Journal: Diabetes

Article Title: Expression and Regulation of Chemokines in Murine and Human Type 1 Diabetes

doi: 10.2337/db11-0853

Figure Lengend Snippet: Chemokine transcripts induced in human islet cells in response to inflammatory stimuli (microarray analysis). Purified human islets obtained from healthy organ donors were cultured for 24 h in the absence (Control) or presence of individual recombinant human cytokines IL-1β, TNFα, or IFNγ or combinations thereof (MIX) prior to microarray analysis as described in . The normalized intensity (log scale) from data obtained on the HG U133 Plus 2.0 Affymetrix chip is shown. Each data point is the mean ± SE of three to four observations. Cytokine cocktail (MIX)-induced expression by a factor of >30 was observed for CCL5 , CCL8 , CCL22 , CX3CL1 , CXCL9 , and CXCL10 (asterisks indicate significant differences between control and cytokine-treated islets).

Article Snippet: For the purpose of the current study, we approved the utility of the following human chemokine–specific antibodies: goat polyclonals αCCL5 (AF278-NA), αCCL8 (AF281-NA), αCXCL9 (AF392), αCXCL10 (AF266-NA), and αCX3CL1 (AF365) as well as polyclonal chicken IgY specific for CCL22 (AF336) (R&D Systems).

Techniques: Microarray, Purification, Cell Culture, Control, Recombinant, Expressing

Journal: Diabetes

Article Title: Expression and Regulation of Chemokines in Murine and Human Type 1 Diabetes

doi: 10.2337/db11-0853

Figure Lengend Snippet: Chemokine expression in the RIP-GP model of virus-induced type 1 diabetes. RIP-GP mice were infected with LCMV, and their pancreata were harvested 7 days later and processed for immunohistological analysis as detailed in . Note the minimal or absent expression of CCL22 and CXCL9, the preferential expression of CCL8 and CXCL10 by β-cells, as well as CX3CL1 production by α-cells; the right-hand column features magnified sections of merged CCL8, CXCL10, and CX3CL1 stains. (A high-quality digital representation of this figure is available in the online issue.)

Article Snippet: For the purpose of the current study, we approved the utility of the following human chemokine–specific antibodies: goat polyclonals αCCL5 (AF278-NA), αCCL8 (AF281-NA), αCXCL9 (AF392), αCXCL10 (AF266-NA), and αCX3CL1 (AF365) as well as polyclonal chicken IgY specific for CCL22 (AF336) (R&D Systems).

Techniques: Expressing, Virus, Infection

Journal: Diabetes

Article Title: Expression and Regulation of Chemokines in Murine and Human Type 1 Diabetes

doi: 10.2337/db11-0853

Figure Lengend Snippet: In situ CXCL10 expression in type 1 diabetic (T1D) and healthy control (Control) pancreata. Combined insulin, glucagon, CD45, and CXCL10 stains were performed as detailed in . Note the absence of CXCL10 staining in the healthy control subjects (case identification no. 6112) but ready presence in type 1 diabetic samples (identification nos. 6087 and 6052), sometimes in close association with infiltrating leukocytes (CD45 + ). To confirm the pattern of exocrine CXCL10 staining, we included a sample from an additional diabetic donor with clinically confirmed pancreatitis (identification no. 6036). (A high-quality digital representation of this figure is available in the online issue.)

Article Snippet: For the purpose of the current study, we approved the utility of the following human chemokine–specific antibodies: goat polyclonals αCCL5 (AF278-NA), αCCL8 (AF281-NA), αCXCL9 (AF392), αCXCL10 (AF266-NA), and αCX3CL1 (AF365) as well as polyclonal chicken IgY specific for CCL22 (AF336) (R&D Systems).

Techniques: In Situ, Expressing, Control, Staining

Journal: FASEB BioAdvances

Article Title: Human inborn errors of long‐chain fatty acid oxidation show impaired inflammatory responses to TLR4 ‐ligand LPS

