A summit on cellular therapy for cancer discussed and presented advances related to the use of adoptive cellular therapy for melanoma and other cancers. The summit revealed that this field is advancing rapidly. Conventional cellular therapies, such as tumor infiltrating lymphocytes (TIL), are becoming more effective and more available. Gene therapy is becoming an important tool in adoptive cell therapy. Lymphocytes are being engineered to express high affinity T cell receptors (TCRs), chimeric antibody-T cell receptors (CARs) and cytokines. T cell subsets with more naïve and stem cell-like characteristics have been shown in pre-clinical models to be more effective than unselected populations and it is now possible to reprogram T cells and to produce T cells with stem cell characteristics. In the future, combinations of adoptive transfer of T cells and specific vaccination against the cognate antigen can be envisaged to further enhance the effectiveness of these therapies. A summit on cellular therapy for cancer was held on November 1 and 2, 2011 at the NIH in Bethesda, Maryland. Advances related to the use of adoptive cellular therapy for melanoma and other cancers were presented and discussed and are summarized in this review.Treatment of melanoma with tumor infiltrating lymphocyte (TIL) cells of high functional avidity has been improved by treating patients with lymphocyte reducing chemotherapy prior to the administration of the TIL cells. Preconditioning the TIL recipient with non-myeloablative chemotherapy has resulted in a objective clinical response rate of 49% with 13% complete responses and preconditioning with non-myeloablative chemotherapy plus 12 Gy total body irradiation (TBI) further improved objective clinical response rate to 72% with 40% complete responses [Several advancements associated with manufacture of TIL have been made and as a result TIL production has become more practical. Traditionally, initial TIL cultures were screened for reactivity to tumor antigens and only the reactive cultures were selectively expanded. Many cell processing laboratories are no longer screening initial TIL colonies for tumor-reactive cells, rather, they are expanding all isolated TIL cells based on the initial finding that nearly 80% of TILs possess autologous tumor reactivity  This hasTIL have been traditionally manufactured using tissue culture plates for initial culture and flasks and bags for rapid expansion protocols (REP). This process, however, results in large final culture volumes: 30 to 60 liters. These large volumes are associated with the use large quantities of media, cytokines, and additives. In addition, harvesting these large volumes is time consuming. In order to simplify and reduce the quantity of reagents and labor associated with TIL production, two new methods are being tested for TIL REP; bioreactors and gas permeable flasks. The WAVE bioreactor can be used for TIL REP . The volThe increased clinical effectiveness and improved production methods are leading to the more widespread use of TIL to treat patients with melanoma.While TIL Therapy is effective, melanoma samples cannot be obtained for TIL production from all patients and, in some cases, TIL cannot be isolated from the resected tumor. Engineered T cells are being used increasingly for patients from whom TIL are not available. Two general approaches involving engineered T cells are being used clinically. Both involve the use of autologous peripheral blood T cells; one involves gene transfer of high affinity T cell receptors (TCR) and the other gene transfer of chimeric antibody-T cell receptors (CAR) .Patients with melanoma have been treated with T cells engineered using recombinant retroviral vectors to express HLA-2 restricted high affinity T cell receptors (TCRs) specific for melanoma antigens MART-1 and gp100 ,8. WhileAnother approach involves the transduction of autologous T cells to express CARs made up of the variable region a tumor specific antibody fused to an intracellular signaling domain capable of activating T cells. Typically, a CAR is comprised of an extracellular scFv portion of a monoclonal antibody and an intracellular CD3 zeta chain in tandem with a co-stimulatory signaling domain, such as CD28. In addition, some CARs include other stimulatory factors such as 4-1BB or OX-40, alone or in combination with CD28 . Since CA clinical protocol that uses the autologous T cells expressing CAR specific for the folate receptor -alpha (FRα) is beingThe reprogramming of adult cells in order to produce more primitive cells or stem cells is becoming an important part of cellular therapy of cancer. Adult cells can be reprogrammed to produce induced pluripotent stem cells (IPSC) which have properties similar to embryonic stem cells. Investigators are now working to reprogram T cells to produce "stem-like" T cells that are more effective in adoptive cell therapy.Methods to reprogram stem cells have improved greatly since Yamanaka first demonstrated that the transfer of 4 transcription factors, Oct4, Klf4, Sox2 and cMyc, into fibroblasts can produce IPSCs . IPSCs d+ T cell (TE) populations derived from central memory T cells (TCM) rather than effecter memory T cells (TEM) retained the ability to survive long-term in the circulation, bone marrow, and lymph nodes [CM-derived TE cells differentiated to both TCM and TEM phenotypes in vivo and responded efficiently to antigen challenge [Several studies have found that T cell phenotype affects their effectiveness for adoptive cell therapy. Comparison of TIL cells from patients responding to therapy and those that did not has found that clinical responses were associated with TIL that expressed co-stimulatory molecules CD27 and CD28, have longer telomeres and persist longer in vivo. Several investigators have been exploring methods to produce cytotoxic T cells which persist longer and are more effectively clinically . Using Cph nodes . Of notehallenge . This wohallenge .+ T cells have stem cell-like qualities. Th17 have superior anti-tumour activities than their Th1 counterparts, are resistant to apoptosis and persist long-term after adoptive cell transfer. Most importantly, they have the stem cell-like properties of self-renewal and multipotency [A major new area reviewed at the meeting was the idea that mature, post-thymic lymphocytes have stem cell-like qualities. Restifo et al. have recently found that Th17-polarized CD4ipotency .In addition, Gattinoni et al. have identified a subpopulation of circulating T cells with both naïve and memory T cell properties with a CD45RO-, CCR7+, CD45RA+, CD62L+, CD27+, CD28+ and IL-7Rα+ phenotype which they have called stem central memory T (Tscm) cells . These Tin vitro when naïve T cells are stimulated in the presence of a Wnt pathway activator, TWS119 [ex vivo for use in adoptive cell therapy coupling TCR or CAR engineering with pharmacological modulation of T cell differentiation.While Tscm cells are potentially very effective in adoptive cellular therapy, very few Tscm cells are present in the circulation. Several laboratories have been investigating methods to reprogram T cells in order to produce the large quantities of Tscm cells that would be needed for adoptive cell therapy. Wnt signaling/β-catenin and mTor signaling pathways have been found to be important in T cell maturation ,26. The , TWS119 ,24. In tAn alternative to adoptive transfer of T cells is vaccination with DCs loaded with short tumor peptides that bind exactly to specific HLA epitopes, however the effectiveness of these therapies have been limited by insufficiently consistent and robust effector T cell responses. Vaccination with longer tumor peptides (28-35 amino acids) results in more efficient peptide processing and presentation than short peptides. The treatment of 20 women with high grade vulvar intraepithelial neoplasia with 3 subcutaneous Human Papilloma Virus 16 (HPV 16) E6 and E7 synthetic peptide vaccines resulted in clinical responses in 15 of 19 patients at 12 months of follow-up . HPV 16-ex vivo by 100-1000 fold and used to treat patients with CLL, colon cancer and renal cell carcinoma (RCC). Patients are first treated with the proteasome inhibitor bortezomib to increase tumor sensitivity to NK cell cytotoxicity mediated by TNF-related apoptosis-inducing ligand (TRAIL) prior to infusion of expanded autologous NK cells with low dose subcutaneous IL-2 administered twice daily for 1 week following infusion [8 NK cells/kg having already been established to be safe, with preliminary evidence for anti-tumor effects being observed against tumors such as RCC and CLL.Autologous NK cells are being expanded infusion . Phase Iin vivo expansion by using pretreatment lymphoreduction therapy and post-infusion IL-2 therapy [Allogeneic NK cells are being used to treat hematological malignancies. These allogeneic NK cells protocols make use of  therapy . As an aA critical part of the treatment of cancer with adoptive cellular therapies is the monitoring of recipients following treatment. Clinical trials of cellular therapies for cancer should include biomarker studies in an integrated, quality, supported, and meta-analyzeable manner . For T chttp://www.fda.gov/biologicsbloodvaccines/newsevents/ucm232821.htm and http://www.fda.gov/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/OtherRecommendationsforManufacturers/ucm094338.htm).Most clinical studies involving adoptive cellular therapies are conducted under an Investigational New Drug (IND) application. FDA encourages early preparation for critical points in the IND process, such as moving into initial clinical studies and transitioning to pivotal clinical trials. Proper preparation allows for an easier transition, a better designed study, and a higher likelihood of success. FDA staff encourages sponsors and investigators to take advantage of formal meetings, such as pre-IND meetings, and is often willing to speak with sponsors and investigators through direct informal interactions. In addition, numerous FDA websites contain information useful for investigators (The field of adoptive cell therapy is advancing rapidly. Conventional cellular therapies, such as TIL, are becoming more effective and more available. Gene therapy is becoming an important tool in adoptive cell therapy. Autologous lymphocytes are being engineered to express TCRs, CARs and cytokines. T cell subsets with more naïve and stem cell-like characteristics have been shown in pre clinical models to be more effective than unselected populations. In the future combination of adoptive transfer of T cells and specific vaccination against the cognate antigen can be envisaged to further enhance the effectiveness of treatment.


