PMVEC monolayer permeability under basal/resting and septic conditions (cytomix: equimolar solution of TNF[alpha], IL1[beta], and IFN[gamma] used to mimic a septic response, 0.3-100 ng/mL, PeproTech, Rocky Hill, NJ) was assessed over a time course (2-24 h) using three techniques: (i) TEER (as above), (ii) FITC-labelled dextran flux (4kDa), and (iii) EB-labelled albumin flux (67kDa).
For these studies, inhibitors were administered simultaneously with the septic stimulus (cytomix).
For these studies, PMVEC were fixed in 10% formalin following basal and septic (cytomix) stimulation and then permeabilized with a 1% [Na.sup.+] citrate/0.1% Triton X-100 solution.
For these studies, PMVEC were stimulated with PBS or cytomix, lifted by trypsinization, and stained with Annex in V and PI in binding buffer (0.1 M HEPES pH 7.4; 1.4 M NaCl; 25 mM CaCl2).
To identify the concentration of cytomix required to induce maximal PMVEC permeability as indicated by two complementary techniques, TEER and EB-albumin flux, PMVEC were treated with a range of cytomix concentrations.
The tachyzoites were resuspended in 800 [micro]l Cytomix
(120 mM KCl, 0.15 mM Ca[Cl.sub.2], 10 mM [K.sub.2]HP[O.sub.4]/ K[H.sub.2]P[O.sub.4], 25 mM Hepes, 2mM EGTA, 5mM Mg[Cl.sub.2], pH7.6).
Immediately afterwards the cytokines IL-1[beta], TNF-[alpha], and IFN[gamma], alone and in combination (cytomix), were added to untreated and treated wells.
IL-1[beta], TNF-[alpha] and IFN[gamma], alone and in combination (cytomix), stimulated significant CXCL10 release from the asthmatic and non-asthmatic ASM cells (Table 1).
When tryptase (1nM) was added to the ASM cells 30 minutes after the cytokines IL-1[beta], TNF-[alpha], IFN[gamma], or cytomix, the CXCL10 levels detected in non-asthmatic and asthmatic ASM cell CM were very much lower than the respective cytokine control (Table 2).
Nevertheless, differences in transcriptional pathways following ASM cell stimulation with the individual cytokines and cytomix may contribute to the differential effects of histamine indirectly.
Cytokines, IFN-[alpha], IL-6, TNF-[alpha], IL-10, IL-1[beta], CXCL-8, CXCL-10, and IL-12p40 in the supernatants were measured by Flow Cytomix (BD Pharmingen) as per the manufacturer's protocol.
After stimulation with CPn for 24 h, supernatants were collected and assayed with Flow Cytomix and/or ELISA to quantify cytokine secretion.