Chronic postsurgical pain in Europe: An observational study.

BACKGROUND Chronic postsurgical pain (CPSP) is an important clinical problem. Prospective studies of the incidence, characteristics and risk factors of CPSP are needed. OBJECTIVES The objective of this study is to evaluate the incidence and risk factors of CPSP. DESIGN A multicentre, prospective, observational trial. SETTING Twenty-one hospitals in 11 European countries. PATIENTS Three thousand one hundred and twenty patients undergoing surgery and enrolled in the European registry PAIN OUT. MAIN OUTCOME MEASURES Pain-related outcome was evaluated on the first postoperative day (D1) using a standardised pain outcome questionnaire. Review at 6 and 12 months via e-mail or telephonic interview used the Brief Pain Inventory (BPI) and the DN4 (Douleur Neuropathique four questions). Primary endpoint was the incidence of moderate to severe CPSP (numeric rating scale, NRS ≥3/10) at 12 months. RESULTS For 1044 and 889 patients, complete data were available at 6 and 12 months. At 12 months, the incidence of moderate to severe CPSP was 11.8% (95% CI 9.7 to 13.9) and of severe pain (NRS ≥6) 2.2% (95% CI 1.2 to 3.3). Signs of neuropathic pain were recorded in 35.4% (95% CI 23.9 to 48.3) and 57.1% (95% CI 30.7 to 83.4) of patients with moderate and severe CPSP, respectively. Functional impairment (BPI) at 6 and 12 months increased with the severity of CPSP (P < 0.01) and presence of neuropathic characteristics (P < 0.001). Multivariate analysis identified orthopaedic surgery, preoperative chronic pain and percentage of time in severe pain on D1 as risk factors. A 10% increase in percentage of time in severe pain was associated with a 30% increase of CPSP incidence at 12 months. CONCLUSION The collection of data on CPSP was feasible within the European registry PAIN OUT. The incidence of moderate to severe CPSP at 12 months was 11.8%. Functional impairment was associated with CPSP severity and neuropathic characteristics. Risk factors for CPSP in the present study were chronic preoperative pain, orthopaedic surgery and percentage of time in severe pain on D1. TRIAL REGISTRATION Clinicaltrials.gov identifier: NCT01467102.

Human DMTF1β antagonizes DMTF1α regulation of the p14(ARF) tumor suppressor and promotes cellular proliferation

The human DMTF1 (DMP1) transcription factor, a DNA binding protein that interacts with cyclin D, is a positive regulator of the p14ARF (ARF) tumor suppressor. Our earlier studies have shown that three differentially spliced human DMP1 mRNAs, α, β and γ, arise from the human gene. We now show that DMP1α, β and γ isoforms differentially regulate ARF expression and promote distinct cellular functions. In contrast to DMP1α, DMP1β and γ did not activate the ARF promoter, whereas only β resulted in a dose-dependent inhibition of DMP1α-induced transactivation of the ARF promoter. Ectopic expression of DMP1β reduced endogenous ARF mRNA levels in human fibroblasts. The DMP1β- and γ-isoforms share domains necessary for the inhibitory function of the β-isoform. That DMP1β may interact with DMP1α to antagonize its function was shown in DNA binding assays and in cells by the close proximity of DMP1α/β in the nucleus. Cells stably expressing DMP1β, as well as shRNA targeting all DMP1 isoforms, disrupted cellular growth arrest induced by serum deprivation or in PMA-derived macrophages in the presence or absence of cellular p53. DMP1 mRNA levels in acute myeloid leukemia samples, as compared to granulocytes, were reduced. Treatment of acute promyelocytic leukemia patient samples with all-trans retinoic acid promoted differentiation to granulocytes and restored DMP1 transcripts to normal granulocyte levels. Our findings imply that DMP1α- and β-ratios are tightly regulated in hematopoietic cells and DMP1β antagonizes DMP1α transcriptional regulation of ARF resulting in the alteration of cellular control with a gain in proliferation.

Impact of Different Illumination Conditions on Visual Caries Detection with ICDAS

The aim of this study was to test the influence of different degrees of additional illumination on visual caries detection using the International Caries Detection and Assessment System (ICDAS). Two calibrated examiners assessed 139 occlusal surfaces of extracted permanent molars using a standard operation lamp with or without an additional headlamp providing three default brightness intensities. Histology served as the gold standard. Pooled data showed no differences in sensitivities. Specificities were not influenced by additional light. The area under the curve for the Marthaler classification D3 threshold was significantly lower when an additional strong headlamp was used (0.59 compared to 0.69-0.72 when reduced illumination intensities were used). One of the two examiners also had a significantly lower sensitivity for the D1 threshold when an additional headlamp was used. The use of additional white light led to a reduced detection of dentine lesions.

