Adherence Phenotype Research Articles

A phagocytosis assay for oxidized low-density lipoprotein versus immunoglobulin G-coated microbeads in human U937 macrophages

The human monocyte cell line U937 was differentiated into an adherent macrophage phenotype using phorbol 12-myristate 13-acetate (PMA) to assay the phagocytosis of oxidized low-density lipoprotein (oxLDL) that may play a role in atherosclerosis. Microbeads were coated with the inflammatory ligand oxLDL to create a novel phagocytosis assay that models the binding of macrophages to oxLDL in the solid phase such as found in the fatty streaks of the arteries. The oxLDL was prepared with LDL from human ethylenediaminetetraacetic acid (EDTA) plasma oxidized with an excess (5 mM) of the strong oxidizing agent CuSO4 and characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis with Western blot. The binding of the oxLDL to the beads was confirmed by DilC18–oxLDL staining and confocal microscopy in addition to trypsin digestion of the microbeads for liquid chromatography, electrospray ionization, and tandem mass spectrometry. Phagocytosis of the oxLDL versus human bulk immunoglobulin G1 (IgG1)-coated microbeads was assayed over time, in the presence and absence of serum factors, by pulse chase and with enzyme inhibitor treatments. The ligand beads were then stained with specific antibodies to oxLDL versus human IgG to differentially stain external versus engulfed ligand microbeads. The phagocytosis of oxLDL and IgG ligand microbeads was abolished by the actin polymerization inhibitors cytochalasin D and latrunculin. Pharmacological inhibitors of the receptor enzymes JAK, SRC, and PLC prevented both IgG and oxLDL receptor function. In contrast, the function of the oxLDL phagocytic receptor complex was more sensitive to inhibition of PTK2, PKC, and SYK activity.

Genome-based Definition of an Inflammatory Bowel Disease-associated Adherent-Invasive Escherichia coli Pathovar.

Mucosal-associated Escherichia coli are commonly found in inflamed tissues during inflammatory bowel disease (IBD). These bacteria often possess an adherent and invasive phenotype but lack virulence-associated features of well-described intestinal E. coli pathogens, and are of diverse serology and phylotypes, making it difficult to correlate strain characteristics with exacerbations of disease. The genome sequences of 14 phenotypically assigned adherent-invasive Escherichia coli (AIEC) isolates obtained from intestinal biopsies of patients with IBD were compared with the genome sequences of 37 other pathogenic and commensal E. coli available from public databases. Core genome-based phylogenetic analyses and genome-wide comparison of genetic content established the existence of a closely related cluster of AIEC strains with 3 distinct genetic insertions differentiating them from commensal E. coli. These strains are of the B2 phylotype have a variant type VI secretion system (T6SS-1), and are highly related to extraintestinal pathogenic E. coli, suggesting that these 2 clinically distinct pathovars have common virulence strategies. Four other mucosally adherent E. coli strains from patients with IBD were of diverse phylogenetic origins and lacked the 3 genetic features, suggesting that they are not related to the B2 AIEC cluster. Although AIEC are often considered as having a unique association with Crohn's disease, isolates from Crohn's disease and ulcerative colitis were genetically indistinguishable. B2 AIEC thus represent a closely related cluster of IBD-associated E. coli strains that are distinct from normal commensal isolates, and which should be considered separately from the phenotypically similar but genetically distinct non-B2 AIEC strains when considering their association with intestinal pathogenesis.

