tlr 4  (Hycult Biotech)

Journal: Human reproduction (Oxford, England)

Article Title: Toll-like receptor 4-mediated growth of endometriosis by human heat-shock protein 70.

doi: 10.1093/humrep/den195

Figure Lengend Snippet: Figure 1: TLR4 protein and gene expression in macrophages. (A) The immunolocalization of Toll-like receptor 4 (TLR4) in CD68-positive macrophages (Mf) derived from the peritoneal fluid of women with or without endometriosis. (B) Western-blot analysis showing a band of TLR4 of 78 kDa molecular size. A lysate of Ramos, a B-lymphoma cell line was used to show a control band posi- tive for TLR4. For Ramos cells, Lane 1 indicates plasma lysate, Lane 2 indicates 50% diluted fraction of plasma lysate, Lane 3 indicates expression in total cell lysates (B). For Mf, Lane 1 indicates plasma lysate, Lanes 2 and 3 indicate 50 and 100% dilution of plasma lysate and Lane 4 indicates TLR4 expression in total cell lysates. We found more expression of TLR4 in total cell lysates and minimal expression in diluted fraction of plasma lysate. (C) TLR4 mRNA (406 bp) expression was detected by standard RT-PCR. Total RNA was extracted from cultured Mf derived from three women each with endometriosis (endoþ) and without endometriosis (endo2).

Article Snippet: A blocking experiment was performed with anti-TLR4 antibody (10 mg/ml) (HTA-125, HyCult Biotechnology) 20 min prior to the treatment with recombinant human Hsp70 (10 mg/ml) in order to examine any change in the secretion of cytokines and growth factors in culture media without washing the pre-incubated antibodies.

Techniques: Gene Expression, Derivative Assay, Western Blot, Control, Clinical Proteomics, Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture

Journal: Human reproduction (Oxford, England)

Article Title: Toll-like receptor 4-mediated growth of endometriosis by human heat-shock protein 70.

doi: 10.1093/humrep/den195

Figure Lengend Snippet: Figure 3: Neutralizing effect of anti-TLR4 antibody on the levels of HGF, VEGF, IL-6 and TNFa in the culture media of Mf (105 cells /well) derived from the peritoneal fluid of women with endometriosis. Mf were pre-treated with anti-TLR4 antibody (10 mg/ml) (black bar) and without antibody (white bar) for 20 min and then further treated with and without Hsp70 for a period of 24 h. Pre-treatment of cells with anti-TLR4 antibody was able to significantly decrease all these macromol- ecules when compared with non-pre-treatment group (P , 0.05 for each). All these data are expressed as mean+SEM of three separate exper- iments for each group and were normalized with same number of cells.

Article Snippet: A blocking experiment was performed with anti-TLR4 antibody (10 mg/ml) (HTA-125, HyCult Biotechnology) 20 min prior to the treatment with recombinant human Hsp70 (10 mg/ml) in order to examine any change in the secretion of cytokines and growth factors in culture media without washing the pre-incubated antibodies.

Techniques: Derivative Assay

Journal: Human reproduction (Oxford, England)

Article Title: Toll-like receptor 4-mediated growth of endometriosis by human heat-shock protein 70.

doi: 10.1093/humrep/den195

Figure Lengend Snippet: Figure 4: HGF gene expression in macrophages by human Hsp70. (A) The effect of a variable concentration of Hsp70 (0–10 mg/ml) and anti-TLR4 antibody (10 mg/ml) on the mRNA expression of HGF in per- itoneal Mf derived from women with or without endometriosis was studied by standard RT-PCR. (B) The individual mRNA band (505 bp) of HGF was normalized with the corresponding band of internal control (b-actin) and is represented by the fold increase of their corresponding control (without treatment with Hsp70). Values of each transcript after single treatment with Hsp70 or pre-treatment with anti-TLR4 antibody (10 mg/ml) were normalized to 1 (dose 0). For HGF, a significance of P , 0.05 was found at the dose of 1 and 10 mg/ml of Hsp70 (endometriosis versus non-endometriosis) and P , 0.05 was found when compared with anti-TLR4 antibody non-treated Mf. The results are expressed as mean+ SEM of three different experiments derived from three separate patients.

Article Snippet: A blocking experiment was performed with anti-TLR4 antibody (10 mg/ml) (HTA-125, HyCult Biotechnology) 20 min prior to the treatment with recombinant human Hsp70 (10 mg/ml) in order to examine any change in the secretion of cytokines and growth factors in culture media without washing the pre-incubated antibodies.

Techniques: Gene Expression, Concentration Assay, Expressing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Human reproduction (Oxford, England)

Article Title: Toll-like receptor 4-mediated growth of endometriosis by human heat-shock protein 70.

doi: 10.1093/humrep/den195

Figure Lengend Snippet: Figure 5: Single and combined effect of exogenous Hsp70 and LPS on the proliferation of stromal cells derived from the eutopic endome- tria of women with endometriosis (black bar) and without endometrio- sis (white bar) as measured by 5-bromo-2-deoxyuridine incorporation. Different concentrations of recombinant human Hsp70 (0, 1, 5, 10 mg/ ml) were applied to stromal cells as shown in (A). The single and com- bined treatment of stromal cells with Hsp70 (10 mg/ml), LPS (10 ng/ ml) and pre-treatment of these cells with either polymyxin B (1 mg/ ml) or anti-TLR4 antibody (10 mg/ml) are shown in (B). The results are represented as percentage of control (without any treatment) expressed as mean+ SEM of three different experiments derived from three separate patients. A. P , 0.05 versus non-treated cells; B. P , 0.05 (Hsp70 versus control), P , 0.05 (LPS versus control), P , 0.05 (LPS alone versus LPS þ polymyxin B); P , 0.05 (com- bined Hsp70 þ LPS versus control); P , 0.05 (anti-TLR4 pre-treated cells versus without anti-TLR4 pre-treated cells).

Article Snippet: A blocking experiment was performed with anti-TLR4 antibody (10 mg/ml) (HTA-125, HyCult Biotechnology) 20 min prior to the treatment with recombinant human Hsp70 (10 mg/ml) in order to examine any change in the secretion of cytokines and growth factors in culture media without washing the pre-incubated antibodies.

Techniques: Derivative Assay, Recombinant, Control