The glutamate/aspartate transporter EAAT1 is crucial for T-cell acute lymphoblastic leukemia proliferation and survival.

T-cell acute lymphoblastic leukemia (T-ALL) is a cancer of the immune system. Approximately 20% of paediatric and 50% of adult T-ALL patients have refractory disease or relapse and die from the disease. To improve patient outcome new therapeutics are needed. With the aim to identify new therapeutic targets, we combined the analysis of T-ALL gene expression and metabolism to identify the metabolic adaptations that T-ALL cells exhibit. We found that glutamine uptake is essential for T-ALL proliferation. Isotope tracing experiments showed that glutamine fuels aspartate synthesis through the TCA cycle and that glutamine and glutamine-derived aspartate together supply three nitrogen atoms in purines and all but one atom in pyrimidine rings. We show that the glutamate-aspartate transporter EAAT1 (SLC1A3), which is normally expressed in the central nervous system, is crucial for glutamine conversion to aspartate and nucleotides and that T-ALL cell proliferation depends on EAAT1 function. Through this work, we identify EAAT1 as a novel therapeutic target for T-ALL treatment.

Crosstalk between AML and stromal cells triggers acetate secretion through the metabolic rewiring of stromal cells.

Acute myeloid leukaemia (AML) cells interact and modulate components of their surrounding microenvironment into their own benefit. Stromal cells have been shown to support AML survival and progression through various mechanisms. Nonetheless, whether AML cells could establish beneficial metabolic interactions with stromal cells is underexplored. By using a combination of human AML cell lines and AML patient samples together with mouse stromal cells and a MLL-AF9 mouse model, here we identify a novel metabolic crosstalk between AML and stromal cells where AML cells prompt stromal cells to secrete acetate for their own consumption to feed the tricarboxylic acid cycle (TCA) and lipid biosynthesis. By performing transcriptome analysis and tracer-based metabolic NMR analysis, we observe that stromal cells present a higher rate of glycolysis when co-cultured with AML cells. We also find that acetate in stromal cells is derived from pyruvate via chemical conversion under the influence of reactive oxygen species (ROS) following ROS transfer from AML to stromal cells via gap junctions. Overall, we present a unique metabolic communication between AML and stromal cells and propose two different molecular targets, ACSS2 and gap junctions, that could potentially be exploited for adjuvant therapy.

Arf-like Protein 2 (ARL2) Controls Microtubule Neogenesis during Early Postnatal Photoreceptor Development.

Arf-like protein 2 (ARL2) is a ubiquitously expressed small GTPase with multiple functions. In a cell culture, ARL2 participates with tubulin cofactor D (TBCD) in the neogenesis of tubulin αß-heterodimers, the building blocks of microtubules. To evaluate this function in the retina, we conditionally deleted ARL2 in mouse retina at two distinct stages, either during the embryonic development (retArl2-/-) or after ciliogenesis specifically in rods (rodArl2-/-). retArl2-/- retina sections displayed distorted nuclear layers and a disrupted microtubule cytoskeleton (MTC) as early as postnatal day 6 (P6). Rod and cone outer segments (OS) did not form. By contrast, the rod ARL2 knockouts were stable at postnatal day 35 and revealed normal ERG responses. Cytoplasmic dynein is reduced in retArl2-/- inner segments (IS), suggesting that dynein may be unstable in the absence of a normal MTC. We investigated the microtubular stability in the absence of either ARL2 (retARL2-/-) or DYNC1H1 (retDync1h1-/-), the dynein heavy chain, and found that both the retArl2-/- and retDync1h1-/- retinas exhibited reduced microtubules and nuclear layer distortion. The results suggest that ARL2 and dynein depend on each other to generate a functional MTC during the early photoreceptor development.

Conditional Deletion of Cytoplasmic Dynein Heavy Chain in Postnatal Photoreceptors.