Effect of polystyrene and fluoropolymer-based culture vessels on monocyte-based immunotherapy products

Effect of polystyrene and fluoropolymer-based culture vessels on monocyte-based immunotherapy products

Isolation, Culture and Identification of Choriocarcinoma Stem-Like Cells from the Human Choriocarcinoma Cell-Line JEG-3

Background/Aims: Cancer stem cells (CSCs) exhibit enhanced proliferative capacity and resistance to chemotherapy; however, choriocarcinoma CSCs have not yet been reported. In this study the human choriocarcinoma cell line JEG-3 was cultured in serum free media, and the characteristics of suspension and parental adherent JEG-3 cells were compared. Methods: Cell proliferation, colony-formation, soft agar clonogenicity, and transwell invasion assays were performed in vitro, and tumor xenografts in BALB/c nude mice were used to evaluate stem cell properties. Results: In serum-supplemented medium (SSM), JEG-3 cells were 4.51 ± 1.71% CD44+, 7.67 ± 2.67% CD133+, and 13.85 ± 2.95% ABCG2+. In serum-free medium (SFM), the expression of these markers increased to 53.08 ± 3.15%, 47.40 ± 2.67%, and 78.70 ± 7.16%, respectively. Moreover, suspension JEG-3 cells exhibited enhanced colony-formation capability as well as invasive and proliferative ability in vitro, alongside enhanced tumorigenic properties in vivo. Suspension JEG-3 cells also exhibited resistance to the chemotherapeutic drugs methotrexate, fluorouracil and etoposide. When seeded in serum supplemented medium, suspension JEG-3 cells readopted an adherent phenotype and continued to differentiate with no significant difference in the morphology between suspension and parent cells. Conclusion: In this study, choriocarcinoma stem-like cells (CSLCs) were isolated from the human choriocarcinoma JEG-3 cell line by SFM culture and characterized.

EPIG-06ATRX DEFICIENCY PROMOTES MIGRATORY BEHAVIOR IN GLIOMA CELLS OF ORIGIN BY DYSREGULATING GENOME-WIDE CHROMATIN ACCESSIBILITY AND MODULATING TRANSCRIPTIONAL PROGRAMS

Comprehensive genomic profiling in cancer continues to reveal frequent alterations in epigenetic regulators, firmly implicating chromatin biology in the oncogenic process. We and others recently found that inactivating mutations in the SWI/SNF family member ATRX represent defining molecular alterations in diffusely infiltrating gliomas, where they invariably pair with mutations in TP53 and IDH1/2. A number of studies have linked ATRX deficiency to a wide spectrum of physiological dysfunction, including aberrant gene regulation, abnormal telomere maintenance, genomic instability, and aneuploidy. Nevertheless, the molecular mechanism(s) by which ATRX deficiency promotes oncogenesis are still largely unestablished. To understand the role of Atrx loss-of-function mutations in gliomagenesis, we inactivated Atrx in Tp53-/- murine neuroepithelial progenitor cells (NPCs), which have been implicated as potential glioma cells of origin by several prior studies. Interestingly, Atrx loss induced NPCs to stop growing as neurospheres and adopt an adherent phenotype, while also exhibiting significantly increased cellular motility by transwell migration assay, the latter an established pathogenic feature of diffusely infiltrating gliomas. These phenotypes correlated with altered gene expression profiles involving molecular networks implicated in development, regulation of signal transduction, and cellular motility and invasion. Integrating these transcriptional changes with shifts in chromatin accessibility occurring with Atrx deficiency and genome-wide Atrx distribution, as determined by ChIP-seq, revealed highly significant spatial correlations between differentially expressed genes, regions of altered chromatin compaction, and genomic sites normally occupied by Atrx. Taken together, these findings demonstrate that Atrx deficiency induces widespread disruptions in chromatin organization, which in turn lead to dramatic shifts in gene expression and acquisition of disease-relevant phenotypes in putative glioma cells of origin. We are now in the process of identifying and characterizing the precise transcriptional and epigenetic events mediating the acquisition of Atrx-dependent behaviors in NPCs.

