Publications by authors named "Louise E Glover"

33 Publications

Creatine Supplementation for Patients with Inflammatory Bowl Diseases: A Scientific Rationale for a Clinical Trial.

Nutrients 2021 Apr 23;13(5). Epub 2021 Apr 23.

Comparative Immunology Group, School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, D2 Dublin, Ireland.

Based on theoretical considerations, experimental data with cells in vitro, animal studies in vivo, as well as a single case pilot study with one colitis patient, a consolidated hypothesis can be put forward, stating that "oral supplementation with creatine monohydrate (Cr), a pleiotropic cellular energy precursor, is likely to be effective in inducing a favorable response and/or remission in patients with inflammatory bowel diseases (IBD), like ulcerative colitis and/or Crohn's disease". A current pilot clinical trial that incorporates the use of oral Cr at a dose of 2 × 7 g per day, over an initial period of 2 months in conjunction with ongoing therapies (NCT02463305) will be informative for the proposed larger, more long-term Cr supplementation study of 2 × 3-5 g of Cr per day for a time of 3-6 months. This strategy should be insightful to the potential for Cr in reducing or alleviating the symptoms of IBD. Supplementation with chemically pure Cr, a natural nutritional supplement, is well tolerated not only by healthy subjects, but also by patients with diverse neuromuscular diseases. If the outcome of such a clinical pilot study with Cr as monotherapy or in conjunction with metformin were positive, oral Cr supplementation could then be used in the future as potentially useful adjuvant therapeutic intervention for patients with IBD, preferably together with standard medication used for treating patients with chronic ulcerative colitis and/or Crohn's disease.
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http://dx.doi.org/10.3390/nu13051429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8145094PMC
April 2021

Mid-luteal uterine artery Doppler indices in the prediction of pregnancy outcome in nulliparous women undergoing assisted reproduction.

Hum Fertil (Camb) 2021 Jan 13:1-11. Epub 2021 Jan 13.

Department of Reproductive Medicine, Merrion Fertility Clinic, Dublin, D2, Ireland.

Traditionally, the assessment of endometrial receptivity at transvaginal ultrasound scan has been based on the thickness and the morphological appearance of the endometrium. The objective of this study was to prospectively evaluate endometrial thickness (ET), endometrial morphology and uterine artery Doppler parameters prior to assisted reproduction treatment (ART) in the prediction of pregnancy outcome. This was a prospective cohort study. ET, morphology and uterine artery Doppler (UtAD) pulsatility index (PI) and resistance index (RI) were measured in the mid-luteal stage of the menstrual cycle ultrasonographically, timed with urinary luteinizing hormone testing. A total of 50 women were included in the analysis. The clinical pregnancy rate (CPR) per embryo transfer was 42.0% ( = 21/50). Twenty nine women (58.0%) had an unsuccessful outcome. There were no differences in mean ± SD endometrial thickness (ET) (10.0 ± 1.8 mm vs. 10.5 ± 2.4;  = 0.43), or endometrial morphology (100% ( = 21) vs 100% ( = 29);  = 1.00) between the pregnant and not pregnant groups. Similarly, there were no differences in mean ± SD UtAD PI (2.17 ± 0.83 vs. 2.07 ± 0.81;  = 0.67 or mean ± SD UtAD RI (0.84 ± 0.10 vs. 0.81 ± 0.10;  = 0.30). Ultrasonographic endometrial assessment did not differentiate between those who would have a subsequent clinical pregnancy.
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http://dx.doi.org/10.1080/14647273.2021.1872111DOI Listing
January 2021

The impact of accurately timed mid-luteal endometrial injury in nulligravid women undergoing their first or second embryo transfer.

Ir J Med Sci 2020 Oct 22. Epub 2020 Oct 22.

Merrion Fertility Clinic, 60 Mount Street, Dublin 2, Ireland.

Introduction: Endometrial injury or 'scratch' preceding an assisted reproductive therapy (ART) cycle has recently been shown not to improve livebirth rates among women undergoing ART. The objective of this study was to compare pregnancy outcomes in nulliparous women who underwent an accurately timed mid-luteal scratch biopsy prior to ART with those who did not.

Methods: This was a prospective cohort study. Women were recruited between October 2016 and February 2018 inclusive. Women who met the inclusion criteria and who did not undergo an endometrial scratch in the study period were used as a comparison group. Patients underwent a cycle of ART in the menstrual cycle following endometrial scratch.

Results: Ninety-eight women were eligible for participation in the study. There were no differences in rates of implantation (35.7% (n = 20/56) vs. 35.4% (n = 17/48); p = 1.00), clinical pregnancy (40.0% (n = 20/50) vs. 39.5% (n = 17/43); p = 1.00) or live birth (34.0% (n = 17/50) vs. 25.6% (n = 11/43); p = 0.50) per embryo transfer between those who underwent a scratch and those who did not.

Conclusion: Endometrial scratch is a simple, inexpensive and low-risk procedure. However, in this relatively small cohort study, no differences in rates of implantation, clinical pregnancy or live birth in women with primary infertility were determined between those who underwent a scratch and those who did not.
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http://dx.doi.org/10.1007/s11845-020-02414-0DOI Listing
October 2020

Epithelial HIF-1α/claudin-1 axis regulates barrier dysfunction in eosinophilic esophagitis.

J Clin Invest 2019 07 2;129(8):3224-3235. Epub 2019 Jul 2.

Gastrointestinal Eosinophilic Diseases Program, Department of Pediatrics, University of Colorado School of Medicine; Digestive Health Institute, Children's Hospital Colorado; Aurora, Colorado, USA.

