Diabetes is associated with increased risk of heart failure and development of a cardiomyopathy whose etiology is only partially understood. Ca2+ entry through the voltage-dependent L-type Ca2+ channel CaV1.2 initiates the contractile cycle in cardiac myocytes. Decreased cardiac contractility and depressed CaV1.2 function have been reported in obese type 2 diabetic db/db mice. Here we demonstrate that a reduction in phosphoinositide 3-kinase (PI3K) signaling is a major contributor to the altered function of CaV1.2 in db/db cardiac myocytes. Using the whole-cell patch clamp technique, we determined that intracellular infusion of cardiac myocytes from db/db mice with phosphatidylinositol 3,4,5-trisphosphate (PIP3), the second messenger produced by PI3K, increased the L-type Ca2+ current (ICa,L) density nearly to the level seen in wildtype cells. PIP3 also reversed the positive shift in the voltage dependence of steady-state current activation observed in db/db myocytes. Infusion of protein kinases that act downstream of PI3K also affected ICa,L. Akt1 and Akt2 were as effective as PIP3 in restoring ICa,L density in db/db myocytes, but did not affect the voltage dependence of current activation. Infusion of atypical PKC-Ι (the human homolog of mouse PKC-λ) caused a small but significant increase in ICa,L density and completely reversed the shift in voltage dependence of steady-state current activation. These results indicate that a defect in PI3K/PIP3/Akt/PKC-λ signaling is mainly responsible for the depressed CaV1.2 function in the heart of type 2 diabetic db/db mice.
Diabetes; L-type Ca2+ current; cardiac myocytes; phosphoinositide 3-kinase (PI3K); Akt; PKC
PIK3C3/Vps34 is the class III PtdIns3K that is evolutionarily conserved from yeast to mammals. Its central role in mammalian autophagy has been suggested through the use of pharmacological inhibitors and the study of its binding partners. However, the precise role of PIK3C3 in mammals is not clear. Using mouse strains that allow tissue-specific deletion of PIK3C3, we have described an essential role of PIK3C3 in regulating autophagy, and liver and heart function.
autophagy; cancer; endocytosis; heart; liver; PIK3C3; PtdIns3K; PtdIns3P; Vps34
Therapy-related myeloid neoplasm (t-MN) is a subtype of acute myeloid leukemia with adverse cytogenetics and poor overall prognosis despite intensive induction chemotherapy and allogeneic hematopoietic cell transplantation (allo-HCT). It is increasingly recognized as a late complication of chronic immunosuppression in patients who have received solid organ transplantation. In this paper, we describe a case of t-MN following orthotopic cardiac transplantation and its treatment with allo-HCT. We discuss molecular and biological challenges and considerations in double solid organ and bone marrow transplantation and review similar cases at our institution. Our experience suggests general feasibility and safety of allo-HCT in patients who have received solid organ transplantation.
Cardiac excitation-contraction (E-C) coupling links action potentials to muscle contraction and is in essence a process of calcium ion mobilization.1 The central mechanism governing this process in ventricular myocytes is Ca2+- induced Ca2+ release, or CICR. It has been established for more than 20 years that CICR operates in a local control mode, taking place in a restricted junctional space of ≈ 12 to 15 nm between the transverse (T)-tubule and sarcoplasmic reticulum (SR) membranes, namely, the junctional membrane complexes or cardiac dyads.2,3 Within this dyadic “fuzzy space,”4 clusters of ryanodine receptor (RyR) Ca2+ release channels on the SR constitute the calcium release apparatus together with the directly apposed voltage-gated L-type Ca2+ channels (LTCCs) located primarily on the T-tubule membrane. 5 On membrane depolarization, a small amount of Ca2+ influx through the opening of LTCCs locally activates adjacent RyRs to release a much larger (≈10 times) amount of Ca2+ from the SR.6,7 The normal, functional cross-talk between LTCCs and RyRs depends on a stable local ultrastructure— the cardiac dyad.
