Search

Rotavirus infection is highly prevalent in children, and the most severe effects are diarrhea and vomiting. It is well accepted that the enteric nervous system (ENS) is activated and plays an important role, but knowledge of how rotavirus activates nerves within ENS and to the vomiting center is lacking. Serotonin is released during rotavirus infection, and antagonists to the serotonin receptor subtype 3 (5-HT3 receptor) can attenuate rotavirus-induced diarrhea. In this study, we used a 5-HT3 receptor knockout (KO) mouse model to investigate the role of this receptor in rotavirus-induced diarrhea, motility, electrolyte secretion, inflammatory response, and vomiting reflex. The number of diarrhea days (P = 0.03) and the number of mice with diarrhea were lower in infected 5-HT3 receptor KO than wild-type pups. In vivo investigation of fluorescein isothiocyanate (FITC)-dextran transit time showed that intestinal motility was lower in the infected 5-HT3 receptor KO compared to wild-type mice (P = 0.0023). Ex vivo Ussing chamber measurements of potential difference across the intestinal epithelia showed no significant difference in electrolyte secretion between the two groups. Immediate early gene cFos expression level showed no difference in activation of the vomiting center in the brain. Cytokine analysis of the intestine indicated a low effect of inflammatory response in rotavirus-infected mice lacking the 5-HT3 receptor. Our findings indicate that the 5-HT3 receptor is involved in rotavirus-induced diarrhea via its effect on intestinal motility and that the vagus nerve signaling to the vomiting center occurs also in the absence of the 5-HT3 receptor. IMPORTANCE The mechanisms underlying rotavirus-induced diarrhea and vomiting are not yet fully understood. To better understand rotavirus pathophysiology, characterization of nerve signaling within the ENS and through vagal efferent nerves to the brain, which have been shown to be of great importance to the disease, is necessary. Serotonin (5-HT), a mediator of both diarrhea and vomiting, has been shown to be released from enterochromaffin cells in response to rotavirus infection and the rotavirus enterotoxin NSP4. Here, we investigated the role of the serotonin receptor 5-HT3, which is known to be involved in the nerve signals that regulate gut motility, intestinal secretion, and signal transduction through the vagus nerve to the brain. We show that the 5-HT3 receptor is involved in rotavirus-induced diarrhea by promoting intestinal motility. The findings shed light on new treatment possibilities for rotavirus diarrhea.

Rotavirus infection is highly prevalent in children, and the most severe effects are diarrhea and vomiting. It is well accepted that the enteric nervous system (ENS) is activated and plays an important role, but knowledge of how rotavirus activates nerves within ENS and to the vomiting center is lacking. Serotonin is released during rotavirus infection, and antagonists to the serotonin receptor subtype 3 (5-HT(3) receptor) can attenuate rotavirus-induced diarrhea. In this study, we used a 5-HT(3) receptor knockout (KO) mouse model to investigate the role of this receptor in rotavirus-induced diarrhea, motility, electrolyte secretion, inflammatory response, and vomiting reflex. The number of diarrhea days (P = 0.03) and the number of mice with diarrhea were lower in infected 5-HT(3) receptor KO than wild-type pups. In vivo investigation of fluorescein isothiocyanate (FITC)-dextran transit time showed that intestinal motility was lower in the infected 5-HT(3) receptor KO compared to wild-type mice (P = 0.0023). Ex vivo Ussing chamber measurements of potential difference across the intestinal epithelia showed no significant difference in electrolyte secretion between the two groups. Immediate early gene cFos expression level showed no difference in activation of the vomiting center in the brain. Cytokine analysis of the intestine indicated a low effect of inflammatory response in rotavirus-infected mice lacking the 5-HT(3) receptor. Our findings indicate that the 5-HT(3) receptor is involved in rotavirus-induced diarrhea via its effect on intestinal motility and that the vagus nerve signaling to the vomiting center occurs also in the absence of the 5-HT(3) receptor. IMPORTANCE The mechanisms underlying rotavirus-induced diarrhea and vomiting are not yet fully understood. To better understand rotavirus pathophysiology, characterization of nerve signaling within the ENS and through vagal efferent nerves to the brain, which have been shown to be of great importance to the disease, is necessary. Serotonin (5-HT), a mediator of both diarrhea and vomiting, has been shown to be released from enterochromaffin cells in response to rotavirus infection and the rotavirus enterotoxin NSP4. Here, we investigated the role of the serotonin receptor 5-HT(3), which is known to be involved in the nerve signals that regulate gut motility, intestinal secretion, and signal transduction through the vagus nerve to the brain. We show that the 5-HT(3) receptor is involved in rotavirus-induced diarrhea by promoting intestinal motility. The findings shed light on new treatment possibilities for rotavirus diarrhea.

The formation of nanodomains in the plasma membrane are thought to be part of membrane proteins regulation and signaling. Plasma membrane proteins are often investigated by analyzing the lateral mobility. k-space ICS (kICS) is a powerful image correlation spectroscopy (ICS) technique and a valuable supplement to fluorescence correlation spectroscopy (FCS). Here, we study the diffusion of aquaporin-9 (AQP9) in the plasma membrane, and the effect of different membrane and cytoskeleton affecting drugs, and therefore nanodomain perturbing, using kICS. We measured the diffusion coefficient of AQP9 after addition of these drugs using live cell Total Internal Reflection Fluorescence imaging on HEK-293 cells. The actin polymerization inhibitors Cytochalasin D and Latrunculin A do not affect the diffusion coefficient of AQP9. Methyl-beta-Cyclodextrin decreases GFP-AQP9 diffusion coefficient in the plasma membrane. Human epidermal growth factor led to an increase in the diffusion coefficient of AQP9. These findings led to the conclusion that kICS can be used to measure diffusion AQP9, and suggests that the AQP9 is not part of nanodomains. Funding Agencies|Villum Foundation [19105]