Epithelial barrier dysfunction is a significant factor in many allergic diseases, including eosinophilic esophagitis (EoE). Infiltrating leukocytes and tissue adaptations increase metabolic demands and decrease oxygen availability at barrier surfaces. Understanding of how these processes impact barrier is limited, particularly in allergy. Here, we identified a regulatory axis whereby the oxygen-sensing transcription factor HIF-1α orchestrated epithelial barrier integrity, selectively controlling tight junction CLDN1 (claudin-1). Prolonged experimental hypoxia or HIF1A knockdown suppressed HIF-1α-dependent claudin-1 expression and epithelial barrier function, as documented in 3D organotypic epithelial cultures. L2-IL5OXA mice with EoE-relevant allergic inflammation displayed localized eosinophil oxygen metabolism, tissue hypoxia, and impaired claudin-1 barrier via repression of HIF-1α/claudin-1 signaling, which was restored by transgenic expression of esophageal epithelial-targeted stabilized HIF-1α. EoE patient biopsy analysis identified a repressed HIF-1α/claudin-1 axis, which was restored via pharmacologic HIF-1α stabilization ex vivo. Collectively, these studies reveal HIF-1α's critical role in maintaining barrier and highlight the HIF-1α/claudin-1 axis as a potential therapeutic target for EoE.
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http://dx.doi.org/10.1172/JCI126744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6668670PMC
July 2019

Hypoxanthine is a checkpoint stress metabolite in colonic epithelial energy modulation and barrier function.

J Biol Chem 2018 04 27;293(16):6039-6051. Epub 2018 Feb 27.

From the Department of Medicine and the Mucosal Inflammation Program, University of Colorado School of Medicine, Aurora, Colorado 80045 and

Intestinal epithelial cells form a selectively permeable barrier to protect colon tissues from luminal microbiota and antigens and to mediate nutrient, fluid, and waste flux in the intestinal tract. Dysregulation of the epithelial cell barrier coincides with profound shifts in metabolic energy, especially in the colon, which exists in an energetically depleting state of physiological hypoxia. However, studies that systematically examine energy flux and adenylate metabolism during intestinal epithelial barrier development and restoration after disruption are lacking. Here, to delineate barrier-related energy flux, we developed an HPLC-based profiling method to track changes in energy flux and adenylate metabolites during barrier development and restoration. Cultured epithelia exhibited pooling of phosphocreatine and maintained ATP during barrier development. EDTA-induced epithelial barrier disruption revealed that hypoxanthine levels correlated with barrier resistance. Further studies uncovered that hypoxanthine supplementation improves barrier function and wound healing and that hypoxanthine appears to do so by increasing intracellular ATP, which improved cytoskeletal G- to F-actin polymerization. Hypoxanthine supplementation increased the adenylate energy charge in the murine colon, indicating potential to regulate adenylate energy charge-mediated metabolism in intestinal epithelial cells. Moreover, experiments in a murine colitis model disclosed that hypoxanthine loss during active inflammation correlates with markers of disease severity. In summary, our results indicate that hypoxanthine modulates energy metabolism in intestinal epithelial cells and is critical for intestinal barrier function.
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http://dx.doi.org/10.1074/jbc.RA117.000269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5912467PMC
April 2018

Uterine natural killer cell progenitor populations predict successful implantation in women with endometriosis-associated infertility.

Am J Reprod Immunol 2018 03 30;79(3). Epub 2018 Jan 30.

School of Biochemistry & Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland.

Problem: Uterine natural killer (uNK) cells play a critical role early in gestation. As we previously identified altered uNK cell development in endometriosis-associated infertility, we herein sought to characterize natural killer (NK) cell profiles in endometriosis that may predict embryo implantation.

Method Of Study: Study participants had a surgical diagnosis of endometriosis-associated infertility. Endometrial tissue and peripheral blood were obtained from 58 women. Thirty-three patients underwent artificial reproductive technology (IVF, ICSI, or IUI) within a mean of 9.5 months of surgery. NK and hematopoietic progenitor cells from endometrium and blood were analyzed by flow cytometry. Successful implantation was defined as a positive pregnancy test.

Results: In successful implantation, populations of endometrial CD34 hematopoietic stem cells were higher (3.97% vs 0.69%; P < .0004), and coexpression of NK cell marker CD56 was increased (81.1% vs 60.9%; P < .034) compared with patients who had failed implantation. In contrast, levels of blood NK progenitors were similar in both groups.

Conclusion: Our study revealed that uterine NK progenitor cell populations are markedly different in patients with endometriosis who proceed to successful or failed embryo implantation and may define a novel predictor of implantation success. Our findings also highlight the fundamental differences inherent in NK cell repertoires between blood and uterine compartments.
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http://dx.doi.org/10.1111/aji.12817DOI Listing
March 2018

Epithelial Barrier Regulation by Hypoxia-Inducible Factor.

Ann Am Thorac Soc 2017 Sep;14(Supplement_3):S233-S236

1 Department of Medicine and.

Mucosal tissues represent surfaces that are exposed to the outside world and provide a conduit for internal and external communication. Tissues such as the intestine and the lung are lined by layer(s) of epithelial cells that, when organized in three dimensions, provide a critical barrier to the flux of luminal contents. This selective barrier is provided through the regulated expression of junctional proteins and mucins. Tissue oxygen metabolism is central to the maintenance of homeostasis in the mucosa. In some organs (e.g., the colon), low baseline Po determines tissue metabolism and results in basal expression of the transcription factor, hypoxia-inducible factor (HIF), which is enhanced after ischemia/inflammation. Recent studies have indicated that HIF contributes fundamentally to the expression of barrier-related genes and in the regulation of barrier-adaptive responses within the mucosa. Here, we briefly review recent literature on the topic of hypoxia and HIF regulation of barrier in mucosal health and during disease.
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http://dx.doi.org/10.1513/AnnalsATS.201608-610MGDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5711342PMC
September 2017

What women want? A scoping survey on women's knowledge, attitudes and behaviours towards ovarian reserve testing and egg freezing.

Eur J Obstet Gynecol Reprod Biol 2017 Oct 19;217:71-76. Epub 2017 Aug 19.

Merrion Fertility Clinic (MFC), Dublin 2, Ireland; Department of Gynaecology, National Maternity Hospital, Dublin 2, Ireland; School of Medicine, University College Dublin, Dublin 4, Ireland.

Objective: We aimed to investigate women's knowledge, attitudes and behaviours towards ovarian reserve testing and egg freezing for non-medical reasons in the general population.