Purpose: Cytoplasmic dynein-1 (henceforth dynein) moves cargo in conjunction with dynactin toward the minus end of microtubules. The dynein heavy chain, DYNC1H1, comprises the backbone of dynein, a retrograde motor. Deletion of Dync1h1 abrogates dynein function. The purpose of this communication is to demonstrate effects of photoreceptor dynein inactivation during late postnatal development and in adult retina. Methods: We mated Dync1h1F/F mice with iCre75 and Prom1-CreERT2 mice to generate conditional rod and tamoxifen-induced knockout in rods and cones, respectively. We documented retina degeneration with confocal microscopy at postnatal day (P) 10 to P30 for the iCre75 line and 1 to 4 weeks post tamoxifen induction (wPTI) for the Prom1-CreERT2 line. We performed scotopic and photopic electroretinography (ERG) at P16 to P30 in the iCre75 line and at 1-week increments in the Prom1-CreERT2 line. Results were evaluated statistically using Student's t-test, two-factor ANOVA, and Welch's ANOVA. Results: Cre-induced homologous recombination of Dync1h1F/F mice truncated DYNC1H1 after exon 23. rodDync1h1-/- photoreceptors degenerated after P14, reducing outer nuclear layer (ONL) thickness and combined inner segment/outer segment (IS/OS) length significantly by P18. Scotopic ERG a-wave amplitudes decreased by P16 and were extinguished at P30. Cones were stable under rod-knockout conditions until P21 but inactive at P30. In tamDync1h1-/- photoreceptors, the IS/OS began shortening by 3wPTI and were nearly eliminated by 4wPTI. The ONL shrank significantly over this interval, indicating rapid photoreceptor degeneration following the loss of dynein. Conclusions: Our results demonstrate dynein is essential for the secretory pathway, formation of outer segments, and photoreceptor maintenance.

Effect of conditional deletion of cytoplasmic dynein heavy chain DYNC1H1 on postnatal photoreceptors.

Cytoplasmic dynein (dynein 1), a major retrograde motor of eukaryotic cells, is a 1.4 MDa protein complex consisting of a pair of heavy chains (DYNC1H1) and a set of heterodimeric noncatalytic accessory components termed intermediate, light intermediate and light chains. DYNC1H1 (4644 amino acids) is the dynein backbone encoded by a gene consisting of 77 exons. We generated a floxed Dync1h1 allele that excises exons 24 and 25 and truncates DYNC1H1 during Six3Cre-induced homologous recombination. Truncation results in loss of the motor and microtubule-binding domain. Dync1h1F/F;Six3Cre photoreceptors degenerated rapidly within two postnatal weeks. In the postnatal day 6 (P6) Dync1h1F/F;Six3Cre central retina, outer and inner nuclear layers were severely disorganized and lacked a recognizable outer plexiform layer (OPL). Although the gene was effectively silenced by P6, DYNC1H1 remnants persisted and aggregated together with rhodopsin, PDE6 and centrin-2-positive centrosomes in the outer nuclear layer. As photoreceptor degeneration is delayed in the Dync1h1F/F;Six3Cre retina periphery, retinal lamination and outer segment elongation are in part preserved. DYNC1H1 strongly persisted in the inner plexiform layer (IPL) beyond P16 suggesting lack of clearance of the DYNC1H1 polypeptide. This persistence of DYNC1H1 allows horizontal, rod bipolar, amacrine and ganglion cells to survive past P12. The results show that cytoplasmic dynein is essential for retina lamination, nuclear positioning, vesicular trafficking of photoreceptor membrane proteins and inner/outer segment elaboration.

Screening Tool for the Assessment of Malnutrition in Pediatrics (STAMP) in the Electronic Health Record: A Validation Study.

