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To achieve a better understanding of the “vinegar syndrome” phenomenon, which has caused serious damage to triacetyl cellulose-based motion picture films, the white powder obtained from damaged film surfaces was analysed in this study. The powder was found to be soluble in acetone, diethyl ether, dimethylformamide, and chloroform, but insoluble in water. From the results of 1H, 13C and 31P nuclear magnetic resonance spectroscopy, mass spectrometry, and X-ray fluorescence measurements, it was concluded that the white precipitate had a molecular weight of 326 amu and was composed of triphenyl phosphate (C18H15O4P).

Diabetes Mellitus (DM) affects 34.2 million Americans. DM impairs the body’s reparative machinery leading to early onset chronic illness (peripheral arterial disease (PAD), neuropathy, nephropathy, and retinopathy, the latter being hallmarks of microvascular disease). Patients with DM+PAD have increased risk of major amputation. Mesenchymal stem cells (MSCs) are reparative cells found in all tissues providing paracrine and trophic support for new tissue. This study’s objective was to identify intracellular and epigenetic mechanisms of how DM impairs MSC function and test if these defects are modifiable with culture rejuvenation (CR). MSCs obtained from bone marrow of 13 consecutive, male Veterans undergoing lower limb major amputation were cultured in 10% fetal bovine serum or 5% human platelet lysate (PL) for CR. Groups were DM+PAD (n=8) and PAD no DM (n=5). Intracellular signaling was analyzed with multiplexed ELISA. Epigenetic differences were identified by ATAC-sequencing. DM+PAD MSCs had modifiable AKT signaling defects with PL (Fig 1) and a discrete DNA profile identified in their introns and intergenic regions (Fig 2). MSCs from PAD alone had increased transcription factor binding at Wnt and cGMP-PKG pathways (p=0.04). DM+PAD MSCs had increased binding at MAPK (p=0.01) and Rap1 (p=0.01) pathways. DM is a complex disease disrupting reparative mechanisms and can lead to major complications. MSC dysfunction in DM may have common mechanisms throughout the body. We have identified potentially druggable pathways that may provide therapeutic solutions to relieve chronic illness for endogenous MSCs and to expand MSC donor pool for regenerative medicine strategies.

Molecular events activating the PI3K pathway are frequently detected in human tumors and the activation of PI3K signaling alters numerous cellular processes including tumor cell proliferation, survival, and motility. More recent studies have highlighted the impact of PI3K signaling on the cellular response to interferons and other immunologic processes relevant to antitumor immunity. Given the ability of IFNγ to regulate antigen processing and presentation and the pivotal role of MHC class I (MHCI) and II (MHCII) expression in T-cell–mediated antitumor immunity, we sought to determine the impact of PI3K signaling on MHCI and MHCII induction by IFNγ. We found that the induction of cell surface MHCI and MHCII molecules by IFNγ is enhanced by the clinical grade PI3K inhibitors dactolisib and pictilisib. We also found that PI3K inhibition increases STAT1 protein levels following IFNγ treatment and increases accessibility at genomic STAT1-binding motifs. Conversely, we found that pharmacologic activation of PI3K signaling can repress the induction of MHCI and MHCII molecules by IFNγ, and likewise, the loss of PTEN attenuates the induction of MHCI, MHCII, and STAT1 by IFNγ. Consistent with these in vitro studies, we found that within human head and neck squamous cell carcinomas, intratumoral regions with high phospho-AKT IHC staining had reduced MHCI IHC staining. Implications: Collectively, these findings demonstrate that MHC expression can be modulated by PI3K signaling and suggest that activation of PI3K signaling may promote immune escape via effects on antigen presentation.

Propranolol is a widely used beta blocker that consists of a racemic mixture of R and S stereoisomers. Only the S stereoisomer has significant activity against the beta-adrenergic receptor. A fortuitous clinical observation was made in an infant who received propranolol for cardiac disease, and regression of a hemangioma of infancy was noted. This has led to the widespread use of propranolol for the treatment of large and life-threatening hemangiomas of infancy. Infants receiving propranolol require monitoring to ensure that they do not suffer from side effects related to beta blockade. The exact mechanism of activity of propranolol in hemangioma of infancy is unknown. In this study, we treated hemangioma stem cells with both beta blockade active S- and inactive R-propranolol and looked for genes that were coordinately regulated by this treatment. Among the genes commonly downregulated, Angiopoietin-like 4 (ANGPTL4) was among the most regulated. We confirmed that propranolol isomers downregulated ANGPTL4 in endothelial cells, with greater downregulation of ANGPTL4 using the beta blockade inactive R-propranolol. ANGPTL4 is present in human hemangiomas of infancy. Finally, R-propranolol inhibited the growth of bEnd.3 hemangioma cells in vivo. The implication of this is that hemangioma growth can be blocked without the side effects of beta blockade. Given that humans have been exposed to racemic propranolol for decades and thus to R-propranolol, clinical development of R-propranolol for hemangiomas of infancy and other angiogenic diseases is warranted.