Finding Regulators Associated with the Expression of the Long Polar Fimbriae in Enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) is a human pathogen that requires initial adhesion to the intestine in order to cause disease. Multiple adhesion factors have been identified in E. coli strains, among them the long polar fimbriae (Lpf), a colonization factor associated with intestinal adhesion. The conditions of Lpf expression are well understood in enterohemorrhagic E. coli (EHEC); however, the expression of EPEC lpf has been found to be repressed under any in vitro condition tested. Therefore, we decided to identify those factors silencing expression of EPEC lpf. Because histone-like nucleoid structuring protein (H-NS) is a known repressor of EHEC lpf, we tested it and found that H-NS is a repressor of EPEC lpf. We also found that the adhesion of the EPEC Δhns strain was significantly enhanced compared to the wild-type strain. Because lpf expression was modestly increased in the hns mutant, transposon mutagenesis was performed to find a strain displaying higher lpf expression than EPEC Δhns. One Tn5 insertion was identified within the yhjX gene, and further in vitro characterization revealed increased lpf expression and adhesion to Caco-2 cells compared with EPEC Δhns. However, in a murine model of intestinal infection, the EPEC Δhns and EPEC Δhns Tn5 mutants had only a slight change in colonization pattern compared to the wild-type strain. Our data showed that EPEC Lpf is transcribed, but its role in EPEC intestinal colonization requires further analysis. Data are presented demonstrating that the long polar fimbriae (lpf) operon in enteropathogenic E. coli (EPEC) is highly regulated; however, derepression occurs by mutagenizing two proteins associated with its control. The study demonstrates that the EPEC lpf operon can be expressed and, therefore, participates in the EPEC adherence phenotype.

Chemorepulsion from the Quorum Signal Autoinducer-2 Promotes Helicobacter pylori Biofilm Dispersal.

ABSTRACTThe gastric pathogen Helicobacter pylori forms biofilms on abiotic and biotic surfaces. We have shown previously that H. pylori perceives the quorum signal autoinducer-2 (AI-2) as a chemorepellent. We report here that H. pylori chemorepulsion from endogenous AI-2 influences the proportions and spatial organization of cells within biofilms. Strains that fail to produce AI-2 (∆luxS strains) or are defective for chemotaxis (∆cheA strains) formed more spatially homogenous biofilms with a greater proportion of adherent versus planktonic cells than wild-type biofilms. Reciprocally, a strain that overproduced AI-2 (luxSOP) formed biofilms with proportionally fewer adherent cells. Along with the known AI-2 chemoreceptor, TlpB, we identified AibA and AibB, two novel periplasmic binding proteins that are required for the AI-2 chemorepulsion response. Disruptions in any of the proteins required for AI-2 chemotaxis recapitulated the biofilm adherence and spatial organization phenotype of the ∆luxS mutant. Furthermore, exogenous administration of AI-2 was sufficient to decrease the proportion of adherent cells in biofilms and promote dispersal of cells from biofilms in a chemotaxis-dependent manner. Finally, we found that disruption of AI-2 production or AI-2 chemotaxis resulted in increased clustering of cells in microcolonies on cultured epithelial cells. We conclude that chemotaxis from AI-2 is a determinant of H. pylori biofilm spatial organization and dispersal.

Effects of cell detachment methods on the dielectric properties of adherent and suspension cells.

Cell analyses such as flow cytometry, dielectrophoresis, and some patch-clamp techniques require that cells be in monodisperse suspension in order for analysis to occur. Where cells have a normally adherent phenotype in vivo, this requires that cells be removed from the surface of the culture flask or vessel. This can be achieved in many ways, but most commonly either by the use of a dissociation medium of some form, or by scraping the cells from the surface. Both methods have potential drawbacks; chemical methods may alter the properties of the cells to such a degree that the measurement might be regarded as meaningless, whilst scraping could cause physical damage to the structure of the cells. In this paper, we use dielectrophoresis to analyse the electrical properties of two adherent cell lines detached by multiple methods, and compare these against a control cell line of suspension cells, examined in both the native state and when subject to the same chemical treatments. The results indicate that most chemical agents do not alter the electrophysiology of cells directly, though they may trigger some potential cell deterioration processes such as apoptosis in the cells. This can be observed in the production of apoptotic body-like particles and the alteration of cytoplasmic conductivity (which has been associated with apoptotic water efflux). However, cells detached by scraping exhibited statistically significant differences in their electrophysiological properties when compared to those detached by the chemical methods, indicating that this method is unsuitable for detachment of adherent cells prior to analysis of isolates suspension cells.

