Sample records for cells elicits distinct

  1. Different Vaccine Vectors Delivering the Same Antigen Elicit CD8plus T Cell Responses with Distinct Clonotype and Epitope Specificity

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    M Honda; R Wang; W Kong; M Kanekiyo; Q Akahata; L Xu; K Matsuo; K Natarajan; H Robinson; et al.


    Prime-boost immunization with gene-based vectors has been developed to generate more effective vaccines for AIDS, malaria, and tuberculosis. Although these vectors elicit potent T cell responses, the mechanisms by which they stimulate immunity are not well understood. In this study, we show that immunization by a single gene product, HIV-1 envelope, with alternative vector combinations elicits CD8{sup +} cells with different fine specificities and kinetics of mobilization. Vaccine-induced CD8{sup +} T cells recognized overlapping third V region loop peptides. Unexpectedly, two anchor variants bound H-2D{sup d} better than the native sequences, and clones with distinct specificities were elicited by alternative vectors. X-ray crystallography revealed major differences in solvent exposure of MHC-bound peptide epitopes, suggesting that processed HIV-1 envelope gave rise to MHC-I/peptide conformations recognized by distinct CD8{sup +} T cell populations. These findings suggest that different gene-based vectors generate peptides with alternative conformations within MHC-I that elicit distinct T cell responses after vaccination.

  2. Distinct gut-derived lactic acid bacteria elicit divergent dendritic cell-mediated NK cell responses

    DEFF Research Database (Denmark)

    Fink, Lisbeth Nielsen; Zeuthen, Louise Hjerrild; Christensen, Hanne


    Lactic acid bacteria (LAB) are abundant in the gastrointestinal tract where they continuously regulate the immune system. NK cells are potently activated by dendritic cells (DCs) matured by inflammatory stimuli, and NK cells are present in the gut epithelium and in mesenteric lymph nodes...

  3. Mast cell degranulation distinctly activates trigemino-cervical and lumbosacral pain pathways and elicits widespread tactile pain hypersensitivity. (United States)

    Levy, Dan; Kainz, Vanessa; Burstein, Rami; Strassman, Andrew M


    Mast cells (MCs) are tissue resident immune cells that participate in a variety of allergic and other inflammatory conditions. In most tissues, MCs are found in close proximity to nerve endings of primary afferent neurons that signal pain (i.e. nociceptors). Activation of MCs causes the release of a plethora of mediators that can activate these nociceptors and promote pain. Although MCs are ubiquitous, conditions associated with systemic MC activation give rise primarily to two major types of pain, headache and visceral pain. In this study we therefore examined the extent to which systemic MC degranulation induced by intraperitoneal administration of the MC secretagogue compound 48/80 activates pain pathways that originate in different parts of the body and studied whether this action can lead to development of behavioral pain hypersensitivity. Using c-fos expression as a marker of central nervous system neural activation, we found that intraperitoneal administration of 48/80 leads to the activation of dorsal horn neurons at two specific levels of the spinal cord; one responsible for processing cranial pain, at the medullary/C2 level, and one that processes pelvic visceral pain, at the caudal lumbar/rostral sacral level (L6-S2). Using behavioral sensory testing, we found that this nociceptive activation is associated with development of widespread tactile pain hypersensitivity within and outside the body regions corresponding to the activated spinal levels. Our data provide a neural basis for understanding the primacy of headache and visceral pain in conditions that involve systemic MC degranulation. Our data further suggest that MC activation may lead to widespread tactile pain hypersensitivity.

  4. Deviating the level of proliferating cell nuclear antigen in Trypanosoma brucei elicits distinct mechanisms for inhibiting proliferation and cell cycle progression. (United States)

    Valenciano, Ana L; Ramsey, Aaron C; Mackey, Zachary B


    The DNA replication machinery is spatially and temporally coordinated in all cells to reproduce a single exact copy of the genome per division, but its regulation in the protozoan parasite Trypanosoma brucei is not well characterized. We characterized the effects of altering the levels of proliferating cell nuclear antigen, a key component of the DNA replication machinery, in bloodstream form T. brucei. This study demonstrated that tight regulation of TbPCNA levels was critical for normal proliferation and DNA replication in the parasite. Depleting TbPCNA mRNA reduced proliferation, severely diminished DNA replication, arrested the synthesis of new DNA and caused the parasites to accumulated in G2/M. Attenuating the parasite by downregulating TbPCNA caused it to become hypersensitive to hydroxyurea. Overexpressing TbPCNA in T. brucei arrested proliferation, inhibited DNA replication and prevented the parasite from exiting G2/M. These results indicate that distinct mechanisms of cell cycle arrest are associated with upregulating or downregulating TbPCNA. The findings of this study validate deregulating intra-parasite levels of TbPCNA as a potential strategy for therapeutically exploiting this target in bloodstream form T. brucei.

  5. Dendritic Cells Pulsed with Intact Streptococcus pneumoniae Elicit both Protein- and Polysaccharide-specific Immunoglobulin Isotype Responses In Vivo through Distinct Mechanisms (United States)


    response to TNP-haptenated antigens such as TNP-Ficoll, TNP-LP, and TNP- Brucella abortus , have been reported (8, 9). Dendritic cells (DCs) are...anti- CD8b.2), and negatively selected by magnetic bead cell sorting using a mixture of affinity purified polyclonal goat anti–rat IgG and goat anti...clonal goat anti–mouse IgM (Southern Biotechnology Associates, Inc.) were used to detect surface Cps14. Controls included pulsed BMDCs stained with

  6. Structurally distinct nicotine immunogens elicit antibodies with non-overlapping specificities (United States)

    Pravetoni, M; Keyler, DE; Pidaparthi, RR; Carroll, FI; Runyon, SP; Murtaugh, MP; Earley, CA; Pentel, PR


    Nicotine conjugate vaccine efficacy is limited by the concentration of nicotine-specific antibodies that can be reliably generated in serum. Previous studies suggest that the concurrent use of 2 structurally distinct nicotine immunogens in rats can generate additive antibody responses by stimulating distinct B cell populations. In the current study we investigated whether it is possible to identify a third immunologically distinct nicotine immunogen. The new 1′-SNic immunogen (2S)-N,N′-(disulfanediyldiethane-2,1-diyl)bis[4-(2-pyridin-3-ylpyrrolidin-1-yl)butanamide] conjugated to keyhole limpet hemocyanin (KLH) differed from the existing immunogens 3′-AmNic-rEPA and 6-CMUNic-BSA in linker position, linker composition, conjugation chemistry, and carrier protein. Vaccination of rats with 1′-SNic-KLH elicited high concentrations of high affinity nicotine-specific antibodies. The antibodies produced in response to 1′-SNic-KLH did not appreciably cross-react in ELISA with either 3′-AmNic-rEPA or 6-CMUNic-BSA or vice-versa, showing that the B cell populations activated by each of these nicotine immunogens were non-overlapping and distinct. Nicotine retention in serum was increased and nicotine distribution to brain substantially reduced in rats vaccinated with 1′-SNic-KLH compared to controls. Effects of 1′-SNic-KLH on nicotine distribution were comparable to those of 3′-AmNic-rEPA which has progressed to late stage clinical trials as an adjunct to smoking cessation. These data show that it is possible to design multiple immunogens from a small molecule such as nicotine which elicit independent immune responses. This approach could be applicable to other addiction vaccines or small molecule targets as well. PMID:22100986

  7. Distinct contractile and cytoskeletal protein patterns in the Antarctic midge are elicited by desiccation and rehydration. (United States)

    Li, Aiqing; Benoit, Joshua B; Lopez-Martinez, Giancarlo; Elnitsky, Michael A; Lee, Richard E; Denlinger, David L


    Desiccation presents a major challenge for the Antarctic midge, Belgica antarctica. In this study, we use proteomic profiling to evaluate protein changes in the larvae elicited by dehydration and rehydration. Larvae were desiccated at 75% relative humidity (RH) for 12 h to achieve a body water loss of 35%, approximately half of the water that can be lost before the larvae succumb to dehydration. To evaluate the rehydration response, larvae were first desiccated, then rehydrated for 6 h at 100% RH and then in water for 6 h. Controls were held continuously at 100% RH. Protein analysis was performed using 2-DE and nanoscale capillary LC/MS/MS. Twenty-four identified proteins changed in abundance in response to desiccation: 16 were more abundant and 8 were less abundant; 84% of these proteins were contractile or cytoskeletal proteins. Thirteen rehydration-regulated proteins were identified: 8 were more abundant and 5 were less abundant, and 69% of these proteins were also contractile or cytoskeletal proteins. Additional proteins responsive to desiccation and rehydration were involved in functions including stress responses, energy metabolism, protein synthesis, glucogenesis and membrane transport. We conclude that the major protein responses elicited by both desiccation and rehydration are linked to body contraction and cytoskeleton rearrangements.

  8. Object-location training elicits an overlapping but temporally distinct transcriptional profile from contextual fear conditioning. (United States)

    Poplawski, Shane G; Schoch, Hannah; Wimmer, Mathieu E; Hawk, Joshua D; Walsh, Jennifer L; Giese, Karl P; Abel, Ted


    Hippocampus-dependent learning is known to induce changes in gene expression, but information on gene expression differences between different learning paradigms that require the hippocampus is limited. The bulk of studies investigating RNA expression after learning use the contextual fear conditioning task, which couples a novel environment with a footshock. Although contextual fear conditioning has been useful in discovering gene targets, gene expression after spatial memory tasks has received less attention. In this study, we used the object-location memory task and studied gene expression at two time points after learning in a high-throughput manner using a microfluidic qPCR approach. We found that expression of the classic immediate-early genes changes after object-location training in a fashion similar to that observed after contextual fear conditioning. However, the temporal dynamics of gene expression are different between the two tasks, with object-location memory producing gene expression changes that last at least 2 hours. Our findings indicate that different training paradigms may give rise to distinct temporal dynamics of gene expression after learning.

  9. Inflammation-induced anorexia and fever are elicited by distinct prostaglandin dependent mechanisms, whereas conditioned taste aversion is prostaglandin independent. (United States)

    Nilsson, Anna; Wilhelms, Daniel Björk; Mirrasekhian, Elahe; Jaarola, Maarit; Blomqvist, Anders; Engblom, David


    Systemic inflammation evokes an array of brain-mediated responses including fever, anorexia and taste aversion. Both fever and anorexia are prostaglandin dependent but it has been unclear if the cell-type that synthesizes the critical prostaglandins is the same. Here we show that pharmacological inhibition or genetic deletion of cyclooxygenase (COX)-2, but not of COX-1, attenuates inflammation-induced anorexia. Mice with deletions of COX-2 selectively in brain endothelial cells displayed attenuated fever, as demonstrated previously, but intact anorexia in response to peripherally injected lipopolysaccharide (10μg/kg). Whereas intracerebroventricular injection of a cyclooxygenase inhibitor markedly reduced anorexia, deletion of COX-2 selectively in neural cells, in myeloid cells or in both brain endothelial and neural cells had no effect on LPS-induced anorexia. In addition, COX-2 in myeloid and neural cells was dispensable for the fever response. Inflammation-induced conditioned taste aversion did not involve prostaglandin signaling at all. These findings collectively show that anorexia, fever and taste aversion are triggered by distinct routes of immune-to-brain signaling.

  10. Single and mixed-species trypanosome and microsporidia infections elicit distinct, ephemeral cellular and humoral immune responses in honey bees. (United States)

    Schwarz, Ryan S; Evans, Jay D


    Frequently encountered parasite species impart strong selective pressures on host immune system evolution and are more apt to concurrently infect the same host, yet molecular impacts in light of this are often overlooked. We have contrasted immune responses in honey bees to two common eukaryotic endoparasites by establishing single and mixed-species infections using the long-associated parasite Crithidia mellificae and the emergent parasite Nosema ceranae. Quantitative polymerase chain reaction was used to screen host immune gene expression at 9 time points post inoculation. Systemic responses in abdomens during early stages of parasite establishment revealed conserved receptor (Down syndrome cell adhesion molecule, Dscam and nimrod C1, nimC1), signaling (MyD88 and Imd) and antimicrobial peptide (AMP) effector (Defensin 2) responses. Late, established infections were distinct with a refined 2 AMP response to C. mellificae that contrasted starkly with a 5 AMP response to N. ceranae. Mixed species infections induced a moderate 3 AMPs. Transcription in gut tissues highlighted important local roles for Dscam toward both parasites and Imd signaling toward N. ceranae. At both systemic and local levels Dscam, MyD88 and Imd transcription was consistently correlated based on clustering analysis. Significant gene suppression occurred in two cases from midgut to ileum tissue: Dscam was lowered during mixed infections compared to N. ceranae infections and both C. mellificae and mixed infections had reduced nimC1 transcription compared to uninfected controls. We show that honey bees rapidly mount complex immune responses to both Nosema and Crithidia that are dynamic over time and that mixed-species infections significantly alter local and systemic immune gene transcription.

  11. Nickel elicits a fast antioxidant response in Coffea arabica cells. (United States)

    Gomes-Junior, R A; Moldes, C A; Delite, F S; Gratão, P L; Mazzafera, P; Lea, P J; Azevedo, R A


    The antioxidant responses of coffee (Coffea arabica L.) cell suspension cultures to nickel (Ni) were investigated. Ni was very rapidly accumulated in the cells and the accumulation could be directly correlated with the increase of NiCl(2) concentration in the medium. At 0.05 mM NiCl(2) growth was stimulated, but at 0.5 mM NiCl(2), the growth rate was reduced. An indication of alterations in the presence of reactive oxygen species was detected by an increase in lipid peroxidation at 0.5 mM NiCl(2). Catalase (CAT; EC, glutathione reductase (GR; EC, ascorbate peroxidase (APX; EC, guaiacol peroxidase (GOPX; EC and superoxide dismutase (SOD; EC activities were increased, particularly at earlier NiCl(2) exposure times and the activities were higher at 0.5 mM NiCl(2) for most of exposure times tested. Non-denaturing PAGE revealed one CAT isoenzyme, nine SOD isoenzymes and four GR isoenzymes. The SOD isoenzymes were differentially affected by NiCl(2) treatment and one GR isoenzyme was increased by NiCl(2). NiCl(2) at 0.05 mM did not induce lipid peroxidation and the main response appeared to be via the induction of SOD, CAT, GOPX and APX activities for the removal of the reactive oxygen species and through the induction of GR to ensure the availability of reduced glutathione.

  12. Elicitation Phenolic Compounds in Cell Culture of Vitis vinifera L. by Phaeomoniella chlamydospora

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    Sák Martin


    Full Text Available The in vitro cell cultures of Vitis vinifera L. cv. St. Laurent were treated with two elicitors - synthetic methyl jasmonate and natural, prepared from grapevine plant infected with the Phaeomoniella chlamydospora, the agent causing the Esca disease of grapevine. Efficiency of phenolic compounds production after elicitation of cell culture was analysed immediately after treatment (15 min, 30 min, 60 min and later (after 24, 48, and 72 hours. The cell growth and content of phenolic compounds (+-catechin, (--epicatechin, p-coumaric acid, syringaldehyde, rutin, vanillic acid, and trans-resveratrol were analysed in cultivated cells as well as in cultivation medium. Pch-treatment increased production of total polyphenols the most significantly 15 min after the elicitation and in optimal time was 2.86 times higher than in nonelicited culture and 1.44 times higher than in MeJa induced cell culture.

  13. Secondary metabolite production in Hypericum perforatum L. cell suspensions upon elicitation with fungal mycelia from Aspergillus flavus

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    Gadzovska-Simic Sonja


    Full Text Available We investigated the production of phenylpropanoids (phenolic compounds, flavanols, flavonols and anthocyanins and naphtodianthrones (hypericins in elicited Hypericum perforatum L. cell suspensions. To determine whether secondary metabolite production could be enhanced, Hypericum cell suspensions were exposed to mycelia extract from the fungus Aspergillus flavus. Elicited Hypericum cell suspension cultures displayed reduced growth and viability and a modification of secondary metabolites production. Anthocyanins were only stimulated in fungal-elicited cell suspensions. Secondary metabolite production in elicited Hypericum cells revealed an antagonism between the flavonoid/naphtodianthrone and anthocyanin pathways. The data suggest a modification of the channeling of the phenylpropanoid compounds. Together, these results represent useful data for monitoring the channeling in different secondary metabolite pathways during the scaled-up production of naphtodianthrones for medicinal uses.

  14. Nanomolar ouabain elicits apoptosis through a direct action on HeLa cell mitochondria. (United States)

    Alonso, Elba; Cano-Abad, María F; Moreno-Ortega, Ana J; Novalbos, Jesús; Milla, Juan; García, Antonio G; Ruiz-Nuño, Ana


    The steroid Na(+)/K(+) ATPase (NKA) blocker ouabain has been shown to exhibit pro-apoptotic effects in various cell systems; however, the mechanism involved in those effects is unclear. Here, we have demonstrated that incubation of HeLa cells during 24h with nanomolar concentrations of ouabain or digoxin causes apoptotic death of 30-50% of the cells. Ouabain caused the activation of caspases-3/7 and -9; however, caspase-8 was unaffected. The fact that compound Z-LEHD-FMK reduced both apoptosis and caspase-9 activation elicited by ouabain, suggest a mitochondrially-mediated pathway. This was strengthened by the fact that ouabain caused ATP depletion and the release of mitochondrial cytochrome c into the cytosol. Furthermore, upon ouabain treatment mitochondrial disruption and redistribution into the cytosol were observed. A mitochondrial site of action for ouabain was further corroborated by tight co-localisation of fluorescent ouabain with mitochondria. Finally, in ouabain-treated cells the histamine-elicited elevation of cytosolic Ca(2+) concentration ([Ca(2+)]c) suggests an additional effect on the endoplasmic reticulum (ER) leading to Ca(2+) store depletion. We conclude that fluorescent ouabain is taken up and tightly co-localises with mitochondria of HeLa cells. This indicates that apoptosis may be triggered by a direct action of ouabain on mitochondria.

  15. [Gastric signet ring cell adenocarcinoma: A distinct entity]. (United States)

    Tabouret, Tessa; Dhooge, Marion; Rouquette, Alexandre; Brezault, Catherine; Beuvon, Frédéric; Chaussade, Stanislas; Coriat, Romain


    Gastric signet ring cell carcinoma (GSRC) is a distinct entity. Their incidence is increasing. The pathologist plays a central role in the identification of this entity. Diagnosis is based on an adenocarcinoma containing a majority of signet ring cells (above 50 %). The prognosis of GSRC is the same as gastric adenocarcinoma while GSRC appeared more aggressive. Signet ring cells present a low sensitivity to chemotherapy. This review aimed to discuss the histological, the prognostic and the therapeutic aspect of this entity.

  16. Effect of Agrobacterium rhizogenes and elicitation on the asiaticoside production in cell cultures of Centella asiatica

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    Komar Ruslan


    Full Text Available Background: Centella asiatica (L. Urb. (Apiaceae is an important medicinal plant, and it has been using to prepare herbal medicines. The compounds responsible for the biological activity of C. asiatica are triterpenoids such as asiaticoside. Asiaticoside is also important as a marker for standardization of C. asiatica. Due to the low content, there is a need to enhance the production of asiaticoside of C. asiatica. The biotechnological approach is one of the methods that can be used to enhance its production. Objectives: This study was designed to enhance the production of asiaticoside from C. asiatica using A. rhizogenes and elicitation experiments. Materials and Methods : Callus cultures were initiated using Murashige and Skoog (MS medium supplemented with 1.0 mg/L indole-3-acetic acid (IAA and 1.0 mg/L 6-benzylaminopurin (BAP. All media were supplemented with 4% (w/w sucrose and solidified with 0.9% agar. Elicitations were done using pectin, methyl jasmonate, and Cu 2+ ions. Transformed hairy root cultures were performed using A. rhizogenes. Results: Callus culture of C. asiatica was successfully initiated. Enhancement of the production of asiaticoside in the callus culture by elicitors pectin was up to 31%; methyl jasmonate (50 ΅M in cell suspension cultures at day 14 was up to 171% compared to explant and 494% compared to control callus; copper ion (25 ΅M at day 21 was up to 144% compared to explant, and 676% compared to control cell suspension cultures. While enhancement by genetic transformation using A. rhizogenes was 166-172% compare to untransformed roots Conclusion: Elicitation and genetically transformed hairy root cultures of C. asiatica produced asiaticoside up to 172% higher than untreated callus.

  17. Distinct human stem cell populations in small and large intestine. (United States)

    Cramer, Julie M; Thompson, Timothy; Geskin, Albert; LaFramboise, William; Lagasse, Eric


    The intestine is composed of an epithelial layer containing rapidly proliferating cells that mature into two regions, the small and the large intestine. Although previous studies have identified stem cells as the cell-of-origin for intestinal epithelial cells, no studies have directly compared stem cells derived from these anatomically distinct regions. Here, we examine intrinsic differences between primary epithelial cells isolated from human fetal small and large intestine, after in vitro expansion, using the Wnt agonist R-spondin 2. We utilized flow cytometry, fluorescence-activated cell sorting, gene expression analysis and a three-dimensional in vitro differentiation assay to characterize their stem cell properties. We identified stem cell markers that separate subpopulations of colony-forming cells in the small and large intestine and revealed important differences in differentiation, proliferation and disease pathways using gene expression analysis. Single cells from small and large intestine cultures formed organoids that reflect the distinct cellular hierarchy found in vivo and respond differently to identical exogenous cues. Our characterization identified numerous differences between small and large intestine epithelial stem cells suggesting possible connections to intestinal disease.

  18. Distinct human stem cell populations in small and large intestine.

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    Julie M Cramer

    Full Text Available The intestine is composed of an epithelial layer containing rapidly proliferating cells that mature into two regions, the small and the large intestine. Although previous studies have identified stem cells as the cell-of-origin for intestinal epithelial cells, no studies have directly compared stem cells derived from these anatomically distinct regions. Here, we examine intrinsic differences between primary epithelial cells isolated from human fetal small and large intestine, after in vitro expansion, using the Wnt agonist R-spondin 2. We utilized flow cytometry, fluorescence-activated cell sorting, gene expression analysis and a three-dimensional in vitro differentiation assay to characterize their stem cell properties. We identified stem cell markers that separate subpopulations of colony-forming cells in the small and large intestine and revealed important differences in differentiation, proliferation and disease pathways using gene expression analysis. Single cells from small and large intestine cultures formed organoids that reflect the distinct cellular hierarchy found in vivo and respond differently to identical exogenous cues. Our characterization identified numerous differences between small and large intestine epithelial stem cells suggesting possible connections to intestinal disease.

  19. Vaccination with a recombinant protein encoding the tumor-specific antigen NY-ESO-1 elicits an A2/157-165-specific CTL repertoire structurally distinct and of reduced tumor reactivity than that elicited by spontaneous immune responses to NY-ESO-1-expressing Tumors. (United States)

    Bioley, Gilles; Guillaume, Philippe; Luescher, Immanuel; Bhardwaj, Nina; Mears, Gregory; Old, Lloyd; Valmori, Danila; Ayyoub, Maha


    In a recent vaccination trial assessing the immunogenicity of an NY-ESO-1 (ESO) recombinant protein administered with Montanide and CpG, we have obtained evidence that this vaccine induces specific cytolytic T lymphocytes (CTL) in half of the patients. Most vaccine-induced CTLs were directed against epitopes located in the central part of the protein, between amino acids 81 and 110. This immunodominant region, however, is distinct from another ESO CTL region, 157-165, that is a frequent target of spontaneous CTL responses in A2+ patients bearing ESO tumors. In this study, we have investigated the CTL responses to ESO 157-165 in A2+ patients vaccinated with the recombinant protein. Our data indicate that after vaccination with the protein, CTL responses to ESO 157-165 are induced in some, but not all, A2+ patients. ESO 157-165-specific CTLs induced by vaccination with the ESO protein were functionally heterogeneous in terms of tumor recognition and often displayed decreased tumor reactivity as compared with ESO 157-165-specific CTLs isolated from patients with spontaneous immune responses to ESO. Remarkably, protein-induced CTLs used T-cell receptors similar to those previously isolated from patients vaccinated with synthetic ESO peptides (Vbeta4.1) and distinct from those used by highly tumor-reactive CTLs isolated from patients with spontaneous immune responses (Vbeta1.1, Vbeta8.1, and Vbeta13.1). Together, these results demonstrate that vaccination with the ESO protein elicits a repertoire of ESO 157-165-specific CTLs bearing T-cell receptors that are structurally distinct from those of CTLs found in spontaneous immune responses to the antigen and that are heterogeneous in terms of tumor reactivity, being often poorly tumor reactive.

  20. A microarray analysis of two distinct lymphatic endothelial cell populations

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    Bernhard Schweighofer


    Full Text Available We have recently identified lymphatic endothelial cells (LECs to form two morphologically different populations, exhibiting significantly different surface protein expression levels of podoplanin, a major surface marker for this cell type. In vitro shockwave treatment (IVSWT of LECs resulted in enrichment of the podoplaninhigh cell population and was accompanied by markedly increased cell proliferation, as well as 2D and 3D migration. Gene expression profiles of these distinct populations were established using Affymetrix microarray analyses. Here we provide additional details about our dataset (NCBI GEO accession number GSE62510 and describe how we analyzed the data to identify differently expressed genes in these two LEC populations.

  1. Timing the generation of distinct retinal cells by homeobox proteins.

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    Sarah Decembrini


    Full Text Available The reason why different types of vertebrate nerve cells are generated in a particular sequence is still poorly understood. In the vertebrate retina, homeobox genes play a crucial role in establishing different cell identities. Here we provide evidence of a cellular clock that sequentially activates distinct homeobox genes in embryonic retinal cells, linking the identity of a retinal cell to its time of generation. By in situ expression analysis, we found that the three Xenopus homeobox genes Xotx5b, Xvsx1, and Xotx2 are initially transcribed but not translated in early retinal progenitors. Their translation requires cell cycle progression and is sequentially activated in photoreceptors (Xotx5b and bipolar cells (Xvsx1 and Xotx2. Furthermore, by in vivo lipofection of "sensors" in which green fluorescent protein translation is under control of the 3' untranslated region (UTR, we found that the 3' UTRs of Xotx5b, Xvsx1, and Xotx2 are sufficient to drive a spatiotemporal pattern of translation matching that of the corresponding proteins and consistent with the time of generation of photoreceptors (Xotx5b and bipolar cells (Xvsx1 and Xotx2. The block of cell cycle progression of single early retinal progenitors impairs their differentiation as photoreceptors and bipolar cells, but is rescued by the lipofection of Xotx5b and Xvsx1 coding sequences, respectively. This is the first evidence to our knowledge that vertebrate homeobox proteins can work as effectors of a cellular clock to establish distinct cell identities.

  2. Vaginal immunization to elicit primary T-cell activation and dissemination.

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    Elena Pettini

    Full Text Available Primary T-cell activation at mucosal sites is of utmost importance for the development of vaccination strategies. T-cell priming after vaginal immunization, with ovalbumin and CpG oligodeoxynucleotide adjuvant as model vaccine formulation, was studied in vivo in hormone-synchronized mice and compared to the one induced by the nasal route. Twenty-four hours after both vaginal or nasal immunization, antigen-loaded dendritic cells were detected within the respective draining lymph nodes. Vaginal immunization elicited a strong recruitment of antigen-specific CD4(+ T cells into draining lymph nodes that was more rapid than the one observed following nasal immunization. T-cell clonal expansion was first detected in iliac lymph nodes, draining the genital tract, and proliferated T cells disseminated towards distal lymph nodes and spleen similarly to what observed following nasal immunization. T cells were indeed activated by the antigen encounter and acquired homing molecules essential to disseminate towards distal lymphoid organs as confirmed by the modulation of CD45RB, CD69, CD44 and CD62L marker expression. A multi-type Galton Watson branching process, previously used for in vitro analysis of T-cell proliferation, was applied to model in vivo CFSE proliferation data in draining lymph nodes 57 hours following immunization, in order to calculate the probabilistic decision of a cell to enter in division, rest in quiescence or migrate/die. The modelling analysis indicated that the probability of a cell to proliferate was higher following vaginal than nasal immunization. All together these data show that vaginal immunization, despite the absence of an organized mucosal associated inductive site in the genital tract, is very efficient in priming antigen-specific CD4(+ T cells and inducing their dissemination from draining lymph nodes towards distal lymphoid organs.

  3. Activated RET/PTC oncogene elicits immediate early and delayed response genes in PC12 cells. (United States)

    Califano, D; Monaco, C; de Vita, G; D'Alessio, A; Dathan, N A; Possenti, R; Vecchio, G; Fusco, A; Santoro, M; de Franciscis, V


    The expression of the receptor-like tyrosine kinase RET is associated with tumors, tissues or cell lines of neural crest origin. In addition RET products (Ret) are involved in determining cell fate during the differentiation of the enteric nervous system and during renal organogenesis. However, as yet, no direct evidence exists to indicate that the Ret kinase activity might interfere in a specific way with cellular differentiation, or proliferation, of a neural crest derived cell line. By using two constitutively activated forms of RET (RET/PTC1 and RET/PTC3) in transient transfection experiments, we have obtained evidence that active RET could reprogramme the gene expression pattern in the rat pheochromocytoma PC12 cell line. Transcription driven by gene promoters, such as NGFI-A and vgf, which belong, respectively, to primary and delayed response genes to nerve growth factor (NGF), and by the neuron-specific enolase (NSE) promoter, is rapidly induced by the expression of activated RET oncogenes. This induction is not elicited in other non neural derived cell types tested. We also demonstrate that endogenous ras activity is required for RET induction of these neural markers. Finally, in the RET/PTC transfected PC12 cells, NGF is unable to induce further their transcription. This suggests that RET/PTC could share an intracellular signalling pathway with the NGF-receptor.

  4. Synthetic B-Cell Epitopes Eliciting Cross-Neutralizing Antibodies: Strategies for Future Dengue Vaccine (United States)

    Poh, Chit Laa; Kirk, Kristin; McBride, William John Hannan; Aaskov, John; Grollo, Lara


    Dengue virus (DENV) is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction approach. The selected peptides representing B-cell epitopes were attached to a known dengue T-helper epitope and evaluated for their vaccine potency. Immunization of mice revealed two novel synthetic vaccine constructs that elicited good humoral immune responses and produced cross-reactive neutralising antibodies against DENV-1, 2 and 3. The findings indicate new directions for epitope mapping and contribute towards the future development of multi-epitope based synthetic peptide vaccine. PMID:27223692

  5. Synthetic B-Cell Epitopes Eliciting Cross-Neutralizing Antibodies: Strategies for Future Dengue Vaccine.

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    Babu Ramanathan

    Full Text Available Dengue virus (DENV is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction approach. The selected peptides representing B-cell epitopes were attached to a known dengue T-helper epitope and evaluated for their vaccine potency. Immunization of mice revealed two novel synthetic vaccine constructs that elicited good humoral immune responses and produced cross-reactive neutralising antibodies against DENV-1, 2 and 3. The findings indicate new directions for epitope mapping and contribute towards the future development of multi-epitope based synthetic peptide vaccine.

  6. Synthetic B-Cell Epitopes Eliciting Cross-Neutralizing Antibodies: Strategies for Future Dengue Vaccine. (United States)

    Ramanathan, Babu; Poh, Chit Laa; Kirk, Kristin; McBride, William John Hannan; Aaskov, John; Grollo, Lara


    Dengue virus (DENV) is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction approach. The selected peptides representing B-cell epitopes were attached to a known dengue T-helper epitope and evaluated for their vaccine potency. Immunization of mice revealed two novel synthetic vaccine constructs that elicited good humoral immune responses and produced cross-reactive neutralising antibodies against DENV-1, 2 and 3. The findings indicate new directions for epitope mapping and contribute towards the future development of multi-epitope based synthetic peptide vaccine.

  7. Distinct T cell signatures define subsets of patients with multiple sclerosis (United States)

    Johnson, Mark C.; Pierson, Emily R.; Spieker, Andrew J.; Nielsen, A. Scott; Posso, Sylvia; Kita, Mariko; Buckner, Jane H.


    Objective: We investigated T cell responses to myelin proteins in the blood of healthy controls and 2 groups of patients with relapsing-remitting multiple sclerosis (RRMS) who exhibited lesions either predominantly in the brain or predominantly in the spinal cord in order to assess whether distinct neuroinflammatory patterns were associated with different myelin protein–specific T cell effector function profiles and whether these profiles differed from healthy controls. Methods: Peripheral blood mononuclear cells were obtained from patients with brain-predominant RRMS, patients with spinal cord–predominant RRMS, and age-matched healthy controls and analyzed by enzyme-linked immunosorbent spot assays to quantify interferon gamma–secreting (Th1) and interleukin 17–secreting (Th17) cells responding directly ex vivo to myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG). Results: Although MBP and MOG elicited different responses, patients with multiple sclerosis (MS) who had spinal cord–predominant lesions exhibited significantly higher Th17:Th1 ratios in response to both MBP and MOG compared to patients with brain-predominant MS. Incorporating the cytokine responses to both antigens into logistic regression models showed that these cytokine responses were able to provide good discrimination between patients with distinct neuroinflammatory patterns. Conclusions: Our findings suggest that the localization of lesions within the brain vs the spinal cord in patients with MS is associated with different effector T cell responses to myelin proteins. Further investigation of the relationship between T cell effector function, antigen specificities, and lesion sites may reveal features of pathogenic pathways that are distinct to patients with different neuroinflammatory patterns. PMID:27606354

  8. Host plant peptides elicit a transcriptional response to control the Sinorhizobium meliloti cell cycle during symbiosis. (United States)

    Penterman, Jon; Abo, Ryan P; De Nisco, Nicole J; Arnold, Markus F F; Longhi, Renato; Zanda, Matteo; Walker, Graham C


    The α-proteobacterium Sinorhizobium meliloti establishes a chronic intracellular infection during the symbiosis with its legume hosts. Within specialized host cells, S. meliloti differentiates into highly polyploid, enlarged nitrogen-fixing bacteroids. This differentiation is driven by host cells through the production of defensin-like peptides called "nodule-specific cysteine-rich" (NCR) peptides. Recent research has shown that synthesized NCR peptides exhibit antimicrobial activity at high concentrations but cause bacterial endoreduplication at sublethal concentrations. We leveraged synchronized S. meliloti populations to determine how treatment with a sublethal NCR peptide affects the cell cycle and physiology of bacteria at the molecular level. We found that at sublethal levels a representative NCR peptide specifically blocks cell division and antagonizes Z-ring function. Gene-expression profiling revealed that the cell division block was produced, in part, through the substantial transcriptional response elicited by sublethal NCR treatment that affected ∼15% of the genome. Expression of critical cell-cycle regulators, including ctrA, and cell division genes, including genes required for Z-ring function, were greatly attenuated in NCR-treated cells. In addition, our experiments identified important symbiosis functions and stress responses that are induced by sublethal levels of NCR peptides and other antimicrobial peptides. Several of these stress-response pathways also are found in related α-proteobacterial pathogens and might be used by S. meliloti to sense host cues during infection. Our data suggest a model in which, in addition to provoking stress responses, NCR peptides target intracellular regulatory pathways to drive S. meliloti endoreduplication and differentiation during symbiosis.