Background Immune-checkpoint inhibitors (ICIs) have been increasingly used for non-small cell lung cancer (NSCLC) treatment in recent years. Although insufficient, the rate of programmed death-ligand 1 expression has been adopted as a predictor of ICI efficacy. We evaluated tumor growth rate as a clinically easy-to-use predictor of the therapeutic effect of ICIs. Methods This study is a single-institution retrospective study in Japan. NSCLC patients treated with nivolumab, pembrolizumab, or atezolizumab at Saitama Medical Center from January 1, 2016 to December 31, 2018 were enrolled, and followed until December 31, 2020. We defined and calculated the initial rapidity of tumor progression (IRP) as: the increase in the sum of the diameters of intrathoracic tumors and lymph nodes on two series of chest computed tomography (CT) scans (one obtained at an initial checkup and the other obtained immediately before the first treatment) divided by the number of days between these CT scans. Two coefficients were calculated: the maximal information coefficient (MIC) between IRP and time to treatment failure (TTF) using the Python package with minepy library, and the Spearman's rank correlation coefficient. Results A total of 55 patients (median age, 70 years; 47 men) were enrolled. The median TTF with ICIs was 126 days, and four patients continued to receive ICI treatment at the end of the follow-up. The MIC between IRP and TTF was 0.302 with weak correlation, and the Spearman's rank correlation coefficient was -0.347 (P=0.00938). Conclusions The initial tumor growth rate had a negative linear correlation with the therapeutic effect of ICIs.

Simple Summary Metastatic melanoma is one of the most aggressive and lethal malignancies with a poor prognosis. Several data underline the crucial role of amoeboid motility in the dissemination of metastatic melanoma cells. Thus, targeting this phenomenon could represent a promising strategy to prevent metastasis formation and improve metastatic melanoma patient’s survival. With this aim, we investigated the effect of Claisened Hexafluoro, a chemical analogue of Honokiol, on human metastatic melanoma cells. We demonstrated that Claisened Hexafluoro, by deregulating the mitochondrial activity, inhibits amoeboid motility and impairs many steps of the dissemination process, finally decreasing the in vivo metastatic spreading. Collectively, these data suggest possible future applications of Claisened Hexafluoro for the treatment of metastatic melanoma. Abstract Metastatic melanoma is characterized by poor prognosis and a low free-survival rate. Thanks to their high plasticity, melanoma cells are able to migrate exploiting different cell motility strategies, such as the rounded/amoeboid-type motility and the elongated/mesenchymal-type motility. In particular, the amoeboid motility strongly contributes to the dissemination of highly invasive melanoma cells and no treatment targeting this process is currently available for clinical application. Here, we tested Claisened Hexafluoro as a novel inhibitor of the amoeboid motility. Reported data demonstrate that Claisened Hexafluoro specifically inhibits melanoma cells moving through amoeboid motility by deregulating mitochondrial activity and activating the AMPK signaling. Moreover, Claisened Hexafluoro is able to interfere with the adhesion abilities and the stemness features of melanoma cells, thus decreasing the in vivo metastatic process. This evidence may contribute to pave the way for future possible therapeutic applications of Claisened Hexafluoro to counteract metastatic melanoma dissemination.

The authors wish to make the following corrections to this paper [...]

Simple Summary Mutations in GNAQ underlie vascular malformations, including Sturge-Weber disease. In order to develop novel therapies for lesions with mutant GNAQ, we introduced mutant GNAQ into MS1 endothelial cells. Mutant GNAQ conferred a novel phenotype of progressive vascular malformations in mice. Chromatin analysis revealed upregulation of C-Kit in the vascular endothelial cells, and we found C-Kit to be highly expressed in Sturge-Weber disease. Given that imatinib is an FDA approved multikinase inhibitor that blocks C-Kit, we evaluated it in our mouse model, and showed that imatinib had activity against these vascular malformations. Repurposing imatinib should be evaluated in clinical trials, including Sturge-Weber disease. Abstract GNAQ is mutated in vascular and melanocytic lesions, including vascular malformations and nevi. No in vivo model of GNAQ activation in endothelial cells has previously been described. We introduce mutant GNAQ into a murine endothelial cell line, MS1. The resultant transduced cells exhibit a novel phenotype in vivo, with extensive vasoformative endothelial cells forming aberrant lumens similar to those seen in vascular malformations. ATAC-seq analysis reveals activation of c-Kit in the novel vascular malformations. We demonstrate that c-Kit is expressed in authentic human Sturge–Weber vascular malformations, indicating a novel druggable target for Sturge–Weber syndrome. Since c-Kit is targeted by the FDA-approved drug imatinib, we tested the ability of imatinib on the phenotype of the vascular malformations in vivo. Imatinib treated vascular malformations are significantly smaller and have decreased supporting stromal cells surrounding the lumen. Imatinib may be useful in the treatment of human vascular malformations that express c-Kit, including Sturge–Weber syndrome.