DNA methylation aberration and microRNA (miRNA) deregulation have been observed in many types of cancers. A systematic study of methylome and transcriptome in bladder urothelial carcinoma has never been reported.The DNA methylation was profiled by modified methylation-specific digital karyotyping (MMSDK) and the expression of mRNAs and miRNAs was analyzed by digital gene expression (DGE) sequencing in tumors and matched normal adjacent tissues obtained from 9 bladder urothelial carcinoma patients. We found that a set of significantly enriched pathways disrupted in bladder urothelial carcinoma primarily related to “neurogenesis” and “cell differentiation” by integrated analysis of -omics data. Furthermore, we identified an intriguing collection of cancer-related genes that were deregulated at the levels of DNA methylation and mRNA expression, and we validated several of these genes  by Bisulfite Sequencing PCR and Reverse Transcription qPCR in a panel of 33 bladder cancer samples.We characterized the profiles between methylome and transcriptome in bladder urothelial carcinoma, identified a set of significantly enriched key pathways, and screened four aberrantly methylated and expressed genes. Conclusively, our findings shed light on a new avenue for basic bladder cancer research. As genetic and epigenetic abnormalities have been observed in the development of cancer cells Bladder cancer is one of the most common cancers in the world. In 2008, an estimated 386,300 new cases were diagnosed, and 150,200 people died from bladder cancer Based on above observations, we hypothesized that a systematic analysis on profiles of DNA methylation and expression of both mRNA and microRNA in bladder cancer would help to identify key alterations of bladder cancer. Thus, we carried out a genome-wide methylation analysis in matched tumor and normal adjacent tissues from 9 bladder urothelial carcinoma patients using modified methylation-specific digital karyotyping (MMSDK), combined with mRNA and miRNA expression data  sequencing, a total of 14,787,388 and 12,754,928 high-quality reads were obtained for tumor and normal tissues, respectively. In total, 16,236 genes and 543 miRNAs were detected expressed in at least one of the tumor or normal adjacent tissue samples.MluI genomic loci and the mRNA expression level between cancer tissues and normal controls, Furthermore, a global up-regulation of miRNAs in bladder cancer was observed.To obtain an overview of the differences in the DNA methylation patterns with regard to the gene expression profiles in the bladder urothelial carcinomas and their matched normal adjacent tissues, the log2-transformed, average-fold changes between tumor and normal tissues for the three types of tags were plotted for the entire genome . At a thTo further examine the methylation status at different genomic regions, we compared the methylation profiles of promoters with or without CpG islands (CGIs), exons and introns of bladder urothelial carcinoma tissues and the matched normal adjacent tissues. A similar level of overall DNA methylation in the examined genomic regions was distributed between two types of tissues   . Of the of 93.8% .Between bladder cancer tissues and the matched normal controls, 2975 coding genes were detected as differentially expressed including 1560 up-regulated genes and 1415 down-regulated genes, and 196 miRNAs were differentially expressed including 156 up-regulated and 40 down-regulated miRNAs  . As showWe performed integrated analysis of selected genes between DNA methylation and gene expression. In total, 4772 genes with both DNA methylation data and gene expression data were identified. As shown in supplementary To generate further insight view of pathway perturbation underlying bladder cancer, we performed GO and KEGG pathway analysis on gene sets containing differentially methylated sites, differentially expressed genes identified from mRNA sequencing and the target genes of deregulated miRNAs.Among all the genes containing divergent DNA methylation loci, the GO analysis for “biological processes” and “molecular function” showed a significant enrichment of 19 and 13 GO terms, respectively, using a FDR P-value cutoff of 0.05. The most significant enriched GO terms in “biological process” were “neuron development”, suggesting a critical role for differential methylation in neurogenesis. Similarly, only the “axon guidance” pathway was significantly enriched  via KEGG pathway analysis, reflecting a concordance with GO analysis result. This result suggests an important mechanism of epigenetic abnormality in neuron genes that play a role in bladder cancer, as cancer-related axonogenesis and neurogenesis has been observed in prostate cancer For differentially expressed genes, 226 GO terms in the category of “biological process” and 8 GO terms in the category of “molecular function” were significantly enriched at a FDR threshold of P-value≤0.05 . From thFor the target genes of all the deregulated miRNAs, 149 GO terms were enriched from the “biological processes” analysis using the similar threshold. Generally, cellular process, biological and metabolic process terms were found to be enriched among the miRNA-target genes that were deregulated in our study. The “molecular function” analysis revealed 11 GO terms that were significantly enriched by a FDR threshold of P-value≤0.05. The most significantly enriched GO term was “protein kinase activity”, suggesting a role in kinase activity regulation. In addition, pathway analysis of the targets of the deregulated miRNAs also showed a significant perturbation of cell cycle, axon guidance and MAPK signaling pathways  .To further scrutinize the connection among the three categories of -omics, the GO terms of molecular functions and biological processes, as well as the KEGG pathways that were enriched in more than one category were further analyzed. The GO and KO terms enriched in more than one category are presented in Through the GO term enrichment and KEGG pathway analysis of selected differentially regulated genes, we observed that certain genes were both differentially methylated and expressed which were involved in significant perturbation pathways, and these pathways were closely connected to various tumorigenesis processes. For instance, SLIT2 was involved in Axon guidance pathway, and CCND1 was involved in cell cycle and p53 signaling pathway. Besides, we identified several canonical cancer-associated genes that were not enriched in the significantly disturbed pathways, such as ADAMTS9, HIC1, RASAL1, in both the methylation statuses and expression data .As DNA methylation aberration in gene promoters is reported to influence their expression levels, those methylation sites located in promoter regions were analyzed in details. Hypermethylated in cancer 1 (HIC1) and slit homolog 2 (SLIT2) were identified as having differentially up-regulated methylation status of their promoter regions and dramatically down-regulated expression level across all the bladder urothelial carcinoma tissue samples. Besides, we found that RAS protein activator like 1 (RASAL1) and keratin 17 (KRT17) were clearly hypomethylated in the promoter region and over-expressed in the tumor samples compared to the controls. These four genes have been identified as potential tumor suppressor genes or oncogenes in studies of other tumor types To further investigate the methylation and expression alterations of HIC1, SLIT2, RASAL1, and KRT17, massive bisulfite sequencing PCR and reverse transcription qPCR were performed in matched pairs of cancer and normal adjacent tissues from additional ∼33 bladder urothelial carcinoma patients (see methods for details). As shown in MluI loci in our study. DGE sequencing is also a useful and sensitive tool that allows for accurate detection of a wide range of expression levels of both mRNAs and miRNAs and thus allows for differential expression analysis of a greater range of transcripts MMSDK technology provides an efficient and cost-effective method to analyze genome-wide DNA methylation profiles with a relatively high sensitivity In general, a similar genome-wide methylation pattern was found between bladder tumor and normal adjacent tissues. Methylation levels at CpG rich and poor promoters, exons, and introns were also similar between tumor and normal tissues, supporting the above findings regarding the similar global methylation patterns. In gene level, it's well known that inactivation of tumor-suppressor genes by site-specific hypermethylation contributes to the initiation and progression of human malignancies The Slit gene family  is a recently characterized family of secreted repellents in axon guidance and neuronal migration during the development of the central nervous system MluI enzyme digestion sites to be found in the promoters of these genes, thus we couldn't appropriately address the methylation levels of promoters in these commonly methylated genes due to technological limitations. However, we found that MLH1 and HOXA9, but not the others of these commonly methylated genes, were significantly under-expressed in bladder cancer, which were in agreement with previous studies Previous studies identified hypermethyled promoter regions in some commonly methylated genes  in different types of cancers. We tried to confirm such results in bladder urothelial carcinoma as well. Unfortunately, there were no In contrast to hypermethylation, DNA hypomethylation contributes to tumorigenesis by activating proto-oncogenes Keratin 17, also known as cytokeratin 17, is involved in the formation and maintenance of various skin appendages, especially in determining the shape and orientation of hair. The expression of KRT17 has been found to be up-regulated in cancerous tissues in cervical Our study also highlights the importance of investigating the functional roles of mRNAs and miRNAs in the tumorigenesis of bladder urothelial carcinoma. Met proto-oncogene (MET), a hepatocyte growth factor receptor (HGFR) that has been suggested to play an important role in the development of various epithelial and nonepithelial tumors, was found to