Human adenovirus 41 (HAdV-41) causes acute gastroenteritis in young children. The main characteristics of HAdV-41 infection are diarrhea and vomiting. Nevertheless, the precise mechanism of HAdV-41-induced diarrhea is unknown, as a suitable small-animal model has not been described. In this study, we used the human midgut carcinoid cell line GOT1 to investigate the effect of HAdV-41 infection and the individual HAdV-41 capsid proteins on serotonin release by enterochromaffin cells and on enteric glia cell (EGC) activation. We first determined that HAdV-41 could infect the enterochromaffin cells. Immunofluorescence staining revealed that the cells expressed HAdV-41-specific coxsackievirus and adenovirus receptor (CAR); flow cytometry analysis supported these findings. HAdV-41 infection of the enterochromaffin cells induced serotonin secretion dose dependently. In contrast, control infection with HAdV-5 did not induce serotonin secretion in the cells. Confocal microscopy studies of enterochromaffin cells infected with HAdV-41 revealed decreased serotonin immunofluorescence compared to that in uninfected cells. Incubation of the enterochromaffin cells with purified HAdV-41 short fiber knob and hexon proteins increased the serotonin levels in the harvested cell supernatant significantly. HAdV-41 infection could also activate EGCs, as shown in the significantly altered expression of glia fibrillary acidic protein (GFAP) in EGCs incubated with HAdV-41. The EGCs were also activated by serotonin alone, as shown in the significantly increased GFAP staining intensity. Likewise, EGCs were activated by the cell supernatant of HAdV-41-infected enterochromaffin cells. IMPORTANCE The nonenveloped human adenovirus 41 causes diarrhea, vomiting, dehydration, and low-grade fever mainly in children under 2 years of age. Even though acute gastroenteritis is well described, how human adenovirus 41 causes diarrhea is unknown. In our study, we analyzed the effect of human adenovirus 41 infection on human enterochromaffin cells and found it stimulates serotonin secretion in the cells, which is involved in regulation of intestinal secretion and gut motility and can also activate enteric glia cells, which are found in close proximity to enterochromaffin cells in vivo . This disruption of gut barrier homeostasis as maintained by these cells following human adenovirus 41 infection might be a mechanism in enteric adenovirus pathogenesis in humans and could indicate a possible serotonin-dependent cross talk between human adenovirus 41, enterochromaffin cells, and enteric glia cells.

Normal epithelial and endothelial renewal and healing after bacterial and viral challenges are essential for homeostasis along the intestine and the blood and lymphatic vessels. We thus investigated whether and how virus affects migration of human epithelial cells and specifically how the nucleocapsid protein (N) modulates the cellular proteome and interactome using human Caco-2 cells in a wound-healing assay with Hazara virus as a model. Here, Hazara virus blocked cell migration in a dose- and time-dependent manner, disrupted the actin cytoskeleton and specifically reduced the expression of the IQ-motif-containing GTPase-activating protein 1 (IQGAP1) and water channel aquaporin 6 (AQP6) that regulate cytoskeletal organization, water homeostasis and vesicle communication. Moreover, in the Caco-2 cell proteome, we identified several distinct groups of molecules associating with N upon Hazara virus infection, being involved in the ensemble of important cellular processes, e.g., chaperone activity, metabolism, cellular defense against infections, cell morphology, and migration. These events do not only facilitate the virus life cycle, but they are also crucial for membrane and cytoskeleton dynamics, cellular self-renewal and wound healing, being so essential for body integrity and homeostasis.

Celiac disease (CD) is a chronic small intestinal enteropathy triggered by gluten in genetically predisposed individuals. The susceptibility is strongly associated with certain human leukocyte antigen (HLA)-genes, but efforts are being made in trying to find non-HLA genes that are predictive for the disease. The criteria for diagnosing CD were previously based primarily on histologic evaluation of small intestinal biopsies, but nowadays are often based only on blood tests and symptoms. In this context, we elucidated the accuracy of three diagnostic indicators for CD, alone or in combination. Genetic analyses of HLA-type and nine single nucleotide polymorphisms (SNPs) known to be associated with CD were performed in 177 children previously investigated for the suspicion of CD. CD was confirmed in 109 children, while 68 were considered non-celiacs. The antibodies and urinary nitrite/nitrate concentrations of all of them were measured. The combinations of all the variables used in the study would classify 93% of the study population in the correct diagnostic group. The single best predictors were antibodies (i.e., anti-endomysium immunoglobulin A (IgA) (EMA) and transglutaminase IgA (TGA)), followed by HLA-type and nitric oxide (NO)-metabolites. The nine SNPs used did not contribute to the right diagnoses. Although our control group consisted of children with mostly gastrointestinal symptoms, the presented methodology predicted a correct classification in more than 90% of the cases.

Crimean–Congo hemorrhagic fever virus (CCHFV) is an arthropod-borne pathogen that causes infectious disease with severe hemorrhagic manifestations in vascular system in humans. The proper function of the cells in the vascular system is critically regulated by aquaporins (AQP), water channels that facilitate fluxes of water and small solutes across membranes. With Hazara virus as a model for CCHFV, we investigated the effects of viruses on AQP6 and the impact of AQP6 on virus infectivity in host cells, using transiently expressed GFP-AQP6 cells, immunofluorescent assay for virus detection, epifluorescent imaging of living cells and confocal microscopy. In GFP-AQP6 expressing cells, Hazara virus reduced both the cellular and perinuclear AQP6 distribution and changed the cell area. Infection of human cell with CCHFV strain IbAR 10200 downregulated AQP6 expression at mRNA level. Interestingly, the overexpression of AQP6 in host cells decreased the infectivity of Hazara virus, speaking for a protective role of AQP6. We suggest the possibility for AQP6 being a novel player in the virus–host interactions, which may lead to less severe outcomes of an infection.

In gram-negative bacteria, cell-cell communication based on HSL QS molecules is known to coordinate the production of virulence factors and biofilms. These bacterial signals can also modulate human immune cell behavior. Using a Transwell migration assay, we found that human primary neutrophils are strongly stimulated by 3O-C12-HSL and -C10-HSL but not C4-HSL in a concentration-dependent manner. Moreover, 3O-C12-HSL and -C10-HSL activate PLCγ1 but not -γ2, mobilize intracellular calcium, and up-regulate IP3R. These changes were paralleled by F-actin accumulation, primarily in the leading edge of neutrophils, as evidenced by phalloidin staining and confocal microscopy. F- and G-actin isolation and quantification by immunoblotting revealed that the F/G-actin ratio was increased significantly after treatment with all three HSLs. Furthemore, 3O-C12-HSL- and 3O-C10-HSL treatment resulted in phosphorylation of Rac1 and Cdc42. In contrast, C4-HSL had negligible influence on the phosphorylation status of PLC and Rac1/Cdc42 and failed to attract neutrophils and induce calcium release. The calcium inhibitor thapsigargin, which blocks ER calcium uptake, strongly prevented neutrophil migration toward 3O-C12-HSL and -C10-HSL. These findings show that the bacterial QS molecules 3O-C12-HSL and -C10-HSL may attract human neutrophils to the sites of bacterial infection and developing biofilms. Indeed, recognition of HSL QS signals by neutrophils may play a critical role in their recruitment during infections.

