Journal: Pharmaceutics

Article Title: Effects of the Interleukin-6 Receptor Blocker Sarilumab on Metabolic Activity and Differentiation Capacity of Primary Human Osteoblasts

doi: 10.3390/pharmaceutics14071390

Figure Lengend Snippet: Concentrations of soluble IL-6 receptor (sIL-6R) and soluble gp130 (sgp130) secreted by human osteoblasts (passage 3) after a 72 h stimulation with different concentrations of IL-6 ( n = 5 independent donors (female: n = 4, male: n = 1); mean age: 77.8 ± 4.4 year). Protein contents were analyzed via ELISA and related to total protein in the cell culture supernatants. All results are shown as median and minimum/maximum values.

Article Snippet: This was followed by stimulation with IL-6 (Peprotech, Hamburg, Germany), rh sIL-6R (CD126; ImmunoTools, Friesoythe; Germany), and sarilumab (Kevzara ® , Sanofi-Aventis, Montpellier, France).

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Pharmaceutics

Article Title: Effects of the Interleukin-6 Receptor Blocker Sarilumab on Metabolic Activity and Differentiation Capacity of Primary Human Osteoblasts

doi: 10.3390/pharmaceutics14071390

Figure Lengend Snippet: Influence of different IL-6 and sIL-6R concentrations on osteoblastic viability and differentiation capacity after 24 h ( a – c , g – i ) and 72 h ( d – f , j – l ) of exposure. ( a , d ) Cell viability was tested via water-soluble tetrazolium salt (WST) 1 assay ( n = 6 independent donors (passage 3; female: n = 1, male: n = 5); mean age: 71.5 ± 11.6 year). ( b , e ) Alkaline phosphatase (ALP) activity of osteoblasts, which was quantified via hydrolysis of p-nitrophenylphosphate ( n = 6 independent donors (passage 3; female: n = 1, male: n = 5); mean age: 71.5 ± 11.6 year). ( c , f ) Release of c-terminal propeptide of collagen type 1 (C1CP) in the cell culture supernatant ( n = 6 independent donors (passage 3; female: n = 1, male: n = 5); mean age: 71.5 ± 11.6 year). ( g – l ) Gene expression analysis of IL6, IL6R, and IL6ST in human osteoblasts (24 h: n = 4–6 independent donors, 72 h: n = 6 independent donors (passage 3; female: n = 1, male: n = 5); mean age: 71.5 ± 11.6 year). Results were calculated by 2 −ΔΔCt method (% unstimulated control). All data are shown as box plots. Statistical significance was determined via two-way ANOVA or repeated-measures two-way ANOVA with Bonferroni’s post hoc test: * p < 0.05, ** p < 0.01; *** p < 0.001, **** p < 0.0001 (significance between IL-6 concentrations); § p < 0.05 (significantly different from sample with same IL-6 concentration at 5 ng/mL sIL-6R); # p < 0.05; ## p < 0.01; ### p < 0.001 (significantly different from sample with same IL-6 concentration at 50 ng/mL sIL-6R).

Article Snippet: This was followed by stimulation with IL-6 (Peprotech, Hamburg, Germany), rh sIL-6R (CD126; ImmunoTools, Friesoythe; Germany), and sarilumab (Kevzara ® , Sanofi-Aventis, Montpellier, France).

Techniques: WST-1 Assay, Activity Assay, Cell Culture, Expressing, Concentration Assay

Journal: Pharmaceutics

Article Title: Effects of the Interleukin-6 Receptor Blocker Sarilumab on Metabolic Activity and Differentiation Capacity of Primary Human Osteoblasts

doi: 10.3390/pharmaceutics14071390

Figure Lengend Snippet: Cell morphology of human osteoblasts (passage 3, male donor, 68 year) after exposure to IL-6, sIL-6R ± sarilumab for 72 h. ( a – c ) Fluorescence stain of the actin cytoskeleton (green fluorescence) and cell nuclei (blue fluorescence). ( d – f ) Overlay of the stained actin cytoskeleton, nuclei, and enhanced darkfield microscopy to visualize intra- and extracellular structures. Bar: 50 µm.

Article Snippet: This was followed by stimulation with IL-6 (Peprotech, Hamburg, Germany), rh sIL-6R (CD126; ImmunoTools, Friesoythe; Germany), and sarilumab (Kevzara ® , Sanofi-Aventis, Montpellier, France).