doi: 10.1096/fba.2024-00060

Figure Lengend Snippet: Inborn errors in ETFDH cause impaired responses to LPS. Primary dermal fibroblasts from healthy controls (C1‐2) and primary dermal fibroblasts derived from MADD patients (P1‐2) were (A) blotted for basal expression level of ETF‐QO. The cells were stimulated with LPS 400 ng/mL, and analyzed for (B, C) IL‐6, IL‐8 secretion at 24 h by ELISA or (D–H) cytokines mRNA (IL‐6, CCL2, CXCL10, IL‐1β, and IL‐1α) expression by RT‐QPCR at 6 h. Control dermal fibroblasts (C2) transfected with scrambled siRNA or ETFDH siRNA for 72 h were stimulated with LPS 400 ng/mL for (I–K) 24 h, blotted and quantified for ETF‐QO, and analyzed for IL‐6 secretion or (L–P) 6 h, and analyzed for cytokines mRNA (IL‐6, CCL2, CXCL10, IL‐1β, and TNF) expression by RT‐QPCR. Blots in figures (A and I) are representative of three independent experiments. Data in figures (B–D) and (K–P) are presented as mean ± SEM of four cell culture experiments, while data in figures (E–H) represent mean ± SEM of two cell culture experiments. * p < 0.05, ** p < 0.01 *** p < 0.001, and **** p < 0.0001; compared to untreated control cells (unpaired t test). MADD, Multiple Acyl‐CoA Dehydrogenase Deficiency; ETFDH, Electron Transfer Flavoprotein Dehydrogenase.

Article Snippet: Cytokines in the patient and control dermal fibroblast culture supernatants were measured using enzyme‐linked immunosorbent assay (ELISA) for quantitative detection of IL‐1β (R&D Systems Cat. No. DY201), tumor necrosis factor TNF‐α (BioLegend® Cat. No. 430204), IL‐6 (BioLegend® Cat. No. 430504 and R&D Systems Cat. No. DY206), IL‐8 (Thermo Scientific 88–8086), IL‐10 (BioLegend® Cat. No. 430604 and R&D Systems Cat. No. DY217B), and CXCL10 (R&D Systems Cat. No. DY266), all according to the manufacturer's instructions.

Techniques: Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Control, Transfection, Cell Culture

Journal: FASEB BioAdvances

Article Title: Human inborn errors of long‐chain fatty acid oxidation show impaired inflammatory responses to TLR4 ‐ligand LPS

doi: 10.1096/fba.2024-00060

Figure Lengend Snippet: Reduced LPS responsiveness in severe VLCADD patient cells. Primary dermal fibroblasts from healthy controls (C4‐6) and primary dermal fibroblasts derived from severe VLCADD patients (P5‐8) stimulated with LPS 400 ng/mL were analyzed for (A–D) cytokines mRNA (IL‐6, CCL2, IL‐1β, and IL‐1α) expression by RT‐QPCR at 6 h and (E, F) IL‐6, IL‐8 secretion at 24 h. (G, H) Primary dermal fibroblasts from a healthy control (C4) transfected with scrambled siRNA or ACADVL siRNA for 72 h were stimulated with LPS 400 ng/mL, blotted and quantified for VLCAD expression. Then the cells were analyzed for (I, J) cytokines secretion (IL‐6, IL‐8) at 24 h, (K‐P) cytokines mRNA ( IL‐6 , CCL2 , IL‐1α , IL‐1β , CXCL10 , TNF ), and (Q) TLR4 mRNA expression by RT‐QPCR at 6 h. (R, S) Primary dermal fibroblasts from healthy controls (C4‐6) and primary dermal fibroblasts derived from severe VLCADD patients (P5‐8) were analyzed for basal level TLR4 mRNA expression. (T, U) The cells were stimulated with LPS 400 ng/mL for 24 h, blotted and quantified for JunB. Data in figures (A–F) represent mean ± SEM of two cell culture experiments, while data in figures (I–Q) are presented as mean ± SEM of four cell culture experiments. Blots in figures (G and T) were performed twice in independent experiments. *Represents significance compared to healthy controls or untreated control cells (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, unpaired t test).

Article Snippet: Cytokines in the patient and control dermal fibroblast culture supernatants were measured using enzyme‐linked immunosorbent assay (ELISA) for quantitative detection of IL‐1β (R&D Systems Cat. No. DY201), tumor necrosis factor TNF‐α (BioLegend® Cat. No. 430204), IL‐6 (BioLegend® Cat. No. 430504 and R&D Systems Cat. No. DY206), IL‐8 (Thermo Scientific 88–8086), IL‐10 (BioLegend® Cat. No. 430604 and R&D Systems Cat. No. DY217B), and CXCL10 (R&D Systems Cat. No. DY266), all according to the manufacturer's instructions.

Techniques: Derivative Assay, Expressing, Quantitative RT-PCR, Control, Transfection, Cell Culture