Pandgt;Background Patients with collagenous colitis have an impaired mucosal barrier. Moreover, collagenous colitis is associated with bile acid malabsorption. Bile acids can increase bacterial mucosal uptake in humans. Mucosal barrier function was investigated by exposing colonic biopsies to chenodeoxycholic acid (CDCA) or deoxycholic acid (DCA) in Ussing chamber experiments. Aim To find if low levels of bile acids increase bacterial uptake in colonic biopsies from collagenous colitis patients. Methods The study comprised 33 individuals; 25 with collagenous colitis (14 in clinical remission without treatment, 11 with active disease and 10 examined in clinical remission resulting from treatment with 6 mg budesonide); eight healthy individuals undergoing screening colonoscopy served as controls. Endoscopic biopsies from the sigmoid colon were mounted in modified Ussing chambers and assessed for short-circuit current (Isc), potential difference, trans-epithelial resistance and transmucosal passage of Escherichia coli K12 after adding 100 mu mol/L CDCA or DCA. Results When adding 100 mu mol/L CDCA or DCA, bacterial uptake increased fourfold in biopsies of patients in remission; CDCA 6.5 units [2.5-9.8] and DCA 6.2 units [2.1-22] (median [IQR]), compared with uptake in biopsies without added bile acids 1.6 units [1.1-3] (P = 0.004 and P = 0.01 respectively). In active disease and in patients in remission due to budesonide treatment, bile acids did not affect bacterial uptake. Confocal microscopy revealed trans-epithelial passage of E. coli K12 within 30 min. Conclusions Low concentrations of dihydroxy-bile acids exacerbate mucosal barrier dysfunction in colonic biopsies of patients with collagenous colitis in remission. This allows a substantially increased bacterial uptake, which may contribute to recurrence of inflammation.

C/EBPα and PU.1 are the basic transcription factors that control differentiation-related genes, including granulocyte- colony-stimulating factor (G-CSFR) and human neutrophil elastase (HNE). Here, we analyzed a role of C/EBPα and PU.1 in human acute leukemia cell lines, HL-60 and NB4, in association with a modified chromatin structure by histone deacetylase inhibitors, FK228, sodium phenyl butyrate and vitamin B3. We found that sodium phenyl butyrate alone and 6h-pretreatment with phenyl butyrate or FK228 before the induction of differentiation with all-trans-retinoic acid in the presence of vitamin B3 effectively accelerated and enhanced differentiation to granulocytes in HL-60 but not in NB4 cells as detected by NBT test and the expression of CD11b and CD114 (G-CSFR) using flow cytometric analysis. HDACIs induced a time- and dose-dependent accumulation of hyper-acetylated histone H4 in both cell lines with the delay in NB4 cells. Time-dependent different induction of HL-60 and NB4 cell differentiation was paralleled by the activation of C/EBPα and PU.1 binding to the G-CSFR and the HNE promoters in electrophoretic mobility shift assay. Chromatin immunoprecipitation analysis revealed histone H4 acetylation in the G-CSF receptor promoter at the C/EBPα binding site in HL-60 but not in NB4 cells under the combined treatment. The results indicate that epigenetic events, such as histone acetylation, are involved in the activity modulation of the key transcription factors responsible for the induction of granulocytic differentiation in promyelocytic leukemia cells.

Current cell therapy protocols require considerable numbers of mesenchymal stromal cells (MSCs), which can be obtained only by in vitro expansion. The most important issue is a choice of optimal growth supplements for cell culture. Ideally, these should be of known composition, free of animal components and allow production of large homogenic populations of MSCs in a considerably short period of time. Since this standard has not been achieved to date, we aimed to assess the molecular responses of MSCs to different growth supplements commonly in use. MSCs were isolated from breast or abdominal adipose tissue and plated into DMEM supplemented with one of four different sera: fetal calf serum (FCS), pretested fetal calf serum (FCS-Sp), human allogeneic serum (HS) or artificial serum substitute (AS). MSCs cultivated with different serum supplements demonstrated distinct morphologies, high adipogenic and osteogenic differentiation potential and expressed characteristic antigens. Using real-time PCR, we found a large increase in PPARγ and Msx2 gene expression in both lines of proliferating MSCs cultivated with AS. We found that MSCs cultivated in the presence of different sera had similar global proteomic expression patterns, but comparisons of identified proteins revealed most differences in the MSCs cultivated with AS. Our results indicate that MSCs cultivated in the presence of FCS and HS display similar growth, differentiation, immunophenotypic and proteomic properties, while AS induces more profound changes in the physiology of MSCs, suggesting that further fundamental studies should be done before its introduction into clinical practice.

We have previously shown that protein kinase C (PKC) activation has distinct effects on the structure and barrier properties of cultured epithelial cells (HT29 and MDCK I). Since the claudin family of tight junction (TJ)-associated proteins is considered to be crucial for the function of mature TJ, we assessed their expression patterns and cellular destination, detergent solubility and phosphorylation upon PKC stimulation for 2 or 18 h with phorbol myristate acetate (PMA). In HT29 cells, claudins 1, 3, 4 and 5 and possibly claudin 2 were redistributed to apical cell-cell contacts after PKC activation and the amounts of claudins 1, 3 and 5, but not of claudin 2, were increased in cell lysates. By contrast, in MDCK I cells, PMA treatment resulted in redistribution of claudins 1, 3, 4 and 5 from the TJ and in reorganization of the proteins into more insoluble complexes. Claudins 1 and 4 were phosphorylated in both MDCK I and HT29 cells, but PKC-induced changes in claudin phosphorylation state were detected only in MDCK I cells. A major difference between HT29 and MDCK I cells, which have low and high basal transepithelial electrical resistance, respectively, was the absence of claudin 2 in the latter. Our findings show that PKC activation targets in characteristic ways the expression patterns, destination, detergent solubility and phosphorylation state of claudins in epithelial cells with different capacities to form an epithelial barrier.