Influence of contact points on the performance of caries detection methods in approximal surfaces of primary molars: an in vivo study

This in vivo study aimed to evaluate the influence of contact points on the approximal caries detection in primary molars, by comparing the performance of the DIAGNOdent pen and visual-tactile examination after tooth separation to bitewing radiography (BW). A total of 112 children were examined and 33 children were selected. In three periods (a, b, and c), 209 approximal surfaces were examined: (a) examiner 1 performed visual-tactile examination using the Nyvad criteria (EX1); examiner 2 used DIAGNOdent pen (LF1) and took BW; (b) 1 week later, after tooth separation, examiner 1 performed the second visual-tactile examination (EX2) and examiner 2 used DIAGNOdent again (LF2); (c) after tooth exfoliation, surfaces were directly examined using DIAGNOdent (LF3). Teeth were examined by computed microtomography as a reference standard. Analyses were based on diagnostic thresholds: D1: D 0 = health, D 1 –D 4 = disease; D2: D 0 , D 1 = health, D 2 –D 4 = disease; D3: D 0 –D 2 = health, D 3 , D 4 = disease. At D1, the highest sensitivity/specificity were observed for EX1 (1.00)/LF3 (0.68), respectively. At D2, the highest sensitivity/ specificity were observed for LF3 (0.69)/BW (1.00), respectively. At D3, the highest sensitivity/specificity were observed for LF3 (0.78)/EX1, EX2 and BW (1.00). EX1 showed higher accuracy values than LF1, and EX2 showed similar values to LF2. We concluded that the visual-tactile examination showed better results in detecting sound surfaces and approximal caries lesions without tooth separation. However, the effectiveness of approximal caries lesion detection of both methods was increased by the absence of contact points. Therefore, regardless of the method of detection, orthodontic separating elastics should be used as a complementary tool for the diagnosis of approximal noncavitated lesions in primary molars.

Integrin mediated growth and apoptosis in human gastric adenocarcinoma

Integrins are important as the primary cell adhesion molecule providing information about the extracellular microenvironment to the interior of the cell to influence cellular behavior such as differentiation, proliferation and apoptosis. Apoptotic death due to loss of adhesion is termed anoikis. In this study we have obtained a parental human gastric adenocarcinoma cell line that yielded two variant lines that had differing responses to lack of adhesion. The STAD.APO cell line undergoes apoptosis when denied adherence and the STAD.ARR cell line enters into cell cycle arrest under the identical suspended conditions. We have shown that cyclin A and cyclin D mRNA and protein are down regulated when cells are denied adherence for 24 hours in tissue culture wells previously coated with poly-HEMA. To test whether cyclin A was able to rescue cells from cell cycle arrest and/or anoikis by overriding the cell cycle machinery we transfected the full length cDNA in to each cell type. Surprisingly we found that anoikis and cell cycle arrest due to suspended conditions was not affected by overexpression of cyclin A protein, but that growth under adhered conditions was reduced compared to vector alone control transfectants. Further, we transfected other cell lines; ST7, gastric cancer, MDA-MB-4.35, breast cancer, and HPB T-cell leukemic and in no case were suspended culturing conditions overcome by cyclin A. This result indicates an additional level of regulation for the cell cycle machinery. Additionally, soluble collagen was shown to be able to save from anoikis and also from cell cycle arrest while the β1 specific mAb 33B6 was only able to save from anoikis. Immunofluorescent studies show that soluble collagen creates clusters of β1 with FAK and also β1 with actin in the STAD.ARR cells but does not in the STAD.APO cells. This result indicates that the phenotypes under suspended conditions between these cell lines may diverge at their requirements for integrin ligation. Additionally we characterized the nature of anoikis by showing cytochrome c release, caspase 3, p21 and p53 activation in STAD.APO cells. Thus, our results have implications in the understanding of integrin biology and neoplastic progression. ^

