Kpl 1

To determine if AipA was important for A. phagocytophilum recognition of sLe^x-capped PSGL-1, DC organisms were incubated with antiserum targeting AipA_1–87, AipA_249–355, OmpA (Ojogun et al., 2012 (link)), or preimmune control serum as described above. Next, the treated bacteria were incubated with PSGL-1 CHO cells or untransfected CHO cells for 1 h, followed by two rounds of washing with PBS to remove unbound bacteria, and enumeration of bound organisms using spinning disk confocal microscopy as described (Troese et al., 2009 (link)). As positive controls for inhibition of bacterial adherence to sLe^x-capped PSGL-1, PSGL-1 CHO cells were incubated with the PSGL-1 N-terminus-specific antibody, KPL-1 (BD Biosciences, San Jose, CA), or the sLe^x-specific antibody, CSLEX1 (BD Biosciences) for 30 min prior to the addition of bacteria. Mouse IgG and mouse IgM served as isotype controls for KPL-1 and CSLEX1, respectively.

Venous blood into EDTA or ACD drawn immediately pre- and 24±4 hours post- infusion was evaluated by flow cytometry for adhesion markers on CD16b+ (ID3, Beckman Coulter) neutrophils: activated Mac-1 (CBRM1/5, eBioscience) or activated β2 integrin (ab13219, abcam), Mac-1 (ICRF44, BD Pharmingen), CD44 (G44-26, BD Pharmingen), E-selectin-Fc chimera (R&D Systems), L-selectin (DREG-56, eBioscience), LFA-1 (HI111 BD Pharmingen), and PSGL-1 (KPL-1, BD Pharmingen). Activated Mac-1 Ab detects the functionally active form of Mac-1(was performed on 200–800 mg/kg cohorts) and activated β2 integrin recognizes the high affinity conformation of β2 integrin, the β subunit of LFA-1 and Mac-1 (was performed on 100 mg/kg cohort).
With late afternoon or evening study drug administration, samples were stored overnight as needed (range 8–24 hours) at 4°C prior to analysis. When stored overnight, all paired pre-IVIG and 24 hour post-IVIG samples per patient were stored for uniform lengths of time. No adhesion markers except activated Mac-1 show expression increase at 24 hr compared to immediate analysis (Figure 1B). Since the time interval to analysis was constant for each subject’s two samples, the ratio between the two samples (Figure 1A) was unaffected by storage.