Study Design: This was a cross-sectional survey study of 663 women aged 18-44 years which assessed female perception of ovarian reserve testing and oocyte cryopreservation. An online forum was used to deliver the survey through the use of two social media sites. Participants were recruited through the technique of "snowballing", whereby existing study subjects recruited others from among their acquaintances. The data collected was analyzed using SPSS to explore descriptive statistics and frequencies relating to the participants' knowledge, attitudes and behaviour towards the practices of ovarian reserve testing and oocyte cryopreservation. Categorical variables were analyzed using Chi-squared; a p-value of <0.05 was considered significant.

Results: A majority (60%) of women surveyed had knowledge of ovarian reserve testing. 64.8% would be interested in having testing performed. Younger women (<30 years of age) were more interested in checking their ovarian reserve (75.8% vs. 59.1%, p<0.0001). Single women were also more likely to be interested, (73.6% v's 62.1%, p=0.022). 89.7% of women surveyed were aware of oocyte cryopreservation. 72.2% agreed that they would consider freezing their eggs to preserve fertility. There was no significant difference in the numbers of single women compared to women in a relationship who would consider egg freezing to preserve fertility (75.7% v's 71.2%, p=0.347, or in younger (<30years) compared to older women, (74.7% v's 71.1%, p=0.387). A majority (62.1%) of study participants believed that it is a woman's right to postpone pregnancy for social reasons and to freeze her eggs, with no significant difference in options noted between younger and older women.

Conclusions: Knowledge of ovarian reserve testing and oocyte cryopreservation for non-medical reasons were higher than in previous studies, possibly reflecting increasing awareness of these issues among the general public. Additionally, we demonstrated that the women, in our study, were very open to the use of these modern technologies in an attempt to avoid unintended childlessness.
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http://dx.doi.org/10.1016/j.ejogrb.2017.08.024DOI Listing
October 2017

Inflammatory Bowel Disease: Influence and Implications in Reproduction.

Inflamm Bowel Dis 2016 11;22(11):2724-2732

*Mucosal Inflammation Program, University of Colorado, Anschutz Medical Campus, Aurora, Colorado; †Department of Medicine, University of Colorado, Anschutz Medical Campus, Aurora, Colorado; and ‡Merrion Fertility Clinic (MFC), National Maternity Hospital and Department of Obstetrics and Gynaecology, University College Dublin, Dublin, Ireland.

The incidence and prevalence of inflammatory bowel disease (IBD) continues to rise with time, signifying its emergence as a global disease. Clinical onset of IBD, comprising Crohn's disease and ulcerative colitis, typically occurs before or at peak reproductive age. Although active disease in female patients is associated with reduced fertility and adverse obstetric outcomes in pregnancy, the molecular mechanisms underlying this altered reproductive course, and its impact on IBD transmission to offspring, remain poorly understood. Clinical and experimental studies have now begun to elucidate the hormonal, environmental, and microbial factors that modulate immune-reproductive cross talk in IBD and define their impact on maternal health, fetal development, and heritability of disease risk. Evolving insight into maternal-fetal imprinting in IBD has important implications for patient counseling and disease management during pregnancy and may help predict clinical outcomes for both mother and child.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5069142PMC
http://dx.doi.org/10.1097/MIB.0000000000000884DOI Listing
November 2016

Perturbation of neddylation-dependent NF-κB responses in the intestinal epithelium drives apoptosis and inhibits resolution of mucosal inflammation.

Mol Biol Cell 2016 Sep 28. Epub 2016 Sep 28.

Mucosal Inflammation Program, Department of Medicine and Immunology, University of Colorado School of Medicine, Aurora, CO 80045 Eastern Colorado Veterans Affairs Hospital, Denver, CO

Recent work has revealed a central role for neddylation (the conjugation of a Nedd8-moiety to Cullin proteins) in the fine tuning of the NF-κB response (via Cullin-1). In the present study, we investigated the contribution of Cullin-1 neddylation and NF-κB signaling to mucosal inflammatory responses in vitro and in vivo. Initial in vitro studies using cultured intestinal epithelial cells revealed that the neddylation inhibitor MLN4924 prominently induces the deneddylation of Cullin-1. Parallel western blot, luciferase reporter and gene target assays identified MLN4924 as a potent inhibitor of intestinal epithelial NF-κB. Subsequent studies revealed that MLN4924 potently induces epithelial apoptosis but only in the presence of additional inflammatory stimuli. In vivo administration of MLN4924 (3 mg/kg/d) in a TNBS-induce colitis model significantly accentuated disease severity. Indeed, MLN4924 resulted in worsened clinical scores and increased mortality early in the inflammatory response. Histologic analysis of the colon revealed that neddylation inhibition results in increased tissue damage and significantly increased mucosal apoptosis as determined by TUNEL and cleaved caspase-3 staining, particularly prominent within the epithelium. Extensions of these studies revealed that ongoing inflammation is associated with significant loss of deneddylase-1 (SENP8) expresssion. These studies reveal that intact Cullin-1 neddylation is central to resolution of acute inflammation.
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http://dx.doi.org/10.1091/mbc.E16-05-0273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5170552PMC
September 2016

Creatine kinase in ischemic and inflammatory disorders.

Clin Transl Med 2016 Dec 15;5(1):31. Epub 2016 Aug 15.

Mucosal Inflammation Program, University of Colorado, Anschutz Medical Campus, 12700 East 19th Ave. MS B-146, Aurora, CO, 80045, USA.