  9. OVCAR-3 spheroid-derived cells display distinct metabolic profiles.

    Directory of Open Access Journals (Sweden)

    Kathleen A Vermeersch

    in an ovarian cancer stem cell line is distinct from that of more differentiated isogenic cancer cells, supporting the potential importance of metabolism in the differences between cancer cells and cancer stem cells.

  10. Recombinant yellow fever viruses elicit CD8+ T cell responses and protective immunity against Trypanosoma cruzi.

    Directory of Open Access Journals (Sweden)

    Raquel Tayar Nogueira

    Full Text Available Chagas' disease is a major public health problem affecting nearly 10 million in Latin America. Despite several experimental vaccines have shown to be immunogenic and protective in mouse models, there is not a current vaccine being licensed for humans or in clinical trial against T. cruzi infection. Towards this goal, we used the backbone of Yellow Fever (YF 17D virus, one of the most effective and well-established human vaccines, to express an immunogenic fragment derived from T. cruzi Amastigote Surface Protein 2 (ASP-2. The cDNA sequence of an ASP-2 fragment was inserted between E and NS1 genes of YF 17D virus through the construction of a recombinant heterologous cassette. The replication ability and genetic stability of recombinant YF virus (YF17D/ENS1/Tc was confirmed for at least six passages in Vero cells. Immunogenicity studies showed that YF17D/ENS1/Tc virus elicited neutralizing antibodies and gamma interferon (IFN-γ producing-cells against the YF virus. Also, it was able to prime a CD8(+ T cell directed against the transgenic T. cruzi epitope (TEWETGQI which expanded significantly as measured by T cell-specific production of IFN-γ before and after T. cruzi challenge. However, most important for the purposes of vaccine development was the fact that a more efficient protective response could be seen in mice challenged after vaccination with the YF viral formulation consisting of YF17D/ENS1/Tc and a YF17D recombinant virus expressing the TEWETGQI epitope at the NS2B-3 junction. The superior protective immunity observed might be due to an earlier priming of epitope-specific IFN-γ-producing T CD8(+ cells induced by vaccination with this viral formulation. Our results suggest that the use of viral formulations consisting of a mixture of recombinant YF 17D viruses may be a promising strategy to elicit protective immune responses against pathogens, in general.

  11. Distinct Pattern of Human Vδ1 T Cells Recognizing MICA

    Institute of Scientific and Technical Information of China (English)

    Jianqiang Li; Lianxian Cui; Wei He


    γδ T cells represent one unique recognition pattern, the limited recognition, which distinguishes from the specific recognition for αβ T cells and pattern recognition for macrophages. Vδ1 γδ T cell is the major subset of human γδT cells, which predominates in mucosal tissue including the intestinal epithelia. Presently, a few antigens that human Vδ1TCR can recognize have been identified. Among them, MHC class Ⅰ chain-related molecules A (MICA)have been studied most intensively. Besides Vδ1TCR, MICA is also the ligand of NKG2D, a C-type lectin-like activating immunoreceptor. In human, only Vδ1 cells can simultaneously express both types of receptors of MICA while NK cells, αβ T cells and other subsets of γδ T cells likewise express NKG2D. Although the precise mechanisms are still enigmatic, this distinct pattern of Vδ1 cells recognizing MICA predicts unique biological significance of Vδ1 cells in immune defense. Recent years, some progresses have been made in this issue. In this review we summarize the related reports and put forward some novel views based on our group's studies.

  12. Different activation signals induce distinct mast cell degranulation strategies (United States)

    Sibilano, Riccardo; Marichal, Thomas; Reber, Laurent L.; Cenac, Nicolas; McNeil, Benjamin D.; Dong, Xinzhong; Hernandez, Joseph D.; Sagi-Eisenberg, Ronit; Hammel, Ilan; Roers, Axel; Valitutti, Salvatore; Tsai, Mindy


    Mast cells (MCs) influence intercellular communication during inflammation by secreting cytoplasmic granules that contain diverse mediators. Here, we have demonstrated that MCs decode different activation stimuli into spatially and temporally distinct patterns of granule secretion. Certain signals, including substance P, the complement anaphylatoxins C3a and C5a, and endothelin 1, induced human MCs rapidly to secrete small and relatively spherical granule structures, a pattern consistent with the secretion of individual granules. Conversely, activating MCs with anti-IgE increased the time partition between signaling and secretion, which was associated with a period of sustained elevation of intracellular calcium and formation of larger and more heterogeneously shaped granule structures that underwent prolonged exteriorization. Pharmacological inhibition of IKK-β during IgE-dependent stimulation strongly reduced the time partition between signaling and secretion, inhibited SNAP23/STX4 complex formation, and switched the degranulation pattern into one that resembled degranulation induced by substance P. IgE-dependent and substance P–dependent activation in vivo also induced different patterns of mouse MC degranulation that were associated with distinct local and systemic pathophysiological responses. These findings show that cytoplasmic granule secretion from MCs that occurs in response to different activating stimuli can exhibit distinct dynamics and features that are associated with distinct patterns of MC-dependent inflammation. PMID:27643442

  13. L-Amino Acids Elicit Diverse Response Patterns in Taste Sensory Cells: A Role for Multiple Receptors.

    Directory of Open Access Journals (Sweden)

    Shreoshi Pal Choudhuri

    Full Text Available Umami, the fifth basic taste, is elicited by the L-amino acid, glutamate. A unique characteristic of umami taste is the response potentiation by 5' ribonucleotide monophosphates, which are also capable of eliciting an umami taste. Initial reports using human embryonic kidney (HEK cells suggested that there is one broadly tuned receptor heterodimer, T1r1+T1r3, which detects L-glutamate and all other L-amino acids. However, there is growing evidence that multiple receptors detect glutamate in the oral cavity. While much is understood about glutamate transduction, the mechanisms for detecting the tastes of other L-amino acids are less well understood. We used calcium imaging of isolated taste sensory cells and taste cell clusters from the circumvallate and foliate papillae of C57BL/6J and T1r3 knockout mice to determine if other receptors might also be involved in detection of L-amino acids. Ratiometric imaging with Fura-2 was used to study calcium responses to monopotassium L-glutamate, L-serine, L-arginine, and L-glutamine, with and without inosine 5' monophosphate (IMP. The results of these experiments showed that the response patterns elicited by L-amino acids varied significantly across taste sensory cells. L-amino acids other than glutamate also elicited synergistic responses in a subset of taste sensory cells. Along with its role in synergism, IMP alone elicited a response in a large number of taste sensory cells. Our data indicate that synergistic and non-synergistic responses to L-amino acids and IMP are mediated by multiple receptors or possibly a receptor complex.

  14. Proteome adaptation in cell reprogramming proceeds via distinct transcriptional networks. (United States)

    Benevento, Marco; Tonge, Peter D; Puri, Mira C; Hussein, Samer M I; Cloonan, Nicole; Wood, David L; Grimmond, Sean M; Nagy, Andras; Munoz, Javier; Heck, Albert J R


    The ectopic expression of Oct4, Klf4, c-Myc and Sox2 (OKMS) transcription factors allows reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). The reprogramming process, which involves a complex network of molecular events, is not yet fully characterized. Here we perform a quantitative mass spectrometry-based analysis to probe in-depth dynamic proteome changes during somatic cell reprogramming. Our data reveal defined waves of proteome resetting, with the first wave occurring 48 h after the activation of the reprogramming transgenes and involving specific biological processes linked to the c-Myc transcriptional network. A second wave of proteome reorganization occurs in a later stage of reprogramming, where we characterize the proteome of two distinct pluripotent cellular populations. In addition, the overlay of our proteome resource with parallel generated -omics data is explored to identify post-transcriptionally regulated proteins involved in key steps during reprogramming.

  15. Distinct T helper cell dependence of memory B-cell proliferation versus plasma cell differentiation. (United States)

    Zabel, Franziska; Fettelschoss, Antonia; Vogel, Monique; Johansen, Pål; Kündig, Thomas M; Bachmann, Martin F


    Several memory B-cell subclasses with distinct functions have been described, of which the most effective is the class-switched (CS) memory B-cell population. We have previously shown, using virus-like particles (VLPs), that the proliferative potential of these CS memory B cells is limited and they fail to re-enter germinal centres (GCs). However, VLP-specific memory B cells quickly differentiated into secondary plasma cells (PCs) with the virtue of elevated antibody production compared with primary PCs. Whereas the induction of VLP(+) memory B cells was strongly dependent on T helper cells, we were wondering whether re-stimulation of VLP(+) memory B cells and their differentiation into secondary PCs would also require T helper cells. Global absence of T helper cells led to strongly impaired memory B cell proliferation and PC differentiation. In contrast, lack of interleukin-21 receptor-dependent follicular T helper cells or CD40 ligand signalling strongly affected proliferation of memory B cells, but differentiation into mature secondary PCs exhibiting increased antibody production was essentially normal. This contrasts with primary B-cell responses, where a strong dependence on CD40 ligand but limited importance of interleukin-21 receptor was seen. Hence, T helper cell dependence differs between primary and secondary B-cell responses as well as between memory B-cell proliferation and PC differentiation.

  16. Ecdysteroids Elicit a Rapid Ca2+ Flux Leading to Akt Activation and Increased Protein Synthesis in Skeletal Muscle Cells


    Gorelick-Feldman, Jonathan; Cohick, Wendie; Raskin, Ilya


    Phytoecdysteroids, structurally similar to insect molting hormones, produce a range of effects in mammals, including increasing growth and physical performance. In skeletal muscle cells, phytoecdysteroids increase protein synthesis. In this study we show that in a mouse skeletal muscle cell line, C2C12, 20-hydroxyecdysone (20HE), a common phytoecdysteroid in both insects and plants, elicited a rapid elevation in intracellular calcium, followed by sustained Akt activation and increased protein...

  17. Candida-elicited murine Th17 cells express high Ctla-4 compared with Th1 cells and are resistant to costimulation blockade. (United States)

    Krummey, Scott M; Floyd, Tamara L; Liu, Danya; Wagener, Maylene E; Song, Mingqing; Ford, Mandy L


    Effector and memory T cells may cross-react with allogeneic Ags to mediate graft rejection. Whereas the costimulation properties of Th1 cells are well studied, relatively little is known about the costimulation requirements of microbe-elicited Th17 cells. The costimulation blocker CTLA-4 Ig has been ineffective in the treatment of several Th17-driven autoimmune diseases and is associated with severe acute rejection following renal transplantation, leading us to investigate whether Th17 cells play a role in CD28/CTLA-4 blockade-resistant alloreactivity. We established an Ag-specific model in which Th1 and Th17 cells were elicited via Mycobacterium tuberculosis and Candida albicans immunization, respectively. C. albicans immunization elicited a higher frequency of Th17 cells and conferred resistance to costimulation blockade following transplantation. Compared with the M. tuberculosis group, C. albicans-elicited Th17 cells contained a higher frequency of IL-17(+)IFN-γ(+) producers and a lower frequency of IL-10(+) and IL-10(+)IL-17(+) cells. Importantly, Th17 cells differentially regulated the CD28/CTLA-4 pathway, expressing similarly high CD28 but significantly greater amounts of CTLA-4 compared with Th1 cells. Ex vivo blockade experiments demonstrated that Th17 cells are more sensitive to CTLA-4 coinhibition and therefore less susceptible to CTLA-4 Ig. These novel insights into the differential regulation of CTLA-4 coinhibition on CD4(+) T cells have implications for the immunomodulation of pathologic T cell responses during transplantation and autoimmunity.

  18. Frequency locking in hair cells: Distinguishing between distinct resonant mechanisms

    CERN Document Server

    Edri, Yuval; Yochelis, Arik


    The auditory system displays remarkable mechanical sensitivity and frequency discrimination. These attributes have been shown to rely on an amplification process, which requires biochemical feedback loops. In some systems, the active process was shown to lead to spontaneous oscillations of hair cell bundles. In the last decade, models that display proximity to an oscillatory onset (a.k.a. Hopf bifurcation) have gained increasing support due to many advantages in explaining the hearing phenomenology. Particularly, they exhibit resonant responses to distinct frequencies of incoming sound waves. Unlike previous studies, two types of driving forces are being examined: additive, in which the external forcing term does not couple directly on the systems observable (passive coupling), and parametric, in which the forcing term directly affects the observable and thus intrinsically modifies the systems properties (active coupling). By applying universal principles near the Hopf bifurcation onset, we find several funda...

  19. Elicitation of gymnemic acid production in cell suspension cultures of Gymnema sylvestre R.Br. through endophytic fungi. (United States)

    Netala, Vasudeva Reddy; Kotakadi, Venkata Subbaiah; Gaddam, Susmila Aparna; Ghosh, Sukhendu Bikash; Tartte, Vijaya


    The enhancement of plant secondary metabolite production in cell suspension cultures through biotic or abiotic elicitation has become a potential biotechnological approach for commercialization or large-scale production of bioactive compounds. Gymnema sylvestre R.Br. is an important medicinal plant, rich in a group of oleanane triterpenoid saponins called gymnemic acid, well known for its anti-diabetic activity. Two endophytic fungal strains were isolated from the leaves of G. sylvestre and identified as Polyancora globosa and Xylaria sp. based on the PCR amplification and internal transcribed spacer (ITS 1-5.8S-ITS 2) sequencing of 18S rRNA gene. The process of elicitation of cell suspension cultures of G. sylvestre with dried powder of fungal mycelia (DPFM) and extracellular culture filtrate (ECF) of endophytic fungi consistently enhanced the accumulation of gymnemic acid and the DPFM was proved to be an effective elicitor when compared to the ECF. The DPFM elicited the gymnemic acid content in the range of 2.57-10.45-fold, while the ECF elicited the gymnemic acid content in the range of 2.39-7.8-fold. P. globosa, a novel and a rare endophytic fungal strain, has shown a great influence on the production of gymnemic acid. Cell suspension cultures elicited with DPFM of P. globosa produced higher amount of gymnemic acid content (124.23 mg/g dried cell weight) compared to the cultures elicited with DPFM of Xylaria sp. (102.24 mg/g DCW). But the cultures treated with consortium of DPFM of both fungi showed great influence on the production of gymnemic acid (139.98 mg/g DCW) than the cultures treated with DPFM alone. Similarly, cultures treated with consortium of ECF of both fungi produced more gymnemic acid content (94.86 mg/g DCW) compared with cultures treated with ECF of Xylaria sp. (77.93 mg/g DCW) and ECF of P. globosa (78.65 mg/g DCW) alone.

  20. [Enhanced production of taxuyunnanine c in cell suspension cultures of Taxus chinensis by methyl jasmonate elicitation and in situ absorption]. (United States)

    Gao, Mingbo; Zhang, Wei; Yu, Xingju


    A bioprocess intensification strategy that combines both elicitation and in situ absorption was developed to improve the production of taxuyunnanine c (Tc) in cell suspension cultures of Taxus chinensis. When 100 micromol/L methyl jasmonate was added as an elicitor on Day 7, the Tc content and yield increased 3.6 and 3.3 times respectively, however the cell growth was reduced by 10%-30%. Significant improvement in Tc yield was observed when an absorbent XAD-7 was added on different time of the culture period. The optimum Tc yield was achieved when 100 g/L XAD-7 was added simultaneously with 100 micromol/L methyl jasmonate on Day 7. The maximum Tc yield of 477.4 mg/L was obtained on Day 21 of the culture, being 6.3-fold of the control and 1.9-fold of the 100 micromol/L methyl jasmonate treatment alone. In the combined treatment, 94% of the Tc produced was secreted outside of the cells and absorbed on XAD-7 absorbents. The results demonstrated that the process strategy combining elicitation and in situ absorption was effective to intensify the Tc biosynthesis via elicitation with the removal of product feedback inhibition via absorption, presenting a great potential in commercial applications.

  1. Specific localization and measurement of hydrogen peroxide in Arabidopsis thaliana cell suspensions and protoplasts elicited by COS-OGA. (United States)

    Ledoux, Quentin; Van Cutsem, Pierre; Markό, Istvan E; Veys, Pascal


    H2O2 acts as an important signaling molecule during plant/pathogen interactions but its study remains a challenge due to the current shortcomings in H2O2-responsive probes. In this work, ContPY1, a new molecular probe developed to specifically detect H2O2 was used to study the elicitation of Arabidopsis thaliana cells by a complex of chitosan oligomers (COS) and oligogalacturonides (OGA). The comparison of cell suspensions, protoplasts of cell suspensions and leaf protoplasts treated with different inhibitors gave indications on the potential sources of hydrogen peroxide in plant cells. The relative contribution of the cell wall, of membrane dehydrogenases and of peroxidases depended on cell type and treatment and proved to be variable. Our present protocol can be used to study hydrogen peroxide production in a large variety of plant species by simple protocol adaptation.

  2. Targeted antigen delivery to dendritic cells elicits robust antiviral T cell-mediated immunity in the liver (United States)

    Volckmar, Julia; Gereke, Marcus; Ebensen, Thomas; Riese, Peggy; Philipsen, Lars; Lienenklaus, Stefan; Wohlleber, Dirk; Klopfleisch, Robert; Stegemann-Koniszewski, Sabine; Müller, Andreas J.; Gruber, Achim D.; Knolle, Percy; Guzman, Carlos A.; Bruder, Dunja


    Hepatotropic viruses such as hepatitis C virus cause life-threatening chronic liver infections in millions of people worldwide. Targeted in vivo antigen-delivery to cross-presenting dendritic cells (DCs) has proven to be extraordinarily efficient in stimulating antigen-specific T cell responses. To determine whether this approach would as well be suitable to induce local antiviral effector T cells in the liver we compared different vaccine formulations based on either the targeting of DEC-205 or TLR2/6 on cross-presenting DCs or formulations not involving in vivo DC targeting. As read-outs we used in vivo hepatotropic adenovirus challenge, histology and automated multidimensional fluorescence microscopy (MELC). We show that targeted in vivo antigen delivery to cross-presenting DCs is highly effective in inducing antiviral CTLs capable of eliminating virus-infected hepatocytes, while control vaccine formulation not involving DC targeting failed to induce immunity against hepatotropic virus. Moreover, we observed distinct patterns of CD8+ T cell interaction with virus-infected and apoptotic hepatocytes in the two DC-targeting groups suggesting that the different vaccine formulations may stimulate distinct types of effector functions. Our findings represent an important step toward the future development of vaccines against hepatotropic viruses and the treatment of patients with hepatic virus infection after liver transplantation to avoid reinfection. PMID:28266658

  3. Interdisciplinary Evaluation of Broadly-Reactive HLA Class II Restricted Epitopes Eliciting HIV-Specific CD4+T Cell Responses

    DEFF Research Database (Denmark)

    Buggert, M.; Norström, M.; Lundegaard, Claus


    , the functional and immunodominant discrepancies of CD4+ T cell responses targeting promiscuous MHC II restricted HIV epitopes remains poorly defined. Thus, utilization of interdisciplinary approaches might aid revealing broadly- reactive peptides eliciting CD4 + T cell responses. Methods: We utilized the novel...... bioinformatic prediction program NetMHCIIpan to select 64 optimized MHC II restricted epitopes located in the HIV Gag, Pol, Env, Nef and Tat regions. The epitopes were selected to cover the global diversity of the virus (multiple subtypes) and the human immune system(diverse MHC II types). Optimized...

  4. Measuring melanoma-specific cytotoxic T lymphocytes elicited by dendritic cell vaccines with a tumor inhibition assay in vitro. (United States)

    Paczesny, Sophie; Shi, Honhgzhen; Saito, Hiroaki; Mannoni, Patrice; Fay, Joseph; Banchereau, Jacques; Palucka, A Karolina


    Improving cancer vaccines depends on assays measuring elicited tumor-specific T-cell immunity. Cytotoxic effector cells are essential for tumor clearance and are commonly evaluated using 51Cr release from labeled target cells after a short (4 hours) incubation with T cells. The authors used a tumor inhibition assay (TIA) that assesses the capacity of cytotoxic T lymphocytes (CTLs) to control the survival/growth of EGFP-labeled tumor cell lines. TIA was validated using CD8+ T cells primed in vitro against melanoma and breast cancer cells. TIA was then used to assess the CTL function of cultured CD8+ T cells isolated from patients with metastatic melanoma who underwent vaccination with peptide-pulsed CD34+ HPCs-derived DCs. After the DC vaccination, T cells from six of eight patients yielded CTLs that could inhibit the survival/growth of melanoma cells. The results of TIA correlated with killing of tumor cells in a standard 4-hour 51Cr release assay, yet TIA allowed detection of CTL activities that appeared marginal in the 51Cr release assay. Thus, TIA might prove valuable for measuring spontaneous and induced antigen-specific cytotoxic T cells.

  5. Fetal and adult hematopoietic stem cells give rise to distinct T cell lineages in humans. (United States)

    Mold, Jeff E; Venkatasubrahmanyam, Shivkumar; Burt, Trevor D; Michaëlsson, Jakob; Rivera, Jose M; Galkina, Sofiya A; Weinberg, Kenneth; Stoddart, Cheryl A; McCune, Joseph M


    Although the mammalian immune system is generally thought to develop in a linear fashion, findings in avian and murine species argue instead for the developmentally ordered appearance (or "layering") of distinct hematopoietic stem cells (HSCs) that give rise to distinct lymphocyte lineages at different stages of development. Here we provide evidence of an analogous layered immune system in humans. Our results suggest that fetal and adult T cells are distinct populations that arise from different populations of HSCs that are present at different stages of development. We also provide evidence that the fetal T cell lineage is biased toward immune tolerance. These observations offer a mechanistic explanation for the tolerogenic properties of the developing fetus and for variable degrees of immune responsiveness at birth.

  6. Metabolic analysis of elicited cell suspension cultures of Cannabis sativa L. by (1)H-NMR spectroscopy. (United States)

    Pec, Jaroslav; Flores-Sanchez, Isvett Josefina; Choi, Young Hae; Verpoorte, Robert


    Cannabis sativa L. plants produce a diverse array of secondary metabolites. Cannabis cell cultures were treated with jasmonic acid (JA) and pectin as elicitors to evaluate their effect on metabolism from two cell lines using NMR spectroscopy and multivariate data analysis. According to principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA), the chloroform extract of the pectin-treated cultures were more different than control and JA-treated cultures; but in the methanol/water extract the metabolome of the JA-treated cells showed clear differences with control and pectin-treated cultures. Tyrosol, an antioxidant metabolite, was detected in cannabis cell cultures. The tyrosol content increased after eliciting with JA.

  7. Signal transduction and metabolic flux of beta-thujaplicin and monoterpene biosynthesis in elicited Cupressus lusitanica cell cultures. (United States)

    Zhao, Jian; Matsunaga, Yoko; Fujita, Koki; Sakai, Kokki


    beta-Thujaplicin is an antimicrobial tropolone derived from geranyl pyrophosphate(GPP) and monoterpene intermediate. Yeast elicitor-treated Cupressus lusitanica cell cultures accumulate high levels of beta-thujaplicin at early stages and other monoterpenes at later stages post-elicitation. The different regulation of beta-thujaplicin and monoterpene biosynthesis and signal transduction directing metabolic flux to beta-thujaplicin firstly and then shifting metabolic flow from beta-thujaplicin to other monoterpene biosynthesis were investigated. The earlier rapid induction of beta-thujaplicin accumulation and a later stimulation of monoterpene biosynthesis by yeast elicitor are in well agreement with elicitor-induced changes in activity of three monoterpene biosynthetic enzymes including isopentenyl pyrophosphate isomerase, GPP synthase, and monoterpene synthase. Yeast elicitor induces an earlier and stronger beta-thujaplicin production and monoterpene biosynthetic enzyme activity than methyl jasmonate (MeJA) does. Profiling all monoterpenes produced by C. lusitanica cell cultures under different conditions reveals that beta-thujaplicin biosynthesis parallels with other monoterpenes and competes for common precursor pools. Yet beta-thujaplicin is produced pre-dominantly at early stage of elicitation whereas other monoterpenes are mainly accumulated at late stage while beta-thujaplicin is metabolized. It is suggested that yeast elicitor-treated C. lusitanica cells preferentially accumulate beta-thujaplicin as a primary defense and other monoterpenes as a secondary defense. Inhibitor treatments suggest that immediate production of beta-thujaplicin post-elicitation largely depends on pre-existing enzymes and translation of pre-existing transcripts as well as recruitment of precursor pools from both the cytosol and plastids. The later beta-thujaplicin and other monoterpene accumulation strictly depends on active transcription and translation. Induction of beta

  8. Mechanism of eliciting host immunity against cancer cells treated with silica-phthalocyanine-based near infrared photoimmunotherapy (Conference Presentation) (United States)

    Kobayashi, Hisataka


    Near infrared (NIR) photoimmunotherapy (PIT) is a new type of molecularly-targeted cancer photo-therapy based on conjugating a near infrared silica-phthalocyanine dye, IR700, to a monoclonal antibody (MAb) targeting cancer-specific cell-surface molecules. When exposed to NIR light, the conjugate induces a highly-selective necrotic/ immunogenic cell death (ICD) only in receptor-positive, MAb-IR700-bound cancer cells. This cell death occurs as early as 1 minute after exposure to NIR light. Meanwhile, immediately adjacent receptor-negative cells including immune cells are unharmed. Therefore, we hypothesized that NIR-PIT could efficiently elicit host immunity against treated cancer cells. Three-dimensional dynamic quantitative phase contrast microscopy and selective plane illumination microscopy of tumor cells undergoing PIT showed rapid swelling in treated cells immediately after light exposure suggesting rapid water influx into cells, followed by irreversible morphologic changes such as bleb formation, and rupture of vesicles. Furthermore, biological markers of ICD including relocation of HSP70/90 and calreticulin, and release of ATP and High Mobility Group Box 1 (HMGB1), were clearly detected immediately after NIR-PIT. When NIR-PIT was performed in a mixture of cancer cells and immature dendritic cells, maturation of immature dendritic cells was strongly induced rapidly after NIR-PIT. In summary, NIR-PIT can induce necrotic/ immunogenic cell death that promotes rapid maturation of immature dendritic cells adjacent to dying cancer cells. Therefore, NIR-PIT could efficiently initiate host immune response against NIR-PIT treated cancer cells growing in patients.

  9. Plasmodium falciparum apical membrane antigen 1 vaccine elicits multifunctional CD4 cytokine-producing and memory T cells. (United States)

    Huaman, Maria Cecilia; Mullen, Gregory E D; Long, Carole A; Mahanty, Siddhartha


    The Plasmodium falciparum apical membrane antigen 1 (AMA1) is a leading vaccine candidate and was tested for safety and immunogenicity in human Phase I Clinical Trials. PBMC from vaccine recipients were analyzed by flow cytometric methods to determine the nature of T-cell responses and AMA1-reactive memory T cells. Both CD4 and CD8 T cells produced a number of cytokines following AMA1 re-stimulation, with IL-5-producing cells at the highest frequency, consistent with a Th2 bias. The relative frequency of multifunctional cells synthesizing Th1 cytokines IFN-gamma, IL-2 and TNF-alpha changed after each vaccination. Interestingly, median fluorescence intensity measurements revealed that cells producing more than one cytokine contributed greater quantities of each cytokine than cell populations that produced each of the cytokines alone. AMA1 vaccination also elicited the development of memory cell populations, and both central and effector memory T cells were identified concurrently after the AMA1 vaccination. The detailed profile of multifunctional T-cell responses to AMA1 presented here will advance our ability to assess the immunogenicity of human malarial vaccines.

  10. A Distinct Lung-Interstitium-Resident Memory CD8+ T Cell Subset Confers Enhanced Protection to Lower Respiratory Tract Infection

    Directory of Open Access Journals (Sweden)

    Pavlo Gilchuk


    Full Text Available The nature and anatomic location of the protective memory CD8+ T cell subset induced by intranasal vaccination remain poorly understood. We developed a vaccination model to assess the anatomic location of protective memory CD8+ T cells and their role in lower airway infections. Memory CD8+ T cells elicited by local intranasal, but not systemic, vaccination with an engineered non-replicative CD8+ T cell-targeted antigen confer enhanced protection to a lethal respiratory viral challenge. This protection depends on a distinct CXCR3LO resident memory CD8+ T (Trm cell population that preferentially localizes to the pulmonary interstitium. Because they are positioned close to the mucosa, where infection occurs, interstitial Trm cells act before inflammation can recruit circulating memory CD8+ T cells into the lung tissue. This results in a local protective immune response as early as 1 day post-infection. Hence, vaccine strategies that induce lung interstitial Trm cells may confer better protection against respiratory pathogens.

  11. Pulsed Electromagnetic Field with Temozolomide Can Elicit an Epigenetic Pro-apoptotic Effect on Glioblastoma T98G Cells. (United States)

    Pasi, Francesca; Fassina, Lorenzo; Mognaschi, Maria Evelina; Lupo, Giuseppe; Corbella, Franco; Nano, Rosanna; Capelli, Enrica


    Treatment with pulsed electromagnetic fields (PEMFs) is emerging as an interesting therapeutic option for patients with cancer. The literature has demonstrated that low-frequency/low-energy electromagnetic fields do not cause predictable effects on DNA; however, they can epigenetically act on gene expression. The aim of the present work was to study a possible epigenetic effect of a PEMF, mediated by miRNAs, on a human glioblastoma cell line (T98G). We tested a PEMF (maximum magnetic induction, 2 mT; frequency, 75 Hz) that has been demonstrated to induce autophagy in glioblastoma cells. In particular, we studied the effect of PEMF on the expression of genes involved in cancer progression and a promising synergistic effect with temozolomide, a frequently used drug to treat glioblastoma multiforme. We found that electromagnetic stimulation in combination with temozolomide can elicit an epigenetic pro-apoptotic effect in the chemo- and radioresistant T98G glioblastoma cell line.

  12. The Distinctive Sensitivity to Microgravity of Immune Cell Subpopulations (United States)

    Chen, Hui; Luo, Haiying; Liu, Jing; Wang, Peng; Dong, Dandan; Shang, Peng; Zhao, Yong


    Immune dysfunction in astronauts is well documented after spaceflights. Microgravity is one of the key factors directly suppressing the function of immune system. However, it is unclear which subpopulations of immune cells including innate and adaptive immune cells are more sensitive to microgravity We herein investigated the direct effects of modeled microgravity (MMg) on different immune cells in vitro. Mouse splenocytes, thymocytes and bone marrow cells were exposed to MMg for 16 hrs. The survival and the phenotypes of different subsets of immune cells including CD4+T cells, CD8+T cells, CD4+Foxp3+ regulatory T cells (Treg), B cells, monocytes/macrophages, dendritic cells (DCs), natural killer cells (NK) were determined by flow cytometry. After splenocytes were cultured under MMg for 16h, the cell frequency and total numbers of monocytes, macrophages and CD4+Foxp3+T cells were significantly decreased more than 70 %. MMg significantly decreased the cell numbers of CD8+ T cells, B cells and neutrophils in splenocytes. The cell numbers of CD4+T cells and NK cells were unchanged significantly when splenocytes were cultured under MMg compared with controls. However, MMg significantly increased the ratio of mature neutrophils to immature neutrophils in bone marrow and the cell number of DCs in splenocytes. Based on the cell survival ability, monocytes, macrophages and CD4+Foxp3+Treg cells are most sensitive to microgravity; CD4+T cells and NK cells are resistant to microgravity; CD8+T cells and neutrophils are impacted by short term microgravity exposure. Microgravity promoted the maturation of neutrophils and development of DCs in vitro. The present studies offered new insights on the direct effects of MMg on the survival and homeostasis of immune cell subsets.

