Journal: Frontiers in Immunology

Article Title: Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

doi: 10.3389/fimmu.2021.764390

Figure Lengend Snippet: Primer names for PCR reactions.

Article Snippet: Surface expression of TLR2 was confirmed by flow cytometry using either human anti-bovine CD282 (anti-TLR2) antibody or mouse anti-human CD282 antibody, both conjugated with Alexa Flour 647 (Bio-Rad, USA).

Techniques: Sequencing

Journal: Frontiers in Immunology

Article Title: Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

doi: 10.3389/fimmu.2021.764390

Figure Lengend Snippet: Identified SNP variants within the coding sequence of bovine tlr2 in HF and BS breeds animals.

Article Snippet: Surface expression of TLR2 was confirmed by flow cytometry using either human anti-bovine CD282 (anti-TLR2) antibody or mouse anti-human CD282 antibody, both conjugated with Alexa Flour 647 (Bio-Rad, USA).

Techniques: Sequencing, Residue

Journal: Frontiers in Immunology

Article Title: Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

doi: 10.3389/fimmu.2021.764390

Figure Lengend Snippet: Genotype frequency of Bos taurus and Bos indicus cattle breeds for TLR2 selected candidate SNP rs68343167 (H326Q).

Article Snippet: Surface expression of TLR2 was confirmed by flow cytometry using either human anti-bovine CD282 (anti-TLR2) antibody or mouse anti-human CD282 antibody, both conjugated with Alexa Flour 647 (Bio-Rad, USA).

Techniques:

Journal: Frontiers in Immunology

Article Title: Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

doi: 10.3389/fimmu.2021.764390

Figure Lengend Snippet: Location of identified SNPs. Gene model for TLR2 from assembly UMD 3.1.1 with annotated SNPs. Missense SNPs are located on the first track below the gene model, with synonymous SNPS on the second track. Reading from left to right, exons and coding sequence (CDS; thick line) are shown. All mutations occur in the second exon, which codes for ligand binding side.

Article Snippet: Surface expression of TLR2 was confirmed by flow cytometry using either human anti-bovine CD282 (anti-TLR2) antibody or mouse anti-human CD282 antibody, both conjugated with Alexa Flour 647 (Bio-Rad, USA).

Techniques: Sequencing, Ligand Binding Assay

Journal: Frontiers in Immunology

Article Title: Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

doi: 10.3389/fimmu.2021.764390

Figure Lengend Snippet: TLR2-Ligand-Dependent NF-κB Activity and CXCL8 Secretion by HEK 293 cells Expressing Bovine and Human TLR2 Sequence Variants. HEK 293 cells, harboring NF-κB-induced SEAP reporter genes were transfected with either empty pTracer vector (mock); bovine or human TLR2 CDS constructs, or a bovine: human chimera in pDuo-mcs. Cells were stimulated with either FSL-1 at 100 ng mL -1 (A, C) , Pam3CSK4 at 1 mg mL -1 (B, D) for 24 hrs. SEAP activity was measured by quantifying optical density of cell culture supernatant at 635 nm (A, B) . OD values were normalised against values for PMA stimulation at 200 ng mL -1 . Additionally, supernatants were assessed for CXCL8 concentration by ELISA (C, D) . Error bars represent standard deviation from the mean of triplicate technical replicates and are representative of three independent repeats. (*=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001) and are only shown in relation to bovine TLR2 and additionally between the human and the chimeric bov: hu TLR 2 receptor. All data analysed by two-way ANOVA with multiple comparisons using GraphPad Prism V8 (GraphPad Inc., USA).

Article Snippet: Surface expression of TLR2 was confirmed by flow cytometry using either human anti-bovine CD282 (anti-TLR2) antibody or mouse anti-human CD282 antibody, both conjugated with Alexa Flour 647 (Bio-Rad, USA).

Techniques: Activity Assay, Expressing, Sequencing, Transfection, Plasmid Preparation, Construct, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

doi: 10.3389/fimmu.2021.764390

Figure Lengend Snippet: M. bovis BCG and M. tuberculosis -Dependent NF-κB Activity Secretion by HEK 293 cells Expressing Bovine and Human TLR2 Sequence Variants. HEK 293 cells, harbouring NF-κB-induced SEAP reporter genes were transfected with either empty pTracer vector (mock); bovine or human TLR2 CDS constructs, or a bovine: human chimera in pDUO-mcs. Cells were stimulated with either live M. bovis BCG (MOI of 10) (A) or M. tuberculosis H37Rv (MOI of 5) (B) for 24 h. SEAP activity was measured by quantifying optical density of cell culture supernatant at 635 nm. OD values were normalised against values for PMA stimulation at 200 ng/ml. Error bars represent standard deviation from the mean of triplicate technical replicates and are representative of three independent repeats. (**=p<0.01, ***=p<0.001, ****=p<0.0001) and are only shown in relation to bovine TLR2 and additionally between the human and the chimeric bov: hu TLR 2 receptor. All data analysed by two-way ANOVA with multiple comparisons using GraphPad Prism V8 (GraphPad Inc., USA).

Article Snippet: Surface expression of TLR2 was confirmed by flow cytometry using either human anti-bovine CD282 (anti-TLR2) antibody or mouse anti-human CD282 antibody, both conjugated with Alexa Flour 647 (Bio-Rad, USA).

Techniques: Activity Assay, Expressing, Sequencing, Transfection, Plasmid Preparation, Construct, Cell Culture, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Single Nucleotide Polymorphisms in the Bovine TLR2 Extracellular Domain Contribute to Breed and Species-Specific Innate Immune Functionality

doi: 10.3389/fimmu.2021.764390

Figure Lengend Snippet: CXCL8 production by bovine MØ in response to FSL-1, M. bovis BCG or MTB Ligand Stimulation. Bovine MØ of BS (heterozygous for TLR2 H326Q SNP) and HF MØ (homozygous for the wild-type TLR2 sequence) were stimulated with either FSL-1 at 100 ng mL -1 (A) , 19 kDa lipoprotein antigen (represents Rv3763 or Mb3789) (EMC microcollections, Germany) or infected with M. bovis BCG at MOI = 5 (B) for 24 hr. CXCL8 concentration in cell culture supernatant was assessed by ELISA. A total of n=8 animals per breed were stimulated with FSL-1 and n=6 animals per breed were infected with M. bovis BCG. Error bars represent standard deviation from the mean. Data was analysed with two-way ANOVA and Tukey’s post hoc comparison in GraphPad Prism V8 (GraphPad Inc., USA) (*=p<0.05, ***=p<0.001).

Article Snippet: Surface expression of TLR2 was confirmed by flow cytometry using either human anti-bovine CD282 (anti-TLR2) antibody or mouse anti-human CD282 antibody, both conjugated with Alexa Flour 647 (Bio-Rad, USA).

Techniques: Sequencing, Infection, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation, Comparison