Two novel EHEC/EAEC hybrid strains isolated from human infections.

The so far highest number of life-threatening hemolytic uremic syndrome was associated with a food-borne outbreak in 2011 in Germany which was caused by an enterohemorrhagic Escherichia coli (EHEC) of the rare serotype O104:H4. Most importantly, the outbreak strain harbored genes characteristic of both EHEC and enteroaggregative E. coli (EAEC). Such strains have been described seldom but due to the combination of virulence genes show a high pathogenicity potential. To evaluate the importance of EHEC/EAEC hybrid strains in human disease, we analyzed the EHEC strain collection of the German National Reference Centre for Salmonella and other Bacterial Enteric Pathogens (NRC). After exclusion of O104:H4 EHEC/EAEC strains, out of about 2400 EHEC strains sent to NRC between 2008 and 2012, two strains exhibited both EHEC and EAEC marker genes, specifically were stx2 and aatA positive. Like the 2011 outbreak strain, one of the novel EHEC/EAEC harbored the Shiga toxin gene type stx2a. The strain was isolated from a patient with bloody diarrhea in 2010, was serotyped as O59:H−, belonged to MLST ST1136, and exhibited genes for type IV aggregative adherence fimbriae (AAF). The second strain was isolated from a patient with diarrhea in 2012, harbored stx2b, was typed as Orough:H−, and belonged to MLST ST26. Although the strain conferred the aggregative adherence phenotype, no known AAF genes corresponding to fimbrial types I to V were detected. In summary, EHEC/EAEC hybrid strains are currently rarely isolated from human disease cases in Germany and two novel EHEC/EAEC of rare serovars/MLST sequence types were characterized.

Serum IL-6 Levels Show a Direct Correction With Circulating Tumor Cells (CTCs) and Poor Prognosis: Molecular Biology and Therapeutics

Distant metastases in head and neck cancer patients almost invariably herald a poor prognosis. Five year survival rates for early stage localized head and neck cancers are over 80% but this drop to 40% where disease has spread to neck nodes, and to below 20% for patients with distant metastatic disease. A key process in this metastatic cascade is the transition of tumor cells from an adherent epithelial phenotype into a highly motile and invasive mesenchymal phenotype. We have recently demonstrated that IL-6 is a potent inducer of epithelial-mesenchymal transition (EMT) in HNSCC.

The isolation and cultivation of bone marrow stem cells and evaluation of differences for neural-like cells differentiation under the induction with neurotrophic factors

The bone marrow represents the most common source from which to isolate mesenchymal stem cells (MSCs). They can be obtained directly from patients and successfully induced to form various differentiated cell types. In addition, cell-based transplantation therapies have been proven to be promising strategies for curing disease of the nerve system. Therefore, it was particularly important to establish an easy and feasible method for the isolation, purification, and differentiation of bone marrow stromal cells (BMSCs). The aim of this study was to isolate and characterize putative bone marrow derived MSCs from Sprague-Dawley (SD) rats. Furthermore, differentiation effects were compared between the GDNF-induction group and the BDNF-induction group. Of these, BMSCs were isolated from the SD rats in a traditional manner, and identified based on plastic adherence, morphology, and surface phenotype assays. After induction with GDNF and BDNF, viability of BMSCs was detected by MTT assay and neuronal differentiation of BMSCs was confirmed by using immunofluorescence and Western blotting. Besides, the number of BMSCs that obviously exhibited neuronal morphology was counted and the results were compared between the GDNF-induction group and BDNF-induction groups. Our results indicate that direct adherence was a simple and convenient method for isolation and cultivation of BMSCs. Furthermore, BMSCs can be induced in vitro to differentiate into neuronal cells by using GDNF, which could achieve a more persistent and stable inducing effect than when using BDNF.