An interferon-inducible protein, p202, in cancer gene therapy

Interferons (IFNs) have been shown to exert antiviral, cell growth regulatory, and immunomodulatory effects on target cells. Both type I (α and β) and type II (γ) IFNs regulate cellular activities by specifically inducing the expression or activation of endogenous proteins that perform distinct biological functions. p202 is a 52 kDa nuclear phosphoprotein known to be induced by IFNs. p202 interacts with a variety of cellular transcription and growth regulatory factors and affects their functions. ^ In this report, we showed that the expression of p202 was associated with an anti-proliferative effect on human prostate cancer cells. Cells that expressed p202 showed reduced ability to grow in soft-agar, indicating a loss of transformation phenotype. More importantly, p202 expression reduced the tumorigenicity of human prostate cancer cells. p202-expressing cells exhibit an elevated level of hypophosphorylated form of pRb, and reduced level of cyclin B1 and p55CDC. ^ Our data suggest that p202 is a growth inhibitor gene in prostate cancer cells and its expression may also suppress transformation phenotype and tumorigenicity of prostate cancer cells. ^ In addition to inhibiting in vitro cell growth, suppressing the tumorigenicity of breast cancer cells in vivo, p202 expression could sensitize breast cancer cells to apoptosis induced by TNF-α treatment. One possible mechanism contributing to this sensitization is the inactivation of NF-κB by its interaction with p202. These results provide a scientific basis for a novel therapeutic strategy that combines p202 and TNF-α treatment against breast cancer. ^ It has been reported that NF-κB is constitutively active in human pancreatic cancer cells. Since p202 interacts with NF-κB and inhibits its activity, we examined a potential p202-mediated anti-tumor activity in pancreatic cancer. We used both ectopic and orthotopic xenograft models and demonstrated that p202 expression is associated with multiple anti-tumor activities that include inhibition of tumor growth, reduced tumorigenicity, prolonged survival, and remarkably, suppression of metastasis and angiogenesis. In vitro invasion assay also showed that p202-expressing pancreatic cancer cells are less invasive than those without p202 expression. That observation was supported by the findings that p202-expressing tumors showed reduced expression of angiogenic factors such as IL-8, and VEGF by inhibiting their transcription, and p202-expressing pancreatic cancer cells have reduced level of MAP-2 activity, a secreted protease activity important for metastasis. Together, our results strongly suggest that p202 expression mediates multiple anti-tumor activities against pancreatic cancer, and that may provide a scientific basis for developing a p202-based gene therapy in pancreatic cancer treatment. ^ Importantly, we demonstrated a treatment efficacy by using p202/SN2 liposome complex in a nude mice orthotopic breast cancer, and an ectopic pancreatic cancer xenograft model, through systemic and intra-tumor injection respectively. These results suggest a feasibility of using p202/SN2 liposome in future pre-clinical gene therapy experiments. ^

4HPR-X radiation mechanisms of interactions in non-small cell lung cancer (NSCLC) cells in vitro

Lung cancer is the leading cause of cancer death. However, poor survival using conventional therapies fuel the search for more rational interventions. The objective of this study was to design and implement a 4HPR-radiation interaction model in NSCLC, employing a traditional clinical modality (radiation), a relatively new, therapeutically unexplored agent (4HPR) and rationally combining them based on molecular mechanistic findings pertaining to their interactions. To test the hypothesis that 4HPR sensitizes cells to radiation-induced cell death via G2+M accumulation, we designed a working model consisting of H522 adenocarcinoma cells (p53, K-ras mutated) derived from an NSCLC patient; 4HPR at concentrations up to 10 μM; and X radiation up to 6 Gy generated by a patient-dedicated Phillips RT-250 X ray unit at 250 KV, 15 mA, 1.85 Gy/min. We found that 4HPR produced time- and dose-dependent morphological changes, growth inhibition, and DNA damage-inducing enhancement of reactive oxygen species. A transient G2+M accumulation of cells maximal at 24 h of continuous 4HPR exposure was used for irradiation time scheduling. Our data demonstrated enhanced cell death (both apoptotic and necrotic) in irradiated cells pre-treated with 4HPR versus those with either stressor alone. 4HPR's effect of increased NSCLC cells' radioresponse was confirmed by clonogenic assay. To explore these practical findings from a molecular mechanistic perspective, we further investigated and showed that levels of cyclin B1 and p34cdc2 kinase—both components of the mitosis promoting factor (MPF) regulating the G2/M transition—did not change following 4HPR treatment. Likewise, cdc25C phosphatase was not altered. However, enhanced p34cdc2 phosphorylation on its Thr14Tyr15 residues—indicative of its inactivation and increased expression of MPF negative regulators chk1 and wee1 kinases—were supportive of explaining 4HPR-treated cells' accumulation. Hence, p34cdc2 phosphorylation, chk1, and wee1 warrant further evaluation as potential molecular targets for 4HPR-X radiation combination. In summary, we (1) demonstrated that 4HPR not only induces cell death by itself, but also increases NSCLC cells' subsequent radioresponse, indicative of potential clinical applicability, and (2) for the first time, shed light on deciphering 4HPR-X radiation molecular mechanisms of interaction, including the finding of 4HPR's role as a p34cdc2 inactivator via Thr14Tyr15 phosphorylation. ^