The creatine/phosphocreatine pathway plays a conserved and central role in energy metabolism. Compartmentalization of specific creatine kinase enzymes permits buffering of local high energy phosphates in a thermodynamically favorable manner, enabling both rapid energy storage and energy transfer within the cell. Augmentation of this metabolic pathway by nutritional creatine supplementation has been shown to elicit beneficial effects in a number of diverse pathologies, particularly those that incur tissue ischemia, hypoxia or oxidative stress. In these settings, creatine and phosphocreatine prevent depletion of intracellular ATP and internal acidification, enhance post-ischemic recovery of protein synthesis and promote free radical scavenging and stabilization of cellular membranes. The creatine kinase energy system is itself further regulated by hypoxic signaling, highlighting the existence of endogenous mechanisms in mammals that can enhance creatine metabolism during oxygen deprivation to promote tissue resolution and homeostasis. Here, we review recent insights into the creatine kinase pathway, and provide rationale for dietary creatine supplementation in human ischemic and inflammatory pathologies.
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http://dx.doi.org/10.1186/s40169-016-0114-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987751PMC
December 2016

Oxygen metabolism and barrier regulation in the intestinal mucosa.

J Clin Invest 2016 10 8;126(10):3680-3688. Epub 2016 Aug 8.

Mucosal surfaces are lined by epithelial cells and provide an important barrier to the flux of antigens from the outside. This barrier is provided at a number of levels, including epithelial junctional complexes, mucus production, and mucosa-derived antimicrobials. Tissue metabolism is central to the maintenance of homeostasis in the mucosa. In the intestine, for example, baseline pO2 levels are uniquely low due to counter-current blood flow and the presence of large numbers of bacteria. As such, hypoxia and HIF signaling predominates normal intestinal metabolism and barrier regulation during both homeostasis and active inflammation. Contributing factors that elicit important adaptive responses within the mucosa include the transcriptional regulation of tight junction proteins, metabolic regulation of barrier components, and changes in autophagic flux. Here, we review recent literature around the topic of hypoxia and barrier function in health and during disease.
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http://dx.doi.org/10.1172/JCI84429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5096807PMC
October 2016

HIF-dependent regulation of claudin-1 is central to intestinal epithelial tight junction integrity.

Mol Biol Cell 2015 Jun 22;26(12):2252-62. Epub 2015 Apr 22.

Mucosal Inflammation Program, University of Colorado Anschutz Medical Campus, Aurora, CO 80045 Department of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045

Intestinal epithelial cells (IECs) are exposed to profound fluctuations in oxygen tension and have evolved adaptive transcriptional responses to a low-oxygen environment. These adaptations are mediated primarily through the hypoxia-inducible factor (HIF) complex. Given the central role of the IEC in barrier function, we sought to determine whether HIF influenced epithelial tight junction (TJ) structure and function. Initial studies revealed that short hairpin RNA-mediated depletion of the HIF1β in T84 cells resulted in profound defects in barrier and nonuniform, undulating TJ morphology. Global HIF1α chromatin immunoprecipitation (ChIP) analysis identified claudin-1 (CLDN1) as a prominent HIF target gene. Analysis of HIF1β-deficient IEC revealed significantly reduced levels of CLDN1. Overexpression of CLDN1 in HIF1β-deficient cells resulted in resolution of morphological abnormalities and restoration of barrier function. ChIP and site-directed mutagenesis revealed prominent hypoxia response elements in the CLDN1 promoter region. Subsequent in vivo analysis revealed the importance of HIF-mediated CLDN1 expression during experimental colitis. These results identify a critical link between HIF and specific tight junction function, providing important insight into mechanisms of HIF-regulated epithelial homeostasis.
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http://dx.doi.org/10.1091/mbc.E14-07-1194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4462943PMC
June 2015

Crosstalk between Microbiota-Derived Short-Chain Fatty Acids and Intestinal Epithelial HIF Augments Tissue Barrier Function.

Cell Host Microbe 2015 May 9;17(5):662-71. Epub 2015 Apr 9.

Mucosal Inflammation Program, University of Colorado, Aurora, CO 80045, USA; Department of Medicine, University of Colorado, Aurora, CO 80045, USA. Electronic address:

Interactions between the microbiota and distal gut are fundamental determinants of human health. Such interactions are concentrated at the colonic mucosa and provide energy for the host epithelium through the production of the short-chain fatty acid butyrate. We sought to determine the role of epithelial butyrate metabolism in establishing the austere oxygenation profile of the distal gut. Bacteria-derived butyrate affects epithelial O2 consumption and results in stabilization of hypoxia-inducible factor (HIF), a transcription factor coordinating barrier protection. Antibiotic-mediated depletion of the microbiota reduces colonic butyrate and HIF expression, both of which are restored by butyrate supplementation. Additionally, germ-free mice exhibit diminished retention of O2-sensitive dyes and decreased stabilized HIF. Furthermore, the influences of butyrate are lost in cells lacking HIF, thus linking butyrate metabolism to stabilized HIF and barrier function. This work highlights a mechanism where host-microbe interactions augment barrier function in the distal gut.
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http://dx.doi.org/10.1016/j.chom.2015.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4433427PMC
May 2015

Metabolic regulation of intestinal epithelial barrier during inflammation.

Tissue Barriers 2015 3;3(1-2):e970936. Epub 2015 Apr 3.

Departments of Medicine and the Mucosal Inflammation Program; University of Colorado School of Medicine ; Aurora, CO USA.

The gastrointestinal mucosa has proven to be an interesting tissue for which to investigate disease-related metabolism. In this review, we outline some evidence that implicates metabolic signaling as important features of barrier in the healthy and disease. Studies from cultured cell systems, animal models and human patients have revealed that metabolites generated within the inflammatory microenvironment are central to barrier regulation. These studies have revealed a prominent role for hypoxia and hypoxia-inducible factor (HIF) at key steps in adenine nucleotide metabolism and within the creatine kinase pathway. Results from animal models of intestinal inflammation have demonstrated an almost uniformly beneficial influence of HIF stabilization on disease outcomes and barrier function. Studies underway to elucidate the contribution of immune responses will provide additional insight into how metabolic changes contribute to the complexity of the gastrointestinal tract and how such information might be harnessed for therapeutic benefit.
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http://dx.doi.org/10.4161/21688362.2014.970936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4372015PMC
April 2015

Stabilization of HIF through inhibition of Cullin-2 neddylation is protective in mucosal inflammatory responses.

FASEB J 2015 Jan 17;29(1):208-15. Epub 2014 Oct 17.