calcium signaling; excitation-contraction coupling; gene transcription; T-tubules
Many drugs, including some commonly used medications, can cause abnormal heart rhythms and sudden death, as manifest by a prolonged QT interval in the electrocardiogram. Cardiac arrhythmias caused by drug-induced long QT syndrome are thought to result mainly from reductions in the delayed rectifier potassium ion (K+) current IKr. Here, we report a mechanism for drug-induced QT prolongation that involves changes in multiple ion currents caused by a decrease in phosphoinositide 3-kinase (PI3K) signaling. Treatment of canine cardiac myocytes with inhibitors of tyrosine kinases or PI3Ks caused an increase in action potential duration that was reversed by intracellular infusion of phosphatidylinositol 3,4,5-trisphosphate. The inhibitors decreased the delayed rectifier K+ currents IKr and IKs, the L-type calcium ion (Ca2+) current ICa,L, and the peak sodium ion (Na+) current INa and increased the persistent Na+ current INaP. Computer modeling of the canine ventricular action potential showed that the drug-induced change in any one current accounted for less than 50% of the increase in action potential duration. Mouse hearts lacking the PI3K p110α catalytic subunit exhibited a prolonged action potential and QT interval that were at least partly a result of an increase in INaP. These results indicate that down-regulation of PI3K signaling directly or indirectly via tyrosine kinase inhibition prolongs the QT interval by affecting multiple ion channels. This mechanism may explain why some tyrosine kinase inhibitors in clinical use are associated with increased risk of life-threatening arrhythmias.
Septic shock heterogeneity has important implications for clinical trial implementation and patient management. We previously addressed this heterogeneity by identifying 3 putative subclasses of children with septic shock based exclusively on a 100-gene expression signature. Here we attempted to prospectively validate the existence of these gene expression-based subclasses in a validation cohort.
Prospective observational study involving microarray-based bioinformatics.
Multiple pediatric intensive care units in the United States.
Separate derivation (n=98) and validation (n=82) cohorts of children with septic shock.
None other than standard care.
Measurements and Main Results
Gene expression mosaics of the 100 class-defining genes were generated for 82 individual patients in the validation cohort. Using computer-based image analysis, patients were classified into 1 of 3 subclasses (“A”, “B”, or “C”) based on color and pattern similarity relative to reference mosaics generated from the original derivation cohort. After subclassification, the clinical database was mined for phenotyping. Subclass A patients had higher illness severity relative to subclasses B and C, as measured by maximal organ failure, fewer ICU-free days, and a higher PRISM score. Patients in subclass A were characterized by repression of genes corresponding to adaptive immunity and glucocorticoid receptor signaling. Separate subclass assignments were conducted by 21 individual clinicians, using visual inspection. The consensus classification of the clinicians had modest agreement with the computer algorithm.
We have validated the existence of subclasses of children with septic shock based on a biologically relevant, 100-gene expression signature. The subclasses have relevant clinical differences.
microarray; gene expression; stratification; staging; septic shock; pediatrics
Differentiating between sterile inflammation and bacterial infection in critically ill patients with fever and other signs of the systemic inflammatory response syndrome (SIRS) remains a clinical challenge. The objective of our study was to mine an existing genome-wide expression database for the discovery of candidate diagnostic biomarkers to predict the presence of bacterial infection in critically ill children.
Genome-wide expression data were compared between patients with SIRS having negative bacterial cultures (n = 21) and patients with sepsis having positive bacterial cultures (n = 60). Differentially expressed genes were subjected to a leave-one-out cross-validation (LOOCV) procedure to predict SIRS or sepsis classes. Serum concentrations of interleukin-27 (IL-27) and procalcitonin (PCT) were compared between 101 patients with SIRS and 130 patients with sepsis. All data represent the first 24 hours of meeting criteria for either SIRS or sepsis.
