Identification and Interpretation of Longitudinal Gene Expression Changes in Trauma

The relationship between leukocyte gene expression and recovery of respiratory function after injury may provide information on the etiology of multiple organ dysfunction.To find a list of genes for which expression after injury predicts respiratory recovery, and to identify which networks and pathways characterize these genes.Blood was sampled at 12 hours and at 1, 4, 7, 21 and 28 days from 147 patients who had been admitted to the hospital after blunt trauma. Leukocyte gene expression was measured using Affymetrix oligonucleotide arrays. A linear model, fit to each probe-set expression value, was used to impute the gene expression trajectory over the entire follow-up period. The proportional hazards model score test was used to calculate the statistical significance of each probe-set trajectory in predicting respiratory recovery. A list of genes was determined such that the expected proportion of false positive results was less than 10%. These genes were compared to the Gene Ontology for 'response to stimulus' and, using Ingenuity software, were mapped into networks and pathways.The median time to respiratory recovery was 6 days. There were 170 probe-sets representing 135 genes that were found to be related to respiratory recovery. These genes could be mapped to nine networks. Two known pathways that were activated were antigen processing and presentation and JAK-signaling.The examination of the relationship of gene expression over time with a patient's clinical course can provide information which may be useful in determining the mechanism of recovery or lack of recovery after severe injury

Early Elevation of Matrix Metalloproteinase-8 and -9 in Pediatric ARDS Is Associated with an Increased Risk of Prolonged Mechanical Ventilation

BACKGROUND: Matrix metalloproteinases (MMP) -8 and -9 may play key roles in the modulation of neutrophilic lung inflammation seen in pediatric Acute Respiratory Distress Syndrome (ARDS). We aimed to perform a comprehensive analysis of MMP-8 and MMP-9 activity in tracheal aspirates of pediatric ARDS patients compared with non-ARDS controls, testing whether increased MMP-8 and -9 activities were associated with clinical outcomes. METHODS: Tracheal aspirates were collected from 33 pediatric ARDS patients and 21 non-ARDS controls at 48 hours of intubation, and serially for those who remained intubated greater than five days. MMPs, tissue inhibitor of metalloproteinases (TIMPs), human neutrophil elastase (HNE) and myeloperoxidase (MPO) activity were measured by ELISA, and correlated with clinical indicators of disease severity such as PRISM (Pediatric Risk of Mortality) scores, oxygen index (OI), multi-organ system failure (MOSF) and clinical outcome measures including length of intubation, ventilator-free days (VFDs) and mortality in the Pediatric Intensive Care Unit (PICU). RESULTS: Active MMP-9 was elevated early in pediatric ARDS subjects compared to non-ARDS controls. Higher MMP-8 and active MMP-9 levels at 48 hours correlated with a longer course of mechanical ventilation (r = 0.41, p = 0.018 and r = 0.75, p<0.001; respectively) and fewer number of VFDs (r = -0.43, p = 0.013 and r = -0.76, p<0.001; respectively), independent of age, gender and severity of illness. Patients with the highest number of ventilator days had the highest levels of active MMP-9. MMP-9 and to a lesser extent MMP-8 activities in tracheal aspirates from ARDS subjects were sensitive to blockade by small molecule inhibitors. CONCLUSIONS: Higher MMP-8 and active MMP-9 levels at 48 hours of disease onset are associated with a longer duration of mechanical ventilation and fewer ventilator-free days among pediatric patients with ARDS. Together, these results identify early biomarkers predictive of disease course and potential therapeutic targets for this life threatening disease

Multimarker RT–PCR assay for the detection of minimal residual disease in sentinel lymph nodes of breast cancer patients

The presence of metastases in lymph nodes is the most powerful prognostic factor in breast cancer patients. Routine histological examination of lymph nodes has limited sensitivity for the detection of breast cancer metastases. The aim of the present study was to develop a multimarker reverse transcriptase–polymerase chain reaction (RT—PCR) assay for the detection of minimal residual disease in sentinel nodes of breast cancer patients. RNA was extracted from 30 sentinel lymph nodes (SLN) obtained from 28 patients, three primary breast cancers (positive controls), three lymph nodes from patients with benign diseases, and peripheral blood lymphocytes of 10 healthy volunteers (negative controls). RT–PCR was performed using the following markers; cytokeratin (CK)-19, NY-BR-1 and mammaglobin B. RT–PCR results were compared to enhanced histopathologic examination and immunohistochemistry (IHC). All three positive controls showed strong PCR amplification for all three markers. None of the 13 negative controls was amplified by any of the three markers. Among the 30 SLN analysed, breast cancer metastases were detected in six SLNs by routine histology, in eight by IHC and in 15 by RT–PCR. We conclude that a multimarker RT–PCR assay probing for NY-BR-1, mammaglobin-B, and CK-19 is more sensitive compared to enhanced pathologic examination. This method may prove to be of value in breast cancer staging and prognosis evaluation

