Journal: Journal of Human Immunity

Article Title: Viral infection and brain inflammation with seizures in PARK7 deficiency

doi: 10.70962/jhi.20250044

Figure Lengend Snippet: Hyperinflammatory cytokine and IFN responses in patient PBMCs. (A–H) PBMCs from the patient (red) and three controls (grey) were left untreated (UT), stimulated with TLR4 agonist LPS, TLR7/8 agonist R848, transfected with PolyIC for TLR3/MDA5/RIG-I stimulation, or mock transfected (transf ctrl). Cells were harvested 24 h after treatment, and RNA was isolated and analyzed for IFNB1 (B), CXCL10 (C), IL6 (D), and TNF (E) gene transcription by RT-PCR, and cell culture supernatants were collected and analyzed for CXCL10 (F), IL6 (G), and TNF (H) protein by ELISA. Bars indicate mean values ± standard deviation (SD) of a single experiment performed in triplicates. Statistics were calculated using the ordinary One-way ANOVA with Šídák’s multiple comparisons test. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001, and ns = not significant.

Article Snippet: RT-PCR was performed with the applied Biosystems TaqMan RNA to CT One Step Kit (4392938; Thermo Fisher Scientific) with primers: TBP(Hs00427620_m1), IFNB1(Hs01077958_s1), CXCL10(Hs01124251_g1), TNF(Hs00174128_m1), IL6(Hs00174131_m1), MX1(Hs00895608_m1), and IFNL1(Hs00601677_g1).

Techniques: Transfection, Isolation, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Journal of Human Immunity

Article Title: Viral infection and brain inflammation with seizures in PARK7 deficiency

doi: 10.70962/jhi.20250044

Figure Lengend Snippet: Inflammatory cytokine and IFN responses in PBMCs following RSV infection. PBMCs from the patient (P, red) and three controls (C, grey) were mock infected (mock) or infected with RSV (RSV-A strain Long, MOI 1). (A–G) Cells were harvested 24 h after treatment, and RNA was isolated and analyzed for intracellular RSV RNA (A), IFNB1 (B), CXCL10 (C), IL6 (D), and TNF (E) gene expression by RT-PCR. Cell culture supernatants were harvested and analyzed for CXCL10 (F) and TNF (G) protein by ELISA. Bars indicate the mean ± SD values of a single experiment performed in triplicate. Statistics were calculated using the ordinary two-way ANOVA with Šídák’s multiple comparisons. ns = not significant. SD, standard deviation.

Article Snippet: RT-PCR was performed with the applied Biosystems TaqMan RNA to CT One Step Kit (4392938; Thermo Fisher Scientific) with primers: TBP(Hs00427620_m1), IFNB1(Hs01077958_s1), CXCL10(Hs01124251_g1), TNF(Hs00174128_m1), IL6(Hs00174131_m1), MX1(Hs00895608_m1), and IFNL1(Hs00601677_g1).

Techniques: Infection, Isolation, Gene Expression, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Journal of Human Immunity

Article Title: Viral infection and brain inflammation with seizures in PARK7 deficiency

doi: 10.70962/jhi.20250044

Figure Lengend Snippet: Generation of PARK7-deficient A549 and SH-SY5Y cells and cytokine responses to RSV infection. (A and B) Immunoblot and RT-PCR demonstrating absence of RSV replication in primary fibroblasts. Primary fibroblasts from a healthy donor were infected with RSV (RSV-A strain Long, MOI 1), and cells were collected at indicated time points. Whole cell lysates were analyzed by immunoblotting for expression of RSV matrix protein (RSV-M2) and compared to vinculin (VCL) as loading control (A), and cells were collected for RNA extraction and quantification of RSV RNA by RT-PCR (B). (C and D) Immunoblot demonstrating KO of PARK7 in A549 pulmonary cells (C) and PARK7 KO in neuronal SH-SY5Y cells (D). Vinculin (VCL, C) and GAPDH (D) were used as loading control. (E) Repeat of experiment shown in , showing additional replicates of two experiments performed in duplicate of human pulmonary A594 cells deficient in PARK7 (red) and control AAVS1 KO (grey) mock treated or infected with RSV (RSV-A strain Long, MOI 1) for 24 and 48 h, and cells were collected for RNA isolation and analysis of RSV RNA by RT-PCR. (F–M) PARK7 KO (red) and control AAVS1 KO (grey) A549 cells were mock treated or infected with RSV (RSV-A, strain Long, MOI 1) for 24 h. (F–I) Cells were collected for RNA isolation, and quantification of IFNL1 (F), CXCL10 (G), IL6 (H), and TNF (I) gene transcription was performed by RT-PCR. (J–M) Cell culture supernatants were collected for IFNλ1 (J), CXLC10 (K), IL6 (L), and TNF (M) protein quantification by ELISA. (N–U) Repeats of experiment shown in , showing an additional two repeat experiments performed in triplicate (N–Q shows the second experiment and R–U shows the third experiment) where neuronal SH-SY5Y cells deficient in PARK7 or control AAVS1 KO were mock transfected (transf ctrl) or transfected with PolyIC (500 ng/ml). Cells were collected after 6 h, and RNA was isolated, and IFNB1 (N and R), CXCL10 (O and S), IL6 (P and T), and MX1 (Q and U) gene transcription quantification by RT-qPCR. Bars indicate mean ± SD values. Statistics were calculated using the two-way ANOVA with multiple comparisons. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, and ns = not significant. SD, standard deviation. Source data are available for this figure: .