be significantly over-expressed in our study of bladder cancer, consistent with previous reports on bladder cancers It is widely acknowledged that disrupted pathways, not just deregulated individual genes, drive the tumorigenesis process. In our search for functional relevance of altered methylation levels and gene expression, we found that KEGG “axon guidance” pathway was significantly enriched in the differentially expressed genes (mRNAs and miRNAs) and that there were overlaps with the pathways enriched for relevant genes with differentially methylated loci. Axon guidance represents a key stage during which axons extend to their correct targets during the formation of neuronal networks and related molecules were thought to be widely expressed and involved in tumor development, angiogenesis and metastasis In summary, we systematically profiled the DNA methylation and expression patterns of both miRNAs and mRNAs on a genome-wide scale in bladder urothelial carcinoma using deep sequencing technology. We characterized the profiles between methylome and transcriptome in bladder urothelial carcinoma, identified a set of significantly enriched key pathways, and screened four simultaneously aberrantly methylated and expressed genes. It is noted that the number of bladder cancer patients in our study was relatively small. This observation requires further confirmation in a larger panel of tumors. Our results provide a new perspective for the application of integrated analysis of multi-omics to identify complex multi-level regulation means involved in cancer.The tumor tissues and matched normal adjacent tissues from the patients with transitional cell carcinoma of the bladder in this study were obtained from the biobank (CXC201005260001A) of Urinogenital Cancer Genomics Consortium (UCGC) in China. Detailed information of the 42 patients  is summarized in Genomic DNA from bladder urothelial carcinomas and normal adjacent tissues were prepared using DNeasy Blood & Tissue Kit  according to the manufacture's protocol, DNA was then dissolved in DNase-free water and the concentrations were determined before being stored at −80°C. Total RNA was extracted from bladder cancer and normal adjacent tissues using TRIZOL  according to the manufacture's protocol and evaluated using Agilent 2100 Bioanalyzer .MluI (recognition sequence: ACGCGT) . The MluI-digested genomic DNA was ligated to biotinylated linkers and fragmented by NlaIII cleavage . DNA fragments were captured with streptavidin-conjugated magnetic beads and then bound to the first adapter, which contains a MmeI restriction enzyme recognition site. Bounded DNA was then digested with MmeI , which generated a 17–18 nt library, followed by ligating to the P7 adapter. DNA fragments were amplified by PCR for 15 cycles using Phusion DNA polymerase  with universal primer and index primers. Gel electrophoresis was used to isolate a 130–150 bp gel slice, and the isolated fragment was gel-purified using Spin-X Centrifuge Tube Filters . Each MMSDK library was examined by Agilent 2100 Bioanalyzer  and real-time PCR for quality control, after which cluster generation and a standard sequencing were performed on the Illumina Cluster Station and Genome Analyzer IIx  following manufacturer's protocol.MMSDK combines the use of methylation-sensitive restriction enzyme digestion and the second generation sequencing technique  MluI and NlaIII digested DNA fragment library was constructed as a reference genome. Digital simulated analysis showed 21038 MluI recognition sites in the human genome sequence (reference build hg18). A total of 42126 virtual fragments were obtained from simulated digestion with MluI and NlaIII under the hypothesis that all cytosines are unmethylated. Only 694 MluI fragments lacked an internal NlaIII recognition site. We found 2942 genomic loci to be located in CGIs defined by the following three criteria: GC content ≥50%, ratio of the observed CpGs to the expected CpGs>0.6, length>200 bp. The corresponding CpG Islands annotation file was downloaded from the UCSC Table Browsers MluI recognition site. In our experiment, the tags represent unmethylated sites. The corresponding gene annotation of those genomic loci was simultaneously found using the UCSC Genome Browser (http://genome.ucsc.edu/). The averaged tag numbers were normalized  and were used to compared the methylation level between tumors and normal adjacent tissues. The Poisson distribution was used to calculate the P-value for each MluI recognition site in the genome to detect differentially methylation level between bladder cancer tissues and matched normal adjacent tissues 2Ratio|≥1 were defined as being differentially methylated.We firstly filtered the data to generate effective reads by removing the low-quality sequences, as well as 3′ adaptor sequence, leaving tags of 17–18 nt. A virtual 18 beads , the cDNA was digested with NlaIII and ligated to the first adapter (GEX adapter 1) that contained a restriction site for MmeI. After dephosphorylation with the alkaline phosphatase, CIAP , the purified MmeI-digested products were linked to the second adaptor (GEX index adapter) containing degenerate 2 bp 3′ overhangs. The double adapter-flanked tags from the mRNAs were then amplified by PCR using Phusion DNA polymerase  and GEX PCR primers following the manufacturer's protocol. The resulting ∼85-bp PCR products were ethanol precipitated and purified by gel electrophoresis and Spin-X filter columns . Finally, the mRNA libraries were sequenced on the Illumina Cluster Station and Genome Analyzer IIx  following the manufacturer's protocol.To detect the expression of mRNA in the tumor tissues and matched normal adjacent tissues, 4 µg of the total RNA isolated from each sample was used for DGE sequencing. Briefly, after synthesis of double-stranded cDNA using oligo(dT)NlaIII restriction sites in the human reference genome (hg18) and the CATG restriction enzyme-digested site were extracted and concatenated to form a new reference sequence 2Ratio using the TPM values. We subsequently performed Poisson distribution test to determine the differentially expressed genes 2Ratio|≥1 were defined as differentially expressed mRNAs.Prior to the statistical analysis, potentially erroneous tags  were filtered out. All of the 17 bp sequences next to the potential For miRNA sequencing, 5 µg of total RNA from each sample was ligated to both a 5′ adapter and 3′ adapter for reverse transcription using Superscript II . Subsequently, the reverse transcripted products were amplified by PCR and evaluated by gel electrophoresis. After obtaining a ∼92 bp DNA band from a 6% PAGE gel, the PCR products were ethanol precipitated and purified using Spin-X filter columns . Finally, miRNA libraries were performed to cluster generation and standard cycle of sequencing on the Illumina Cluster Station and Genome Analyzer IIx following manufacturer's protocol.http://www.ncbi.nih.gov). The same pipeline used for the DGE mRNA differential expression analysis was also used for the miRNA expression analysis.Low quality reads were trimmed and the adapter sequences were accurately clipped using a dynamic programming algorithm prior to subsequent statistical analysis. After elimination of the duplicate reads, the remaining reads of at least 18 nt were mapped to a human reference genome (hg18) using SOAP V2.0. We removed tags originating from protein-coding genes, repeat sequences, rRNA, tRNA, snRNA, and snoRNA by mapping the short read tags to UCSC RefGene, RepeatMasker and NCBI Refseq, as well as our in-house ncRNA annotation datasets compiled from the NCBI GenBank database  with the ClueGo V1.3 plug-in GO and KEGG pathway analysis was performed by Cytoscape V2.7 (http://www.urogene.org/methprimer/index1.html). The bisulfite conversion products were amplified using the following PCR program: 94°C for 4 min, followed by 35 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec and then extension at 72°C for 10 min. Then, bisulfite PCR products were cloned into a T vector  after purification, and 30 clones were selected to further Sanger sequenced for at each validated gene.To validate the methylation status of genes detected as being differentially methylated by deep sequencing, we combined BSP and clone sequencing for 16 differentially methylated genes in both tumor and normal adjacent tissue samples from the 9 bladder cancer patients. In brief, 1 µg of genome DNA from each sample was bisulfite converted according to the manufacturer's protocol . Primers for BSP were designed by MethPrimer  . The reverse transcription products were amplified using the following PCR program: 50°C for 2 min,94°C for 2 min, followed by 40 cycles of 94°C for 30 sec, 55°C for 20 sec, and 72°C for 15 sec. All of the primers used in the validation assays are listed in Table S1Differentially methylated genomic loci located in genic regions.(XLS)Click here for additional data file.Table S2mRNAs that are differentially expressed in bladder cancer.(XLS)Click here for additional data file.Table S3miRNAs that are differentially expressed in bladder cancer.(XLS)Click here for additional data file.Table S4Differently expressed miRNAs and miRNA targets in bladder cancer.(XLS)Click here for additional data file.Table S5Differentially methylated and expressed genes in bladder cancer.(XLS)Click here for additional data file.Table S6Enriched GO terms and KEGG pathways in differentially methylated genomic loci in bladder cancer.(XLS)Click here for additional data file.Table S7Enriched GO terms and KEGG pathways in differentially expressed genes in bladder cancer.(XLS)Click here for additional data file.Table S8Enriched GO terms and KEGG pathways in targets of differentially expressed miRNAs in bladder cancer.(XLS)Click here for additional data file.Table S9GO and KO terms enriched in multiple data types  in bladder cancer.(XLS)Click here for additional data file.Table S10Clinical information on the 42 patients sequenced or validated in this study.(DOC)Click here for additional data file.Table S11BSP and RT-qPCR primers used in the validation assays.(DOC)Click here for additional data file.