Characterization of E2F1's oncogenic, apoptotic and tumor-suppressive activities utilizing the K5 mouse model

Though E2F1 is deregulated in most human cancers by mutations of the p16-cyclin D-Rb pathway, it also exhibits tumor suppressive activity. A transgenic mouse model overexpressing E2F1 under the control of the bovine keratin 5 (K5) promoter exhibits epidermal hyperplasia and spontaneously develops tumors in the skin and other epithelial tissues after one year of age. In a p53-deficient background, aberrant apoptosis in K5 E2F1 transgenic epidermis is reduced and tumorigenesis is accelerated. In sharp contrast, K5 E2F1 transgenic mice are resistant to papilloma formation in the DMBA/TPA two-stage carcinogenesis protocol. K5 E2F4 and K5 DP1 transgenic mice were also characterized and both display epidermal hyperplasia but do not develop spontaneous tumors even in cooperation with p53 deficiency. These transgenic mice do not have increased levels of apoptosis in their skin and are more susceptible to papilloma formation in the two-stage carcinogenesis model. These studies show that deregulated proliferation does not necessarily lead to tumor formation and that the ability to suppress skin carcinogenesis is unique to E2F1. E2F1 can also suppress skin carcinogenesis when okadaic acid is used as the tumor promoter and when a pre-initiated mouse model is used, demonstrating that E2F1's tumor suppressive activity is not specific for TPA and occurs at the promotion stage. E2F1 was thought to induce p53-dependent apoptosis through upregulation of p19ARF tumor suppressor, which inhibits mdm2-mediated p53 degradation. Consistent with in vitro studies, the overexpression of E2F1 in mouse skin results in the transcriptional activation of the p19ARF and the accumulation of p53. Inactivation of either p19ARF or p53 restores the sensitivity of K5 E2F1 transgenic mice to DMBA/TPA carcinogenesis, demonstrating that an intact p19ARF-p53 pathway is necessary for E2F1 to suppress carcinogenesis. Surprisingly, while p53 is required for E2F1 to induce apoptosis in mouse skin, p19ARF is not, and inactivation of p19ARF actually enhances E2F1-induced apoptosis and proliferation in transgenic epidermis. This indicates that ARF is important for E2F1-induced tumor suppression but not apoptosis. Senescence is another potential mechanism of tumor suppression that involves p53 and p19ARF. K5 E2F1 transgenic mice initiated with DMBA and treated with TPA show an increased number of senescence cells in their epidermis. These experiments demonstrate that E2F1's unique tumor suppressive activity in two-stage skin carcinogenesis can be genetically separated from E2F1-induced apoptosis and suggest that senescence utilizing the p19ARF-p53 pathway plays a role in tumor suppression by E2F1. ^

14-3-3zeta overexpression in multiple human cancers plays a critical role in cancer progression