Intestinal epithelial cells form a barrier that is critical in protecting the host from the hostile luminal environment. Previously, we showed that lysophosphatidic acid (LPA) receptor 1 regulates proliferation of intestinal epithelial cells, such that the absence of LPA1 mitigates the epithelial wound healing process. This study provides evidence that LPA1 is important for the maintenance of epithelial barrier integrity. The epithelial permeability, determined by fluorescently labeled dextran flux and transepithelial resistance, is increased in the intestine of mice with global deletion of Lpar1, Lpar1−/− (Lpa1−/−). Serum liposaccharide level and bacteria loads in the intestinal mucosa and peripheral organs were elevated in Lpa1−/− mice. Decreased claudin-4, caudin-7, and E-cadherin expression in Lpa1−/− mice further suggested defective apical junction integrity in these mice. Regulation of LPA1 expression in Caco-2 cells modulated epithelial permeability and the expression levels of junctional proteins. The increased epithelial permeability in Lpa1−/− mice correlated with increased susceptibility to an experimental model of colitis. This resulted in more severe inflammation and increased mortality compared with control mice. Treatment of Caco-2 cells with tumor necrosis factor-α and interferon-γ significantly increased paracellular permeability, which was blocked by cotreatment with LPA, but not LPA1 knockdown cells. Similarly, orally given LPA blocked tumor necrosis factor–mediated intestinal barrier defect in mice. LPA1 plays a significant role in maintenance of epithelial barrier in the intestine via regulation of apical junction integrity.

IGF1R and CD44 are overexpressed in most advanced melanomas so we designed chemotherapeutic nanoparticles to target those receptors. Tris(dibenzylideneacetone)dipalladium (Tris DBA-Pd) is a novel inhibitor of N-myristoyltransferase 1 (NMT-1) and has proven in vivo activity against melanoma. However, poor solubility impairs its effectiveness. To improve its therapeutic efficacy and overcome drug resistance in advanced melanomas, we synthesized Tris DBA-Pd hyaluronic acid nanoparticles (Tris DBA-Pd HANP) and evaluated them against in vivo xenografts of LM36R, an aggressive BRAF mutant human melanoma resistant to BRAF inhibitors. We treated xenografted mice in four arms: empty HANPs, free Tris DBA-Pd, Tris DBA-Pd HANPs, and Tris DBA-Pd HANPs with IGF1R antibody. The Tris DBA-Pd HANP group was the most responsive to treatment and showed the greatest depletion of CD44-positive cells on IHC. Surprisingly, the HANP containing IGF1R antibody was less effective than particles without antibody, possibly due to steric hindrance of IGF1R and CD44 binding. Tris DBA-Pd nanoparticles are an effective therapy for CD44-positive tumors like melanoma, and further development of these nanoparticles should be pursued.

Two pharmacologic approaches that are currently at the forefront of treating advanced cancer are those that center on disrupting critical growth/survival signaling pathways within tumor cells (commonly referred to as "targeted therapies") and those that center on enhancing the capacity of a patient's immune system to mount an antitumor response (immunotherapy). Maximizing responses to both of these approaches requires an understanding of the oncogenic events present in a given patient's tumor and the nature of the tumor-immune microenvironment. Although these 2 modalities were developed and initially used independently, combination regimens are now being tested in clinical trials, underscoring the need to understand how targeted therapies influence immunologic events. Translational studies and preclinical models have demonstrated that targeted therapies can influence immune cell trafficking, the production of and response to chemokines and cytokines, antigen presentation, and other processes relevant to antitumor immunity and immune homeostasis. Moreover, because these and other effects of targeted therapies occur in nonmalignant cells, targeted therapies are being evaluated for use in applications outside of oncology.

Lesions with driver mutations, including atypical nevi and seborrheic keratoses, are very common in dermatology, and are prone to senescence. The molecular events that prevent senescent lesions from becoming malignant are not well understood. We have developed a model of vascular proliferation using a temperaturesensitive, large T antigen and oncogenic HRas. By elevating the temperature to 39 &deg, C, we can turn off large T antigen and study the molecular events in cells with the Ras driver mutation. To assess the signaling events associated with the switch from a proliferative to a nonproliferative state in the constant presence of a driver oncogene, SVR cells were cultivated for 24 and 48 hours and compared with SVR cells at 37 &deg, C. Cells were evaluated by Western Blot (WB) gene chip microarray (GC) and quantitative reverse transcription polymerase chain reaction (RT-qPCR). Upon evaluation, a novel phenotype was observed in endothelial cells after switching off the large T antigen. This phenotype was characterized by Notch activation, downregulation of p38 phosphorylation, downregulation of the master immune switch IRF7, and downregulation of hnRNP A0 . Switching off proliferative signaling may result in immune privilege and Notch activation, which may account, in part, for the survival of common skin lesions.

We investigated the proteome of a female Crested Ibis (Nipponia nippon, ID#162) that died on March 10, 2010 at the Sado Japanese Crested Ibis Conservation Center. Protein preparations from the brain, trachea, liver, heart, lung, proventriculus, muscular stomach, small intestine, duodenum, ovary and neck muscle were subjected to in-solution shotgun mass spectrometry (MS)/MS analyses using an LTQ Orbitrap XL mass spectrometer. A search of the National Center for Biotechnology Information Gallus gallus databases revealed 4253 GI (GenInfo Identifier) numbers with the sum of the same 11 tissues examined in the Crested Ibis. To interpret the obtained proteomics data, it was verified in detail with the data obtained from the brain of the Crested Ibis. It has been reported that drebrin A is specifically expressed in adult chicken brain. In the shotgun proteomic analyses of the Crested Ibis, we identified drebrin A as a brain-specific protein. Furthermore, Western blotting analysis of the protein preparations from 10 tissues of the Crested Ibis and 150-day-old hens using anti-drebrin antibodies showed intensive expression of approximately 110 kDa polypeptides of drebrin in both brains. We believe firmly that the present data will contribute to initial and fundamental steps toward understanding the Crested Ibis proteome.