A Quantitative Comparison of Human HT-1080 Fibrosarcoma Cells and Primary Human Dermal Fibroblasts Identifies a 3D Migration Mechanism with Properties Unique to the Transformed Phenotype

Here, we describe an engineering approach to quantitatively compare migration, morphologies, and adhesion for tumorigenic human fibrosarcoma cells (HT-1080s) and primary human dermal fibroblasts (hDFs) with the aim of identifying distinguishing properties of the transformed phenotype. Relative adhesiveness was quantified using self-assembled monolayer (SAM) arrays and proteolytic 3-dimensional (3D) migration was investigated using matrix metalloproteinase (MMP)-degradable poly(ethylene glycol) (PEG) hydrogels (“synthetic extracellular matrix” or “synthetic ECM”). In synthetic ECM, hDFs were characterized by vinculin-containing features on the tips of protrusions, multipolar morphologies, and organized actomyosin filaments. In contrast, HT-1080s were characterized by diffuse vinculin expression, pronounced β1-integrin on the tips of protrusions, a cortically-organized F-actin cytoskeleton, and quantitatively more rounded morphologies, decreased adhesiveness, and increased directional motility compared to hDFs. Further, HT-1080s were characterized by contractility-dependent motility, pronounced blebbing, and cortical contraction waves or constriction rings, while quantified 3D motility was similar in matrices with a wide range of biochemical and biophysical properties (including collagen) despite substantial morphological changes. While HT-1080s were distinct from hDFs for each of the 2D and 3D properties investigated, several features were similar to WM239a melanoma cells, including rounded, proteolytic migration modes, cortical F-actin organization, and prominent uropod-like structures enriched with β1-integrin, F-actin, and melanoma cell adhesion molecule (MCAM/CD146/MUC18). Importantly, many of the features observed for HT-1080s were analogous to cellular changes induced by transformation, including cell rounding, a disorganized F-actin cytoskeleton, altered organization of focal adhesion proteins, and a weakly adherent phenotype. Based on our results, we propose that HT-1080s migrate in synthetic ECM with functional properties that are a direct consequence of their transformed phenotype.

RcsB determines the locus of enterocyte effacement (LEE) expression and adherence phenotype of Escherichia coli O157 : H7 spinach outbreak strain TW14359 and coordinates bicarbonate-dependent LEE activation with repression of motility

The 2006 US spinach outbreak of Escherichia coli O157 : H7, characterized by unusually severe disease, has been attributed to a strain (TW14359) with enhanced pathogenic potential, including elevated virulence gene expression, robust adherence and the presence of novel virulence factors. This study proposes a mechanism for the unique virulence expression and adherence phenotype of this strain, and further expands the role for regulator RcsB in control of the E. coli locus of enterocyte effacement (LEE) pathogenicity island. Proteomic analysis of TW14359 revealed a virulence proteome consistent with previous transcriptome studies that included elevated levels of the LEE regulatory protein Ler and type III secretion system (T3SS) proteins, secreted T3SS effectors and Shiga toxin 2. Basal levels of the LEE activator and Rcs phosphorelay response regulator, RcsB, were increased in strain TW14359 relative to O157 : H7 strain Sakai. Deletion of rcsB eliminated inherent differences between these strains in ler expression, and in T3SS-dependent adherence. A reciprocating regulatory pathway involving RcsB and LEE-encoded activator GrlA was identified and predicted to co-ordinate LEE activation with repression of the flhDC flagellar regulator and motility. Overexpression of grlA was shown to increase RcsB levels, but did not alter expression from promoters driving rcsB transcription. Expression of rcsDB and RcsB was determined to increase in response to physiological levels of bicarbonate, and bicarbonate-dependent stimulation of the LEE was shown to be dependent on an intact Rcs system and ler activator grvA. The results of this study significantly broaden the role for RcsB in enterohaemorrhagic E. coli virulence regulation.

