Patients were evaluated, on average, every 4 months. At each visit an interval history of any relapse, Incapacity Status Scale, Functional Systems Scale, Expanded Disability Status Scale (EDSS), and a proprietary graded neurologic examinations were obtained.
Annual MRI of the brain using a contrast-enhanced MS protocol was also obtained for most patients.
Breakthrough disease was defined as continued clinical relapses, new T2 or enhanced lesions on MRI, or worsening of EDSS or neurologic examination.
Five patients did not tolerate the increase in frequency of administration. IFNβ neutralizing antibody testing was performed on 25 patients while on twice-weekly dosing, and one patient who failed twice-weekly IFNβ had consistently elevated titers on two determinations (4%).
African-American patients, patients with a higher EDSS score when switching, and patients with a longer duration of stability on once-weekly treatment were less likely to respond..
Freshly harvested heart tissue sections (10 mg) were snap-frozen in liquid nitrogen and homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA; weight/volume ratio, 1:10). Freshly cultured cells (106/well, 6-well plates) were directly homogenized in TRIzol reagent. Total RNA was extracted by chloroform/isopropanol/ethanol method, treated with RNase free DNase I (NEB, Beverly MA), and assessed for quality (OD260/280 ratio ? 2.0) and quantity (OD260 of 1 = 40 ?g/ml RNA). Total RNA (2 ?g) was reverse transcribed by using poly (dT)18 oligonucleotide with an iScript kit (Bio-Rad, Hercules CA). The cDNA was utilized as a template, and quantitative real-time PCR was performed on an iCycler Thermal Cycler with SYBR-Green supermix (Bio-Rad) and gene-specific oligonucleotide pairs. The PCR Base Line Subtracted Curve Fit mode was applied for threshold cycle (Ct), Ct values for target mRNAs were normalized to GAPDH mRNA, and the relative expression level of each target gene was calculated as 2??Ct (?Ct = Ct sample—Ct control) . The oligonucleotide pairs used for amplifying the mRNAs are listed in Table A in S1 Table.