Background: The PTEN/PIK3 pathway plays a critical role in the cellular response to growth factors. Furthermore, aberrant activation of PI3K signaling is a known hallmark of many forms of cancer. While the role of PI3K signaling in the context of growth factor biology has been well studied, less is known about its activity in the cellular response to cytokines such as interferons (IFNs). We examined how PI3K activation by PTEN inhibition would affect interferon signaling pathways using an in vitro experimental model system. Methods: Cultured human HCT 116 colorectal carcinoma cells and isogenic PTEN (-/-) cells were treated with escalating doses of IFNγ, IFNα2β, and IFNλ. The response to IFNs was evaluated by measuring mRNA levels via RT-PCR and cell surface expression by flow cytometry of MHC Class I and Class II at sequential timepoints. Western blotting analysis was performed to measure levels of total AKT, phospho-AKT (pAKT), PTEN, STAT1, and pSTAT1. Additional pharmacologic studies were performed using cultured HPV-negative SqCC/Y1 oSCC cells treated with IFN, the PTEN inhibitor VO-OHpic, and AKT activator SC79. Further experiments were also done in an isogenic PTEN knockdown BRAFV600E mutant a375 melanoma cell line. Clinical correlates utilizing immunohistochemistry (IHC) on human head and neck cancers were conducted. Results: IFN-γ- induced surface expression of MHC II in PTEN (-/-) HCT 116 cells was markedly reduced by greater than 50% vs parental cells. PTEN (-/-) HCT 116 cells also expressed lower basal levels of MHC I, with similar attenuation in induction by IFNs. These reductions in surface expression correlated with lower MHC I/II mRNA levels. IHC studies in patient head & neck cancer biopsy samples demonstrated an inverse relationship between pAKT and MHC I expression. Additional studies were performed comparing PTEN knockout and the expression of STAT1, a downstream target of IFN signaling. We observed that PTEN/PIK3 signaling implicates STAT1, and that PTEN inhibition consistently modulated total and phosphorylated levels of STAT1 protein in PTEN (-/-) HCT 116 cells. Similar findings were also observed in melanoma cells. Conclusions: We aim to further characterize the link between oncogenic signaling and anti-tumor immunity. We demonstrate, using an in vitro model, that genetic and pharmacologic activation of the PI3K pathway modulates the immune response to IFNs and represses MHC induction and expression. Importantly, similar inverse patterns of PI3K signaling and MHC expression were seen in patient biopsy samples. We also present a novel mechanism linking the PTEN/PIK3 and STAT1 pathways to further explain our findings. Additional characterization of this interaction in in vivo models is necessary. Citation Format: Sanjay Chandrasekaran, Maiko Sasaki, Brian P. Pollack. Linking growth factors and immune actors: PTEN/PI3K signaling in cancer and the response to cytokines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4510.

35 Background: The PTEN/PIK3 pathway plays an important role in the cellular response to growth factors and aberrant activation of PI3K signaling is a hallmark of many forms of cancer. While the role of PI3K signaling has been well studied in the context of growth factor biology, its role in the cellular response to cytokines such as interferons (IFNs) is less well understood. We examined the impact of PI3K activation on interferon signaling pathways using an in vitro experimental model system. Methods: Cultured human HCT 116 colorectal carcinoma cells and isogenic PTEN (-/-) cells were treated with escalating doses of IFNg, IFNa2b, and IFNl. IFN response was evaluated by measuring MHC Class I and Class II mRNA levels and cell surface expression via RT-PCR and flow cytometry, respectively. Western blots for total AKT, phospho-AKT (pAKT), PTEN, STAT1, and pSTAT1 were performed. Corroborating pharmacologic studies were performed using cultured HPV-negative SqCC/Y1 oSCC cells treated with IFN and the PTEN inhibitor VO-OHpic and AKT activator SC79. Clinical correlates utilizing immunohistochemistry (IHC) on human head and neck cancers were conducted. Results: PTEN (-/-) cells expressed lower basal levels of MHC I and the induction by IFNs were likewise attenuated. Dramatically, PTEN knockout reduced IFN-g- induced surface expression of MHC II by > 50%. Corresponding reductions in MHC I/II mRNA were seen. IHC in patient biopsy samples showed an inverse relationship between phospho-AKT and MHC I expression. We identified a potential explanation of this effect wherein PTEN/PIK3 signaling implicates STAT1, a downstream target of IFN signaling. PTEN inhibition consistently modulated total and phosphorylated levels of STAT1 protein. Conclusions: Much remains unknown about the link between growth factor pathways and the tumor immune response. We demonstrate using an in vitro model that genetic and pharmacologic activation of the PI3K pathway attenuates the immune response to IFNs and represses MHC induction and expression. We present a novel mechanism for explaining this phenomenon linking the PTEN/PIK3 and STAT1 pathways. Further characterization of this interaction in in vitro models is necessary.

