Journal: Frontiers in Immunology

Article Title: Transplanted gene-modified placental cells boost FVIII activity in pediatric sheep without eliciting immunity, toxicity, or adverse events

doi: 10.3389/fimmu.2025.1716950

Figure Lengend Snippet: PLC-mcoET3 can be detected in tissues of pediatric sheep after IP and IV administration at 7–18 months post-administration by dPCR and by RT-qPCR. dPCR using human Alu-specific primers was performed in triplicate on DNA extracted from liver, lung, spleen, and thymus. All the tissues analyzed contained human DNA; the same tissue samples from control non-treated animals showed no target amplification. RT-qPCR was performed in triplicate using primers specific for mcoET3 and sheep GAPDH. Percentage of engraftment was determined by comparing ΔCt values to a standard curve consisting of increasing percentages of mcoET3-PLC (0, 0.01, 0.1, 1, 5, and 10%) in sheep stromal cells. (AH) Percentages of human cells present in liver (A, E) , lung (B, F) , spleen (C, G) , and thymus (D, H) as determined dPCR (A–D) and RT-qPCR (E–H) using human Alu- and mcoET3-specific primers, respectively; (I) H&E staining was also performed on sections from each tissue to determine whether engraftment of PLC-mcoET3 in each of the different tissues caused any untoward effects. Slides of the tissue sections were sent to a certified animal pathologist. Results showed no evidence of any lentivector-related toxicity in any of the tissues examined. Moreover, there were no changes in tissue architecture, ectopic tissue formation, gross tumor development, or areas of atypical cells or foci of hyperplasia or neoplasia. All images were acquired with a Leica DM4000 B microscope using a 10x objective. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ns, not significant.

Article Snippet: Each reaction contained 200nM of Alu and RPL13A primers and 250nM of their respective probes in a final concentration of 1X Absolute QTM DNA Digital PCR Master Mix (ThermoFisher Scientific, A52490).

Techniques: Quantitative RT-PCR, Control, Amplification, Staining, Microscopy

Journal: Frontiers in Immunology

Article Title: Transplanted gene-modified placental cells boost FVIII activity in pediatric sheep without eliciting immunity, toxicity, or adverse events

doi: 10.3389/fimmu.2025.1716950

Figure Lengend Snippet: Treatment of animals with pre-existing inhibitors with PLC-mcoET3 results in an increase in FVIII activity in two of three animals at most of the time points analyzed despite the rise in titers of inhibitory antibodies. Human PLC-mcoET3, producing 3.6 IU/10 6 cells/24hr of FVIII/ET3, were administered to 3 animals on 3 consecutive weeks (20IU/kg/24h) to reach a total dose of 60IU/kg/24h at 14–23 months after the administration of ET3 and/or FVIII protein. (A) Animals were chosen because they continued to harbor FVIII and ET3 inhibitors at the time of PLC-mcoET3 infusion; (B) PLC-mcoET3 infusion triggered an increase in inhibitors against ET3 (p<0.05) and FVIII (p<0.05) at W3, when compared to W0 in all 3 animals; (C) FVIII activity increased in two out of three animals at most of the time points analyzed, despite the rise in titers of inhibitory antibodies; (D) dPCR and (E) RT-qPCR were performed as described above, and PLC-mcoET3 were found to be present in all tissues analyzed. *p<0.05, **p<0.01, ***p<0.001. ns, not significant.

Article Snippet: Each reaction contained 200nM of Alu and RPL13A primers and 250nM of their respective probes in a final concentration of 1X Absolute QTM DNA Digital PCR Master Mix (ThermoFisher Scientific, A52490).

Techniques: Activity Assay, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Interplay Between 3D Chromatin Architecture and Gene Regulation at the APOE Locus Contributes to Alzheimer’s Disease Risk

doi: 10.3390/ijms27010302

Figure Lengend Snippet: Cell type-specific chromatin interaction between the APOE exon 4 CpG island (CGI) locus and the APOC1 promoter, and its relationship to APOC1 transcription. ( A ) Chromatin interaction (CI) strengths were quantified in HepG2, U87, HMC3, and SH-SY5Y cells using the CIS-dPCR assay. ( B ) APOC1 mRNA levels were measured by RT-qPCR (TaqMan assay) in the same cell lines. Expression was normalized using the ∆∆Ct method, where a higher ∆∆Ct value indicates greater mRNA abundance. APOC1 ∆Ct was calculated relative to a housekeeping gene ACTB , and ∆∆Ct values were derived using SH-SY5Y cells as the baseline (set to 1).

Article Snippet: The dPCR reaction was performed using the Absolute Q Digital PCR System (ThermoFisher) with a 10 μL reaction mixture, including 10 ng of 3C-generated template DNA, 2 μL of 5× Absolute Q Universal DNA Digital PCR Master Mix (ThermoFisher), and 1 μL of 10× digital PCR assay comprising 8 μM of each primer and 4 μM probe.

Techniques: Quantitative RT-PCR, TaqMan Assay, Expressing, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Interplay Between 3D Chromatin Architecture and Gene Regulation at the APOE Locus Contributes to Alzheimer’s Disease Risk

doi: 10.3390/ijms27010302

Figure Lengend Snippet: Chromatin interaction (CI) strength and APOC1 mRNA levels in PMB samples. ( A ) Data stratified by disease status (control (Ctrl), n = 10; AD, n = 15). CIS-dPCR assay revealed increased CI strength at the APOE – APOC1 locus in AD compared to in control ( p < 0.05), accompanied by elevated APOC1 mRNA levels ( p < 0.005) measured by RT-qPCR using the ΔΔCt method, normalized to ACTB and a reference PMB sample. CI strength and APOC1 expression showed divergent correlations in Ctrl (r = −0.56, p = 0.09) and AD (r = 0.39, p = 0.15). ( B ) Data stratified by APOE genotypes (ε3/ε3, n = 10; ε3/ε4, n = 10; ε4/ε4, n = 5). CI strength varied by APOE genotype, with highest levels in ε3/ε4 carriers ( p < 0.05 vs. ε3/ε3). APOC1 mRNA levels exhibited genotype-dependent trends, though not statistically significant ( p > 0.05). Genotype-stratified correlations revealed a positive trend in ε3/ε4 carriers (r = 0.61, p = 0.06), with no correlation observed in ε3/ε3 or ε4/ε4 groups. An independent samples t -test was used to compare across the biological variables. Statistical significance is indicated as follows: *, p < 0.05; **, p < 0.005.

Article Snippet: The dPCR reaction was performed using the Absolute Q Digital PCR System (ThermoFisher) with a 10 μL reaction mixture, including 10 ng of 3C-generated template DNA, 2 μL of 5× Absolute Q Universal DNA Digital PCR Master Mix (ThermoFisher), and 1 μL of 10× digital PCR assay comprising 8 μM of each primer and 4 μM probe.

Techniques: Control, Quantitative RT-PCR, Expressing