14-3-3 is a family of highly conserved and ubiquitously expressed proteins in eukaryotic organisms. 14-3-3 isoforms bind in a phospho-serine/threonine-dependent manner to a host of proteins involved in essential cellular processes including cell cycle, signal transduction and apoptosis. We fortuitously discovered 14-3-3 zeta overexpression in many human primary cancers, such as breast, lung, and sarcoma, and in a majority of cancer cell lines. To determine 14-3-3 zeta involvement in breast cancer progression, we used immunohistochemical analysis to examine 14-3-3 zeta expression in human primary invasive breast carcinomas. High 14-3-3 zeta expression was significantly correlated with poor prognosis of breast cancer patients. Increased expression of 14-3-3 zeta was also significantly correlated with elevated PKB/Akt activation in patient samples. Thus, 14-3-3 zeta is a marker of poor prognosis in breast cancers. Furthermore, up-regulation of 14-3-3 zeta enhanced malignant transformation of cancer cells in vitro. ^ To determine the biological significance of 14-3-3 zeta in human cancers, small interfering RNAs (siRNA) were used to specifically block 14-3-3 zeta expression in cancer cells. 14-3-3 zeta siRNA inhibited cellular proliferation by inducing a G1 arrest associated with up-regulation of p27 KIP1 and p21CIP1 cyclin dependent kinase inhibitors. Reduced 14-3-3 zeta inhibited PKB/Akt activation while stimulating the p38 signaling pathway. Silencing 14-3-3 zeta expression also increased stress-induced apoptosis by caspase activation. Notably, 14-3-3 zeta siRNA inhibited transformation related properties of breast cancer cells in vitro and inhibited tumor progression of breast cancer cells in vivo. 14-3-3 zeta may be a key regulatory factor controlling multiple signaling pathways leading to tumor progression. ^ The data indicate 14-3-3 zeta is a major regulator of cell growth and apoptosis and may play a critical role in the development of multiple cancer types. Hence, blocking 14-3-3 zeta may be a promising therapeutic approach for numerous cancers. ^

Cellular mechanisms of operant and classical conditioning

The ability to associate a predictive stimulus with a subsequent salient event (i.e., classical conditioning) and the ability to associate an expressed behavior with the consequences (i.e., operant conditioning) allow for a predictive understanding of a changing environment. Although they are operationally distinct, there has been considerable debate whether at some fundamental level classical and operant conditioning are mechanistically distinct or similar. Feeding behavior of Aplysia (i.e., biting) was chosen as the model system and was successfully conditioned with appetitive forms of both operant and classical conditioning. The neuronal circuitry responsible for feeding is well understood and is suitable for cellular analyses, thus providing for a mechanistic comparison between these two forms of associative learning. ^ Neuron B51 is part of the feeding circuitry of Aplysia and is critical for the expression of ingestive behaviors. B51 also is a locus of plasticity following both operant and classical conditioning. Both in vivo and in vitro operant conditioning increased the input resistance and the excitability of B51. No pairing-specific changes in the input resistance were observed following both in vivo and in vitro classical conditioning. However, classical conditioning decreased the excitability of B51. Thus, both operant and classical conditioning modified the threshold level for activation of neuron B51, but in opposite directions, revealing key differences in the cellular mechanisms underlying these two forms of associative learning. ^ Next, the cellular mechanisms underlying operant conditioning were investigated in more detail using a single-cell analogue. The single-cell analogue successfully recapitulated the previous in vivo and in vitro operant conditioning results by increasing the input resistance and the excitability of B51. Both PKA and PKC were necessary for operant conditioning. Dopamine appears to be the transmitter mediating the reinforcement signal in this form of conditioning. A D1 dopamine receptor antibody revealed that the D1receptor localizes to the axon hillock, which is also the region that gives the strongest response when iontophoresing dopamine. ^ The studies presented herein, thus, provide for a greater understanding of the mechanisms underlying both of these forms of associative learning and demonstrate that they likely operate through distinct cellular mechanisms. ^

The regulation of JAB1 and its role in herceptin resistance

The Jun activation domain-binding protein (JAB1) is a c-Jun co-activator and a member of the COP9 signalosome. Additionally, it has recently been named a key negative regulator of the cyclin-dependent kinase inhibitor, p27. JAB1 overexpression has been observed in breast cancer and correlates with low p27 levels as well as poor prognosis, yet the mechanism of JAB1 deregulation is unknown. Data from our laboratory suggest that constitutive transcriptional activation of the jab1 gene is responsible for JAB1 protein overexpression. Therefore, we hypothesized that overexpression of JAB1 in breast cancer can be attributed to increased transcriptional activity. To identify potential positive regulators of JAB1, we characterized the promoter and found a 128 bp region that was critical for jab1 transcriptional activation. Our studies show that two oncogenic transcription factors, C/EBPβ and STAT3, play an important role in modulating jab1 transcription. Further, we have identified jab1 as a direct target gene of the SRC/STAT3 pathway. These studies provide insight to the mechanism of JAB1 overexpression in breast cancer and open up possibilities for therapies to inhibit its expression. ^ The development of the humanized monoclonal antibody, Herceptin (trastuzumab) targeting the HER2 (ErbB2) receptor has provided promising treatment to patients with aggressive HER2 positive breast cancer. However, many patients are resistant to Herceptin and additional therapies are needed to overcome resistance. Recent findings indicate that one mechanism of resistance involves AKT phosphorylation and subsequent mislocalization of the cyclin dependent kinase inhibitor, p27. We examined whether JAB1 facilitated degradation of p27 may be another mechanism of resistance to Herceptin. Our studies show that overexpression of JAB1 inhibited Herceptin induced G1-arrest and p27 accumulation. Interestingly, increased JAB1 levels were observed in two BT-474 Herceptin resistant clones. Targeted silencing of JAB1 increased p27 protein levels, reinstated a G1 checkpoint, and reduced cellular proliferation in the resistant clones. Our studies have demonstrated that inhibition of JAB1 sensitizes Herceptin resistant cells to treatment. Therefore, inhibition of JAB1 could provide a novel method of sensitizing resistant tumors to Herceptin-induced tumor growth arrest. ^