Techniques: Fluorescence, Staining, Microscopy

Journal: Pharmaceutics

Article Title: Effects of the Interleukin-6 Receptor Blocker Sarilumab on Metabolic Activity and Differentiation Capacity of Primary Human Osteoblasts

doi: 10.3390/pharmaceutics14071390

Figure Lengend Snippet: Release of soluble gp130 (sgp130), osteoblastic viability, and alkaline phosphatase (ALP) activity after exposure to IL-6, sIL-6R ± sarilumab for 72 h. ( a ) Secreted sgp130 was determined in supernatants of exposed osteoblasts in passage 3 ( n = 4 independent donor (female: n = 3, male: n = 1; mean age: 77.5 ± 5.1 year)— n = 5 independent donors (female: n = 4, male: n = 1); mean age: 77.8 ± 4.4 year). ( b ) Cell viability was tested via water-soluble tetrazolium salt (WST-)1 assay ( n = 12 independent donors in passage 3 (female: n = 8, male: n = 4); mean age: 67.3 ± 16.1 year). ( c ) Alkaline phosphatase (ALP) activity of osteoblasts, which was quantified via hydrolysis of p-nitrophenyl phosphate ( n = 12 independent donors in passage 3 (female: n = 8, male: n = 4); mean age: 67.3 ± 16.1 year). ( d ) ALP activity related to metabolic activity ( n = 12 independent donors in passage 3 (female: n = 8, male: n = 4); mean age: 67.3 ± 16.1 year). All data are shown as box plots. Statistical significance was determined via repeated-measures two-way ANOVA with Bonferroni’s post hoc test: ** p < 0.05; *** p < 0.001, **** p < 0.0001 (significantly different from samples with sarilumab costimulation); a p < 0.05 (significance between IL-6 concentrations).

Article Snippet: This was followed by stimulation with IL-6 (Peprotech, Hamburg, Germany), rh sIL-6R (CD126; ImmunoTools, Friesoythe; Germany), and sarilumab (Kevzara ® , Sanofi-Aventis, Montpellier, France).

Techniques: Activity Assay, WST-1 Assay

Journal: Pharmaceutics

Article Title: Effects of the Interleukin-6 Receptor Blocker Sarilumab on Metabolic Activity and Differentiation Capacity of Primary Human Osteoblasts

doi: 10.3390/pharmaceutics14071390

Figure Lengend Snippet: Concentrations of the c-terminal propeptide of collagen 1 (C1CP; n = 12 independent donors in passage 3 (female: n = 8, male: n = 4); mean age: 67.3 ± 16.1 year) and osteopontin ( n = 4 independent donor (female: n = 3, male: n = 1; mean age: 77.5 ± 5.1 year)— n = 5 independent donors (female: n = 4, male: n = 1; mean age: 77.8 ± 4.4 year) after stimulation with IL-6, sIL-6R, and sarilumab. Protein contents were analyzed via ELISA and related to total protein. All results are shown as median and minimum/maximum values.

Article Snippet: This was followed by stimulation with IL-6 (Peprotech, Hamburg, Germany), rh sIL-6R (CD126; ImmunoTools, Friesoythe; Germany), and sarilumab (Kevzara ® , Sanofi-Aventis, Montpellier, France).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Pharmaceutics

Article Title: Effects of the Interleukin-6 Receptor Blocker Sarilumab on Metabolic Activity and Differentiation Capacity of Primary Human Osteoblasts