Mx1 gene. We sequenced the coding regions of Mx1 in the eight CC founder strains, and identified a novel Mx1 allele that showed reduced ability to inhibit viral replication, while maintaining protection from weight loss.Genetic variation contributes to host responses and outcomes following infection by influenza A virus or other viral infections. Yet narrow windows of disease symptoms and confounding environmental factors have made it difficult to identify polymorphic genes that contribute to differential disease outcomes in human populations. Therefore, to control for these confounding environmental variables in a system that models the levels of genetic diversity found in outbred populations such as humans, we used incipient lines of the highly genetically diverse Collaborative Cross (CC) recombinant inbred (RI) panel (the pre-CC population) to study how genetic variation impacts influenza associated disease across a genetically diverse population. A wide range of variation in influenza disease related phenotypes including virus replication, virus-induced inflammation, and weight loss was observed. Many of the disease associated phenotypes were correlated, with viral replication and virus-induced inflammation being predictors of virus-induced weight loss. Despite these correlations, pre-CC mice with unique and novel disease phenotype combinations were observed. We also identified sets of transcripts (modules) that were correlated with aspects of disease. In order to identify how host genetic polymorphisms contribute to the observed variation in disease, we conducted quantitative trait loci (QTL) mapping. We identified several QTL contributing to specific aspects of the host response including virus-induced weight loss, titer, pulmonary edema, neutrophil recruitment to the airways, and transcriptional expression. Existing whole-genome sequence data was applied to identify high priority candidate genes within QTL regions. A key host response QTL was located at the site of the known anti-influenza  in vivo experimental model was used that recapitulates many of the genetic characteristics of an outbred population, such as humans. By controlling viral dose, environment and demographic variables, we were able to focus on the role that host genetic variation plays in influenza virus infection. Both the range of disease phenotypes and the combinations of sets of disease phenotypes at 4 days post infection across this population exhibited a large amount of diversity, reminiscent of the variation seen across the human population. Multiple host genome regions were identified that contributed to different aspects of the host response to influenza infection. Taken together, these results emphasize the critical role of host genetics in the response to infectious diseases. Given the breadth of host responses seen within this population, several new models for unique host responses to infection were identified.Host responses to an infectious agent are highly variable across the human population, however, it is not entirely clear how various factors such as pathogen dose, demography, environment and host genetic polymorphisms contribute to variable host responses and infectious outcomes. In this study, a new  Worldwide influenza infections cause several million cases annually, with severe pandemics (such as the 1918 pandemic) causing high levels of morbidity and mortality Influenza A virus (IAV)  octo-parental RI panel, known as the pre-CC population n = 5–11) from each of the eight CC founder strains. Mice were infected with a dose of the mouse adapted A/PR/8/34 (PR8) IAV that was known to cause severe disease in several of the CC founder strains , and we assessed IAV-induced weight loss (measured as a percentage of starting weight) and clinical disease daily through four days post infection (D4), at which point the mice were euthanized and lung tissue assessed for viral replication, virus-induced inflammation and pathology, and (pre-CC mice only) transcriptional profiles by microarray analysis within the lungs  of D4 weight  were significant predictors . A total of 11,700 genes passed quality control processing, and did not have a SNP across the eight founder lines which could impact their intensity on the array. Out of these 11,700, we identified the 6000 most variable and interconnected genes across this population and used weighted gene co-expression network analysis (WCGNA) to cluster these transcripts into twelve modules, labeled A–L . Seven mMx1, on chromosome 16, and our a priori expectation (see below) was that we would identify a QTL over Mx1, validating our mapping approaches within the CC. Consistent with this, we identified a highly significant QTL on chromosome 16, HrI1 (Host response to Influenza) that contributed to a number of these disease-associated phenotypes . HrI1 explained 41.67% of the variation in weight loss , and similar amounts of variation in other phenotypes, and was located in a 0.71 Mb region  annotated as containing 10 genes and one non-coding RNA, including the known anti-influenza gene Mx1. We also conducted QTL mapping on the eigengenes of the expression modules to identify mQTL (module QTL). Three modules  had QTL that overlapped with HrI1  and in the Sanger Institute's Mouse Genomes sequencing project Mx1 exons in each of the eight founder strains by sequencing the Mx1 exons from each strain , and a single amino acid substitution identified in CAST/EiJ relative to NZO/HILtJ and PWK/PhJ (Gly83Arg). Although we cannot preclude transcriptional differences at different time points during infection from having a role in the functional differences between the three Mx1 alleles, the nonsynonymous substitution that is unique to the CAST/EiJ haplotypes is a strong candidate to explain the intermediate phenotype of the CAST/EiJ Mx1 allele. These results demonstrate our ability to identify: 1) a known IAV resistance locus with only one mouse per line, 2) the multiple phenotypes regulated by this locus, and 3) previously unidentified allelic variants due to the multiple alleles segregating within the pre-CC population.The sequence variation at the rain see . We founrain see . The mosMx1 allele could contribute to protection from weight loss while showing high titers and severe inflammatory responses. Of the 11,700 transcripts that passed QA/QC and were not SNP impacted, we determined that 2156 transcripts (18.4%) had their expression levels significantly impacted by genotype at the most significant Mx1 marker  CAST/EiJ Mx1 alleles grouped with the resistant PWK/PhJ or NZO/HILtJ Mx1 alleles, or (b) where CAST/EiJ allele grouped with susceptible A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ and WSB/EiJ Mx1 alleles. A total of 307 transcripts with an eQTL at Mx1 (14.2%) had allele effects consistent with CAST/EiJ grouping with the resistant PWK/PhJ and NZO/HILtJ alleles, while 1207 transcripts with an eQTL at Mx1 (55.9%) had allele effects consistent with CAST/EiJ grouping with the susceptible A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ and WSB/EiJ alleles.We returned to our transcriptional data in an attempt to better understand how the CAST/EiJ Those transcripts where CAST/EiJ grouped with the susceptible alleles showed significant enrichments for a large number of GO terms , includiMx1 on influenza response, there was large phenotypic variation within both the functional and non-functional Mx1 allele classes. This suggested the presence of modifier alleles segregating in the pre-CC population. To find these modifiers, we conducted additional genome scans after accounting for genotype at the most significant HrI1 marker , that explained 9.7% of the variation in D4 weight . Even in the absence of a large effect resistance gene, Mx1, we were able to group transcripts into functionally relevant co-expression modules. In total, this analysis identified eleven modules labeled M-W (Mx1 region (see below) indicates that, as expected, the effect of the Mx1 locus on the coexpression network has been ameliorated in this population. This indicates, along with the continued, albeit, weaker associations between the modules and phenotypes that performing a WGCNA analysis conditioning on the Mx1 allele group provides a way to highlight additional diseases-associated genetic regulation of transcript expression.RNA of high quality was recovered from 60 mice within this eled M-W . Again, eled M-W . Eight meled M-W . AbsenceMx1-/- subpopulation, we identified a significant QTL, HrI3, which explained 29.73% of the variation in Pulmonary Edema on chromosome 1  annotated as containing 24 genes and 11 non-coding RNA , a 9.19 Mb region annotated as containing 206 genes and 35 non-coding RNAs . We examined the severity of pulmonary edema in these animals at four days post infection. Founder strain alleles at HrI3 significantly affected pulmonary edema , from 7.31 Mb to 1.01 Mb, containing 10/24 genes and 1/10 annotated non-coding RNAs  was similarly reduced from 9.19 Mb to 91 kb, including 12/206 genes and 2/35 non-coding RNAs  or susceptibility (129S1/SvImJ) contrasted with the other six strains showing an intermediate phenotype. We were therefore looking for individual SNPs (and not regions of difference) that differentiated A/J or 129S1/SvImJ from the other strains. We identified 144 private A/J SNPs or small in/dels, and 611 private 129S1/SvImJ SNPs or small in/dels . These SNPs occur in or near 28 genes  corresponding to that allele, contrasted with the other alleles in the cross. In the simplest case of one allele contrasted with the other seven this means that a private genome feature in the single strain is causative for the QTL. Under more complex scenarios (e.g. two strains contrasted with six), because CC mice share common ancestry due to their natural history and the unique history of laboratory mice ing RNAs . HrI4 . While reverse genetics approaches have afforded insight into the role of viral genes in infection Mx1 was not surprising. Mx1 is known to strongly inhibit influenza virus replication, limiting the resultant IAV-induced disease symptoms in mice Mx1 alleles reduced viral titers, weight loss and clinical disease, inflammation and pathology. Mx1 also acted to influence the expression levels of a large number of transcripts. While it is unlikely a direct transcriptional regulator, Mx1's potent ability to inhibit IAV replication likely alters the signaling environments and host response pathways triggered in response to infection.Host genetic polymorphisms have been shown to contribute to differential disease outcomes, and evidence exists for influenza Mx1. A total of 5 distinct Mx1 haplotypes exist in the pre-CC population, and they can be grouped into three functionally distinct alleles based on their effects during influenza infection. Of particular interest is the CAST/EiJ allele, which disassociates the effects of Mx1 on control of viral replication from its' ability to protect from a clinical disease aspects. We utilized the large number of transcripts that had an eQTL at Mx1 to better understand potential ways in which the CAST/EiJ allele might provide clinical protection while being unable to control IAV replication. We identified a set of transcripts that were significantly upregulated in those individuals with defective Mx1s, but were downregulated in individuals with CAST/EiJ, PWK/PhJ and NZO/HILtJ Mx1 allele). These transcripts included sets of inflammatory and immune related transcripts, such as SOCS3, Irf1, and Interferon-gamma. GTPases, such as Mx1, are important in a number of signaling and protein production activities Mx1 are responsible for regulating specific transcripts, such as those mentioned above, in limiting clinical disease independent of Mx1's previously described anti-IAV activities. However, additional studies are needed to better define whether the differential effect of the CAST/EiJ Mx1 allele are due to Mx1-associated signaling or more subtle effects on viral replication which subsequently affect inflammation and disease.Within human populations, it is possible