  13. Single-cell RNA sequencing identifies distinct mouse medial ganglionic eminence cell types (United States)

    Chen, Ying-Jiun J.; Friedman, Brad A.; Ha, Connie; Durinck, Steffen; Liu, Jinfeng; Rubenstein, John L.; Seshagiri, Somasekar; Modrusan, Zora


    Many subtypes of cortical interneurons (CINs) are found in adult mouse cortices, but the mechanism generating their diversity remains elusive. We performed single-cell RNA sequencing on the mouse embryonic medial ganglionic eminence (MGE), the major birthplace for CINs, and on MGE-like cells differentiated from embryonic stem cells. Two distinct cell types were identified as proliferating neural progenitors and immature neurons, both of which comprised sub-populations. Although lineage development of MGE progenitors was reconstructed and immature neurons were characterized as GABAergic, cells that might correspond to precursors of different CINs were not identified. A few non-neuronal cell types were detected, including microglia. In vitro MGE-like cells resembled bona fide MGE cells but expressed lower levels of Foxg1 and Epha4. Together, our data provide detailed understanding of the embryonic MGE developmental program and suggest how CINs are specified. PMID:28361918

  14. Yellow fever vaccination elicits broad functional CD4+ T cell responses that recognize structural and nonstructural proteins. (United States)

    James, Eddie A; LaFond, Rebecca E; Gates, Theresa J; Mai, Duy T; Malhotra, Uma; Kwok, William W


    Yellow fever virus (YFV) can induce acute, life-threatening disease that is a significant health burden in areas where yellow fever is endemic, but it is preventable through vaccination. The live attenuated 17D YFV strain induces responses characterized by neutralizing antibodies and strong T cell responses. This vaccine provides an excellent model for studying human immunity. While several studies have characterized YFV-specific antibody and CD8(+) T cell responses, less is known about YFV-specific CD4(+) T cells. Here we characterize the epitope specificity, functional attributes, and dynamics of YFV-specific T cell responses in vaccinated subjects by investigating peripheral blood mononuclear cells by using HLA-DR tetramers. A total of 112 epitopes restricted by seven common HLA-DRB1 alleles were identified. Epitopes were present within all YFV proteins, but the capsid, envelope, NS2a, and NS3 proteins had the highest epitope density. Antibody blocking demonstrated that the majority of YFV-specific T cells were HLA-DR restricted. Therefore, CD4(+) T cell responses could be effectively characterized with HLA-DR tetramers. Ex vivo tetramer analysis revealed that YFV-specific T cells persisted at frequencies ranging from 0 to 100 cells per million that are detectable years after vaccination. Longitudinal analysis indicated that YFV-specific CD4(+) T cells reached peak frequencies, often exceeding 250 cells per million, approximately 2 weeks after vaccination. As frequencies subsequently declined, YFV-specific cells regained CCR7 expression, indicating a shift from effector to central memory. Cells were typically CXCR3 positive, suggesting Th1 polarization, and produced gamma interferon and other cytokines after reactivation in vitro. Therefore, YFV elicits robust early effector CD4(+) T cell responses that contract, forming a detectable memory population.

  15. An NK Cell Perforin Response Elicited via IL-18 Controls Mucosal Inflammation Kinetics during Salmonella Gut Infection.

    Directory of Open Access Journals (Sweden)

    Anna A Müller


    Full Text Available Salmonella Typhimurium (S.Tm is a common cause of self-limiting diarrhea. The mucosal inflammation is thought to arise from a standoff between the pathogen's virulence factors and the host's mucosal innate immune defenses, particularly the mucosal NAIP/NLRC4 inflammasome. However, it had remained unclear how this switches the gut from homeostasis to inflammation. This was studied using the streptomycin mouse model. S.Tm infections in knockout mice, cytokine inhibition and -injection experiments revealed that caspase-1 (not -11 dependent IL-18 is pivotal for inducing acute inflammation. IL-18 boosted NK cell chemoattractants and enhanced the NK cells' migratory capacity, thus promoting mucosal accumulation of mature, activated NK cells. NK cell depletion and Prf-/- ablation (but not granulocyte-depletion or T-cell deficiency delayed tissue inflammation. Our data suggest an NK cell perforin response as one limiting factor in mounting gut mucosal inflammation. Thus, IL-18-elicited NK cell perforin responses seem to be critical for coordinating mucosal inflammation during early infection, when S.Tm strongly relies on virulence factors detectable by the inflammasome. This may have broad relevance for mucosal defense against microbial pathogens.

  16. An NK Cell Perforin Response Elicited via IL-18 Controls Mucosal Inflammation Kinetics during Salmonella Gut Infection. (United States)

    Müller, Anna A; Dolowschiak, Tamas; Sellin, Mikael E; Felmy, Boas; Verbree, Carolin; Gadient, Sandra; Westermann, Alexander J; Vogel, Jörg; LeibundGut-Landmann, Salome; Hardt, Wolf-Dietrich


    Salmonella Typhimurium (S.Tm) is a common cause of self-limiting diarrhea. The mucosal inflammation is thought to arise from a standoff between the pathogen's virulence factors and the host's mucosal innate immune defenses, particularly the mucosal NAIP/NLRC4 inflammasome. However, it had remained unclear how this switches the gut from homeostasis to inflammation. This was studied using the streptomycin mouse model. S.Tm infections in knockout mice, cytokine inhibition and -injection experiments revealed that caspase-1 (not -11) dependent IL-18 is pivotal for inducing acute inflammation. IL-18 boosted NK cell chemoattractants and enhanced the NK cells' migratory capacity, thus promoting mucosal accumulation of mature, activated NK cells. NK cell depletion and Prf-/- ablation (but not granulocyte-depletion or T-cell deficiency) delayed tissue inflammation. Our data suggest an NK cell perforin response as one limiting factor in mounting gut mucosal inflammation. Thus, IL-18-elicited NK cell perforin responses seem to be critical for coordinating mucosal inflammation during early infection, when S.Tm strongly relies on virulence factors detectable by the inflammasome. This may have broad relevance for mucosal defense against microbial pathogens.

  17. Distinct iPS Cells Show Different Cardiac Differentiation Efficiency. (United States)

    Ohno, Yohei; Yuasa, Shinsuke; Egashira, Toru; Seki, Tomohisa; Hashimoto, Hisayuki; Tohyama, Shugo; Saito, Yuki; Kunitomi, Akira; Shimoji, Kenichiro; Onizuka, Takeshi; Kageyama, Toshimi; Yae, Kojiro; Tanaka, Tomofumi; Kaneda, Ruri; Hattori, Fumiyuki; Murata, Mitsushige; Kimura, Kensuke; Fukuda, Keiichi


    Patient-specific induced pluripotent stem (iPS) cells can be generated by introducing transcription factors that are highly expressed in embryonic stem (ES) cells into somatic cells. This opens up new possibilities for cell transplantation-based regenerative medicine by overcoming the ethical issues and immunological problems associated with ES cells. Despite the development of various methods for the generation of iPS cells that have resulted in increased efficiency, safety, and general versatility, it remains unknown which types of iPS cells are suitable for clinical use. Therefore, the aims of the present study were to assess (1) the differentiation potential, time course, and efficiency of different types of iPS cell lines to differentiate into cardiomyocytes in vitro and (2) the properties of the iPS cell-derived cardiomyocytes. We found that high-quality iPS cells exhibited better cardiomyocyte differentiation in terms of the time course and efficiency of differentiation than low-quality iPS cells, which hardly ever differentiated into cardiomyocytes. Because of the different properties of the various iPS cell lines such as cardiac differentiation efficiency and potential safety hazards, newly established iPS cell lines must be characterized prior to their use in cardiac regenerative medicine.

  18. Distinct iPS Cells Show Different Cardiac Differentiation Efficiency

    Directory of Open Access Journals (Sweden)

    Yohei Ohno


    Full Text Available Patient-specific induced pluripotent stem (iPS cells can be generated by introducing transcription factors that are highly expressed in embryonic stem (ES cells into somatic cells. This opens up new possibilities for cell transplantation-based regenerative medicine by overcoming the ethical issues and immunological problems associated with ES cells. Despite the development of various methods for the generation of iPS cells that have resulted in increased efficiency, safety, and general versatility, it remains unknown which types of iPS cells are suitable for clinical use. Therefore, the aims of the present study were to assess (1 the differentiation potential, time course, and efficiency of different types of iPS cell lines to differentiate into cardiomyocytes in vitro and (2 the properties of the iPS cell-derived cardiomyocytes. We found that high-quality iPS cells exhibited better cardiomyocyte differentiation in terms of the time course and efficiency of differentiation than low-quality iPS cells, which hardly ever differentiated into cardiomyocytes. Because of the different properties of the various iPS cell lines such as cardiac differentiation efficiency and potential safety hazards, newly established iPS cell lines must be characterized prior to their use in cardiac regenerative medicine.

  19. IL25 elicits a multipotent progenitor cell population that promotes TH2 cytokine responses (United States)

    CD4+ T helper 2 (TH2) cells secrete interleukin (IL)4, IL5 and IL13, and are required for immunity to gastrointestinal helminth infections. However, TH2 cells also promote chronic inflammation associated with asthma and allergic disorders. The non-haematopoietic-cell-derived cytokines thymic stromal...

  20. The ethanol response gene Cab45 can modulate the impairment elicited by ethanol and ultraviolet in PC12 cells

    Institute of Scientific and Technical Information of China (English)

    Yunfeng Zhu; Quanli Wang; Wangru Xu; Sha Li


    High consumption of ethanolic beverages facilitates neurodegeneration,but the mechanism of this process still remained elusive.Suppression subtractive hybridization (SSH) is a technique for detection of rare transcripts.With SSH approach,we identified one ethanol response gene Cab45,which was down-regulated by ethanol with time-dependent manner in B104 cells.The full-length sequence of Cab45 gene was obtained by 5'-RACE (5'Rapid Amplification of cDNA Ends) for the first time in rat.Based on the sequence of deduced amino acid of rat Cab45,the alignment was conducted with its counterparts in different species and displayed a high conservation.Using different tissues in rat and cell lines,Cab45 was characterized by a ubiquitous expression and differentiation dependent down-regulation.Given that ethanol facilitates some cell differentiation,we hypothesize that Cab45 is involved in ethanol-mediated differentiation.With transient transfection,the function of Cab45 was investigated by up-regulation and down-regulation in PC12 cells.Ethanol treatment and UV exposure were conducted subsequently and cell proliferations were detected by MTT (Methyl Thiazolyl Tetrazolium) approach.It revealed that the up-regulation of Cab45 modulated the impairment elicited by ethanol and UV in transfected cells.As a member of new calcium binding protein family,the exact role of Cab45 still remains unclear.

  1. Functional cell types in taste buds have distinct longevities.

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    Isabel Perea-Martinez

    Full Text Available Taste buds are clusters of polarized sensory cells embedded in stratified oral epithelium. In adult mammals, taste buds turn over continuously and are replenished through the birth of new cells in the basal layer of the surrounding non-sensory epithelium. The half-life of cells in mammalian taste buds has been estimated as 8-12 days on average. Yet, earlier studies did not address whether the now well-defined functional taste bud cell types all exhibit the same lifetime. We employed a recently developed thymidine analog, 5-ethynil-2'-deoxyuridine (EdU to re-evaluate the incorporation of newly born cells into circumvallate taste buds of adult mice. By combining EdU-labeling with immunostaining for selected markers, we tracked the differentiation and lifespan of the constituent cell types of taste buds. EdU was primarily incorporated into basal extragemmal cells, the principal source for replenishing taste bud cells. Undifferentiated EdU-labeled cells began migrating into circumvallate taste buds within 1 day of their birth. Type II (Receptor taste cells began to differentiate from EdU-labeled precursors beginning 2 days after birth and then were eliminated with a half-life of 8 days. Type III (Presynaptic taste cells began differentiating after a delay of 3 days after EdU-labeling, and they survived much longer, with a half-life of 22 days. We also scored taste bud cells that belong to neither Type II nor Type III, a heterogeneous group that includes mostly Type I cells, and also undifferentiated or immature cells. A non-linear decay fit described these cells as two sub-populations with half-lives of 8 and 24 days respectively. Our data suggest that many post-mitotic cells may remain quiescent within taste buds before differentiating into mature taste cells. A small number of slow-cycling cells may also exist within the perimeter of the taste bud. Based on their incidence, we hypothesize that these may be progenitors for Type III cells.

  2. Distinct stem cells contribute to mammary gland development and maintenance. (United States)

    Van Keymeulen, Alexandra; Rocha, Ana Sofia; Ousset, Marielle; Beck, Benjamin; Bouvencourt, Gaëlle; Rock, Jason; Sharma, Neha; Dekoninck, Sophie; Blanpain, Cédric


    The mammary epithelium is composed of several cell lineages including luminal, alveolar and myoepithelial cells. Transplantation studies have suggested that the mammary epithelium is maintained by the presence of multipotent mammary stem cells. To define the cellular hierarchy of the mammary gland during physiological conditions, we performed genetic lineage-tracing experiments and clonal analysis of the mouse mammary gland during development, adulthood and pregnancy. We found that in postnatal unperturbed mammary gland, both luminal and myoepithelial lineages contain long-lived unipotent stem cells that display extensive renewing capacities, as demonstrated by their ability to clonally expand during morphogenesis and adult life as well as undergo massive expansion during several cycles of pregnancy. The demonstration that the mammary gland contains different types of long-lived stem cells has profound implications for our understanding of mammary gland physiology and will be instrumental in unravelling the cells at the origin of breast cancers.

  3. Elicitation triterpene yield in Alstonia scholaris cell cultures via synergetic organisms

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    Ercan Arıcan


    Full Text Available Cell cultures of Alstonia scholaris were treated with homogenates of Candida albicans, Fusarium oxysporum, Penicillium avelanium and Saccharomyces cerevisiae. The impact caused by the concentration, exposure time and the type of synergetic organisms on the accumulation of pentacyclic triterpenes was observed. When exposed to biotic elicitors for longer periods, some cell lines doubled the production of those triterpenes. S. cerevisiae homogenate was the best elicitor of triterpenes in all cell lines investigated.

  4. CD34+ cells in human intestine are fibroblasts adjacent to, but distinct from, interstitial cells of Cajal

    DEFF Research Database (Denmark)

    Vanderwinden, J M; Rumessen, J J; De Laet, M H;


    and confocal microscopy. CD34 immunoreactivity identified previously unrecognized cells closely adjacent to, but distinct from, the Kit immunoreactive ICC. These CD34 immunoreactive cells expressed the fibroblast marker prolyl 4-hydroxylase-whereas ICC did not-and were also distinct from smooth muscle cells...

  5. Proteome adaptation in cell reprogramming proceeds via distinct transcriptional networks

    NARCIS (Netherlands)

    Benevento, Marco; Tonge, Peter D; Puri, Mira C; Hussein, Samer M I; Cloonan, Nicole; Wood, David L; Grimmond, Sean M; Nagy, Andras; Munoz, Javier; Heck, Albert J R


    The ectopic expression of Oct4, Klf4, c-Myc and Sox2 (OKMS) transcription factors allows reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). The reprogramming process, which involves a complex network of molecular events, is not yet fully characterized. Here we perform a quan

  6. Involvement of distinct PKC gene products in T cell functions

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    Gottfried eBaier


    Full Text Available It is well established that members of the Protein kinase C(PKC family seem to have important roles in T cells. Focusing on the physiological and non-redundant PKC functions established in primary mouse T cells via germline gene-targeting approaches, our current knowledge defines two particularly critical PKC gene products, PKCθ and PKCα, as the flavor of PKC in T cells that appear to have a positive role in signaling pathways that are necessary for full antigen receptor-mediated T cell activation ex vivo and T cell-mediated immunity in vivo. Consistently, in spite of the current dogma that PKCθ inhibition might be sufficient to achieve complete immunosuppressive effects, more recent results have indicated that the pharmacological inhibition of PKCθ, and additionally, at least PKCα, appears to be needed to provide a successful approach for the prevention of allograft rejection and treatment of autoimmune diseases.

  7. Cell-Surface Protein Profiling Identifies Distinctive Markers of Progenitor Cells in Human Skeletal Muscle

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    Akiyoshi Uezumi


    Full Text Available Skeletal muscle contains two distinct stem/progenitor populations. One is the satellite cell, which acts as a muscle stem cell, and the other is the mesenchymal progenitor, which contributes to muscle pathogeneses such as fat infiltration and fibrosis. Detailed and accurate characterization of these progenitors in humans remains elusive. Here, we performed comprehensive cell-surface protein profiling of the two progenitor populations residing in human skeletal muscle and identified three previously unrecognized markers: CD82 and CD318 for satellite cells and CD201 for mesenchymal progenitors. These markers distinguish myogenic and mesenchymal progenitors, and enable efficient isolation of the two types of progenitors. Functional study revealed that CD82 ensures expansion and preservation of myogenic progenitors by suppressing excessive differentiation, and CD201 signaling favors adipogenesis of mesenchymal progenitors. Thus, cell-surface proteins identified here are not only useful markers but also functionally important molecules, and provide valuable insight into human muscle biology and diseases.

  8. Elicitation of silymarin in cell cultures of Silybum marianum: effect of subculture and repeated addition of methyl jasmonate. (United States)

    Sánchez-Sampedro, Maria Angeles; Fernández-Tárrago, Jorge; Corchete, Purificación


    Production of silymarin and the effect of the elicitor, methyl jasmonate (MeJA), was monitored in cell cultures of Silybum marianum over 4 years. Silymarin concentrations gradually declined after prolonged subculture, making the success of elicitor strategy limited in long-term cultures. The continuous presence of MeJA in cultures for an extended period was necessary for induction of silymarin accumulation. A repeated elicitor strategy was not a good option for improving silymarin productivity in batch cultures. Removal of medium from elicited cultures and addition of fresh medium avoided the toxic effects of elicitor accumulation, allowing the system to respond to a repeated MeJA treatment without loss of productivity.

  9. slan/M-DC8+ cells constitute a distinct subset of dendritic cells in human tonsils. (United States)

    Micheletti, Alessandra; Finotti, Giulia; Calzetti, Federica; Lonardi, Silvia; Zoratti, Elisa; Bugatti, Mattia; Stefini, Stefania; Vermi, William; Cassatella, Marco A


    Human blood dendritic cells (DCs) include three main distinct subsets, namely the CD1c+ and CD141+ myeloid DCs (mDCs) and the CD303+ plasmacytoid DCs (pDCs). More recently, a population of slan/M-DC8+ cells, also known as "slanDCs", has been described in blood and detected even in inflamed secondary lymphoid organs and non-lymphoid tissues. Nevertheless, hallmarks of slan/M-DC8+ cells in tissues are poorly defined. Herein, we report a detailed characterization of the phenotype and function of slan/M-DC8+ cells present in human tonsils. We found that tonsil slan/M-DC8+ cells represent a unique DC cell population, distinct from their circulating counterpart and also from all other tonsil DC and monocyte/macrophage subsets. Phenotypically, slan/M-DC8+ cells in tonsils display a CD11c+HLA-DR+CD14+CD11bdim/negCD16dim/negCX3CR1dim/neg marker repertoire, while functionally they exhibit an efficient antigen presentation capacity and a constitutive secretion of TNFα. Notably, such DC phenotype and functions are substantially reproduced by culturing blood slan/M-DC8+ cells in tonsil-derived conditioned medium (TDCM), further supporting the hypothesis of a full DC-like differentiation program occurring within the tonsil microenvironment. Taken together, our data suggest that blood slan/M-DC8+ cells are immediate precursors of a previously unrecognizedcompetent DC subset in tonsils, and pave the way for further characterization of slan/M-DC8+ cells in other tissues.

  10. CD4 and CD8 T cells mediate distinct lethal meningoencephalitis in mice challenged with Tacaribe arenavirus (United States)

    Ireland, Derek DC; Tami, Cecilia; Pedras-Vasconcelos, Joao; Verthelyi, Daniela


    Neonates are at increased risk of viral encephalopathies that can result in neurological dysfunction, seizures, permanent disability and even death. The neurological damage results from the combined effect of the virus and the immune response it elicits, thus finding tools to facilitate viral clearance from central nervous system (CNS) while minimizing neuron damage remains a critical challenge. Neonatal mice inoculated intraperitoneally with Tacaribe virus (TCRV) develop seizures, hindlimb paralysis and death within 15 days of inoculation. TCRV localizes to the CNS within days of challenge, primarily infecting astrocytes in the cerebellum and brain stem. We show that infection leads to inflammation, T cell and monocyte infiltration into the cerebellar parenchyma, apoptosis of astrocytes, neuronal degeneration and loss of Purkinje cells. Infiltrating antigen-specific T cells fail to clear the virus but drive the disease, as T-cell-deficient CD3ɛ KO mice survive TCRV infection with minimal inflammation or clinical manifestations despite no difference in CNS viral loads in comparison with T-cell sufficient mice. CD8+ T cells drive the pathology, which even in the absence of CD4+ T-cell help, infiltrate the parenchyma and mediate the apoptotic loss of cerebellar astrocytes, neurodegeneration and loss of Purkinje cells resulting in loss of balance, paralysis and death. CD4+ T cells are also pathogenic inducing gliosis and inflammation in the cerebellum and cerebrum that are associated with wasting and death several weeks after CD4+ T-cell transfer. These data demonstrate distinct pathogenic effects of CD4+ and CD8+ T cells and identify them as possible therapeutic targets. PMID:27569560

  11. Myf5 haploinsufficiency reveals distinct cell fate potentials for adult skeletal muscle stem cells. (United States)

    Gayraud-Morel, Barbara; Chrétien, Fabrice; Jory, Aurélie; Sambasivan, Ramkumar; Negroni, Elisa; Flamant, Patricia; Soubigou, Guillaume; Coppée, Jean-Yves; Di Santo, James; Cumano, Ana; Mouly, Vincent; Tajbakhsh, Shahragim


    Skeletal muscle stem cell fate in adult mice is regulated by crucial transcription factors, including the determination genes Myf5 and Myod. The precise role of Myf5 in regulating quiescent muscle stem cells has remained elusive. Here we show that most, but not all, quiescent satellite cells express Myf5 protein, but at varying levels, and that resident Myf5 heterozygous muscle stem cells are more primed for myogenic commitment compared with wild-type satellite cells. Paradoxically however, heterotypic transplantation of Myf5 heterozygous cells into regenerating muscles results in higher self-renewal capacity compared with wild-type stem cells, whereas myofibre regenerative capacity is not altered. By contrast, Pax7 haploinsufficiency does not show major modifications by transcriptome analysis. These observations provide a mechanism linking Myf5 levels to muscle stem cell heterogeneity and fate by exposing two distinct and opposing phenotypes associated with Myf5 haploinsufficiency. These findings have important implications for how stem cell fates can be modulated by crucial transcription factors while generating a pool of responsive heterogeneous cells.

  12. Intracellular angiotensin II elicits Ca2+ increases in A7r5 vascular smooth muscle cells

    NARCIS (Netherlands)

    Filipeanu, CM; Brailoiu, E; Kok, JW; Henning, RH; De Zeeuw, D; Nelemans, SA


    Recent studies show that angiotensin II can act within the cell, possibly via intracellular receptors pharmacologically different from typical plasma membrane angiotensin II receptors. The signal transduction of intracellular angiotensin LI is unclear. Therefore. we investigated the effects of intra

  13. Host plant peptides elicit a transcriptional response to control the Sinorhizobium meliloti cell cycle during symbiosis


    Penterman, Jon; Abo, Ryan P.; De Nisco, Nicole J.; Markus F F Arnold; Longhi, Renato; ZANDA, Matteo; Walker, Graham C.


    Sinorhizobium meliloti and its legume hosts establish a symbiosis in which bacterial fixed nitrogen is exchanged for plant carbon compounds. We study this symbiosis because it is agriculturally and ecologically important and to identify mechanisms used in host–microbe interactions. S. meliloti is internalized in specialized host nodule cells that then use small, cysteine-rich peptides to drive their differentiation into polyploid cells that fix nitrogen. We found that a representative host pe...

  14. Suboptimal B-cell antigen receptor signaling activity in vivo elicits germinal center counterselection mechanisms. (United States)

    Königsberger, Sebastian; Weis, Vanessa; Prodöhl, Jan; Stehling, Martin; Hobeika, Elias; Reth, Michael; Kiefer, Friedemann


    Syk and Zap-70 constitute a closely related nonreceptor protein tyrosine kinase family, of which both members are functionally indispensable for conferring their respective antigen receptors with enzymatic activity. In this study, we analyze the impact of altering BCR signaling output on B-cell germinal center (GC) fate selection by constitutive, as well as inducible, monoallelic Syk kinase loss in the presence of a Zap-70 knock-in rescue allele. Cre-mediated Syk deletion in Syk(flox/Zap-70) B cells lowers pErk, but not pAkt-mediated signaling. Surprisingly, the use of a B-cell-specific constitutive mb1-cre deleter mouse model showed that a small cohort of peripheral Syk(flox/Zap-70);mb1-cre B cells efficiently circumvents deletion, which ultimately favors these Syk-sufficient cells to contribute to the GC reaction. Using a developmentally unbiased Syk(flox/Zap-70);mb1-creER(T2) approach in combination with an inducible tdRFP allele, we further demonstrate that this monoallelic deletion escape is not fully explained by leakiness of Cre expression, but is possibly the result of differential Syk locus accessibility in maturing B cells. Altogether, this underscores the importance of proper Syk kinase function not only during central and peripheral selection processes, but also during GC formation and maintenance.

  15. Anaplasma phagocytophilum and Anaplasma marginale Elicit Different Gene Expression Responses in Cultured Tick Cells

    Directory of Open Access Journals (Sweden)

    Zorica Zivkovic


    Full Text Available The genus Anaplasma (Rickettsiales: Anaplasmataceae includes obligate tick-transmitted intracellular organisms, Anaplasma phagocytophilum and Anaplasma marginale that multiply in both vertebrate and tick host cells. Recently, we showed that A. marginale affects the expression of tick genes that are involved in tick survival and pathogen infection and multiplication. However, the gene expression profile in A. phagocytophilum-infected tick cells is currently poorly characterized. The objectives of this study were to characterize tick gene expression profile in Ixodes scapularis ticks and cultured ISE6 cells in response to infection with A. phagocypthilum and to compare tick gene expression responses in A. phagocytophilum- and A. marginale-infected tick cells by microarray and real-time RT-PCR analyses. The results of these studies demonstrated modulation of tick gene expression by A. phagocytophilum and provided evidence of different gene expression responses in tick cells infected with A. phagocytophilum and A. marginale. These differences in Anaplasma-tick interactions may reflect differences in pathogen life cycle in the tick cells.

  16. Salicylic-acid elicited phospholipase D responses in Capsicum chinense cell cultures. (United States)

    Rodas-Junco, B A; Muñoz-Sánchez, J A; Vázquez-Flota, F; Hernández-Sotomayor, S M T


    The plant response to different stress types can occur through stimulus recognition and the subsequent signal transduction through second messengers that send information to the regulation of metabolism and the expression of defense genes. The phospholipidic signaling pathway forms part of the plant response to several phytoregulators, such as salicylic acid (SA), which has been widely used to stimulate secondary metabolite production in cell cultures. In this work, we studied the effects of SA treatment on [(32)-P]Pi phospholipid turnover and phospholipase D (PLD) activity using cultured Capsicum chinense cells. In cultured cells, the PIP2 turnover showed changes after SA treatment, while the most abundant phospholipids (PLs), such as phosphatidylcholine (PC), did not show changes during the temporal course. SA treatment significantly increased phosphatidic acid (PA) turnover over time compared to control cells. The PA accumulation in cells treated with 1-butanol showed a decrease in messengers; at the same time, there was a 1.5-fold increase in phosphatidylbutanol. These results suggest that the participation of the PLD pathway is a source of PA production, and the activation of this mechanism may be important in the cell responses to SA treatment.

  17. Biophysical characterization of hematopoietic cells from normal and leukemic sources with distinct primitiveness (United States)

    Tan, Youhua; Fung, Tsz-Kan; Wan, Haixia; Wang, Kaiqun; Leung, Anskar Y. H.; Sun, Dong


    This letter reported the biophysical characterization of immunophenotypically distinct hematopoietic cells from normal and leukemic sources, through manipulation with optical tweezers at single cell level. The results show that the percentage of cells that are stretchable and their deformability are significantly higher in the more primitive cell populations. This study provides the evidence that normal and leukemic hematopoietic cell populations with distinct primitiveness exhibit differential biophysical properties. These findings raise a hypothesis that the high deformability may be related to the unique functions and activities of primitive hematopoietic cells.

  18. Inflammation-Induced CCR7 Oligomers Form Scaffolds to Integrate Distinct Signaling Pathways for Efficient Cell Migration. (United States)

    Hauser, Mark A; Schaeuble, Karin; Kindinger, Ilona; Impellizzieri, Daniela; Krueger, Wolfgang A; Hauck, Christof R; Boyman, Onur; Legler, Daniel F


    Host defense depends on orchestrated cell migration guided by chemokines that elicit selective but biased signaling pathways to control chemotaxis. Here, we showed that different inflammatory stimuli provoked oligomerization of the chemokine receptor CCR7, enabling human dendritic cells and T cell subpopulations to process guidance cues not only through classical G protein-dependent signaling but also by integrating an oligomer-dependent Src kinase signaling pathway. Efficient CCR7-driven migration depends on a hydrophobic oligomerization interface near the conserved NPXXY motif of G protein-coupled receptors as shown by mutagenesis screen and a CCR7-SNP demonstrating super-oligomer characteristics leading to enhanced Src activity and superior chemotaxis. Furthermore, Src phosphorylates oligomeric CCR7, thereby creating a docking site for SH2-domain-bearing signaling molecules. Finally, we identified CCL21-biased signaling that involved the phosphatase SHP2 to control efficient cell migration. Collectively, our data showed that CCR7 oligomers serve as molecular hubs regulating distinct signaling pathways.

  19. Recognition of enteroinvasive Escherichia coli and Shigella flexneri by dendritic cells: distinct dendritic cell activation states

    Directory of Open Access Journals (Sweden)

    Ana Carolina Ramos Moreno


    Full Text Available The innate and adaptive immune responses of dendritic cells (DCs to enteroinvasive Escherichia coli (EIEC infection were compared with DC responses to Shigella flexneri infection. EIEC triggered DCs to produce interleukin (IL-10, IL-12 and tumour necrosis factor (TNF-α, whereas S. flexneri induced only the production of TNF-α. Unlike S. flexneri, EIEC strongly increased the expression of toll like receptor (TLR-4 and TLR-5 in DCs and diminished the expression of co-stimulatory molecules that may cooperate to inhibit CD4+ T-lymphocyte proliferation. The inflammation elicited by EIEC seems to be related to innate immunity both because of the aforementioned results and because only EIEC were able to stimulate DC transmigration across polarised Caco-2 cell monolayers, a mechanism likely to be associated with the secretion of CC chemokine ligands (CCL20 and TNF-α. Understanding intestinal DC biology is critical to unravelling the infection strategies of EIEC and may aid in the design of treatments for infectious diseases.

  20. The effect of beta-interferon therapy on myelin basic protein-elicited CD4+ T cell proliferation and cytokine production in multiple sclerosis

    DEFF Research Database (Denmark)

    Hedegaard, Chris J; Krakauer, Martin; Bendtzen, Klaus


    Interferon (IFN)-beta therapy has well-established clinical benefits in multiple sclerosis (MS), but the underlying modulation of cytokine responses to myelin self-antigens remains poorly understood. We analysed the CD4+ T cell proliferation and cytokine responses elicited by myelin basic protein...... (MBP) and a foreign recall antigen, tetanus toxoid (TT), in mononuclear cell cultures from fourteen MS patients undergoing IFN-beta therapy. The MBP-elicited IFN-gamma-, TNF-alpha- and IL-10 production decreased during therapy (p...

  1. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade (United States)

    McGranahan, Nicholas; Furness, Andrew J. S.; Rosenthal, Rachel; Ramskov, Sofie; Lyngaa, Rikke; Saini, Sunil Kumar; Jamal-Hanjani, Mariam; Wilson, Gareth A.; Birkbak, Nicolai J.; Hiley, Crispin T.; Watkins, Thomas B. K.; Shafi, Seema; Murugaesu, Nirupa; Mitter, Richard; Akarca, Ayse U.; Linares, Joseph; Marafioti, Teresa; Henry, Jake Y.; Van Allen, Eliezer M.; Miao, Diana; Schilling, Bastian; Schadendorf, Dirk; Garraway, Levi A.; Makarov, Vladimir; Rizvi, Naiyer A.; Snyder, Alexandra; Hellmann, Matthew D.; Merghoub, Taha; Wolchok, Jedd D.; Shukla, Sachet A.; Wu, Catherine J.; Peggs, Karl S.; Chan, Timothy A.; Hadrup, Sine R.; Quezada, Sergio A.; Swanton, Charles


    As tumors grow, they acquire mutations, some of which create neoantigens that influence the response of patients to immune checkpoint inhibitors. We explored the impact of neoantigen intratumor heterogeneity (ITH) on antitumor immunity. Through integrated analysis of ITH and neoantigen burden, we demonstrate a relationship between clonal neoantigen burden and overall survival in primary lung adenocarcinomas. CD8+ tumor-infiltrating lymphocytes reactive to clonal neoantigens were identified in early-stage non–small cell lung cancer and expressed high levels of PD-1. Sensitivity to PD-1 and CTLA-4 blockade in patients with advanced NSCLC and melanoma was enhanced in tumors enriched for clonal neoantigens. T cells recognizing clonal neoantigens were detectable in patients with durable clinical benefit. Cytotoxic chemotherapy–induced subclonal neoantigens, contributing to an increased mutational load, were enriched in certain poor responders. These data suggest that neoantigen heterogeneity may influence immune surveillance and support therapeutic developments targeting clonal neoantigens. PMID:26940869

  2. Amidase, a cell wall hydrolase, elicits protective immunity against Staphylococcus aureus and S. epidermidis. (United States)

    Nair, Nisha; Vinod, Vivek; Suresh, Maneesha K; Vijayrajratnam, Sukhithasri; Biswas, Lalitha; Peethambaran, Reshmi; Vasudevan, Anil Kumar; Biswas, Raja


    The morbidity and the mortality associated with Staphylococcus aureus and S. epidermidis infections have greatly increased due to the rapid emergence of highly virulent and antibiotic resistant strains. Development of a vaccine-based therapy is greatly desired. However, no staphylococcal vaccine is available till date. In this study, we have identified Major amidase (Atl-AM) as a prime candidate for future vaccine design against these pathogens. Atl-AM is a multi-functional non-covalently cell wall associated protein which is involved in staphylococcal cell separation after cell division, host extracellular matrix adhesion and biofilm formation. Atl-AM is present on the surface of diverse S. aureus and S. epidermidis strains. When used in combination with Freund's adjuvant, Atl-AM generated a mixed Th1 and Th2 mediated immune response which is skewed more toward Th1; and showed increased production of opsonophagocytic IgG2a and IgG2b antibodies. Significant protective immune response was observed when vaccinated mice were challenged with S. aureus or S. epidermidis. Vaccination prevented the systemic dissemination of both organisms. Our results demonstrate the remarkable efficacy of Atl-AM as a vaccine candidate against both of these pathogens.