Expression of the domain cassette 8 Plasmodium falciparum erythrocyte membrane protein 1 is associated with cerebral malaria in Benin.

Background Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP-1) is a highly polymorphic adherence receptor expressed on the surface of infected erythrocytes. Based on sequence homology PfEMP-1 variants have been grouped into three major groups A-C, the highly conserved VAR2CSA variants, and semi-conserved types defined by tandem runs of specific domains (“domain cassettes” (DC)). The PfEMP-1 type expressed determines the adherence phenotype, and is associated with clinical outcome of infection.MethodsParasite isolates from Beninese children or women presenting with, respectively, CM or PAM were collected along with samples from patients with uncomplicated malaria (UM). We assessed the transcript level of var genes by RT-qPCR and the expression of PfEMP-1 proteins by LC-MS/MS.Results Var genes encoding DC8 and Group A PfEMP-1 were transcribed more often and at higher levels in cerebral malaria vs. uncomplicated malaria patients. LC-MS/MS identified peptides from group A, DC8 PfEMP-1 more frequently in cerebral malaria than in uncomplicated malaria and pregnancy-associated malaria samples.ConclusionThis is the first study to show association between PfEMP-1 subtype and disease outcome by direct analysis of parasites proteome. The results corroborate that group A and specifically the PfEMP-1 types DC8 are universally associated with cerebral malaria. This is a crucial observation for promoting studies on malaria pathogenesis.

Lability of the pAA Virulence Plasmid in Escherichia coli O104:H4: Implications for Virulence in Humans

Background Escherichia coli O104:H4 that caused the large German outbreak in 2011 is a highly virulent hybrid of enterohemorrhagic (EHEC) and enteroaggregative (EAEC) E. coli. The strain displays “stacked-brick” aggregative adherence to human intestinal epithelial cells mediated by aggregative adherence fimbriae I (AAF/I) encoded on the pAA plasmid. The AAF/I-mediated augmented intestinal adherence might facilitate systemic absorption of Shiga toxin, the major virulence factor of EHEC, presumably enhancing virulence of the outbreak strain. However, the stability of pAA in the outbreak strain is unknown. We therefore tested outbreak isolates for pAA, monitored pAA loss during infection, and determined the impact of pAA loss on adherence and clinical outcome of infection.Methodology/Principal Findings E. coli O104:H4 outbreak isolates from 170 patients (128 with hemolytic uremic syndrome [HUS] and 42 with diarrhea without HUS) were tested for pAA using polymerase chain reaction and plasmid profiling. pAA-harboring bacteria in stool samples were quantified using colony blot hybridization, and adherence to HCT-8 cells was determined. Isolates from 12 (7.1%) patients lacked pAA. Analyses of sequential stool samples demonstrated that the percentages of pAA-positive populations in the initial stools were significantly higher than those in the follow-up stools collected two to eight days later in disease (P≤0.01). This indicates a rapid loss of pAA during infections of humans. The pAA loss was associated with loss of the aggregative adherence phenotype and significantly reduced correlation with HUS (P = 0.001).Conclusions/SignificanceThe pAA plasmid can be lost by E. coli O104:H4 outbreak strain in the human gut in the course of disease. pAA loss might attenuate virulence and diminish the ability to cause HUS. The pAA instability has clinical, diagnostic, epidemiologic, and evolutionary implications.

Exposure of Bifidobacterium longum subsp. infantis to Milk Oligosaccharides Increases Adhesion to Epithelial Cells and Induces a Substantial Transcriptional Response.