Objective Regenerative medicine seeks to stall or to reverse the pathologic consequences of chronic diseases. Many people with diabetes have peripheral arterial disease (PAD), which increases their already high risk of major amputation. Cellular therapies are a promising regenerative medicine approach to PAD that can be used to focally inject regenerative cells to endangered tissue beds. Mesenchymal stem cells (MSCs) are known to promote tissue regeneration through stromal support and paracrine stimulation of new blood vessels (angiogenesis). Whereas little is known about human diabetic MSCs (dMSCs), particularly those from patients with PAD, dMSCs have a limited expansion capacity but can be improved with human platelet lysate (PL) supplementation. PL is rich in many growth factors, including epidermal growth factor (EGF), which is known to be important to cell proliferation and survival signaling pathways. We hypothesize that dMSCs have a reversible defect in EGF receptor pathways. The objective of this work was to test this hypothesis using dMSCs from PAD patients. Methods The secretome expression of EGF and prominent angiogens was characterized from bone marrow (BM)-derived and adipose tissue-derived (ATD) dMSCs from five patients (six limbs) undergoing major amputation. Western blot was used to characterize the AKT and extracellular signal-regulated protein kinases 1 and 2 expression in dMSCs under standard culture (5% fetal bovine serum plus fibroblast growth factor 2 [FGF2]), 5% human PL, or 5% fetal bovine serum plus EGF. Healthy donor MSCs were control cells. The angiogenic activity of BM- and ATD-dMSCs was tested on human umbilical vein endothelial cells (ECs). Paired t-test, analysis of variance, and Kruskal-Wallis tests were used as appropriate. Results Both BM- and ATD-dMSCs had typical MSC surface marker expression and similar expansion profiles, and they did not express EGF in their secretome. PL supplementation of dMSCs improved AKT signaling, but they were resistant to FGF2 activation of extracellular signal-regulated protein kinases 1 and 2. EGF supplementation led to similar AKT expression as with PL, but PL had greater phosphorylation of AKT at 30 and 60 minutes. The conditioned media from both BM- and ATD-dMSCs had robust levels of prominent angiogens (vascular endothelial growth factor, monocyte chemoattractant protein 1, hepatocyte growth factor), which stimulated EC proliferation and migration, and the co-culture of dMSCs with ECs led to significantly longer EC sprouts in three-dimensional gel than EC-alone pellets. Conclusions PL and EGF supplementation improves AKT expression in dMSCs over that of FGF2, but PL improved pAKT over that of EGF. Thus, PL supplementation strategies may improve AKT signaling, which could be important to MSC survival in cellular therapies. Furthermore, BM- and ATD-dMSCs have similar secretomes and robust in vitro angiogenic activity, which supports pursuing dMSCs from both reservoirs in regenerative medicine strategies.

e24119Background: PD-L1 expression in oral squamous cell carcinoma (oSCC) is well characterized and is one potential mechanism for suppression of the anti-tumor immune response. Enhanced phosphoino...

e24143Background: Antigen presentation plays a central role in anti-tumor immunity and the response to immunotherapy. Recent studies have underscored the importance of MHC class I (MHC I) and class...

Lymphocyte inhibitory factor A (lifA) in Citrobacter rodentium encodes the large toxin lymphostatin, which contains two enzymatic motifs associated with bacterial pathogenesis, a glucosyltransferase and a protease. Our aim was to determine the effects of each lymphostatin motif on intestinal epithelial-barrier function. In-frame mutations of C. rodentium lifA glucosyltransferase (CrGlM21) and protease (CrPrM5) were generated by homologous recombination. Infection of both model intestinal epithelial monolayers and mice with C. rodentium wild type resulted in compromised epithelial barrier function and mislocalization of key intercellular junction proteins in the tight junction and adherens junction. In contrast, CrGlM21 was impaired in its ability to reduce barrier function and influenced the tight junction proteins ZO-1 and occludin. CrPrM5 demonstrated decreased effects on the adherens junction proteins beta-catenin and E-cadherin. Analysis of the mechanisms revealed that C. rodentium wild type differentially influenced Rho GTPase activation, suppressed Cdc42 activation, and induced Rho GTPase activation. CrGlM21 lost its suppressive effects on Cdc42 activation, whereas CrPrM5 was unable to activate Rho signaling. Rescue experiments using constitutively active Cdc42 or C3 exotoxin to inhibit Rho GTPase supported a role of Rho GTPases in the epithelial barrier compromise induced by C. rodentium. Taken together, our results suggest that lymphostatin is a bacterial virulence factor that contributes to the disruption of intestinal epithelial-barrier function via the modulation of Rho GTPase activities.