BACKGROUND: The impact of malnutrition on pediatric patients in the acute care setting is significant. Hospitalized patients with malnutrition have been shown to have poor clinical outcomes. Nutrition screening is the first critical step in identifying and treating malnutrition. Although several pediatric nutrition screening tools exist, none incorporate both electronic health record (EHR) compatibility and the recommended indicators of pediatric malnutrition, a gap recently identified in a systematic review by the Academy of Nutrition and Dietetics. The aim of this study was to prove the validity of a new version of Screening Tool for the Assessment of Malnutrition in Pediatrics (STAMP), EHR-STAMP, modified for incorporation into the EHR and inclusion of updated pediatric malnutrition indicators. METHODS: An interprofessional team modified the existing STAMP for integration into the EHR. Audits were performed by the research dietitian to assess accuracy and provide feedback for continuous improvement of the tool design. RESULTS: A total of 3553 pediatric inpatients were studied from August 2017 to May 2019. Accuracy, sensitivity, and specificity improved with each modification to the EHR-STAMP. The final version of the EHR-STAMP found 85% accuracy, 89% sensitivity, and 97% specificity, with a positive predictive value of 60% and a negative predictive value of 94%. CONCLUSION: The EHR-STAMP is a highly reliable tool in the screening of nutrition risk for pediatric hospitalized patients. The tool is easy to use, EHR compatible, and incorporates the current indicators recommended for assessing pediatric malnutrition.

Epithelial CD47 is critical for mucosal repair in the murine intestine in vivo.

CD47 is a ubiquitously expressed transmembrane glycoprotein that regulates inflammatory responses and tissue repair. Here, we show that normal mice treated with anti-CD47 antibodies, and Cd47-null mice have impaired intestinal mucosal wound healing. Furthermore, intestinal epithelial cell (IEC)-specific loss of CD47 does not induce spontaneous immune-mediated intestinal barrier disruption but results in defective mucosal repair after biopsy-induced colonic wounding or Dextran Sulfate Sodium (DSS)-induced mucosal damage. In vitro analyses using primary cultures of CD47-deficient murine colonic IEC or human colonoid-derived IEC treated with CD47-blocking antibodies demonstrate impaired epithelial cell migration in wound healing assays. Defective wound repair after CD47 loss is linked to decreased epithelial ß1 integrin and focal adhesion signaling, as well as reduced thrombospondin-1 and TGF-ß1. These results demonstrate a critical role for IEC-expressed CD47 in regulating mucosal repair and raise important considerations for possible alterations in wound healing secondary to therapeutic targeting of CD47.

Formyl peptide receptor 2 regulates monocyte recruitment to promote intestinal mucosal wound repair.

Mucosal wound repair is coordinated by dynamic crosstalk between endogenous and exogenous mediators and specific receptors on epithelial cells and infiltrating immune cells. One class of such receptor-ligand pairs involves formyl peptide receptors (FPRs) that have been shown to influence inflammatory response and repair. Here we explored the role of murine Fpr2/3, an ortholog of human FPR2/receptor for lipoxin A4 (ALX), in orchestrating intestinal mucosal repair. Compared with wild-type (WT) mice, Fpr2/3-/- mice exhibited delayed recovery from acute experimental colitis and perturbed repair after biopsy-induced colonic mucosal injury. Decreased numbers of infiltrating monocytes were observed in healing wounds from Fpr2/3-/- mice compared with WT animals. Bone marrow transplant experiments revealed that Fpr2/3-/- monocytes showed a competitive disadvantage when infiltrating colonic wounds. Moreover, Fpr2/3-/- monocytes were defective in chemotactic responses to the chemokine CC chemokine ligand (CCL)20, which is up-regulated during early phases of inflammation. Analysis of Fpr2/3-/- monocytes revealed altered expression of the CCL20 receptor CC chemokine receptor (CCR)6, suggesting that Fpr2/3 regulates CCL20-CCR6-mediated monocyte chemotaxis to sites of mucosal injury in the gut. These findings demonstrate an important contribution of Fpr2/3 in facilitating monocyte recruitment to sites of mucosal injury to influence wound repair.-Birkl, D., O'Leary, M. N., Quiros, M., Azcutia, V., Schaller, M., Reed, M., Nishio, H., Keeney, J., Neish, A. S., Lukacs, N. W., Parkos, C. A., Nusrat, A. Formyl peptide receptor 2 regulates monocyte recruitment to promote intestinal mucosal wound repair.

Quantitative Isotopomer Rates in Real-Time Metabolism of Cells Determined by NMR Methods.