Quantification of Age-Dependent Somatic CAG Repeat Instability in Hdh CAG Knock-In Mice Reveals Different Expansion Dynamics in Striatum and Liver

Age at onset of Huntington's disease (HD) is largely determined by the CAG trinucleotide repeat length in the HTT gene. Importantly, the CAG repeat undergoes tissue-specific somatic instability, prevalent in brain regions that are disease targets, suggesting a potential role for somatic CAG repeat instability in modifying HD pathogenesis. Thus, understanding underlying mechanisms of somatic CAG repeat instability may lead to discoveries of novel therapeutics for HD. Investigation of the dynamics of the CAG repeat size changes over time may provide insights into the mechanisms underlying CAG repeat instability.To understand how the HTT CAG repeat length changes over time, we quantified somatic instability of the CAG repeat in Huntington's disease CAG knock-in mice from 2-16 months of age in liver, striatum, spleen and tail. The HTT CAG repeat in spleen and tail was very stable, but that in liver and striatum expanded over time at an average rate of one CAG per month. Interestingly, the patterns of repeat instability were different between liver and striatum. Unstable CAG repeats in liver repeatedly gained similar sizes of additional CAG repeats (approximately two CAGs per month), maintaining a distinct population of unstable repeats. In contrast, unstable CAG repeats in striatum gained additional repeats with different sizes resulting in broadly distributed unstable CAG repeats. Expanded CAG repeats in the liver were highly enriched in polyploid hepatocytes, suggesting that the pattern of liver instability may reflect the restriction of the unstable repeats to a unique cell type.Our results are consistent with repeat expansion occurring as a consequence of recurrent small repeat insertions that differ in different tissues. Investigation of the specific mechanisms that underlie liver and striatal instability will contribute to our understanding of the relationship between instability and disease and the means to intervene in this process

Control of hyperglycaemia in paediatric intensive care (CHiP): study protocol.

BACKGROUND: There is increasing evidence that tight blood glucose (BG) control improves outcomes in critically ill adults. Children show similar hyperglycaemic responses to surgery or critical illness. However it is not known whether tight control will benefit children given maturational differences and different disease spectrum. METHODS/DESIGN: The study is an randomised open trial with two parallel groups to assess whether, for children undergoing intensive care in the UK aged <or= 16 years who are ventilated, have an arterial line in-situ and are receiving vasoactive support following injury, major surgery or in association with critical illness in whom it is anticipated such treatment will be required to continue for at least 12 hours, tight control will increase the numbers of days alive and free of mechanical ventilation at 30 days, and lead to improvement in a range of complications associated with intensive care treatment and be cost effective. Children in the tight control group will receive insulin by intravenous infusion titrated to maintain BG between 4 and 7.0 mmol/l. Children in the control group will be treated according to a standard current approach to BG management. Children will be followed up to determine vital status and healthcare resources usage between discharge and 12 months post-randomisation. Information regarding overall health status, global neurological outcome, attention and behavioural status will be sought from a subgroup with traumatic brain injury (TBI). A difference of 2 days in the number of ventilator-free days within the first 30 days post-randomisation is considered clinically important. Conservatively assuming a standard deviation of a week across both trial arms, a type I error of 1% (2-sided test), and allowing for non-compliance, a total sample size of 1000 patients would have 90% power to detect this difference. To detect effect differences between cardiac and non-cardiac patients, a target sample size of 1500 is required. An economic evaluation will assess whether the costs of achieving tight BG control are justified by subsequent reductions in hospitalisation costs. DISCUSSION: The relevance of tight glycaemic control in this population needs to be assessed formally before being accepted into standard practice

Real-time RT–PCR detection of disseminated tumour cells in bone marrow has superior prognostic significance in comparison with circulating tumour cells in patients with breast cancer