  3. Equal modulation of endothelial cell function by four distinct tissue-specific mesenchymal stem cells. (United States)

    Lin, Ruei-Zeng; Moreno-Luna, Rafael; Zhou, Bin; Pu, William T; Melero-Martin, Juan M


    Mesenchymal stem cells (MSCs) can generate multiple end-stage mesenchymal cell types and constitute a promising population of cells for regenerative therapies. Additionally, there is increasing evidence supporting other trophic activities of MSCs, including the ability to enable formation of vasculature in vivo. Although MSCs were originally isolated from the bone marrow, the presence of these cells in the stromal vascular fraction of multiple adult tissues has been recently recognized. However, it is unknown whether the capacity to modulate vasculogenesis is ubiquitous to all MSCs regardless of their tissue of origin. Here, we demonstrated that tissue-resident MSCs isolated from four distinct tissues have equal capacity to modulate endothelial cell function, including formation of vascular networks in vivo. MSCs were isolated from four murine tissues, including bone marrow, white adipose tissue, skeletal muscle, and myocardium. In culture, all four MSC populations secreted a plethora of pro-angiogenic factors that unequivocally induced proliferation, migration, and tube formation of endothelial colony-forming cells (ECFCs). In vivo, co-implantation of MSCs with ECFCs into mice generated an extensive network of blood vessels with ECFCs specifically lining the lumens and MSCs occupying perivascular positions. Importantly, there were no differences among all four MSCs evaluated. Our studies suggest that the capacity to modulate the formation of vasculature is a ubiquitous property of all MSCs, irrespective of their original anatomical location. These results validate multiple tissues as potential sources of MSCs for future cell-based vascular therapies.

  4. Elicitation, an Effective Strategy for the Biotechnological Production of Bioactive High-Added Value Compounds in Plant Cell Factories

    Directory of Open Access Journals (Sweden)

    Karla Ramirez-Estrada


    Full Text Available Plant in vitro cultures represent an attractive and cost-effective alternative to classical approaches to plant secondary metabolite (PSM production (the “Plant Cell Factory” concept. Among other advantages, they constitute the only sustainable and eco-friendly system to obtain complex chemical structures biosynthesized by rare or endangered plant species that resist domestication. For successful results, the biotechnological production of PSM requires an optimized system, for which elicitation has proved one of the most effective strategies. In plant cell cultures, an elicitor can be defined as a compound introduced in small concentrations to a living system to promote the biosynthesis of the target metabolite. Traditionally, elicitors have been classified in two types, abiotic or biotic, according to their chemical nature and exogenous or endogenous origin, and notably include yeast extract, methyl jasmonate, salicylic acid, vanadyl sulphate and chitosan. In this review, we summarize the enhancing effects of elicitors on the production of high-added value plant compounds such as taxanes, ginsenosides, aryltetralin lignans and other types of polyphenols, focusing particularly on the use of a new generation of elicitors such as coronatine and cyclodextrins.

  5. Distinct regulatory functions of calpain 1 and 2 during neural stem cell self-renewal and differentiation.

    Directory of Open Access Journals (Sweden)

    Daniela M Santos

    Full Text Available Calpains are calcium regulated cysteine proteases that have been described in a wide range of cellular processes, including apoptosis, migration and cell cycle regulation. In addition, calpains have been implicated in differentiation, but their impact on neural differentiation requires further investigation. Here, we addressed the role of calpain 1 and calpain 2 in neural stem cell (NSC self-renewal and differentiation. We found that calpain inhibition using either the chemical inhibitor calpeptin or the endogenous calpain inhibitor calpastatin favored differentiation of NSCs. This effect was associated with significant changes in cell cycle-related proteins and may be regulated by calcium. Interestingly, calpain 1 and calpain 2 were found to play distinct roles in NSC fate decision. Calpain 1 expression levels were higher in self-renewing NSC and decreased with differentiation, while calpain 2 increased throughout differentiation. In addition, calpain 1 silencing resulted in increased levels of both neuronal and glial markers, β-III Tubulin and glial fibrillary acidic protein (GFAP. Calpain 2 silencing elicited decreased levels of GFAP. These results support a role for calpain 1 in repressing differentiation, thus maintaining a proliferative NSC pool, and suggest that calpain 2 is involved in glial differentiation.

  6. Antigen-Specific lgA B Memory Cell Responses to Shigella Antigens Elicited in Volunteers Immunized with Live Attenuated Shigella flexneri 2a Oral Vaccine Candidates (United States)


    cell responses to Shigella antigens elicited in volunteers immunized with live attenuated Shigella flexneri 2a oral vaccine candidates J.K. Simona,b... Shigella ;. B cell memory; Immunoglobulin lgA; Mucosal immunity Abstract We studied the induction of antigen-specific lgA memory B cells (BM) in...volunteers who received live attenuated Shigella flexneri 2a vaccines. Subjects ingested a single oral dose of 107 , 108 or 109 CFU of S. flexneri 2a with

  7. Th22 cells represent a distinct human T cell subset involved in epidermal immunity and remodeling. (United States)

    Eyerich, Stefanie; Eyerich, Kilian; Pennino, Davide; Carbone, Teresa; Nasorri, Francesca; Pallotta, Sabatino; Cianfarani, Francesca; Odorisio, Teresa; Traidl-Hoffmann, Claudia; Behrendt, Heidrun; Durham, Stephen R; Schmidt-Weber, Carsten B; Cavani, Andrea


    Th subsets are defined according to their production of lineage-indicating cytokines and functions. In this study, we have identified a subset of human Th cells that infiltrates the epidermis in individuals with inflammatory skin disorders and is characterized by the secretion of IL-22 and TNF-alpha, but not IFN-gamma, IL-4, or IL-17. In analogy to the Th17 subset, cells with this cytokine profile have been named the Th22 subset. Th22 clones derived from patients with psoriasis were stable in culture and exhibited a transcriptome profile clearly separate from those of Th1, Th2, and Th17 cells; it included genes encoding proteins involved in tissue remodeling, such as FGFs, and chemokines involved in angiogenesis and fibrosis. Primary human keratinocytes exposed to Th22 supernatants expressed a transcriptome response profile that included genes involved in innate immune pathways and the induction and modulation of adaptive immunity. These proinflammatory Th22 responses were synergistically dependent on IL-22 and TNF-alpha. Furthermore, Th22 supernatants enhanced wound healing in an in vitro injury model, which was exclusively dependent on IL-22. In conclusion, the human Th22 subset may represent a separate T cell subset with a distinct identity with respect to gene expression and function, present within the epidermal layer in inflammatory skin diseases. Future strategies directed against the Th22 subset may be of value in chronic inflammatory skin disorders.

  8. Distinct mechanical behavior of HEK293 cells in adherent and suspended states. (United States)

    Haghparast, Seyed Mohammad Ali; Kihara, Takanori; Miyake, Jun


    The mechanical features of individual animal cells have been regarded as indicators of cell type and state. Previously, we investigated the surface mechanics of cancer and normal stromal cells in adherent and suspended states using atomic force microscopy. Cancer cells possessed specific mechanical and actin cytoskeleton features that were distinct from normal stromal cells in adherent and suspended states. In this paper, we report the unique mechanical and actin cytoskeletal features of human embryonic kidney HEK293 cells. Unlike normal stromal and cancer cells, the surface stiffness of adherent HEK293 cells was very low, but increased after cell detachment from the culture surface. Induced actin filament depolymerization revealed that the actin cytoskeleton was the underlying source of the stiffness in suspended HEK293 cells. The exclusive mechanical response of HEK293 cells to perturbation of the actin cytoskeleton resembled that of adherent cancer cells and suspended normal stromal cells. Thus, with respect to their special cell-surface mechanical features, HEK293 cells could be categorized into a new class distinct from normal stromal and cancer cells.

  9. Bystander effects elicited by single-cell photo-oxidative blue-light stimulation in retinal pigment epithelium cell networks (United States)

    Ishii, Masaaki; Rohrer, Bärbel


    ‘Bystander effect’ refers to the induction of biological effects in cells not directly targeted. The retinal pigment epithelium consists of hexagonal cells, forming a monolayer interconnected by gap junctions (GJs). Oxidative stress initiated in an individual cell by photostimulation (488 nm) triggered changes in reactive oxygen species (ROS), Ca2+ and mitochondrial membrane potential (ψm). The Ca2+ signal was transmitted to neighboring cells slowly and non-uniformly; the ROS signal spread fast and radially. Increased Ca2+ levels were associated with a loss in ψm. GJ blockers prevented the spreading of the Ca2+, but not the ROS-related signal. The GJ-mediated Ca2+ wave was associated with cell death by 24 h, requiring endoplasmic reticulum–mitochondria Ca2+ transfer. Ensuing cell death was correlated with baseline Ca2+ levels, and baseline Ca2+ levels were correlated with pigmentation. Hence, local oxidative stress in a donor cell can trigger changes in certain connected recipient cells, a signal that required GJ communication and an ROS-Ca2+ dual-hit. Finally, damage apparently occurred in susceptible cells, which correlated with baseline Ca2+ levels. PMID:28179989

  10. Affinity for self antigen selects Treg cells with distinct functional properties. (United States)

    Wyss, Lena; Stadinski, Brian D; King, Carolyn G; Schallenberg, Sonja; McCarthy, Nicholas I; Lee, Jun Young; Kretschmer, Karsten; Terracciano, Luigi M; Anderson, Graham; Surh, Charles D; Huseby, Eric S; Palmer, Ed


    The manner in which regulatory T cells (Treg cells) control lymphocyte homeostasis is not fully understood. We identified two Treg cell populations with differing degrees of self-reactivity and distinct regulatory functions. We found that GITR(hi)PD-1(hi)CD25(hi) (Triple(hi)) Treg cells were highly self-reactive and controlled lympho-proliferation in peripheral lymph nodes. GITR(lo)PD-1(lo)CD25(lo) (Triple(lo)) Treg cells were less self-reactive and limited the development of colitis by promoting the conversion of CD4(+) Tconv cells into induced Treg cells (iTreg cells). Although Foxp3-deficient (Scurfy) mice lacked Treg cells, they contained Triple(hi)-like and Triple(lo)-like CD4(+) T cells zsuper> T cells infiltrated the skin, whereas Scurfy Triple(lo)CD4(+) T cells induced colitis and wasting disease. These findings indicate that the affinity of the T cell antigen receptor for self antigen drives the differentiation of Treg cells into distinct subsets with non-overlapping regulatory activities.

  11. Different modes of endothelial-smooth muscle cell interaction elicit differential β-catenin phosphorylations and endothelial functions. (United States)

    Chang, Shun-Fu; Chen, Li-Jing; Lee, Pei-Ling; Lee, Ding-Yu; Chien, Shu; Chiu, Jeng-Jiann


    β-Catenin phosphorylation plays important roles in modulating its functions, but the effects of different phosphorylated forms of β-catenin in response to heterocellular interaction are unclear. Here we investigated whether distinct modes of phosphorylation on β-catenin could be triggered through heterocellular interactions between endothelial cells (ECs) and smooth muscle cells (SMCs), and the consequent modulation of EC functions. ECs were cocultured with SMCs to initiate direct contact and paracrine interaction. EC-SMC coculture induced EC β-catenin phosphorylations simultaneously at tyrosine 142 (Tyr142) and serine 45/threonine 41 (Ser45/Thr41) at the cytoplasm/nuclei and the membrane, respectively. Treating ECs with SMC-conditional medium induced β-catenin phosphorylation only at Ser45/Thr41. These findings indicate that different phosphorylation effects of EC-SMC coculture were induced through heterocellular direct contact and paracrine effects, respectively. Using specific blocking peptides, antagonists, and siRNAs, we found that the β-catenin Tyr142-phosphorylation was mediated by connexin 43/Fer and that the β-catenin Ser45/Thr41-phosphorylation was mediated by SMC-released bone morphogenetic proteins through VE-cadherin and bone morphogenetic protein receptor-II/Smad5. Transfecting ECs with β-catenin-Tyr142 or -Ser45 mutants showed that these two phosphorylated forms of β-catenin modulate differential EC function: The Tyr142-phosphorylated β-catenin stimulates vascular cell-adhesion molecule-1 expression to increase EC-monocytic adhesion, but the Ser45/Thr41-phosphorylated β-catenin attenuates VE-cadherin-dependent junction structures to increase EC permeability. Our findings provide new insights into the understanding of regulatory complexities of distinct modes of β-catenin phosphorylations under EC-SMC interactions and suggest that different phosphorylated forms of β-catenin play important roles in modulating vascular pathophysiology

  12. Early- and late-born parvalbumin basket cell subpopulations exhibiting distinct regulation and roles in learning. (United States)

    Donato, Flavio; Chowdhury, Ananya; Lahr, Maria; Caroni, Pico


    Brain networks can support learning by promoting acquisition of task-relevant information or by adhering to validated rules, but the mechanisms involved are poorly understood. Upon learning, local inhibitory parvalbumin (PV)-expressing Basket cell networks can switch to opposite configurations that either favor or interfere with further learning, but how this opposite plasticity is induced and relates to distinct learning requirements has remained unclear. Here, we show that PV Basket cells consist of hitherto unrecognized subpopulations, with distinct schedules of neurogenesis, input connectivities, output target neurons, and roles in learning. Plasticity of hippocampal early-born PV neurons was recruited in rule consolidation, whereas plasticity of late-born PV neurons was recruited in new information acquisition. This involved regulation of early-born neuron plasticity specifically through excitation, and of late-born neuron plasticity specifically through inhibition. Therefore, opposite learning requirements are implemented by distinct local networks involving PV Basket cell subpopulations specifically regulated through inhibition or excitation.

  13. Genes and Gene Networks Involved in Sodium Fluoride-Elicited Cell Death Accompanying Endoplasmic Reticulum Stress in Oral Epithelial Cells

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    Yoshiaki Tabuchi


    Full Text Available Here, to understand the molecular mechanisms underlying cell death induced by sodium fluoride (NaF, we analyzed gene expression patterns in rat oral epithelial ROE2 cells exposed to NaF using global-scale microarrays and bioinformatics tools. A relatively high concentration of NaF (2 mM induced cell death concomitant with decreases in mitochondrial membrane potential, chromatin condensation and caspase-3 activation. Using 980 probe sets, we identified 432 up-regulated and 548 down-regulated genes, that were differentially expressed by >2.5-fold in the cells treated with 2 mM of NaF and categorized them into 4 groups by K-means clustering. Ingenuity® pathway analysis revealed several gene networks from gene clusters. The gene networks Up-I and Up-II included many up-regulated genes that were mainly associated with the biological function of induction or prevention of cell death, respectively, such as Atf3, Ddit3 and Fos (for Up-I and Atf4 and Hspa5 (for Up-II. Interestingly, knockdown of Ddit3 and Hspa5 significantly increased and decreased the number of viable cells, respectively. Moreover, several endoplasmic reticulum (ER stress-related genes including, Ddit3, Atf4 and Hapa5, were observed in these gene networks. These findings will provide further insight into the molecular mechanisms of NaF-induced cell death accompanying ER stress in oral epithelial cells.

  14. Characterization and heterologous expression of hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase from elicited cell cultures of carnation, Dianthus caryophyllus L. (United States)

    Yang, Q; Reinhard, K; Schiltz, E; Matern, U


    Benzoyl-CoA:anthranilate N-benzoyltransferase catalyzes the first committed reaction of phytoalexin biosynthesis in carnation (Dianthus caryophyllus L.), and the product N-benzoylanthranilate is the precursor of several sets of dianthramides. The transferase activity is constitutively expressed in suspension-cultured carnation cells and can be rapidly induced by the addition of yeast extract. The enzyme was purified to homogeneity from yeast-induced carnation cells and shown to consist of a single polypeptide chain of 53 kDa. Roughly 20% of the sequence was identified by micro-sequencing of tryptic peptides, and some of these sequences differed in a few amino acid residues only suggesting the presence of isoenzymes. A specific 0.8 kb cDNA probe was generated by RT-PCR, employing degenerated oligonucleotide primers complementary to two of the tryptic peptides and using poly(A)+ RNA from elicited carnation cells. Five distinct benzoyltransferase clones were isolated from a cDNA library, and three cDNAs, pchcbt1-3, were sequenced and shown to encode full-size N-benzoyltransferases. The translated peptide sequences revealed more than 95% identity among these three clones. The additional two clones harbored insert sequences mostly homologous with pchcbt 1 but differing in the 3'-flanking regions due to variable usage of poly(A) addition sites. The identity of the clones was confirmed by matching the translated polypeptides with the tryptic enzyme sequences as well as by the activity of the benzoyltransferase expressed in Escherichia coli. Therefore, carnation encodes a small family of anthranilate N-benzoyltransferase genes. In vitro, the benzoyltransferases exhibited narrow substrate specificity for anthranilate but accepted a variety of aromatic acyl-CoAs. Catalytic rates with cinnamoyl- or 4-coumaroyl-CoA exceeded those observed with benzoyl-CoA, although the corresponding dianthramides did not accumulate in vivo. Thus the cDNAs described represent also the first

  15. KDM6B Elicits Cell Apoptosis by Promoting Nuclear Translocation of FOXO1 in Non-Small Cell Lung Cancer

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    Jun Ma


    Full Text Available Background/Aims: Non-small cell lung carcinoma (NSCLC is the most common type of lung cancer and the cause of most cancer-related deaths. The molecular mechanisms that are involved in NSCLC development are currently not well understood. Accumulating evidence shows that histone demethylases play important roles in the regulation of pathological developmental processes in many diseases, including various types of cancers. Methods: Mitochondrial membrane potential assays, migration and invasion assays, caspase-3 and caspase-9 activity assays and western blot analysis were used in this research. Results: We found that overexpression of KDM6B, a demethylase that acts on histone H3 at lysine 27 (H3K27, inhibited cell growth by initiating mitochondria-dependent apoptosis and by attenuating the invasion-metastasis cascade in NSCLC cells. Moreover, our results showed that KDM6B directly interacted with FOXO1 and that overexpression of KDM6B promoted nuclear accumulation of FOXO1. The effects of KDM6B on cell apoptosis and metastasis were weakened by knockdown of FOXO1 expression. On the contrary, knocking down expression of KDM6B inhibited cell apoptosis and promoted cell growth by mitigating the nuclear translocation of FOXO1 in NSCLC cells. Conclusions: These findings suggest that KDM6B may act in a pro-apoptotic role in NSCLC by causing the nuclear translocation of FOXO1.

  16. Osmotically induced cell swelling versus cell shrinking elicits specific changes in phospholipid signals in tobacco pollen tubes

    NARCIS (Netherlands)

    L.E. Zonia; T. Munnik


    Pollen tube cell volume changes rapidly in response to perturbation of the extracellular osmotic potential. This report shows that specific phospholipid signals are differentially stimulated or attenuated during osmotic perturbations. Hypo-osmotic stress induces rapid increases in phosphatidic acid

  17. MeHg Developing Exposure Causes DNA Double-Strand Breaks and Elicits Cell Cycle Arrest in Spinal Cord Cells (United States)

    Ferreira, Fabiana F.; Ammar, Dib; Bourckhardt, Gilian F.; Kobus-Bianchini, Karoline; Müller, Yara M. R.; Nazari, Evelise M.


    The neurotoxicity caused by methylmercury (MeHg) is well documented; however, the developmental neurotoxicity in spinal cord is still not fully understood. Here we investigated whether MeHg affects the spinal cord layers development. Chicken embryos at E3 were treated in ovo with 0.1 μg MeHg/50 μL saline solution and analyzed at E10. Thus, we performed immunostaining using anti-γ-H2A.X to recognize DNA double-strand breaks and antiphosphohistone H3, anti-p21, and anti-cyclin E to identify cells in proliferation and cell cycle proteins. Also, to identify neuronal cells, we used anti-NeuN and anti-βIII-tubulin antibodies. After the MeHg treatment, we observed the increase on γ-H2A.X in response to DNA damage. MeHg caused a decrease in the proliferating cells and in the thickness of spinal cord layers. Moreover, we verified that MeHg induced an increase in the number of p21-positive cells but did not change the cyclin E-positive cells. A significantly high number of TUNEL-positive cells indicating DNA fragmentation were observed in MeHg-treated embryos. Regarding the neuronal differentiation, MeHg induced a decrease in NeuN expression and did not change the expression of βIII-tubulin. These results showed that in ovo MeHg exposure alters spinal cord development by disturbing the cell proliferation and death, also interfering in early neuronal differentiation. PMID:26793240

  18. MeHg Developing Exposure Causes DNA Double-Strand Breaks and Elicits Cell Cycle Arrest in Spinal Cord Cells

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    Fabiana F. Ferreira


    Full Text Available The neurotoxicity caused by methylmercury (MeHg is well documented; however, the developmental neurotoxicity in spinal cord is still not fully understood. Here we investigated whether MeHg affects the spinal cord layers development. Chicken embryos at E3 were treated in ovo with 0.1 μg MeHg/50 μL saline solution and analyzed at E10. Thus, we performed immunostaining using anti-γ-H2A.X to recognize DNA double-strand breaks and antiphosphohistone H3, anti-p21, and anti-cyclin E to identify cells in proliferation and cell cycle proteins. Also, to identify neuronal cells, we used anti-NeuN and anti-βIII-tubulin antibodies. After the MeHg treatment, we observed the increase on γ-H2A.X in response to DNA damage. MeHg caused a decrease in the proliferating cells and in the thickness of spinal cord layers. Moreover, we verified that MeHg induced an increase in the number of p21-positive cells but did not change the cyclin E-positive cells. A significantly high number of TUNEL-positive cells indicating DNA fragmentation were observed in MeHg-treated embryos. Regarding the neuronal differentiation, MeHg induced a decrease in NeuN expression and did not change the expression of βIII-tubulin. These results showed that in ovo MeHg exposure alters spinal cord development by disturbing the cell proliferation and death, also interfering in early neuronal differentiation.

  19. Distinct and shared transcriptomes are regulated by microphthalmia-associated transcription factor isoforms in mast cells. (United States)

    Shahlaee, Amir H; Brandal, Stephanie; Lee, Youl-Nam; Jie, Chunfa; Takemoto, Clifford M


    The Microphthalmia-associated transcription factor (Mitf) is an essential basic helix-loop-helix leucine zipper transcription factor for mast cell development. Mice deficient in Mitf harbor a severe mast cell deficiency, and Mitf-mutant mast cells cultured ex vivo display a number of functional defects. Therefore, an understanding of the genetic program regulated by Mitf may provide important insights into mast cell differentiation. Multiple, distinct isoforms of Mitf have been identified in a variety of cell types; we found that Mitf-a, Mitf-e, and Mitf-mc were the major isoforms expressed in mast cells. To determine the physiologic function of Mitf in mast cells, we restored expression of these isoforms in primary mast cells from Mitf(-/-) mice. We found that these isoforms restored granular morphology and integrin-mediated migration. By microarray analysis, proteases, signaling molecules, cell surface receptor, and transporters comprised the largest groups of genes up-regulated by all isoforms. Furthermore, we found that isoforms also regulated distinct genes sets, suggesting separable biological activities. This work defines the transcriptome regulated by Mitf in mast cells and supports its role as master regulator of mast cell differentiation. Expression of multiple isoforms of this transcription factor may provide for redundancy of biological activities while also allowing diversity of function.

  20. Human Lymphoid Tissues Harbor a Distinct CD69+CXCR6+ NK Cell Population. (United States)

    Lugthart, Gertjan; Melsen, Janine E; Vervat, Carly; van Ostaijen-Ten Dam, Monique M; Corver, Willem E; Roelen, Dave L; van Bergen, Jeroen; van Tol, Maarten J D; Lankester, Arjan C; Schilham, Marco W


    Knowledge of human NK cells is based primarily on conventional CD56(bright) and CD56(dim) NK cells from blood. However, most cellular immune interactions occur in lymphoid organs. Based on the coexpression of CD69 and CXCR6, we identified a third major NK cell subset in lymphoid tissues. This population represents 30-60% of NK cells in marrow, spleen, and lymph node but is absent from blood. CD69(+)CXCR6(+) lymphoid tissue NK cells have an intermediate expression of CD56 and high expression of NKp46 and ICAM-1. In contrast to circulating NK cells, they have a bimodal expression of the activating receptor DNAX accessory molecule 1. CD69(+)CXCR6(+) NK cells do not express the early markers c-kit and IL-7Rα, nor killer cell Ig-like receptors or other late-differentiation markers. After cytokine stimulation, CD69(+)CXCR6(+) NK cells produce IFN-γ at levels comparable to CD56(dim) NK cells. They constitutively express perforin but require preactivation to express granzyme B and exert cytotoxicity. After hematopoietic stem cell transplantation, CD69(+)CXCR6(+) lymphoid tissue NK cells do not exhibit the hyperexpansion observed for both conventional NK cell populations. CD69(+)CXCR6(+) NK cells constitute a separate NK cell population with a distinct phenotype and function. The identification of this NK cell population in lymphoid tissues provides tools to further evaluate the cellular interactions and role of NK cells in human immunity.


    Institute of Scientific and Technical Information of China (English)


    Objective To ascertain whether the growth inhibitor in conditioned medium from cultured rabbit arte rial cells is distinct from TGF-β. Methods Rabbit aortic smooth muscle ceils were grown from explained segments of the aorta. Conditioned medium from cultured rabbit aortic smooth muscle ceils and anti-TGF-β were employed in this study. Smooth muscle cell proliferation was measured by XTT detection (Boehringer Mannheim). Results Acidified conditioned medium from smooth muscle ceils had significantly stronger effects of growth inhibition than controls, and anti-TGF-β did not affect the growth inhibitory effect of conditioned medium from cultured rabbit arterial smooth muscle cells. Conclusion The growth inhibiting substance in conditioned medium from cultured rabbit aortic smooth muscle cells is distinct from TGF-β.

  2. Polyfunctional and IFN-γ monofunctional human CD4+ T cell populations are molecularly distinct (United States)

    Burel, Julie G.; Apte, Simon H.; Groves, Penny L.; McCarthy, James S.; Doolan, Denise L.


    Pathogen-specific polyfunctional T cell responses have been associated with favorable clinical outcomes, but it is not known whether molecular differences exist between polyfunctional and monofunctional cytokine-producing T cells. Here, we report that polyfunctional CD4+ T cells induced during Plasmodium falciparum (P. falciparum) blood-stage infection in humans have a unique transcriptomic profile compared with IFN-γ monofunctional CD4+ T cells and, thus, are molecularly distinct. The 14-gene signature revealed in P. falciparum–reactive polyfunctional T cells is associated with cytokine signaling and lymphocyte chemotaxis, and systems biology analysis identified IL-27 as an upstream regulator of the polyfunctional gene signature. Importantly, the polyfunctional gene signature is largely conserved in Influenza-reactive polyfunctional CD4+ T cells, suggesting that polyfunctional T cells have core characteristics independent of pathogen specificity. This study provides the first evidence to our knowledge that consistent molecular differences exist between polyfunctional and monofunctional CD4+ T cells. PMID:28194431

  3. Borrelia burgdorferi elicited-IL-10 suppresses the production of inflammatory mediators, phagocytosis, and expression of co-stimulatory receptors by murine macrophages and/or dendritic cells.

    Directory of Open Access Journals (Sweden)

    Yutein Chung

    Full Text Available Borrelia burgdorferi (Bb is a tick-borne spirochete that is the causative agent for Lyme disease. Our previous studies indicate that virulent Bb can potently enhance IL-10 production by macrophages (MØs and that blocking IL-10 production significantly enhances bacterial clearance. We hypothesize that skin-associated APC types, such as MØs and dendritic cells (DCs are potent producers of IL-10 in response to Bb, which may act in autocrine fashion to suppress APC responses critical for efficient Bb clearance. Our goal is to delineate which APC immune functions are dysregulated by Bb-elicited IL-10 using a murine model of Lyme disease. Our in vitro studies indicated that both APCs rapidly produce IL-10 upon exposure to Bb, that these levels inversely correlate with the production of many Lyme-relevant proinflammatory cytokines and chemokines, and that APCs derived from IL-10(-/- mice produced greater amounts of these proinflammatory mediators than wild-type APCs. Phagocytosis assays determined that Bb-elicited IL-10 levels can diminish Bb uptake and trafficking by MØs, suppresses ROS production, but does not affect NO production; Bb-elicited IL-10 had little effect on phagocytosis, ROS, and NO production by DCs. In general, Bb exposure caused little-to-no upregulation of several critical surface co-stimulatory markers by MØs and DCs, however eliminating Bb-elicited IL-10 allowed a significant upregulation in many of these co-stimulatory receptors. These data indicate that IL-10 elicited from Bb-stimulated MØs and DCs results in decreased production of proinflammatory mediators and co-stimulatory molecules, and suppress phagocytosis-associated events that are important for mediating both innate and adaptive immune responses by APCs.

  4. Phenotypic and functional distinctions between the TH2+ and JRA+ T cell subsets in man. (United States)

    Reinherz, E L; Strelkauskas, A J; O'Brien, C; Schlossman, S F


    Prior work has demonstrated the existence of distinct human peripheral blood T cell subsets by utilizing heterologous as well as autoimmune antisera. In the present study, the relationship between the TH2+ and JRA+ T cell subsets was examined. T cells were purified with Sephadex G-200 anti-F(ab)2' affinity chromatography and E-rosetting technique, and subsequently fractionated into TH2+ and TH2- subsets by utilizing indirect immunofluorescence on FACS. Approximately 40 to 45% of the TH2- subset was shown to be JRA+, whereas less than 5% of the TH2+ subset was JRA+. In reciprocal studies, T cells were fractionated into JRA+ and JRA- subsets and reacted with heterologous antisera with anti-TH2+ specificity and indirect immunofluorescence. FACS analysis demonstrated that the JRA+ population contained no TH2+ T cells. In contrast, the JRA- population contained TH2+ T cells and accounted for the entire TH2+ subset found in the unfractionated T cell population. Functional studies showed that the TH2+ subset, and not the JRA+ subset, contain the effector population for cell-mediated lympholysis. It is concluded that the TH2+ and JRA+ T cell subsets define distinct and different T cell populations in man.

  5. Naive and primed murine pluripotent stem cells have distinct miRNA expression profiles


    Jouneau, A.; Ciaudo, C.; Sismeiro, O.; Brochard, V.; Jouneau, L.; Vandormael-Pournin, S; Coppee, J.-Y.; Zhou, Q.; Heard, E.; Antoniewski, C.; Cohen-Tannoudji, M.


    Over the last years, the microRNA (miRNA) pathway has emerged as a key component of the regulatory network of pluripotency. Although clearly distinct states of pluripotency have been described in vivo and ex vivo, differences in miRNA expression profiles associated with the developmental modulation of pluripotency have not been extensively studied. Here, the authors performed deep sequencing to profile miRNA expression in naive (embryonic stem cell [ESC]) and primed (epiblast stem cell [EpiSC...

  6. A DNA vaccine encoding multiple HIV CD4 epitopes elicits vigorous polyfunctional, long-lived CD4+ and CD8+ T cell responses.

    Directory of Open Access Journals (Sweden)

    Daniela Santoro Rosa

    Full Text Available T-cell based vaccines against HIV have the goal of limiting both transmission and disease progression by inducing broad and functionally relevant T cell responses. Moreover, polyfunctional and long-lived specific memory T cells have been associated to vaccine-induced protection. CD4(+ T cells are important for the generation and maintenance of functional CD8(+ cytotoxic T cells. We have recently developed a DNA vaccine encoding 18 conserved multiple HLA-DR-binding HIV-1 CD4 epitopes (HIVBr18, capable of eliciting broad CD4(+ T cell responses in multiple HLA class II transgenic mice. Here, we evaluated the breadth and functional profile of HIVBr18-induced immune responses in BALB/c mice. Immunized mice displayed high-magnitude, broad CD4(+/CD8(+ T cell responses, and 8/18 vaccine-encoded peptides were recognized. In addition, HIVBr18 immunization was able to induce polyfunctional CD4(+ and CD8(+ T cells that proliferate and produce any two cytokines (IFNγ/TNFα, IFNγ/IL-2 or TNFα/IL-2 simultaneously in response to HIV-1 peptides. For CD4(+ T cells exclusively, we also detected cells that proliferate and produce all three tested cytokines simultaneously (IFNγ/TNFα/IL-2. The vaccine also generated long-lived central and effector memory CD4(+ T cells, a desirable feature for T-cell based vaccines. By virtue of inducing broad, polyfunctional and long-lived T cell responses against conserved CD4(+ T cell epitopes, combined administration of this vaccine concept may provide sustained help for CD8(+ T cells and antibody responses- elicited by other HIV immunogens.