In Pseudomonas aeruginosa, cell-cell communication based on acyl-homoserine lactone (HSL) quorum sensing molecules is known to coordinate the production of virulence factors and biofilms by the bacterium. Incidentally, these bacterial signals can also modulate mammalian cell behaviour. We demonstrate here that 3O-C(12)-HSL can induce changes in calcium signalling through influx and release of calcium from thapsigargin-sensitive stores and delocalization of inositol 1,4,5-trisphosphate receptors (IP(3)R), but not of ryanodine receptors (RyR). In parallel, P. aeruginosa 3O-C(12)-HSL disrupts junctions in human Caco-2 cells as evidenced by a reduction of the expression and distribution of ZO-3 and JAM-A. Using co-immunoprecipitation we also found an alteration in the binding of ZO-3 to JAM-A in protein complexes. Moreover, 3O-C(12)-HSL-treatment resulted in tyrosine hyperphosphorylation of ZO-3 and JAM-A. On the contrary, serine and threonine residues of ZO-1 and JAM-A became less phosphorylated after exposition of 3O-C(12)-HSL. The 3O-C(12)-HSL-induced intracellular calcium signalling and alteration in the phosphorylation status of junction proteins furthermore correlated with changes in the association between JAM-A-ZO-3. The calcium inhibitors thapsigargin, xestospongin C, and dantrolene partly prevented the 3O-C(12)-HSL-induced decreases in TER and increases in the paracellular flux of 10kDa dextran. These findings clearly suggest that P. aeruginosa 3O-C(12)-HSL can cause the loss of epithelial barrier function via calcium signalling and further alteration in the phosphorylation status of junction proteins; and that bacterial quorum sensing signals represent inter-kingdom signalling.

Objective. The aim of this study was to investigate the metabolic function of intestinal microflora in children with screening-detected celiac disease (CD) to see if there is an aberrant gut flora in screening-detected CD similar to symptomatic CD and contrary to healthy controls. Materials and methods. As part of a Swedish multicenter screening for CD, 912 12-year-old children were screened with serum anti-human tissue transglutaminase-IgA. Small bowel biopsy specimens from children with positive serology revealed 17 individuals with CD. The functional status of the intestinal microflora was evaluated by gas liquid chromatography of short chain fatty acids (SCFAs) in fecal samples. Our previously published findings in children with symptomatic CD and healthy controls were used as comparison. Results. The children with screening-detected CD had a similar fecal SCFA profile to children with symptomatic CD, but differed significantly from that in healthy children. Conclusions. This is the first study on SCFA patterns in fecal samples from children with screening-detected CD. The similarity of the fecal SCFA profile in screening-detected and symptomatic CD indicates common pathogenic mechanisms. This could open the way for new therapeutic or prophylactic measures based on novel biological principles. Original Publication:Bo Tjellstrom, Lars Stenhammar, Lotta Högberg, Karin Fälth-Magnusson, Karl-Eric Magnusson, Tore Midtvedt, Tommy Sundqvist and Elisabeth Norin, Screening-detected and symptomatic untreated celiac children show similar gut microflora-associated characteristics, 2010, SCANDINAVIAN JOURNAL OF GASTROENTEROLOGY, (45), 9, 1059-1062.http://dx.doi.org/10.3109/00365521.2010.483738Copyright: Informa Healthcarehttp://informahealthcare.com/

Actin-binding protein α-actinin-4 is a member of the spectrin superfamily; it is located in both the cytoplasm and nucleus. However, nuclear functions of α-actinin-4 are still not clear. In this study, we analyzed the composition of nuclear protein complexes associated with α-actinin-4 in A431 cells. Using 2D electrophoresis, we found that about 50 different proteins may be associated with nuclear α-actinin-4. Major proteins of these complexes were analyzed by mass spectrometry and the following proteins have been identified: β-actin, α- and β-tubulins, ribonucleoprotein A2/B1 (regulates splicing and is associated with β-actin), peroxiredoxin-1 (involved in oxidative stress), and glycolytic enzyme D-3-phosphoglycerate dehydrogenase. The detection of these proteins in nuclear complexes with α-actinin-4 may suggest that α-actinin-4 is implicated in transcription and splicing. The presence of β-actin in these complexes was confirmed by tandem mass spectrometry (MALDI-TOF-TOF). The immunoprecipitation of nuclear proteins with antibodies against α-tubulin proved the association of α-actinin-4 with α-tubulin in the protein complex. Nuclear α-actinin-4 consists of a 105-kDa, full-size isoform and two truncated isoforms of 65 and 75 kDa; only truncated isoforms have been found in nuclear complexes with α-tubulin. These data suggest that α-actinin-4 is associated with a number of various nuclear protein complexes that may be able to carry out different functions in the cell nucleus.

The extracellular matrix (ECM) is a highly organized multimolecular structure essential for the vital functions of any organism. Although much of the data of extracellular matrix components has been accumulated, the isolation of an entire set of these proteins remains a complex procedure due to the high content of fibrillar proteins and proteoglycans, which form multidomain, netlike structures. In the study presented, we developed a method for isolating ECM proteins from cell cultures. Human epidermoid carcinoma cells A431 and fibroblasts obtained from normal and scar human skin were used. We showed that EDTA solution removed cells from culture plates without destroying the cell membranes. Subsequent treatment of remaining ECM proteins with acetic acid in order to dissociate collagen fibers significantly improved the fractioning of ECM proteins. The extraction of remaining proteins from the surface of the culture plate was preformed by a buffer developed based on Laemmli probe buffer. Using this method, we isolated ECM proteins synthesized by cultured cells, and the extracted proteins were suitable for future analysis by SDS PAGE and two-dimentional electrophoresis, as well as for identifying individual proteins by mass spectrometry. This study may allow us to compare assortments of ECM proteins isolated from different sources, and elucidate impact of various proteins on structure and property of extracellular matrix of investigated cells.

Acute promyelocytic leukemia KG1 cells with t(11;17) PLZF-RARalpha respond poorly to the differentiation inducer all-trans retinoic acid (RA), and the reason for the RA resistance is the recruitment of histone deacetylase by PLZF-RARalpha. Here, we demonstrate that histone deacetylase inhibitors (HDACIs), FK228, BML-210, phenyl butyrate, and vitamin B3, in different combinations with RA, act as KG1 cell growth inhibitors. Partial differentiation to granulocytes was induced by 3 micromol/L RA, and its combination with HDAC inhibitors did not enhance RA-induced but potentiated apoptosis. HDACIs induced accumulation of hyperacetylated histone H4. Chromatin immunoprecipitation analysis has revealed phenyl butyrate and its combinations with RA and vitamin B3 cause histone H4 acetylation in the p21 promoter regions corresponding to p53 and/or Sp1 sites. This was coincident with the activation of the transcription factor p53-binding activity to the p21 promoter in electrophoretic mobility shift assay. The results indicate the possibility of using the combination of agents for therapeutic strategy in RA-resistant acute myeloid leukemia to produce both differentiation and apoptosis.