Article Snippet: RT-PCR was performed with the applied Biosystems TaqMan RNA to CT One Step Kit (4392938; Thermo Fisher Scientific) with primers: TBP(Hs00427620_m1), IFNB1(Hs01077958_s1), CXCL10(Hs01124251_g1), TNF(Hs00174128_m1), IL6(Hs00174131_m1), MX1(Hs00895608_m1), and IFNL1(Hs00601677_g1).

Techniques: Infection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Control, RNA Extraction, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Transfection, Quantitative RT-PCR, Standard Deviation

Journal: Journal of Human Immunity

Article Title: Viral infection and brain inflammation with seizures in PARK7 deficiency

doi: 10.70962/jhi.20250044

Figure Lengend Snippet: Enhanced cellular stress pathways and hyperinflammatory cytokine responses in PARK7-deficient neuronal SH-SY5Y cells. (A) Human pulmonary A594 cells deficient in PARK7 (red) and control AAVS1 KO (grey) were mock treated or infected with RSV (RSV-A strain Long, MOI 1) for 24 and 48 h, and cells were collected for RNA isolation and analysis of RSV RNA by RT-PCR. Bars indicate mean ± SD values of two experiments, performed in duplicate. Repeats of the experiments are shown in . (B and C) Whole cell lysates from SH-SY5Y with PARK7 KO (red) or AAVS1 KO (control, grey) were analyzed for phosphorylation of ASK1/2 (pASK1/2) and expression of total ASK1 and vinculin (VCL) as loading control by immunoblotting (B). Graph depicting the densitometric quantification of pASK1/2 relative to total ASK1 (C). Bars indicate the mean ± SD values of three experiments. (D–G) Neuronal SH-SY5Y cells deficient in PARK7 or control AAVS1 KO were mock transfected (transf ctrl) or transfected with PolyIC (tPolyIC, 500 ng/ml). Cells were collected after 6 h, and RNA was isolated, and IFNB1 (D), MX1 (E), CXCL10 (F), and IL6 (G) gene transcription quantification by RT-PCR. Bars indicate mean ± SD values of a representative experiment performed in triplicate. Repeats of the experiment are shown in . (H–J) Neuronal SH-SY5Y cells deficient in PARK7 or AAVS KO (control) were left untransduced (−) or were transduced with lentiviral vectors to express PARK7 or GFP (control). Cells were collected after 48 h for RNA isolation to determine PARK7 mRNA expression (H), and whole cell lysates were analyzed by immunoblotting for GFP and PARK7 expression with VCL as loading control (I). (J) Cells were transfected with PolyIC (tPolyIC, 500 ng/ml) and collected after 6 h for quantification of IFNB1 transcription by RT-PCR. Bars indicate mean ± SD values of a representative experiment performed in duplicate. (K–M) Neuronal SH-SY5Y cells deficient for PARK7 were transduced with lentiviral vectors to express PARK7 WT, the patient PARK7 variant R28*, the R98Q variant frequent in the healthy population (MAF>10 −3 ), and the L166P variant associated with Parkinson’s disease. Cells were collected for RNA isolation to determine PARK7 mRNA expression (K), and whole cell lysates were analyzed by immunoblotting for PARK7 expression with VCL as loading control (L). (M) Cells were transfected with PolyIC (tPolyIC, 500 ng/ml) and collected after 6 h for quantification of IFNB1 transcription by RT-PCR. Bars indicate mean ± SD values of a representative experiment performed in duplicate. (N–P) Primary fibroblasts from the patient (P) were left untransduced (−) or were transduced with lentiviral vectors to express GFP, PARK7WT, PARK7 R28*, PARK7 R98Q, or PARK7 L166P. As control (C), primary fibroblasts from a healthy donor were left untransduced or were transduced to express GFP. Cells were collected for RNA isolation to determine PARK7 mRNA expression (N), and whole cell lysates were analyzed by immunoblotting for GFP and PARK7 expression with VCL as loading control (O). Cells were infected with mock virus (mock) or RSV (RSV-A strain Long, MOI 3, 24 h) and stained with Annexin-V (apoptosis) and 7-AAD (viability dye) to analyze induction of noninflammatory apoptotic cell death by flow cytometry (P). Bars show mean ± SD values of a single experiment performed in duplicate and triplicate. Statistics were calculated using the multiple paired t test (A), the unpaired t test (C), the one-way ANOVA (H and K), and ordinary two-way ANOVA with Šídák’s multiple comparisons (J, M, and P). * = P < 0.05, ** = P < 0.01, *** = P < 0.001, and ns = not significant. SD, standard deviation. Source data are available for this figure: .

Article Snippet: RT-PCR was performed with the applied Biosystems TaqMan RNA to CT One Step Kit (4392938; Thermo Fisher Scientific) with primers: TBP(Hs00427620_m1), IFNB1(Hs01077958_s1), CXCL10(Hs01124251_g1), TNF(Hs00174128_m1), IL6(Hs00174131_m1), MX1(Hs00895608_m1), and IFNL1(Hs00601677_g1).

Techniques: Control, Infection, Isolation, Reverse Transcription Polymerase Chain Reaction, Phospho-proteomics, Expressing, Western Blot, Transfection, Transduction, Variant Assay, Virus, Staining, Flow Cytometry, Standard Deviation