Identification of essential apoptotic genes in Drosophila

Apoptosis is a normal physiological cell suicide process which is essential for tissue homeostasis and normal development of metazoans. Misregulation of apoptosis is associated with many developmental defects and human diseases. The genes involved in the regulation and execution of apoptosis are highly conserved in humans and flies. Caspases are the executioners of cell suicide. Because of the unavailability of specific fly mutants, the developmental function of many caspase genes and genetic relationship between caspases and apoptotic components were undefined in Drosophila. We isolated several mutant alleles of the initiator caspase gene dronc, the effector casase drICE, and the Mediator component Cyclin C from the GMR-hid eyFLP/FRT screens which is designed to isolate mutants of recessive cell death genes in Drosophila melanogaster. Characterization of these mutants defined that they are essential for developmental cell death in Drosophila. dronc is required for most, but not all, cell death in Drosophila. drICE is required for apoptosis in many cells and it shares redundancy with another effector caspase gene, dcp-1, in a subset of cells in Drosophila. The genetic relationship between caspases and other apoptotic components was established through mutant analysis. We found that the pro-apoptotic protein Hid induces transcription of the initiator caspase gene dronc and the GMR-induced dronc transcripts are dependent on activated effector casapses, revealing a novel regulatory mechanism to promote caspase activity in Drosophila. Cyclin C and its kinase partner Cdk8 are required for prompt transcriptional induction of dronc in cell killing contexts. In short, we define the essential pro-apoptoic function of dronc, drICE, and Cyclin C in Drosophila and reveal a novel mechanism for regulation of dronc transcription. In the long run, these studies will help us decipher the complicated regulatory mechanism of cell death in humans. ^

Prediction of sepsis in the burn intensive care unit patient

Sepsis is a significant cause for multiple organ failure and death in the burn patient, yet identification in this population is confounded by chronic hypermetabolism and impaired immune function. The purpose of this study was twofold: 1) determine the ability of the systemic inflammatory response syndrome (SIRS) and American Burn Association (ABA) criteria to predict sepsis in the burn patient; and 2) develop a model representing the best combination of clinical predictors associated with sepsis in the same population. A retrospective, case-controlled, within-patient comparison of burn patients admitted to a single intensive care unit (ICU) was conducted for the period January 2005 to September 2010. Blood culture results were paired with clinical condition: "positive-sick"; "negative-sick", and "screening-not sick". Data were collected for the 72 hours prior to each blood culture. The most significant predictors were evaluated using logistic regression, Generalized Estimating Equations (GEE) and ROC area under the curve (AUC) analyses to assess model predictive ability. Bootstrapping methods were employed to evaluate potential model over-fitting. Fifty-nine subjects were included, representing 177 culture periods. SIRS criteria were not found to be associated with culture type, with an average of 98% of subjects meeting criteria in the 3 days prior. ABA sepsis criteria were significantly different among culture type only on the day prior (p = 0.004). The variables identified for the model included: heart rate>130 beats/min, mean blood pressure<60 mmHg, base deficit<-6 mEq/L, temperature>36°C, use of vasoactive medications, and glucose>150 mg/d1. The model was significant in predicting "positive culture-sick" and sepsis state, with AUC of 0.775 (p < 0.001) and 0.714 (p < .001), respectively; comparatively, the ABA criteria AUC was 0.619 (p = 0.028) and 0.597 (p = .035), respectively. SIRS criteria are not appropriate for identifying sepsis in the burn population. The ABA criteria perform better, but only for the day prior to positive blood culture results. The time period useful to diagnose sepsis using clinical criteria may be limited to 24 hours. A combination of predictors is superior to individual variable trends, yet algorithms or computer support will be necessary for the clinician to find such models useful. ^