Ganoderma sinensis has been used widely in Oriental countries for the prevention and treatment of various diseases including cancer. Previous studies have shown that the lipid extract from Ganoderma exhibits direct cytotoxicity against tumor cells. Here, it is reported that the lipid extract from germinating G. sinensis spores, at lower concentrations that have no direct tumoricidal activity, induce potent antitumor immune responses in human monocytes/macrophages. Upon stimulation with the lipid extract, monocytes/macrophages exhibited markedly increased production of proinflammatory cytokines and surface expression of costimulatory molecules. Conditioned medium from stimulated cells effectively suppressed the growth of tumor cells. Apparently, the lipid extract triggered macrophage activation via a mechanism different from that associated with LPS. Moreover, it was observed that the lipid extract could partially re-establish the antitumor activity of the immunosuppressive tumor-associated macrophages. These results indicated that in addition to its direct tumoricidal activity, the lipid extract from G. sinensis spores could exert antitumor activity by stimulating the activation of human monocytes/macrophages.

Transmembrane water fluxes through aquaporins (AQPs) are suggested to play pivotal roles in cell polarization and directional cell motility. Local dilution by water influences the dynamics of the subcortical actin polymerization and directs the formation of nascent membrane protrusions. In this paper, recent evidence is discussed in support of such a central role of AQP in membrane protrusion formation and cell migration as a basis for our understanding of the underlying molecular mechanisms of directional motility. Specifically, AQP9 in a physiological context controls transmembrane water fluxes driving membrane protrusion formation, as an initial cellular response to a chemoattractant or other migratory signals. The importance of AQP-facilitated water fluxes in directional cell motility is underscored by the observation that blocking or modifying specific sites in AQP9 also interferes with the molecular machinery that govern actin-mediated cellular shape changes.

Recurrent Crohn's disease originates with small erosions in the follicle-associated epithelium overlying the Peyer's patches. Animal studies have illustrated mucosal immune regulation by dendritic cells located in the subepithelial dome. The aim of this study was to characterize the dendritic cells at this specific site in patients with Crohn's disease. Ileal tissues were obtained after surgery performed on Crohn's patients; ileal samples from noninflammatory bowel disease and ulcerative colitis served as standard and inflammatory controls, respectively. Flow cytometry of isolated intestinal mononuclear cells showed a larger subset of dendritic cells in Crohn's samples compared with controls. This finding was corroborated by confocal microscopy, showing enhanced infiltrates of cells positive for the dendritic cell markers, DC-SIGN(+) and CD83(+), in the subepithelial dome. Moreover, the CD83(+) cells in Crohn's tissues showed reduced expression of the lymph node migratory receptor, CCR7, possibly contributing to the high numbers of dendritic cells. After exposure to nonpathogenic Escherichia coli in Ussing chambers, dendritic cells in the subepithelial dome of Crohn's disease demonstrated increased co-localization with translocated bacteria. Immunohistochemical results revealed that DC-SIGN(+) cells in Crohn's tissues were found to express toll-like receptor 4 and produce tumor necrosis factor-alpha. In conclusion, nonmigrating dendritic cells that accumulate in the subepithelial dome and internalize nonpathogenic bacteria may be important for the onset and perpetuation of mucosal inflammation in Crohn's disease.

In Pseudomonas aeruginosa, cell-cell communication based on acyl-homoserine lactone (HSL) quorum sensing molecules is known to coordinate the production of virulence factors and biofilms by the bacterium. Incidentally, these bacterial signals can also modulate mammalian cell behaviour. We report that 3O-C(12)-HSL can disrupt adherens junctions in human epithelial Caco-2 cells as evidenced by a reduction of the expression and distribution of E-cadherin and beta-catenin. Using co-immunoprecipitation we also found that P. aeruginosa 3O-C(12)-HSL-treatment resulted in tyrosine hyperphosphorylation of E-cadherin, beta-catenin, occludin and ZO-1. Similarly, serine and threonine residues of E-cadherin and ZO-1 became more phosphorylated after 3O-C(12)-HSL treatment. On the contrary, occludin and beta-catenin underwent dephosphorylation on serine and threonine residues after exposition of 3O-C(12)-HSL. These changes in the phosphorylation state were paralleled by alteration in the structure of junction complexes and increased paracellular permeability. Moreover, pre-treatment of the Caco-2 cells with protein phosphatase and kinase inhibitors prevented 3O-C(12)-HSL-induced changes in paracellular permeability and interactions between occludin-ZO-1 and the E-cadherin-beta-catenin. These findings clearly suggest that an alteration in the phosphorylation status of junction proteins are involved in the changes in cell junction associations and enhanced paracellular permeability, and that bacterial signals are indeed sensed by the host cells.

Crohn's disease is characterized by a defect in intestinal barrier function, where bacteria are considered the most important inflammation-driving factor. Enteric bacteria, including E. coli and Yersinia spp, affect tight junctions in enterocytes, but little is known about bacterial effects on the transcellular pathway. Our objective was to study the short-term effects of Y. pseudotuberculosis on uptake of nanoparticles across human villus epithelium. Monolayers of human colon epithelium-derived Caco-2 cells and biopsies of normal human ileum were studied after 2 h exposure to Y. pseudotuberculosis expressing (inv+) or lacking (inv-) the bacterial adhesion molecule, invasin. Transepithelial transport of fluorescent nanoparticles (markers of transcytosis) was quantified by flow cytometry, and mechanisms explored by using inhibitors of endocytosis. Epithelial expressions of beta1-integrin and particle uptake pathways were studied by confocal microscopy. The paracellular pathway was assessed by electrical resistance (TER), mannitol flux, and expression of tight junction proteins occludin and caludin-4 by confocal microscopy. Inv+ Y. pseudotuberculosis adhered to the apical surface of epithelial cells and induced transcytosis of exogenous nanoparticles across Caco-2 monolayers (30-fold increase, P

Chemotaxis is the stimulated directional migration of cells in response to chemotactic factors, manifested for instance during leukocyte interaction with chemoattractants in inflammation. The N-formyl-Met-Leu-Phe (fMLF) bacterial peptide family is particularly potent in attracting and activating neutrophilic granulocytes. To accomplish defined circumstances for recruitment and activation of cells, we fabricated semitransparent gold-coated glass coverslips functionalized with chemoattractant fMLF receptor peptide agonist analogues. Peptides based on a common leading four-amino-acid sequence Gly-Gly-Gly-Cys were thus coupled to two potent fMLF receptor agonists, N-formyl-Tyr-Nle-Phe-Leu- Nle-Gly-Gly-Gly-Cys and N-formyl-Met-Leu-Phe-Gly-Gly-Gly-Cys, and a formylated control peptide, N-formyl-Gly-Gly-Gly-Cys. They were anchored via the SH group of Cys either directly to the gold surface or a mixed self-assembled monolayer composed of maleimide- and hydroxyl-terminated oligo(ethylene glycol) alkyldisulfides. The overall peptide immobilization procedure was characterized with ellipsometry, contact angle measurement, and infrared spectroscopy. When exposed to granulocytes, the agonist surface rapidly recruited neutrophils and the cells responded with extensive spreading and intracellular calcium transients within minutes. The reference peptide generated no such activation, and the cells maintained a more spherical morphology, suggesting that we have been able to immobilize chemoattractant receptor agonist peptides with retained bioactivity. This is a crucial step in designing surfaces with specific effects on cellular behavior. © 2008 American Chemical Society.

The NF-kappaB/RelA family of transcription factors regulates inducible transcription of a large number of genes in response to diverse stimuli. Little is known, however, about the location of NF-kappaB in the cytoplasm and the transport mechanism to the nucleus. We found that NF-kappaB is associated with the actin-binding protein alpha-actinin-4. NF-kappaB and alpha-actinin-4 co-localized along actin stress fibers and in membrane lamellae in A431 cells. After a 30-min stimulation with EGF or TNF-alpha, alpha-actinin-4 and p65 were found in the nucleus. Disruption of cytoskeleton by cytochalasin D prior to treatment with TNF-alpha led to increase of p65 nuclear translocation. Antibodies to p65 subunit of NF-kappaB co-immunoprecipitated alpha-actinin-4 from A431 cell lysates and nuclear extracts, but alpha-actinin-1 and beta-actin were not found in the precipitates. Affinity chromatography experiments displayed that p65 and p50 subunits of NF-kappaB can bind to matrix-bound chicken gizzard alpha-actinin. We suggest that the alpha-actinin-4 is important for the NF-kappaB nuclear translocation and its functions inside the nucleus.

The protective immune response against the parasite, including the role of dendritic cells (DC) in the course of infection, plays a fundamental role. This study shows that wild-type (WT) Leishmania promastigotes and specifically the phosphoglycans family of virulence-associated antigens inhibit human monocyte-derived dendritic cells (MoDC) maturation and detachment to distinct surfaces. Immature phagocytosis of Leishmania donovani promastigotes by immature MoDC results in the increased expression of CD11b and CD51, and inhibition of cell detachment to distinct surfaces, which was dependent on the presence of phosphoglycans. These findings demonstrate that phosphoglycans of WT L. donovani might also inhibit human DC migration to lymphoid organs.

Quorum sensing (QS) communication allows Pseudomonas aeruginosa to collectively control its population density and the production of biofilms and virulence factors. QS signal molecules, like N-3-oxo-dodecanoyl-L-homoserine lactone (30-C-12-HSL), can also affect the behavior of host cells, e.g., by modulating the chemotaxis, migration, and phagocytosis of human leukocytes. Moreover, host water homeostasis and water channels aquaporins (AQP) are critical for cell morphology and functions as AQP interact indirectly with the cell cytoskeleton and signaling cascades. Here, we investigated how P aeruginosa 30-C-12-HSL affects cell morphology, area, volume and AQP9 expression and distribution in human primary macrophages, using quantitative PCR, immunoblotting, two- and three-dimensional live imaging, confocal and nanoscale imaging. Thus, 30-C-12-HSL enhanced cell volume and area and induced cell shape and protrusion fluctuations in macrophages, processes tentatively driven by fluxes of water across cell membrane through AQP9, the predominant AQP in macrophages. Moreover, 30-C-12-HSL upregulated the expression of AQP9 at both the protein and mRNA levels. This was accompanied with enhanced whole cell AQP9 fluorescent intensity and redistribution of AQP9 to the leading and trailing regions, in parallel with increased cell area in the macrophages. Finally, nanoscopy imaging provided details on AQP9 dynamics and architecture within the lamellipodial area of 30-C-12-HSL-stimulated cells. We suggest that these novel events in the interaction between P aeruginosa and macrophage may have an impact on the effectiveness of innate immune cells to fight bacteria, and thereby resolve the early stages of infections and inflammations. Funding Agencies|Swedish Research Council [2010-3045]; European Science foundation (TraPPs Euromembrane project); Magnus Bergvall Foundation; Faculty of Medicine and Health Sciences, Linkoping University

Rotavirus (RV) has been shown to infect and stimulate secretion of serotonin from human enterochromaffin (EC) cells and to infect EC cells in the small intestine of mice. It remains to identify which intracellularly expressed viral protein(s) is responsible for this novel property and to further establish the clinical role of serotonin in RV infection. First, we found that siRNA specifically silencing NSP4 (siRNA(NSP4)) significantly attenuated secretion of serotonin from Rhesus rotavirus (RRV) infected EC tumor cells compared to siRNA(VP4), siRNA(VP6) and siRNA(VP7). Second, intracellular calcium mobilization and diarrhoeal capacity from virulent and avirulent porcine viruses correlated with the capacity to release serotonin from EC tumor cells. Third, following administration of serotonin, all (10/10) infants, but no (0/8) adult mice, responded with diarrhoea. Finally, blocking of serotonin receptors using Ondansetron significantly attenuated murine RV (strain EDIM) diarrhoea in infant mice (2.9 vs 4.5 days). Ondansetron-treated mice (n = 11) had significantly (p amp;lt; 0.05) less diarrhoea, lower diarrhoea severity score and lower total diarrhoea output as compared to mock-treated mice (n = 9). Similarly, Ondansetron-treated mice had better weight gain than mock-treated animals (p amp;lt; 0.05). A most surprising finding was that the serotonin receptor antagonist significantly (p amp;lt; 0.05) also attenuated total viral shedding. In summary, we show that intracellularly expressed NSP4 stimulates release of serotonin from human EC tumor cells and that serotonin participates in RV diarrhoea, which can be attenuated by Ondansetron. Funding Agencies|Swedish Research Council [320301]; Diarrhoeal Disease Center, Linkoping University