  7. CD8+ T-cells expressing interferon gamma or perforin play antagonistic roles in heart injury in experimental Trypanosoma cruzi-elicited cardiomyopathy. (United States)

    Silverio, Jaline Coutinho; Pereira, Isabela Resende; Cipitelli, Márcio da Costa; Vinagre, Nathália Ferreira; Rodrigues, Maurício Martins; Gazzinelli, Ricardo Tostes; Lannes-Vieira, Joseli


    In Chagas disease, CD8(+) T-cells are critical for the control of Trypanosoma cruzi during acute infection. Conversely, CD8(+) T-cell accumulation in the myocardium during chronic infection may cause tissue injury leading to chronic chagasic cardiomyopathy (CCC). Here we explored the role of CD8(+) T-cells in T. cruzi-elicited heart injury in C57BL/6 mice infected with the Colombian strain. Cardiomyocyte lesion evaluated by creatine kinase-MB isoenzyme activity levels in the serum and electrical abnormalities revealed by electrocardiogram were not associated with the intensity of heart parasitism and myocarditis in the chronic infection. Further, there was no association between heart injury and systemic anti-T. cruzi CD8(+) T-cell capacity to produce interferon-gamma (IFNγ) and to perform specific cytotoxicity. Heart injury, however, paralleled accumulation of anti-T. cruzi cells in the cardiac tissue. In T. cruzi infection, most of the CD8(+) T-cells segregated into IFNγ(+) perforin (Pfn)(neg) or IFNγ(neg)Pfn(+) cell populations. Colonization of the cardiac tissue by anti-T. cruzi CD8(+)Pfn(+) cells paralleled the worsening of CCC. The adoptive cell transfer to T. cruzi-infected cd8(-/-) recipients showed that the CD8(+) cells from infected ifnγ(-/-)pfn(+/+) donors migrate towards the cardiac tissue to a greater extent and caused a more severe cardiomyocyte lesion than CD8(+) cells from ifnγ(+/+)pfn(-/-) donors. Moreover, the reconstitution of naïve cd8(-/-) mice with CD8(+) cells from naïve ifnγ(+/+)pfn(-/-) donors ameliorated T. cruzi-elicited heart injury paralleled IFNγ(+) cells accumulation, whereas reconstitution with CD8(+) cells from naïve ifnγ(-/-)pfn(+/+) donors led to an aggravation of the cardiomyocyte lesion, which was associated with the accumulation of Pfn(+) cells in the cardiac tissue. Our data support a possible antagonist effect of CD8(+)Pfn(+) and CD8(+)IFNγ(+) cells during CCC. CD8(+)IFNγ(+) cells may exert a beneficial role

  8. CD8+ T-Cells Expressing Interferon Gamma or Perforin Play Antagonistic Roles in Heart Injury in Experimental Trypanosoma Cruzi-Elicited Cardiomyopathy (United States)

    Cipitelli, Márcio da Costa; Vinagre, Nathália Ferreira; Rodrigues, Maurício Martins; Gazzinelli, Ricardo Tostes; Lannes-Vieira, Joseli


    In Chagas disease, CD8+ T-cells are critical for the control of Trypanosoma cruzi during acute infection. Conversely, CD8+ T-cell accumulation in the myocardium during chronic infection may cause tissue injury leading to chronic chagasic cardiomyopathy (CCC). Here we explored the role of CD8+ T-cells in T. cruzi-elicited heart injury in C57BL/6 mice infected with the Colombian strain. Cardiomyocyte lesion evaluated by creatine kinase-MB isoenzyme activity levels in the serum and electrical abnormalities revealed by electrocardiogram were not associated with the intensity of heart parasitism and myocarditis in the chronic infection. Further, there was no association between heart injury and systemic anti-T. cruzi CD8+ T-cell capacity to produce interferon-gamma (IFNγ) and to perform specific cytotoxicity. Heart injury, however, paralleled accumulation of anti-T. cruzi cells in the cardiac tissue. In T. cruzi infection, most of the CD8+ T-cells segregated into IFNγ+ perforin (Pfn)neg or IFNγnegPfn+ cell populations. Colonization of the cardiac tissue by anti-T. cruzi CD8+Pfn+ cells paralleled the worsening of CCC. The adoptive cell transfer to T. cruzi-infected cd8−/− recipients showed that the CD8+ cells from infected ifnγ−/−pfn+/+ donors migrate towards the cardiac tissue to a greater extent and caused a more severe cardiomyocyte lesion than CD8+ cells from ifnγ+/+pfn−/− donors. Moreover, the reconstitution of naïve cd8−/− mice with CD8+ cells from naïve ifnγ+/+pfn−/− donors ameliorated T. cruzi-elicited heart injury paralleled IFNγ+ cells accumulation, whereas reconstitution with CD8+ cells from naïve ifnγ−/−pfn+/+ donors led to an aggravation of the cardiomyocyte lesion, which was associated with the accumulation of Pfn+ cells in the cardiac tissue. Our data support a possible antagonist effect of CD8+Pfn+ and CD8+IFNγ+ cells during CCC. CD8+IFNγ+ cells may exert a beneficial role, whereas CD8+Pfn+ may play a detrimental role

  9. CD8+ T-cells expressing interferon gamma or perforin play antagonistic roles in heart injury in experimental Trypanosoma cruzi-elicited cardiomyopathy.

    Directory of Open Access Journals (Sweden)

    Jaline Coutinho Silverio

    Full Text Available In Chagas disease, CD8(+ T-cells are critical for the control of Trypanosoma cruzi during acute infection. Conversely, CD8(+ T-cell accumulation in the myocardium during chronic infection may cause tissue injury leading to chronic chagasic cardiomyopathy (CCC. Here we explored the role of CD8(+ T-cells in T. cruzi-elicited heart injury in C57BL/6 mice infected with the Colombian strain. Cardiomyocyte lesion evaluated by creatine kinase-MB isoenzyme activity levels in the serum and electrical abnormalities revealed by electrocardiogram were not associated with the intensity of heart parasitism and myocarditis in the chronic infection. Further, there was no association between heart injury and systemic anti-T. cruzi CD8(+ T-cell capacity to produce interferon-gamma (IFNγ and to perform specific cytotoxicity. Heart injury, however, paralleled accumulation of anti-T. cruzi cells in the cardiac tissue. In T. cruzi infection, most of the CD8(+ T-cells segregated into IFNγ(+ perforin (Pfn(neg or IFNγ(negPfn(+ cell populations. Colonization of the cardiac tissue by anti-T. cruzi CD8(+Pfn(+ cells paralleled the worsening of CCC. The adoptive cell transfer to T. cruzi-infected cd8(-/- recipients showed that the CD8(+ cells from infected ifnγ(-/-pfn(+/+ donors migrate towards the cardiac tissue to a greater extent and caused a more severe cardiomyocyte lesion than CD8(+ cells from ifnγ(+/+pfn(-/- donors. Moreover, the reconstitution of naïve cd8(-/- mice with CD8(+ cells from naïve ifnγ(+/+pfn(-/- donors ameliorated T. cruzi-elicited heart injury paralleled IFNγ(+ cells accumulation, whereas reconstitution with CD8(+ cells from naïve ifnγ(-/-pfn(+/+ donors led to an aggravation of the cardiomyocyte lesion, which was associated with the accumulation of Pfn(+ cells in the cardiac tissue. Our data support a possible antagonist effect of CD8(+Pfn(+ and CD8(+IFNγ(+ cells during CCC. CD8(+IFNγ(+ cells may exert a beneficial role, whereas CD8(+Pfn

  10. Gastrin: a distinct fate of neurogenin3 positive progenitor cells in the embryonic pancreas.

    Directory of Open Access Journals (Sweden)

    Yaron Suissa

    Full Text Available Neurogenin3(+ (Ngn3(+ progenitor cells in the developing pancreas give rise to five endocrine cell types secreting insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. Gastrin is a hormone produced primarily by G-cells in the stomach, where it functions to stimulate acid secretion by gastric parietal cells. Gastrin is expressed in the embryonic pancreas and is common in islet cell tumors, but the lineage and regulators of pancreatic gastrin(+ cells are not known. We report that gastrin is abundantly expressed in the embryonic pancreas and disappears soon after birth. Some gastrin(+ cells in the developing pancreas co-express glucagon, ghrelin or pancreatic polypeptide, but many gastrin(+ cells do not express any other islet hormone. Pancreatic gastrin(+ cells express the transcription factors Nkx6.1, Nkx2.2 and low levels of Pdx1, and derive from Ngn3(+ endocrine progenitor cells as shown by genetic lineage tracing. Using mice deficient for key transcription factors we show that gastrin expression depends on Ngn3, Nkx2.2, NeuroD1 and Arx, but not Pax4 or Pax6. Finally, gastrin expression is induced upon differentiation of human embryonic stem cells to pancreatic endocrine cells expressing insulin. Thus, gastrin(+ cells are a distinct endocrine cell type in the pancreas and an alternative fate of Ngn3+ cells.

  11. Embryonic Stem Cell Culture Conditions Support Distinct States Associated with Different Developmental Stages and Potency

    DEFF Research Database (Denmark)

    Martin Gonzalez, Javier; Morgani, Sophie M; Bone, Robert A;


    Embryonic stem cells (ESCs) are cell lines derived from the mammalian pre-implantation embryo. Here we assess the impact of derivation and culture conditions on both functional potency and ESC transcriptional identity. Individual ESCs cultured in either two small-molecule inhibitors (2i....... Conversely, the transcriptome of serum-cultured ESCs correlated with later stages of development (E4.5), at which point embryonic cells are more restricted in their developmental potential. Thus, ESC culture systems are not equivalent, but support cell types that resemble distinct developmental stages. Cells......) or with knockout serum replacement (KOSR), but not serum, can generate high-level chimeras regardless of how these cells were derived. ESCs cultured in these conditions showed a transcriptional correlation with early pre-implantation embryos (E1.5-E3.5) and contributed to development from the 2-cell stage...

  12. The satellite cell in male and female, developing and adult mouse muscle: distinct stem cells for growth and regeneration.

    Directory of Open Access Journals (Sweden)

    Alice Neal

    Full Text Available Satellite cells are myogenic cells found between the basal lamina and the sarcolemma of the muscle fibre. Satellite cells are the source of new myofibres; as such, satellite cell transplantation holds promise as a treatment for muscular dystrophies. We have investigated age and sex differences between mouse satellite cells in vitro and assessed the importance of these factors as mediators of donor cell engraftment in an in vivo model of satellite cell transplantation. We found that satellite cell numbers are increased in growing compared to adult and in male compared to female adult mice. We saw no difference in the expression of the myogenic regulatory factors between male and female mice, but distinct profiles were observed according to developmental stage. We show that, in contrast to adult mice, the majority of satellite cells from two week old mice are proliferating to facilitate myofibre growth; however a small proportion of these cells are quiescent and not contributing to this growth programme. Despite observed changes in satellite cell populations, there is no difference in engraftment efficiency either between satellite cells derived from adult or pre-weaned donor mice, male or female donor cells, or between male and female host muscle environments. We suggest there exist two distinct satellite cell populations: one for muscle growth and maintenance and one for muscle regeneration.

  13. Systematic analysis of reportedly distinct populations of multipotent bone marrow-derived stem cells reveals a lack of distinction. (United States)

    Lodie, Tracey A; Blickarz, Courtney E; Devarakonda, Tara J; He, Chufa; Dash, Ajeeta B; Clarke, Jennifer; Gleneck, Kristen; Shihabuddin, Lamya; Tubo, Ross


    Adult human bone marrow-derived stem cells, having the ability to differentiate into cells of multiple lineages, have been isolated and propagated by varied protocols, including positive (CD105(+))/negative (CD45(-)GlyA(-)) selection with immunomagnetic beads, or direct plating into selective culture media. Each substratum-adherent cell population was subjected to a systematic analysis of their cell surface markers and differentiation potential. In the initial stages of culture, each cell population proliferated slowly, reaching confluence in 10-14 days. Adherent cells proliferated at similar rates whether cultured in serum-free medium supplemented with basic fibroblast growth factor, medium containing 2% fetal bovine serum (FBS) supplemented with epidermal growth factor and platelet-derived growth factor, or medium containing 10% FBS alone. Cell surface marker analysis revealed that more than 95% of the cells were positive for CD105/endoglin, a putative mesenchymal stem cell marker, and negative for CD34, CD31, and CD133, markers of hematopoietic, endothelial, and neural stem cells, respectively, regardless of cell isolation and propagation method. CD44 expression was variable, apparently dependent on serum concentration. Functional similarity of the stem cell populations was also observed, with each different cell population expressing the cell type-specific markers beta-tubulin, type II collagen, and desmin, and demonstrating endothelial tube formation when cultured under conditions favoring neural, cartilage, muscle, and endothelial cell differentiation, respectively. On the basis of these data, adult human bone marrow-derived stem cells cultured in adherent monolayer are virtually indistinguishable, both physically and functionally, regardless of the method of isolation or proliferative expansion.

  14. Cell culture and animal infection with distinct Trypanosoma cruzi strains expressing red and green fluorescent proteins. (United States)

    Pires, S F; DaRocha, W D; Freitas, J M; Oliveira, L A; Kitten, G T; Machado, C R; Pena, S D J; Chiari, E; Macedo, A M; Teixeira, S M R


    Different strains of Trypanosoma cruzi were transfected with an expression vector that allows the integration of green fluorescent protein (GFP) and red fluorescent protein (RFP) genes into the beta-tubulin locus by homologous recombination. The sites of integration of the GFP and RFP markers were determined by pulse-field gel electrophoresis and Southern blot analyses. Cloned cell lines selected from transfected epimastigote populations maintained high levels of fluorescent protein expression even after 6 months of in vitro culture of epimastigotes in the absence of drug selection. Fluorescent trypomastigotes and amastigotes were observed within Vero cells in culture as well as in hearts and diaphragms of infected mice. The infectivity of the GFP- and RFP-expressing parasites in tissue culture cells was comparable to wild type populations. Furthermore, GFP- and RFP-expressing parasites were able to produce similar levels of parasitemia in mice compared with wild type parasites. Cell cultures infected simultaneously with two cloned cell lines from the same parasite strain, each one expressing a distinct fluorescent marker, showed that at least two different parasites are able to infect the same cell. Double-infected cells were also detected when GFP- and RFP-expressing parasites were derived from strains belonging to two distinct T. cruzi lineages. These results show the usefulness of parasites expressing GFP and RFP for the study of various aspects of T. cruzi infection including the mechanisms of cell invasion, genetic exchange among parasites and the differential tissue distribution in animal models of Chagas disease.

  15. Telomere dysfunction and cell survival: roles for distinctTIN2-containing complexes

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sahn-Ho; Davalos, Albert R.; Heo, Seok-Jin; Rodier, Francis; Beausejour, Christian; Kaminker, Patrick; Campisi, Judith


    Telomeres are maintained by three DNA binding proteins, TRF1, TRF2 and POT1, and several associated factors. One factor, TIN2, binds TRF1 and TRF2 directly and POT1 indirectly. These and two other proteins form a soluble complex that may be the core telomere-maintenance complex. It is not clear whether subcomplexes exist or function in vivo. Here, we provide evidence for two TIN2 subcomplexes with distinct functions in human cells. TIN2 ablation by RNA interference caused telomere uncapping and p53-independent cell death in all cells tested. However, we isolated two TIN2 complexes from cell lysates, each selectively sensitive to a TIN2 mutant (TIN2-13, TIN2-15C). In cells with wild-type p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere uncapping and eventual growth arrest. In cells lacking p53 function, TIN215C more than TIN2-13 caused genomic instability and cell death. Thus, TIN2 subcomplexes likely have distinct functions in telomere maintenance, and may provide selective targets for eliminating cells with mutant p53.

  16. Distinct populations of innate CD8+ T cells revealed in a CXCR3 reporter mouse. (United States)

    Oghumu, Steve; Dong, Ran; Varikuti, Sanjay; Shawler, Todd; Kampfrath, Thomas; Terrazas, Cesar A; Lezama-Davila, Claudio; Ahmer, Brian M M; Whitacre, Caroline C; Rajagopalan, Sanjay; Locksley, Richard; Sharpe, Arlene H; Satoskar, Abhay R


    CXCR3, expressed mainly on activated T and NK cells, is implicated in a host of immunological conditions and can contribute either to disease resolution or pathology. We report the generation and characterization of a novel CXCR3 internal ribosome entry site bicistronic enhanced GFP reporter (CIBER) mouse in which enhanced GFP expression correlates with surface levels of CXCR3. Using CIBER mice, we identified two distinct populations of innate CD8(+) T cells based on constitutive expression of CXCR3. We demonstrate that CXCR3(+) innate CD8(+) T cells preferentially express higher levels of Ly6C and CD122, but lower levels of CCR9 compared with CXCR3(-) innate CD8(+) T cells. Furthermore, we show that CXCR3(+) innate CD8(+) T cells express higher transcript levels of antiapoptotic but lower levels of proapoptotic factors, respond more robustly to IL-2 and IL-15, and produce significantly more IFN-γ and granzyme B. Interestingly, CXCR3(+) innate CD8(+) T cells do not respond to IL-12 or IL-18 alone, but produce significant amounts of IFN-γ on stimulation with a combination of these cytokines. Taken together, these findings demonstrate that CXCR3(+) and CXCR3(-) innate CD8(+) T cells are phenotypically and functionally distinct. These newly generated CIBER mice provide a novel tool for studying the role of CXCR3 and CXCR3-expressing cells in vivo.

  17. Isolation (from a basal cell carcinoma) of a functionally distinct fibroblast-like cell type that overexpresses Ptch. (United States)

    Dicker, Anthony J; Serewko, Magdalena M; Russell, Terry; Rothnagel, Joseph A; Strutton, Geoff M; Dahler, Alison L; Saunders, Nicholas A


    In this study we report on the isolation and characterization of a nonepithelial, nontumorigenic cell type (BCC1) derived from a basal cell carcinoma from a patient. The BCC1 cells share many characteristics with dermal fibroblasts, such as the expression of vimentin, lack of expression of cytokeratins, and insensitivity to agents that cause growth inhibition and differentiation of epithelial cells; however, significant differences between BCC1 cells and fibroblasts also exist. For example, BCC1 cells are stimulated to undergo DNA synthesis in response to interferon-gamma, whereas dermal fibroblasts are not. More over, BCC1 cells overexpress the basal cell carcinoma-specific genes ptch and ptch2. These data indicate that basal cell carcinomas are associated with a functionally distinct population of fibroblast-like cells that overexpress known tumor-specific markers (ptch and ptch2).

  18. Dispersed cells represent a distinct stage in the transition from bacterial biofilm to planktonic lifestyles

    DEFF Research Database (Denmark)

    Chua, Song Lin; Liu, Yang; Yam, Joey Kuok Hoong


    Bacteria assume distinct lifestyles during the planktonic and biofilm modes of growth. Increased levels of the intracellular messenger c-di-GMP determine the transition from planktonic to biofilm growth, while a reduction causes biofilm dispersal. It is generally assumed that cells dispersed from...... biofilms immediately go into the planktonic growth phase. Here we use single-nucleotide resolution transcriptomic analysis to show that the physiology of dispersed cells from Pseudomonas aeruginosa biofilms is highly different from those of planktonic and biofilm cells. In dispersed cells, the expression...... of the small regulatory RNAs RsmY and RsmZ is downregulated, whereas secretion genes are induced. Dispersed cells are highly virulent against macrophages and Caenorhabditis elegans compared with planktonic cells. In addition, they are highly sensitive towards iron stress, and the combination of a biofilm...

  19. Strategy for eliciting antigen-specific CD8+ T cell-mediated immune response against a cryptic CTL epitope of merkel cell polyomavirus large T antigen

    Directory of Open Access Journals (Sweden)

    Gomez Bianca P


    Full Text Available Abstract Background Merkel cell carcinoma (MCC is a relatively new addition to the expanding category of oncovirus-induced cancers. Although still comparably rare, the number of cases has risen dramatically in recent years. Further complicating this trend is that MCC is an extremely aggressive neoplasm with poor patient prognosis and limited treatment options for advanced disease. The causative agent of MCC has been identified as the merkel cell polyomavirus (MCPyV. The MCPyV-encoded large T (LT antigen is an oncoprotein that is theorized to be essential for virus-mediated tumorigenesis and is therefore, an excellent MCC antigen for the generation of antitumor immune responses. As a foreign antigen, the LT oncoprotein avoids the obstacle of immune tolerance, which normally impedes the development of antitumor immunity. Ergo, it is an excellent target for anti-MCC immunotherapy. Since tumor-specific CD8+ T cells lead to better prognosis for MCC and numerous other cancers, we have generated a DNA vaccine that is capable of eliciting LT-specific CD8+ T cells. The DNA vaccine (pcDNA3-CRT/LT encodes the LT antigen linked to a damage-associated molecular pattern, calreticulin (CRT, as it has been demonstrated that the linkage of CRT to antigens promotes the induction of antigen-specific CD8+ T cells. Results The present study shows that DNA vaccine-induced generation of LT-specific CD8+ T cells is augmented by linking CRT to the LT antigen. This is relevant since the therapeutic effects of the pcDNA3-CRT/LT DNA vaccine is mediated by LT-specific CD8+ T cells. Mice vaccinated with the DNA vaccine produced demonstrably more LT-specific CD8+ T cells. The DNA vaccine was also able to confer LT-specific CD8+ T cell-mediated protective and therapeutic effects to prolong the survival of mice with LT-expressing tumors. In the interest of determining the LT epitope which most MCC-specific CD8+ T cells recognize, we identified the amino acid sequence of the

  20. Non-replicating Mycobacterium tuberculosis elicits a reduced infectivity profile with corresponding modifications to the cell wall and extracellular matrix.

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    Joanna Bacon

    Full Text Available A key feature of Mycobacterium tuberculosis is its ability to become dormant in the host. Little is known of the mechanisms by which these bacilli are able to persist in this state. Therefore, the focus of this study was to emulate environmental conditions encountered by M. tuberculosis in the granuloma, and determine the effect of such conditions on the physiology and infectivity of the organism. Non-replicating persistent (NRP M. tuberculosis was established by the gradual depletion of nutrients in an oxygen-replete and controlled environment. In contrast to rapidly dividing bacilli, NRP bacteria exhibited a distinct phenotype by accumulating an extracellular matrix rich in free mycolate and lipoglycans, with increased arabinosylation. Microarray studies demonstrated a substantial down-regulation of genes involved in energy metabolism in NRP bacteria. Despite this reduction in metabolic activity, cells were still able to infect guinea pigs, but with a delay in the development of disease when compared to exponential phase bacilli. Using these approaches to investigate the interplay between the changing environment of the host and altered physiology of NRP bacteria, this study sheds new light on the conditions that are pertinent to M. tuberculosis dormancy and how this organism could be establishing latent disease.

  1. Choriodecidual Cells from Term Human Pregnancies Show Distinctive Functional Properties Related to the Induction of Labor (United States)

    Castillo-Castrejon, Marisol; Meraz-Cruz, Noemí; Gomez-Lopez, Nardhy; Flores-Pliego, Arturo; Beltrán-Montoya, Jorge; Viveros-Alcaráz, Martín; Vadillo-Ortega, Felipe


    Problem Human parturition is associated with an intrauterine pro-inflammatory environment in the choriodecidua. Evidence that some mediators of this signaling cascade also elicit responses leading to labor prompted us to characterize the cellular sources of these mediators in the human choriodecidua. Method of study Leukocyte-enriched preparations from human choriodecidua (ChL) and intervillous placental blood leukocytes (PL) were maintained in culture. Secretions of inflammatory cytokines, chemokines and MMP-9 were documented. Leukocyte phenotype of ChL and PL was determined by flow cytometry using specific fluorochrome-conjugated antibodies. Results and Conclusions ChL showed a distinct pro-inflammatory secretion pattern of cytokines and chemokines when compared with PL, including higher amounts of TNF-α and IL-6, and decreased secretions of IL-4 and IL-1ra. ChL also secreted more MIP-1α and MCP-1 and MMP-9 than PL. No significant differences were found in leukocytes subsets between compartments. Based on our findings, we propose that ChL isolated from fetal membranes at term are functionally different from PL and may collaborate to modulate the microenvironment linked to induction and progression of human labor. PMID:24286217

  2. Telomere dysfunction and cell survival: Roles for distinct TIN2-containing complexes

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sahn-ho; Davalos, Albert R.; Heo, Seok-Jin; Rodier, Francis; Zou, Ying; Beausejour, Christian; Kaminker, Patrick; Yannone, Steven M.; Campisi, Judith


    Telomeres are maintained by three DNA binding proteins (TRF1, TRF2 and POT1), and several associated factors. One factor, TIN2, binds TRF1 and TRF2 directly and POT1 indirectly. Along with two other proteins, TPP1 and hRap1, these form a soluble complex that may be the core telomere maintenance complex. It is not clear whether sub-complexes also exist in vivo. We provide evidence for two TIN2 sub-complexes with distinct functions in human cells. We isolated these two TIN2 sub-complexes from nuclear lysates of unperturbed cells and cells expressing TIN2 mutants TIN2-13, TIN2-15C, which cannot bind TRF2 or TRF1, respectively. In cells with wild-type p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere uncapping and eventual growth arrest. In cells lacking p53 function, TIN2-15C was more potent than TIN2-13 in causing telomere dysfunction and cell death. Our findings suggest that distinct TIN2 complexes exist, and that TIN2-15C-sensitive subcomplexes are particularly important for cell survival in the absence of functional p53.

  3. Distinct mesenchymal alterations in N-cadherin and E-cadherin positive primary renal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Christof Keller

    Full Text Available BACKGROUND: Renal tubular epithelial cells of proximal and distal origin differ markedly in their physiological functions. Therefore, we hypothesized that they also differ in their capacity to undergo epithelial to mesenchymal alterations. RESULTS: We used cultures of freshly isolated primary human tubular cells. To distinguish cells of different tubular origin we took advantage of the fact that human proximal epithelial cells uniquely express N-cadherin instead of E-cadherin as major cell-cell adhesion molecule. To provoke mesenchymal alteration we treated these cocultures with TGF-β for up to 6 days. Within this time period, the morphology of distal tubular cells was barely altered. In contrast to tubular cell lines, E-cadherin was not down-regulated by TGF-β, even though TGF-β signal transduction was initiated as demonstrated by nuclear localization of Smad2/3. Analysis of transcription factors and miRNAs possibly involved in E-cadherin regulation revealed high levels of miRNAs of the miR200-family, which may contribute to the stability of E-cadherin expression in human distal tubular epithelial cells. By contrast, proximal tubular epithelial cells altered their phenotype when treated with TGF-β. They became elongated and formed three-dimensional structures. Rho-kinases were identified as modulators of TGF-β-induced morphological alterations. Non-specific inhibition of Rho-kinases resulted in stabilization of the epithelial phenotype, while partial effects were observed upon downregulation of Rho-kinase isoforms ROCK1 and ROCK2. The distinct reactivity of proximal and distal cells was retained when the cells were cultured as polarized cells. CONCLUSIONS: Interference with Rho-kinase signaling provides a target to counteract TGF-β-mediated mesenchymal alterations of epithelial cells, particularly in proximal tubular epithelial cells. Furthermore, primary distal tubular cells differed from cell lines by their high phenotypic stability

  4. Further evidence for a human B cell activating factor distinct from IL-4. (United States)

    Diu, A; Février, M; Mollier, P; Charron, D; Banchereau, J; Reinherz, E L; Thèze, J


    Supernatants from activated human T cell clones were previously shown to contain B cell-activating factor (BCAF), an activity which results in polyclonal resting B cell stimulation. In the present study, we investigate the relationship between this activity and human interleukin-4 which was also shown to act on resting B cells. The supernatant of the T cell clone TT9 contains IL-4 but anti-IL-4 antiserum does not affect the response of B cells as measured by thymidine uptake or cell volume increase. Furthermore, IL-4 induces Fc epsilon-receptor (CD23) expression on 30% of unstimulated human B cells, whereas BCAF-containing supernatants from clone P2, that do not contain detectable amounts of IL-4, promote B cell proliferation without inducing CD23 expression. Our results therefore establish that IL-4 and BCAF are distinct activities and suggest that they trigger different activation pathways in human B cells. In addition, culture of B cells with T cell supernatants for 72 hr induces a three- to fourfold increase in the expression of HLA-DR, -DP, and -DQ antigens in 50% of B cells. The addition of inhibiting concentrations of anti-IFN-gamma, LT, or IL-4 antisera to the cultures does not change these results. Finally, 30% of B cells cultured with T cell supernatants leave the G1 phase of the cell cycle and 20% reach mitosis. Taken together, our findings further support the existence of a B cell-activating factor responsible for the activation of resting human B cells.

  5. Differences in TCDD-elicited gene expression profiles in human HepG2, mouse Hepa1c1c7 and rat H4IIE hepatoma cells

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    Burgoon Lyle D


    Full Text Available Abstract Background 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD is an environmental contaminant that elicits a broad spectrum of toxic effects in a species-specific manner. Current risk assessment practices routinely extrapolate results from in vivo and in vitro rodent models to assess human risk. In order to further investigate the species-specific responses elicited by TCDD, temporal gene expression responses in human HepG2, mouse Hepa1c1c7 and rat H4IIE cells were compared. Results Microarray analysis identified a core set of conserved gene expression responses across species consistent with the role of AhR in mediating adaptive metabolic responses. However, significant species-specific as well as species-divergent responses were identified. Computational analysis of the regulatory regions of species-specific and -divergent responses suggests that dioxin response elements (DREs are involved. These results are consistent with in vivo rat vs. mouse species-specific differential gene expression, and more comprehensive comparative DRE searches. Conclusions Comparative analysis of human HepG2, mouse Hepa1c1c7 and rat H4IIE TCDD-elicited gene expression responses is consistent with in vivo rat-mouse comparative gene expression studies, and more comprehensive comparative DRE searches, suggesting that AhR-mediated gene expression is species-specific.

  6. Hepatocyte Growth Factor-mediated satellite cells niche perturbation promotes development of distinct sarcoma subtypes. (United States)

    Morena, Deborah; Maestro, Nicola; Bersani, Francesca; Forni, Paolo Emanuele; Lingua, Marcello Francesco; Foglizzo, Valentina; Šćepanović, Petar; Miretti, Silvia; Morotti, Alessandro; Shern, Jack F; Khan, Javed; Ala, Ugo; Provero, Paolo; Sala, Valentina; Crepaldi, Tiziana; Gasparini, Patrizia; Casanova, Michela; Ferrari, Andrea; Sozzi, Gabriella; Chiarle, Roberto; Ponzetto, Carola; Taulli, Riccardo


    Embryonal Rhabdomyosarcoma (ERMS) and Undifferentiated Pleomorphic Sarcoma (UPS) are distinct sarcoma subtypes. Here we investigate the relevance of the satellite cell (SC) niche in sarcoma development by using Hepatocyte Growth Factor (HGF) to perturb the niche microenvironment. In a Pax7 wild type background, HGF stimulation mainly causes ERMS that originate from satellite cells following a process of multistep progression. Conversely, in a Pax7 null genotype ERMS incidence drops, while UPS becomes the most frequent subtype. Murine EfRMS display genetic heterogeneity similar to their human counterpart. Altogether, our data demonstrate that selective perturbation of the SC niche results in distinct sarcoma subtypes in a Pax7 lineage-dependent manner, and define a critical role for the Met axis in sarcoma initiation. Finally, our results provide a rationale for the use of combination therapy, tailored on specific amplifications and activated signaling pathways, to minimize resistance emerging from sarcomas heterogeneity.

  7. Two developmentally distinct populations of neural crest cells contribute to the zebrafish heart. (United States)

    Cavanaugh, Ann M; Huang, Jie; Chen, Jau-Nian


    Cardiac neural crest cells are essential for outflow tract remodeling in animals with divided systemic and pulmonary circulatory systems, but their contributions to cardiac development in animals with a single-loop circulatory system are less clear. Here we genetically labeled neural crest cells and examined their contribution to the developing zebrafish heart. We identified two populations of neural crest cells that contribute to distinct compartments of zebrafish cardiovascular system at different developmental stages. A stream of neural crest cells migrating through pharyngeal arches 1 and 2 integrates into the myocardium of the primitive heart tube between 24 and 30 h post fertilization and gives rise to cardiomyocytes. A second wave of neural crest cells migrating along aortic arch 6 envelops the endothelium of the ventral aorta and invades the bulbus arteriosus after three days of development. Interestingly, while inhibition of FGF signaling has no effect on the integration of neural crest cells to the primitive heart tube, it prevents these cells from contributing to the outflow tract, demonstrating disparate responses of neural crest cells to FGF signaling. Furthermore, neural crest ablation in zebrafish leads to multiple cardiac defects, including reduced heart rate, defective myocardial maturation and a failure to recruit progenitor cells from the second heart field. These findings add to our understanding of the contribution of neural crest cells to the developing heart and provide insights into the requirement for these cells in cardiac maturation.

  8. Two distinct secretory vesicle–priming steps in adrenal chromaffin cells


    Liu, Yuanyuan; Schirra, Claudia; Edelmann, Ludwig; Matti, Ulf; Rhee, JeongSeop; Hof, Detlef; Bruns, Dieter; Brose, Nils; Rieger, Heiko; Stevens, David R.; Rettig, Jens


    Priming of large dense-core vesicles (LDCVs) is a Ca2+-dependent step by which LDCVs enter a release-ready pool, involving the formation of the soluble N-ethyl-maleimide sensitive fusion protein attachment protein (SNAP) receptor complex consisting of syntaxin, SNAP-25, and synaptobrevin. Using mice lacking both isoforms of the calcium-dependent activator protein for secretion (CAPS), we show that LDCV priming in adrenal chromaffin cells entails two distinct steps. CAPS is required for primin...

  9. Long-term label retaining cells localize to distinct regions within the female reproductive epithelium. (United States)

    Patterson, Amanda L; Pru, James K


    distinct populations of epithelial cells that exhibit stem/progenitor cell qualities. Distinct stem/progenitor-like cells localize to the oviduct, endometrium, and cervix.

