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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

Heparins alone are not immunogenic in assay cultures. MIMIC® PTE (lower graph) and total PBMC (upper graph) cultures were treated with 10 μg/mL heparins (Lot 1) or LPS/R848 for 48 hours and 24 hours, respectively. Thereafter, the cells were harvested and evaluated for their expression of CD86, CD25, CD14, and HLA-DR by flow cytometry. The plotted values represent the mean ± SD for 10 donors; each experiment was repeated twice. Ag indicates antigen; APC, antigen-presenting cell; MIMIC-PTE, Modular IMmune In vitro Construct system-peripheral tissue equivalent; PBMC, peripheral blood mononuclear cells; SD, standard deviation; LPS+R848, 100 ng/mL of TLR4 agonist (LPS): lipopolysaccharide plus 10 µg/mL of the TLR7/8 agonist R848.
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fig1-1076029614562037: Heparins alone are not immunogenic in assay cultures. MIMIC® PTE (lower graph) and total PBMC (upper graph) cultures were treated with 10 μg/mL heparins (Lot 1) or LPS/R848 for 48 hours and 24 hours, respectively. Thereafter, the cells were harvested and evaluated for their expression of CD86, CD25, CD14, and HLA-DR by flow cytometry. The plotted values represent the mean ± SD for 10 donors; each experiment was repeated twice. Ag indicates antigen; APC, antigen-presenting cell; MIMIC-PTE, Modular IMmune In vitro Construct system-peripheral tissue equivalent; PBMC, peripheral blood mononuclear cells; SD, standard deviation; LPS+R848, 100 ng/mL of TLR4 agonist (LPS): lipopolysaccharide plus 10 µg/mL of the TLR7/8 agonist R848.

Mentions: The purpose of this report was to evaluate the immunostimulatory potential of branded and generic enoxaparins in an in vitro model of the human innate immune system, termed the MIMIC® PTE construct. As published reports using human PBMCs and animal models suggest heparins in a noncomplexed state are not immunostimulatory,10,12,21–23 our first objective in this study was to confirm these observations in the MIMIC® PTE construct. In a 10-donor experiment, no changes in expression of 2 APC T-cell stimulatory ligands, CD86 and CD25, were detected in the MIMIC® based assay following treatment with branded or US-generic enoxaparins (Figure 1B). In contrast, both markers were strongly upregulated on APCs cultured in the presence of LPS and R848 (positive control) in the same experiment (Figure 1B). An analysis of total PBMCs, which serves as a standard of in vitro human immune evaluations and was included in this experiment to permit the benchmarking of the MIMIC® PTE results against published literature, generated similar results following LMWH treatment (Figure 1A). Of note, the positive TLR agonist control induced stronger upregulation of CD25 than CD86 in this assay (Figure 1A). To verify the observations shown in Figure 1 were not an artifact of the 24-hour PBMC and 48-hour MIMIC® PTE assay incubation periods being too short to generate APC phenotype changes, the PBMC assay was extended to 5 days but again showed no obvious effect of the heparins (data not shown).


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)

Heparins alone are not immunogenic in assay cultures. MIMIC® PTE (lower graph) and total PBMC (upper graph) cultures were treated with 10 μg/mL heparins (Lot 1) or LPS/R848 for 48 hours and 24 hours, respectively. Thereafter, the cells were harvested and evaluated for their expression of CD86, CD25, CD14, and HLA-DR by flow cytometry. The plotted values represent the mean ± SD for 10 donors; each experiment was repeated twice. Ag indicates antigen; APC, antigen-presenting cell; MIMIC-PTE, Modular IMmune In vitro Construct system-peripheral tissue equivalent; PBMC, peripheral blood mononuclear cells; SD, standard deviation; LPS+R848, 100 ng/mL of TLR4 agonist (LPS): lipopolysaccharide plus 10 µg/mL of the TLR7/8 agonist R848.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1-1076029614562037: Heparins alone are not immunogenic in assay cultures. MIMIC® PTE (lower graph) and total PBMC (upper graph) cultures were treated with 10 μg/mL heparins (Lot 1) or LPS/R848 for 48 hours and 24 hours, respectively. Thereafter, the cells were harvested and evaluated for their expression of CD86, CD25, CD14, and HLA-DR by flow cytometry. The plotted values represent the mean ± SD for 10 donors; each experiment was repeated twice. Ag indicates antigen; APC, antigen-presenting cell; MIMIC-PTE, Modular IMmune In vitro Construct system-peripheral tissue equivalent; PBMC, peripheral blood mononuclear cells; SD, standard deviation; LPS+R848, 100 ng/mL of TLR4 agonist (LPS): lipopolysaccharide plus 10 µg/mL of the TLR7/8 agonist R848.
Mentions: The purpose of this report was to evaluate the immunostimulatory potential of branded and generic enoxaparins in an in vitro model of the human innate immune system, termed the MIMIC® PTE construct. As published reports using human PBMCs and animal models suggest heparins in a noncomplexed state are not immunostimulatory,10,12,21–23 our first objective in this study was to confirm these observations in the MIMIC® PTE construct. In a 10-donor experiment, no changes in expression of 2 APC T-cell stimulatory ligands, CD86 and CD25, were detected in the MIMIC® based assay following treatment with branded or US-generic enoxaparins (Figure 1B). In contrast, both markers were strongly upregulated on APCs cultured in the presence of LPS and R848 (positive control) in the same experiment (Figure 1B). An analysis of total PBMCs, which serves as a standard of in vitro human immune evaluations and was included in this experiment to permit the benchmarking of the MIMIC® PTE results against published literature, generated similar results following LMWH treatment (Figure 1A). Of note, the positive TLR agonist control induced stronger upregulation of CD25 than CD86 in this assay (Figure 1A). To verify the observations shown in Figure 1 were not an artifact of the 24-hour PBMC and 48-hour MIMIC® PTE assay incubation periods being too short to generate APC phenotype changes, the PBMC assay was extended to 5 days but again showed no obvious effect of the heparins (data not shown).

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