Alpha-actinins are actin-binding proteins of non-muscle cells, which can participate in the regulation of transcription factor activity. We describe the distribution of alpha-actinin-1 and -4 depending on different actin cytoskeleton formed as a result of cell adhesion to extracellular matrix proteins, such as fibronectin and laminin 2/4. Immunofluorescent studies show a difference in the distribution of alpha-actinin and -4. Both isoforms localise along stress-fibres, but alpha-actinin-1 localises in the perinuclear region more abundantly than alpha-actinin-4. Western blot analysis demonstrated existence of truncated forms of both isoforms. Truncated alpha-actinin-1 appears in cells spread on fibronectin or laminin. Cell spreading also correlated with more tight association of alpha-actinin-4 with chromatin. Basing on our previous finding of an interaction of alpha-actinin-4 with p65 subunit of the NF-kappaB, we checked the possible influence of immobilised ligands on its redistribution in nuclear complexes containing p65. alpha-Actinin-4 seems to be present in some but not all nuclear complexes containing p65. Immobilised ligands may affect the interaction of alpha-actinin-4/p65 complexes with chromatin. The data suggest that adhesion to extra-cellular matrix may interfere in cellular reactions mediated by alpha-actinin-1 and -4.

Intracellular transport of organelles, vesicles and proteins is crucial in all eukaryotic cells, and is accomplished by motor proteins that move along cytoskeletal filaments. A widely used model of intracellular transport is Xenopus laevis melanophores. These cells help the frog to change color by redistributing melanin-containing organelles in the cytoplasm. The high contrast of the pigment organelles permits changes in distribution to be observed by ordinary light microscopy; other intracellular transport systems often require fluorescence labeling. Here we have developed white light Image Correlation Spectroscopy (ICS) to monitor aggregation and dispersion of pigment. Hitherto in ICS, images of fluorescent particles from Confocal Laser Scanning Microscopy (CLSM) have been used to calculate autocorrelation functions from which the density can be obtained. In the present study we show that ICS can be modified to enable analysis of light-microscopy images; it can be used to monitor pigment aggregation and dispersion, and distinguish between different stimuli. This new approach makes ICS applicable not only to fluorescent but also to black-and-white images from light or electron microscopy, and is thus very versatile in different studies of movement of particles on the membrane or in the cytoplasm of cells without potentially harmful fluorescence labeling and activation.

Helicobacter pylori produces outer membrane vesicles (OMV), delivering bacterial substances including the oncogenic cytotoxin-associated CagA protein to their surroundings. We investigated the effects of H. pylori OMV carrying CagA (OMV-CagA) on cell junctions and ATP-binding proteome of epithelial monolayers, using proteomics, mass spectrometry and imaging. OMV-CagA localized in close vicinity of ZO-1 tight junction protein and induced histone H1 binding to ATP. We suggest the expression of novel events in the interactions between H. pylori OMV and epithelia, which may have an influence on host gene transcription and lead to different outcomes of an infection and development of cancer.

Water-soluble vitamin B3, niacin, and its related compounds were suggested to be applicable for medical use. In this article, we examined the anti-leukemic effects of two distinct histone deacetylase (HDACI and Sir2) inhibitors, sodium phenyl butyrate (PB) and vitamin B3, respectively, on human promyelocytic leukemia cells HL-60, using HDACIs alone and in combination with all trans retinoic acid (RA). We demonstrated that the HDACI combinations exert different effects on cell cycle arrest and differentiation as determined by nitro blue reduction and the expression of the early myeloid differentiation marker CD11b. The most beneficial effects were found by use of 6-h pretreatment with PB and vitamin B3 before the exposition to RA alone or in combination with vitamin B3, showing significant acceleration and a high level of granulocytic differentiation. The effects were associated with a rapid histone 114 acetylation and later histone H3 modifications. Our results suggest that the use of two HDACI altogether before the induction of differentiation and acting via chromatin remodeling may be promising for the treatment of acute promyelocytic leukemia.

Human papillomavirus (HPV) infection is believed to be the central cause of cervical cancer. The viral proteins E6 and E7 from high-risk HPV types prevent cells from differentiating apoptosis and inducing hyperproliferative lesions. Human cervical carcinoma HeLa cells contain integrated human papillomavirus type 18 (HPV-18). Retinoic acid (RA) is a key regulator of epithelial cell differentiation and a growth inhibitor in vitro of HeLa cervical carcinoma cells. Cellular responses to RA are mediated by nuclear retinoic acid receptors (RARs) and retinoid X receptors. On the other hand, histone deacetylase inhibitors have been shown to be chemopreventive agents for the treatment of cancer cells. In this article, we have examined the antiproliferative effect of RA and histone deacetylase inhibitor BML-210 on HeLa cells, and particularly the effects on protein expression that may be involved in the cell cycle control and apoptosis. Our data suggest that a combination of RA and BML-210 leads to cell growth inhibition with subsequent apoptosis in a treatment time-dependent manner. We confirm that BML-210 alone or in combination with RA causes a marked increase in the level of p21. The changes in the p53 level are under the influence of p38 phosphorylation. We also discovered that the histone deacetylase inhibitor BML-210 causes increased levels of anti-apoptotic protein Bcl-2 and phosphorylated p38 MAP Kinase; the latter link in cell cycle arrest with response to extracellular stimuli. Our results suggest that RA and BML-210 are involved in different signaling pathways that regulate cell cycle arrest and lead to apoptosis of HeLa cells.