Two hundred twenty one gene probes were differentially regulated between patients with SIRS and patients with sepsis. The LOOCV procedure correctly predicted 86% of the SIRS and sepsis classes, and Epstein-Barr virus-induced gene 3 (EBI3) had the highest predictive strength. Computer-assisted image analyses of gene-expression mosaics were able to predict infection with a specificity of 90% and a positive predictive value of 94%. Because EBI3 is a subunit of the heterodimeric cytokine, IL-27, we tested the ability of serum IL-27 protein concentrations to predict infection. At a cut-point value of ≥5 ng/ml, serum IL-27 protein concentrations predicted infection with a specificity and a positive predictive value of >90%, and the overall performance of IL-27 was generally better than that of PCT. A decision tree combining IL-27 and PCT improved overall predictive capacity compared with that of either biomarker alone.
Genome-wide expression analysis has provided the foundation for the identification of IL-27 as a novel candidate diagnostic biomarker for predicting bacterial infection in critically ill children. Additional studies will be required to test further the diagnostic performance of IL-27.
The microarray data reported in this article have been deposited in the Gene Expression Omnibus under accession number GSE4607.
Clinicians have long appreciated the distinct phenotype of systemic juvenile idiopathic arthritis (SJIA) compared to polyarticular juvenile idiopathic arthritis (POLY). We hypothesized that gene expression profiles of peripheral blood mononuclear cells (PBMC) from children with each disease would reveal distinct biological pathways when analyzed for significant associations with elevations in two markers of JIA activity, erythrocyte sedimentation rate (ESR) and number of affected joints (joint count, JC).
PBMC RNA from SJIA and POLY patients was profiled by kinetic PCR to analyze expression of 181 genes, selected for relevance to immune response pathways. Pearson correlation and Student's t-test analyses were performed to identify transcripts significantly associated with clinical parameters (ESR and JC) in SJIA or POLY samples. These transcripts were used to find related biological pathways.
Combining Pearson and t-test analyses, we found 91 ESR-related and 92 JC-related genes in SJIA. For POLY, 20 ESR-related and 0 JC-related genes were found. Using Ingenuity Systems Pathways Analysis, we identified SJIA ESR-related and JC-related pathways. The two sets of pathways are strongly correlated. In contrast, there is a weaker correlation between SJIA and POLY ESR-related pathways. Notably, distinct biological processes were found to correlate with JC in samples from the earlier systemic plus arthritic phase (SAF) of SJIA compared to samples from the later arthritis-predominant phase (AF). Within the SJIA SAF group, IL-10 expression was related to JC, whereas lack of IL-4 appeared to characterize the chronic arthritis (AF) subgroup.
The strong correlation between pathways implicated in elevations of both ESR and JC in SJIA argues that the systemic and arthritic components of the disease are related mechanistically. Inflammatory pathways in SJIA are distinct from those in POLY course JIA, consistent with differences in clinically appreciated target organs. The limited number of ESR-related SJIA genes that also are associated with elevations of ESR in POLY implies that the SJIA associations are specific for SJIA, at least to some degree. The distinct pathways associated with arthritis in early and late SJIA raise the possibility that different immunobiology underlies arthritis over the course of SJIA.
Arthritis; Inflammation; Juvenile idiopathic arthritis (JIA); Systemic JIA; Polyarticular JIA; Transcriptional analysis
The intrinsic heterogeneity of clinical septic shock is a major challenge. For clinical trials, individual patient management, and quality improvement efforts, it is unclear which patients are least likely to survive and thus benefit from alternative treatment approaches. A robust risk stratification tool would greatly aid decision-making. The objective of our study was to derive and test a multi-biomarker-based risk model to predict outcome in pediatric septic shock.
Twelve candidate serum protein stratification biomarkers were identified from previous genome-wide expression profiling. To derive the risk stratification tool, biomarkers were measured in serum samples from 220 unselected children with septic shock, obtained during the first 24 hours of admission to the intensive care unit. Classification and Regression Tree (CART) analysis was used to generate a decision tree to predict 28-day all-cause mortality based on both biomarkers and clinical variables. The derived tree was subsequently tested in an independent cohort of 135 children with septic shock.
The derived decision tree included five biomarkers. In the derivation cohort, sensitivity for mortality was 91% (95% CI 70 - 98), specificity was 86% (80 - 90), positive predictive value was 43% (29 - 58), and negative predictive value was 99% (95 - 100). When applied to the test cohort, sensitivity was 89% (64 - 98) and specificity was 64% (55 - 73). In an updated model including all 355 subjects in the combined derivation and test cohorts, sensitivity for mortality was 93% (79 - 98), specificity was 74% (69 - 79), positive predictive value was 32% (24 - 41), and negative predictive value was 99% (96 - 100). False positive subjects in the updated model had greater illness severity compared to the true negative subjects, as measured by persistence of organ failure, length of stay, and intensive care unit free days.
The pediatric sepsis biomarker risk model (PERSEVERE; PEdiatRic SEpsis biomarkEr Risk modEl) reliably identifies children at risk of death and greater illness severity from pediatric septic shock. PERSEVERE has the potential to substantially enhance clinical decision making, to adjust for risk in clinical trials, and to serve as a septic shock-specific quality metric.
To determine whether the phosphoinositide 3-kinase (PI3K) catalytic subunits p110-α and p110-β play a role in liver steatosis induced by a high-fat diet (HFD).
RESEARCH DESIGN AND METHODS
Liver-specific p110-α and p110-β knockout mice and control animals for each group were fed an HFD or normal chow for 8 weeks. Biochemical assays and quantitative real-time PCR were used to measure triglyceride, expression of lipogenic and gluconeogenic genes, and activity of protein kinases downstream of PI3K in liver lysates. Fatty acid uptake and incorporation into triglycerides were assessed in isolated hepatocytes.
Hepatic triglyceride levels in HFD-fed p110-α−/− mice were 84 ± 3% lower than in p110-α+/+ mice, whereas the loss of p110-β did not significantly alter liver lipid accumulation. p110-α−/− livers also showed a reduction in atypical protein kinase C activity and decreased mRNA and protein expression of several lipogenic genes. Hepatocytes isolated from p110-α−/− mice exhibited decreased palmitate uptake and reduced fatty acid incorporation into triglycerides as compared with p110-α+/+ cells, and hepatic expression of liver fatty acid binding protein was lower in p110-α−/− mice fed the HFD as compared with controls. Ablation of neither p110-α nor p110-β ameliorated glucose intolerance induced by the HFD, and genes involved in gluconeogenesis were upregulated in the liver of both knockout animals.
PI3K p110-α, and not p110-β, promotes liver steatosis in mice fed an HFD. p110-α might exert this effect in part through activation of atypical protein kinase C, upregulation of lipogenesis, and increased uptake of fatty acids.
Autophagy is critically controlled by phosphatidylinositol 3-kinases (PtdIns3Ks). The common understanding for mammalian autophagy is that class I PtdIns3Ks inhibit autophagy by activating the Akt-TOR kinase cascade, whereas the class III PtdIns3K (Vps34) promotes autophagy by generating the phospholipid PtdIns(3)P. However, direct genetic evidence for a role of class I PtdIns3Ks in autophagy has been lacking. Using mice with a conditional deletion of the class I PtdIns3K catalytic subunit isoform p110α or p110β, we revealed an unexpected function of p110β as a positive regulator of autophagy.
autophagy; PtdIns3K; p110α; p110β; Vps34; PtdIns(3)P
Septic-shock-associated acute kidney injury (SSAKI) carries high morbidity in the pediatric population. Effective treatment strategies are lacking, in part due to poor detection and prediction. There is a need to identify novel candidate biomarkers of SSAKI. The objective of our study was to determine whether microarray data from children with septic shock could be used to derive a panel of candidate biomarkers for predicting SSAKI.
A retrospective cohort study compared microarray data representing the first 24 hours of admission for 179 children with septic shock with those of 53 age-matched normal controls. SSAKI was defined as a >200% increase of baseline serum creatinine, persistent to 7 days after admission.
Patients with SSAKI (n = 31) and patients without SSAKI (n = 148) were clinically similar, but SSAKI carried a higher mortality (45% vs. 10%). Twenty-one unique gene probes were upregulated in SSAKI patients versus patients without SSAKI. Using leave-one-out cross-validation and class prediction modeling, these probes predicted SSAKI with a sensitivity of 98% (95% confidence interval (CI) = 81 to 100) and a specificity of 80% (95% CI = 72 to 86). Serum protein levels of two specific genes showed high sensitivity for predicting SSAKI: matrix metalloproteinase-8 (89%, 95% CI = 64 to 98) and elastase-2 (83%, 95% CI = 58 to 96). Both biomarkers carried a negative predictive value of 95%. When applied to a validation cohort, although both biomarkers carried low specificity (matrix metalloproteinase-8: 41%, 95% CI = 28 to 50; and elastase-2: 49%, 95% CI = 36 to 62), they carried high sensitivity (100%, 95% CI = 68 to 100 for both).
Gene probes upregulated in critically ill pediatric patients with septic shock may allow for the identification of novel candidate serum biomarkers for SSAKI prediction.
Septic shock is a frequent and costly problem among patients in the pediatric intensive care unit (PICU) and is associated with high mortality and devastating survivor morbidity. Genome-wide expression patterns can provide molecular granularity of the host response and offer insight into why large variations in outcomes exist. We derived whole-blood genome-wide expression patterns within 24 h of PICU admission from children with septic shock. We compared the transcriptome between septic shock developmental-age groups defined as neonates (≤28 d, n = 17), infants (1 month to 1 year, n = 62), toddlers (2–5 years, n = 54) and school-age (≥6 years, n = 47) and age-matched controls. Direct intergroup comparisons demonstrated profound changes in neonates, relative to older children. Neonates with septic shock demonstrated reduced expression of genes representing key pathways of innate and adaptive immunity. In contrast to the largely upregulated transcriptome in all other groups, neonates exhibited a predominantly downregulated transcriptome when compared with controls. Neonates and school-age subjects had the most uniquely regulated genes relative to controls. Age-specific studies of the host response are necessary to identify developmentally relevant translational opportunities that may lead to improved sepsis outcomes.
Pulmonary Arterial Hypertension (PAH) remains a therapeutic challenge, and the search continues for more effective drugs and drug combinations. We recently reported that deletion of the vasoactive intestinal peptide (VIP) gene caused the spontaneous expression of a PH phenotype that was fully corrected by VIP. The objectives of this investigation were to answer the questions: 1) Can VIP protect against PH in other experimental models? and 2) Does combining VIP with an endothelin (ET) receptor antagonist bosentan enhance its efficacy?
Within 3 weeks of a single injection of monocrotaline (MCT, s.c.) in Sprague Dawley rats, PAH developed, manifested by pulmonary vascular remodeling, lung inflammation, RV hypertrophy, and death within the next 2 weeks. MCT-injected animals were either untreated, treated with bosentan (p.o.) alone, with VIP (i.p.) alone, or with both together. We selected this particular combination upon finding that VIP down-regulates endothelin receptor expression which is further suppressed by bosentan. Therapeutic outcomes were compared as to hemodynamics, pulmonary vascular pathology, and survival.
Treatment with VIP, every other day for 3 weeks, begun on the same day as MCT, almost totally prevented PAH pathology, and eliminated mortality for 45 days. Begun 3 weeks after MCT, however, VIP only partially reversed PAH pathology, though more effectively than bosentan. Combined therapy with both drugs fully reversed the pathology, while preventing mortality for at least 45 days.
1) VIP completely prevented and significantly reversed MCT-induced PAH; 2) VIP was more effective than bosentan, probably because it targets a wider range of pro-remodeling pathways; and 3) combination therapy with VIP plus bosentan was more effective than either drug alone, probably because both drugs synergistically suppressed ET-ET receptor pathway.
Pulmonary Hypertension; Vasoactive Intestinal Peptide; Endothelin Receptor Antagonist; Pulmonary Vascular Remodeling; Monocrotaline
Phosphoinositide 3-kinases (PI3Ks) regulate numerous physiological processes including some aspects of cardiac function. Although regulation of cardiac contraction by individual PI3K isoforms has been studied, little is known about the cardiac consequences of downregulating multiple PI3Ks concurrently.
Methods and Results
Genetic ablation of both p110α and p110β in cardiac myocytes throughout development or in adult mice caused heart failure and death. Ventricular myocytes from double knockout animals showed transverse tubule (T-tubule) loss and disorganization, misalignment of L-type Ca2+ channels in the T-tubules with ryanodine receptors in the sarcoplasmic reticulum, and reduced Ca2+ transients and contractility. Junctophilin-2, which is thought to tether T-tubules to the sarcoplasmic reticulum, was mislocalized in the double PI3K-null myocytes without a change in expression level.
PI3K p110α and p110β are required to maintain the organized network of T-tubules that is vital for efficient Ca2+-induced Ca2+ release and ventricular contraction. PI3Ks maintain T-tubule organization by regulating junctophilin-2 localization. These results could have important medical implications because several PI3K inhibitors that target both isoforms are being used to treat cancer patients in clinical trials.
Determine the association between prenatal cocaine exposure and postnatal environmental adversity on salivary cortisol stress reactivity in school aged children.
Subjects included 743 11 year old children (n=320 cocaine exposed; 423 comparison) followed since birth in a longitudinal prospective multisite study. Saliva samples were collected to measure cortisol at baseline and after a standardized procedure to induce psychological stress. Children were divided into those who showed an increase in cortisol from baseline to post stress and those who showed a decrease or blunted cortisol response. Covariates measured included site, birthweight, maternal pre and postnatal use of alcohol, tobacco or marijuana, social class, changes in caretakers, maternal depression and psychological symptoms, domestic and community violence, child abuse and quality of the home.
With adjustment for confounding variables, cortisol reactivity to stress was more likely to be blunted in children with prenatal cocaine exposure. Cocaine exposed children exposed to domestic violence showed the strongest effects.
The combination of prenatal cocaine exposure and an adverse postnatal environment could down regulate the hypothalamic-pituitary-adrenal axis (HPA) resulting in the blunted cortisol response to stress possibly increasing risk for later psychopathology and adult disease.
prenatal cocaine exposure; cortisol reactivity; environmental adversity
p110-β associates with the Vps34–Vps15–Beclin 1–Atg14L complex and facilitates generation of PtdIns(3)P to promote autophagy.
Autophagy is an evolutionarily conserved cell renewal process that depends on phosphatidylinositol 3-phosphate (PtdIns(3)P). In metazoans, autophagy is inhibited by PtdIns(3,4,5)P3, the product of class IA PI3Ks, which mediates the activation of the Akt–TOR kinase cascade. However, the precise function of class IA PI3Ks in autophagy remains undetermined. Class IA PI3Ks are heterodimeric proteins consisting of an 85-kD regulatory subunit and a 110-kD catalytic subunit. Here we show that the class IA p110-β catalytic subunit is a positive regulator of autophagy. Genetic deletion of p110-β results in impaired autophagy in mouse embryonic fibroblasts, liver, and heart. p110-β does not promote autophagy by affecting the Akt–TOR pathway. Rather, it associates with the autophagy-promoting Vps34–Vps15–Beclin 1–Atg14L complex and facilitates the generation of cellular PtdIns(3)P. Our results unveil a previously unknown function for p110-β as a positive regulator of autophagy in multicellular organisms.
Phosphoinositide 3-kinase (PI3K) p110α plays a key role in insulin action and tumorigenesis. Myocyte contraction is initiated by an inward Ca2+ current (ICa,L) through the voltage-dependent L-type Ca2+ channel (LTCC). The aim of this study was to evaluate if p110α also controls cardiac contractility by regulating the LTCC.
Methods and Results
Genetic ablation of p110α (also known as Pik3ca), but not p110β (also known as Pik3cb), in cardiac myocytes of adult mice reduced ICa,L and blocked insulin signaling in the heart. p110α-null myocytes had a reduced number of LTCCs on the cell surface and a contractile defect that decreased cardiac function in vivo. Similarly, pharmacological inhibition of p110α decreased ICa,L and contractility in canine myocytes. Inhibition of p110β did not reduce ICa,L.
PI3K p110α but not p110β regulates the LTCC in cardiac myocytes. Decreased signaling to p110α reduces the number of LTCCs on the cell surface and thus attenuates ICa,L and contractility.
calcium; ion channels; signal transduction; myocyte; contractility
To advance our biological understanding of pediatric septic shock, we measured the genome-level expression profiles of critically ill children representing the systemic inflammatory response syndrome (SIRS), sepsis, and septic shock spectrum.
Prospective observational study involving microarray-based bioinformatics.
Multiple pediatric intensive care units in the United States.
Children ≤10 years of age: 18 normal controls, 22 meeting criteria for SIRS, 32 meeting criteria for sepsis, and 67 meeting criteria for septic shock on day 1. The available day 3 samples included 20 patients still meeting sepsis criteria, 39 patients still meeting septic shock criteria, and 24 patients meeting the exclusive day 3 category, SIRS resolved.
None other than standard care.
Measurements and Main Results
Longitudinal analyses were focused on gene expression relative to control samples and patients having paired day 1 and day 3 samples. The longitudinal analysis focused on up-regulated genes revealed common patterns of up-regulated gene expression, primarily corresponding to inflammation and innate immunity, across all patient groups on day 1. These patterns of up-regulated gene expression persisted on day 3 in patients with septic shock, but not to the same degree in the other patient classes. The longitudinal analysis focused on down-regulated genes demonstrated gene repression corresponding to adaptive immunity-specific signaling pathways and was most prominent in patients with septic shock on days 1 and 3. Gene network analyses based on direct comparisons across the SIRS, sepsis, and septic shock spectrum, and all available patients in the database, demonstrated unique repression of gene networks in patients with septic shock corresponding to major histocompatibility complex antigen presentation. Finally, analyses focused on repression of genes corresponding to zinc-related biology demonstrated that this pattern of gene repression is unique to patients with septic shock.
Although some common patterns of gene expression exist across the pediatric SIRS, sepsis, and septic shock spectrum, septic shock is particularly characterized by repression of genes corresponding to adaptive immunity and zinc-related biology. (Crit Care Med 2009; 37:1558–1566)
microarray; T cell; antigen presentation; children; inflammation; zinc
Mammalian target of rapamycin (mTOR) is a protein kinase that controls cell growth, proliferation, and survival. mTOR signaling is often upregulated in cancer and there is great interest in developing drugs that target this enzyme. Rapamycin and its analogs bind to a domain separate from the catalytic site to block a subset of mTOR functions. These drugs are extremely selective for mTOR and are already in clinical use for treating cancers, but they could potentially activate an mTOR-dependent survival pathway that could lead to treatment failure. By contrast, small molecules that compete with ATP in the catalytic site would inhibit all of the kinase-dependent functions of mTOR without activating the survival pathway. Several non-selective mTOR kinase inhibitors have been described and here we review their chemical and cellular properties. Further development of selective mTOR kinase inhibitors holds the promise of yielding potent anticancer drugs with a novel mechanism of action.
mTOR; Rictor; Raptor; Rapamycin; Phosphatidylinositol 3-kinase; Akt
Human septic shock involves multiple genome-level perturbations. We have conducted microarray analyses in children with septic shock within 24 hours of intensive care unit admission, using whole-blood derived RNA. Based on sequential statistical and expression filters, there were 2,482 differentially regulated gene probes (1,081 upregulated and 1,401 downregulated) between patients with septic shock (n = 42) and controls (n = 15). Both gene lists encompassed several biologically relevant gene ontologies and canonical pathways. Notably, many of the genes downregulated in the patients with septic shock, relative to the controls, participate in gene ontologies related to metal or zinc homeostasis. Comparison of septic shock survivors (n = 33) and nonsurvivors (n = 9) demonstrated differential regulation of 63 gene probes. Among the 63 gene probes differentially regulated between septic shock survivors and nonsurvivors, two isoforms of metallothionein (MT) demonstrated increased expression in the nonsurvivors. Consistent with the ability of MT to sequester zinc in the intracellular compartment, nonsurvivors had lower serum zinc levels compared to survivors. In a corroborating study of murine sepsis, MT-null mice demonstrated a survival advantage compared to wild-type mice. These data represent the largest reported cohort of pediatric patients with septic shock, which has undergone genome-level expression profiling based on microarray. The data are biologically plausible and demonstrate that genome-level alterations of zinc homeostasis may be prevalent in clinical pediatric septic shock.
The dopamine (DA) transporter (DAT) is a major molecular target of the psychostimulant amphetamine (AMPH). AMPH, as a result of its ability to reverse DAT-mediated inward transport of DA, induces DA efflux thereby increasing extracellular DA levels. This increase is thought to underlie the behavioral effects of AMPH. We have demonstrated previously that insulin, through phosphatidylinositol 3-kinase (PI3K) signaling, regulates DA clearance by fine-tuning DAT plasma membrane expression. PI3K signaling may represent a novel mechanism for regulating DA efflux evoked by AMPH, since only active DAT at the plasma membrane can efflux DA. Here, we show in both a heterologous expression system and DA neurons that inhibition of PI3K decreases DAT cell surface expression and, as a consequence, AMPH-induced DA efflux.
dopamine transporter (DAT); amphetamine (AMPH); dopamine (DA); phosphatidylinositol 3-kinase (PI3K); insulin; drug abuse; transient current
Septic shock is a heterogeneous syndrome within which probably exist several biological subclasses. Discovery and identification of septic shock subclasses could provide the foundation for the design of more specifically targeted therapies. Herein we tested the hypothesis that pediatric septic shock subclasses can be discovered through genome-wide expression profiling.
Genome-wide expression profiling was conducted using whole blood-derived RNA from 98 children with septic shock, followed by a series of bioinformatic approaches targeted at subclass discovery and characterization.
Three putative subclasses (subclasses A, B, and C) were initially identified based on an empiric, discovery-oriented expression filter and unsupervised hierarchical clustering. Statistical comparison of the three putative subclasses (analysis of variance, Bonferonni correction, P < 0.05) identified 6,934 differentially regulated genes. K-means clustering of these 6,934 genes generated 10 coordinately regulated gene clusters corresponding to multiple signaling and metabolic pathways, all of which were differentially regulated across the three subclasses. Leave one out cross-validation procedures indentified 100 genes having the strongest predictive values for subclass identification. Forty-four of these 100 genes corresponded to signaling pathways relevant to the adaptive immune system and glucocorticoid receptor signaling, the majority of which were repressed in subclass A patients. Subclass A patients were also characterized by repression of genes corresponding to zinc-related biology. Phenotypic analyses revealed that subclass A patients were younger, had a higher illness severity, and a higher mortality rate than patients in subclasses B and C.
Genome-wide expression profiling can identify pediatric septic shock subclasses having clinically relevant phenotypes.
We previously generated genome-wide expression data (microarray) from children with septic shock having the potential to lead the field into novel areas of investigation. Herein we seek to validate our data through a bioinformatic approach centered on a validation patient cohort. Forty-two children with a clinical diagnosis of septic shock and 15 normal controls served as the training data set, while 30 separate children with septic shock and 14 separate normal controls served as the test data set. Class prediction modeling using the training data set and the previously reported genome-wide expression signature of pediatric septic shock correctly identified 95 to 100% of controls and septic shock patients in the test data set, depending on the class prediction algorithm and the gene selection method. Subjecting the test data set to an identical filtering strategy as that used for the training data set, demonstrated 75% concordance between the two gene lists. Subjecting the test data set to a purely statistical filtering strategy, with highly stringent correction for multiple comparisons, demonstrated less than 50% concordance with the previous gene filtering strategy. However, functional analysis of this statistics-based gene list demonstrated similar functional annotations and signaling pathways as that seen in the training data set. In particular, we validated that pediatric septic shock is characterized by large scale repression of genes related to zinc homeostasis and lymphocyte function. These data demonstrate that the previously reported genome-wide expression signature of pediatric septic shock is applicable to a validation cohort of patients.