Limits...
Evaluation of Immunostimulatory Potential of Branded and US-Generic Enoxaparins in an In Vitro Human Immune System Model.

Luna E, Agrawal P, Mehta R, Vernhes C, Viskov C, Amiral J, Warren WL, Drake DR - Clin. Appl. Thromb. Hemost. (2014)

Bottom Line: Low-molecular-weight heparins (LMWHs) have several positive therapeutic effects and can also form immunostimulatory complexes with plasma proteins, such as platelet factor 4 (PF4).Production of tissue factor pathway inhibitor (TFPI), a physiologic heparin-induced inhibitor of tissue factor-induced coagulation that was used as a functional readout of biological activity of enoxaparins in these assays, was heightened in the presence of branded enoxaparin complexes, but its levels were variable in cultures treated with complexes containing US-generic enoxaparins.Analytical analyses suggest that the heightened immunostimulatory potential of some of the US-generic enoxaparin product lots could be tied to their capacity to form ultra-large and/or more stable complexes with PF4 than the other LMWHs included in this study.

View Article: PubMed Central - PubMed

Affiliation: Sanofi Pasteur, VaxDesign Campus, Orlando, FL, USA.

No MeSH data available.


Related in: MedlinePlus

Demonstration of differential immune responses induced against PF4 complexes with UFH, LMWH (branded enoxaparin), and ULMWH in MIMIC® PTE assays. MIMIC® PTE assays were treated with a broad range of PF4–heparin complexes as indicated in the figure labels. After 48 hours, the reverse-transmigrated APCs were harvest and labeled with markers specific for a variety of parameters by flow cytometry. A, Cell viability was measured by LIVE/DEAD Aqua staining over the entire dose range and (B) statistical analysis of the viability data was calculated at the highest treatment dose. C, APC activation was measured by CD86 upregulation on the CD14+HLA-DR+ DC population; (D) a statistical assessment of CD86 expression is shown for the highest treatment dose. The plotted values represent the mean ± SD for 14 donors studied in 5 distinct experiments. Ag indicates antigen; APC, antigen-presenting cell; DC, dendritic cell; LMWH, low-molecular-weight heparin; MIMIC® PTE, Modular IMmune In vitro Construct system-peripheral tissue equivalent; PF4, platelet factor 4; SD, standard deviation; UFH, unfractionated heparin; ULMWH, ultra-low-molecular-weight heparin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2 - License 3
getmorefigures.php?uid=PMC4401814&req=5

fig2-1076029614562037: Demonstration of differential immune responses induced against PF4 complexes with UFH, LMWH (branded enoxaparin), and ULMWH in MIMIC® PTE assays. MIMIC® PTE assays were treated with a broad range of PF4–heparin complexes as indicated in the figure labels. After 48 hours, the reverse-transmigrated APCs were harvest and labeled with markers specific for a variety of parameters by flow cytometry. A, Cell viability was measured by LIVE/DEAD Aqua staining over the entire dose range and (B) statistical analysis of the viability data was calculated at the highest treatment dose. C, APC activation was measured by CD86 upregulation on the CD14+HLA-DR+ DC population; (D) a statistical assessment of CD86 expression is shown for the highest treatment dose. The plotted values represent the mean ± SD for 14 donors studied in 5 distinct experiments. Ag indicates antigen; APC, antigen-presenting cell; DC, dendritic cell; LMWH, low-molecular-weight heparin; MIMIC® PTE, Modular IMmune In vitro Construct system-peripheral tissue equivalent; PF4, platelet factor 4; SD, standard deviation; UFH, unfractionated heparin; ULMWH, ultra-low-molecular-weight heparin.

Mentions: Given that complexes of heparins and positively charged proteins, such as PF4, are thought to be more immunostimulatory than heparins alone,12,24 we anticipated the evaluation of heparin innate immune system engagement might be better achieved by comparing variant PF4–heparin complexes in the MIMIC® PTE construct. Therefore, in the pilot study (see Materials and Methods section and Table 1 for additional details), graded doses of PF4–heparin complexes (200 to 12.5 µg/mL PF4 at a constant ratio to heparin) formed with UFH, branded enoxaparin, and ULMWH were examined for their impact on APC viability and activation in the MIMIC® PTE construct. (Both UFH and ULMWH were particularly crucial for this proof-of-concept study, since they were expected to trigger strong and weak immune responses, respectively, based on their anticipated capacities to form complexes with PF4.) As shown in Figure 2A, PF4–heparin complexes prepared with UFH triggered a steep reduction in cell numbers in a dose-dependent manner; for example, at the highest concentration, cell viability was decreased to 18.65% ± 14.5% from 61.6% ± 6.6% in the no-complex control (ANOVA, P ≤ .0001; Figure 2B). In contrast, even at the highest treatment dose, PF4–ULMWH complexes failed to trigger a statistically significant decrease in cell numbers (55.06% ± 7.2%; ANOVA, P > .05). Although they were not completely inert in these experiments, PF4–branded enoxaparin triggered only minimal (not statistically significant) reductions in cell viability (Figure 2A and B).


