Romano for advice in the statistical evaluation of the data and Dr. KIAA0558 response to therapeutic proteins. Using lipopolysaccharide (LPS) and CpG ODN as IIRMIs we showed that trace levels of these impurities synergized to induce IgM, IFN, TNF and IL-6 expression. and LPS (0111:B4) were purchased from InvivoGen (San Diego, CA) and used per manufacturer’s instructions. The stated potency of the endotoxin is 1EU/ng. Ultrapure Ovalbumin (Ovalbumin grade V, OVA) was obtained from Sigma-Aldrich. Ovalbumin used to immunize mice was a kind gift from Brian Kelsall (NIAID, NIH) and contained less than 0.025EU of endotoxin per 5 g OVA as assessed by the LAL assay. Human Erythropoietin (rhuEPO) was purchased from GenScript Corporation (Piscataway, NJ). Endotoxin content in rhuEPO was measured using LAL Assay as above. Mice and experimental design Animal studies were conducted under protocol 2006-43 as approved by the White Oak Animal care and Use Committee of the FDA. Mice used for study were obtained from the National Cancer Institute (Frederick, MD). In one study, 2 month old female BALB/c mice (n?=?5/group) were immunized with 5 g of OVA alone or together with different amounts of CpG ODN or LPS as specified for each experiment. The animals were boosted 3 weeks later with the same preparation. Mice that received saline were used as controls. Mice were tail-bled before the immunization and weekly thereafter. For another study, two month old female C57BL/6 mice (n?=?5/group) received 5 g of rhuEPO alone or together with the TLR agonists (i.p.). Mice that received saline served as controls to establish the natural variability of the hematocrit and the impact of serial tail bleeding. After first dose, blood was collected and HCT was MBP146-78 measured weekly/biweekly. Animals were re-treated 14 and 62 days after first injection. Percent of increased/reduced hematocrit was calculated relative to the baseline for each individual mouse. Quantitative Real-Time PCR (q-RT-PCR) Total RNA was prepared from splenocytes using TRIzol (Invitrogen, Paisley, Scotland, UK) as per manufacturer instructions and then purified with RNAeasy (Qiagen, Valencia, CA). Subsequently, RNA (500 ng/sample) was reverse transcribed into cDNA using a High MBP146-78 Capacity cDNA Reverse Transcription Kit (Applied Biosystems Inc., Foster City, CA, USA) as per manufacturer’s instructions. cDNA samples were treated RNase H (Invitrogen) for 30 minutes at 37C and stored at ?20C until used for q-RT-PCR. Expression values were calculated using the 2 2?method [27]. For the mRNA array, total RNA was then reverse transcribed and subsequently analyzed on TaqMan Low Density Array cards by TaqMan PCR using a 7900HT MBP146-78 (ABI) as per manufacturer’s instruction. Cytokine and Antibody Assays Supernatants: 5105 splenocytes/well were cultured in RPMI 10% FCS media at 37C for 48 hr. IFN, IL-6, and Ig were assessed by using 96-well plates (Immunolon, Thermo LabSystems, Franklin MA) coated with cytokine-specific antibody or antigen and then blocked with PBS-1% bovine serum albumin (Sigma, St Louis, MO). After washing, the plates were overlaid with the supernatant for 3 hours, then further washed, and treated with the appropriate biotinylated secondary antibody, followed by AKP-conjugated avidin (BD, Biosciences, Franklin Lakes, NJ). Absorbance was read at 405nm. Statistical analysis Changes in antibody MBP146-78 or cytokine expression were analyzed by t test, ANOVA or repeated measure ANOVA as appropriate. Differences in hematocrit were evaluated by SAS using a mixed model with repeated measures where HCT?=?treat+day+treat*day+treat*day2+treat*day3. P values 0.05 were considered significant. Results Substimulatory levels of LPS and CpG ODN synergize to induce an immune response Endotoxin and host cell DNA are impurities routinely monitored in the manufacture of biologics. Both can stimulate the innate immune system via the TLR system and function as adjuvants enhancing the immunogenicity of proteins by activating antigen MBP146-78 presenting cells, inducing cytokine secretion and directing polyclonal B cell activation [11]. a reduction in the hematocrit. Further, in our model, the reduction in hematocrit was more severe following a second or third.