In this study, we tested the hypothesis that milk oligosaccharides may contribute not only to selective growth of bifidobacteria, but also to their specific adhesive ability. Human milk oligosaccharides (3′sialyllactose and 6′sialyllactose) and a commercial prebiotic (Beneo Orafti P95; oligofructose) were assayed for their ability to promote adhesion of Bifidobacterium longum subsp. infantis ATCC 15697 to HT-29 and Caco-2 human intestinal cells. Treatment with the commercial prebiotic or 3′sialyllactose did not enhance adhesion. However, treatment with 6′sialyllactose resulted in increased adhesion (4.7 fold), while treatment with a mixture of 3′- and 6′-sialyllactose substantially increased adhesion (9.8 fold) to HT-29 intestinal cells. Microarray analyses were subsequently employed to investigate the transcriptional response of B. longum subsp. infantis to the different oligosaccharide treatments. This data correlated strongly with the observed changes in adhesion to HT-29 cells. The combination of 3′- and 6′-sialyllactose resulted in the greatest response at the genetic level (both in diversity and magnitude) followed by 6′sialyllactose, and 3′sialyllactose alone. The microarray data was further validated by means of real-time PCR. The current findings suggest that the increased adherence phenotype of Bifidobacterium longum subsp. infantis resulting from exposure to milk oligosaccharides is multi-faceted, involving transcription factors, chaperone proteins, adhesion-related proteins, and a glycoside hydrolase. This study gives additional insight into the role of milk oligosaccharides within the human intestine and the molecular mechanisms underpinning host-microbe interactions.

Repression of Type-1 Fimbriae in Shiga Toxin-Producing Escherichia coli O91:H21 Isolated from Asymptomatic Human Carriers in Korea

Seventy-four Shiga toxin-producing Escherichia coli (STEC) isolates belonging to the serotype O91:H21 were isolated from 1,643 asymptomatic human carriers in a STEC outbreak at Gwangju in Korea. Although the isolates did not cause any symptoms, all of them produced Shiga toxins 1 (Stx1) and 2 (Stx2). In order to determine why these strains cause no symptoms, we explored the differences in virulence potential between the asymptomatic STEC O91:H21 isolates and symptomatic STEC O91:H21 strains (ATCC 51435 and ATCC 51434). The asymptomatic STEC O91:H21 isolates showed strongly reduced cytopathic effects compared with the symptomatic strains when intact bacterial cells were used as an inoculant. Moreover, we found a reduced adherence phenotype when testing asymptomatic strains on HeLa cells. Real-time quantitative PCR results suggest that transcriptional repression of the genes encoding type-1 fimbriae occurs in the asymptomatic isolates but not in the symptomatic strains.

Lipids: Free Fatty Acids, Eicosanoids, and Lysophospholipids and the Pro-Inflammatory Effects of Transfusion