Mucosal Inflammation Program, Department of Medicine, and

There is interest in understanding post-translational modifications of proteins in inflammatory disease. Neddylation is the conjugation of the molecule neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) to promote protein stabilization. Cullins are a family of NEDD8 targets important in the stabilization and degradation of proteins, such as hypoxia-inducible factor (HIF; via Cullin-2). Here, we elucidate the role of human deneddylase-1 (DEN-1, also called SENP8) in inflammatory responses in vitro and in vivo and define conditions for targeting neddylation in models of mucosal inflammation. HIF provides protection in inflammatory models, so we examined the contribution of DEN-1 to HIF stabilization. Pharmacologic targeting of neddylation activity with MLN4924 (IC50, 4.7 nM) stabilized HIF-1α, activated HIF promoter activity by 2.5-fold, and induced HIF-target genes in human epithelial cells up to 5-fold. Knockdown of DEN-1 in human intestinal epithelial cells resulted in increased kinetics in barrier formation, decreased permeability, and enhanced barrier restitution by 2 ± 0.5-fold. Parallel studies in vivo revealed that MLN4924 abrogated disease severity in murine dextran sulfate sodium colitis, including weight loss, colon length, and histologic severity. We conclude that DEN-1 is a regulator of cullin neddylation and fine-tunes the inflammatory response in vitro and in vivo. Pharmacologic inhibition of cullin neddylation may provide a therapeutic opportunity in mucosal inflammatory disease.
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http://dx.doi.org/10.1096/fj.14-259663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4285538PMC
January 2015

Transmigrating neutrophils shape the mucosal microenvironment through localized oxygen depletion to influence resolution of inflammation.

Immunity 2014 Jan 9;40(1):66-77. Epub 2014 Jan 9.

Mucosal Inflammation Program, University of Colorado, Anschutz Medical Campus, Aurora, CO 80045, USA.

Acute intestinal inflammation involves early accumulation of neutrophils (PMNs) followed by either resolution or progression to chronic inflammation. Based on recent evidence that mucosal metabolism influences disease outcomes, we hypothesized that transmigrating PMNs influence the transcriptional profile of the surrounding mucosa. Microarray studies revealed a cohort of hypoxia-responsive genes regulated by PMN-epithelial crosstalk. Transmigrating PMNs rapidly depleted microenvironmental O2 sufficiently to stabilize intestinal epithelial cell hypoxia-inducible factor (HIF). By utilizing HIF reporter mice in an acute colitis model, we investigated the relative contribution of PMNs and the respiratory burst to "inflammatory hypoxia" in vivo. CGD mice, lacking a respiratory burst, developed accentuated colitis compared to control, with exaggerated PMN infiltration and diminished inflammatory hypoxia. Finally, pharmacological HIF stabilization within the mucosa protected CGD mice from severe colitis. In conclusion, transcriptional imprinting by infiltrating neutrophils modulates the host response to inflammation, via localized O2 depletion, resulting in microenvironmental hypoxia and effective inflammatory resolution.
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http://dx.doi.org/10.1016/j.immuni.2013.11.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3951457PMC
January 2014

Identification of hypoxia-inducible factor HIF-1A as transcriptional regulator of the A2B adenosine receptor during acute lung injury.

J Immunol 2014 Feb 3;192(3):1249-56. Epub 2014 Jan 3.

Mucosal Inflammation Program, Department of Anesthesiology, University of Colorado, Aurora, CO 80045.

Although acute lung injury (ALI) contributes significantly to critical illness, resolution often occurs spontaneously through endogenous pathways. We recently found that mechanical ventilation increases levels of pulmonary adenosine, a signaling molecule known to attenuate lung inflammation. In this study, we hypothesized a contribution of transcriptionally controlled pathways to pulmonary adenosine receptor (ADOR) signaling during ALI. We gained initial insight from microarray analysis of pulmonary epithelia exposed to conditions of cyclic mechanical stretch, a mimic for ventilation-induced lung disease. Surprisingly, these studies revealed a selective induction of the ADORA2B. Using real-time RT-PCR and Western blotting, we confirmed an up to 9-fold induction of the ADORA2B following cyclic mechanical stretch (A549, Calu-3, or human primary alveolar epithelial cells). Studies using ADORA2B promoter constructs identified a prominent region within the ADORA2B promoter conveying stretch responsiveness. This region of the promoter contained a binding site for the transcription factor hypoxia-inducible factor (HIF)-1. Additional studies using site-directed mutagenesis or transcription factor binding assays demonstrated a functional role for HIF-1 in stretch-induced increases of ADORA2B expression. Moreover, studies of ventilator-induced lung injury revealed induction of the ADORA2B during ALI in vivo that was abolished following HIF inhibition or genetic deletion of Hif1a. Together, these studies implicate HIF in the transcriptional control of pulmonary adenosine signaling during ALI.
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http://dx.doi.org/10.4049/jimmunol.1100593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3946986PMC
February 2014

IFN-γ-mediated induction of an apical IL-10 receptor on polarized intestinal epithelia.

J Immunol 2014 Feb 23;192(3):1267-76. Epub 2013 Dec 23.

Department of Anesthesiology and Perioperative Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO 80045;

Cytokines secreted at sites of inflammation impact the onset, progression, and resolution of inflammation. In this article, we investigated potential proresolving mechanisms of IFN-γ in models of inflammatory bowel disease. Guided by initial microarray analysis, in vitro studies revealed that IFN-γ selectively induced the expression of IL-10R1 on intestinal epithelia. Further analysis revealed that IL-10R1 was expressed predominantly on the apical membrane of polarized epithelial cells. Receptor activation functionally induced canonical IL-10 target gene expression in epithelia, concomitant with enhanced barrier restitution. Furthermore, knockdown of IL-10R1 in intestinal epithelial cells results in impaired barrier function in vitro. Colonic tissue isolated from murine colitis revealed that levels of IL-10R1 and suppressor of cytokine signaling 3 were increased in the epithelium and coincided with increased tissue IFN-γ and IL-10 cytokines. In parallel, studies showed that treatment of mice with rIFN-γ was sufficient to drive expression of IL-10R1 in the colonic epithelium. Studies of dextran sodium sulfate colitis in intestinal epithelial-specific IL-10R1-null mice revealed a remarkable increase in disease susceptibility associated with increased intestinal permeability. Together, these results provide novel insight into the crucial and underappreciated role of epithelial IL-10 signaling in the maintenance and restitution of epithelial barrier and of the temporal regulation of these pathways by IFN-γ.
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http://dx.doi.org/10.4049/jimmunol.1301757DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4042245PMC
February 2014

Control of creatine metabolism by HIF is an endogenous mechanism of barrier regulation in colitis.