for multiple alleles to exist at any given locus. Similarly, at any locus within the pre-CC population, up to eight distinct alleles exist. In addition to increasing the probability of having functionally variant alleles segregating within the population, multiple alleles at a locus can give rise to distinct phenotypic outcomes across the population. The effects of this allelic variation can clearly be seen when considering Mx1 allele we found in the pre-CC population and the alternate hypothesis that CAST/EiJ's Mx1 was completely non-functional, and that there was another polymorphic gene within CAST/EiJ that provided some protection from clinical disease. It is only through the recombination present within the CC, and the use of large populations of unique lines that can be evaluated within the CC that such hypothesis can be differentiated. This lack of structure across the genomes also allowed us to gain new insight into the relationships between coexpressed transcripts and disease outcomes, as well as the relationships between specific disease processes (see population-wide pattern section below).In a more general sense, these results illustrate the advantages provided by using a system such as the CC compared to using classical inbred strains such as the founder strains. Because of the inherent genome structure of the founder strains Mx1 served to validate our mapping study, the presence of a large effect allele within genetically variable populations can mask those with smaller effect sizes. Indeed, this genetic architecture is common to pathogen resistance, as several other large effect genes have been identified for viral (e.g. flaviviruses Mx1-/- models). It is likely that there are specific alleles influencing disease processes that only act in the context of the presence or absence of major resistance alleles . To address this issue, we conducted further genome mapping while accounting for Mx1 status using two complementary approaches: asking whether there are loci that act in addition to Mx1 in regulating disease-associated phenotypes (HrI2), and also asking if there are loci that act only in the highly susceptible Mx1 negative population . By doing so, we were able to identify three additional loci influencing weight loss, pulmonary edema and neutrophil infiltration into the airways, further validating the role of genetic variation at HrI3 in contributing to pulmonary edema differences in a separate set of fully inbred CC animals. The development of pulmonary edema Though the identification of Mx1-/- animals also allowed us to compare our study to other studies using QTL mapping within Mx1-/- panels Mx1-/- animals in the pre-CC population, due to sample size, it is likely that similar QTL can be identified in future work using animals with functional Mx1.In addition to allowing us to identify novel QTL that impacted the host response to IAV, our mapping using only Mx1 provides a cautionary note for regions of the genome in that there is a large in/del differentiating the eight CC founders. In fact, C57BL/6J, and thus the assembly, has the deletion. In such regions, functional genomic features may be misannotated and more importantly the genetic variants present in founders without the deletion is currently not annotated. This lack of annotation makes it difficult to conclusively dissect the polymorphisms within these regions that might cause phenotypic variation across the population, and currently requires more intensive sequencing efforts on a case-by-case basis. The Mx1 result illustrates the importance of improving the annotation of genetic variants in the mouse genome. It also suggests that in addition of the processed lists of SNPs and in/dels available at the Mouse Genome Projects from the Sanger Institute, the analysis of the allele effect in QTL intervals should be analyzed by searching for signatures of structural variation that might be present in the raw reads. As further QTL analyses are undertaken within the CC system, approaches to narrow down onto candidate genes and polymorphisms will need to be further developed, likely integrating both transcriptional and refined analysis of sequence data to account for other potential causative genome features. These approaches will be facilitated by the interrogation of transcriptional activity at multiple time points in completely inbred CC lines.Ultimately, the goal of QTL mapping studies is to identify the causal polymorphic genes or genome features responsible for variation in disease processes. A variety of studies have used transcriptional data The pre-CC experiment also allowed us to identify specific CC lines that might be useful models for specific disease phenotypes , as well as other interesting relationships between disease components across the pre-CC population. For example, while lung hemorrhage is a clinically important influenza-associated phenotype In addition, we identified transcriptional modules that correlated with overall disease severity, or with specific aspects of the host response to infection . Recent efforts from a number of groups in vivo studies as to the importance of virus-induced inflammation Mx1-/- subpopulation of pre-CC animals, we again found that both viral replication and levels of inflammation together were better predictors of disease outcome than either was alone. Consistent with this analysis, we identified CC lines that showed high levels of replication and weight loss, but little inflammation as well as lines with excessive inflammation and weight loss, but low viral titers suggesting that multiple pathways can lead to similar clinical disease outcomes across a genetically diverse population. Future studies utilizing this model with fully inbred CC lines, will allow us to more fully evaluate how the kinetics, magnitude, and duration of viral replication and inflammation contribute to disease outcome over the temporal course of the infection. These results illustrate the potential and the power of using genetically diverse mice to study the relative contribution of specific aspects of the pathogen and host response which together drive disease outcome.The findings of this study also provide new insights into the relative contribution of viral replication versus virus-induced inflammation in the pathogenesis of influenza infection. There is conflicting evidence from a variety of Mx1, even when using a single mouse/time point, suggests that the CC lines represent a robust system for identifying polymorphic genes that regulate host responses to infectious diseases. As the CC can be recreated and manipulated, it will increasingly become a useful tool to a) identify candidate genes and pathways for more targeted association studies within human populations, and b) allow us to increase our understanding of how critical demographic and environmental factors, as well as specific genetic subpopulations impact some of the variability in GWAS studies of acute infectious diseases in humans.There is an increased appreciation for the role that host polymorphisms play in the host response to infectious diseases in vivo model represents a useful system for modeling pathogen interactions within genetically diverse populations and identifying novel genetic loci controlling multiple aspects of disease pathogenesis. Though this study had clear limitations, the pre-CC population provided the appropriate framework to develop the methodological approaches that resulted in the identification and prioritization of genes within novel disease loci. These results strongly support the hypothesis that studies using the fully inbred CC lines, with the use of replicate animals and evaluation of phenotypic variation during influenza infection over time, will be even more successful in identifying polymorphic genes that regulate multiple disease associated phenotypes including those phenotypes associated with adaptive immune responses and disease recovery. Furthermore, through careful selection of CC lines, studies can be designed to specifically investigate how interactions between allelic variants in two or more genes interact to influence complex phenotypic outcomes during infection.Host genetic variation clearly plays an important role in regulating differential response phenotypes to infectious disease progression. Herein, we provide proof of concept and a framework for identifying the role of polymorphic genes on microbial pathogenesis using a genetically diverse population: underlying relationships between different disease phenotypes, genetic control of phenotypes following infection , and transcriptional profiles that related to specific disease-associated phenotypes. In summary, this study shows that a genetically complex Mouse studies were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All mouse studies were performed at the University of North Carolina  using protocols approved by the UNC Institutional Animal Care and Use Committee (IACUC). All studies were performed in a manner designed to minimize pain and suffering in infected animals, and any animals that exhibited severe disease signs was euthanized immediately in accordance with IACUC approved endpoints.8–16 week old female animals from the 8 founder strains  were derived from the Jackson labs (jax.org), and bred at UNC Chapel Hill under specific pathogen free conditions. 8–12 week old female Pre-CC mice were bred at Oak Ridge National Laboratories under specific pathogen free conditions, and transferred directly into a BSL-3 containment laboratory at UNC Chapel Hill. Inbred CC mice were bred at UNC Chapel Hill under specific pathogen free conditions. All experiments were approved by the UNC Chapel Hill Institutional Animal Care and Use Committee.The mouse adapted influenza A strain A/PR/8/34 (H1N1) was used for all infection studies. A/PR/8/34 stocks were made by infection of 10-day old embryonated chicken eggs. MDCK cells grown in high glucose Dulbecco's modified Eagle's medium  were used for titering virus.Animals were lightly anesthetized via inhalation with Isoflurane . Following anesthesia, animals were infected intranasally with 5×10∧2 pfu of PR8 in 50 µL of phosphate buffered saline (PBS), while mock infected animals received only 50 µL of PBS. Animals were assayed daily for morbidity (determined as % weight loss), mortality and clinical disease scores. At 4 days post infection, animals were euthanized via Isoflurane overdose and cardiac puncture, animals were assessed for gross pathology (lung hemorrhage and edema) and tissues were taken for various assays.st column of the plate, and 146 uL of lung homogenate in DMEM was added to these wells . Serial dilutions of 46 µL (0.5 log dilutions) were carried out across the plate. Plates were incubated at 37 degrees C for 1 hour, inoculum was removed and 150 µL of serum free DMEM with 1 µg/mL of trypsin was added to each well. Plates were then incubated at 37oC for 3 days. Media was then removed, and wells were stained with a 1% Crystal Violet solution. The stain was washed off with water. Titer is determined as follows:MDCK cells were seeded into 96 well plates at a density of 1.5×10∧5 cells/well in DMEM  and incubated at 37 degrees overnight. Cells were washed 2 times with PBS, before addition of 100 µL of DMEM to each well. Media was removed from all wells in the 1The right lung was removed and submerged in 10% buffered formalin (Fischer) without inflation for 1 week before being submitted to the UNC Linberger Comprehensive Cancer Center histopathology core for processing. Two 5 micron thick Hematoxylin and Eosin stained lung sections (step-separated by 100 microns) were blind-scored by microscopic evaluation performed by two independent scorers for a variety of metrics relating to the extent and severity of immune cell infiltration and pathological damage on a 0–3  scale.For detection of influenza virus antigen, we used serial sections from formalin-fixed, paraffin-embedded lung samples. After deparaffinization and rehydration, antigen retrieval was performed using 0.1% protease (10 min at 37°C). Endogenous peroxidase