  10. Motor neurons and oligodendrocytes arise from distinct cell lineages by progenitor recruitment. (United States)

    Ravanelli, Andrew M; Appel, Bruce


    During spinal cord development, ventral neural progenitor cells that express the transcription factors Olig1 and Olig2, called pMN progenitors, produce motor neurons and then oligodendrocytes. Whether motor neurons and oligodendrocytes arise from common or distinct progenitors in vivo is not known. Using zebrafish, we found that motor neurons and oligodendrocytes are produced sequentially by distinct progenitors that have distinct origins. When olig2(+) cells were tracked during the peak period of motor neuron formation, most differentiated as motor neurons without further cell division. Using time-lapse imaging, we found that, as motor neurons differentiated, more dorsally positioned neuroepithelial progenitors descended to the pMN domain and initiated olig2 expression. Inhibition of Hedgehog signaling during motor neuron differentiation blocked the ventral movement of progenitors, the progressive initiation of olig2 expression, and oligodendrocyte formation. We therefore propose that the motor neuron-to-oligodendrocyte switch results from Hedgehog-mediated recruitment of glial-fated progenitors to the pMN domain subsequent to neurogenesis.

  11. Connectivity from OR37 expressing olfactory sensory neurons to distinct cell types in the hypothalamus

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    Andrea eBader


    Full Text Available Olfactory sensory neurons which express a member from the OR37 subfamily of odorant receptor genes are wired to the main olfactory bulb in a unique monoglomerular fashion; from these glomeruli an untypical connectivity into higher brain centers exists. In the present study we have investigated by DiI and transsynaptic tracing approaches how the connection pattern from these glomeruli into distinct hypothalamic nuclei is organized. The application of DiI onto the ventral domain of the bulb which harbors the OR37 glomeruli resulted in the labeling of fibers within the paraventricular and supraoptic nucleus of the hypothalamus; some of these fibers were covered with varicose-like structures. No DiI-labeled cell somata were detectable in these nuclei. The data indicate that projection neurons which originate in the OR37 region of the main olfactory bulb form direct connections into these nuclei. The cells that were labeled by the transsynaptic tracer WGA in these nuclei were further characterized. Their distribution pattern in the paraventricular nucleus was reminiscent of cells which produce distinct neuropeptides. Double labeling experiments confirmed that they contained vasopressin, but not the related neuropeptide oxytocin. Morphological analysis revealed that they comprise of magno- and parvocellular cells. A comparative investigation of the WGA-positive cells in the supraoptic nucleus demonstrated that these were vasopressin-positive, as well, whereas oxytocin-producing cells of this nucleus also contained no transsynaptic tracer. Together, the data demonstrate a connectivity from OR37 expressing sensory neurons to distinct hypothalamic neurons with the same neuropeptide content.

  12. Human Airway Primary Epithelial Cells Show Distinct Architectures on Membrane Supports Under Different Culture Conditions. (United States)

    Min, Kyoung Ah; Rosania, Gus R; Shin, Meong Cheol


    To facilitate drug development for lung delivery, it is highly demanding to establish appropriate airway epithelial cell models as transport barriers to evaluate pharmacokinetic profiles of drug molecules. Besides the cancer-derived cell lines, as the primary cell model, normal human bronchial epithelial (NHBE) cells have been used for drug screenings because of physiological relevance to in vivo. Therefore, to accurately interpret drug transport data in NHBE measured by different laboratories, it is important to know biophysical characteristics of NHBE grown on membranes in different culture conditions. In this study, NHBE was grown on the polyester membrane in a different medium and its transport barrier properties as well as cell architectures were fully characterized by functional assays and confocal imaging throughout the days of cultures. Moreover, NHBE cells on inserts in a different medium were subject to either of air-interfaced culture (AIC) or liquid-covered culture (LCC) condition. Cells in the AIC condition were cultivated on the membrane with medium in the basolateral side only, whereas cells with medium in apical and basolateral sides under the LCC condition. Quantitative microscopic imaging with biophysical examination revealed distinct multilayered architectures of differentiated NHBE cells, suggesting NHBE as functional cell barriers for the lung-targeting drug transport.

  13. Plumbagin elicits differential proteomic responses mainly involving cell cycle, apoptosis, autophagy, and epithelial-to-mesenchymal transition pathways in human prostate cancer PC-3 and DU145 cells

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    Qui JX


    critical role in the regulation of cell cycle, apoptosis, autophagy, epithelial to mesenchymal transition (EMT, and reactive oxygen species generation. The proteomic study showed substantial differences in response to PLB treatment between PC-3 and DU145 cells. PLB treatment significantly modulated the expression of critical proteins that regulate cell cycle, apoptosis, and EMT signaling pathways in PC-3 cells but not in DU145 cells. Consistently, our Western blotting analysis validated the bioinformatic and proteomic data and confirmed the modulating effects of PLB on important proteins that regulated cell cycle, apoptosis, autophagy, and EMT in PC-3 and DU145 cells. The data from the Western blot assay could not display significant differences between PC-3 and DU145 cells. These findings indicate that PLB elicits different proteomic responses in PC-3 and DU145 cells involving proteins and pathways that regulate cell cycle, apoptosis, autophagy, reactive oxygen species production, and antioxidation/oxidation homeostasis. This is the first systematic study with integrated computational, proteomic, and functional analyses revealing the networks of signaling pathways and differential proteomic responses to PLB treatment in prostate cancer cells. Quantitative proteomic analysis using SILAC represents an efficient and highly sensitive approach to identify the target networks of anticancer drugs like PLB, and the data may be used to discriminate the molecular and clinical subtypes, and to identify new therapeutic targets and biomarkers, for prostate cancer. Further studies are warranted to explore the potential of quantitative proteomic analysis in the identification of new targets and biomarkers for prostate cancer.Keywords: EMT, proteomics, SILAC

  14. DNA vaccine encoding prostatic acid phosphatase (PAP) elicits long-term T-cell responses in patients with recurrent prostate cancer. (United States)

    Becker, Jordan T; Olson, Brian M; Johnson, Laura E; Davies, James G; Dunphy, Edward J; McNeel, Douglas G


    Prostatic acid phosphatase (PAP) is a tumor antigen in prostate cancer and the target of several anti-tumor vaccines in earlier clinical trials. Ultimately, the goal of anti-tumor vaccines is to elicit a sustainable immune response, able to eradicate a tumor, or at least restrain its growth. We have investigated plasmid DNA vaccines and have previously conducted a phase 1 trial in which patients with recurrent prostate cancer were vaccinated with a DNA vaccine encoding PAP. In this study, we investigated the immunologic efficacy of subsequent booster immunizations, and conducted more detailed longitudinal immune analysis, to answer several questions aimed at guiding optimal schedules of vaccine administration for future clinical trials. We report that antigen-specific cytolytic T-cell responses were amplified after immunization in 7 of 12 human leukocyte antigen-A2-expressing individuals, and that multiple immunizations seemed necessary to elicit PAP-specific interferon-gamma-secreting immune responses detectable by enzyme-linked immunosorbent spot assay. Moreover, among individuals who experienced a >/=200% increase in prostate-specific antigen doubling time, long-term PAP-specific interferon-gamma-secreting T-cell responses were detectable in 6 of 8, but in only 1 of 14 individuals without an observed change in prostate-specific antigen doubling time (P=0.001). Finally, we identified that immune responses elicited could be further amplified by subsequent booster immunizations. These results suggest that future trials using this DNA vaccine, and potentially other anti-tumor DNA vaccines, could investigate ongoing schedules of administration with periodic booster immunizations. Moreover, these results suggest that DNA vaccines targeting PAP could potentially be combined in heterologous immunization strategies with other vaccines to further augment PAP-specific T-cell immunity.

  15. Distinct types of fibrocyte can differentiate from mononuclear cells in the presence and absence of serum.

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    S John Curnow

    Full Text Available BACKGROUND: Fibrocytes are bone-marrow derived cells, expressing both haematopoietic and stromal cell markers, which contribute to tissue repair as well as pathological fibrosis. The differentiation of fibrocytes remains poorly characterised and this has limited understanding of their biology and function. In particular two methods are used to generate fibrocytes in vitro that differ fundamentally by the presence or absence of serum. METHODOLOGY/PRINCIPAL FINDINGS: We show here that fibrocytes grown in the absence of serum (SF differentiate more efficiently from peripheral blood mononuclear cells than CD14(+ monocytes, and respond to serum by losing their spindle-shaped fibrocyte morphology. Although fibrocytes generated in the presence of serum (SC express the same range of markers, they differentiate more efficiently from CD14(+ monocytes and do not change their morphology in response to serum. Transcriptional analysis revealed that both types of fibrocyte are distinct from each other, fibroblasts and additional monocyte-derived progeny. The gene pathways that differ significantly between SF and SC fibrocytes include those involved in cell migration, immune responses and response to wounding. CONCLUSIONS/SIGNIFICANCE: These data show that SF and SC fibrocytes are distinct but related cell types, and suggest that they will play different roles during tissue repair and fibrosis where changes in serum proteins may occur.

  16. Distinct and Overlapping Sarcoma Subtypes Initiated from Muscle Stem and Progenitor Cells

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    Jordan M. Blum


    Full Text Available Rhabdomyosarcoma (RMS is the most common soft tissue sarcoma in children, whereas undifferentiated pleomorphic sarcoma (UPS is one of the most common soft tissue sarcomas diagnosed in adults. To investigate the myogenic cell(s of origin of these sarcomas, we used Pax7-CreER and MyoD-CreER mice to transform Pax7+ and MyoD+ myogenic progenitors by expressing oncogenic KrasG12D and deleting Trp53 in vivo. Pax7-CreER mice developed RMS and UPS, whereas MyoD-CreER mice developed UPS. Using gene set enrichment analysis, RMS and UPS each clustered specifically within their human counterparts. These results suggest that RMS and UPS have distinct and overlapping cells of origin within the muscle lineage. Taking them together, we have established mouse models of soft tissue sarcoma from muscle stem and progenitor cells.

  17. Infrared Signatures of Bacillus Bacteria: Clear IR Distinctions Between Sporulated and Vegetative Cells with Chemical Assighments

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, Timothy J.; Williams, Stephen D.; Valentine, Nancy B.; Su, Yin-Fong; Kreuzer-Martin, Helen W.; Wahl, Karen L.; Forrester, Joel B.


    This paper highlights the distinctions between the infrared (IR) absorption spectra of vegetative versus sporulated Bacillus bacteria. It is observed that there are unique signatures clearly associated with either the sporulated or the vegetative state, and that vegetative cells (and associated debris) can contribute to the spore spectra. A distinct feature at ~1739 cm-1 appears to be unique to vegetative cell spectra, and can also be used as an indicator of vegetative cells or cell debris in the spore spectra. The data indicate the band is caused by a phospholipid carbonyl bond and are consistent with, but do not prove it to be, either phosphatidyl ethanolamine (PE) or phosphatidyl glycerol (PG), the two major classes of phospholipids found in vegetative cells of Bacillus species. The endospore spectra show characteristic peaks at 1441, 1277, and 1015 cm-1 along with a distinct quartet of peaks at 766, 725, 701, and 659 cm-1. These are clearly associated with calcium dipicolinate trihydrate, CaDP•3H2O. We emphasize that the spore peaks, especially the quartet, arise from the calcium dipicolinate trihydrate and not from dipicolinic acid or other dipicolinate hydrate salts. The CaDP•3H2O vibrational peaks and the effects of hydration were studied using quantum chemistry in the PQS software package. The quartet is associated with many motions including contributions from the Ca2+ counterion and hydration waters including Ca-O-H bends, H2O-Ca-O torsions and O-C-O bends. The 1441 and 1015 cm-1 modes are planar pyridine modes with the 1441 mode primarily a ring C-N stretch and the 1015 mode primarily a ring C-C stretch.

  18. Infrared signatures of Bacillus bacteria: Clear IR distinctions between sporulated and vegetative cells with chemical assignments

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, Timothy J.; Williams, Stephen D.; Valentine, Nancy B.; Su, Yin-Fong; Kreuzer-Martin, Helen W.; Wahl, Karen L.; Forrester, Joel B.


    This paper highlights the distinctions between the infrared (IR) absorption spectra of vegetative versus sporulated Bacillus bacteria. It is observed that there are unique signatures clearly associated with either the sporulated or the vegetative state, and that vegetative cells (and associated debris) can contribute to the spore spectra. A distinct feature at ~1739 cm-1 appears to be unique to vegetative cell spectra, and can also be used as an indicator of vegetative cells or cell debris in the spore spectra. The data indicate the band is caused by a phospholipid carbonyl bond and are consistent with, but do not prove it to be, either phosphatidyl ethanolamine (PE) or phosphatidyl glycerol (PG), the two major classes of phospholipids found in vegetative cells of Bacillus species. The endospore spectra show characteristic peaks at 1441, 1277, and 1015 cm-1 along with a distinct quartet of peaks at 766, 725, 701, and 659 cm-1. These are clearly associated with calcium dipicolinate trihydrate, CaDP•3H2O. We emphasize that the spore peaks, especially the quartet, arise from the calcium dipicolinate trihydrate and not from dipicolinic acid or other dipicolinate hydrate salts. The CaDP•3H2O vibrational peaks and the effects of hydration were studied using quantum chemistry in the PQS software package. The quartet is associated with many motions including contributions from the Ca2+ counterion and hydration waters including Ca-O-H bends, H2O-Ca-O torsions and O-C-O bends. The 1441 and 1015 cm-1 modes are planar pyridine modes with the 1441 mode primarily a ring C-N stretch and the 1015 mode primarily a ring C-C stretch.

  19. Two distinct subpopulations of nestin-positive cells in adult mouse dentate gyrus. (United States)

    Fukuda, Satoshi; Kato, Fusao; Tozuka, Yusuke; Yamaguchi, Masahiro; Miyamoto, Yusei; Hisatsune, Tatsuhiro


    Neurogenesis in the dentate gyrus of the adult mammalian hippocampus has been proven in a series of studies, but the differentiation process toward newborn neurons is still unclear. In addition to the immunohistochemical study, electrophysiological membrane recordings of precursor cells could provide an alternative view to address this differentiation process. In this study, we performed green fluorescent protein (GFP)-guided selective recordings of nestin-positive progenitor cells in adult dentate gyrus by means of nestin-promoter GFP transgenic mice, because nestin is a typical marker for precursor cells in the adult dentate gyrus. The patch-clamp recordings clearly demonstrated the presence of two distinct subpopulations (type I and type II) of nestin-positive cells. Type I cells had a lower input resistance value of 77.1 M(Omega) (geometric mean), and their radial processes were stained with anti-glial fibrillary acidic protein antibody. On the other hand, type II nestin-positive cells had a higher input resistance value of 2110 MOmega and expressed voltage-dependent sodium current. In most cases, type II cells were stained with anti-polysialylated neural cell adhesion molecule. Taken together with a bromodeoxyuridine pulse-chase analysis, our results may reflect a rapid and dynamic cell conversion of nestin-positive progenitor, from type I to type II, at an early stage of adult neurogenesis in the dentate gyrus.

  20. Interleukin-1 exerts distinct actions on different cell types of the brain in vitro

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    Ying An


    Full Text Available Ying An, Qun Chen, Ning QuanDepartment of Oral Biology, Ohio State University, Columbus, OH, USAAbstract: Interleukin-1 (IL-1 is a critical neuroinflammatory mediator in the central nervous system (CNS. In this study, we investigated the effect of IL-1 on inducing inflammation-related gene expression in three astrocyte, two microglial, and one brain endothelial cell line. Interleukin-1 beta (IL-1β is found to be produced by the two microglial cell lines constitutively, but these cells do not respond to IL-1β stimulation. The three astrocyte cell lines responded to IL-1ß stimulation by expressing MCP-1, CXCL-1, and VCAM-1, but different subtypes of astrocytes exhibited different expression profiles after IL-1β stimulation. The brain endothelial cells showed strongest response to IL-1β by producing MCP-1, CXCL-1, VCAM-1, ICAM-1, IL-6, and COX-2 mRNA. The induction of endothelial COX-2 mRNA is shown to be mediated by p38 MAPK pathway, whereas the induction of other genes is mediated by the NF-κB pathway. These results demonstrate that IL-1 exerts distinct cell type-specific action in CNS cells and suggest that IL-1-mediated neuroinflammation is the result of the summation of multiple responses from different cell types in the CNS to IL-1.Keywords: astrocyte, microglia, endothelial cells, signal transduction pathways, gene expression 

  1. Comparative expression profiling of distinct T cell subsets undergoing oxidative stress.

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    Rudolf Lichtenfels

    Full Text Available The clinical outcome of adoptive T cell transfer-based immunotherapies is often limited due to different escape mechanisms established by tumors in order to evade the hosts' immune system. The establishment of an immunosuppressive micromilieu by tumor cells along with distinct subsets of tumor-infiltrating lymphocytes is often associated with oxidative stress that can affect antigen-specific memory/effector cytotoxic T cells thereby substantially reducing their frequency and functional activation. Therefore, protection of tumor-reactive cytotoxic T lymphocytes from oxidative stress may enhance the anti-tumor-directed immune response. In order to better define the key pathways/proteins involved in the response to oxidative stress a comparative 2-DE-based proteome analysis of naïve CD45RA(+ and their memory/effector CD45RO(+ T cell counterparts in the presence and absence of low dose hydrogen peroxide (H(2O(2 was performed in this pilot study. Based on the profiling data of these T cell subpopulations under the various conditions, a series of differentially expressed spots were defined, members thereof identified by mass spectrometry and subsequently classified according to their cellular function and localization. Representative targets responding to oxidative stress including proteins involved in signaling pathways, in regulating the cellular redox status as well as in shaping/maintaining the structural cell integrity were independently verified at the transcript and protein level under the same conditions in both T cell subsets. In conclusion the resulting profiling data describe complex, oxidative stress-induced, but not strictly concordant changes within the respective expression profiles of CD45RA(+ and CD45RO(+ T cells. Some of the differentially expressed genes/proteins might be further exploited as potential targets toward modulating the redox capacity of the distinct lymphocyte subsets thereby providing the basis for further studies

  2. Overexpressed Genes/ESTs and Characterization of Distinct Amplicons on 17823 in Breast Cancer Cells

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    Ayse E. Erson


    Full Text Available 17823 is a frequent site of gene amplification in breast cancer. Several lines of evidence suggest the presence of multiple amplicons on 17823. To characterize distinct amplicons on 17823 and localize putative oncogenes, we screened genes and expressed sequence tags (ESTs in existing physical and radiation hybrid maps for amplification and overexpression in breast cancer cell lines by semiquantitative duplex PCR, semiquantitative duplex RT-PCR, Southern blot, Northern blot analyses. We identified two distinct amplicons on 17823, one including TBX2 and another proximal region including RPS6KB1 (PS6K and MUL. In addition to these previously reported overexpressed genes, we also identified amplification and overexpression of additional uncharacterized genes and ESTs, some of which suggest potential oncogenic activity. In conclusion, we have further defined two distinct regions of gene amplification and overexpression on 17823 with identification of new potential oncogene candidates. Based on the amplification and overexpression patterns of known and as of yet unrecognized genes on 17823, it is likely that some of these genes mapping to the discrete amplicons function as oncogenes and contribute to tumor progression in breast cancer cells.

  3. Distinct impact of targeted actin cytoskeleton reorganization on mechanical properties of normal and malignant cells. (United States)

    Efremov, Yu M; Dokrunova, A A; Efremenko, A V; Kirpichnikov, M P; Shaitan, K V; Sokolova, O S


    The actin cytoskeleton is substantially modified in cancer cells because of changes in actin-binding protein abundance and functional activity. As a consequence, cancer cells have distinctive motility and mechanical properties, which are important for many processes, including invasion and metastasis. Here, we studied the effects of actin cytoskeleton alterations induced by specific nucleation inhibitors (SMIFH2, CK-666), cytochalasin D, Y-27632 and detachment from the surface by trypsinization on the mechanical properties of normal Vero and prostate cancer cell line DU145. The Young's modulus of Vero cells was 1300±900 Pa, while the prostate cancer cell line DU145 exhibited significantly lower Young's moduli (600±400 Pa). The Young's moduli exhibited a log-normal distribution for both cell lines. Unlike normal cells, cancer cells demonstrated diverse viscoelastic behavior and different responses to actin cytoskeleton reorganization. They were more resistant to specific formin-dependent nucleation inhibition, and reinforced their cortical actin after detachment from the substrate. This article is part of a Special Issue entitled: Mechanobiology.

  4. Peptide Immunization Elicits Polyomavirus-Specific MHC Class Ib-Restricted CD8 T Cells in MHC Class Ia Allogeneic Mice (United States)

    Hofstetter, Amelia R.; Evavold, Brian D.


    Abstract Unlike the polymorphic MHC class Ia molecules, MHC class Ib molecules are oligomorphic or nonpolymorphic. We recently discovered a protective CD8 T cell response to mouse polyomavirus (MPyV) in H-2b haplotype mice that is restricted by H2-Q9, a member of the Qa-2 MHC class Ib family. Here, we demonstrate that immunization with a peptide corresponding to a virus capsid-derived peptide presented by Q9 also elicits MHC class Ib-restricted MPyV-specific CD8 T cells in mice of H-2s and H-2g7 strains. These findings support the concept that immunization with a single MHC class Ib-restricted peptide can expand CD8 T cells in MHC class Ia allogeneic hosts. PMID:23374150

  5. Leptin differentially regulates NPY secretion in hypothalamic cell lines through distinct intracellular signal transduction pathways. (United States)

    Dhillon, Sandeep S; Belsham, Denise D


    Leptin acts as a key peripheral hormone in distinct neurons in the hypothalamus to modulate both reproductive function and energy homeostasis. The control of neuropeptide Y (NPY) secretion is an example of a process that can be differentially regulated by leptin. In order to further understand these distinct modulatory effects, we have used immortalized, neuronal hypothalamic cell lines expressing NPY, mHypoE-38 and mHypoE-46. We found that these cell lines express the endogenous leptin receptor, ObRb, and secrete detectable levels of NPY. We exposed the neurons to 100nM leptin for 1h and determined that the basal levels of NPY in the cell lines were differentially regulated: NPY secretion was inhibited in mHypoE-46 neurons, whereas NPY secretion was induced in the mHypoE-38 neurons. In order to determine the mechanisms involved in the divergent regulation of NPY release, we analyzed the activity of a number of signaling components using phospho-specific antibodies directed towards specific proteins in the MAP kinase, PI3K, and AMPK pathways, among others. We found that leptin activated a different combination of second messengers in each cell line. Importantly, we could link the regulation of NPY secretion to different signaling pathways, AMPK in the mHypoE-46 and both MAPK and PI3K in the mHypoE-38 neurons. This is the first demonstration that leptin can specifically regulate individual NPY neuron secretory responses through distinct signaling pathways.

  6. Immunostaining for substance P receptor labels GABAergic cells with distinct termination patterns in the hippocampus. (United States)

    Acsády, L; Katona, I; Gulyás, A I; Shigemoto, R; Freund, T F


    A specific antiserum against substance P receptor (SPR) labels nonprincipal neurons in the cerebral cortex of the rat (T. Kaneko et al. [1994], Neuroscience 60:199-211; Y. Nakaya et al. [1994], J. Comp. Neurol. 347:249-274). In the present study, we aimed to identify the types of SPR-immunoreactive neurons in the hippocampus according to their content of neurochemical markers, which label interneuron populations with distinct termination patterns. Markers for perisomatic inhibitory cells, parvalbumin and cholecystokinin (CCK), colocalized with SPR in pyramidallike basket cells in the dentate gyrus and in large multipolar or bitufted cells within all hippocampal subfields respectively. A dense meshwork of SPR-immunoreactive spiny dendrites in the hilus and stratum lucidum of the CA3 region belonged largely to inhibitory cells terminating in the distal dendritic region of granule cells, as indicated by the somatostatin and neuropeptide Y (NPY) content. In addition, SPR and NPY were colocalized in numerous multipolar interneurons with dendrites branching close to the soma. Twenty-five percent of the SPR-immunoreactive cells overlapped with calretinin-positive neurons in all hippocampal subfields, showing that interneurons specialized to contact other gamma-aminobutyric acid-ergic cells may also contain SPR. On the basis of the known termination pattern of the colocalized markers, we conclude that SPR-positive interneurons are functionally heterogeneous and participate in different inhibitory processes: (1) perisomatic inhibition of principal cells (CCK-containing cells, and parvalbumin-positive cells in the dentate gyrus), (2) feedback dendritic inhibition in the entorhinal termination zone (somatostatin and NPY-containing cells), and (3) innervation of other interneurons (calretinin-containing cells).

  7. Mosaic vaccines elicit CD8+ T cell responses in monkeys that confer immune coverage of diverse HIV strains

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    Fischer, Will [Los Alamos National Laboratory; Korber, Bette [Los Alamos National Laboratory


    Creation of a successful HIV vaccine will require the development of a strategy to generate cellular immunity with sufficient cross-clade breadth to deal with the extreme genetic diversity of the virus. Polyvalent mosaic immunogens derived from in silica recombination of natural strains of HIV are designed to induce cellular immune responses that maximally cover the sequence diversity of circulating virus isolates. Immunization of rhesus monkeys with plasmid DNA and recombinant vaccinia virus vaccine constructs expressing either consensus immunogens or polyvalent mosaic immunogens elicited a CD4+ T lymphocyte-biased response with comparably broad epitope-specific total T lymphocyte specificities. However, immunization with the mosaic immunogens induced HIV-specific CD8+ T lymphocyte responses with markedly greater depth and breadth. Therefore, the use of polyvalent mosaic immunogens is a promising strategy for a global vaccine for HIV.

  8. Accumulation of distinct prelamin A variants in human diploid fibroblasts differentially affects cell homeostasis

    Energy Technology Data Exchange (ETDEWEB)

    Candelario, Jose; Borrego, Stacey [Department of Molecular Microbiology and Immunology, Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033 (United States); Reddy, Sita, E-mail: [Department of Biochemistry and Molecular Biology, Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033 (United States); Comai, Lucio, E-mail: [Department of Molecular Microbiology and Immunology, Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033 (United States)


    Lamin A is a component of the nuclear lamina that plays a major role in the structural organization and function of the nucleus. Lamin A is synthesized as a prelamin A precursor which undergoes four sequential post-translational modifications to generate mature lamin A. Significantly, a large number of point mutations in the LMNA gene cause a range of distinct human disorders collectively known as laminopathies. The mechanisms by which mutations in lamin A affect cell function and cause disease are unclear. Interestingly, recent studies have suggested that alterations in the normal lamin A pathway can contribute to cellular dysfunction. Specifically, we and others have shown, at the cellular level, that in the absence of mutations or altered splicing events, increased expression of wild-type prelamin A results in a growth defective phenotype that resembles that of cells expressing the mutant form of lamin A, termed progerin, associated with Hutchinson-Gilford Progeria syndrome (HGPS). Remarkably, the phenotypes of cells expressing elevated levels of wild-type prelamin A can be reversed by either treatment with farnesyltransferase inhibitors or overexpression of ZMPSTE24, a critical prelamin A processing enzyme, suggesting that minor increases in the steady-state levels of one or more prelamin A intermediates is sufficient to induce cellular toxicity. Here, to investigate the molecular basis of the lamin A pathway toxicity, we characterized the phenotypic changes occurring in cells expressing distinct prelamin A variants mimicking specific prelamin A processing intermediates. This analysis demonstrates that distinct prelamin A variants differentially affect cell growth, nuclear membrane morphology, nuclear distribution of lamin A and the fundamental process of transcription. Expression of prelamin A variants that are constitutively farnesylated induced the formation of lamin A aggregates and dramatic changes in nuclear membrane morphology, which led to reduced

  9. MR1-restricted MAIT cells display ligand discrimination and pathogen selectivity through distinct T cell receptor usage

    DEFF Research Database (Denmark)

    Gold, Marielle C.; McLaren, James E.; Reistetter, Joseph A.


    Mucosal-associated invariant T (MAIT) cells express a semi-invariant T cell receptor (TCR) that detects microbial metabolites presented by the nonpolymorphic major histocompatibility complex (MHC)-like molecule MR1. The highly conserved nature of MR1 in conjunction with biased MAIT TCRα chain usage...... is widely thought to indicate limited ligand presentation and discrimination within a pattern-like recognition system. Here, we evaluated the TCR repertoire of MAIT cells responsive to three classes of microbes. Substantial diversity and heterogeneity were apparent across the functional MAIT cell repertoire...... with this interpretation, MAIT cell clones with distinct TCRs responded differentially to a riboflavin metabolite. These results suggest that MAIT cells can discriminate between pathogen-derived ligands in a clonotype-dependent manner, providing a basis for adaptive memory via recruitment of specific repertoires shaped...

  10. MR1-restricted MAIT cells display ligand discrimination and pathogen selectivity through distinct T cell receptor usage

    DEFF Research Database (Denmark)

    Gold, Marielle C.; McLaren, James E.; Reistetter, Joseph A.


    Mucosal-associated invariant T (MAIT) cells express a semi-invariant T cell receptor (TCR) that detects microbial metabolites presented by the nonpolymorphic major histocompatibility complex (MHC)-like molecule MR1. The highly conserved nature of MR1 in conjunction with biased MAIT TCRα chain usage...... is widely thought to indicate limited ligand presentation and discrimination within a pattern-like recognition system. Here, we evaluated the TCR repertoire of MAIT cells responsive to three classes of microbes. Substantial diversity and heterogeneity were apparent across the functional MAIT cell repertoire...... with this interpretation, MAIT cell clones with distinct TCRs responded differentially to a riboflavin metabolite. These results suggest that MAIT cells can discriminate between pathogen-derived ligands in a clonotype-dependent manner, providing a basis for adaptive memory via recruitment of specific repertoires shaped...

  11. Glutamic acid decarboxylase 67 expression by a distinct population of mouse vestibular supporting cells

    Directory of Open Access Journals (Sweden)

    Giancarlo eRusso


    Full Text Available The function of the enzyme glutamate decarboxylase (GAD is to convert glutamate in -aminobutyric acid (GABA.GAD exists as two major isoforms, termed GAD65 and GAD67,.that are usually expressed in GABA-containing neurons in the central nervous system. GAD65 has been proposed to be associated with GABA exocytosis whereas GAD67 with GABA metabolism. In the present immunofluorescence study, we have investigated the presence of the two GAD isoforms in the semicircular canal cristae of wild type and GAD67-GFP knock-in mice. While no evidence for GAD65 expression was found, GAD67 was detected in a distinct population of peripherally-located supporting cells, but not in hair cells or in centrally-located supporting cells. GABA, on the other hand, was found in all supporting cells. The present result indicate that only a discrete population of supporting cells use GAD67 to synthesize GABA. This is the first report of a marker that allows to distinguish two populations of supporting cells in the vestibular epithelium. On the other hand, the lack of GABA and GAD enzymes in hair cells excludes its involvement in afferent transmission.

  12. MR1-restricted MAIT cells display ligand discrimination and pathogen selectivity through distinct T cell receptor usage. (United States)

    Gold, Marielle C; McLaren, James E; Reistetter, Joseph A; Smyk-Pearson, Sue; Ladell, Kristin; Swarbrick, Gwendolyn M; Yu, Yik Y L; Hansen, Ted H; Lund, Ole; Nielsen, Morten; Gerritsen, Bram; Kesmir, Can; Miles, John J; Lewinsohn, Deborah A; Price, David A; Lewinsohn, David M


    Mucosal-associated invariant T (MAIT) cells express a semi-invariant T cell receptor (TCR) that detects microbial metabolites presented by the nonpolymorphic major histocompatibility complex (MHC)-like molecule MR1. The highly conserved nature of MR1 in conjunction with biased MAIT TCRα chain usage is widely thought to indicate limited ligand presentation and discrimination within a pattern-like recognition system. Here, we evaluated the TCR repertoire of MAIT cells responsive to three classes of microbes. Substantial diversity and heterogeneity were apparent across the functional MAIT cell repertoire as a whole, especially for TCRβ chain sequences. Moreover, different pathogen-specific responses were characterized by distinct TCR usage, both between and within individuals, suggesting that MAIT cell adaptation was a direct consequence of exposure to various exogenous MR1-restricted epitopes. In line with this interpretation, MAIT cell clones with distinct TCRs responded differentially to a riboflavin metabolite. These results suggest that MAIT cells can discriminate between pathogen-derived ligands in a clonotype-dependent manner, providing a basis for adaptive memory via recruitment of specific repertoires shaped by microbial exposure.

  13. Chemoprophylaxis with sporozoite immunization in P. knowlesi rhesus monkeys confers protection and elicits sporozoite-specific memory T cells in the liver (United States)

    Spring, Michele D.; Yongvanitchit, Kosol; Kum-Arb, Utaiwan; Limsalakpetch, Amporn; Im-Erbsin, Rawiwan; Ubalee, Ratawan; Vanachayangkul, Pattaraporn; Remarque, Edmond J.; Angov, Evelina; Smith, Philip L.; Saunders, David L.