Tracer-based metabolism is becoming increasingly important for studying metabolic mechanisms in cells. NMR spectroscopy offers several approaches to measure label incorporation in metabolites, including 13 C- and 1 H-detected spectra. The latter are generally more sensitive, but quantification depends on the proton-carbon 1 JCH coupling constant, which varies significantly between different metabolites. It is therefore not possible to have one experiment optimised for all metabolites, and quantification of 1 H-edited spectra such as HSQCs requires precise knowledge of coupling constants. Increasing interest in tracer-based and metabolic flux analysis requires robust analyses with reasonably small acquisition times. Herein, we compare 13 C-filtered and 13 C-edited methods for quantification and show the applicability of the methods for real-time NMR spectroscopy of cancer-cell metabolism, in which label incorporations are subject to constant flux. We find an approach using a double filter to be most suitable and sufficiently robust to reliably obtain 13 C incorporations from difference spectra. This is demonstrated for JJN3 multiple myeloma cells processing glucose over 24 h. The proposed method is equally well suited for calculating the level of label incorporation in labelled cell extracts in the context of metabolic flux analysis.

A framework for tracer-based metabolism in mammalian cells by NMR.

Metabolism changes extensively during the normal proliferation and differentiation of mammalian cells, and in cancer and inflammatory diseases. Since changes in the metabolic network reflect interactions between genetic, epigenetic and environmental changes, it is helpful to study the flow of label from isotopically labelled precursors into other metabolites rather than static metabolite levels. For this Nuclear Magnetic Resonance (NMR) spectroscopy is an attractive technique as it can quantify site-specific label incorporation. However, for applications using human cells and cell lines, the challenge is to optimize the process to maximize sensitivity and reproducibility. Here we present a new framework to analyze metabolism in mammalian cell lines and primary cells, covering the workflow from the preparation of cells to the acquisition and analysis of NMR spectra. We have applied this new approach in hematological and liver cancer cell lines and confirm the feasibility of tracer-based metabolism in primary liver cells.

Metabonomics uncovers a reversible proatherogenic lipid profile during infliximab therapy of inflammatory bowel disease.

BACKGROUND: One-third of inflammatory bowel disease (IBD) patients show no response to infliximab (IFX) induction therapy, and approximately half of patients responding become unresponsive over time. Thus, identification of potential treatment response biomarkers are of great clinical significance. This study employs spectroscopy-based metabolic profiling of serum from patients with IBD treated with IFX and healthy subjects (1) to substantiate the use of spectroscopy as a semi-invasive diagnostic tool, (2) to identify potential biomarkers of treatment response and (3) to characterise the metabolic changes during management of patients with tumour necrosis factor-α inhibitors. METHODS: Successive serum samples collected during IFX induction treatment (weeks 0, 2, 6 and 14) from 87 IBD patients and 37 controls were analysed by 1H nuclear magnetic resonance (NMR) spectroscopy. Data were analysed with principal components analysis and orthogonal projection to latent structures discriminant analysis using SIMCA-P+ v12 and MATLAB. RESULTS: Metabolic profiles were significantly different between active ulcerative colitis and controls, active Crohn's disease and controls, and quiescent Crohn's disease and controls. Metabolites holding differential power belonged primarily to lipids and phospholipids with proatherogenic characteristics and metabolites in the pyruvate metabolism, suggestive of an intense inflammation-driven energy demand. IBD patients not responding to IFX were identified as a potentially distinct group based on their metabolic profile, although no applicable response biomarkers could be singled out in the current setting. CONCLUSION: 1H NMR spectroscopy of serum samples is a powerful semi-invasive diagnostic tool in flaring IBD. With its use, we provide unique insights into the metabolic changes taking place during induction treatment with IFX. Of distinct clinical relevance is the identification of a reversible proatherogenic lipid profile in IBD patients with active disease, which partially explains the increased risk of cardiovascular disease associated with IBD.

Metabolomic Evidence for a Field Effect in Histologically Normal and Metaplastic Tissues in Patients with Esophageal Adenocarcinoma.