was blocked with 3% hydrogen peroxide and slides were briefly washed with phosphate-buffered saline (PBS)/0.05% Tween 20. Mouse anti- influenza virus nucleoprotein  and horseradish peroxidase labeled goat anti-mouse IgG2a were used for 1 h at room temperature. Peroxidase activity was revealed by incubating slides in 3-amino-9-ethylcarbazole  for 10 minutes, resulting in a bright red precipitate, followed by counterstaining with hematoxylin. Tissue sections from non-infected BALB/c mice and mouse IgG2a isotype antibody (R&D) were used as negative controls. The extent of influenza viral antigen spread across these slides was then scored in a blinded fashion on a 0–3 scale.At 4 days after infection, mice were killed and lung tissue harvested and placed in RNAlater  and stored at −80°. The tissues were subsequently homogenized, and RNA extracted as previously described www.bioconductor.org). The probes were filtered requiring that all probes meet specific QC requirements  for all samples. Differential expression analysis was performed using the LIMMA Bioconductor package P-value distribution for this analysis to address dependencies inherent from the hierarchical/nested structure of the GO categories.The Agilent arrays were background corrected by applying the Normal-Exponential convolution model For both the full analysis and the Mx1-/- analysis, six thousand probes were chosen to be entered into the analysis based on both high variability across samples as well as a measure of how connected they were Genotyping and haplotype reconstruction were done as described in Genome scans were run as described in Mx1 status, whereby the null model included the haplotype information from the most significant marker at the Mx1 locus (JAX00072951). LOD scores are then computed for each haplotype interval based on the increase in fit of genotype to phenotype when Mx1 haplotype is already taken into account.A more complex model was also used to control for st nucleotide after the last inconsistent SNP until the last nucleotide before the next inconsistent SNP, and also had to exceed 100 base pairs in length. We identified all annotated genes and non-coding RNAs that were within 500 bases of, or in consistent regions and classify these as our likely candidates.For the likely regions of identified QTL peaks, SNP data for the eight founder strains from the Sanger mouse genomes project was downloaded, and filtered to include only homozygous calls. In the case where a single founder strain allele drives a QTL peak, all private SNPs for that strain are candidates for the observed phenotype. In the case where multiple founder strains drive a QTL peak, the most likely hypothesis is that the causative polymorphism exists in a region of shared ancestry between these founder strains. SNPs were categorized into 3 classes: Consistent with a shared ancestry (SNPs where the driver strains share a private SNP), Inconsistent with a shared ancestry , and Uninformative with regards to ancestry (SNPs private to a single strain and SNPs shared by driver strains as well as others). Candidate regions were defined as regions containing at least one consistent SNP, and were bounded by the 1Mx1 locus we combined data from the mouse genome assembly, the Sanger Institute's Mouse Genomes sequencing project and a mouse full-length Mx1 cDNA clone (CT010406). By aligning the cDNA to the Mx1 genomic locus we identified three missing exons . We used this information to design primers to amplify and sequence every exon and the span the deletion boundaries in each strain , and following their protocol. One microgram of total isolated RNA from each sample was reverse transcribed using MMLV-RT , and following their protocol. We ran TaqMan real time PCR with two primer-probe pairs : Mm01217999_m1 to amplify the 5′ gene region of Mx1 transcripts, and Hs03928985_g1 to amplify 18s mRNA. Fold-induction was calculated as the difference in expression levels for infected animals as compared to their strain-matched mock animals.Dataset S1Phenotypes of Pre-CC animals.(CSV)Click here for additional data file.Dataset S2Genotypes of Pre-CC animals.(ZIP)Click here for additional data file.Figure S1Variation in weight loss curves across the eight founder strains of the Collaborative Cross. Following infection with IAV, n = 5–12 animals from each of the eight founder strains had their weights recorded through four days post infection. Shown are weight loss curves (with standard deviations) for these eight strains (A/J (n = 11) = yellow, C57BL/6J (n = 6) = grey, 129S1/SvImJ (n = 5) = pink, NOD/ShiLtJ (n = 5) = dk. Blue, NZO/HILtJ (n = 12) = lt. blue, CAST/EiJ (n = 5) = green, PWK/PhJ (n = 5) = red, WSB/EiJ (n = 5) = purple).). The high variation present within NOD/ShiLtJ and CAST/EiJ was due to bimodal weight loss responses in each strain, and repeated in multiple experiments,(TIF)Click here for additional data file.Figure S2QTL underlying weight loss following influenza infection. (A) QTL scans showing LOD score (Y-axis) and genome position (X-axis) for weight loss. HrI1 on chromosome 16 (dark peak) was identified and influenced many disease phenotypes We remapped weight loss after accounting for the large effect of HrI1 in our QTL model, and identified another QTL, HrI2 on chromosome 7 (light grey peak). Allele effects plot for (B) HrI1 and (C) HrI2, showing the estimated contributions of each founder strain allele (y-axis) across the likely region for these loci  on weight loss. A phenotype by haplotype plot (D) showing the weight loss phenotypes for those animals that were homozygous for founder strain alleles at HrI2 .(TIF)Click here for additional data file.Figure S3QTL underlying pulmonary edema following influenza infection. (A) QTL scans within the Mx1 -/- subpopulation (light grey line) identified a QTL, HrI3 on chromosome 1, influencing pulmonary edema that was not detectable within the whole pre-CC population (black line). (B) Allele effects plot for HrI3, showing the estimated contributions of each founder strain allele across this locus on pulmonary edema. A phenotype by haplotype plot (C) showing the pulmonary edema score for those animals that were homozygous for founder strain alleles at HrI3 .(TIF)Click here for additional data file.Figure S4QTL underlying neutrophil infiltration following influenza infection. (A) QTL scans within the Mx1 -/- subpopulation (light grey line) identified a QTL, HrI4 on chromosome 15, influencing neutrophil infiltration that was not detectable within the whole pre-CC population (black line). (B) Allele effects plot for HrI4, showing the estimated contributions of each founder strain allele across this locus on neutrophil infiltration. A phenotype by haplotype plot (C) showing the neutrophil infiltrate score for those animals that were homozygous for founder strain alleles at HrI4 .(TIF)Click here for additional data file.Table S1Mean (range) of IAV associated phenotypes comparing founder strains and the Pre-CC.(DOCX)Click here for additional data file.Table S2Phenotypic correlations across the pre-CC population.(DOCX)Click here for additional data file.Table S3Significance of predictors of D4 weight.(DOCX)Click here for additional data file.Table S4Transcripts within expression modules.(DOCX)Click here for additional data file.Table S5GO-term enrichment by module.(DOCX)Click here for additional data file.Table S6Mx1 sequence variants.(DOCX)Click here for additional data file.Table S7Transcripts with an eQTL at Mx1.(DOCX)Click here for additional data file.Table S8GO-Term enrichment of eQTL that differentiate the CAST/EiJ Mx1 allele's effects.(DOCX)Click here for additional data file.Table S9Phenotypic mean (ranges) between entire pre-CC population and the Mx1-/- subpopulation.(DOCX)Click here for additional data file.Table S10Phenotypic correlations across the Mx1-/- pre-CC subpopulation.(DOCX)Click here for additional data file.Table S11Transcripts within modules in the Mx1-/- subpopulation.(DOCX)Click here for additional data file.Table S12GO-term enrichment by module in Mx1-/- subpopulation.(DOCX)Click here for additional data file.Table S13Transcriptional module correlates with influenza phenotypes in the Mx1-/- subpopulation.(DOCX)Click here for additional data file.Table S14Primers used in sequencing Mx1.(DOCX)Click here for additional data file.


MX1, HERC5, and OAS1, were not upregulated, suggesting a mechanism by which viral antagonism of immunity results in a profound inflammatory response. These data provide an important first description of the response to a natural herpesvirus infection in humans.Epstein-Barr Virus (EBV) causes infectious mononucleosis and establishes lifelong infection associated with cancer and autoimmune disease. To better understand immunity to EBV, we performed a prospective study of natural infection in healthy humans. Transcriptome analysis defined a striking and reproducible expression profile during acute infection but no lasting gene changes were apparent during latent infection. Comparing the EBV response profile to multiple other acute viral infections, including influenza A (influenza), respiratory syncytial virus (RSV), human rhinovirus (HRV), attenuated yellow fever virus (YFV), and Dengue fever virus (DENV), revealed similarity only to DENV. The signature shared by EBV and DENV was also present in patients with hemophagocytic syndromes, suggesting these two viruses cause uncontrolled inflammatory responses. Interestingly, while EBV induced a strong type I interferon response, a subset of interferon induced genes, including  Epstein-Barr virus (EBV) is a herpesvirus that causes lifelong infection in humans. EBV is estimated to be a causative agent in 1% of all human cancers, in vitroIn order to gain insight into EBV pathogenesis, we examined the host response to EBV using a genomic approach. Systems biology is emerging as an important tool for understanding the human response to infection that can provide novel means of diagnosis and insight into disease mechanisms. All participants gave written informed consent and the University of Minnesota Institutional Review Board approved all protocols used.We recruited 546 healthy undergraduate volunteers from the University of Minnesota residence halls in 2006 and 2007 as recently described. 7 cells/ml aliquots in a cryopreservative solution containing 90% FBS and 10% dimethysulfoxide (DMSO) (Sigma-Aldrich). Samples were allowed to slowly freeze at −80°C overnight and then transferred to liquid nitrogen for storage until needed. Cells were rapidly thawed in a 37°C water bath, diluted to 10 ml in RPNK media supplemented with 50U/ml benzonase (Novagen) (RPNK media: RPMI 1640 (Cellgro) supplemented with 10% FBS , 2% Penicillin – Streptomycin  and 1% L-glutamine ). Cells were then counted using a hemocytometer and divided into separate fractions for flow cytometry or RNA processing.Peripheral blood samples were obtained from subjects via venipuncture and collected in 10 ml purple-top EDTA Vacutainer® tubes (Fisher Scientific). 