Proc Natl Acad Sci U S A 2013 Dec 18;110(49):19820-5. Epub 2013 Nov 18.

Mucosal Inflammation Program, Department of Medicine, Department of Anesthesiology and Perioperative Medicine, and Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, CO 80045.

Mucosal surfaces of the lower gastrointestinal tract are subject to frequent, pronounced fluctuations in oxygen tension, particularly during inflammation. Adaptive responses to hypoxia are orchestrated largely by the hypoxia-inducible transcription factors (HIFs). As HIF-1α and HIF-2α are coexpressed in mucosal epithelia that constitute the barrier between the lumen and the underlying immune milieu, we sought to define the discrete contribution of HIF-1 and HIF-2 transactivation pathways to intestinal epithelial cell homeostasis. The present study identifies creatine kinases (CKs), key metabolic enzymes for rapid ATP generation via the phosphocreatine-creatine kinase (PCr/CK) system, as a unique gene family that is coordinately regulated by HIF. Cytosolic CKs are expressed in a HIF-2-dependent manner in vitro and localize to apical intestinal epithelial cell adherens junctions, where they are critical for junction assembly and epithelial integrity. Supplementation with dietary creatine markedly ameliorated both disease severity and inflammatory responses in colitis models. Further, enzymes of the PCr/CK metabolic shuttle demonstrate dysregulated mucosal expression in a subset of ulcerative colitis and Crohn disease patients. These findings establish a role for HIF-regulated CK in epithelial homeostasis and reveal a fundamental link between cellular bioenergetics and mucosal barrier.
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http://dx.doi.org/10.1073/pnas.1302840110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3856803PMC
December 2013

HIF-dependent regulation of AKAP12 (gravin) in the control of human vascular endothelial function.

FASEB J 2014 Jan 12;28(1):256-64. Epub 2013 Sep 12.

1Mucosal Inflammation Program, University of Colorado, 12700 E. 19th Ave, Aurora, CO 80045, USA.

Hypoxia has been widely implicated in many pathological conditions, including those associated with inflammation and tumorigenesis. A number of recent studies have implicated hypoxia in the control of vasculogenesis and permeability, the basis for which is not fully understood. Here we examine the transcriptional regulation of angiogenesis and permeability by hypoxia in endothelial cells. Guided by a global profiling approach in cultured endothelial cells, these studies revealed the selective induction of human gravin (protein kinase A anchoring protein 12) by hypoxia. Analysis of the cloned gravin promoter identified a functional hypoxia-responsive region including 2 binding sites for hypoxia-inducible factor (HIF). Site-directed mutagenesis identified the most distal HIF-binding site as essential for the induction of gravin by hypoxia. Further studies examining gravin gain and loss of function confirmed strong dependence of gravin in control of microvascular endothelial tube formation, wherein gravin functions as a "braking" system for angiogenesis. Additional studies in confluent endothelia revealed that gravin functionally couples to control endothelial barrier function in response to protein kinase A (PKA) agonists. Taken together, these results demonstrate transcriptional coordination of gravin by HIF-1α and amplified PKA-dependent endothelial responses. These findings provide an important link between hypoxia and metabolic conditions associated with inflammation and angiogenesis.
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http://dx.doi.org/10.1096/fj.13-238741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3868838PMC
January 2014

Effects of nitric oxide and reactive oxygen species on HIF-1α stabilization following clostridium difficile toxin exposure of the Caco-2 epithelial cell line.

Cell Physiol Biochem 2013 27;32(2):417-30. Epub 2013 Aug 27.

Gastrointestinal Research Group at the Snyder Institute for Chronic Disease, Departments of Biochemistry & Molecular Biology, University of Calgary, Calgary, AB, Canada.

Background/aims: Stabilization of the hypoxia-inducible factor (HIF-1α) is proposed to provide a protective host-response to C. difficile intoxication. Here, we aimed to elucidate whether nitric oxide and/or reactive oxygen species produced during C. difficile toxin exposure could influence HIF-1α stability and initiate protection against epithelial cell damage.

Methods/results: HIF-1α and inducible nitric oxide synthase (iNOS) proteins were up-regulated whereas factor-inhibiting HIF-1 (FIH-1) protein was down-regulated in Caco-2 epithelial cell monolayers with in vitro toxin exposure. We demonstrate using the biotin-switch assay that the stabilization of HIF-1α protein occurred via iNOS-dependent nitrosylation. Inhibition of iNOS activity by selective inhibitor (1400W) attenuated HIF-1α stabilization and exacerbated toxin-dependent disruptions in Caco-2 monolayer morphology and tight junctional integrity in vitro. Treatment of Caco-2 cell monolayers with N-actylcysteine (NAC), a scavenger of reactive oxygen species (ROS), attenuated toxin-dependent increases in iNOS and HIF-1α protein levels but had no effect on FIH-1 responses. In addition, mice that were exposed to C. difficile toxin in vivo also demonstrated a significant increase in HIF-1α protein and nitrosylation levels.

Conclusion: Taken together, these data suggest that important synergistic actions exist between nitric oxide and ROS to stabilize HIF-1α and its innate, protective actions in the context of C. difficile toxin-mediated epithelial injury.
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http://dx.doi.org/10.1159/000354448DOI Listing
April 2014

Central role for endothelial human deneddylase-1/SENP8 in fine-tuning the vascular inflammatory response.