    Whole malaria sporozoite vaccine regimens are promising new strategies, and some candidates have demonstrated high rates of durable clinical protection associated with memory T cell responses. Little is known about the anatomical distribution of memory T cells following whole sporozoite vaccines, and immunization of nonhuman primates can be used as a relevant model for humans. We conducted a chemoprophylaxis with sporozoite (CPS) immunization in P. knowlesi rhesus monkeys and challenged via mosquito bites. Half of CPS immunized animals developed complete protection, with a marked delay in parasitemia demonstrated in the other half. Antibody responses to whole sporozoites, CSP, and AMA1, but not CelTOS were detected. Peripheral blood T cell responses to whole sporozoites, but not CSP and AMA1 peptides were observed. Unlike peripheral blood, there was a high frequency of sporozoite-specific memory T cells observed in the liver and bone marrow. Interestingly, sporozoite-specific CD4+ and CD8+ memory T cells in the liver highly expressed chemokine receptors CCR5 and CXCR6, both of which are known for liver sinusoid homing. The majority of liver sporozoite-specific memory T cells expressed CD69, a phenotypic marker of tissue-resident memory (TRM) cells, which are well positioned to rapidly control liver-stage infection. Vaccine strategies that aim to elicit large number of liver TRM cells may efficiently increase the efficacy and durability of response against pre-erythrocytic parasites. PMID:28182750

  14. Sepsis Induces Hematopoietic Stem Cell Exhaustion and Myelosuppression through Distinct Contributions of TRIF and MYD88

    Directory of Open Access Journals (Sweden)

    Huajia Zhang


    Full Text Available Toll-like receptor 4 (TLR4 plays a central role in host responses to bacterial infection, but the precise mechanism(s by which its downstream signaling components coordinate the bone marrow response to sepsis is poorly understood. Using mice deficient in TLR4 downstream adapters MYD88 or TRIF, we demonstrate that both cell-autonomous and non-cell-autonomous MYD88 activation are major causes of myelosuppression during sepsis, while having a modest impact on hematopoietic stem cell (HSC functions. In contrast, cell-intrinsic TRIF activation severely compromises HSC self-renewal without directly affecting myeloid cells. Lipopolysaccharide-induced activation of MYD88 or TRIF contributes to cell-cycle activation of HSC and induces rapid and permanent changes in transcriptional programs, as indicated by persistent downregulation of Spi1 and CebpA expression after transplantation. Thus, distinct mechanisms downstream of TLR4 signaling mediate myelosuppression and HSC exhaustion during sepsis through unique effects of MyD88 and TRIF.

  15. Highly efficient derivation of ventricular cardiomyocytes from induced pluripotent stem cells with a distinct epigenetic signature

    Institute of Scientific and Technical Information of China (English)

    Huansheng Xu; Ibrahim J Domian; Erding Hu; Robert Willette; John Lepore; Alexander Meissner; Zhong Wang; Kenneth R Chien; B Alexander Yi; Hao Wu; Christoph Bock; Hongcang Gu; Kathy O Lui; Joo-Hye C Park; Ying Shao; Alyssa K Riley


    Cardiomyocytes derived from pluripotent stem cells can be applied in drug testing,disease modeling and cellbased therapy.However,without procardiogenic growth factors,the efficiency of cardiomyogenesis from pluripotent stem cells is usually low and the resulting cardiomyocyte population is heterogeneous.Here,we demonstrate that induced pluripotent stem cells (iPSCs) can be derived from murine ventricular myocytes (VMs),and consistent with other reports of iPSCs derived from various somatic cell types,VM-derived iPSCs (ViPSCs) exhibit a markedly higher propensity to spontaneously differentiate into beating cardiomyocytes as compared to genetically matched embryonic stem cells (ESCs) or iPSCs derived from tail-tip fibroblasts.Strikingly,the majority of ViPSC-derived cardiomyocytes display a ventricular phenotype.The enhanced ventricular myogenesis in ViPSCs is mediated via increased numbers of cardiovascular progenitors at early stages of differentiation.In order to investigate the mechanism of enhanced ventricular myogenesis from ViPSCs,we performed global gene expression and DNA methylation analysis,which revealed a distinct epigenetic signature that may be involved in specifying the VM fate in pluripotent stem cells.

  16. Nonpolarized signaling reveals two distinct modes of 3D cell migration. (United States)

    Petrie, Ryan J; Gavara, Núria; Chadwick, Richard S; Yamada, Kenneth M


    We search in this paper for context-specific modes of three-dimensional (3D) cell migration using imaging for phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and active Rac1 and Cdc42 in primary fibroblasts migrating within different 3D environments. In 3D collagen, PIP3 and active Rac1 and Cdc42 were targeted to the leading edge, consistent with lamellipodia-based migration. In contrast, elongated cells migrating inside dermal explants and the cell-derived matrix (CDM) formed blunt, cylindrical protrusions, termed lobopodia, and Rac1, Cdc42, and PIP3 signaling was nonpolarized. Reducing RhoA, Rho-associated protein kinase (ROCK), or myosin II activity switched the cells to lamellipodia-based 3D migration. These modes of 3D migration were regulated by matrix physical properties. Specifically, experimentally modifying the elasticity of the CDM or collagen gels established that nonlinear elasticity supported lamellipodia-based migration, whereas linear elasticity switched cells to lobopodia-based migration. Thus, the relative polarization of intracellular signaling identifies two distinct modes of 3D cell migration governed intrinsically by RhoA, ROCK, and myosin II and extrinsically by the elastic behavior of the 3D extracellular matrix.

  17. Temporal dynamics of distinct CA1 cell populations during unconscious state induced by ketamine.

    Directory of Open Access Journals (Sweden)

    Hui Kuang

    Full Text Available Ketamine is a widely used dissociative anesthetic which can induce some psychotic-like symptoms and memory deficits in some patients during the post-operative period. To understand its effects on neural population dynamics in the brain, we employed large-scale in vivo ensemble recording techniques to monitor the activity patterns of simultaneously recorded hippocampal CA1 pyramidal cells and various interneurons during several conscious and unconscious states such as awake rest, running, slow wave sleep, and ketamine-induced anesthesia. Our analyses reveal that ketamine induces distinct oscillatory dynamics not only in pyramidal cells but also in at least seven different types of CA1 interneurons including putative basket cells, chandelier cells, bistratified cells, and O-LM cells. These emergent unique oscillatory dynamics may very well reflect the intrinsic temporal relationships within the CA1 circuit. It is conceivable that systematic characterization of network dynamics may eventually lead to better understanding of how ketamine induces unconsciousness and consequently alters the conscious mind.

  18. Cells Respond to Distinct Nanoparticle Properties with Multiple Strategies As Revealed by Single-Cell RNA-Seq

    Energy Technology Data Exchange (ETDEWEB)

    Mitchell, Hugh D.; Markillie, Lye Meng; Chrisler, William B.; Gaffrey, Matthew J.; Hu, Dehong; Szymanski, Craig J.; Xie, Yumei; Melby, Eric S.; Dohnalkova, Alice; Taylor, Ronald C.; Grate, Eva K.; Cooley, Scott K.; McDermott, Jason E.; Heredia-Langner, Alejandro; Orr, Galya


    The impact of distinct nanoparticle (NP) properties on cellular response and ultimately human health is unclear. This gap is partially due to experimental difficulties in achieving uniform NP loads in the studied cells, creating heterogeneous populations with some cells “overloaded” while other cells are loaded with few or no NPs. Yet gene expression studies have been conducted in the population as a whole, identifying generic responses, while missing unique responses due to signal averaging across many cells, each carrying different loads. Here we applied single-cell RNA-Seq to alveolar epithelial cells carrying defined loads of aminated or carboxylated quantum dots (QDs), showing higher or lower toxicity, respectively. Interestingly, cells carrying lower loads responded with multiple strategies, mostly with upregulated processes, which were nonetheless coherent and unique to each QD type. In contrast, cells carrying higher loads responded more uniformly, with mostly downregulated processes that were shared across QD types. Strategies unique to aminated QDs showed strong upregulation of stress responses, coupled in some cases with regulation of cell cycle, protein synthesis and organelle activities. In contrast, strategies unique to carboxylated QDs showed upregulation of DNA repair and RNA activities, and decreased regulation of cell division, coupled in some cases with upregulation of stress responses and ATP related functions. Together, our studies suggest scenarios where higher NP loads lock cells into uniform responses, mostly shutdown of cellular processes, whereas lower loads allow for unique responses to each NP type that are more diversified, proactive defenses or repairs of the NP insults.

  19. Functional melanocytes are readily reprogrammable from multilineage-differentiating stress-enduring (muse) cells, distinct stem cells in human fibroblasts. (United States)

    Tsuchiyama, Kenichiro; Wakao, Shohei; Kuroda, Yasumasa; Ogura, Fumitaka; Nojima, Makoto; Sawaya, Natsue; Yamasaki, Kenshi; Aiba, Setsuya; Dezawa, Mari


    The induction of melanocytes from easily accessible stem cells has attracted attention for the treatment of melanocyte dysfunctions. We found that multilineage-differentiating stress-enduring (Muse) cells, a distinct stem cell type among human dermal fibroblasts, can be readily reprogrammed into functional melanocytes, whereas the remainder of the fibroblasts do not contribute to melanocyte differentiation. Muse cells can be isolated as cells positive for stage-specific embryonic antigen-3, a marker for undifferentiated human embryonic stem cells, and differentiate into cells representative of all three germ layers from a single cell, while also being nontumorigenic. The use of certain combinations of factors induces Muse cells to express melanocyte markers such as tyrosinase and microphthalmia-associated transcription factor and to show positivity for the 3,4-dihydroxy-L-phenylalanine reaction. When Muse cell-derived melanocytes were incorporated into three-dimensional (3D) cultured skin models, they localized themselves in the basal layer of the epidermis and produced melanin in the same manner as authentic melanocytes. They also maintained their melanin production even after the 3D cultured skin was transplanted to immunodeficient mice. This technique may be applicable to the efficient production of melanocytes from accessible human fibroblasts by using Muse cells, thereby contributing to autologous transplantation for melanocyte dysfunctions, such as vitiligo.

  20. Human memory B cells originate from three distinct germinal center-dependent and -independent maturation pathways. (United States)

    Berkowska, Magdalena A; Driessen, Gertjan J A; Bikos, Vasilis; Grosserichter-Wagener, Christina; Stamatopoulos, Kostas; Cerutti, Andrea; He, Bing; Biermann, Katharina; Lange, Johan F; van der Burg, Mirjam; van Dongen, Jacques J M; van Zelm, Menno C


    Multiple distinct memory B-cell subsets have been identified in humans, but it remains unclear how their phenotypic diversity corresponds to the type of responses from which they originate. Especially, the contribution of germinal center-independent responses in humans remains controversial. We defined 6 memory B-cell subsets based on their antigen-experienced phenotype and differential expression of CD27 and IgH isotypes. Molecular characterization of their replication history, Ig somatic hypermutation, and class-switch profiles demonstrated their origin from 3 different pathways. CD27⁻IgG⁺ and CD27⁺IgM⁺ B cells are derived from primary germinal center reactions, and CD27⁺IgA⁺ and CD27⁺IgG⁺ B cells are from consecutive germinal center responses (pathway 1). In contrast, natural effector and CD27⁻IgA⁺ memory B cells have limited proliferation and are also present in CD40L-deficient patients, reflecting a germinal center-independent origin. Natural effector cells at least in part originate from systemic responses in the splenic marginal zone (pathway 2). CD27⁻IgA⁺ cells share low replication history and dominant Igλ and IgA2 use with gut lamina propria IgA+ B cells, suggesting their common origin from local germinal center-independent responses (pathway 3). Our findings shed light on human germinal center-dependent and -independent B-cell memory formation and provide new opportunities to study these processes in immunologic diseases.

  1. Distinctive effects of rat fibroblast growth factor-2 isoforms on PC12 and Schwann cells. (United States)

    Müller-Ostermeyer, F; Claus, P; Grothe, C


    Fibroblast growth factor-2 (FGF-2) is an important modulator of cell growth and differentiation and stimulates cell survival of various cells including neurons. Rat FGF-2 occurs in three isoforms, a low molecular weight 18 kD and two high molecular weight forms (21, 23 kD), representing alternative translation products from a single mRNA. The 18 kD isoform shows mainly cytoplasmatic localization, whereas the 21/23 kD FGF-2 are localized in the nucleus. In addition, the FGF-2 isoforms are differentially regulated in the sensory ganglia and peripheral nerve following nerve injury and in the adrenal medulla during post-natal development and after hormonal stimuli. The distinct intracellular distribution and differential regulation of the different FGF-2 isoforms indicate that they have unique biological roles, however, little is known about the biological effects of the high molecular weight FGF-2 isoforms. Immortalized Schwann cells and PC12 cells, which stably overexpress the different FGF-2 isoforms, showed that the different endogenous-overexpressed FGF-2 isoforms lead to dramatic modifications in cell proliferation and survival, when tested in serum-free and serum-containing medium. In contrast, application of recombinant FGF-2 isoforms on normal PC12 and immortalized Schwann cells results in similar biological effects on the proliferation and survival of the cells. Furthermore, we investigated the potential regulatory effects of endogenous-overexpressed and exogenous-applied FGF-2 isoforms on the mRNA level of the FGF-2 receptors and, additionally, on the tyrosin hydroxylase mRNA expression in PC12 cells.

  2. “Dermal dendritic cells” comprise two distinct populations: CD1+ dendritic cells and CD209+ macrophages


    Ochoa,Maria Teresa; Loncaric, Anya; Krutzik, Stephan R.; Becker, Todd C.; Modlin, Robert L.


    A key cell type of the resident skin immune system is the dendritic cell, which in normal skin is located in two distinct microanatomical compartments: Langerhans cells (LC) mainly in the epidermis and dermal dendritic cells (DDC) in the dermis. Here, the lineage of dermal dendritic cells was investigated using monoclonal antibodies and immunohistology. We provide evidence that “dermal dendritic cells” comprise at least two major phenotypic populations of dendritic appearing cells: immature D...

  3. Mechanism for multiplicity of steady states with distinct cell concentration in continuous culture of mammalian cells. (United States)

    Yongky, Andrew; Lee, Jongchan; Le, Tung; Mulukutla, Bhanu Chandra; Daoutidis, Prodromos; Hu, Wei-Shou


    Continuous culture for the production of biopharmaceutical proteins offers the possibility of steady state operations and thus more consistent product quality and increased productivity. Under some conditions, multiplicity of steady states has been observed in continuous cultures of mammalian cells, wherein with the same dilution rate and feed nutrient composition, steady states with very different cell and product concentrations may be reached. At those different steady states, cells may exhibit a high glycolysis flux with high lactate production and low cell concentration, or a low glycolysis flux with low lactate and high cell concentration. These different steady states, with different cell concentration, also have different productivity. Developing a mechanistic understanding of the occurrence of steady state multiplicity and devising a strategy to steer the culture toward the desired steady state is critical. We establish a multi-scale kinetic model that integrates a mechanistic intracellular metabolic model and cell growth model in a continuous bioreactor. We show that steady state multiplicity exists in a range of dilution rate in continuous culture as a result of the bistable behavior in glycolysis. The insights from the model were used to devise strategies to guide the culture to the desired steady state in the multiple steady state region. The model provides a guideline principle in the design of continuous culture processes of mammalian cells.

  4. Singling out Drosophila tendon cells: a dialogue between two distinct cell types. (United States)

    Volk, T


    The precise match between somatic muscles and their epidermal attachment cells is achieved through a continuous dialogue between these two cell types. Whereas tendon cells direct myotube migration and final patterning, the muscles are essential for the maintenance of the fate of tendon cells. The Drosophila neuregulin-like ligand, Vein, and its receptor, the epidermal growth factor receptor (Egfr), are critical components in the inductive signaling process that takes place between muscles and tendon cells. Additional gene products that relay the Vein-Egfr effect in Drosophila are conserved in the vertebrate neuregulin-mediated cascade. This review describes genetic and molecular aspects of the muscle-tendon inductive processes in Drosophila, and compares them with the relevant mechanisms in the vertebrate embryo.

  5. Tumor and endothelial cell-derived microvesicles carry distinct CEACAMs and influence T-cell behavior.

    Directory of Open Access Journals (Sweden)

    Harrison T Muturi

    Full Text Available Normal and malignant cells release a variety of different vesicles into their extracellular environment. The most prominent vesicles are the microvesicles (MVs, 100-1000 nm in diameter, which are shed of the plasma membrane, and the exosomes (70-120 nm in diameter, derivates of the endosomal system. MVs have been associated with intercellular communication processes and transport numerous proteins, lipids and RNAs. As essential component of immune-escape mechanisms tumor-derived MVs suppress immune responses. Additionally, tumor-derived MVs have been found to promote metastasis, tumor-stroma interactions and angiogenesis. Since members of the carcinoembryonic antigen related cell adhesion molecule (CEACAM-family have been associated with similar processes, we studied the distribution and function of CEACAMs in MV fractions of different human epithelial tumor cells and of human and murine endothelial cells. Here we demonstrate that in association to their cell surface phenotype, MVs released from different human epithelial tumor cells contain CEACAM1, CEACAM5 and CEACAM6, while human and murine endothelial cells were positive for CEACAM1 only. Furthermore, MVs derived from CEACAM1 transfected CHO cells carried CEACAM1. In terms of their secretion kinetics, we show that MVs are permanently released in low doses, which are extensively increased upon cellular starvation stress. Although CEACAM1 did not transmit signals into MVs it served as ligand for CEACAM expressing cell types. We gained evidence that CEACAM1-positive MVs significantly increase the CD3 and CD3/CD28-induced T-cell proliferation. All together, our data demonstrate that MV-bound forms of CEACAMs play important roles in intercellular communication processes, which can modulate immune response, tumor progression, metastasis and angiogenesis.

  6. Distinct modulation of allergic T cell responses by subcutaneous vs. sublingual allergen-specific immunotherapy

    DEFF Research Database (Denmark)

    Schulten, V; Tripple, V; Aasbjerg, Kristian


    injections or SLIT tablets or neither. PBMCs were tested for Timothy grass (TG)-specific cytokine production by ELISPOT after in vitro expansion with TG-peptide pools. Phenotypic characterization of cytokine-producing cells was performed by FACS. RESULTS: In the SCIT group, decreased IL-5 production...... was observed starting 10 months after treatment commenced. At 24 months, T cell responses showed IL-5 levels significantly below the before-treatment baseline. No significant reduction of IL-5 was observed in the SLIT or untreated group. However, a significant transient increase in IL-10 production after 10......: The most dominant immunological changes on a cellular level were a decrease in IL-5 in the SCIT group and a significant, transient increase of IL-10 observed after 10 months of treatment in both treated groups. The distinct routes of AIT administration may induce different immunomodulatory mechanisms...

  7. Distinct Functions of Specialized Dendritic Cell Subsets in Atherosclerosis and the Road Ahead

    Directory of Open Access Journals (Sweden)

    Alma Zernecke


    Full Text Available Atherosclerotic vascular disease is modulated by immune mechanisms. Dendritic cells (DCs and T cells are present within atherosclerotic lesions and function as central players in the initiation and modulation of adaptive immune responses. In previous years, we have studied the functional contribution of distinct DC subsets in disease development, namely, that of CCL17-expressing DCs as well as that of plasmacytoid DCs that play specialized roles in disease development. This review focuses on important findings gathered in these studies and dissects the multifaceted contribution of CCL17-expressing DCs and pDCs to the pathogenesis of atherosclerosis. Furthermore, an outlook on future challenges faced when studying DCs in this detrimental disease are provided, and hurdles that will need to be overcome in order to enable a better understanding of the contribution of DCs to atherogenesis are discussed, a prerequisite for their therapeutic targeting in atherosclerosis.

  8. Neural Stem Cell Differentiation Is Dictated by Distinct Actions of Nuclear Receptor Corepressors and Histone Deacetylases

    Directory of Open Access Journals (Sweden)

    Gonçalo Castelo-Branco


    Full Text Available Signaling factors including retinoic acid (RA and thyroid hormone (T3 promote neuronal, oligodendrocyte, and astrocyte differentiation of cortical neural stem cells (NSCs. However, the functional specificity of transcriptional repressor checkpoints controlling these differentiation programs remains unclear. Here, we show by genome-wide analysis that histone deacetylase (HDAC2 and HDAC3 show overlapping and distinct promoter occupancy at neuronal and oligodendrocyte-related genes in NSCs. The absence of HDAC3, but not HDAC2, initiated a neuronal differentiation pathway in NSCs. The ablation of the corepressor NCOR or HDAC2, in conjunction with T3 treatment, resulted in increased expression of oligodendrocyte genes, revealing a direct HDAC2-mediated repression of Sox8 and Sox10 expression. Interestingly, Sox10 was required also for maintaining the more differentiated state by repression of stem cell programming factors such as Sox2 and Sox9. Distinct and nonredundant actions of NCORs and HDACs are thus critical for control of lineage progression and differentiation programs in neural progenitors.

  9. Xnrs and activin regulate distinct genes during Xenopus development: activin regulates cell division.

    Directory of Open Access Journals (Sweden)

    Joana M Ramis

    Full Text Available BACKGROUND: The mesoderm of the amphibian embryo is formed through an inductive interaction in which vegetal cells of the blastula-staged embryo act on overlying equatorial cells. Candidate mesoderm-inducing factors include members of the transforming growth factor type beta family such as Vg1, activin B, the nodal-related proteins and derrière. METHODOLOGY AND PRINCIPLE FINDINGS: Microarray analysis reveals different functions for activin B and the nodal-related proteins during early Xenopus development. Inhibition of nodal-related protein function causes the down-regulation of regionally expressed genes such as chordin, dickkopf and XSox17alpha/beta, while genes that are mis-regulated in the absence of activin B tend to be more widely expressed and, interestingly, include several that are involved in cell cycle regulation. Consistent with the latter observation, cells of the involuting dorsal axial mesoderm, which normally undergo cell cycle arrest, continue to proliferate when the function of activin B is inhibited. CONCLUSIONS/SIGNIFICANCE: These observations reveal distinct functions for these two classes of the TGF-beta family during early Xenopus development, and in doing so identify a new role for activin B during gastrulation.

  10. Morphological Variability and Distinct Protein Profiles of Cultured and Endosymbiotic Symbiodinium cells Isolated from Exaiptasia pulchella. (United States)

    Pasaribu, Buntora; Weng, Li-Chi; Lin, I-Ping; Camargo, Eddie; Tzen, Jason T C; Tsai, Ching-Hsiu; Ho, Shin-Lon; Lin, Mong-Rong; Wang, Li-Hsueh; Chen, Chii-Shiarng; Jiang, Pei-Luen


    Symbiodinium is a dinoflagellate that plays an important role in the physiology of the symbiotic relationships of Cnidarians such as corals and sea anemones. However, it is very difficult to cultivate free-living dinoflagellates after being isolated from the host, as they are very sensitive to environmental changes. How these symbiont cells are supported by the host tissue is still unclear. This study investigated the characteristics of Symbiodinium cells, particularly with respect to the morphological variability and distinct protein profiles of both cultured and endosymbiotic Symbiodinium which were freshly isolated from Exaiptasia pulchella. The response of the cellular morphology of freshly isolated Symbiodinium cells kept under a 12 h L:12 h D cycle to different temperatures was measured. Cellular proliferation was investigated by measuring the growth pattern of Symbiodinium cells, the results of which indicated that the growth was significantly reduced in response to the extreme temperatures. Proteomic analysis of freshly isolated Symbiodinium cells revealed twelve novel proteins that putatively included transcription translation factors, photosystem proteins, and proteins associated with energy and lipid metabolism, as well as defense response. The results of this study will bring more understandings to the mechanisms governing the endosymbiotic relationship between the cnidarians and dinoflagellates.

  11. Butyrate and deoxycholic acid play common and distinct roles in HCT116 human colon cell proliferation. (United States)

    Zeng, Huawei; Claycombe, Kate J; Reindl, Katie M


    Consumption of a high-fat diet causes an increase in bile acid deoxycholic acid (DCA) in colon lumen and colon cancer risk, while butyrate, an intestinal microbiota metabolite of dietary fiber, has been shown to exhibit colon cancer-preventive effects. To distinguish these opposing effects of DCA and butyrate (two major metabolites in colon lumen), we examined the effects of physiologically relevant doses of butyrate (0.5-2 mmol/l) and DCA (0.05-0.3 mmol/l) on colon cell proliferation. We hypothesize that butyrate and DCA each modulates the cell cycle and apoptosis via common and distinct cellular signaling targets. In this study, we demonstrated that both butyrate and DCA inhibited cell proliferation by up to 89% and 92% and increased cell apoptosis rate by up to 3.1- and 4.5-fold, respectively. Cell cycle analyses revealed that butyrate led to an increase in G1 and G2 fractions with a concomitant drop in the S-phase fraction, but DCA induced an increase in only G1 fraction with a concomitant drop in the S-phase fraction when compared with the untreated cells. The examination of early cellular signaling revealed that DCA but not butyrate increased intracellular reactive oxygen species, genomic DNA breakage, the activation of ERK1/2, caspase-3 and PARP. In contrast, DCA decreased activated Rb protein level, and butyrate but not DCA increased p21 expression. Collectively, although both butyrate and DCA inhibit colonic cell proliferation, butyrate increases tumor suppressor gene expression, whereas DCA decreases tumor suppressor activation in cell cycle and apoptosis pathways.

  12. Quantification of cell edge velocities and traction forces reveals distinct motility modules during cell spreading.

    Directory of Open Access Journals (Sweden)

    Benjamin J Dubin-Thaler

    Full Text Available Actin-based cell motility and force generation are central to immune response, tissue development, and cancer metastasis, and understanding actin cytoskeleton regulation is a major goal of cell biologists. Cell spreading is a commonly used model system for motility experiments -- spreading fibroblasts exhibit stereotypic, spatially-isotropic edge dynamics during a reproducible sequence of functional phases: 1 During early spreading, cells form initial contacts with the surface. 2 The middle spreading phase exhibits rapidly increasing attachment area. 3 Late spreading is characterized by periodic contractions and stable adhesions formation. While differences in cytoskeletal regulation between phases are known, a global analysis of the spatial and temporal coordination of motility and force generation is missing. Implementing improved algorithms for analyzing edge dynamics over the entire cell periphery, we observed that a single domain of homogeneous cytoskeletal dynamics dominated each of the three phases of spreading. These domains exhibited a unique combination of biophysical and biochemical parameters -- a motility module. Biophysical characterization of the motility modules revealed that the early phase was dominated by periodic, rapid membrane blebbing; the middle phase exhibited continuous protrusion with very low traction force generation; and the late phase was characterized by global periodic contractions and high force generation. Biochemically, each motility module exhibited a different distribution of the actin-related protein VASP, while inhibition of actin polymerization revealed different dependencies on barbed-end polymerization. In addition, our whole-cell analysis revealed that many cells exhibited heterogeneous combinations of motility modules in neighboring regions of the cell edge. Together, these observations support a model of motility in which regions of the cell edge exhibit one of a limited number of motility modules

  13. Spontaneously immortalised bovine mammary epithelial cells exhibit a distinct gene expression pattern from the breast cancer cells

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    Li Qianqian


    exhibit non-malignant transformation. Although this cell line displays altered patterns of gene expression, it is clearly distinct from malignant breast cancer cell line. It showed that co-inhibition of cellular senescence and mitochondrial apoptosis pathways coordinates BME65Cs cells immortalisation. Additionally, mechanisms other than gene mutation are likely to be involved in regulation of cellular functions. This study provides an insight into the relationship between cell senescence and immortalisation. BME65Cs cells will be useful in future studies of cellular senescence and tumorigenesis.

  14. Differential expression of adhesion moleculesshaping the T-cell subset prevalence during the early phase of autoimmune and Trypanosoma cruzi-elicited myocarditis

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    Ana Paula MP Marino


    Full Text Available The participation of cell adhesion molecules (CAMs in the establishment of autoimmune and infectious myocarditis is an important matter of investigation and may have therapeutic implication. Trypanosoma cruzi infection induces a CD8-mediated myocarditis in patients with severe cardiomyopathy and experimental animals. Previously, we have proposed that this predominance of CD8+ T-cells is, at least in part, consequence of the differential expression of CAMs on circulating CD8+ lymphocytes. In the present study we investigated the participation of CAMs in shaping the phenotypic nature of the autoimmune CD4-mediated myosin-induced and the CD8-mediated T. cruzi-elicited myocarditis. We provide evidence that the prevalence of a certain T-cell subset inside the inflamed heart reflects the differential profile of the adhesion molecules VLA-4, LFA-1, and ICAM-1 displayed on a large proportion of this particular T-cell population in peripheral blood during the early phase of inflammation. Further, the expression of VCAM-1, ligand for VLA-4, and ICAM-1, counter-receptor for LFA-1, was up-regulated on vascular endothelium and paralleled the entrance of inflammatory cells into the cardiac tissue. Thus, this up-regulated expression of receptors-counter-receptors that regulate T-cell transmigration through the vascular endothelium may have an important role in the pathogenesis of the early phase of both autoimmune and infectious myocarditis.

  15. Induction of extracellular defense-related proteins in suspension cultured-cells of Daucus carota elicited with cyclodextrins and methyl jasmonate. (United States)

    Sabater-Jara, Ana B; Almagro, Lorena; Pedreño, María A


    Suspension cultured-cells (SCC) of Daucus carota were used to evaluate the effect of methyl jasmonate and cyclodextrins, separately or in combination, on the induction of defense responses, particularly the accumulation of pathogenesis-related proteins. A comparative study of the extracellular proteome (secretome) between control and elicited carrot SCC pointed to the presence of amino acid sequences homologous to glycoproteins which have inhibitory activity against the cell-wall-degrading enzymes secreted by pathogens and/or are induced when carrot cells are exposed to a pathogen elicitor. Other amino acid sequences were homologous to Leucine-Rich Repeat domain-containing proteins, which play an essential role in defense against pathogens, as well as in the recognition of microorganisms, making them important players in the innate immunity of this plant. Also, some tryptic peptides were shown to be homologous to a thaumatin-like protein, showing high specificity to abiotic stress and to different reticuline oxidase-like proteins that displayed high levels of antifungal activity, suggesting that methyl jasmonate and cyclodextrins could play a role in mediating defense-related gene product expression in SCC of D. carota. Apart from these elicitor-inducible proteins, we observed the presence of PR-proteins in both control and elicited carrot SCC, suggesting that their expression is mainly constitutive. These PR-proteins are putative class IV chitinases, which also have inhibitory activity against pathogen growth and the class III peroxidases that participate in response to environmental stress (e.g. pathogen attack and oxidative), meaning that they are involved in defense responses triggered by both biotic and abiotic factors.

  16. Potent antitumor effect elicited by gp96-peptide complexes pulsed by dendritic cell on mice of H22 liver cancer

    Institute of Scientific and Technical Information of China (English)

    YANG Wei; CAO Chun-xia; CHU Yong-lie; LIU Qing-guang; YU Liang; PAN Cheng-en


    Objective: To improve DC-based tumor vaccination, we studied whether dendritic cells (DCs) which cocultured with H22 liver cancer cells-derived heat shock protein (HSP) glycoprotein 96(gp96) affect the T cell-activating potential in vitro and the induction of tumor immunity in vivo. Methods: Maturation of murine bone marrow-derived DC was induced by GM-CSF plus IL-4, which mimiced the immunostimulatory effect of DC. Cocultured DC and gp96-peptide complexes were used to vaccine H22liver cancer cells of mice. Using murine models we compared the immunogenecity of DC modified by gp96-peptides complexes derived from murine liver cancer cells alone or inactive tumor cells. To verify the specificity of the vaccine, in vitro assays were executed. Serum cytokine levels were quantified to explore the supposed pathway of DC modified by gp96 peptide complexes and its effect on antitumor immune response.Results: DC modified by gp96-peptide complexes can activate spleen lymphocyte and the latter can specifically kill H22 cells but not Ehrilich ascites carcinoma cells. Modified DC can induce potent tumor-antigenspecific immune response, augment the proliferation of Th1 cells, and inhibit tumor growth. Conclusion:In this study, we have developed a novel DC-mediated tumor vaccine by combing the gp96 antigenic peptides complexes and inducing immune response against specific tumor cells. gp96 can be identified as a potent DC activator.

  17. Brucella invasion of human intestinal epithelial cells elicits a weak proinflammatory response but a significant CCL20 secretion. (United States)

    Ferrero, Mariana C; Fossati, Carlos A; Rumbo, Martín; Baldi, Pablo C


    In spite of the frequent acquisition of Brucella infection by the oral route in humans, the interaction of the bacterium with cells of the intestinal mucosa has been poorly studied. Here, we show that different Brucella species can invade human colonic epithelial cell lines (Caco-2 and HT-29), in which only smooth species can replicate efficiently. Infection with smooth strains did not produce a significant cytotoxicity, while the rough strain RB51 was more cytotoxic. Infection of Caco-2 cells or HT-29 cells with either smooth or rough strains of Brucella did not result in an increased secretion of TNF-α, IL-1β, MCP-1, IL-10 or TGF-β as compared with uninfected controls, whereas all the infections induced the secretion of IL-8 and CCL20 by both cell types. The MCP-1 response to flagellin from Salmonella typhimurium was similar in Brucella-infected or uninfected cells, ruling out a bacterial inhibitory mechanism as a reason for the weak proinflammatory response. Infection did not modify ICAM-1 expression levels in Caco-2 cells, but increased them in HT-29 cells. These results suggest that Brucella induces only a weak proinflammatory response in gut epithelial cells, but produces a significant CCL20 secretion. The latter may be important for bacterial dissemination given the known ability of Brucella to survive in dendritic cells.