Patients with Barrett's esophagus (BO) are at increased risk of developing esophageal adenocarcinoma (EAC). Most Barrett's patients, however, do not develop EAC, and there is a need for markers that can identify those most at risk. This study aimed to see if a metabolic signature associated with the development of EAC existed. For this, tissue extracts from patients with EAC, BO, and normal esophagus were analyzed using 1H nuclear magnetic resonance. Where possible, adjacent histologically normal tissues were sampled in those with EAC and BO. The study included 46 patients with EAC, 7 patients with BO, and 68 controls who underwent endoscopy for dyspeptic symptoms with normal appearances. Within the cancer cohort, 9 patients had nonneoplastic Barrett's adjacent to the cancer suitable for biopsy. It was possible to distinguish between histologically normal, BO, and EAC tissue in EAC patients [area under the receiver operator curve (AUROC) 1.00, 0.86, and 0.91] and between histologically benign BO in the presence and absence of EAC (AUROC 0.79). In both these cases, sample numbers limited the power of the models. Comparison of histologically normal tissue proximal to EAC versus that from controls (AUROC 1.00) suggests a strong field effect which may develop prior to overt EAC and hence be useful for identifying patients at high risk of developing EAC. Excellent sensitivity and specificity were found for this model to distinguish histologically normal squamous esophageal mucosa in EAC patients and healthy controls, with 8 metabolites being very significantly altered. This may have potential diagnostic value if a molecular signature can detect tissue from which neoplasms subsequently arise.

Malonate as a ROS product is associated with pyruvate carboxylase activity in acute myeloid leukaemia cells.

BACKGROUND: The role of anaplerotic nutrient entry into the Krebs cycle via pyruvate carboxylase has been the subject of increased scrutiny and in particular whether this is dysregulated in cancer. Here, we use a tracer-based NMR analysis involving high-resolution (1)H-(13)C-HSQC spectra to assess site-specific label incorporation into a range of metabolite pools, including malate, aspartate and glutamate in the acute myeloid leukaemia cell line K562. We also determine how this is affected following treatment with the redeployed drug combination of the lipid-regulating drug bezafibrate and medroxyprogesterone (BaP). RESULTS: Using the tracer-based approach, we assessed the contribution of pyruvate carboxylase (PC) vs. pyruvate dehydrogenase (PDH) activity in the derivation of Krebs cycle intermediates. Our data show that PC activity is indeed high in K562 cells. We also demonstrate a branched entry to the Krebs cycle of K562 cells with one branch running counterclockwise using PC-derived oxaloacetate and the other clockwise from the PDH activity. Finally, we show that the PC activity of K562 cells exclusively fuels the ROS-induced decarboxylation of oxaloacetate to malonate in response to BaP treatment; resulting in further Krebs cycle disruption via depletion of oxaloacetate and malonate-mediated inhibition of succinate dehydrogenase (SDH) resulting in a twofold reduction of fumarate. CONCLUSIONS: This study extends the interest in the PC activity in solid cancers to include leukaemias and further demonstrates the value of tracer-based NMR approaches in generating a more accurate picture of the flow of carbons and metabolites within the increasingly inappropriately named Krebs cycle. Moreover, our studies indicate that the PC activity in cancer cells can be exploited as an Achilles heel by using treatments, such as BaP, that elevate ROS production.

Tracer-Based Metabolic NMR-Based Flux Analysis in a Leukaemia Cell Line.

High levels of reactive oxygen species (ROS) have a profound impact on acute myeloid leukaemia cells and can be used to specifically target these cells with novel therapies. We have previously shown how the combination of two redeployed drugs, the contraceptive steroid medroxyprogesterone and the lipid-regulating drug bezafibrate exert anti-leukaemic effects by producing ROS. Here we report a 13C-tracer-based NMR metabolic study to understand how these drugs work in K562 leukaemia cells. Our study shows that [1,2-13C]glucose is incorporated into ribose sugars, indicating activity in oxidative and non-oxidative pentose phosphate pathways alongside lactate production. There is little label incorporation into the tricarboxylic acid cycle from glucose, but much greater incorporation arises from the use of [3-13C]glutamine. The combined medroxyprogesterone and bezafibrate treatment decreases label incorporation from both glucose and glutamine into α-ketoglutarate and increased that for succinate, which is consistent with ROS-mediated conversion of α-ketoglutarate to succinate. Most interestingly, this combined treatment drastically reduced the production of several pyrimidine synthesis intermediates.

