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

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: Cells

Article Title: Induction of CXCL10-Mediated Cell Migration by Different Types of Galectins

doi: 10.3390/cells10020274

Figure Lengend Snippet: Galectins induce CXCL10 in a cell type-specific manner. ( A ) SDS-polyacrylamide gel electrophoresis of recombinant human galectins (rhGal) stained with Coomassie Blue. Molecular weights of protein standards are indicated on the left. The hemagglutination assay was performed using serial two-fold dilutions of the recombinant protein. The bottom row contains protein incubated with β-lactose, a competitive carbohydrate inhibitor of galectin binding. ( B ) Cultures of human corneal fibroblasts, human corneal epithelial cells, THP-1 monocytes and human umbilical vein endothelial cells (HUVECs) were incubated with 50 µg/mL rhGal-1, -3 and -8 or 50 µg/mL IFN-γ. Quantitative real-time PCR was used to determine the levels of CXCL10 mRNA after 6 h of incubation ( n = 3 independent experiments), whereas ELISA was used to determine the levels of CXCL10 protein in cell culture supernatants after 24 h of incubation ( n = 6 independent experiments). The data represent the mean ± SEM.

Article Snippet: A mouse monoclonal antibody against CXCL10 (1.2 μg/mL; MAB266, R&D Systems) was added to the lower compartment of the chamber in neutralization studies.

Techniques: Polyacrylamide Gel Electrophoresis, Recombinant, Staining, Hemagglutination Assay, Incubation, Binding Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cells

Article Title: Induction of CXCL10-Mediated Cell Migration by Different Types of Galectins

doi: 10.3390/cells10020274

Figure Lengend Snippet: Galectins differentially dictate fibroblast-dependent immune cell chemotaxis. ( A ) Recombinant human galectin (rhGal)-1, -3 and -8 were incubated with fibroblasts for 24 h. The conditioned media were pre-cleared with β-lactose Sepharose beads three times to remove galectins prior to chemotaxis assays. The presence of galectins in pre-cleared conditioned media was evaluated by immunoblotting. ( B ) Quantification by flow cytometry of the migration of CFSE-labeled THP-1 and Jurkat cells to conditioned media from fibroblasts treated for 24 h with increasing concentrations of recombinant galectins or BSA ( n = 3 independent experiments performed in duplicate). ( C ) Migration of CFSE-labeled THP-1 and Jurkat cells to conditioned media from fibroblasts treated for 24 h with 50 μg/mL of recombinant galectins or BSA. A CXCL10 neutralizing antibody was added to the conditioned media in neutralization studies ( n = 4–5 independent experiments in duplicate). The data in ( B ) represent the mean ± SEM. The box and whisker plots show the 25 and 75 percentiles (box), the median, and the minimum and maximum data values (whiskers). Significance was determined using Student’s t test. ** p < 0.01.

Article Snippet: A mouse monoclonal antibody against CXCL10 (1.2 μg/mL; MAB266, R&D Systems) was added to the lower compartment of the chamber in neutralization studies.

Techniques: Chemotaxis Assay, Recombinant, Incubation, Western Blot, Flow Cytometry, Migration, Labeling, Neutralization, Whisker Assay

Journal: Clinical and Vaccine Immunology

Article Title: Increased CXC Ligand 10 Levels and Gene Expression in Type 1 Leprosy Reactions

doi: 10.1128/cvi.00042-11

Figure Lengend Snippet: FIG. 1. Circulating CXCL10 in leprosy patients and healthy con- trols. Each point represents one sample from a different individual. The groups were healthy controls, borderline tuberculoid without re- action (BT), borderline lepromatous without reaction (BL), polar lep- romatous without reaction (LL), BT with reaction (BT plus RR), BL with reaction (BL plus RR), and LL patients with T2R (erythema nodosum leprosum). For patients with reaction (BT plus RR, BL plus RR, and T2R), the sample was taken at the time of reaction, prior to treatment. For LL, n 6; for all other groups, n 10. The horizontal lines indicate median values. The median level of CXCL10 in LL patients with type 2 reaction was not significantly elevated (P 0.9).