Cellular Uptake of Neutrohpil Elastase Links Inflammation to Adaptive Immunity

Many tumors arise from sites of inflammation providing evidence that innate immunity is a critical component in the development and progression of cancer. Neutrophils are primary mediators of the innate immune response. Upon activation, an important function of neutrophils is release of an assortment of proteins from their granules including the serine protease neutrophil elastase (NE). The effect of NE on cancer has been attributed primarily to its ability to degrade the extracellular matrix thereby promoting invasion and metastasis. Recently, it was shown that NE could be taken up by lung cancer cells leading to degradation of insulin receptor substrate-1 thereby promoting hyperactivity of the phosphatidylinositol-3 kinase (PI3K) pathway and tumor cell proliferation. To our knowledge, nobody has investigated uptake of NE by other tumor types. In addition, NE has broad substrate specificity suggesting that uptake of NE by tumor cells could impact processes regulating tumorigenensis other than activation of the PI3K pathway. Neutrophil elastase has been identified in breast cancer specimens where high levels of NE have prognostic significance. These studies have assessed NE levels in whole tumor lysates. Because the major source of NE is from activated neutrophils, we hypothesized that breast cancer cells do not have endogenous NE but may take up NE released by tumor associated neutrophils in the tumor microenvironment and that this could provide a link between the innate immune response to tumors and specific adaptive immune responses. In this thesis, we show that breast cancer cells lack endogenous NE expression and that they are able to take up NE resulting in increased generation of low molecular weight cyclin E (CCNE) and enhanced susceptibility to lysis by CCNE-specific cytotoxic T lymphocytes. We also show that after taking up NE and proteinase 3 (PR3), a second primary granule protease with significant homology to NE, breast cancer cells cross-present the NE- and PR3-derived peptide PR1 rendering them susceptible to PR1-targeted therapies. Taken together, our data support a role for NE uptake in modulating adaptive immune responses against breast cancer.

Functional Analysis of Drosophila Integrator Complex in snRNA 3' End Processing

Uridine-rich small nuclear RNAs (U snRNAs) play essential roles in eukaryotic gene expression by facilitating the removal of introns from mRNA precursors and the processing of the replication-dependent histone pre-mRNAs. Formation of the 3’ end of these snRNAs is carried out by a poorly characterized, twelve-membered protein complex named Integrator Complex. In the effort to understand Integrator Complex function in the formation of the snRNA 3’ end, we performed a functional RNAi screen in Drosophila S2 cells to identify protein factors required for snRNA 3’ end formation. This screen was conducted by using a fluorescence-based reporter that elicits GFP expression in response to a deficiency in snRNA processing. Besides scoring the known Integrator subunits, we identified Asunder and CG4785 as additional core members of the Integrator Complex. Additionally, we also found a conserved requirement for Cyclin C and Cdk8 in both fly and human snRNA 3’ end processing. We have further demonstrated that the kinase activity of Cdk8 is critical for snRNA 3’ end processing and is likely to function independent of its well-documented function within the Mediator Cdk8 module. Taken together, this work functionally defines the Drosophila Integrator Complex and demonstrates a novel function for Cyclin C/Cdk8 in snRNA 3’ end formation. This thesis work has also characterized an important functional interaction mediated by a microdomain within Integrator subunit 12 (IntS12) and IntS1 that is required for the activity of the Integrator Complex in processing the snRNA 3’ end. Through the development of a reporter-based functional RNAi-rescue assay in Drosophila S2 cells, we analyzed domains within IntS12 required for snRNA 3’ end formation. This analysis unexpectedly revealed that an N-terminal 30 amino acid region and not the highly conserved central PHD finger domain, is required for snRNA 3’ end cleavage. The IntS12 microdomain (1-45) functions autonomously, and is sufficient to interact and stabilize the putative scaffold protein IntS1. Our findings provide more details of the Integrator Complex for understanding the molecular mechanism of snRNA 3’ end processing. Moreover, these results lay the foundation for future studies of the complex through the identification of a novel functional domain within one subunit and the identification of additional subunits.