This study assessed the ability of real-time reverse transcription–polymerase chain reaction (RT–PCR) analysis to detect disseminated epithelial cells (DEC) in peripheral blood (PB) and bone marrow (BM) of patients with breast cancer (BC). Detection of DEC in BM is an obvious choice in BC, but blood sampling is more convenient. The aim of this study was to evaluate whether the detection of DEC in either PB or BM predicts overall survival (OS). Peripheral blood and BM samples were collected from 148 patients with primary (stage M0, n=116/78%) and metastatic (stage M+, n=32/21%) BC before the initiation of any local or systemic treatment. Peripheral blood of healthy volunteers and BM of patients with a nonmalignant breast lesion or a haematological malignancy served as the control group. Disseminated epithelial cells was detected by measuring relative gene expression (RGE) for cytokeratin-19 (CK-19) and mammaglobin (MAM), using a quantitative RT–PCR detection method. The mean follow-up time was 786 days (+/− 487). Kaplan–Meier analysis was used for predicting OS. By taking the 95 percentile of the RGE of CK-19 (BM: 26.3 and PB: 58.7) of the control group as cutoff, elevated CK-19 expression was detected in 42 (28%) BM samples and in 22 (15%) PB samples. Mammaglobin expression was elevated in 20% (both PB and BM) of the patients with BC. There was a 68% (CK-19) and 75% (MAM) concordance between PB and BM samples when classifying the results as either positive or negative. Patients with an elevated CK-19 or MAM expression in the BM had a worse prognosis than patients without elevated expression levels (OS: log-rank test, P=0.0045 (CK-19) and P=0.025 (MAM)). For PB survival analysis, no statistical significant difference was observed between patients with or without elevated CK-19 or MAM expression (OS: log-rank test, P=0.551 (CK-19) and P=0.329 (MAM)). Separate analyses of the M0 and M+ patients revealed a marked difference in OS according to the BM CK-19 or MAM status in the M+ patient group, but in the M0 group, only MAM expression was a prognostic marker for OS. Disseminated epithelial cells, measured as elevated CK-19 or MAM mRNA expression, could be detected in both PB and BM of patients with BC. Only the presence of DEC in BM was highly predictive for OS. The occurrence of DEC in the BM is probably less time-dependent and may act as a filter for circulating BC cells. The use of either larger volumes of PB or performing an enrichment step for circulating tumour in blood cells might improve these results

Energy Restriction during Childhood and Early Adulthood and Ovarian Cancer Risk

Dietary energy restriction may protect against cancer. In parts of the Netherlands, mostly in larger cities, periods of chronically impaired nutrition and even severe famine (Hunger Winter 1944–1945) existed during the 1930s and World War II (1940–1945). We studied the association between energy restriction during childhood and early adulthood on the risk of ovarian cancer later in life. In 1986, the Netherlands Cohort Study was initiated. A self-administered questionnaire on dietary habits and other cancer risk factors was completed by 62,573 women aged 55–69 years at baseline. Follow-up for cancer was established by record linkage to the Netherlands Cancer Registry. After 16.3 years of follow-up, 364 invasive epithelial ovarian cancer cases and 2220 subcohort members (sampled from the total cohort directly after baseline) with complete information confounders were available for case-cohort analyses. In multivariable analysis, ovarian cancer risk was lower for participants with an unemployed father during the 1930s (Hazard Ratio (HR), 0.70; 95% Confidence Interval (CI), 0.47–1.06) compared to participants with an employed father as well as for participants living in a city during World War II (HR, 0.69; 95% CI, 0.54–0.90) compared to participants living in the country-side. Residence in a Western City during the famine (Hunger Winter) was not associated with a decreased risk. Our results show a relation between proxy variables for modest energy restriction over a longer period of time during childhood or early adulthood and a reduced ovarian cancer risk

KAI1 suppresses HIF-1α and VEGF expression by blocking CDCP1-enhanced Src activation in prostate cancer

<p>Abstract</p> <p>Background</p> <p>KAI1 was initially identified as a metastasis-suppressor gene in prostate cancer. It is a member of the tetraspan transmembrane superfamily (TM4SF) of membrane glycoproteins. As part of a tetraspanin-enriched microdomain (TEM), KAI1 inhibits tumor metastasis by negative regulation of Src. However, the underlying regulatory mechanism has not yet been fully elucidated. CUB-domain-containing protein 1 (CDCP1), which was previously known as tetraspanin-interacting protein in TEM, promoted metastasis via enhancement of Src activity. To better understand how KAI1 is involved in the negative regulation of Src, we here examined the function of KAI1 in CDCP1-mediated Src kinase activation and the consequences of this process, focusing on HIF-1 α and VEGF expression.</p> <p>Methods</p> <p>We used the human prostate cancer cell line PC3 which was devoid of KAI1 expression. Vector-transfected cells (PC3-GFP clone #8) and KAI1-expressing PC3 clones (PC3-KAI1 clone #5 and #6) were picked after stable transfection with KAI1 cDNA and selection in 800 <it>μ</it>g/ml G418. Protein levels were assessed by immunoblotting and VEGF reporter gene activity was measured by assaying luciferase activitiy. We followed tumor growth <it>in vivo </it>and immunohistochemistry was performed for detection of HIF-1, CDCP1, and VHL protein level.</p> <p>Results</p> <p>We demonstrated that Hypoxia-inducible factor 1α (HIF-1α) and VEGF expression were significantly inhibited by restoration of KAI1 in PC3 cells. In response to KAI1 expression, CDCP1-enhanced Src activation was down-regulated and the level of von Hippel-Lindau (VHL) protein was significantly increased. In an <it>in vivo </it>xenograft model, KAI1 inhibited the expression of CDCP1 and HIF-1α.</p> <p>Conclusions</p> <p>These novel observations may indicate that KAI1 exerts profound metastasis-suppressor activity in the tumor malignancy process via inhibition of CDCP1-mediated Src activation, followed by VHL-induced HIF-1α degradation and, ultimately, decreased VEGF expression.</p