Maria Cecilia Lopez

Incidence, clinical predictors, genomics, and outcome of acute kidney injury among trauma patients

Objective:To determine clinical and genomic characteristics and in-hospital mortality risk associated with acute kidney injury (AKI) in the multicenter prospective cohort of patients with blunt trauma. Summary Background Data:Less severe stages of AKI characterized by small changes in serum creatinine (sCr) are inadequately studied among trauma patients. Methods:We performed a secondary analysis of the “Inflammation and the Host Response to Injury” (Glue Grant) database to include adult blunt trauma patients without history of kidney disease. AKI was defined by the Risk, Injury, Failure, Loss, and End-stage Kidney (RIFLE) classification, which requires a 50% increase in sCr and stratifies patients into following 3 severity stages: risk, injury, and failure. Association between all stages of AKI and in-hospital mortality was analyzed using a multivariable logistic regression analysis. Genome-wide expression analysis was performed on whole blood leukocytes obtained within 12 hours of trauma. Results:AKI occurred in 26% of 982 patients. The adjusted risk for hospital death was 3 times higher for patients with AKI compared with patients without AKI (odds ratio = 3.05) (95% confidence interval, 1.73–5.40). This risk was evident in a dose-response manner and even patients with mild AKI had odds ratio for dying of 2.57 (95% confidence interval, 1.19–5.50) compared with patients without AKI. Genome-wide expression analysis failed to show a significant number of genes whose expression could discriminate among patients with and without AKI. Conclusions:In a multicenter prospective cohort of blunt trauma patients, AKI characterized by small changes in sCr was associated with an independent risk of hospital death.

Postnatal Age Is a Critical Determinant of the Neonatal Host Response to Sepsis

Neonates manifest a unique host response to sepsis even among other children. Preterm neonates may experience sepsis soon after birth or during often-protracted birth hospitalizations as they attain physiologic maturity. We examined the transcriptome using genome-wide expression profiling on prospectively collected peripheral blood samples from infants evaluated for sepsis within 24 h after clinical presentation. Simultaneous plasma samples were examined for alterations in inflammatory mediators. Group designation (sepsis or uninfected) was determined retrospectively on the basis of clinical exam and laboratory results over the next 72 h from the time of evaluation. Unsupervised analysis showed the major node of separation between groups was timing of sepsis episode relative to birth (early, <3 d, or late, ≥3 d). Principal component analyses revealed significant differences between patients with early or late sepsis despite the presence of similar key immunologic pathway aberrations in both groups. Unique to neonates, the uninfected state and host response to sepsis is significantly affected by timing relative to birth. Future therapeutic approaches may need to be tailored to the timing of the infectious event based on postnatal age.

The yeast protein Gcr1p binds to the PGK UAS and contributes to the activation of transcription of the PGK gene

Analysis of the upstream activation sequence (UAS) of the yeast phosphoglycerate kinase gene (PGK) has demonstrated that a number of sequence elements are involved in its activity and two of these sequences are bound by the multifunctional factors Rap1p and Abftp. In this report we show by in vivo footprinting that the regulatory factor encoded by GCR1 binds to two elements in the 3′ half of the PGK UAS. These elements contain the sequence CTTCC, which was previously suggested to be important for the activity of the PGK UAS and has been shown to be able to bind Gcrlp in vitro. Furthermore, we find that Gcr1p positively influences PGK transcription, although it is not responsible for the carbon source dependent regulation of PGK mRNA synthesis. In order to mediate its transcriptional influence we find that Gcrtp requires the Rap1p binding site, in addition to its own, but not the Abf1p site. As neither a Rapip nor a Gcr1p binding site alone is able to activate transcription, we propose that Gcr1p and Rapip interact in an interdependent fashion to activate PGK transcription.

Neutrophil chemotaxis and transcriptomics in term and preterm neonates

&NA; Neutrophils play a crucial role in combating life‐threatening bacterial infections in neonates. Previous studies investigating neonatal cell function have been limited because of restricted volume sampling. Here, using novel microfluidic approaches, we provide the first description of neutrophil chemotaxis and transcriptomics from whole blood of human term and preterm neonates, as well as young adults. Ex vivo percent cell migration, neutrophil velocity, and directionality to N‐formylmethionyl‐leucyl‐phenylalanine were measured from whole blood using time‐lapse imaging of microfluidic chemotaxis. Genome‐wide expression was also evaluated in CD66b+ cells using microfluidic capture devices. Neutrophils from preterm neonates migrated in fewer numbers compared to term neonates (preterm 12.3%, term 30.5%, P = 0.008) and at a reduced velocity compared to young adults (preterm 10.1 &mgr;m/min, adult 12.7 &mgr;m/min, P = 0.003). Despite fewer neutrophils migrating at slower velocities, neutrophil directionality from preterm neonates was comparable to adults and term neonates. 3607 genes were differentially expressed among the 3 groups (P < 0.001). Differences in gene expression between neutrophils from preterm and term neonates were consistent with reduced pathogen recognition and antimicrobial activity but not neutrophil migration, by preterm neonates. In summary, preterm neonates have significant disturbances in neutrophil chemotaxis compared to term neonates and adults, and these differences in phenotype appear at the transcriptional level to target inflammatory pathways in general, rather than in neutrophil migration and chemotaxis.