J Immunol 2013 Jan 3;190(1):392-400. Epub 2012 Dec 3.

Mucosal Inflammation Program, Department of Medicine and Immunology, University of Colorado School of Medicine, Aurora, CO 80045, USA.

A deeper understanding of the mechanisms that control responses to inflammation is critical to the development of effective therapies. We sought to define the most proximal regulators of the Cullin (Cul)-RING ligases, which play a central role in the stabilization of NF-κB and hypoxia-inducible factor (HIF). In these studies, we identify the human deneddylase-1 (SENP8) as a key regulator of Cul neddylation response in vitro and in vivo. Using human microvascular endothelial cells (HMECs), we examined inflammatory responses to LPS or TNF-α by assessing Cul neddylation status, NF-κB and HIF-1α stabilization, and inflammatory cytokine secretion. HMECs with an intact neddylation pathway showed a time-dependent induction of Cul-1 neddylation, nuclear translocation of NF-κB, stabilization of HIF-1α, and increased NF-κB/HIF-α promoter activity in response to LPS. HMECs lacking SENP8 were unable to neddylate Cul-1 and subsequently were unable to activate NF-κB or HIF-1α. Pharmacological targeting of neddylation (MLN4924) significantly abrogated NF-κB responses, induced HIF-1α promoter activity, and reduced secretion of TNF-α-elicited proinflammatory cytokines. MLN4924 stabilized HIF and abrogated proinflammatory responses while maintaining anti-inflammatory IL-10 responses in vivo following LPS administration. These studies identify SENP8 as a proximal regulator of Cul neddylation and provide an important role for SENP8 in fine-tuning the inflammatory response. Moreover, our findings provide feasibility for therapeutic targeting of the Culs during inflammation.
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http://dx.doi.org/10.4049/jimmunol.1202041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3529810PMC
January 2013

Hypoxia-inducible factor-1 alpha-dependent induction of FoxP3 drives regulatory T-cell abundance and function during inflammatory hypoxia of the mucosa.

Proc Natl Acad Sci U S A 2012 Oct 17;109(41):E2784-93. Epub 2012 Sep 17.

Department of Anesthesiology, University of Colorado School of Medicine, Aurora, CO 80045, USA.

Recent studies have demonstrated dramatic shifts in metabolic supply-and-demand ratios during inflammation, a process resulting in localized tissue hypoxia within inflammatory lesions ("inflammatory hypoxia"). As part of the adaptive immune response, T cells are recruited to sites of inflammatory hypoxia. Given the profound effects of hypoxia on gene regulation, we hypothesized that T-cell differentiation is controlled by hypoxia. To pursue this hypothesis, we analyzed the transcriptional consequences of ambient hypoxia (1% oxygen) on a broad panel of T-cell differentiation factors. Surprisingly, these studies revealed selective, robust induction of FoxP3, a key transcriptional regulator for regulatory T cells (Tregs). Studies of promoter binding or loss- and gain-of-function implicated hypoxia-inducible factor (HIF)-1α in inducing FoxP3. Similarly, hypoxia enhanced Treg abundance in vitro and in vivo. Finally, Treg-intrinsic HIF-1α was required for optimal Treg function and Hif1a-deficient Tregs failed to control T-cell-mediated colitis. These studies demonstrate that hypoxia is an intrinsic molecular cue that promotes FoxP3 expression, in turn eliciting potent anti-inflammatory mechanisms to limit tissue damage in conditions of reduced oxygen availability.
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http://dx.doi.org/10.1073/pnas.1202366109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3478644PMC
October 2012

Contributions of neutrophils to resolution of mucosal inflammation.

Immunol Res 2013 Mar;55(1-3):75-82

Department of Medicine and the Mucosal Inflammation Program, University of Colorado School of Medicine, Aurora, CO 80045, USA.

Neutrophil (PMN) recruitment from the blood stream into surrounding tissues involves a regulated series of events central to acute responses in host defense. Accumulation of PMN within mucosal tissues has historically been considered pathognomonic features of both acute and chronic inflammatory conditions. Historically, PMNs have been deemed necessary but detrimental when recruited, given the potential for tissue damage that results from a variety of mechanisms. Recent work, however, has altered our preconceived notions of PMN contributions to inflammatory processes. In particular, significant evidence implicates a central role for the PMN in triggering inflammatory resolution. Such mechanisms involve both metabolic and biochemical crosstalk pathways during the intimate interactions of PMN with other cell types at inflammatory sites. Here, we highlight several recent examples of how PMN coordinate the resolution of ongoing inflammation, with a particular focus on the gastrointestinal mucosa.
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http://dx.doi.org/10.1007/s12026-012-8350-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047712PMC
March 2013

Hypoxia and metabolic factors that influence inflammatory bowel disease pathogenesis.

Gastroenterology 2011 May;140(6):1748-55

Mucosal Inflammation Program, Department of Medicine, University of Colorado Denver, Aurora, Colorado, USA.

The gastrointestinal epithelium is anatomically positioned to provide a selective barrier between the anaerobic lumen and lamina propria, which has a high rate of metabolism. Supported by a complex vasculature, this important barrier is affected by reduced blood flow and resultant tissue hypoxia, particularly during the severe metabolic shifts associated with active inflammation in individuals with inflammatory bowel disease. Activation of hypoxia-inducible factor (HIF) under these conditions promotes resolution of inflammation in mouse models of disease. Protective influences of HIF are attributed, in part, to the complex regulation of barrier protection with the intestinal mucosa. Reagents that activate HIF, via inhibition of the prolyl hydroxylase enzymes, might be developed to induce hypoxia-mediated resolution in patients with intestinal mucosal inflammatory disease.
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http://dx.doi.org/10.1053/j.gastro.2011.01.056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3093411PMC
May 2011

An endogenously anti-inflammatory role for methylation in mucosal inflammation identified through metabolite profiling.

J Immunol 2011 Jun 22;186(11):6505-14. Epub 2011 Apr 22.

Department of Anesthesiology and Perioperative Medicine, University of Colorado Health Science Center, Aurora, CO 80045, USA.