A bout of prolonged aerobic exercise can enhance the sensitivity of muscle glucose uptake to insulin, and this may be mediated by activation of 5'-adenosine monophosphate-activated protein kinase (AMPK). The aim of this study was to examine whether high-intensity short-term exercise resulting in a significantly greater increase in the activation of AMPK is more effective in enhancing muscle insulin sensitivity compared with low-intensity prolonged aerobic exercise. We measured insulin sensitivity after high-intensity intermittent swimming (HIS) or low-intensity continuous swimming (LIS) exercise in rat epitrochlearis muscle. During HIS, the rats underwent eight 20-second bouts of swimming with a weight equal to 18% of body weight. The LIS rats swam with no load for 3 hours. High-intensity intermittent swimming increased (P.05) 2-deoxyglucose uptake approximately 8-fold, whereas LIS increased it (P.05) approximately 2-fold immediately after exercise compared with rested muscle. This response was associated with an increase (P.05) in phosphorylation of AMPK Thr(172) and its downstream target acetyl-coenzyme A carboxylase (ACC) Ser(79) in HIS (13- and 6-fold, respectively) and LIS (2.8- and 2-fold, respectively) immediately after exercise. In contrast, submaximal (30 microU/mL) insulin-stimulated 2-deoxyglucose uptake measured 4 hours after exercise was 73% and 46% higher (P.05) in LIS and HIS, respectively, compared with rest. The HIS exercise resulted in a greater activation of AMPK compared with LIS, but insulin sensitivity was higher after LIS compared with HIS. The results suggest that HIS is not more effective in enhancing insulin sensitivity than LIS. Thus, AMPK activation immediately after exercise may not be the only factor that determines the magnitude of the exercise-induced increase in insulin sensitivity in rat epitrochlearis muscle.

BACKGROUND Epidemiological and genetic studies suggest a role for enteric flora in the pathogenesis of Crohn's disease (CD). Crohn's disease-associated Escherichia coli (CDEC) is characterized by their ability to invade epithelial cells and survive and induce high concentration of TNF-α from infected macrophages. However, the molecular mechanisms of CDEC survival in infected macrophages are not completely understood. METHODS Intracellular survival of CDEC strain LF82 isolated from inflamed ileum tissue, 13I isolated from inflamed colonic tissue, and control E. coli strains were tested in the murine macrophage cell line, J774A.1 by Gentamicin protection assay. Modulation of intracellular cell signaling pathways by the E. coli strains were assessed by western blot analysis and confocal microscopy. RESULTS 13I demonstrated increased survival in macrophages with 2.6-fold higher intracellular bacteria compared with LF82, yet both strains induced comparable levels of TNF-α. LF82 and 13I differentially modulated key mitogen-activated protein kinase pathways during the acute phase of infection; LF82 activated all 3 mitogen-activated protein kinase pathways, whereas 13I activated ERK1/2 pathway but not p38 and JNK pathways. Both 13I and LF82 suppressed nuclear translocation of NFκB compared with noninvasive E. coli strains during the acute phase of infection. However, unlike noninvasive E. coli strains, 13I and LF82 infection resulted in chronic activation of NFκB during the later phase of infection. CONCLUSIONS Our results showed that CDEC survive in macrophages by initially suppressing NFκB activation. However, persistence of bacterial within macrophages induces chronic activation of NFκB, which correlates with increased TNF-α secretion from infected macrophages.

We investigated the proteome of a female Crested Ibis (Nipponia nippon, ID#162) that died on March 10, 2010 at the Sado Japanese Crested Ibis Conservation Center. Protein preparations from the brain, trachea, liver, heart, lung, proventriculus, muscular stomach, small intestine, duodenum, ovary and neck muscle were subjected to in-solution shotgun mass spectrometry (MS)/MS analyses using an LTQ Orbitrap XL mass spectrometer. A search of the National Center for Biotechnology Information Gallus gallus databases revealed 4253 GI (GenInfo Identifier) numbers with the sum of the same 11 tissues examined in the Crested Ibis. To interpret the obtained proteomics data, it was verified in detail with the data obtained from the brain of the Crested Ibis. It has been reported that drebrin A is specifically expressed in adult chicken brain. In the shotgun proteomic analyses of the Crested Ibis, we identified drebrin A as a brain-specific protein. Furthermore, Western blotting analysis of the protein preparations from 10 tissues of the Crested Ibis and 150-day-old hens using anti-drebrin antibodies showed intensive expression of approximately 110 kDa polypeptides of drebrin in both brains. We believe firmly that the present data will contribute to initial and fundamental steps toward understanding the Crested Ibis proteome.

Background Metastasis, the spread and growth of tumor cells to distant organ sites, represents the most devastating attribute and plays a major role in the morbidity and mortality of cancer. Inflammation is crucial for malignant tumor transformation and survival. Thus, blocking inflammation is expected to serve as an effective cancer treatment. Among anti-inflammation therapies, chemokine modulation is now beginning to emerge from the pipeline. CXC chemokine receptor-4 (CXCR4) and its ligand stromal cell-derived factor-1 (CXCL12) interaction and the resulting cell signaling cascade have emerged as highly relevant targets since they play pleiotropic roles in metastatic progression. The unique function of CXCR4 is to promote the homing of tumor cells to their microenvironment at the distant organ sites. Methodology/Principal Findings We describe the actions of N,N′-(1,4-phenylenebis(methylene))dipyrimidin-2-amine (designated MSX-122), a novel small molecule and partial CXCR4 antagonist with properties quite unlike that of any other reported CXCR4 antagonists, which was prepared in a single chemical step using a reductive amination reaction. Its specificity toward CXCR4 was tested in a binding affinity assay and a ligand competition assay using 18F-labeled MSX-122. The potency of the compound was determined in two functional assays, Matrigel invasion assay and cAMP modulation. The therapeutic potential of MSX-122 was evaluated in three different murine models for inflammation including an experimental colitis, carrageenan induced paw edema, and bleomycin induced lung fibrosis and three different animal models for metastasis including breast cancer micrometastasis in lung, head and neck cancer metastasis in lung, and uveal melanoma micrometastasis in liver in which CXCR4 was reported to play crucial roles. Conclusions/Significance We developed a novel small molecule, MSX-122, that is a partial CXCR4 antagonist without mobilizing stem cells, which can be safer for long-term blockade of metastasis than other reported CXCR4 antagonists.

