpaired t-test. ***p<0.001; ****p<0.0001. c. Treating THP-1 cells with the anti-CD33 antibody WM53, which profoundly reduces cell surface expression of CD33, increases CD45-specific phosphatase activity. CD45-specific phosphatase activity was determined by subtracting phosphatase activity measured with the addition of a CD45 specific inhibitor (CD45 Inhibitor VI), capturing all non-CD45 phosphatase activity, from global phosphatase activity. WM53 treatment was compared to IgG control. Each dot represents the average of two technical replicates. Unpaired t-test. ***p<0.001. d. CD45-specific phosphatase activity was measured over a period of 4 hours as described above. WM53 treatment of THP-1 monocytes, which profoundly reduces CD33 surface expression, maintains a consistent increase in CD45-specific phosphatase activity compared to IgG control over the course of the reaction. Each dot represents the mean + SEM of three experiments for WM53 and IgG, and each experiment was the average of two technical replicates. Two-way ANOVA with Sidak’s multiple comparisons test, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: A CD33 encoding lentivirus was obtained from Origene (RC207023L1) and various CD33 mutants were made (R119K, Y340F, Y358F, and Y340F/Y358F) using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs).

Techniques: Activity Assay, Colorimetric Assay, Mutagenesis, Expressing, Control

Journal: bioRxiv

Article Title: CD33-CD45 Interaction Reveals a Mechanistic Link to Alzheimer’s Disease Susceptibility

doi: 10.1101/2025.07.28.667311

Figure Lengend Snippet: a. Primary human monocyte-derived microglia (MDMi) were treated with aggregated Aβ 1-42 for 24 hours, after which proximity ligation assay (PLA) was performed to examine CD33-CD45 interaction. Representative images of PLA signal for untreated and Aβ 1-42 treated cells are shown. b. PLA signal was quantified as the PLA signal area per DAPI+ cell. Each dot represents an individual with paired untreated and Aβ 1-42 treated cells. There was an increase in the number of interactions between CD33 and CD45 after aggregated Aβ 1-42 stress compared to untreated MDMi from the same individuals. N=6 biological replicates. Ratio paired t-test. **p<0.01. c. Representative images of CD33 and CD45 MDMi protein staining following treatment with Aβ 1-42 for 24 hours. d. CD33 and CD45 protein expression was quantified as area of fluorescent signal for each DAPI+ cell. N=3 biological replicates. Ratio paired t-test Aβ 1-42 stress relative to untreated control. *p<0.05.

Article Snippet: A CD33 encoding lentivirus was obtained from Origene (RC207023L1) and various CD33 mutants were made (R119K, Y340F, Y358F, and Y340F/Y358F) using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs).

Techniques: Derivative Assay, Proximity Ligation Assay, Staining, Expressing, Control

Journal: bioRxiv

Article Title: CD33-CD45 Interaction Reveals a Mechanistic Link to Alzheimer’s Disease Susceptibility

doi: 10.1101/2025.07.28.667311

Figure Lengend Snippet: a. We investigated whether the interaction between CD33 and PTPRC gene expression influences AD clinical and pathological traits using the transcriptomic dataset of dorsolateral prefrontal cortex from the ROSMAP cohort. After adjustment for multiple hypothesis testing, CD33-PTPRC interaction was significantly associated with pathological diagnosis of AD and global AD pathology burden, both with negative beta values. b. Median fluorescence intensity of CD33-CD45 proximity ligation assay (PLA) in primary monocytes from individuals with Alzheimer’s disease (AD) or non-AD controls (NAC). Donors were matched for sex and CD33 rs3865444 genotype. N = 10 NAC donors and 9 AD donors. *p<0.05, unpaired t-test. c. Representative overlay flow cytometry histogram of PLA fluorescent signal from one AD and one NAC donor.

Article Snippet: A CD33 encoding lentivirus was obtained from Origene (RC207023L1) and various CD33 mutants were made (R119K, Y340F, Y358F, and Y340F/Y358F) using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs).

Techniques: Gene Expression, Biomarker Discovery, Fluorescence, Proximity Ligation Assay, Flow Cytometry