Abstract SCI-48Stored packed red blood cells (PRBCs) have been implicated in increased morbidity and mortality in critically ill patients, patients requiring cardiothoracic surgery, and injured patients following blunt and/or penetrating trauma. Lipids are generated during the routine storage of cellular blood components and have been implicated in transfusion-related acute lung injury (TRALI) and postinjury multiple organ failure (MOF). These lipids are comprised of two classes, as denoted by their retention times on normal-phase HPLC: 1) nonpolar lipids, including arachidonic acid, 5-hydroxy-eicosotetranoic acid (HETE), 12-HETE, 15-HETE, and 5-oxo-eicosotetranoic acid (5-oxo-ETE); and 2) a mixture of lysophosphatidylcholines (lyso-PCs), including stearoyl-, oleoyl-, or palmitoyl-lyso-PCs as well as C16 or C18 lyso-platelet activating factor. Importantly, all of these lipids were identified by quadrupole LC/MS/MS. The former are derived from red blood cells, because they are generated during the routine storage of prestorage leuko- and platelet-reduced PRBCs, while the latter are platelet-derived, and are generated during storage of platelet concentrates (PC) and unmodified PRBCs but not leukocyte-reduced PRBCs (LR-PRBCs). Generation of the nonpolar lipids requires an active phospholipase, such as peroxiredoxin-6, which accumulates during PRBC and LR-PRBC storage and appears to be active and T-phosphorylated. The nonpolar lipids do not accumulate during PC storage. These lipids are released into the plasma fraction of the stored component and have been shown to: 1) prime the polymorphonuclear leukocytes (PMN) oxidase; 2) activate primed PMNs; and 3) serve as the second event in a two-event model of TRALI in both rodents and sheep. This lipid priming of PMNs occurs through activation of specific receptors, for example G2A, for lyso-PCs, and causes activation of G-protein-linked cellular receptor, G-protein release, and stimulation of kinase cascades. This results in translocation of the cytosolic oxidase components and a change from a resting PMN phenotype to a hyperactive, adherent phenotype. Conversely, stored PRBCs have also been implicated in MOF. However, acute lung injury does not occur until 72 hours postinjury, implicating PRBCs as a first event(s). The nonpolar lipids that accumulate during routine storage activate primary human pulmonary microvascular endothelial cells (HMVECs) and primary human liver sinusoidal endothelial cells (LSECs), as quantified by increased surface expression of intercellular adhesion molecule-1 (ICAM-1) and the synthesis and release of chemokines, for example IL-8. This proinflammatory activation results in PMN adherence, and such HMVEC activation occurs through activation of the BLT2 receptor and activation of a PKC-dependent kinase cascade. Stored PRBCs (day 42) may serve as the first event in a two-event rodent model of acute lung injury. We conclude that lipids generated during the routine storage of cellular blood components have proinflammatory effects in vitro and in vivo, which may explain some of the adverse events of transfusion, including TRALI and postinjury MOF, and that inhibition and avoidance of these compounds may make transfusions safer. Further research is already under way in several prospective trials, including: 1) Age of Blood Components transfused in the PICU (ABC-PICU); 2) red cell storage duration and outcomes in cardiac surgery at the Cleveland Clinic; 3) Red Cell Storage duration Study (RECESS); and 4) the Age of Blood Evaluation trial (ABLE). Disclosures:Silliman:Pall Corporation: Honoraria, Research Funding.

Loss of the Malignant Phenotype of Human Neuroblastoma Cells by a Catalytically Inactive Dominant-Negative hTERT Mutant

Telomerase, a ribonucleoprotein complex mainly composed of the reverse transcriptase catalytic subunit (human telomerase reverse transcriptase, hTERT) and the RNA component (hTR), is a key enzyme of cancer progression. That aggressive stage 4-neuroblastoma expressed high levels of telomerase activity, whereas favorable tumors had no or little telomerase expression and activity, prompted us to investigate the role of this enzyme in this tumor model of altered proliferation, neuronal differentiation, and apoptosis. A human MYCN-amplified neuroblastoma cell line (IGR-N-91) was engineered to stably express either the normal hTERT protein (WT-hTERT) or a catalytically inactive dominant-negative mutant of this protein (DN-hTERT). We showed that DN-hTERT expression inhibited the endogenous hTERT in the malignant neuroblasts without telomere shortening nor loss of in vitro proliferative capacity. Importantly, DN-hTERT expression induced major changes in cell morphology of neuroblasts that switched them from a neuronal to a substrate adherent phenotype, which was more prone to apoptosis and lost their tumorigenic properties in nude mice. These biologic effects arose from modifications in the expression of genes involved in both apoptosis and neuroblastoma biology. Taken together these results highlighted the functional relevance of noncanonical functions of hTERT in the determination of neuroblast cell fate. Therefore, our results envision new therapeutic strategies for metastatic neuroblastoma therapeutic management.

The Role of Autophagy in Crohn’s Disease

(Macro)-autophagy is a homeostatic process by which eukaryotic cells dispose of protein aggregates and damaged organelles. Autophagy is also used to degrade micro-organisms that invade intracellularly in a process termed xenophagy. Genome-wide association scans have recently identified autophagy genes as conferring susceptibility to Crohn’s disease (CD), one of the chronic inflammatory bowel diseases, with evidence suggesting that CD arises from a defective innate immune response to enteric bacteria. Here we review the emerging role of autophagy in CD, with particular focus on xenophagy and enteric E. coli strains with an adherent and invasive phenotype that have been consistently isolated from CD patients with ileal disease.