Evaluation of Immunostimulatory Potential of Branded and US-Generic Enoxaparins in an In Vitro Human Immune System Model.

Luna E, Agrawal P, Mehta R, Vernhes C, Viskov C, Amiral J, Warren WL, Drake DR - Clin. Appl. Thromb. Hemost. (2014)

Demonstration of differential immune responses induced against PF4 complexes with UFH, LMWH (branded enoxaparin), and ULMWH in MIMIC® PTE assays. MIMIC® PTE assays were treated with a broad range of PF4–heparin complexes as indicated in the figure labels. After 48 hours, the reverse-transmigrated APCs were harvest and labeled with markers specific for a variety of parameters by flow cytometry. A, Cell viability was measured by LIVE/DEAD Aqua staining over the entire dose range and (B) statistical analysis of the viability data was calculated at the highest treatment dose. C, APC activation was measured by CD86 upregulation on the CD14+HLA-DR+ DC population; (D) a statistical assessment of CD86 expression is shown for the highest treatment dose. The plotted values represent the mean ± SD for 14 donors studied in 5 distinct experiments. Ag indicates antigen; APC, antigen-presenting cell; DC, dendritic cell; LMWH, low-molecular-weight heparin; MIMIC® PTE, Modular IMmune In vitro Construct system-peripheral tissue equivalent; PF4, platelet factor 4; SD, standard deviation; UFH, unfractionated heparin; ULMWH, ultra-low-molecular-weight heparin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2 - License 3
Show All Figures
getmorefigures.php?uid=PMC4401814&req=5

fig2-1076029614562037: Demonstration of differential immune responses induced against PF4 complexes with UFH, LMWH (branded enoxaparin), and ULMWH in MIMIC® PTE assays. MIMIC® PTE assays were treated with a broad range of PF4–heparin complexes as indicated in the figure labels. After 48 hours, the reverse-transmigrated APCs were harvest and labeled with markers specific for a variety of parameters by flow cytometry. A, Cell viability was measured by LIVE/DEAD Aqua staining over the entire dose range and (B) statistical analysis of the viability data was calculated at the highest treatment dose. C, APC activation was measured by CD86 upregulation on the CD14+HLA-DR+ DC population; (D) a statistical assessment of CD86 expression is shown for the highest treatment dose. The plotted values represent the mean ± SD for 14 donors studied in 5 distinct experiments. Ag indicates antigen; APC, antigen-presenting cell; DC, dendritic cell; LMWH, low-molecular-weight heparin; MIMIC® PTE, Modular IMmune In vitro Construct system-peripheral tissue equivalent; PF4, platelet factor 4; SD, standard deviation; UFH, unfractionated heparin; ULMWH, ultra-low-molecular-weight heparin.
Mentions: Given that complexes of heparins and positively charged proteins, such as PF4, are thought to be more immunostimulatory than heparins alone,12,24 we anticipated the evaluation of heparin innate immune system engagement might be better achieved by comparing variant PF4–heparin complexes in the MIMIC® PTE construct. Therefore, in the pilot study (see Materials and Methods section and Table 1 for additional details), graded doses of PF4–heparin complexes (200 to 12.5 µg/mL PF4 at a constant ratio to heparin) formed with UFH, branded enoxaparin, and ULMWH were examined for their impact on APC viability and activation in the MIMIC® PTE construct. (Both UFH and ULMWH were particularly crucial for this proof-of-concept study, since they were expected to trigger strong and weak immune responses, respectively, based on their anticipated capacities to form complexes with PF4.) As shown in Figure 2A, PF4–heparin complexes prepared with UFH triggered a steep reduction in cell numbers in a dose-dependent manner; for example, at the highest concentration, cell viability was decreased to 18.65% ± 14.5% from 61.6% ± 6.6% in the no-complex control (ANOVA, P ≤ .0001; Figure 2B). In contrast, even at the highest treatment dose, PF4–ULMWH complexes failed to trigger a statistically significant decrease in cell numbers (55.06% ± 7.2%; ANOVA, P > .05). Although they were not completely inert in these experiments, PF4–branded enoxaparin triggered only minimal (not statistically significant) reductions in cell viability (Figure 2A and B).

Bottom Line: Low-molecular-weight heparins (LMWHs) have several positive therapeutic effects and can also form immunostimulatory complexes with plasma proteins, such as platelet factor 4 (PF4).Production of tissue factor pathway inhibitor (TFPI), a physiologic heparin-induced inhibitor of tissue factor-induced coagulation that was used as a functional readout of biological activity of enoxaparins in these assays, was heightened in the presence of branded enoxaparin complexes, but its levels were variable in cultures treated with complexes containing US-generic enoxaparins.Analytical analyses suggest that the heightened immunostimulatory potential of some of the US-generic enoxaparin product lots could be tied to their capacity to form ultra-large and/or more stable complexes with PF4 than the other LMWHs included in this study.

View Article: PubMed Central - PubMed

Affiliation: Sanofi Pasteur, VaxDesign Campus, Orlando, FL, USA.

No MeSH data available.


Related in: MedlinePlus