Factors implicated in the pathophysiology of ulcerative colitis (UC) are an abnormal immune response, defect in intestinal epithelial barrier function, and gut microbiota. Currently, it is unclear whether specific bacterial strains are responsible for the induction of intestinal inflammation, but increased bacterial tissue invasion has been described in affected UC patients. Further, a quantitative and qualitative microbial imbalance in UC, defined as dysbiosis, has been characterized by an increase in Rhodococcus spp., Shigella spp., and Escherichia spp., but a decrease in certain Bacteroides spp.. More specifically, Campylobacter spp., Enterobacteriae, and enterohepatic Helicobacter were more prevalent in tissue sample from UC patients subjected to molecular detection methods, but not controls. In addition, serologic testing identified Fusobacterim varium as a potential contributor to the intestinal inflammation in UC. Interestingly, in-situ hybridization studies have shown anti-inflammatory Lactobacillus spp. and Pediococcus spp. were absent in samples from subjects affected by UC. Therefore, dysbiosis is a factor in the pathogenesis of UC.

It has become increasingly clear that inflammatory bowel disease (IBD) develops on the background of genetic defects in the host, conveying an increased susceptibility to an environmental antigen or antigens. The environmental factor implicated in the pathophysiology of gut inflammation, which is undergoing increased scrutiny, is the intestinal flora. The intestinal flora as a whole and specific bacteria and their products have been found to trigger cytokine expression in various cell types. Consistently, multiple bacterial strains were found to induce tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8) in macrophage and epithelial cell systems, respectively, in particular in Crohn's disease. Interestingly, various cell types from patients with IBD display an increased susceptibility to specific bacterial products, including flagellin, pili, and lipopolysaccharides. It remains to be determined whether additional effector proteins regulate cytokine expression and the aberrant mucosal immune response in IBD.

β-Adrenergic stimulation and exercise up-regulate the mRNA expression of nuclear receptor NR4A3, which is involved in the regulation of glucose and fatty acid utilization genes in skeletal muscle. The objective of our study was to examine the effects of β-adrenergic stimulation and exercise on the expression of NR4A3 protein in rat skeletal muscle. A single subcutaneous injection of clenbuterol, which is a β2-adrenergic receptor (β2-AR) agonist, increased NR4A3 mRNA and protein expression in the fast-twitch glycolytic triceps muscle. On the other hand, an acute 3-h session of either treadmill running or swimming did not increase the NR4A3 protein level in the exercised muscle, although both treadmill running and swimming increased NR4A3 mRNA. Finally, loss of postural contractile activity because of hindlimb immobilization reduced NR4A3 mRNA and protein in the slow-twitch oxidative soleus muscle. These results suggest that: β-adrenergic stimulation up-regulates not only NR4A3 mRNA but also NR4A3 protein in fast-twitch glycolytic muscle; exercise may increase NR4A3 mRNA but not NR4A3 protein in skeletal muscle; and local postural contractile activity plays a crucial role in maintaining NR4A3 protein expression level in postural muscle.

Exercise upregulates the expression of NR4A receptors, which are involved in regulation of glucose and fatty acid utilization genes in skeletal muscle. The aims of our study were 1) to determine the role of local contractile activity on NR4A mRNA expression in skeletal muscle during exercise; and 2) to elucidate the mechanisms underlying the induction of NR4A mRNA expression in response to muscle contractile activity. Rats were subjected to an acute 3-h low-intensity swimming or a 3-h low-intensity treadmill running as a model of endurance exercise. Low-intensity swimming increased NR4A1 and NR4A3 mRNA in triceps but not in soleus muscle. Conversely, low-intensity treadmill running increased NR4A1 and NR4A3 mRNA in soleus but not in triceps muscle. NR4A mRNA increased concomitantly with reduced postexercise muscle glycogen, suggesting that gene expression of NR4A receptors occurs in muscles recruited during exercise. Furthermore, in resting rats, an acute 1-h local electrical stimulation of a motor nerve to the tibialis anterior muscle caused increases in NR4A1 and NR4A3 mRNA relative to the contralateral control muscle of the same animals. On the other hand, after 6 h of hindlimb immobilization, NR4A1 and NR4A3 mRNA were reduced in immobilized soleus muscle relative to contralateral control muscle. In addition, both NR4A1 and NR4A3 mRNA in epitrochlearis muscle were increased after 6-h incubation with 0.5 mM 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside, which activates AMP-activated protein kinase. These results suggest that 1) local muscle contractile activity is required for increased expressions of NR4A1 and NR4A3 mRNA during exercise; and 2) muscle contractile activity-induced increases in NR4A1 and NR4A3 mRNA may be mediated by AMPK activation, at least in part.

