Journal: Journal of Histochemistry and Cytochemistry

Article Title: The Expression of Toll-like Receptors in Normal Human and Murine Gastrointestinal Organs and the Effect of Microbiome and Cancer

doi: 10.1369/0022155416656154

Figure Lengend Snippet: Used TLR Antibodies With Dilutions, Catalog Numbers, and Manufacturer.

Article Snippet: TLR3 , 1:25 , IMG-315A , Imgenex.

Techniques:

Journal: Journal of Histochemistry and Cytochemistry

Article Title: The Expression of Toll-like Receptors in Normal Human and Murine Gastrointestinal Organs and the Effect of Microbiome and Cancer

doi: 10.1369/0022155416656154

Figure Lengend Snippet: Typical Toll-like receptor (TLR) expression patterns from the human ascending colon. TLRs are expressed throughout the epithelium with a diffuse manner. Paired figures from ascending colon organ donor (left) and tumor-adjacent normal epithelium (right) are presented: (A) TLR1, (B) TLR2, (C) TLR3, (D) TLR4, (E) TLR5, (F) TLR6, (G) TLR7, (H) TLR8, and (I) TLR9. 20× magnification was used and 50-µm scale bar is in panel I.

Article Snippet: TLR3 , 1:25 , IMG-315A , Imgenex.

Techniques: Expressing

Journal: Journal of Histochemistry and Cytochemistry

Article Title: The Expression of Toll-like Receptors in Normal Human and Murine Gastrointestinal Organs and the Effect of Microbiome and Cancer

doi: 10.1369/0022155416656154

Figure Lengend Snippet: TLR1 to TLR9 Protein Expression Detected With Immunohistochemistry From Different Anatomic Segments of the Alimentary Tract.

Article Snippet: TLR3 , 1:25 , IMG-315A , Imgenex.

Techniques: Expressing, Immunohistochemistry

Journal: Journal of Histochemistry and Cytochemistry

Article Title: The Expression of Toll-like Receptors in Normal Human and Murine Gastrointestinal Organs and the Effect of Microbiome and Cancer

doi: 10.1369/0022155416656154

Figure Lengend Snippet: Typical Toll-like receptor (TLR) expression patterns from the conventional and germ-free mouse small intestines. TLRs are expressed throughout the epithelium with a diffuse manner. Paired figures from small intestine conventional (left) and germ-free mice (right) are presented: (A) TLR1, (B) TLR2, (C) TLR3, (D) TLR4, (E) TLR5, (F) TLR6, (G) TLR7, (H) TLR8, and (I) TLR9. 10× magnification was used and 50-µm scale bar is in panel I.

Article Snippet: TLR3 , 1:25 , IMG-315A , Imgenex.

Techniques: Expressing

Journal: The Journal of Experimental Medicine

Article Title: Herpes simplex virus encephalitis in a patient with complete TLR3 deficiency: TLR3 is otherwise redundant in protective immunity

doi: 10.1084/jem.20101568

Figure Lengend Snippet: Compound heterozygous mutations in TLR3 in a child with HSE. (A) Family pedigree with allele segregation. The patient, indicated in black, carries the compound mutations P554S (red) and E746X (blue) in TLR3 . The other family members heterozygous for the P554S or E746X mutation are indicated by vertical lines. TLR3 genotypes are indicated under each individual. (B) Compound heterozygous c.1660C>T and c.2236G>T mutations in TLR3 in the patient. The sequences of the PCR products of gDNA from a healthy control (C) and from the patient (P) are shown. The c.1660C>T and c.2236G>T mutations were confirmed in gDNA and cDNA from leukocytes and fibroblasts. (C) Schematic diagram of the human TLR3 gene. The coding exons are numbered with Roman numerals and delimited by a vertical bar. The regions corresponding to the leader sequence (L), leucine-rich repeats (LRR), transmembrane domain (TM), linker region (LR), and the TIR domain are shaded in light gray and are delimited by dark gray lines. The two leucine-rich repeats with an insertion are indicated by asterisks.