Unique transcriptomic response to sepsis is observed among patients of different age groups

Sepsis is a major cause of morbidity and mortality, especially at the extremes of age. To understand the human age-specific transcriptomic response to sepsis, a multi-cohort, pooled analysis was conducted on adults, children, infants, and neonates with and without sepsis. Nine public whole-blood gene expression datasets (636 patients) were employed. Age impacted the transcriptomic host response to sepsis. Gene expression from septic neonates and adults was more dissimilar whereas infants and children were more similar. Neonates showed reductions in inflammatory recognition and signaling pathways compared to all other age groups. Likewise, adults demonstrated decreased pathogen sensing, inflammation, and myeloid cell function, as compared to children. This may help to explain the increased incidence of sepsis-related organ failure and death in adults. The number of dysregulated genes in septic patients was proportional to age and significantly differed among septic adults, children, infants, and neonates. Overall, children manifested a greater transcriptomic intensity to sepsis as compared to the other age groups. The transcriptomic magnitude for adults and neonates was dramatically reduced as compared to children and infants. These findings suggest that the transcriptomic response to sepsis is age-dependent, and diagnostic and therapeutic efforts to identify and treat sepsis will have to consider age as an important variable.

Inhibition of PI3K/Akt/mTOR signaling in PI3KR2-overexpressing colon cancer stem cells reduces tumor growth due to apoptosis

In sporadic colon cancer, colon cancer stem cells (CCSCs) initiate tumorigenesis and may contribute to late disease recurrences and metastases. We previously showed that aldehyde dehydrogenase (ALDH) activity (as indicated by the ALDEFLUOR® assay) is an effective marker for highly enriching CCSCs for further evaluation. Here, we used comparative transcriptome and proteome approaches to identify signaling pathways overrepresented in the CCSC population. We found overexpression of several components of the phosphoinositide 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) signaling pathway, including PI3KR2, a regulatory subunit of PI3K. LY294002, a PI3K inhibitor, defined the contribution of the PI3K/Akt/mTOR signaling pathway in CCSCs. LY294002-treated CCSCs showed decreases in proliferation, sphere formation and self-renewal, in phosphorylation-dependent activation of Akt, and in expression of cyclin D1. Inhibition of PI3K in vivo reduced tumorigenicity, increased detection of cleaved caspase 3, an indicator of apoptosis, and elevated expression of the inflammatory chemokine, CXCL8. Collectively, these results indicate that PI3K/Akt/mTOR signaling controls CCSC proliferation and CCSC survival, and suggests that it would be useful to develop therapeutic agents that target this signaling pathway.

Abstract A05: Phosphoinositide-3-kinase pathway promotes colon cancer stem cell proliferation

Introduction. The cancer stem cell (CSC) model posits that a select cell-subpopulation exists in solid tumors that sustain tumorigenesis. Previously, we have shown that aldehyde-dehydrogenase (ALDH) enriches colon CSCs (CCSC) from sporadic colon cancer. In this study, Affymetrix microarray was used to screen for differences between ALDH hi and ALDH lo cells isolated from sporadic human colon adenocarcinoma. As a result, the phosphoinositide-3-kinase (PI3K) pathway was identified as a potential regulator of CCSC growth and differentiation. Functional assays demonstrate that inhibition of PI3K results in significant reduction in proliferation and colony formation of by CCSCs. Experimental Procedures. Unbiased Expression-Profile Analysis: We implanted human CCSCs into flanks of NOD-SCID mice. Flow assisted cell sorting discriminated resulting tumor epithelia into ALDH hi and ALDH lo groups from 6 individual sporadic human adenocarcinomas, which were analyzed using Affymetrix GeneChip Human Genome U133 Plus 2.0 Array. Proliferation Assay: Bromodeoxyuridine (BrdU) incorporation measured the effect of LY294002 (Calbiochem) (1-100 μM) on proliferation. Colony Assay: Methylcellulose colony assays were used to assess the effect of LY294002 on colony formation. Summary of Data. Expression Profile Analysis revealed 136 genes that were significantly up or down-regulated in the ALDH hi cell groups (>3-fold; p hi vs. ALDH lo CCSCs. Cell Proliferation Assay: LY294002 significantly inhibits CCSC proliferation with IC 50 ~ 50 μM and near complete inhibition at the dose of 100 μM (p Conclusions. We have previously shown that ALDH enriches colon cancer epithelia. Here we show that the PI3K signaling pathway is differentially expressed between ALDH hi and ALDH lo CCSCs derived from primary tumor xenografts. We demonstrate in vitro functional differences in CCSCs with drug-inhibition assays. Citation Format: Sugong Chen, Robert C. Fisher, Anitha K. Shenoy, Maria Cecilia Lopez, Lyle L. Moldawer, Henry V. Baker, Emina H. Huang. Phosphoinositide-3-kinase pathway promotes colon cancer stem cell proliferation. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A05.