FK228 (depsipeptide) is a novel histone deacetylase inhibitor (HDACI) that has shown therapeutical efficacy in clinical trials for malignant lymphoma. In this article, we examined in vitro effects of FK228 on human leukemia cell lines, NB4 and HL-60. FK228 alone (0.2-1 ng/mL) inhibited leukemia cell growth in a dose-dependent manner and induced death by apoptosis. FK228 had selective differentiating effects on two cell lines when used for 6 h before induction of granulocytic differentiation by retinoic acid (RA) or in combination with RA. These effects were accompanied by a time- and dose-dependent histone H4 hyper-acetylation or histone H3 dephosphorylation and alterations in DNA binding of NF-kappaB in association with cell death and differentiation. Pifithrin-alpha (PFT), an inhibitor of p53 transcriptional activity, protected only NB4 cells with functional p53 from FK228-induced apoptosis and did not interfere with antiproliferative activity in p53-negative HL-60 cells. In NB4 cells, PFT inhibited p53 binding to the p21 (Waf1/Cip1) promotor and induced DNA binding of NF-kappaB leading to enhanced cell survival. Thus, beneficial effects of FK228 on human promyelocytic leukemia may be exerted through the induction of differentiation or apoptosis via histone modification and selective involvement of transcription factors, such as NF-kappaB and p53.

Histone deacetylase inhibitors have a potent role in the strategy for the treatment of leukemias. BML-210 (N-(2-Aminophenyl)-N' phenyloctanol diamine) is the novel histone deacetylase inhibitor, and its mechanism of action has not been characterized. In this study, we examined the in vitro effects of BML-210 on the human leukemia cell lines (NB4, HL-60, THP-1, and K562). We found that BML-210 inhibits the growth of all cell lines and promotes apoptosis in a dose- and time-dependent manner. BML-210 alone induces HL-60 and K562 cell differentiation (up to 30%) to granulocytes and erythrocytes, respectively, and in combination with differentiation agents - all-trans retinoic acid and hemin, markedly potentates it. Those treatments cause G1 arrest and histone H4 acetylation, affects transcription factor NF-kappaB and Sp1 binding activity to their consensus sequences, the p21 or the FasL promoters, and influences expression of Sp1, NF-kappaB, p21 and FasL. These findings suggest that BML-210 could be a promising antileukemic agent to induce apoptosis and to modulate differentiation through the modulation of histone acetylation and gene expression.

A novel biosensor for superoxide radical (O2{radical dot}-) detection based on Pseudomonas aeruginosa azurin immobilized on gold electrode was designed. The rate constant of azurin reduction by O2{radical dot}- was found to be 105 M-1 s-1 in solution and five times lower, i.e., 0.2 × 105 M-1 s-1, for azurin coupled to gold by 3,3′-dithiobis(sulfosuccinimidylpropionate) (DTSSP). The electron transfer rate between the protein and the electrode ranged from 2 to 6 s-1. The sensitivity of this biosensor to O2{radical dot}- was 6.8 × 102 A m-2 M-1. The response to the interference substances, such as uric acid, H2O2, and dimethylsulfoxide was negligible below 10 μM. The electrode was applied in three O2{radical dot}- generating systems: (i) xanthine oxidase (XOD), (ii) potassium superoxide (KO2), and (iii) stimulated neutrophil granulocytes. The latter was compared with luminol-amplified chemiluminescence. The biosensor responded to O2{radical dot}- in all three environments, and the signals were antagonized by superoxide dismutase. © 2006 Elsevier B.V. All rights reserved.

The protozoan parasite Leishmania donovani, the causative agent of visceral leishmaniasis, is transmitted to humans through bites by infected sand flies. Flagellated Leishmania promastigotes are phagocytosed by host macrophages, where they survive inside phagosomes (9, 27). Lipophosphoglycan (LPG), which is the major surface glycoconjugate of promastigotes, is essential for intracellular survival (10, 15, 23, 30). Phagosomes containing L. donovani mutants that lack the repeating sugars of LPG (lpg2−/− knockout [KO]) proceed through the phagolysosomal pathway, and the parasites are killed (10). The mechanism by which LPG arrests phagosomal maturation in macrophages is partially clarified and includes accumulation of F-actin around newly formed phagosomes (17). The effects of LPG on actin are in part related to inhibition of protein kinase Cα (PKCα), an enzyme implicated in F-actin depolymerization at the phagosomal membrane (2, 16, 17, 23, 25). Results from our group indicate that LPG from L. donovani interacts with GM1-enriched lipid microdomains (lipid rafts, detergent-resistant membranes) in the macrophage plasma membrane and that active PKCα colocalizes to a lesser degree with GM1 in these cells (unpublished data). Moreover, it was recently shown that LPG causes a disorganization of the phagosomal membrane, preventing normal assembly of GM1-enriched microdomains (8). In line with this, we found that disrupting membrane microdomains by cholesterol depletion, thereby mimicking the effect of LPG, raised the levels of periphagosomal F-actin around LPG-deficient lpg2−/− KO mutants. However, the levels of periphagosomal F-actin in cholesterol-depleted cells did not reach the levels observed around wild-type (WT) promastigotes (unpublished data). The present study investigates the involvement of the Rho family members Cdc42 and Rac1 in the generation of the F-actin shell formed around phagosomes containing L. donovani promastigotes. The Rho family of small GTPases, including Rho, Rac, and Cdc42, has emerged as main regulators of the cytoskeleton (11, 14). GTP-bound Rho GTPases drive cellular processes such as cytoskeletal reorganization, migration, and proliferation (7, 14, 28). Importantly, Rho GTPases play a profound role in phagocytosis of opsonized prey (6, 22, 26, 29); however, their role in nonopsonic phagocytosis is poorly understood. Dominant negative (N17) and constitutively active (V12) forms of Cdc42 and Rac1, expressed as fusion proteins with the human immunodeficiency virus-derived cell permeable peptide TAT, were used to study the effects of these proteins on periphagosomal F-actin and phagosomal maturation in RAW264.7 macrophages. We found that simultaneous introduction of N17Cdc42 and N17Rac1 prevented the accumulation of F-actin around phagosomes with WT L. donovani. Conversely, introduction of V12Cdc42 and V12Rac1 restored the accumulation of periphagosomal F-actin around phagosomes with LPG-defective promastigotes, resulting in inhibition of phagosomal maturation. Interestingly, introduction of N17Cdc42 and N17Rac1 into the cells, which reduced the levels of periphagosomal F-actin around WT L. donovani, did not promote maturation of these phagosomes. This result points toward a direct role for Cdc42 and Rac1 in phagosomal maturation. In conclusion, our results show that L. donovani requires functional Cdc42 and Rac1 to build up a coat of F-actin around its phagosome to prevent phagosomal maturation.