Article Snippet: These polyvinylidene difluoride membranes were then probed with a goat anti–human TLR3 antibody directed against the human TLR3 ectodomain (R&D Systems).

Techniques: Mutagenesis, Control, Sequencing

Journal: The Journal of Experimental Medicine

Article Title: Herpes simplex virus encephalitis in a patient with complete TLR3 deficiency: TLR3 is otherwise redundant in protective immunity

doi: 10.1084/jem.20101568

Figure Lengend Snippet: P554S and E746X TLR3 alleles are loss-of-function. (A) TLR3 mRNA levels were determined by RT-qPCR in P2.1 TLR3-deficient fibrosarcoma cells not transfected (P2.1) or stably transfected with WT TLR3 (P2.1-TLR3 WT), P554S (P2.1-TLR3 P554S) or E746X (P2.1-TLR3 E746X) mutant TLR3 , N284I (P2.1-TLR3 N284I) or L412F (P2.1-TLR3 L412F) TLR3 variant, or mock vector (P2.1-mock). β-Glucuronidase (GUS) was included for normalization. The results shown are representative of three independent experiments. (B) TLR3 expression, assessed by immunoblotting (IB) after immunoprecipitation (IP), in P2.1 TLR3-deficient fibrosarcoma cells not stably transfected (P2.1) or transfected with WT TLR3 , P554S or E746X mutant TLR3 , N284I or L412F TLR3 variant, or mock vector, with an anti-TLR3 N-terminal (N) antibody and an anti-HA C-terminal tag antibody. The experiment shown is representative of three experiments performed. TLR3 protein extracted from HEK293T cells transfected with human WT TLR3 was included as a positive control. We used β-tubulin as an internal expression control for immunoblotting. (C) IL29 (IFN-λ1) mRNA induction, without stimulation (NS) or after 4 h of stimulation with poly(I:C), assessed by RT-qPCR, in P2.1 TLR3-deficient fibrosarcoma cells not transfected (P2.1) or transfected with WT TLR3 , P554S or E746X mutant TLR3 , N284I or L412F TLR3 variant, or mock vector. All transfections generated stable cell lines. β-Glucuronidase was included for normalization. Mean values ± SD were calculated from two independent experiments. (D) IFN-λ production without stimulation (NS) or after 24 h of stimulation with poly(I:C), as assessed by ELISA, in P2.1 TLR3-deficient fibrosarcoma cells not transfected (P2.1) or transfected with WT TLR3 , P554S or E746X mutant TLR3 , N284I or L412F TLR3 variant, or mock vector. All transfections generated stable cell lines. One experiment representative of the three performed is shown. Mean values ± SD were calculated from triplicates in one experiment.

Article Snippet: These polyvinylidene difluoride membranes were then probed with a goat anti–human TLR3 antibody directed against the human TLR3 ectodomain (R&D Systems).

Techniques: Quantitative RT-PCR, Transfection, Stable Transfection, Mutagenesis, Variant Assay, Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation, Positive Control, Control, Generated, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Herpes simplex virus encephalitis in a patient with complete TLR3 deficiency: TLR3 is otherwise redundant in protective immunity