doi: 10.3390/pharmaceutics14071390

Figure Lengend Snippet: Overview of sarilumab-responsive and nonresponsive osteoblasts after 72 h stimulation with IL-6, sIL-6R ± sarilumab. ( a , b ) Cell viability was tested via water-soluble tetrazolium salt (WST-)1 assay. ( c , d ) ALP activity of osteoblasts, which was quantified via hydrolysis of p-nitrophenyl phosphate (pNpp). ( e , f ) ALP activity normalized to metabolic activity. All data are shown as single data points with connecting lines for the same cell donor ( n = 7 independent donors (passage 3) for responder (female: n = 4, male: n = 3; mean age: 70.3 ± 6.0 year) and n = 5 independent donors (passage 3) for nonresponder (female: n = 4, male: n = 1; mean age 65.0 ± 24.0 year) with female donors (green dot and purple square) and male donors (green up-facing triangle and purple down-facing triangle) assigned separate symbols. Statistical significance was determined via repeated-measures two-way ANOVA with Bonferroni’s post hoc test: ** p < 0.01; **** p < 0.0001 (significantly different from samples with sarilumab costimulation); a,b p < 0.05 (same letters indicate significance between different IL-6 concentrations) ( g ) Overview of strongest regulated genes determined via transcriptome analysis. Here, RNA from a total of seven independent donors was pooled and extracted for gene expression analysis via Clariom D Human array. ( h ) Validation of selected genes that were regulated in transcriptome analysis. To this purpose, human osteoblasts of three independent donors in passage 3 (female: n = 1, male: n = 2); mean age: 76.7 ± 9.2 year) were treated with sIL-6R, sarilumab ± IL-6 over a period of 72 h. Gene expression patterns were calculated by 2 −ΔΔCt method (% unstimulated control). Data are shown as means ± SD. Statistical significance was determined via repeated-measures two-way ANOVA with Bonferroni’s post hoc test: * p < 0.05 (significance between IL-6 concentrations); § p < 0.05 (significantly different from samples without sarilumab co-stimulation). Abbreviations: SFRP: Secreted frizzled-related protein; MMP: Matrix metalloprotease; SPP1: Osteopontin; LBP: Lipopolysaccharide binding protein; IBSP: Integrin-Binding Sialoprotein; MGP: Matrix Gla protein; ALP: Alkaline Phosphatase; gp130: Glycoprotein 130.

Article Snippet: This was followed by stimulation with IL-6 (Peprotech, Hamburg, Germany), rh sIL-6R (CD126; ImmunoTools, Friesoythe; Germany), and sarilumab (Kevzara ® , Sanofi-Aventis, Montpellier, France).

Techniques: WST-1 Assay, Activity Assay, Expressing, Binding Assay

Journal: Pharmaceutics

Article Title: Effects of the Interleukin-6 Receptor Blocker Sarilumab on Metabolic Activity and Differentiation Capacity of Primary Human Osteoblasts

doi: 10.3390/pharmaceutics14071390

Figure Lengend Snippet: Cell morphology ( a – f ) and mineralization capacity ( g – l ) of human osteoblasts following exposure to IL-6, sIL-6R ± sarilumab over 72 h in calcium-supplemented medium (passage 3, male donor, 68 year). ( a – c ) Fluorescence stain of the actin cytoskeleton (green fluorescence) and cell nuclei (blue fluorescence; bar: 50 µm). ( d – f ) Overlay of the stained actin cytoskeleton, nuclei, and enhanced darkfield microscopy to visualize intra- and extracellular structures (bar: 50 µm). ( g – l ) Hydroxyapatite portion of bone-like nodules (green fluorescence) deposited by the osteoblasts after 7 and 14 days (bar: 50 µm).

Article Snippet: This was followed by stimulation with IL-6 (Peprotech, Hamburg, Germany), rh sIL-6R (CD126; ImmunoTools, Friesoythe; Germany), and sarilumab (Kevzara ® , Sanofi-Aventis, Montpellier, France).

Techniques: Fluorescence, Staining, Microscopy

Journal: Pharmaceutics

Article Title: Effects of the Interleukin-6 Receptor Blocker Sarilumab on Metabolic Activity and Differentiation Capacity of Primary Human Osteoblasts

doi: 10.3390/pharmaceutics14071390

Figure Lengend Snippet: Mineralization capacity of human osteoblasts ( n = 6 independent donors in passage 3 (female: n = 3, male: n = 3; mean age: 65.3 ± 7.8 year). Cells were stimulated with IL-6, sIL-6R ± sarilumab over a period of 7 or 14 days in calcium-supplemented medium. Afterward, deposition of the hydroxyapatite portion of bone-like nodules was quantified via OsteoImage™. Fluorescence signals of mineralization were detected at excitation/emission wavelengths of 492/520 nm using a microplate reader. Statistical significance was determined via repeated-measures two-way ANOVA with Bonferroni’s post hoc test: **** p < 0.0001 (significantly different from samples of the same stimulation group at 7 days).

Article Snippet: This was followed by stimulation with IL-6 (Peprotech, Hamburg, Germany), rh sIL-6R (CD126; ImmunoTools, Friesoythe; Germany), and sarilumab (Kevzara ® , Sanofi-Aventis, Montpellier, France).

Techniques: Fluorescence