Tissues of the mucosa are lined by an epithelium that provides barrier and transport functions. It is now appreciated that inflammatory responses in inflammatory bowel diseases are accompanied by striking shifts in tissue metabolism. In this paper, we examined global metabolic consequences of mucosal inflammation using both in vitro and in vivo models of disease. Initial analysis of the metabolic signature elicited by inflammation in epithelial models and in colonic tissue isolated from murine colitis demonstrated that levels of specific metabolites associated with cellular methylation reactions are significantly altered by model inflammatory systems. Furthermore, expression of enzymes central to all cellular methylation, S-adenosylmethionine synthetase and S-adenosylhomocysteine hydrolase, are increased in response to inflammation. Subsequent studies showed that DNA methylation is substantially increased during inflammation and that epithelial NF-κB activity is significantly inhibited following treatment with a reversible S-adenosylhomocysteine hydrolase inhibitor, DZ2002. Finally, these studies demonstrated that inhibition of cellular methylation in a murine model of colitis results in disease exacerbation while folate supplementation to promote methylation partially ameliorates the severity of murine colitis. Taken together, these results identify a global change in methylation, which during inflammation, translates to an overall protective role in mucosal epithelia.
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http://dx.doi.org/10.4049/jimmunol.1002805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3383860PMC
June 2011

IFN-γ attenuates hypoxia-inducible factor (HIF) activity in intestinal epithelial cells through transcriptional repression of HIF-1β.

J Immunol 2011 Feb 3;186(3):1790-8. Epub 2011 Jan 3.

Mucosal Inflammation Program, Department of Medicine, University of Colorado Denver, Aurora, CO 80045, USA.

Numerous studies have revealed that hypoxia and inflammation occur coincidentally in mucosal disorders, such as inflammatory bowel disease. During inflammation, epithelial-expressed hypoxia-inducible factor (HIF) serves an endogenously protective function. In this study, we sought to explore how mucosal immune responses influence HIF-dependent end points. Guided by a screen of relevant inflammatory mediators, we identified IFN-γ as a potent repressor of HIF-dependent transcription in human intestinal epithelial cells. Analysis of HIF levels revealed that HIF-1β, but not HIF-1α, is selectively repressed by IFN-γ in a JAK-dependent manner. Cloning and functional analysis of the HIF-1β promoter identified a prominent region for IFN-γ-dependent repression. Further studies revealed that colonic IFN-γ and HIF-1β levels were inversely correlated in a murine colitis model. Taken together, these studies demonstrated that intestinal epithelial HIF is attenuated by IFN-γ through transcriptional repression of HIF-1β. These observations are relevant to the pathophysiology of colitis (i.e., that loss of HIF signaling during active inflammation may exacerbate disease pathogenesis).
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http://dx.doi.org/10.4049/jimmunol.1001442DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4040106PMC
February 2011

Resolvin E1-induced intestinal alkaline phosphatase promotes resolution of inflammation through LPS detoxification.

Proc Natl Acad Sci U S A 2010 Aug 26;107(32):14298-303. Epub 2010 Jul 26.

Mucosal Inflammation Program, Department of Medicine, University of Colorado, Aurora, CO 80045, USA.

Resolvin-E1 (RvE1) has been demonstrated to promote inflammatory resolution in numerous disease models. Given the importance of epithelial cells to coordination of mucosal inflammation, we hypothesized that RvE1 elicits an epithelial resolution signature. Initial studies revealed that the RvE1-receptor (ChemR23) is expressed on intestinal epithelial cells (IECs) and that microarray profiling of cells exposed to RvE1 revealed regulation of inflammatory response gene expression. Notably, RvE1 induced intestinal alkaline phosphatase (ALPI) expression and significantly enhanced epithelial ALPI enzyme activity. One role recently attributed to ALPI is the detoxification of bacterial LPS. In our studies, RvE1-exposed epithelia detoxified LPS (assessed by attenuation of NF-kappaB signaling). Furthermore, in epithelial-bacterial interaction assays, we determined that ALPI retarded the growth of Escherichia coli. To define these features in vivo, we used a murine dextran sulfate sodium (DSS) model of colitis. Compared with vehicle controls, administration of RvE1 resulted in significant improvement of disease activity indices (e.g., body weight, colon length) concomitant with increased ALPI expression in the intestinal epithelium. Moreover, inhibition of ALPI activity resulted in increased severity of colitis in DSS-treated animals and partially abrogated the protective influence of RvE1. Together, these data implicate a previously unappreciated role for ALPI in RvE1-mediated inflammatory resolution.
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http://dx.doi.org/10.1073/pnas.0914730107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2922533PMC
August 2010

Dysferlin overexpression in skeletal muscle produces a progressive myopathy.

Ann Neurol 2010 Mar;67(3):384-93

Day Neuromuscular Research Laboratory, Massachusetts General Hospital, Charlestown, MA.

Objective: The dose-response effects of dysferlin transgenesis were analyzed to determine if the dysferlin-deficient myopathies are good candidates for gene replacement therapy.

Methods: We have generated 3 lines of transgenic mice, expressing low, mid, and high levels of full-length human dysferlin from a muscle-specific promoter. Transgenic skeletal muscle was analyzed and scored for morphological and functional deficits.

Results: Overexpression of dysferlin in mice resulted in a striking phenotype of kyphosis, irregular gait, and reduced muscle mass and strength. Moreover, protein dosage correlated with phenotype severity. In contrast to dysferlin-null skeletal muscle, no evidence of sarcolemmal impairment was revealed. Rather, increased levels of Ca(2+)-regulated, dysferlin-binding proteins and endoplasmic reticulum stress chaperone proteins were observed in muscle lysates from transgenic mice as compared with controls.

Interpretation: Expression levels of dysferlin are important for appropriate function without deleterious or cytotoxic effects. As a corollary, we propose that future endeavors in gene replacement for correction of dysferlinopathy should be tailored to take account of this.
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http://dx.doi.org/10.1002/ana.21926DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3900233PMC
March 2010