Tracer-Based Metabolic NMR-Based Flux Analysis in a Leukaemia Cell Line.

High levels of reactive oxygen species (ROS) have a profound impact on acute myeloid leukaemia cells and can be used to specifically target these cells with novel therapies. We have previously shown how the combination of two redeployed drugs, the contraceptive steroid medroxyprogesterone and the lipid-regulating drug bezafibrate exert anti-leukaemic effects by producing ROS. Here we report a 13 C-tracer-based NMR metabolic study to understand how these drugs work in K562 leukaemia cells. Our study shows that [1,2-13 C]glucose is incorporated into ribose sugars, indicating activity in oxidative and non-oxidative pentose phosphate pathways alongside lactate production. There is little label incorporation into the tricarboxylic acid cycle from glucose, but much greater incorporation arises from the use of [3-13 C]glutamine. The combined medroxyprogesterone and bezafibrate treatment decreases label incorporation from both glucose and glutamine into α-ketoglutarate and increased that for succinate, which is consistent with ROS-mediated conversion of α-ketoglutarate to succinate. Most interestingly, this combined treatment drastically reduced the production of several pyrimidine synthesis intermediates.

IL-27R-mediated regulation of IL-17 controls the development of respiratory syncytial virus-associated pathogenesis.

IL-27 is a heterodimeric cytokine composed of the subunits p28 and Epstein-Barr virus induced gene (EBI)-3 and is known for its effects on T-cell function and differentiation. IL-27 signals through the widely expressed IL-27 receptor (IL-27R), composed of the ligand-specific IL-27Rα chain and gp130. Engagement of the IL-27R activates STAT1 signaling, induces the expression of the type 1 helper T-cell (Th1) cytokine, interferon γ, and suppresses the differentiation of Th2 and Th17 cells. This study investigates the role of IL-27 signaling in respiratory syncytial virus (RSV) infection using IL-27Rα-deficient mice (IL-27rKO). Analysis of lungs from RSV-infected IL-27rKO mice showed exacerbation of mucus secretion compared with wild type, as well as enhanced expression of Muc5ac and Gob5 mRNA, markers of goblet cell metaplasia/hyperplasia. When compared with wild-type mice, RSV-challenged IL-27rKO mice had enhanced expression of Th17-associated cytokine IL-17a and an imbalance between Th1 and Th2 cytokine levels. Neutralization of IL-17 in RSV-infected IL-27rKO mice resulted in a significant decrease in the pulmonary mucus response and inhibition of the Th2-associated cytokines. Interestingly, IL-17 blockage led to an increase in the expression of IL-27 subunits p28 and EBI-3 in the lungs and lymph nodes of RSV-infected mice. Thus, IL-27 functions as a regulatory cytokine during RSV pathogenesis by suppressing the development of Th17 cells, but it also appears to be regulated by IL-17 induced by the virus.

Maslinic acid-enriched diet decreases intestinal tumorigenesis in Apc(Min/+) mice through transcriptomic and metabolomic reprogramming.