200μl of blood was used for DNA extraction and HLA typing. Blood Peripheral blood mononuclear cells (PBMCs) were isolated by Accuspin™ System-Histopaque®-1077 (Sigma-Aldrich) density gradient centrifugation per manufacturer’s instructions. PBMC counts were recorded post-ficoll purification of whole blood during each collection and stored as detailed below. Once pelleted, cells were frozen in 1×106 PMBCs were used for each RNA extraction. Cells were first homogenized using QIAshredder columns (Qiagen) per the manufacturer’s instructions. RNA was then extracted using RNeasy kit (Qiagen) with on column DNase step (Qiagen) per the manufacturer’s instructions. RNA was then quantified using a Nanodrop 2000/2000c spectrophotometer (Thermo Scientific) and kept frozen at −80°C.For each sample, 1–2×10200ng of RNA per sample was amplified for microarray analysis using the Illumina TotalPrep RNA Amplification Kit (Ambion) per the manufacturer’s instructions. Tagged transcripts were then sent to the microarray facility of the BioMedical Genomics Center at the University of Minnesota for hybridization to an Illumina Sentrix BeadChip HumanRef-8 v3. Preinfection and latent samples were hybridized to Illumina HumanHT-12 v4 Expression Beadchips.t-test with Bonferonni multiple tests correction. Studies which used healthy controls were subjected to an unpaired unequal variance t-test (Welch) with Bonferonni multiple tests correction. Log2 transformed fold changes were exported from GeneSpring into a *.txt file. These values were imported into the open source program Cluster 3.0, Raw data from 2 transformed fold changes for all relevant genes were imported into the software using their Illumina probe identifiers and were then associated with their appropriate gene symbol by IPA. The software then used these genes and their fold changes to determine which gene sets had statistically significant importance in each of the cases. Canonical pathways were assigned based on the number of genes present in our geneset for each pathway, the level at which these genes were up or down regulated, and the likelihood these trends would occur by chance by using the multiple test correction Fisher’s exact test. Results are reported in this work as the Benjamini-Hochberg p-value or ratio.Pathway analysis was performed using Ingenuity Pathway Analysis (IPA) software (Ingenuity System). Gene sets were screened to limit the number of genes utilized for analysis. For the acute gene set, the 464 significantly changed genes previously described were used. 53 genes with potential long-term changes were used from the latent analysis. LogGene Set Enrichment Analysis (GSEA) was performed on whole chip acute and latent infection microarray data using GSEA software (Broad Institute). Analysis was performed per Broad Institute instructions.Frozen PBMCs from baseline and acute IM timepoints from 4 subjects were thawed and stained to be sorted into four different subsets: CD8 T cells , NK cells , B cells , and monocytes (CD14/CD16). Subsets were sorted into 1.5 ml tubes pre-coated with media using a FACS Aria under BSL-2 sorting conditions. Cells were washed with PBS and RNA was extracted as described above.100ng of RNA was used with the SuperScript III Platinum Two-Step qRT-PCR Kit (Invitrogen) to generate cDNA. Samples were then stored at −20°C. 43 genes were selected for analysis by PCR from a larger list of changed genes in IM subjects and other acute viral infections that comprised relevant functional groupings as assessed by IPA. 384-well SuperArray plates pre-coated with primers for the desired 43 genes plus 5 controls were obtained as a custom order from SABiosciences. 10 μL of cDNA per subject was used with RT2 Real-time SYBR green/Rox PCR master mix (SABiosciences) for qRT-PCR analysis. Products were detected using an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). The genes ACTB, B2M, and RPL13A were used as housekeeping genes during the calculation of fold changes as they had previously been shown not to change significantly during acute EBV infection (data not shown). Fold changes were calculated as: 2∧ (Δ Acute Housekeeping Control – Baseline Housekeeping Control)/2∧(Δ Acute Gene of Interest – Baseline Gene of Interest).Subject samples for the blinded gene signature study were chosen by H.H.B Jr and RNA extraction was performed by O.A.O to ensure S.K.D was blinded to sample identity. cDNA synthesis, qRT-PCR and analysis were performed by S.K.D. Sample cycle thresholds were imported into Cluster 3.0 as a text file and a K-means clustering was performed on the samples with 3 groupings and 10,000 iterations to provide a sufficient number of successful solutions from the algorithm.t-test or a one-way ANOVA with a p-value less that 0.05 as the cutoff for statistical significance. Pearson correlation coefficients were generated with Prism by comparing pairs of relevant groupings.Except for where Genespring (Agilent) or IPA (Ingenuity Systems) was used, most other statistical analysis was performed using Prism software (Graphpad). MedCalc  was used for comparison of Pearson correlation coefficients. Comparisons between groups were performed with either an unpaired two-tailed Gene expression data is available from the Gene Expression Omnibus. The data are part of superseries GSE45924 composed of two subseries, GSE45918 and GSE45919.EBV naïve subjects were recruited from freshman classes at the University of Minnesota during 2006 and 2007. Enrolled study subjects were screened for exposure to EBV on an ongoing basis by testing blood or throat wash samples taken every four to eight weeks. Samples were evaluated for EBV genomes by qPCR and/or antibodies to viral antigens. Of 143 subjects followed in the study, 66 experienced primary EBV infection during their undergraduate years Due to the long incubation period, the exact date of viral acquisition is not known. Instead, date of symptom onset was used for timing, since this date was well defined for each subject. For acute microarray analysis, we chose eight subjects with peripheral blood samples taken near the onset of symptoms, and compared them to samples taken at least three months before infection. These eight subjects plus an additional two were chosen for analysis of latency, with latent samples being defined as at least >200 days after infection . BaselinMicroarray analysis was performed on peripheral blood mononuclear cells (PBMCs) from subjects described above. Each was compared with his or her own healthy pre-infection sample. Gene expression fold changes greater than 2, with a p-value of ≤0.05 with Bonferonni multiple tests correction were considered significant. For subjects presenting with acute IM, this analysis yielded a list of 464 genes that changed more than two fold . 318 genIFNG, which was shown to be elevated in mice latently infected with a murine gammaherpesvirus Studies in mice showed that latent herpesvirus infection protected hosts from subsequent bacterial infection, suggesting that persistent infection with herpesviruses may have a beneficial role in their human hostsWe chose to perform analysis of whole PBMCs in this study to facilitate the comparison to data published on other infections and disorders (see below). However, we still sought to understand which cellular components were representing the distinct aspects of the signature observed in acute EBV infection. Thus, we measured gene expression in sorted CD8+ T cells, natural killer (NK) cells, monocytes, and B cells  from 4 sAll cell types displayed ≥3 fold upregulation of type I IRGs, suggesting that the interferon response during primary EBV infection is systemic, not limited to a particular cell type . The celHuman peripheral blood has been shown to have different subgroups of monocytes, which may be broadly described as inflammatory M1 monocytes or immunoregulatory M2 monocytes. We compared our data on EBV with published data sets from five other acute viral infections including influenza A virus (Flu), human rhinovirus (HRV), respiratory syncitial virus (RSV), in vitro (data not shown). Not surprisingly, such genes were also upregulated by Poly IC, and were a dominant element of the SLE signature . This downregulation is particularly surprising for IL1B, which is considered a type I IRG and therefore one might expect it would be upregulated during viral infection. Indeed, IL1B was generally upregulated during antiviral responses, including subjects with Flu, HRV, and RSV, and subjects treated with Poly IC or SLE patients (blue asterisk in The heatmap in  and OSM . The dowPRF1) and the syntaxins. The genetic basis of MAS is less well understood but the vast majority of patients first present with sJIA. Due to the surprising difference between the EBV/DENV signature and that observed in other acute viral infections, we theorized that perhaps the signature would more closely resemble that observed during inflammatory syndromes. To this end we examined microarray data obtained from subjects with familial hemophagocytic lymphohistiocytosis (FLH) Although we cannot know precisely when subjects were infected with EBV, we were able to “stage” the subjects relative to the date of symptom onset. In the heatmaps shown, the subjects are ordered according to their symptom onset date. It is apparent that set B genes (IRGs) were upregulated in two of the pre-symptomatic subjects, but the other major aspect of the signature was absent . To deteMX1, which is a classic IFN induced GTP-binding protein, MX1, OAS1, and HERC5 and performed real-time PCR on 70 timepoints from 50 subjects. As expected, these genes were upregulated in every other acute infection data set examined and in SLE  TYMS , and SLAMF8 , are all upregulated by IFNγ consistent with the known effects of IFNγ when given as therapy OASL in particular has been hypothesized to cause fatigue through antagonism of the thyroid receptor Finally, we investigated whether or not there was a correlation between gene expression and severity of illness during acute infection. A hallmark of infectious mononucleosis is the dramatic expansion of CD8 T cells. Indeed, both the total number of CD8 T cells/ml blood or the ratio of CD8 to CD4 T cells in the blood provided the best predictor of disease severity in a large prospective study. This study provides the first gene expression profiling of a natural herpes virus infection in healthy human subjects. One notable aspect of the EBV gene expression profile was the striking upregulation of genes related to cell cycle and proliferation. While proliferation of cells is likely to be part of the immune response to any pathogenic virus, what is striking is the magnitude and duration of these gene expression changes, which allow them to be observed at the level of whole blood gene expression profiling. Most other viral infections did not display significant upregulation of these genes at any time point. The exception was DENV. Indeed the shared profile of EBV and DENV was also observed in hemophagocytic syndromes, emphasizing the exaggerated inflammatory response that ensues in these viral infections. Although other potentially interesting comparisons would be to DNA viruses, particularly CMV, which can cause IM, there are currently no data available on gene expression during primary infection in humans.In contrast to the readily apparent gene signature in acute infection, we were surprised to discover that there were no consistently detectable gene expression changes in human blood during latent infection, despite the sensitivity of our approach. EBV is known to persist in the B cell compartment, albeit at quite low levels (1–50 copies per million B cells). IL1B, which was surprising given that monocytes are activated and proliferating during acute EBV infection. Recent data suggested IL-1β may be downregulated by type I IFN in human monocytic cells in vitro. IL1B would be downregulated by IFN in EBV but upregulated by IFN in most other viral infections. Another possibility was suggested by a recent report, which showed that EBV encodes a microRNA (BART15) that targets the NLRP3 inflammasome, and exosome mediated secretion of this miRNA resulted in downregulation of IL-1β in bystander (uninfected) monocytes in vitro. IL1B was also down regulated in most DENV patients and in about half of subjects given the YFV vaccine Click here for additional data file.Figure S2EBV demonstrates a bias toward a type II IRG expression pattern.(PDF)Click here for additional data file.Figure S3Gene set analysis can successfully segregate the EBV signature from other acute illnesses.(PDF)Click here for additional data file.Figure S4Timecourse of gene changes during EBV infection compared to YFV, Poly IC, and DENV.(PDF)Click here for additional data file.Table S1Characteristics of the subjects chosen for microarray analysis.(DOCX)Click here for additional data file.Table S2List of significantly changed genes during acute IM.(DOCX)Click here for additional data file.Table S3Genes with a fold change ≥3.(DOCX)Click here for additional data file.Table S4EBV “unique” genes.(DOCX)Click here for additional data file.