doi: 10.1084/jem.20101568

Figure Lengend Snippet: Absence of response to TLR3 in the patient’s fibroblasts. (A) Production of IFN-λ, IFN-β, and IL-6 by SV40-fibroblasts after stimulation with various doses of poly(I:C) for 12 or 24 h, as assessed by ELISA, with cells from a healthy control (C), the patient (P), a patient with partial AD TLR3 deficiency (AD TLR3), a patient with complete AR UNC-93B deficiency (UNC-93B −/− ), and a NEMO-deficient patient (NEMO IP). The panels illustrate mean values ± SD for triplicates of one experiment, representative of three performed. (B) TLR3 mRNA levels in SV40-fibroblasts were determined by RT-qPCR on RNA samples from five healthy controls (C), the patient (P), an AD TLR3 patient, and a UNC-93B −/− patient. β-Glucuronidase (GUS) was used for normalization. One representative experiment of three performed is shown. (C) IL29 (IFN-λ1) and IFNB mRNA levels in SV40-fibroblasts from a control, the patient, an AD TLR3 patient, and a UNC-93B −/− patient, unstimulated (NS) and stimulated for 2, 4, 6, and 8 h with poly(I:C). The panels illustrate results from a single experiment, representative of three performed. (D) Production of IFN-λ and IL-6 by SV40-fibroblasts from a control, the patient, an AD TLR3 patient, a UNC-93B −/− patient, and a NEMO IP patient, unstimulated or after stimulation with the TLR3-specific agonist poly(A:U) for 12 and 24 h, as assessed by ELISA. The panels illustrate mean values ± SD for triplicates of one experiment, representative of three performed. (E) IRF-3 monomers and dimers in total cell extracts of SV40-fibroblasts from a control, the patient, an AD TLR3 patient, and a UNC-93B −/− patient, after stimulation with poly(I:C) for 1 and 2 h, as assessed by Western blotting. The results shown are representative of three independent experiments. (F) NF-κB activation was assessed by monitoring expression of the NF-κB luciferase reporter in SV40-fibroblasts from a control, the patient, an AD TLR3 patient, a UNC-93B −/− patient, and a NEMO IP patient, unstimulated or after stimulation with poly(I:C) (top) and IL-1β (bottom) for 6 h. The panels illustrate mean values ± SD for three independent experiments.

Article Snippet: These polyvinylidene difluoride membranes were then probed with a goat anti–human TLR3 antibody directed against the human TLR3 ectodomain (R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Quantitative RT-PCR, Western Blot, Activation Assay, Expressing, Luciferase

Journal: The Journal of Experimental Medicine

Article Title: Herpes simplex virus encephalitis in a patient with complete TLR3 deficiency: TLR3 is otherwise redundant in protective immunity

doi: 10.1084/jem.20101568

Figure Lengend Snippet: WT TLR3 expression rescues responsiveness to TLR3 in the patient’s fibroblasts. (A) Production of IFN-λ and IL-6 unstimulated (NS) or after 24 h of stimulation with poly(I:C) with the presence of Lipofectamine (poly(I:C)+L) or without Lipofectamine (poly(I:C)), as assessed by ELISA, in SV40-fibroblasts from a control (C), a NEMO-deficient patient (NEMO IP), the patient (P), and in SV40-fibroblasts from P transfected with an empty vector (P-mock) or the C-terminal HA-tagged pUNO-TLR3 WT vector (P-TLR3 WT). All transfections generated stable cell lines. The panels illustrate mean values ± SD for triplicates of one experiment, representative of three. (B) TLR3 expression in SV40-fibroblasts from the patient without transfection (P) or after stable transfection with human WT TLR3 (P-TLR3 WT) or mock vector (P-mock) was assessed by immunoblotting (IB) with an anti-HA C-terminally tagged antibody (C) after immunoprecipitation (IP) with an anti-TLR3 N-terminal (N) antibody. One experiment representative of the three performed is shown. TLR3 protein extracted from HEK293T cells transfected with human WT TLR3 was included as a positive control. We used β-tubulin as an internal expression control for immunoblotting.

Article Snippet: These polyvinylidene difluoride membranes were then probed with a goat anti–human TLR3 antibody directed against the human TLR3 ectodomain (R&D Systems).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control, Transfection, Plasmid Preparation, Generated, Stable Transfection, Western Blot, Immunoprecipitation, Positive Control

Journal: The Journal of Experimental Medicine

Article Title: Herpes simplex virus encephalitis in a patient with complete TLR3 deficiency: TLR3 is otherwise redundant in protective immunity

doi: 10.1084/jem.20101568

Figure Lengend Snippet: Genome-wide transcriptional evaluation of the TLR3 pathway in fibroblasts. (A) Cumulative fold change (FC) score (top) and heat maps (bottom) of the transcripts regulated by 2 h (left) or 8 h (right) of stimulation with poly(I:C) in primary fibroblasts from three healthy controls (C), the patient (P), a UNC-93B −/− patient, a patient with AD TLR3 deficiency (AD TLR3), and a patient of MyD88 deficiency (MyD88 −/− ). The cumulative score is the sum of all the fold change values >1.5 (up- or down-regulation). Heat maps show a hierarchical clustering of transcripts differentially expressed upon poly(I:C) stimulation (based on 100 differences in intensity and 1.5-fold changes compared with nonstimulated condition in healthy controls). Changes with respect to the unstimulated condition are shown by a color scale: red, up-regulated; blue, down-regulated; yellow, no change. The probes displaying differences of >100 in intensity were used to calculate the cumulative score. (B) Ranking of the 302 transcripts up-regulated after 8 h pf poly(I:C) stimulation, with a fold change of at least 2 in all three controls tested, in primary fibroblasts from three healthy controls (C), the patient (P), a UNC-93B −/− patient, an AD TLR3 patient, and an MyD88 −/− patient. (C) Networks generated from differentially expressed transcripts (up-regulated) in fibroblasts from control (C), the patient (P), a UNC-93B −/− patient, an AD TLR3 patient, and an MyD88 −/− patient after 8 h of poly(I:C) stimulation with Ingenuity Pathway Analysis software. Eligible genes or gene products regulated by these factors are represented as nodes, and the biological relationship between two nodes is represented as an edge (line). Solid and dashed lines indicate direct and indirect relationships, respectively. All edges are supported by at least one reference from the literature. Nodes are arranged according to the cellular distribution of the corresponding gene products. Up-regulated transcripts are represented in red, and down-regulated transcripts are represented in green.

Article Snippet: These polyvinylidene difluoride membranes were then probed with a goat anti–human TLR3 antibody directed against the human TLR3 ectodomain (R&D Systems).

Techniques: Genome Wide, Generated, Control, Software

Journal: The Journal of Experimental Medicine

Article Title: Herpes simplex virus encephalitis in a patient with complete TLR3 deficiency: TLR3 is otherwise redundant in protective immunity

doi: 10.1084/jem.20101568

Figure Lengend Snippet: IFN production after virus stimulation in fibroblasts from the patient. (A–C) Production of IFN-λ (top) and IFN-β (bottom) after 24 h of stimulation with VSV (A), HSV-1 (B), Para III virus, EMCV, Sindbis virus, and measles virus (MeV; C), or left unstimulated (NS), as assessed by ELISA, in SV40-fibroblasts from a control (C), patient (P), an AD TLR3-deficient patient (AD TLR3), and a UNC-93B −/− patient. The panels illustrate results from a single experiment, representative of three performed. Mean values ± SD were calculated from triplicates in one experiment.

Article Snippet: These polyvinylidene difluoride membranes were then probed with a goat anti–human TLR3 antibody directed against the human TLR3 ectodomain (R&D Systems).

Techniques: Virus, Enzyme-linked Immunosorbent Assay, Control

Journal: The Journal of Experimental Medicine

Article Title: Herpes simplex virus encephalitis in a patient with complete TLR3 deficiency: TLR3 is otherwise redundant in protective immunity

doi: 10.1084/jem.20101568

Figure Lengend Snippet: Impaired IFN-dependent virus control in fibroblasts from the patient. (A) VSV titers, estimated on Vero cells, in SV40-fibroblasts from a healthy control (C), the patient (P), an AD TLR3-deficient patient, a UNC-93B −/− patient, and a patient with complete AR STAT1-deficiency (STAT1 −/− ) at various times after VSV infection, without (left) or with (right) 18 h of prior treatment with IFN-α2b. The panels illustrate results from a single experiment, representative of two performed. (B) HSV-1 replication, quantified by GFP measurement, in SV40-fibroblasts from a healthy control, the patient, an AD TLR3-deficient patient, a UNC-93B −/− patient, and a STAT1 −/− patient at various times after HSV-1 GFP infection, without (left) or with (right) 18 h of prior treatment with IFN-α2b. (C) Viability, estimated by resazurin oxidoreduction, of SV40-fibroblasts from a healthy control, the patient, and a STAT1 −/− patient 24 h after infection with VSV at various MOIs. The cells were not treated (left) or were subjected to prior treatment (right) with recombinant IFN-α2b for 18 h. (D) Viability, estimated by resazurin oxidoreduction, of SV40-fibroblasts from a healthy control, the patient, an AD TLR3-deficient patient, a UNC-93B −/− patient, and a STAT1 −/− patient 72 h after infection with HSV-1 at various MOIs. The cells were not treated (left) or were subjected to prior treatment (right) with recombinant IFN-α2b for 18 h. (B–D) Panels illustrate results from a single experiment, representative of three performed. Mean values calculated from triplicates in one experiment are presented.

Article Snippet: These polyvinylidene difluoride membranes were then probed with a goat anti–human TLR3 antibody directed against the human TLR3 ectodomain (R&D Systems).

Techniques: Virus, Control, Infection, Recombinant

Journal: The Journal of Experimental Medicine

Article Title: Herpes simplex virus encephalitis in a patient with complete TLR3 deficiency: TLR3 is otherwise redundant in protective immunity

doi: 10.1084/jem.20101568

Figure Lengend Snippet: Normal IFN response to poly(I:C) and genome-wide transcriptional evaluation of the poly(I:C) responses in PBMCs. (A) Production of IFN-α after 24 h of stimulation with poly(I:C) in PBMCs from two healthy controls (C) and the patient (P). The PBMCs were incubated in RPMI 1640 medium supplemented with 10% FCS (top) or 1% human serum (SAB; bottom). Mean values ± SD were calculated from three independent experiments. (B) Cumulative fold change (FC) score (top) and heat maps (bottom) of the transcripts regulated by 2 h (left) or 8 h (right) of stimulation with poly(I:C) in PBMCs from three healthy controls (C+) and the patient. The cumulative score is the sum of all the fold change values >1.5 (up- or down-regulation). Heat maps represent a hierarchical clustering of transcripts differentially expressed upon poly(I:C) stimulation (based on a difference in intensity of 100 and a 1.5-fold change with respect to baseline in healthy controls). Changes with respect to unstimulated conditions are represented by a color scale: red, up-regulated; blue, down-regulated; yellow, no change. Probes giving a difference in intensity >100 were used to calculate the cumulative score. (C) Induction of IFIT1, IFIT2, IFIT3, and IL29 mRNA after 2 or 8 h of poly(I:C) stimulation in PBMCs from two healthy controls and the TLR3 −/− patient. Results from one experiment representative of the two performed are shown. (D) Networks generated from differentially expressed transcripts (up-regulated) in control and patient PBMCs after 8 h of poly(I:C) stimulation with Ingenuity Pathway Analysis software. Eligible genes or gene products regulated by these factors are represented as nodes, and the biological relationship between two nodes is represented as an edge (line). Solid and dashed lines indicate direct and indirect relationships, respectively. All edges are supported by at least one reference from the literature. Nodes are arranged according to the cellular distribution of the corresponding gene products. Up-regulated transcripts are represented in red.

Article Snippet: These polyvinylidene difluoride membranes were then probed with a goat anti–human TLR3 antibody directed against the human TLR3 ectodomain (R&D Systems).

Techniques: Genome Wide, Incubation, Generated, Control, Software