Chemoprevention is a pragmatic approach to reduce the risk of colorectal cancer, one of the leading causes of cancer-related death in western countries. In this regard, maslinic acid (MA), a pentacyclic triterpene extracted from wax-like coatings of olives, is known to inhibit proliferation and induce apoptosis in colon cancer cell lines without affecting normal intestinal cells. The present study evaluated the chemopreventive efficacy and associated mechanisms of maslinic acid treatment on spontaneous intestinal tumorigenesis in Apc(Min/+) mice. Twenty-two mice were randomized into 2 groups: control group and MA group, fed with a maslinic acid-supplemented diet for six weeks. MA treatment reduced total intestinal polyp formation by 45% (P<0.01). Putative molecular mechanisms associated with suppressing intestinal polyposis in Apc(Min/+) mice were investigated by comparing microarray expression profiles of MA-treated and control mice and by analyzing the serum metabolic profile using NMR techniques. The different expression phenotype induced by MA suggested that it exerts its chemopreventive action mainly by inhibiting cell-survival signaling and inflammation. These changes eventually induce G1-phase cell cycle arrest and apoptosis. Moreover, the metabolic changes induced by MA treatment were associated with a protective profile against intestinal tumorigenesis. These results show the efficacy and underlying mechanisms of MA against intestinal tumor development in the Apc(Min/+) mice model, suggesting its chemopreventive potential against colorectal cancer.

A lyophilized red grape pomace containing proanthocyanidin-rich dietary fiber induces genetic and metabolic alterations in colon mucosa of female C57BL/6J mice.

Diet plays a decisive role in promoting or preventing colon cancer. However, the specific effects of some nutrients remain unclear. The capacity of fruit and vegetables to prevent cancer has been associated with their fiber and antioxidant composition. We investigated whether consumption of a lyophilized red grape pomace containing proanthocyanidin-rich dietary fiber (grape antioxidant dietary fiber, GADF) by female C57BL/6J mice would affect the serum metabolic profile or colon mucosa gene expression using NMR techniques and DNA microarray, respectively. The mice were randomly assigned to 2 groups that for 2 wk consumed a standard rodent diet and were gavaged with 100 mg/kg body weight GADF suspended in water or an equivalent volume of plain tap water (10 mL/kg body weight). The amount of fiber supplemented was calculated to equal the current recommended daily levels of fiber consumption for humans. The inclusion of dietary GADF induced alterations in the expression of tumor suppressor genes and proto-oncogenes as well as the modulation of genes from pathways, including lipid biosynthesis, energy metabolism, cell cycle, and apoptosis. Overexpression of enzymes pertaining to the xenobiotic detoxifying system and endogenous antioxidant cell defenses was also observed. In summary, the genetic and metabolic profiles induced by GADF were consistent with the preventive effects of fiber and polyphenols. On the basis of these observations, we propose that GADF may contribute to reducing the risk of colon cancer.

Neuronal nicotinic alpha7 receptors modulate early neutrophil infiltration to sites of skin inflammation.

BACKGROUND: A major site of initiation of inflammatory responses upon physical perturbation(s) and infection by invading organisms is the skin. Control of responses in this organ is, in part, modulated by the neuronal nicotinic acetylcholine receptor (nAChR) alpha7. METHODS: To further investigate the role of alpha7 in skin inflammatory responses, a local inflammatory response was induced by topical application of croton oil to the ear skin of wild-type (alpha7WT) and alpha7 knock-out (alpha7KO) mice. Cells infiltrating the inflamed tissue were characterized by flow cytometry and RNA analysis. RESULTS: Six hours following croton oil application, analysis of infiltrating cells showed that the alpha7KO mice exhibited a significantly enhanced number of cells, and specifically, of Ly6G positive neutrophils. Macrophage and lymphocyte infiltration was equivalent in the alpha7KO and alpha7WT mice. RNA analysis showed that IL-1beta and IL-6 were increased significantly in the infiltrating cells of the alpha7KO mouse, although TNF failed to reach significance. In contrast, resident cells of the skin exhibited no differences in these cytokines between genotypes. Both resident and infiltrating cell populations from alpha7KO mice did show elevated message levels for the adhesion protein ICAM1. Measurement of chemokines revealed enhanced expression of the skin-related CCL27 by resident cells in alpha7KO mice. Further, we demonstrate that the population of Ly6G+ neutrophils at the croton oil-inflamed skin site expresses low levels of CCR10, a receptor for CCL27 normally associated with lymphocytes. CONCLUSION: nAChRalpha7 in the skin can impact on early local inflammatory responses mediated through a novel population of neutrophils that are Ly6G+CCR10lo.