Dolph Houben

139 Viability and immune response to VCA 7 Inflammatory response of allotransplant In order to evaluate the local inflammatory response of the allotransplant, we used an existing histologic score of bone inflammation (the Histologic Osteomyelitis Evaluation Score or HOES) for the purpose of analyzing rejection of bone allotransplants [21] . We modified the HOES score by examining the endosteal surface and intramedullary space on the H&E stained bone sections for osteonecrosis, soft tissue fibrosis and inflammatory infiltrate. Each factor was scored on a 0-3-point scale: non-existent= 0, mild= 1, moderate= 2, or severe= 3 in which 1= < 25%, 2= 25- 50%, 3= > 75% of the field of interest. A total score of 9 points could be received for each sample by scoring osteonecrosis, soft-tissue fibrosis, and inflammatory infiltrate. We examined 6 fields selected at random in each sample. In order to quantify osteonecrosis more accurately, we used a bone image analyses system to measure osteocyte counts (OsteoMeasure; OsteoMetrics, Atlanta, GA). The extend of osteonecrosis is defined by the percentage of empty lacunae per field of interest (FOI). Immunogenicity of allotransplant by quantitative real-time-PCR (RT-qPCR) We measured levels of inflammatory cytokines within bone samples harvested at the 20-week survival time by RT-qPCR. The cytokines were selected by relevance to allotransplantation, and included TNF, IL2, IL6, IL8, INFG, CD4, CD8a, and CD28 . GAPDH served as a reference gene (Table 1). The 2mm, previously flash-frozen bone sections were cleared of soft tissue and pulverized in liquid nitrogen using the A11 basic analytical mill (IKA-Werke GmbH & Co. KG, Germany). RNA was extracted from the pulverized bone with the PureLink RNA mini kit, TRIzol reagent and on-column Pure link DNase treatment (Thermo Fisher Scientific, Cat no. 12813018A, 12034977, 12185-010, Carlsbad, CA). RNA purity and quantification were performed on a Nano- drop Spectrometer (ThermoScientific Nano-drop Technologies, Wilmington, DE). The absence of RNA degradation was confirmed by gel electrophoreses before the RNA was converted to cDNA. cDNA was synthesized with the iScript cDNA synthesis kit (Bio-Rad Laboratories Inc., Hercules, CA) following the protocol of the manufacturer with 200 ng of RNA. Quantitative real-time PCR (RT-qPCR) was performed to quantify the expression of target genes with iQ SYBR green Supermix and the CFX384 Real-Time detection system (Bio-Rad Hercules, CA). Transcript quantity measurements were normalized to GAPDH , and gene expression levels quantified using the 2 (- ΔΔ CT) method [22] . Primer sequences are given in table 1 (ThermoScientific, Invitrogen, Wilmington, DE). Real-time PCR was followed by melt curve analyses with the following conditions: 15min denaturation at 95⁰C for one cycle, 20s of denaturation at 95⁰C, 35s of annealing and extension at 72⁰C for 51 cycles followed by generation of a melting curve. Melt curves were performed from 60 ⁰C to 95⁰C with an increment of 0.5⁰C.