Page 169 - Haematologica August 2018

Dexamethasone promotes tolerance to factor VIII
ity of between 25% and 75%. The reported inhibitory activity was calculated from the plasma dilution that resulted in a residual FVIII activity closest to 50%. Samples with no evidence of FVIII inhibitory activity or with inhibitory activity <0.4BU/ml when undiluted were considered negative.
Anti-human VWF IgG ELISA
Ninety-six-well plates (4HBX, Immulon) were coated with FVIII free plasma-derived human VWF (0.1IU/ml, ~1μg/ml) (Biotest) overnight at 4°C, then blocked for two hours at room tempera- ture. Plasma samples were diluted to 1:40 and incubated for two hours at room temperature in duplicate. IgG detection was carried out as described for the anti-FVIII IgG ELISA. The OD cutoff for
positivity was the OD490 of pooled plasma (1:40 dilution) taken from FVIII deficient mice with no previous exogenous VWF expo- sure. Samples with an OD490 below the cutoff at a dilution of 1:40 were considered to have non-detectable anti-human VWF IgG.
Lymphocyte enumeration studies
E16KO mice received HBSS, Dex, FVIII or FVIII+Dex for five consecutive days. Three days or three weeks after the last injec- tion spleen, thymus and blood were collected. Lymphocyte popu- lations were assessed via flow cytometry (MACSQuant Analyzer, Miltenyi) by staining for CD19, CD4, CD8, CD25 and FoxP3 (eBiosciences) as appropriate for the particular organ of origin. Data was analyzed with FlowJoX (Tree Star).
A
B
Figure 1. Short-term treatment protocols. A. E17KO/hMHC mice received FVIII (6IU IV) alone or in combination with Dex (75μg IP) for five consecutive days. At week five blood was collected and plasma anti-FVIII IgG titers were measured. Mice with evidence of anti-FVIII IgG were excluded from the remainder of the study. Mice with no evidence of anti-FVIII IgG were re-exposed to FVIII (6IU IV), alone or in combination with LPS (2μg IV), for three consecutive days. At week nine blood was collected. Plasma anti-FVIII IgG titers and FVIII inhibitory activity were measured. B. E16KO mice received FVIII (6IU IV) alone or in combination with Dex (75μg IP) for five consec- utive days. At week five blood was collected and plasma anti-FVIII IgG titers as well as FVIII inhibitory activity were measured. FVIII: factor VIII; Dex: dexamethasone; LPS: lipopolysaccharide; Wk:
week. : anti-FVIII IgG negative mice; positive mice; : injection; : blood collection.
: anti-FVIII IgG
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