Serial C-reactive protein (CRP) values may be useful for decision-making regarding duration of antibiotics in neonates. However, established standard of practice for its use in preterm very low birth weight  infants are lacking.Evaluate compliance with a CRP-guided computerized decision support (CDS) algorithm and compare characteristics and outcomes of compliant versus non-compliant cases. Measure correlation between CRPs and white blood count (WBC) indices.We examined 3 populations: 1) all preterm VLBW infants born at Vanderbilt 2006–2011 – we assessed provider compliance with CDS algorithm and measured relevant outcomes; 2) all patients with positive blood culture results admitted to the Vanderbilt NICU 2006–2012 – we tested the correlation between CRP and WBC results within 7 days of blood culture phlebotomy; 3) 1,000 randomly selected patients out of the 7,062 patients admitted to the NICU 2006–2012 – we correlated time-associated CRP values and absolute neutrophil counts.Of 636 VLBW infants in cohort 1), 569 (89%) received empiric antibiotics for suspected early-onset sepsis. In 409 infants (72%) the CDS algorithm was followed; antibiotics were discontinued ≤48 hours in 311 (55%) with normal serial CRPs and continued in 98 (17%) with positive CRPs, resulting in significant reduction in antibiotic exposure (p<0.001) without increase in complications or subsequent infections. One hundred sixty (28%) were considered non-compliant because antibiotics were continued beyond 48 hours despite negative serial CRPs and blood cultures. Serial CRPs remained negative in 38 (12%) of 308 blood culture-positive infants from cohort 2, but only 4 patients had clinically probable sepsis with single organisms and no immunodeficiency besides extreme prematurity. Leukopenia of any cell type was not linked with CRPs in cohorts 2 and 3.CDS/CRP-guided antibiotic use is safe and effective in culture-negative VLBW infants. CRP results are not affected by low WBC indices. In the NICU, “suspect sepsis” is by far the most common diagnosis and antibiotics are the most frequently prescribed medications Computerized physician order entry (CPOE) with clinical decision support (CDS) in the NICU setting has been shown to decrease variance in medication administration, improve protocol compliance and reduce mortality Traditionally, WBC indices are used as diagnostic sepsis markers in early- and late-onset sepsis This study has been approved by the Vanderbilt University Institutional Review Board (IRB #120953 and 111600). Consent by the next of kin, caretakers, or guardians on the behalf of the infant participants, for their stored medical records used for research, was specifically waived by the approving IRB. We performed a retrospective chart review on 3 separate sets of patients:a priori was the length of antibiotic treatment of infants with negative blood cultures who were treated based on 2 consecutive CRP values obtained at initial evaluation (t0) and at 48 hours (t48) (“CRP protocol compliant”) and those that were treated outside the protocol (“CRP protocol non-compliant”). Patients for whom antibiotics were never started were excluded from the study (n = 67).Cohort 1: All inborn preterm infants <1500 g admitted at Vanderbilt between January 2006 and December 2011 with negative blood cultures during the first week post-partum. We excluded patients with congenital anomalies and/or surgery performed within the first 3 postnatal days typically resulting in prolonged empiric antibiotic use. We compared the number of antibiotic doses, days on antibiotics, as well as specific morbidities affecting VLBW infants . Our primary outcome designated Staphylococcus (CONS), and cases with and without documented clinical signs of sepsis , and blood culture results within 7 days of diagnostic phlebotomy. We excluded non-viable infants or infants who did not survive past the first 48 hours post-partum, patients no longer hospitalized at Vanderbilt 48 hours after birth, and patients who did not have at least one time-correlated set of CRP and neutrophil data. We stratified CRP and WBC correlations by positive blood cultures with and without coagulase-negative Cohort 3: 1,000 randomly selected patients out of the 7,062 patients admitted to the NICU during the same study period as in cohort 2. We correlated all time-associated CRP values and absolute neutrophil counts that were accessible in the Vanderbilt electronic medical records. The same exclusion criteria as in cohort 2 applied.Candida species. CONS infections were considered confirmed if at least 1 repeat positive culture was present within 72 hours of the initial positive blood culture. A positive CRP result was defined as any value greater than 10 mg/L 3 cells/μL and a normal total neutrophil count as >2,500/μL Confirmed positive blood cultures were defined as any single culture positive for non-contaminant, non-CONS bacteria or for The CRP protocol included automated orders for a blood culture, CRP, WBC with differential at time of antibiotic order followed by a repeat CRP and WBC with differential 48 hours later . If the Continuous variables were described using the median, 25th and 75th percentiles, and categorical variables were described using percentages. The Wilcoxon signed rank test (2 group) or Kruskal-Wallis test (3 groups) were used to determine if the continuous variables differed between compliance groups. Pearson's Chi-squared test was used to evaluate differences by compliance group for categorical variables. Separate multivariable logistic regression were fit to determine if the binary outcomes of death, NEC, PDA, CLD or IVH were associated with compliance group while controlling for gestational age, ventilator days and nasal CPAP days. To evaluate the association between the absolute neutrophil count (ANC) and CRP, we used a linear mixed effects regression model. This regression model included a random intercept term to control for taking repeated observations on subjects over time.We identified 636 culture-negative VLBWs within the 5-year study period of which 569 (89%) received empiric antibiotics in the first week post-partum for possible early-onset sepsis. Antibiotics were discontinued within 48 hours in 311 infants (55%) with negative CRP results and continued in 98 infants (15%) for at least one CRP value >10 mg/L, totaling 409 infants deemed “CRP-protocol compliant” (Comp). Overall compliance with the computerized CRP protocol was 72%, as empiric antibiotics were continued >48 hours in the setting of a negative blood culture with negative serial CRP results in 160 patients (28%). We designated these 160 cases as “CRP-protocol non-compliant” (Non-Comp). Patients in the Non-Comp group were of lower gestational age and birth weight compared to the Comp group . The NonOf all infants in whom antibiotics were continued because of elevated CRP results on admission, only 32% had CRP values >10 mg/L at 48 hours. Of all 126 infants with continued antibiotics because of positive CRP values at either t0 or t48, at 48 hours, 27% had values between 10 and 15 mg/L, 17.5% between 15 and 20 mg/L, 17.6% between 20 and 40 mg/L and 16.8% >40 mg/L .The 409 infants in the Comp group received significantly less doses of ampicillin  and a smaller total dose of ampicillin  compared to the 160 patients in the Non-Comp group . Within Among the 311 VLBW infants where antibiotics were stopped within 48 hours, 204 infants (66%) had abnormal WBC indices. Among these 204 infants, over 80% had low WBC indices and 20% had high WBC indices  cases of reported positive blood cultures had negative (<10 mg/L) CRP values, of which 68 (33%) were deemed to be confirmed positive blood cultures as defined above. The distribution of gram-negative and gram-positive bacteria in patients with negative or positive CRP results at initial evaluation was similar, indicating the relatively low sensitivity of initially negative CRP results .In 38 (12%) infants of 308 with confirmed positive blood cultures, serial CRP values remained negative. A list of 39 organisms identified from 38 patients is presented in To test the hypothesis that false-negative CRP results are more commonly observed in cases of abnormal WBC indices, we compared the frequency of low WBC values between cases with confirmed positive blood cultures and either positive or negative serial CRP results . In 11 oTo determine if the frequency of false-negative CRP results is higher in neutropenic patients compared to infants with normal neutrophil counts, we reviewed 27 cases of persistently negative CRP values despite confirmed positive blood cultures in NICU patients with ANCs >3,500/μL. We found 3 non-CONS cases with clinically probable sepsis as defined in Ref. 19, indicating a similar yet overall very rare incidence of false-negative CRP results in non-neutropenic infants.False negative CRP results in proven clinically significant sepsis have occurred in association with neutropenia only one infant developed non-CONS-positive blood cultures in this time frame. This finding suggests the protocol allowed accurate identification of uninfected infants and thus a safe reduction of antimicrobial exposure. Protocol deviations were more common than expected (28%).The implementation of a computerized CRP-based early sepsis evaluation protocol for VLBW infants was associated with a reduction of antimicrobial days/doses without evidence of increased risk in infants treated per the algorithm. Clinician compliance with the protocol was reasonable (72%) and subsequent suspect sepsis evaluations were infrequent in infants following the week of CRP protocol-guided cessation of antibiotics (16%). In our large cohort While still considered “gold standard” for the diagnosis of neonatal sepsis, blood cultures are known for their low sensitivity in VLBW infants The use of CPOE with CDS has demonstrated utility and patient benefit Because hepatocytes are the primary source of CRP There are noteworthy distinctions and limitations of our study. This study was retrospective and was not adequately powered to definitively determine the risk of common morbidities of prematurity. The lack of a comparison group treated without CRP protocol during the same time period does not allow us to claim overall reduction in antibiotic use. However, our results suggest that implementation of our protocol was associated with safe cessation of antimicrobial treatment in VLBW preterm infants unlikely to be infected. CPOE with CDS was implemented at our institution in 2003. Thus, at the time of CRP protocol initiation, all providers had adapted to the culture of CPOE, which may explain the relative high compliance with the protocol.Any additional laboratory test can increase the risk for anemia in this vulnerable population. While we cannot exclude the possibility for increased anemia since introduction of the CDS/CRP-guided antibiotic use protocol, we do not expect a significant effect on anemia for the following reasons: 1) CRP values were almost always obtained at time of other routine laboratory tests  and 2) we only measured 2 CRP values within 48 hours followed by one additional value at 7 days of antibiotics in case initial CRP values suggested infection and a decision to treat was made. For better integration into provider workflow, the CRP protocol analyzed here used samples at initial diagnostic work-up. Given the lag period of CRP, elevated levels may not be evident until 12 hours after initiation of the inflammatory stimulus The computerized CRP protocol is used for determining the length of empiric antibiotic use in late-onset sepsis at our institution. We plan to evaluate the effectiveness and safety in this population as well but because of the greater heterogeneity of diagnoses and potential reasons to prolong antibiotic use (e.g. peri-operative), this project would fall outside the scope of this report. Another limitation is the definition of “confirmed positive blood culture”. The association of confirmed positive blood cultures and negative serial CRP values in our cohort was 12%. Others have reported negative serial CRP results in 36% of cases with gram-positive blood cultures and 8% with gram-negative blood cultures While cut-off values may have to be lowered for extremely premature infants, we consider CRP a reliable marker for clinically significant positive blood cultures in NICU patients.We developed of a computerized CRP-based sepsis evaluation protocol for VLBW infants that was generally well accepted by health care providers, but WBC results influenced antibiotic duration decision-making despite CRP results. Persistently negative CRP values were rare in clinically probable non-CONS sepsis (1%), and do not occur more frequently in the presence of neutropenia.Table S1Shows the number and timing of medical and surgical necrotizing enterocolitis (NEC) cases and associated deaths by antibiotic group from cohort 1 (continued versus discontinued). Early NEC occurred more often in the antibiotics discontinued group but total number of NEC and total number of associated deaths were higher in the antibiotics continued group. The study was underpowered to test the effect of CRP protocol compliance on timing of NEC.(DOCX)Click here for additional data file.