Crohn's disease (CD) and ulcerative colitis (UC) are idiopathic inflammatory conditions of the gut. Our goal was to investigate if invasive Escherichia coli strains were present in patients with inflammatory bowel disease (IBD). Bacterial strains were isolated from biopsy material obtained from normal controls, and patients with a clinical diagnosis of CD and UC. Invasive bacteria were characterized by gentamicin protection assay and biochemical profiling (Api-20E). Strains were characterized by induction of cytokine expression in epithelial and macrophage cell cultures, measurement of epithelial barrier function, and confocal microscopy. Of all invasive bacterial strains in CD 98.9% were identified as E. coli as opposed to 42.1% in UC and 2.1% in normal controls. Epithelial invasion in vitro was significantly higher for CD-associated E. coli (8.4%, +/-5.5 of initial inoculum (I/O)) in comparison to UC (2.5%, +/-0.4 I/O), but highest for strains from inflamed CD tissue (11.3%, +/-4.3 I/O). Both, CD and UC E. coli strains induced high mean TNF-alpha expression in macrophage cell lines (2604.8 pg/10(5) cells, +/-447.4; 2,402.6 pg/10(5) cells, +/-476.3, respectively), but concentrations were significantly higher for isolates from inflamed CD tissue (3071.3 pg/10(5) cells, +/-226.0). Invasive E. coli from IBD tissue induced similar concentrations of interleukin (IL)-8 in epithelial cell cultures, but strains from inflamed CD tissue induced significantly less epithelial IL-8 (674.1 pg/10(5) cells, +/-58.0 vs 920.5 pg/10(5) cells, +/-94.6). IBD-associated E. coli strains significantly decreased transepithelial resistance, induced disorganization of F-actin and displacement of ZO-1, and E-cadherin from the apical junctional complex (AJC). In comparison to normal controls and UC, E. coli are more prevalent in CD, are highly invasive, and do not encode for known effector proteins. E. coli strains from IBD patients regulate cytokine expression and epithelial barrier function, two pathological features of IBD.

Background & Aims: Kruppel-like factor 5 (KLF5) is a transcription factor that is highly expressed in proliferating crypt cells of the intestinal epithelium. KLF5 has a pro-proliferative effect in vitro and is induced by mitogenic and stress stimuli. To determine whether KLF5 is involved in mediating proliferative responses to intestinal stressors in vivo, we examined its function in a mouse model of transmissible murine colonic hyperplasia triggered by colonization of the mouse colon by the bacteria Citrobacter rodentium. Methods: Heterozygous Klf5 knockout (Klf5+/−) mice were generated from embryonic stem cells carrying an insertional disruption of the Klf5 gene. Klf5+/− mice or wild-type (WT) littermates were infected with C rodentium by oral gavage. At various time points postinfection, mice were killed and distal colons were harvested. Colonic crypt heights were determined morphometrically from sections stained with H&E. Frozen tissues were stained by immunofluorescence using antibodies against Klf5 and the proliferation marker, Ki67, to determine Klf5 expression and numbers of proliferating cells per crypt. Results: Infection of WT mice with C rodentium resulted in a 2-fold increase in colonic crypt heights at 14 days postinfection and was accompanied by a 1.7-fold increase in Klf5 expression. Infection of Klf5+/− mice showed an attenuated induction of Klf5 expression, and hyperproliferative responses to C rodentium were reduced in the Klf5+/− animals as compared with WT littermates. Conclusion: Our study shows that Klf5 is a key mediator of crypt cell proliferation in the colon in response to pathogenic bacterial infection.

Pseudomonas aeruginosa is an opportunistic pathogen that forms biofilms on tissues and other surfaces. We characterized the interaction of purified human neutrophils with P. aeruginosa, growing in biofilms, with regard to morphology, oxygen consumption, phagocytosis, and degranulation. Scanning electron and confocal laser microscopy indicated that the neutrophils retained a round, unpolarized, unstimulated morphology when exposed to P. aeruginosa PAO1 biofilms. However, transmission electron microscopy demonstrated that neutrophils, although rounded on their dorsal side, were phagocytically active with moderate membrane rearrangement on their bacteria-adjacent surfaces. The settled neutrophils lacked pseudopodia, were impaired in motility, and were enveloped by a cloud of planktonic bacteria released from the biofilms. The oxygen consumption of the biofilm/neutrophil system increased 6- and 8-fold over that of the biofilm alone or unstimulated neutrophils in suspension, respectively. H2O2 accumulation was transient, reaching a maximal measured value of 1 μM. Following contact, stimulated degranulation was 20–40% (myeloperoxidase, β-glucuronidase) and 40–80% (lactoferrin) of maximal when compared with formylmethionylleucylphenylalanine plus cytochalasin B stimulation. In summary, after neutrophils settle on P. aeruginosa biofilms, they become phagocytically engorged, partially degranulated, immobilized, and rounded. The settling also causes an increase in oxygen consumption of the system, apparently resulting from a combination of a bacterial respiration and escape response and the neutrophil respiratory burst but with little increase in the soluble concentration of H2O2. Thus, host defense becomes compromised as biofilm bacteria escape while neutrophils remain immobilized with a diminished oxidative potential.