Article Snippet: Tissue sections were stained by an indirect immunoperoxidase method using goat anti-human CXCL10 (R&D Systems, Inc.; 1:40) or rabbit anti-human CXCR3 (Sigma) (1:75) for 60 min, followed by biotinylated donkey anti-goat IgG or goat anti-rabbit IgG (both 1:40; Jackson ImmunoResearch Laboratories, Inc.) for 30 min. Peroxidase-conjugated avidin-biotin complexes (Vectastain Elite ABC Reagent kit) were applied for 30 min. All incubations were followed by three washes with phosphate-buffered saline isotonic buffer, pH 7.4.

Techniques:

Journal: Clinical and Vaccine Immunology

Article Title: Increased CXC Ligand 10 Levels and Gene Expression in Type 1 Leprosy Reactions

doi: 10.1128/cvi.00042-11

Figure Lengend Snippet: FIG. 2. Circulating CXCL10 in repeated samples from leprosy patients who developed T1R. Each graph depicts the measurements made from serum samples obtained at 1 month intervals in borderline tuberculoid (A, no. 1 to 10) and borderline lepromatous (B, no. 11 to 20) patients. All patients with clinical T1R received standard multidrug treatment starting at the first visit. The arrows indicate the visits at which T1R was diagnosed. The number above each time point indicates the dose of prednisone started on that day, tapered monthly as indicated; in some cases, corticosteroids were not used initially but were started 1 to 2 months after the initial clinical diagnosis of T1R. Each serum specimen was obtained before corticosteroid treatment was initiated. CXCL10 was significantly elevated during T1R in the 10 BT patients (A) (P 0.0006) and the 10 BL patients (B) (P 0.0001). In patients 12, 16, and 17 (B), T1R was diagnosed by histopathological criteria only; when they were excluded from the analysis, the association of T1R with CXCL10 remained significant (P 0.05).

Article Snippet: Tissue sections were stained by an indirect immunoperoxidase method using goat anti-human CXCL10 (R&D Systems, Inc.; 1:40) or rabbit anti-human CXCR3 (Sigma) (1:75) for 60 min, followed by biotinylated donkey anti-goat IgG or goat anti-rabbit IgG (both 1:40; Jackson ImmunoResearch Laboratories, Inc.) for 30 min. Peroxidase-conjugated avidin-biotin complexes (Vectastain Elite ABC Reagent kit) were applied for 30 min. All incubations were followed by three washes with phosphate-buffered saline isotonic buffer, pH 7.4.

Techniques: Biomarker Discovery

Journal: Clinical and Vaccine Immunology

Article Title: Increased CXC Ligand 10 Levels and Gene Expression in Type 1 Leprosy Reactions

doi: 10.1128/cvi.00042-11

Figure Lengend Snippet: FIG. 3. Expression levels of CXCL10 and IFN- genes in skin biopsy specimens from leprosy patients. (A to H) Real-time quantitative RT-PCR was performed on cDNA obtained from sequential skin biopsy specimens from individual leprosy patients who were placed on MDT, none of whom had T1R at the time of the first biopsy. Five patients (A to E) had clinical symptoms of T1R at the time of the second biopsy; two of these (C and D) had a third biopsy after the reaction had resolved. Three patients (F to H) had no clinical symptoms of T1R at the time of the initial or second biopsy. Data were obtained using the standard-curve method and were normalized using 18S rRNA values, repeated three times for all determinations. The results are presented as the mean standard deviation.

Article Snippet: Tissue sections were stained by an indirect immunoperoxidase method using goat anti-human CXCL10 (R&D Systems, Inc.; 1:40) or rabbit anti-human CXCR3 (Sigma) (1:75) for 60 min, followed by biotinylated donkey anti-goat IgG or goat anti-rabbit IgG (both 1:40; Jackson ImmunoResearch Laboratories, Inc.) for 30 min. Peroxidase-conjugated avidin-biotin complexes (Vectastain Elite ABC Reagent kit) were applied for 30 min. All incubations were followed by three washes with phosphate-buffered saline isotonic buffer, pH 7.4.

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation