Journal: Frontiers in Nutrition

Article Title: A Cocoa Diet Can Partially Attenuate the Alterations in Microbiota and Mucosal Immunity Induced by a Single Session of Intensive Exercise in Rats

doi: 10.3389/fnut.2022.861533

Figure Lengend Snippet: Changes in small intestine gene expression: Toll-like receptor (TLR) 2 (A) , TLR4 (B) , TLR5 (C) , TLR7 (D) , free fatty acid receptor (FFAR) 2 (E) and FFAR3 (F) , polymeric immunoglobulin receptor (pIgR) (G) , retinoic acid receptor (RAR) α (H) , forkhead box P3 (FoxP3) (I) ; occludin (Ocldn) (J) , claudin-4 (Cldn4) (K) , zonula occludens (ZO) 1 (L) and ZO2 (M) gene expression. Results expressed as fold change with respect to the REF/C group. The control (C) groups are represented by smooth bars and the runner (R) groups by striped bars. REF, reference diet; CF, 5% cocoa fiber-enriched diet; C10, 10% cocoa-enriched diet. Data are expressed as mean ± SEM ( n = 8). Statistical differences (Kruskal-Wallis followed by Dunnet’s test): * p < 0.05 vs. the C group in the same diet; # p < 0.05 vs. the same exercise condition in the REF diet; λ p < 0.05 vs. the same exercise condition in the CF diet.

Article Snippet: Afterward, the Real Time (RT)-PCR was carried out using the ABI Prism 7900 HT quantitative RT-PCR system (AB) and the following specific PCR TaqMan primers (AB): pIgR (Rn00562362_m1, Inventoried, I), RARα (Rn00580551_m1, I), FoxP3 (Rn01525092_m1, I), TLR2 (Rn02133647_s1, I), TLR4 (Rn00569848_m1, I), TLR5 (Rn04219239_s1, I), TLR7 (Rn01771083_m1, I), FFAR2 (Rn02345824_s1, I), FFAR3 (Rn01457614_g1, I), Ocldn (Rn00580064_m1, I), Cldn4 (Rn01196224_s1, I), ZO1 (Rn02116071_s1, I) and ZO2 (Rn01501483_m1, I).

Techniques: Expressing

Journal: Heliyon

Article Title: Activation of TLR-pathway to induce host Th1 immune response against visceral leishmaniasis: Involvement of galactosylated-flavonoids

doi: 10.1016/j.heliyon.2022.e09868

Figure Lengend Snippet: TLR4 and MyD88 are required for Q-gal and K-gal mediated antileishmanial response. A. RAW264.7 cells transfected with siRNAs specific to TLR4, TLR2 and MyD88. Control group of mice was transfected with control siRNA (control). 24 h post-transfection, cells were recuperated and TLR4, TLR2 and MyD88 levels measured by western blots. GAPDH was used as loading controls. Blots are representative of three separate experiments. For the original blots of TLR4, TLR2, MyD88 and GAPDH, refer to supplementary figure number S7A, S7B, S7C & S7D respectively. B. TLR4 transfected, untransfected and TLR2 transfected RAW264.7 cells were infected with LD (APC/parasite 1:20) for 24 h and then treated with 25 μg/mL of Q-gal and K-gal for the next 24 h. Intracellular parasite number was determined by Giemsa staining and expressed as parasites/100 mΦ. The results are demonstrative of three independent experiments and data presented are mean ± SD; n = 3. ∗p values versus corresponding infected control; paired two-tailed Student’s t-test. ∗p < 0.0001, ∗∗p < 0.001 and the other values are not significant compared to P < 0.01. C. RAW264.7 cells transfected with MyD88 and control siRNA (control) and were infected with LD (APC/parasite 1:20) for 6h. RAW264.7 cells were also infected with LD (APC/parasite 1:20) in presence or absence of a TRIF inhibitory peptide, Pepinh-TRIF (100 μM), for six hours. Non-ingested promastigotes were removed by washing, and cells were cultured for another 18 h. Infected APCs were treated with Q-gal and K-gal (25 μg/mL) for twenty-four hours. Intracellular parasites were counted by Giemsa staining. Experiments were performed in triplicate and repeated three times each and one set of demonstrative data is shown. Error bars signify mean ± SD, ∗p < 0.0001 and the other values are not significant compared to p < 0.01; paired two-tailed Student’s t-test.

Article Snippet: 1 μg of appropriate siRNA or control siRNA were added to the RAW264.7 cells to achieve transfection of the desired level, following manufacturer's instructions (Santa Cruz Biotechnology; sc-40261, sc-40257 & sc-45987 for TLR4 siRNA, TLR2 siRNA & MyD88 siRNA, respectively).

Techniques: Transfection, Western Blot, Infection, Staining, Two Tailed Test, Cell Culture

Journal: Heliyon

Article Title: Activation of TLR-pathway to induce host Th1 immune response against visceral leishmaniasis: Involvement of galactosylated-flavonoids

doi: 10.1016/j.heliyon.2022.e09868

Figure Lengend Snippet: In vivo efficacy of Q-gal and K-gal against L. donovani infection through TLR4. Q-gal and K-gal were administered at an amount of 5 mg/kg/day orally for 10 successive days starting at 60th day post-infection in BALB/c mice. Animals were sacrificed 20 days after treatment and splenic (A) and hepatic parasite loads (B) were determined for all groups in LDU as described in the materials and method section previously. Parasite load in bone marrow was determined as parasites/100 nucleated cells (C). Data shown as mean ± SD of ten animals/group, and are demonstrative of three independent experiments. ∗p < 0.0001 and the other values are not significant compared to P < 0.01; related with infected control groups at all time points; paired two-tailed Student’s t-test. 5 mg/kg/day of Q-gal and K-gal orally were given to sixty days AG83 and GE1F8R infected TLR4 deficient C3H/HeJ mice for 10 consecutive days. The parasite burdens in liver and spleen (log 10 LDU, D) and in bone marrow (parasites/100 nucleated cells, E) of each animals were determined at 20 days post-treatment. The results are demonstrative of three independent experiments and data represent as mean ± SD; n = 5. ∗p < 0.0001 vs corresponding infected group; paired two-tailed Student’s t-test.

Article Snippet: 1 μg of appropriate siRNA or control siRNA were added to the RAW264.7 cells to achieve transfection of the desired level, following manufacturer's instructions (Santa Cruz Biotechnology; sc-40261, sc-40257 & sc-45987 for TLR4 siRNA, TLR2 siRNA & MyD88 siRNA, respectively).

Techniques: In Vivo, Infection, Two Tailed Test

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Increased toll‐like receptor 4 ( TLR 4) expression in the myocardium of chronic heart failure ( CHF ) rats. ( A ) TLR 4 mRNA levels in infarct and remote myocardium of sham and CHF rats (n = 6/group). ( B ) Representative Western blot images and ( C ) quantification of TLR 4 proteins in infarct and remote myocardium of sham and CHF rats (n = 4/group). ( D ) Representative immunohistochemistry images of heart sections stained with TLR 4 (green) and CD 45 (red). The yellow box indicates the enlarged area shown on the right (data are means ± SD , * P < 0.05, ** P < 0.01 versus respective sham).

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

Techniques: Expressing, Western Blot, Immunohistochemistry, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Increased toll‐like receptor 4 ( TLR 4) expression in the surviving cardiomyocytes of chronic heart failure ( CHF ) rats. ( A ) Representative immunofluorescent images of TLR 4 in cardiomyocytes isolated from sham and CHF rats. ( B ) TLR 4 mRNA levels in cardiomyocytes isolated from sham and CHF rats. ( C ) Representative Western blot images and ( D ) quantification of TLR 4 proteins in cardiomyocytes isolated from sham and CHF rats (data are means ± SD , n = 6/group, ** P < 0.01 versus sham).

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

Techniques: Expressing, Isolation, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Toll‐like receptor 4 ( TLR 4)‐sh RNA lentivirus reduced myocardial inflammation and improved heart function after myocardial infarction ( MI ). The rats received intra‐myocardial injection of normal saline ( NS ), control‐sh RNA lentivirus or TLR 4‐sh RNA lentivirus (1 × 10 9 TU /ml, 100 μl/heart) just after left anterior descending coronary artery ( LAD ) ligation or sham operation. All examinations were performed after 4 weeks of MI . ( A ) Expression of green fluorescent protein ( GFP ; green), the marker gene carried by TLR 4‐sh RNA lentivirus, in the myocardium. The nuclei were counter‐stained with Hoechst 33258 (blue). ( B ) Representative Western blot images and quantification of TLR 4 proteins in sham and chronic heart failure ( CHF ) myocardium. ( C ) tumour necrosis factor ( TNF )‐α and interleukin ( IL )‐6 protein contents in infarct and remote myocardium. ( D ) Representative images of Masson's trichrome staining (upper panel) and quantification (lower panel) of post‐infarct failing hearts, showing that TLR 4‐sh RNA lentivirus reduced cardiac fibrosis. Cross‐sections were cut at the midhorizontal plane of the fixed paraffin‐embedded heart, and stained with Masson's trichrome reagents. ( E ) Infarct size of post‐infarct failing hearts. ( F ) Fractional shortening (%) of the left ventricle (data are means ± SD , n = 4/group, a P < 0.05, A P < 0.01 versus respective sham‐ NS ; B P < 0.01 versus respective CHF ‐ NS ).

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

Techniques: Injection, Ligation, Expressing, Marker, Staining, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Enhanced binding activity of toll‐like receptor 4 ( TLR 4) in chronic heart failure ( CHF ) cardiomyocytes to lipopolysaccharide ( LPS ) and heat shock protein 60 ( HSP 60). Isolated cardiomyocytes were cultured in a CO 2 incubator at 37°C for 24 hrs, then the binding assay was performed at 4°C for 30 min. To block TLR 4, cultured cardiomyocytes were incubated with TLR 4 neutralizing antibody (anti‐ TLR 4, 5 μg/ml) at 37°C for 15 min., and subsequently incubated with FITC ‐ LPS or OG ‐ HSP 60 at 4°C for 30 min. ( A ) Representative fluorescent images of isolated cardiomyocytes after the incubation with FITC ‐ LPS (green) or OG ‐ HSP 60 (green). ( B ) Binding curves of FITC ‐ LPS to cardiomyocytes. ( C ) Binding curves of OG ‐ HSP 60 to cardiomyocytes.

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

Techniques: Binding Assay, Activity Assay, Isolation, Cell Culture, Blocking Assay, Incubation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Increased cytokine production mediated by toll‐like receptor 4 ( TLR 4) in chronic heart failure ( CHF ) cardiomyocytes. Cultured cardiomocytes from sham and CHF rats were treated with lipopolysaccharide ( LPS ; 1 μg/ml) or heat shock protein 60 ( HSP 60; 1 μg/ml) for 6 hrs. TLR 4 neutralizing antibody (anti‐ TLR 4, 5 μg/ml) was added 15 min before LPS or HSP 60 treatment. ( A ) Tumour necrosis factor ( TNF )‐α and interleukin ( IL )‐6 mRNA levels (n = 6/group). ( B ) The amount of TNF ‐α and IL ‐6 released into culture supernatant (n = 6/group). ( C ) Representative Western blot images and quantification of p65 in the nuclei of cardiomyocytes from three independent experiments (data are means ± SD , a P < 0.05, A P < 0.01 versus respective sham; b P < 0.05, B P < 0.01 versus sham‐blank; c P < 0.05, C P < 0.01 versus CHF ‐blank; d P < 0.05, D P < 0.01 versus respective LPS ; e P < 0.05, E P < 0.01 versus respective HSP60).

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

Techniques: Cell Culture, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Up‐regulated TLR 4 in cardiomyocytes exacerbates heart failure after long‐term myocardial infarction

doi: 10.1111/jcmm.12659

Figure Lengend Snippet: Primers for real‐time PCR

Article Snippet: The membrane was then blocked with 5% non‐fat dried milk, and probed with the primary antibody against TLR4 (Cat. NB100‐56566; Novus Biologicals, Littleton, CO, USA) followed by the peroxidase‐conjugated secondary antibody, at the concentration of 1:500 and 1:1000 respectively.

Techniques: