Clinical Features of Genetic Resilience in Chronic Obstructive Pulmonary Disease.

Introduction: In the personalized risk quantification of chronic obstructive pulmonary disease (COPD), genome-wide association studies and polygenic risk scores (PRS) complement traditional risk factors, such as age and cigarette smoking. However, despite being at considerable levels of risk, some individuals do not develop COPD. Research on COPD resilience remains largely unexplored. Methods: We applied the previously published COPD PRS to whole genome sequencing data from non-Hispanic white and African American individuals in the COPDGene study. We defined genetic resilience as individuals unaffected by COPD with a polygenic risk score above the 90 th percentile. We defined risk-matched case individuals as those with COPD (i.e., FEV 1 /FVC < 0.70) and a PRS above the 90 th percentile. We defined low risk individuals without COPD (i.e., FEV 1 /FVC > 0.70) as a polygenic risk score below the 10 th percentile. We compared genetically resilient individuals to risk-matched individuals with COPD and low risk individuals by demographics, lung function, respiratory symptoms, co-morbidities, and chest CT scan measurements. We also performed survival analyses, differential expression analysis, and matching for sensitivity analyses. Results: We identified 211 resilient individuals without COPD, 605 genetic risk-matched individuals with COPD, and 527 low-risk individuals without COPD. Resilient individuals had higher FEV 1 % predicted and lower percent emphysema. In contrast, resilient individuals had higher airway wall thickness compared to low-risk unaffected individuals. While there was no difference in survival between low-risk and resilient individuals, resilient individuals had higher survival compared to risk matched cases. We also identified two genes that were differentially expressed between low-risk unaffected individuals and resilient individuals. Conclusion: Genetically resilient individuals had a reduced burden of COPD disease-related measures compared to risk-matched cases but had subtly increased measures compared to low-risk unaffected individuals. Further genetic studies will be needed to illuminate the underlying pathobiology of our observations.

Changes in cytokine and cytokine receptor levels during postnatal development of the human dorsolateral prefrontal cortex.

In addition to their traditional roles in immune cell communication, cytokines regulate brain development. Cytokines are known to influence neural cell generation, differentiation, maturation, and survival. However, most work on the role of cytokines in brain development investigates rodents or focuses on prenatal events. Here, we investigate how mRNA and protein levels of key cytokines and cytokine receptors change during postnatal development of the human prefrontal cortex. We find that most cytokine transcripts investigated (IL1B, IL18, IL6, TNF, IL13) are lowest at birth and increase between 1.5 and 5 years old. After 5 years old, transcriptional patterns proceeded in one of two directions: decreased expression in teens and young adults (IL1B, p = 0.002; and IL18, p = 0.004) or increased mean expression with maturation, particularly in teenagers (IL6, p = 0.004; TNF, p = 0.002; IL13, p < 0.001). In contrast, cytokine proteins tended to remain elevated after peaking significantly around 3 years of age (IL1B, p = 0.012; IL18, p = 0.026; IL6, p = 0.039; TNF, p < 0.001), with TNF protein being highest in teenagers. An mRNA-only analysis of cytokine receptor transcripts found that early developmental increases in cytokines were paralleled by increases in their ligand-binding receptor subunits, such as IL1R1 (p = 0.033) and IL6R (p < 0.001) transcripts. In contrast, cytokine receptor-associated signaling subunits, IL1RAP and IL6ST, did not change significantly between age groups. Of the two TNF receptors, the 'pro-death' TNFRSF1A and 'pro-survival' TNFRSF1B, only TNFRSF1B was significantly changed (p = 0.028), increasing first in toddlers and again in young adults. Finally, the cytokine inhibitor, IL13, was elevated first in toddlers (p = 0.006) and again in young adults (p = 0.053). While the mean expression of interleukin-1 receptor antagonist (IL1RN) was highest in toddlers, this increase was not statistically significant. The fluctuations in cytokine expression reported here support a role for increases in specific cytokines at two different stages of human cortical development. The first is during the toddler/preschool period (IL1B, IL18, and IL13), and the other occurs at adolescence/young adult maturation (IL6, TNF and IL13).

Increased prefrontal cortical cells positive for macrophage/microglial marker CD163 along blood vessels characterizes a neuropathology of neuroinflammatory schizophrenia.

Transcript levels of cytokines and SERPINA3 have been used to define a substantial subset (40%) of individuals with schizophrenia with elevated inflammation and worse neuropathology in the dorsolateral prefrontal cortex (DLPFC). In this study, we tested if inflammatory proteins are likewise related to high and low inflammatory states in the human DLFPC in people with schizophrenia and controls. Levels of inflammatory cytokines (IL6, IL1ß, IL18, IL8) and a macrophage marker (CD163 protein) were measured in brains obtained from the National Institute of Mental Health (NIMH) (N = 92). First, we tested for diagnostic differences in protein levels overall, then we determined the percentage of individuals that could be defined as "high" inflammation using protein levels. IL-18 was the only cytokine to show increased expression in schizophrenia compared to controls overall. Interestingly, two-step recursive clustering analysis showed that IL6, IL18, and CD163 protein levels could be used as predictors of "high and low" inflammatory subgroups. By this model, a significantly greater proportion of schizophrenia cases (18/32; 56.25%; SCZ) were identified as belonging to the high inflammatory (HI) subgroup compared to control cases (18/60; 30%; CTRL) [χ2(1) = 6.038, p = 0.014]. When comparing across inflammatory subgroups, IL6, IL1ß, IL18, IL8, and CD163 protein levels were elevated in both SCZ-HI and CTRL-HI compared to both low inflammatory subgroups (all p < 0.05). Surprisingly, TNFα levels were significantly decreased (-32.2%) in schizophrenia compared to controls (p < 0.001), and were most diminished in the SCZ-HI subgroup compared to both CTRL-LI and CTRL-HI subgroups (p < 0.05). Next, we asked if the anatomical distribution and density of CD163+ macrophages differed in those with schizophrenia and high inflammation status. Macrophages were localized to perivascular sites and found surrounding small, medium and large blood vessels in both gray matter and white matter, with macrophage density highest at the pial surface in all schizophrenia cases examined. A higher density of CD163+ macrophages, that were also larger and more darkly stained, was found in the SCZ-HI subgroup (+154% p < 0.05). We also confirmed the rare existence of parenchymal CD163+ macrophages in both high inflammation subgroups (schizophrenia and controls). Brain CD163+ cell density around blood vessels positively correlated with CD163 protein levels. In conclusion, we find a link between elevated interleukin cytokine protein levels, decreased TNFα protein levels, and elevated CD163+ macrophage densities especially along small blood vessels in those with neuroinflammatory schizophrenia.

The extracellular matrix protein fibulin-3/EFEMP1 promotes pleural mesothelioma growth by activation of PI3K/Akt signaling.

Malignant pleural mesothelioma (MPM) is an aggressive tumor with poor prognosis and limited therapeutic options. The extracellular matrix protein fibulin-3/EFEMP1 accumulates in the pleural effusions of MPM patients and has been proposed as a prognostic biomarker of these tumors. However, it is entirely unknown whether fibulin-3 plays a functional role on MPM growth and progression. Here, we demonstrate that fibulin-3 is upregulated in MPM tissue, promotes the malignant behavior of MPM cells, and can be targeted to reduce tumor progression. Overexpression of fibulin-3 increased the viability, clonogenic capacity and invasion of mesothelial cells, whereas fibulin-3 knockdown decreased these phenotypic traits as well as chemoresistance in MPM cells. At the molecular level, fibulin-3 activated PI3K/Akt signaling and increased the expression of a PI3K-dependent gene signature associated with cell adhesion, motility, and invasion. These pro-tumoral effects of fibulin-3 on MPM cells were disrupted by PI3K inhibition as well as by a novel, function-blocking, anti-fibulin-3 chimeric antibody. Anti-fibulin-3 antibody therapy tested in two orthotopic models of MPM inhibited fibulin-3 signaling, resulting in decreased tumor cell proliferation, reduced tumor growth, and extended animal survival. Taken together, these results demonstrate for the first time that fibulin-3 is not only a prognostic factor of MPM but also a relevant molecular target in these tumors. Further development of anti-fibulin-3 approaches are proposed to increase early detection and therapeutic impact against MPM.

Evidence-based use of scalable biomarkers to increase diagnostic efficiency and decrease the lifetime costs of autism.

Challenges associated with the current screening and diagnostic process for autism spectrum disorder (ASD) in the US cause a significant delay in the initiation of evidence-based interventions at an early age when treatments are most effective. The present study shows how implementing a second-order diagnostic measure to high risk cases initially flagged positive from screening tools can further inform clinical judgment and substantially improve early identification. We use two example measures for the purposes of this demonstration; a saliva test and eye-tracking technology, both scalable and easy-to-implement biomarkers recently introduced in ASD research. Results of the current cost-savings analysis indicate that lifetime societal cost savings in special education, medical and residential care are estimated to be nearly $580,000 per ASD child, with annual cost savings in education exceeding $13.3 billion, and annual cost savings in medical and residential care exceeding $23.8 billion (of these, nearly $11.2 billion are attributable to Medicaid). These savings total more than $37 billion/year in societal savings in the US. Initiating appropriate interventions faster and reducing the number of unnecessary diagnostic evaluations can decrease the lifetime costs of ASD to society. We demonstrate the value of implementing a scalable highly accurate diagnostic in terms of cost savings to the US. LAY SUMMARY: This paper demonstrates how biomarkers with high accuracy for detecting autism spectrum disorder (ASD) could be used to increase the efficiency of early diagnosis. Results also show that, if more children with ASD are identified early and referred for early intervention services, the system would realize substantial costs savings across the lifespan.

Distance running alters peripheral microRNAs implicated in metabolism, fluid balance, and myosin regulation in a sex-specific manner.

Microribonucleic acids (miRNAs) mediate adaptive responses to exercise and may serve as biomarkers of exercise intensity/capacity. Expression of miRNAs is altered in skeletal muscle, plasma, and saliva following exertion. Women display unique physiologic responses to endurance exercise, and miRNAs respond to pathologic states in sex-specific patterns. However sex-specific miRNA responses to exercise remain unexplored. This study utilized high-throughput RNA sequencing to measure changes in salivary RNA expression among 25 collegiate runners following a single long-distance run. RNA concentrations in pre- and post-run saliva was assessed through alignment and quantification of 4,694 miRNAs and 27,687 mRNAs. Pair-wise Wilcoxon rank-sum test identified miRNAs with significant [false discovery rate (FDR) < 0.05] post-run changes. Associations between miRNA levels and predicted mRNA targets were explored with Pearson correlations. Differences in miRNA patterns between men ( n = 13) and women ( n = 12) were investigated with two-way analysis of variance. Results revealed 122 salivary miRNAs with post-run changes. The eight miRNAs with the largest changes were miR-3671, miR-5095 (downregulated); and miR-7154-3p, miR-200b-5p, miR-5582-3p, miR-6859-3p, miR-6751-5p, miR-4419a (upregulated). Predicted mRNA targets for these miRNAs represented 15 physiologic processes, including glycerophospholipid metabolism (FDR = 0.042), aldosterone-regulated sodium reabsorption (FDR = 0.049), and arrhythmogenic ventricular cardiomyopathy (FDR = 0.018). Twenty-six miRNA/mRNA pairs had associated changes in post-run levels. Three miRNAs (miR-4675, miR-6745, miR-6746-3p) demonstrated sex-specific responses to exercise. Numerous salivary miRNAs change in response to endurance running and target the expression of genes involved in metabolism, fluid balance, and musculoskeletal adaptations. A subset of miRNAs may differentiate the metabolic response to exercise in men and women.

SHIP1 Deficiency in Inflammatory Bowel Disease Is Associated With Severe Crohn's Disease and Peripheral T Cell Reduction.

In our previous study, we observed a severe reduction in the Src homology 2-containing-inositol-phosphatase-1 (SHIP1) protein in a subpopulation of subjects from a small adult Crohn's Disease (CD) cohort. This pilot study had been undertaken since we had previously demonstrated that engineered deficiency of SHIP1 in mice results in a spontaneous and severe CD-like ileitis. Here, we extend our analysis of SHIP1 expression in peripheral blood mononuclear cells in a second much larger adult Inflammatory Bowel Disease (IBD) cohort, comprised of both CD and Ulcerative Colitis patients, to assess contribution of SHIP1 to the pathogenesis of human IBD. SHIP1 protein and mRNA levels were evaluated from blood samples obtained from IBD subjects seen at UCSF/SFVA, and compared to healthy control samples. Western blot analyses revealed that ~15% of the IBD subjects are severely SHIP1-deficient, with less than 10% of normal SHIP1 protein present in PBMC. Further analyses by flow cytometry and sequencing were performed on secondary samples obtained from the same subjects. Pan-hematolymphoid SHIP1 deficiency was a stable phenotype and was not due to coding changes in the INPP5D gene. A very strong association between SHIP1 deficiency and the presence of a novel SHIP1:ATG16L1 fusion transcript was seen. Similar to SHIP1-deficient mice, SHIP1-deficient subjects had reduced numbers of circulating CD4+ T cell numbers. Finally, SHIP1-deficient subjects with CD had a history of severe disease requiring multiple surgeries. These studies reveal that the SHIP1 protein is crucial for normal T cell homeostasis in both humans and mice, and that it is also a potential therapeutic and/or diagnostic target in human IBD.

Reduced Slc1a1 expression is associated with neuroinflammation and impaired sensorimotor gating and cognitive performance in mice: Implications for schizophrenia.

We previously reported a 84-Kb hemi-deletion copy number variant at the SLC1A1 gene locus that reduces its expression and appeared causally linked to schizophrenia. In this report, we characterize the in vivo and in vitro consequences of reduced expression of Slc1a1 in mice. Heterozygous (HET) Slc1a1+/- mice, which more closely model the hemi-deletion we found in human subjects, were examined in a series of behavioral, anatomical and biochemical assays. Knockout (KO) mice were also included in the behavioral studies for comparative purposes. Both HET and KO mice exhibited evidence of increased anxiety-like behavior, impaired working memory, decreased exploratory activity and impaired sensorimotor gating, but no changes in overall locomotor activity. The magnitude of changes was approximately equivalent in the HET and KO mice suggesting a dominant effect of the haploinsufficiency. Behavioral changes in the HET mice were accompanied by reduced thickness of the dorsomedial prefrontal cortex. Whole transcriptome RNA-Seq analysis detected expression changes of genes and pathways involved in cytokine signaling and synaptic functions in both brain and blood. Moreover, the brains of Slc1a1+/- mice displayed elevated levels of oxidized glutathione, a trend for increased oxidative DNA damage, and significantly increased levels of cytokines. This latter finding was further supported by SLC1A1 knockdown and overexpression studies in differentiated human neuroblastoma cells, which led to decreased or increased cytokine expression, respectively. Taken together, our results suggest that partial loss of the Slc1a1 gene in mice causes haploinsufficiency associated with behavioral, histological and biochemical changes that reflect an altered redox state and may promote the expression of behavioral features and inflammatory states consistent with those observed in schizophrenia.

Analysis of SHIP1 expression and activity in Crohn's disease patients.

BACKGROUND: SH2 domain containing inositol-5-phosphatase (SHIP1) is an important modulator of innate and adaptive immunity. In mice, loss of SHIP1 provokes severe ileitis resembling Crohn's disease (CD), as a result of deregulated immune responses, altered cytokine production and intestinal fibrosis. Recently, SHIP1 activity was shown to be correlated to the presence of a CD-associated single nucleotide polymorphism in ATG16L1. Here, we studied SHIP1 activity and expression in an adult cohort of CD patients. METHODS: SHIP1 activity, protein and mRNA expression in peripheral blood mononuclear cells from CD patients in clinical remission were determined by Malachite green assay, Western blotting and qRT-PCR respectively. Genomic DNA was genotyped for ATG16L1 rs2241880. RESULTS: SHIP1 protein levels are profoundly diminished in a subset of patients; however, SHIP1 activity and expression are not correlated to ATG16L1 SNP status in this adult cohort. CONCLUSIONS: Aberrant SHIP1 activity can contribute to disease in a subset of adult CD patients, and warrants further investigation.

Heterogeneity of p53 dependent genomic responses following ethanol exposure in a developmental mouse model of fetal alcohol spectrum disorder.

Prenatal ethanol exposure can produce structural and functional deficits in the brain and result in Fetal Alcohol Spectrum Disorder (FASD). In rodent models acute exposure to a high concentration of alcohol causes increased apoptosis in the developing brain. A single causal molecular switch that signals for this increase in apoptosis has yet to be identified. The protein p53 has been suggested to play a pivotal role in enabling cells to engage in pro-apoptotic processes, and thus figures prominently as a hub molecule in the intracellular cascade of responses elicited by alcohol exposure. In the present study we examined the effect of ethanol-induced cellular and molecular responses in primary somatosensory cortex (SI) and hippocampus of 7-day-old wild-type (WT) and p53-knockout (KO) mice. We quantified apoptosis by active caspase-3 immunohistochemistry and ApopTag™ labeling, then determined total RNA expression levels in laminae of SI and hippocampal subregions. Immunohistochemical results confirmed increased incidence of apoptotic cells in both regions in WT and KO mice following ethanol exposure. The lack of p53 was not protective in these brain regions. Molecular analyses revealed a heterogeneous response to ethanol exposure that varied depending on the subregion, and which may go undetected using a global approach. Gene network analyses suggest that the presence or absence of p53 alters neuronal function and synaptic modifications following ethanol exposure, in addition to playing a classic role in cell cycle signaling. Thus, p53 may function in a way that underlies the intellectual and behavioral deficits observed in FASD.

Evaluation of cell proliferation, apoptosis, and DNA-repair genes as potential biomarkers for ethanol-induced CNS alterations.

BACKGROUND: Alcohol use disorders (AUDs) lead to alterations in central nervous system (CNS) architecture along with impaired learning and memory. Previous work from our group and that of others suggests that one mechanism underlying these changes is alteration of cell proliferation, apoptosis, and DNA-repair in neural stem cells (NSCs) produced as a consequence of ethanol-induced effects on the expression of genes related to p53-signaling. This study tests the hypothesis that changes in the expression of p53-signaling genes represent biomarkers of ethanol abuse which can be identified in the peripheral blood of rat drinking models and human AUD subjects and posits that specific changes may be correlated with differences in neuropsychological measures and CNS structure. RESULTS: Remarkably, microarray analysis of 350 genes related to p53-signaling in peripheral blood leukocytes (PBLs) of binge-drinking rats revealed 190 genes that were significantly altered after correcting for multiple testing. Moreover, 40 of these genes overlapped with those that we had previously observed to be changed in ethanol-exposed mouse NSCs. Expression changes in nine of these genes were tested for independent confirmation by a custom QuantiGene Plex (QGP) assay for a subset of p53-signaling genes, where a consistent trend for decreased expression of mitosis-related genes was observed. One mitosis-related gene (Pttg1) was also changed in human lymphoblasts cultured with ethanol. In PBLs of human AUD subjects seven p53-signaling genes were changed compared with non-drinking controls. Correlation and principal components analysis were then used to identify significant relationships between the expression of these seven genes and a set of medical, demographic, neuropsychological and neuroimaging measures that distinguished AUD and control subjects. Two genes (Ercc1 and Mcm5) showed a highly significant correlation with AUD-induced decreases in the volume of the left parietal supramarginal gyrus and neuropsychological measures. CONCLUSIONS: These results demonstrate that alcohol-induced changes in genes related to proliferation, apoptosis, and DNA-repair are observable in the peripheral blood and may serve as a useful biomarker for CNS structural damage and functional performance deficits in human AUD subjects.

Increased promoter methylation of the immune regulatory gene SHP-1 in leukocytes of multiple sclerosis subjects.

The protein tyrosine phosphatase, SHP-1, is a negative regulator of proinflammatory signaling and autoimmune disease. We have previously reported reduced SHP-1 expression in peripheral blood leukocytes of subjects with multiple sclerosis (MS). Recent evidence indicates that virus-induced DNA methylation of the SHP-1 promoter is responsible for aberrant silencing of SHP-1 expression and function in hematopoietic cells that might relate to inflammatory diseases. In the present study, bisulfite sequencing of the SHP-1 promoter demonstrated that over a third of MS subjects had abnormally high promoter methylation. As SHP-1 is deficient in MS leukocytes and SHP-1-regulated proinflammatory genes are correspondingly upregulated, we propose that increased SHP-1 promoter methylation may relate in part to decreased SHP-1 expression and increased leukocyte-mediated inflammation in MS.

Regulating the availability of transforming growth factor ß1 in B104 neuroblastoma cells.

Transforming growth factor (TGF) beta1 is a key player in early brain development, hence, its availability (i.e., synthesis and release) affects neuronogenesis. TGFbeta1 moves proliferating cells out of the cell cycle and promotes their subsequent migration. The present study tested the hypothesis that neural progenitors self-regulate TGFbeta1. B104 neuroblastoma cells which can grow in the absence of serum or growth factors were used in systematic studies of transcription, translation, release, and activation. These studies relied on quantitative enzyme-linked immunosorbent assays and real-time polymerase chain reactions. TGFbeta1 positively upregulated its own intracellular expression and promoted increased release of TGFbeta1 from cells. The induction of TGFbeta1 was independent of a change in transcription, but it depended on cycloheximide-inhibited translation. Signaling mediated by downstream Smad2/3 through the TGFbeta receptors and intracellular protein transport were also required for release of TGFbeta1 from B104 cells. Thus, TGFbeta1 production and release were mediated through a feed-forward mechanism and were pivotally regulated at the level of translation. These activities appear to be key for the role of TGFbeta1 in the proliferation and migration of young neurons.

Thrombospondin-1: a proatherosclerotic protein augmented by hyperglycemia.

OBJECTIVE: Diabetes is associated with a more aggressive form of atherosclerosis. Thrombospondin-1 (TSP-1), an extracellular matrix protein, is an acute-phase reactant that induces vascular smooth muscle (VSMC) migration and proliferation in areas of vascular injury and is also up-regulated in VSMCs exposed to hyperglycemia. This study tested the hypothesis that hyperglycemia amplifies the expression of genes induced by TSP-1 in VSMCs. METHODS: Human aortic VSMCs were cultured in Dulbecco Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics. Cells were used between passages three and five. VSMCs were preincubated in DMEM containing 0.2% FBS with 5 mM glucose (normoglycemia), 25 mM glucose (hyperglycemia), 25 mM mannose (osmotic control), TSP-1 (20 microg/mL), 25 mM glucose + TSP-1 (20 microg/mL), or 25 mM mannose + TSP-1 (20 microg/mL). Total RNA was extracted. Microarray analysis was performed and analyzed by analysis of variance. P < .05 was considered significant. Quantitative real-time polymerase chain reaction (rtPCR) was used to confirm selected up-regulated genes. RESULTS: Microarray analysis revealed: (1) hyperglycemia altered 30 genes; (2) TSP-1 altered 212 genes, of which 8 were altered similarly to VSMCs exposed to 25 mM glucose; (3) TSP-1 up-regulated 10 genes associated with atherosclerosis and 4 others with diabetic vascular disease; (4) hyperglycemia combined with TSP-1 altered expression of 2822 genes. The three genes most up-regulated by TSP-1 in a normoglycemic environment were uridine 5'-diphosphoglucose (UDP-glucose) dehydrogenase (UGDH, 127%), transforming growth factor beta-2 (TGFbeta2, 116%), and hyaluronan synthase 2 (HAS2, 113%). Further, TSP-1 altered the expression of genes in 13 canonical pathways; however, when combined with hyperglycemia, 53 canonical pathways were affected. CONCLUSION: Quantitative rtPCR confirmed that genes in several of these pathways for TSP-1 and hyperglycemia combined with TSP-1 were up-regulated. These findings suggest that TSP-1 may be germane to the progression of atherosclerosis and may have a large effect with concurrent hyperglycemia.

The effects of strain and prenatal nicotine exposure on ethanol consumption by adolescent male and female rats.

Two studies of variables affecting voluntary ethanol consumption by adolescent male and female rats are reported. Sprague-Dawley (SD) and spontaneously hypertensive rats (SHRs) were compared in Experiment 1. Starting on postnatal day 30 all had 24-h access to 2%, then 4%, and then 6% ethanol, followed by 1-h access to the 6% until intake stabilized. During the 1-h access SHR females consumed more ethanol than all other groups. In Experiment 2, the same procedure was used to compare SD groups prenatally exposed to nicotine, with controls. Nicotine-exposed females consumed more ethanol during 1-h access than both nicotine-exposed and control males; but after using water intake as a covariate, the differences were not significant. These data show that deprivation conditions need to be considered when generalizing the results of voluntary consumption studies, and that estrogens may be a modulator of addictive behavior.

Microarray cluster analysis of irradiated growth plate zones following laser microdissection.

PURPOSE: Genes and pathways involved in early growth plate chondrocyte recovery after fractionated irradiation were sought as potential targets for selective radiorecovery modulation. MATERIALS AND METHODS: Three groups of six 5-week male Sprague-Dawley rats underwent fractionated irradiation to the right tibiae over 5 days, totaling 17.5 Gy, and then were killed at 7, 11, and 16 days after the first radiotherapy fraction. The growth plates were collected from the proximal tibiae bilaterally and subsequently underwent laser microdissection to separate reserve, perichondral, proliferative, and hypertrophic zones. Differential gene expression was analyzed between irradiated right and nonirradiated left tibia using RAE230 2.0 GeneChip microarray, compared between zones and time points and subjected to functional pathway cluster analysis with real-time polymerase chain reaction to confirm selected results. RESULTS: Each zone had a number of pathways showing enrichment after the pattern of hypothesized importance to growth plate recovery, yet few met the strictest criteria. The proliferative and hypertrophic zones showed both the greatest number of genes with a 10-fold right/left change at 7 days after initiation of irradiation and enrichment of the most functional pathways involved in bone, cartilage, matrix, or skeletal development. Six genes confirmed by real-time polymerase chain reaction to have early upregulation included insulin-like growth factor 2, procollagen type I alpha 2, matrix metallopeptidase 9, parathyroid hormone receptor 1, fibromodulin, and aggrecan 1. CONCLUSIONS: Nine overlapping pathways in the proliferative and hypertrophic zones (skeletal development, ossification, bone remodeling, cartilage development, extracellular matrix structural constituent, proteinaceous extracellular matrix, collagen, extracellular matrix, and extracellular matrix part) may play key roles in early growth plate radiorecovery.

Prevention of hepatocarcinogenesis and increased susceptibility to acetaminophen-induced liver failure in transaldolase-deficient mice by N-acetylcysteine.

Although oxidative stress has been implicated in acute acetaminophen-induced liver failure and in chronic liver cirrhosis and hepatocellular carcinoma (HCC), no common underlying metabolic pathway has been identified. Recent case reports suggest a link between the pentose phosphate pathway (PPP) enzyme transaldolase (TAL; encoded by TALDO1) and liver failure in children. Here, we show that Taldo1-/- and Taldo1+/- mice spontaneously developed HCC, and Taldo1-/- mice had increased susceptibility to acetaminophen-induced liver failure. Oxidative stress in Taldo1-/- livers was characterized by the accumulation of sedoheptulose 7-phosphate, failure to recycle ribose 5-phosphate for the oxidative PPP, depleted NADPH and glutathione levels, and increased production of lipid hydroperoxides. Furthermore, we found evidence of hepatic mitochondrial dysfunction, as indicated by loss of transmembrane potential, diminished mitochondrial mass, and reduced ATP/ADP ratio. Reduced beta-catenin phosphorylation and enhanced c-Jun expression in Taldo1-/- livers reflected adaptation to oxidative stress. Taldo1-/- hepatocytes were resistant to CD95/Fas-mediated apoptosis in vitro and in vivo. Remarkably, lifelong administration of the potent antioxidant N-acetylcysteine (NAC) prevented acetaminophen-induced liver failure, restored Fas-dependent hepatocyte apoptosis, and blocked hepatocarcinogenesis in Taldo1-/- mice. These data reveal a protective role for the TAL-mediated branch of the PPP against hepatocarcinogenesis and identify NAC as a promising treatment for liver disease in TAL deficiency.

Weight regain after Roux-en-Y: a significant 20% complication related to PYY.

OBJECTIVE: Roux-en-Y gastric bypass (RYGB) produces rapid and dramatic weight loss in very heavy obese patients. Up to 20% cannot sustain their weight loss beyond 2 to 3 y after surgery. METHODS: To identify putative etiologic factors producing post-RYGB weight regain, a literature survey of metabolic changes in very obese and a review of our diet-induced obese RYGB rat model data was done. RESULTS: Weight regain suggests an imbalance in physiologic mechanisms regulating appetite and metabolic rate. Weight regain occurred in 25% of our rats, produced by return to presurgical energy intake levels. The 75% of rats that sustained weight loss secreted a significantly larger amount of peptide YY (PYY) while suppressing leptin secretion; those that failed were unable to develop or sustain a sufficiently large plasma PYY:leptin ratio. Metabolic consequences of this failure included reversal of initial postsurgical increases in peripheral fatty acid oxidation, anorexigenic activity in the hypothalamic arcuate nucleus and paraventricular nucleus, and the expression of uncoupling protein-2 in adipose tissues, and decreases in hepatic lipogenesis, free tri-iodothyronine secretion, expression of orexigenic activity in the arcuate nucleus and paraventricular nucleus, expression of adenosine monophosphate kinase in adipose tissues, skeletal muscle mitochondrial mass, and endocannabinoid content and appetite. CONCLUSION: Weight regain after RYGB occurs in approximately 20% of patients and constitutes a serious complication. Weight regain-promoting consequences are attributed to a failure to sustain elevated plasma PYY concentrations, indicating that combining RYGB with pharmacologic stimulation of PYY secretion in patients after RYGB who exhibit inadequate PYY concentration may increase long-term success of surgical weight reduction in morbidly obese adults.

Cyclosporine inhibition of angiogenesis involves the transcription factor HESR1.

PURPOSE: Angiogenesis is critical in normal development and in tumor growth. Experimentally, cyclosporine A (CyA) inhibits angiogenesis in an in vivo mouse model and an in vitro capillary tube model. The mechanisms behind its antiangiogenic effects are not well characterized. To determine which nuclear factor, if any, may be involved in the antiangiogenic effects of CyA, we performed a microarray analysis of human aortic endothelial cells (HAEC) subjected to CyA and another calcineurin inhibitor, FK 506. METHODS: HAEC were divided into four groups: (1) HAEC incubated with CyA 2 microg/mL; (2) HAEC incubated with CyA 10 microg/mL; (3) HAEC incubated with FK 506 1 microg/mLl for 24 h; and (4) HAEC as control. We used Affymetrix GeneChip U133-A for gene expression analysis and validated our results with quantitative reverse transcription-polymerase chain reaction. RESULTS: At a 2 microg/mL dose, CyA treated HAEC revealed a 44-fold increase in the expression of hairy enhancer of split-related protein 1 (HESR1) and 1.73-fold down-regulation of transcripts encoding for the vascular endothelial growth factor (VEGF) receptor (VEGFR2). At 10 microg/mL, the expression of the HESR1 transcript was 57-fold higher than control, and VEGFR2 exhibited a 1.93-fold down-regulation. Quantitative reverse transcription-polymerase chain reaction confirmed a significant (P < 0.0001) increase in expression of HESR1 in CyA treated cells. In contrast, the expression level of HESR1 was not affected by the FK 506 treatment. CONCLUSION: CyA demonstrate antiangiogenic activities linked to an overexpression of HESR1 transcription factor, and down-regulation of VEGFR2. Thus, use of high-dose CyA may provide a novel treatment in angiogenesis dependent disease.

Microarray analysis of perichondral and reserve growth plate zones identifies differential gene expressions and signal pathways.

In the growth plate, the reserve and perichondral zones have been hypothesized to have similar functions, but their exact functions are poorly understood. Our hypothesis was that significant differential gene expression exists between perichondral and reserve chondrocytes that may differentiate the respective functions of these two zones. Normal Sprague-Dawley rat growth plate chondrocytes from the perichondral zone (PC) and reserve zone (RZ) were isolated by laser microdissection and then subjected to microarray analysis. In order to most comprehensively capture the unique features of the two zones, we analyzed both the most highly expressed genes and those that were most significantly different from the proliferative zone (PZ) as a single comparator. Confirmation of the differential expression of selected genes was done by quantitative real-time RT-PCR. A total of 8 transcripts showing high expression unique to the PC included translationally-controlled tumor protein (Tpt1), connective tissue growth factor (Ctgf), mortality factor 4 (Morf4l1), growth arrest specific 6 (Gas6), type V procollagen (Col5a2), frizzled-related protein (Frzb), GDP-dissociation inhibitor 2 (Gdi2) and Jun D proto-oncogene (Jund). In contrast, 8 transcripts showing unique high expression in the RZ included hyaluronan and proteoglycan link protein 1 (Hapln1), hemoglobin beta-2 subunit, type I procollagen (Col1a2), retinoblastoma binding protein 4 (LOC685491), Sparc-related modular calcium binding 2 (Smoc2), and calpastatin (Cast). Other genes were highly expressed in cells from both PC and RZ zones, including collagen II, collagen IX, catenin (cadherin associated protein) beta 1, eukaryotic translation elongation factor, high mobility group, ribosomal protein, microtubule-associated protein, reticulocalbin, thrombospondin, retinoblastoma binding protein, carboxypeptidase E, carnitine palmitoyltransferase 1, cysteine rich glycoprotein, plexin B2 (Plxnb2), and gap junction membrane channel protein. Functional classification of the most highly expressed transcripts were analyzed, and the pathway analysis indicated that ossification, bone remodeling, and cartilage development were uniquely enriched in the PC whereas both the PC and RZ showed pathway enrichment for skeletal development, extracellular matrix structural constituent, proteinaceous extracellular matrix, collagen, extracellular matrix, and extracellular matrix part pathways. We conclude that differential gene expression exists between the RZ and PC chondrocytes and these differentially expressed genes have unique roles to play corresponding to the function of their respective zones.