  18. Leishmania amazonensis Promastigotes Present Two Distinct Modes of Nucleus and Kinetoplast Segregation during Cell Cycle (United States)

    da Silva, Marcelo Santos; Monteiro, Jomar Patrício; Nunes, Vinícius Santana; Vasconcelos, Elton José; Perez, Arina Marina; Freitas-Júnior, Lúcio de Holanda; Elias, Maria Carolina; Cano, Maria Isabel Nogueira


    Here, we show the morphological events associated with organelle segregation and their timing in the cell cycle of a reference strain of Leishmania (L.) amazonensis promastigotes, the main causative agent of Tegumentary leishmaniasis in the Americas. We show evidences that during the cell cycle, L. amazonensis promastigotes present two distinct modes of nucleus and kinetoplast segregation, which occur in different temporal order in different proportions of cells. We used DAPI-staining and EdU-labeling to monitor the segregation of DNA-containing organelles and DNA replication in wild-type parasites. The emergence of a new flagellum was observed using a specific monoclonal antibody. The results show that L. amazonensis cell cycle division is peculiar, with 65% of the dividing cells duplicating the kinetoplast before the nucleus, and the remaining 35% doing the opposite or duplicating both organelles concomitantly. In both cases, the new flagellum appeared during S to G2 phase in 1N1K cells and thus before the segregation of both DNA-containing organelles; however, we could not determine the exact timing of flagellar synthesis. Most of these results were confirmed by the synchronization of parasites using hydroxyurea. Altogether, our data show that during the cell cycle of L. amazonensis promastigotes, similarly to L. donovani, the segregation of nucleus and kinetoplast do not follow a specific order, especially when compared to other trypanosomatids, reinforcing the idea that this characteristic seems to be species-specific and may represent differences in cellular biology among members of the Leishmania genus. PMID:24278433

  19. Distinct and conserved prominin-1/CD133-positive retinal cell populations identified across species.

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    József Jászai

    Full Text Available Besides being a marker of various somatic stem cells in mammals, prominin-1 (CD133 plays a role in maintaining the photoreceptor integrity since mutations in the PROM1 gene are linked with retinal degeneration. In spite of that, little information is available regarding its distribution in eyes of non-mammalian vertebrates endowed with high regenerative abilities. To address this subject, prominin-1 cognates were isolated from axolotl, zebrafish and chicken, and their retinal compartmentalization was investigated and compared to that of their mammalian orthologue. Interestingly, prominin-1 transcripts--except for the axolotl--were not strictly restricted to the outer nuclear layer (i.e., photoreceptor cells, but they also marked distinct subdivisions of the inner nuclear layer (INL. In zebrafish, where the prominin-1 gene is duplicated (i.e., prominin-1a and prominin-1b, a differential expression was noted for both paralogues within the INL being localized either to its vitreal or scleral subdivision, respectively. Interestingly, expression of prominin-1a within the former domain coincided with Pax-6-positive cells that are known to act as progenitors upon injury-induced retino-neurogenesis. A similar, but minute population of prominin-1-positive cells located at the vitreal side of the INL was also detected in developing and adult mice. In chicken, however, prominin-1-positive cells appeared to be aligned along the scleral side of the INL reminiscent of zebrafish prominin-1b. Taken together our data indicate that in addition to conserved expression of prominin-1 in photoreceptors, significant prominin-1-expressing non-photoreceptor retinal cell populations are present in the vertebrate eye that might represent potential sources of stem/progenitor cells for regenerative therapies.

  20. Probiotics-pathogen interactions elicit differential regulation of cutaneous immune responses in epidermal cells of Atlantic cod Gadus morhua. (United States)

    Lazado, Carlo C; Caipang, Christopher Marlowe A


    Little is known on the cutaneous immune responses during probiotics-pathogen interactions in fish. Thus, this study employed Atlantic cod primary epidermal (EP) cell cultures as a model to understand this interaction. The probiotics-pathogen interactions in the EP cell cultures were elucidated using Vibrio anguillarum 2133 (VA) as the pathogen and two host-derived bacteria (GP21 and GP12) as the probiotics. There was a regional size difference on the EP cells; i.e., EP cells from the dorsal region were significantly larger than the EP cells at the ventral side. VA significantly decreased viability of EP cells. In the presence of probiotics, this inhibition was mitigated. The probiotics reduced VA-induced cellular apoptosis and the probiotics-pathogen interactions influenced cellular myeloperoxidase activity during the latter stage of co-incubation. The probiotics-pathogen interactions triggered differential regulation of immune-related genes and the effects of the interaction were dependent on the region where the cells were isolated and the length of the co-incubation period. In most cases, the presence of probiotics alone showed no significant change on the mRNA level of immune genes in the EP cells but triggered immunostimulatory activity when incubated with VA. This study showed that the virulence of VA in EP cells could be modulated by host-derived probiotics and the immunomodulatory characteristics of the two candidate probionts advanced their immune-related probiotic potential.

  1. A distinct microRNA signature for definitive endoderm derived from human embryonic stem cells. (United States)

    Hinton, Andrew; Afrikanova, Ivka; Wilson, Mike; King, Charles C; Maurer, Brian; Yeo, Gene W; Hayek, Alberto; Pasquinelli, Amy E


    Human embryonic stem cells (hESCs) have the potential to differentiate into many adult cell types, and they are being explored as a resource for cell replacement therapies for multiple diseases. In order to optimize in vitro differentiation protocols, it will be necessary to elucidate regulatory mechanisms that contribute to lineage specification. MicroRNAs (miRNAs) are emerging as key regulators of hESC differentiation and embryonic development. In this study, we compare miRNA expression profiles between pluripotent hESCs and definitive endoderm (DE), an early step in the pathway toward the pancreatic lineage. Results from microarray analysis showed that DE can be distinguished by its unique miRNA profile, which consists of 37 significantly down-regulated and 17 up-regulated miRNAs in 2 different cell lines and in the presence/absence of feeder layers. Comparison to other hESC-derived lineages showed that most of the highly up-regulated miRNAs are specific to endoderm in early development. Notably, miR-375, which was previously implicated in regulating development and function of later stages of pancreatic development, is highly and specifically up-regulated during DE formation, suggesting that it may have a distinct role very early in development. Examination of potential mRNA targets showed that TIMM8A is repressed by ectopic miR-375 expression in pluripotent hESCs.

  2. Brief reports: A distinct DNA methylation signature defines breast cancer stem cells and predicts cancer outcome. (United States)

    El Helou, Rita; Wicinski, Julien; Guille, Arnaud; Adélaïde, Jose; Finetti, Pascal; Bertucci, François; Chaffanet, Max; Birnbaum, Daniel; Charafe-Jauffret, Emmanuelle; Ginestier, Christophe


    Self-renewal and differentiation are two epigenetic programs that regulate stem cells fate. Dysregulation of these two programs leads to the development of cancer stem cells (CSCs). Recent evidence suggests that CSCs are relatively resistant to conventional therapies and responsible for metastasis formation. Deciphering these processes will help understand oncogenesis and allow the development of new targeted therapies. Here, we have used a whole genome promoter microarray to establish the DNA methylation portraits of breast cancer stem cells (bCSCs) and non-bCSCs. A total of 68 differentially methylated regions (DMRs) were more hypomethylated in bCSCs than in non-bCSCs. Using a differentiation assay we demonstrated that DMRs are rapidly hypermethylated within the first 6 hours following induction of CSC differentiation whereas the cells reached the steady-state within 6 days, suggesting that these DMRs are linked to early CSC epigenetic regulation. These DMRs were significantly enriched in genes coding for TGF-β signaling-related proteins. Interestingly, DMRs hypomethylation was correlated to an overexpression of TGF-β signaling genes in a series of 109 breast tumors. Moreover, patients with tumors harboring the bCSC DMRs signature had a worse prognosis than those with non-bCSC DMRs signature. Our results show that bCSCs have a distinct DNA methylation landscape with TGF-β signaling as a key epigenetic regulator of bCSCs differentiation.

  3. LIM domain only-2 (LMO2 induces T-cell leukemia by two distinct pathways.

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    Stephen Smith

    Full Text Available The LMO2 oncogene is deregulated in the majority of human T-cell leukemia cases and in most gene therapy-induced T-cell leukemias. We made transgenic mice with enforced expression of Lmo2 in T-cells by the CD2 promoter/enhancer. These transgenic mice developed highly penetrant T-ALL by two distinct patterns of gene expression: one in which there was concordant activation of Lyl1, Hhex, and Mycn or alternatively, with Notch1 target gene activation. Most strikingly, this gene expression clustering was conserved in human Early T-cell Precursor ALL (ETP-ALL, where LMO2, HHEX, LYL1, and MYCN were most highly expressed. We discovered that HHEX is a direct transcriptional target of LMO2 consistent with its concordant gene expression. Furthermore, conditional inactivation of Hhex in CD2-Lmo2 transgenic mice markedly attenuated T-ALL development, demonstrating that Hhex is a crucial mediator of Lmo2's oncogenic function. The CD2-Lmo2 transgenic mice offer mechanistic insight into concordant oncogene expression and provide a model for the highly treatment-resistant ETP-ALL subtype.

  4. Induction of trans-resveratrol and extracellular pathogenesis-related proteins in elicited suspension cultured cells of Vitis vinifera cv Monastrell. (United States)

    Belchí-Navarro, Sarai; Almagro, Lorena; Sabater-Jara, Ana Belén; Fernández-Pérez, Francisco; Bru, Roque; Pedreño, Maria Angeles


    Suspension-cultured cells of Vitis vinifera cv Monastrell were used to investigate the effects of methyljasmonate, ethylene and salicylic acid separately or in combination with cyclodextrins on both trans-resveratrol production and the induction of defense responses. The results showed that the addition of methyljasmonate or ethylene to suspension-cultured cells jointly treated with cyclodextrins and salicylic acid provoked a decrease of trans-resveratrol levels suggesting that salicylic acid has a negative and antagonistic effect with methyljasmonate or ethylene on trans-resveratrol production. Likewise, the exogenous application of these compounds induced the accumulation of pathogenesis-related proteins. Analysis of the extracellular proteome showed the presence of amino acid sequences homologous to an specific β-1,3-glucanase, class III peroxidases and a β-1,4-mannanase, which suggests that these signal molecules could play a role in mediating defense-related gene product expression in V. vinifera cv Monastrell. Apart from these inducible proteins, other proteins were found in both the control and elicited cell cultures of V. vinifera. These included class IV chitinase, polygalacturonase inhibitor protein and reticuline oxidase-like protein, suggesting that their expression is constitutive being involved in the modification of the cell wall architecture during cell culture growth and in the prevention of pathogen attack.

  5. An oral recombinant Salmonella enterica serovar Typhimurium mutant elicits systemic antigen-specific CD8+ T cell cytokine responses in mice

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    Chin'ombe Nyasha


    Full Text Available Abstract Background The induction of antigen-specific CD8+ T cell cytokine responses against an attenuated, oral recombinant Salmonella enterica serovar Typhimurium vaccine expressing a green fluorescent protein (GFP model antigen was investigated. A GFP expression plasmid was constructed in which the gfp gene was fused in-frame with the 5' domain of the Escherichia coli β-galactosidase α-gene fragment with expression under the lac promoter. Groups of mice were orally immunized three times with the bacteria and systemic CD8+ T cell cytokine responses were evaluated. Results High level of the GFP model antigen was expressed by the recombinant Salmonella vaccine vector. Systemic GFP-specific CD8+ T cell cytokine (IFN-γ and IL-4 immune responses were detected after mice were orally vaccinated with the bacteria. It was shown that 226 net IFN-γ and 132 net IL-4 GFP-specific SFUs/10e6 splenocytes were formed in an ELISPOT assay. The level of IFN-γ produced by GFP peptide-stimulated cells was 65.2-fold above background (p Conclusion These results suggested that a high expressing recombinant Salmonella vaccine given orally to mice would elicit antigen-specific CD8+ T cell responses in the spleen. Salmonella bacteria may, therefore, be used as potential mucosal vaccine vectors.

  6. Gadolinium chloride elicits apoptosis in human osteosarcoma U-2 OS cells through extrinsic signaling, intrinsic pathway and endoplasmic reticulum stress. (United States)

    Tsai, Yuh-Feng; Huang, Ching-Wen; Chiang, Jo-Hua; Tsai, Fuu-Jen; Hsu, Yuan-Man; Lu, Chi-Cheng; Hsiao, Chen-Yu; Yang, Jai-Sing


    Gadolinium (Gd) compounds are important as magnetic resonance imaging (MRI) contrast agents, and are potential anticancer agents. However, no report has shown the effect of gadolinium chloride (GdCl3) on osteosarcoma in vitro. The present study investigated the apoptotic mechanism of GdCl3 on human osteosarcoma U-2 OS cells. Our results indicated that GdCl3 significantly reduced cell viability of U-2 OS cells in a concentration-dependent manner. GdCl3 led to apoptotic cell shrinkage and DNA fragmentation in U-2 OS cells as revealed by morphologic changes and TUNEL staining. Colorimetric assay analyses also showed that activities of caspase-3, caspase-8, caspase-9 and caspase-4 occurred in GdCl3-treated U-2 OS cells. Pretreatment of cells with pan-caspase inhibitor (Z-VAD-FMK) and specific inhibitors of caspase-3/-8/-9 significantly reduced cell death caused by GdCl3. The increase of cytoplasmic Ca2+ level, ROS production and the decrease of mitochondria membrane potential (ΔΨm) were observed by flow cytometric analysis in U-2 OS cells after GdCl3 exposure. Western blot analyses demonstrated that the levels of Fas, FasL, cytochrome c, Apaf-1, GADD153 and GRP78 were upregulated in GdCl3-treated U-2 OS cells. In conclusion, death receptor, mitochondria-dependent and endoplasmic reticulum (ER) stress pathways contribute to GdCl3-induced apoptosis in U-2 OS cells. GdCl3 might have potential to be used in treatment of osteosarcoma patients.

  7. Dengue virus infection elicits highly polarized CX3CR1+ cytotoxic CD4+ T cells associated with protective immunity. (United States)

    Weiskopf, Daniela; Bangs, Derek J; Sidney, John; Kolla, Ravi V; De Silva, Aruna D; de Silva, Aravinda M; Crotty, Shane; Peters, Bjoern; Sette, Alessandro


    Dengue virus (DENV) is a rapidly spreading pathogen with unusual pathogenesis, and correlates of protection from severe dengue disease and vaccine efficacy have not yet been established. Although DENV-specific CD8(+) T-cell responses have been extensively studied, the breadth and specificity of CD4(+) T-cell responses remains to be defined. Here we define HLA-restricted CD4(+) T-cell epitopes resulting from natural infection with dengue virus in a hyperepidemic setting. Ex vivo flow-cytometric analysis of DENV-specific CD4(+) T cells revealed that the virus-specific cells were highly polarized, with a strong bias toward a CX3CR1(+) Eomesodermin(+) perforin(+) granzyme B(+) CD45RA(+) CD4 CTL phenotype. Importantly, these cells correlated with a protective HLA DR allele, and we demonstrate that these cells have direct ex vivo DENV-specific cytolytic activity. We speculate that cytotoxic dengue-specific CD4(+) T cells may play a role in the control of dengue infection in vivo, and this immune correlate may be a key target for dengue virus vaccine development.

  8. Distinct effects of EGFR ligands on human mammary epithelial cell differentiation.

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    Chandrani Mukhopadhyay

    Full Text Available Based on gene expression patterns, breast cancers can be divided into subtypes that closely resemble various developmental stages of normal mammary epithelial cells (MECs. Thus, understanding molecular mechanisms of MEC development is expected to provide critical insights into initiation and progression of breast cancer. Epidermal growth factor receptor (EGFR and its ligands play essential roles in normal and pathological mammary gland. Signals through EGFR is required for normal mammary gland development. Ligands for EGFR are over-expressed in a significant proportion of breast cancers, and elevated expression of EGFR is associated with poorer clinical outcome. In the present study, we examined the effect of signals through EGFR on MEC differentiation using the human telomerase reverse transcriptase (hTERT-immortalized human stem/progenitor MECs which express cytokeratin 5 but lack cytokeratin 19 (K5(+K19(- hMECs. As reported previously, these cells can be induced to differentiate into luminal and myoepithelial cells under appropriate culture conditions. K5(+K19(- hMECs acquired distinct cell fates in response to EGFR ligands epidermal growth factor (EGF, amphiregulin (AREG and transforming growth factor alpha (TGFα in differentiation-promoting MEGM medium. Specifically, presence of EGF during in vitro differentiation supported development into both luminal and myoepithelial lineages, whereas cells differentiated only towards luminal lineage when EGF was replaced with AREG. In contrast, substitution with TGFα led to differentiation only into myoepithelial lineage. Chemical inhibition of the MEK-Erk pathway, but not the phosphatidylinositol 3-kinase (PI3K-AKT pathway, interfered with K5(+K19(- hMEC differentiation. The present data validate the utility of the K5(+K19(- hMEC cells for modeling key features of human MEC differentiation. This system should be useful in studying molecular/biochemical mechanisms of human MEC differentiation.

  9. Three distinct subsets of thymic epithelial cells in rats and mice defined by novel antibodies.

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    Yasushi Sawanobori

    Full Text Available AIM: Thymic epithelial cells (TECs are thought to play an essential role in T cell development and have been detected mainly in mice using lectin binding and antibodies to keratins. Our aim in the present study was to create a precise map of rat TECs using antibodies to putative markers and novel monoclonal antibodies (i.e., ED 18/19/21 and anti-CD205 antibodies and compare it with a map from mouse counterparts and that of rat thymic dendritic cells. RESULTS: Rat TECs were subdivided on the basis of phenotype into three subsets; ED18+ED19+/-keratin 5 (K5+K8+CD205+ class II MHC (MHCII+ cortical TECs (cTECs, ED18+ED21-K5-K8+Ulex europaeus lectin 1 (UEA-1+CD205- medullary TECs (mTEC1s, and ED18+ED21+K5+K8dullUEA-1-CD205- medullary TECs (mTEC2s. Thymic nurse cells were defined in cytosmears as an ED18+ED19+/-K5+K8+ subset of cTECs. mTEC1s preferentially expressed MHCII, claudin-3, claudin-4, and autoimmune regulator (AIRE. Use of ED18 and ED21 antibodies revealed three subsets of TECs in mice as well. We also detected two distinct TEC-free areas in the subcapsular cortex and in the medulla. Rat dendritic cells in the cortex were MHCII+CD103+ but negative for TEC markers, including CD205. Those in the medulla were MHCII+CD103+ and CD205+ cells were found only in the TEC-free area. CONCLUSION: Both rats and mice have three TEC subsets with similar phenotypes that can be identified using known markers and new monoclonal antibodies. These findings will facilitate further analysis of TEC subsets and DCs and help to define their roles in thymic selection and in pathological states such as autoimmune disorders.

  10. Distinctive left-sided distribution of adrenergic-derived cells in the adult mouse heart.

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    Kingsley Osuala

    Full Text Available Adrenaline and noradrenaline are produced within the heart from neuronal and non-neuronal sources. These adrenergic hormones have profound effects on cardiovascular development and function, yet relatively little information is available about the specific tissue distribution of adrenergic cells within the adult heart. The purpose of the present study was to define the anatomical localization of cells derived from an adrenergic lineage within the adult heart. To accomplish this, we performed genetic fate-mapping experiments where mice with the cre-recombinase (Cre gene inserted into the phenylethanolamine-n-methyltransferase (Pnmt locus were cross-mated with homozygous Rosa26 reporter (R26R mice. Because Pnmt serves as a marker gene for adrenergic cells, offspring from these matings express the β-galactosidase (βGAL reporter gene in cells of an adrenergic lineage. βGAL expression was found throughout the adult mouse heart, but was predominantly (89% located in the left atrium (LA and ventricle (LV (p<0.001 compared to RA and RV, where many of these cells appeared to have cardiomyocyte-like morphological and structural characteristics. The staining pattern in the LA was diffuse, but the LV free wall displayed intermittent non-random staining that extended from the apex to the base of the heart, including heavy staining of the anterior papillary muscle along its perimeter. Three-dimensional computer-aided reconstruction of XGAL+ staining revealed distribution throughout the LA and LV, with specific finger-like projections apparent near the mid and apical regions of the LV free wall. These data indicate that adrenergic-derived cells display distinctive left-sided distribution patterns in the adult mouse heart.

  11. Rhein Elicits In Vitro Cytotoxicity in Primary Human Liver HL-7702 Cells by Inducing Apoptosis through Mitochondria-Mediated Pathway

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    Guy-Armel Bounda


    Full Text Available Objective. To study rhein-induced apoptosis signaling pathway and to investigate its molecular mechanisms in primary human hepatic cells. Results. Cell viability of HL-7702 cells treated with rhein showed significant decrease in dose-dependent manner. Following rhein treatment (25 μM, 50 μM, and 100 μM for 12 h, the detection of apoptotic cells was significantly analyzed by flow cytometry and nuclear morphological changes by Hoechst 33258, respectively. Fatty degeneration studies showed upregulation level of the relevant hepatic markers (P < 0.01. Caspase activities expressed significant upregulation of caspase-3, caspase-9, and caspase-8. Moreover, apoptotic cells by rhein were significantly inhibited by Z-LEHD-FMK and Z-DEVD-FMK, caspase-9 inhibitor, and caspase-3 inhibitor, respectively. Overproduction of reactive oxygen species, lipid peroxidation, and loss of mitochondrial membrane potential were detected by fluorometry. Additionally, NAC, a ROS scavenger, significantly attenuated rhein-induced oxidative damage in HL-7702 cells. Furthermore, real-time qPCR results showed significant upregulation of p53, PUMA, Apaf-1, and Casp-9 and Casp-3 mRNA, with no significant changes of Fas and Cytochrome-c. Immunoblotting revealed significant Cytochrome-c release from mitochondria into cytosol and no change in Fas expression. Conclusion. Taken together, these observations suggested that rhein could induce apoptosis in HL-7702 cells via mitochondria-mediated signal pathway with involvement of oxidative stress mechanism.

  12. Live Attenuated Influenza Vaccine Strains Elicit a Greater Innate Immune Response than Antigenically-Matched Seasonal Influenza Viruses during Infection of Human Nasal Epithelial Cell Cultures (United States)

    Fischer, William A.; Brighton, Missy; Jaspers, Ilona


    Influenza viruses are global pathogens that infect approximately 10–20% of the world’s population each year. Vaccines, including the live attenuated influenza vaccine (LAIV), are the best defense against influenza infections. The LAIV is a novel vaccine that actively replicates in the human nasal epithelium and elicits both mucosal and systemic protective immune responses. The differences in replication and innate immune responses following infection of human nasal epithelium with influenza seasonal wild type (WT) and LAIV viruses remain unknown. Using a model of primary differentiated human nasal epithelial cell (hNECs) cultures, we compared influenza WT and antigenically-matched cold adapted (CA) LAIV virus replication and the subsequent innate immune response including host cellular pattern recognition protein expression, host innate immune gene expression, secreted pro-inflammatory cytokine production, and intracellular viral RNA levels. Growth curves comparing virus replication between WT and LAIV strains revealed significantly less infectious virus production during LAIV compared with WT infection. Despite this disparity in infectious virus production the LAIV strains elicited a more robust innate immune response with increased expression of RIG-I, TLR-3, IFNβ, STAT-1, IRF-7, MxA, and IP-10. There were no differences in cytotoxicity between hNEC cultures infected with WT and LAIV strains as measured by basolateral levels of LDH. Elevated levels of intracellular viral RNA during LAIV as compared with WT virus infection of hNEC cultures at 33°C may explain the augmented innate immune response via the up-regulation of pattern recognition receptors and down-stream type I IFN expression. Taken together our results suggest that the decreased replication of LAIV strains in human nasal epithelial cells is associated with a robust innate immune response that differs from infection with seasonal influenza viruses, limits LAIV shedding and plays a role in the

  13. Live attenuated influenza vaccine strains elicit a greater innate immune response than antigenically-matched seasonal influenza viruses during infection of human nasal epithelial cell cultures. (United States)

    Fischer, William A; Chason, Kelly D; Brighton, Missy; Jaspers, Ilona


    Influenza viruses are global pathogens that infect approximately 10-20% of the world's population each year. Vaccines, including the live attenuated influenza vaccine (LAIV), are the best defense against influenza infections. The LAIV is a novel vaccine that actively replicates in the human nasal epithelium and elicits both mucosal and systemic protective immune responses. The differences in replication and innate immune responses following infection of human nasal epithelium with influenza seasonal wild type (WT) and LAIV viruses remain unknown. Using a model of primary differentiated human nasal epithelial cell (hNECs) cultures, we compared influenza WT and antigenically-matched cold adapted (CA) LAIV virus replication and the subsequent innate immune response including host cellular pattern recognition protein expression, host innate immune gene expression, secreted pro-inflammatory cytokine production, and intracellular viral RNA levels. Growth curves comparing virus replication between WT and LAIV strains revealed significantly less infectious virus production during LAIV compared with WT infection. Despite this disparity in infectious virus production the LAIV strains elicited a more robust innate immune response with increased expression of RIG-I, TLR-3, IFNβ, STAT-1, IRF-7, MxA, and IP-10. There were no differences in cytotoxicity between hNEC cultures infected with WT and LAIV strains as measured by basolateral levels of LDH. Elevated levels of intracellular viral RNA during LAIV as compared with WT virus infection of hNEC cultures at 33°C may explain the augmented innate immune response via the up-regulation of pattern recognition receptors and down-stream type I IFN expression. Taken together our results suggest that the decreased replication of LAIV strains in human nasal epithelial cells is associated with a robust innate immune response that differs from infection with seasonal influenza viruses, limits LAIV shedding and plays a role in the silent

  14. The nucleocapsid protein of Rift Valley fever virus is a potent human CD8+ T cell antigen and elicits memory responses.

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    Weidong Xu

    Full Text Available There is no licensed human vaccine currently available for Rift Valley Fever Virus (RVFV, a Category A high priority pathogen and a serious zoonotic threat. While neutralizing antibodies targeting the viral glycoproteins are protective, they appear late in the course of infection, and may not be induced in time to prevent a natural or bioterrorism-induced outbreak. Here we examined the immunogenicity of RVFV nucleocapsid (N protein as a CD8(+ T cell antigen with the potential for inducing rapid protection after vaccination. HLA-A*0201 (A2-restricted epitopic determinants were identified with N-specific CD8(+ T cells from eight healthy donors that were primed with dendritic cells transduced to express N, and subsequently expanded in vitro by weekly re-stimulations with monocytes pulsed with 59 15mer overlapping peptides (OLPs across N. Two immunodominant epitopes, VT9 (VLSEWLPVT, N(121-129 and IL9 (ILDAHSLYL, N165-173, were defined. VT9- and IL9-specific CD8(+ T cells identified by tetramer staining were cytotoxic and polyfunctional, characteristics deemed important for viral control in vivo. These peptides induced specific CD8(+ T cell responses in A2-transgenic mice, and more importantly, potent N-specific CD8(+ T cell reactivities, including VT9- and IL9-specific ones, were mounted by mice after a booster vaccination with the live attenuated RVF MP-12. Our data suggest that the RVFV N protein is a potent human T cell immunogen capable of eliciting broad, immunodominant CD8(+ T cell responses that are potentially protective. Understanding the immune responses to the nucleocapsid is central to the design of an effective RVFV vaccine irrespective of whether this viral protein is effective as a stand-alone immunogen or only in combination with other RVFV antigens.

  15. Elicitation of T-cell responses by structural and non-structural proteins of coxsackievirus B4. (United States)

    Bengs, Suvi; Marttila, Jane; Susi, Petri; Ilonen, Jorma


    Coxsackievirus B4 (CV-B4) belongs to the genus Enterovirus within the family Picornaviridae. To investigate target proteins recognized by T-cells in human enterovirus B infections, virus-encoded structural [VP0 (VP4 and VP2), VP1, VP3] and non-structural (2A, 2B, 2C, 3C and 3D) proteins were expressed and purified in Escherichia coli. Peripheral blood of 19 healthy adult donors was used to create enterovirus-specific T-cell lines by repeated stimulation with CV-B4 cell lysate antigen. T-cell lines responded in individual patterns, and responses to all purified proteins were observed. The most often recognized enteroviral protein was VP0, which is the fusion between the most conserved structural proteins, VP4 and VP2. T-cell responses to VP0 were detected in 15 of the 19 (79 %) donor lines. Non-structural 2C protein was recognized in 11 of the 19 (58 %) lines, and 11 of the 19 (58 %) lines also had a response to 3D protein. Furthermore, responses to other non-structural proteins (2A, 2B and 3C) were also detected. T-cell responses did not correlate clearly to the individual HLA-DR-DQ phenotype or the history of past coxsackie B virus infections of the donors.

  16. BMP and Hedgehog Regulate Distinct AGM Hematopoietic Stem Cells Ex Vivo

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    Mihaela Crisan


    Full Text Available Hematopoietic stem cells (HSC, the self-renewing cells of the adult blood differentiation hierarchy, are generated during embryonic stages. The first HSCs are produced in the aorta-gonad-mesonephros (AGM region of the embryo through endothelial to a hematopoietic transition. BMP4 and Hedgehog affect their production and expansion, but it is unknown whether they act to affect the same HSCs. In this study using the BRE GFP reporter mouse strain that identifies BMP/Smad-activated cells, we find that the AGM harbors two types of adult-repopulating HSCs upon explant culture: One type is BMP-activated and the other is a non-BMP-activated HSC type that is indirectly controlled by Hedgehog signaling through the VEGF pathway. Transcriptomic analyses demonstrate that the two HSC types express distinct but overlapping genetic programs. These results revealing the bifurcation in HSC types at early embryonic stages in the AGM explant model suggest that their development is dependent upon the signaling molecules in the microenvironment.

  17. Distinct and synergistic feedforward inhibition of pyramidal cells by basket and bistratified interneurons

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    Michele eFerrante


    Full Text Available Feedforward inhibition (FFI enables pyramidal cells in area CA1 of the hippocampus (CA1PCs to remain easily excitable while faithfully representing a broad range of excitatory inputs without quickly saturating. Despite the cortical ubiquity of FFI, its specific function is not completely understood. FFI in CA1PCs is mediated by two physiologically and morphologically distinct GABAergic interneurons: fast-spiking, perisomatic-targeting basket cells and regular-spiking, dendritic-targeting bistratified cells. These two FFI pathways might create layer-specific computational sub-domains within the same CA1PC, but teasing apart their specific contributions remains experimentally challenging. We implemented a biophysically realistic model of CA1PCs using 40 digitally reconstructed morphologies and constraining synaptic numbers, locations, amplitude, and kinetics with available experimental data. First, we validated the model by reproducing the known combined basket and bistratified FFI of CA1PCs at the population level. We then analyzed how the two interneuron types independently affected the CA1PC spike probability and timing as a function of inhibitory strength. Separate FFI by basket and bistratified respectively modulated CA1PC threshold and gain. Concomitant FFI by both interneuron types synergistically extended the dynamic range of CA1PCs by buffering their spiking response to excitatory stimulation. These results suggest testable hypotheses on the precise effects of GABAergic diversity on cortical computation.

  18. Stromal derived factor-1 exerts differential regulation on distinct cortical cell populations in vitro

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    Zeef Leo


    Full Text Available Abstract Background Stromal derived factor (SDF-1, an alpha chemokine, is a widely known chemoattractant in the immune system. A growing body of evidence now suggests multiple regulatory roles for SDF-1 in the developing nervous system. Results To investigate the role of SDF-1 signaling in the growth and differentiation of cortical cells, we performed numerous in vitro experiments, including gene chip and quantitative RT-PCR analysis. Using SDF-1 medium and AMD3100, a receptor antagonist, we demonstrate that the chemokine signaling regulates key events during early cortical development. First, SDF-1 signaling maintains cortical progenitors in proliferation, possibly through a mechanism involving connexin 43 mediated intercellular coupling. Second, SDF-1 signaling upregulates the differentiation of cortical GABAergic neurons, independent of sonic signaling pathway. Third, SDF-1 enables the elongation and branching of axons of cortical glutamatergic neurons. Finally, cortical cultures derived from CXCR4-/- mutants show a close parallel to AMD3100 treatment with reduced cell proliferation and differentiation of GABAergic neurons. Conclusion Results from this study show that SDF-1 regulates distinct cortical cell populations in vitro.

  19. Pluripotent and Multipotent Stem Cells Display Distinct Hypoxic miRNA Expression Profiles (United States)

    Agrawal, Rahul; Dale, Tina P.; Al-Zubaidi, Mohammed A.; Benny Malgulwar, Prit; Forsyth, Nicholas R.; Kulshreshtha, Ritu


    MicroRNAs are reported to have a crucial role in the regulation of self-renewal and differentiation of stem cells. Hypoxia has been identified as a key biophysical element of the stem cell culture milieu however, the link between hypoxia and miRNA expression in stem cells remains poorly understood. We therefore explored miRNA expression in hypoxic human embryonic and mesenchymal stem cells (hESCs and hMSCs). A total of 50 and 76 miRNAs were differentially regulated by hypoxia (2% O2) in hESCs and hMSCs, respectively, with a negligible overlap of only three miRNAs. We found coordinate regulation of precursor and mature miRNAs under hypoxia suggesting their regulation mainly at transcriptional level. Hypoxia response elements were located upstream of 97% of upregulated hypoxia regulated miRNAs (HRMs) suggesting hypoxia-inducible-factor (HIF) driven transcription. HIF binding to the candidate cis-elements of specific miRNAs under hypoxia was confirmed by Chromatin immunoprecipitation coupled with qPCR. Role analysis of a subset of upregulated HRMs identified linkage to reported inhibition of differentiation while a downregulated subset of HRMs had a putative role in the promotion of differentiation. MiRNA-target prediction correlation with published hypoxic hESC and hMSC gene expression profiles revealed HRM target genes enriched in the cytokine:cytokine receptor, HIF signalling and pathways in cancer. Overall, our study reveals, novel and distinct hypoxia-driven miRNA signatures in hESCs and hMSCs with the potential for application in optimised culture and differentiation models for both therapeutic application and improved understanding of stem cell biology. PMID:27783707

  20. Adipose stromal cells contain phenotypically distinct adipogenic progenitors derived from neural crest.

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    Yoshihiro Sowa

    Full Text Available Recent studies have shown that adipose-derived stromal/stem cells (ASCs contain phenotypically and functionally heterogeneous subpopulations of cells, but their developmental origin and their relative differentiation potential remain elusive. In the present study, we aimed at investigating how and to what extent the neural crest contributes to ASCs using Cre-loxP-mediated fate mapping. ASCs harvested from subcutaneous fat depots of either adult P0-Cre/or Wnt1-Cre/Floxed-reporter mice contained a few neural crest-derived ASCs (NCDASCs. This subpopulation of cells was successfully expanded in vitro under standard culture conditions and their growth rate was comparable to non-neural crest derivatives. Although NCDASCs were positive for several mesenchymal stem cell markers as non-neural crest derivatives, they exhibited a unique bipolar or multipolar morphology with higher expression of markers for both neural crest progenitors (p75NTR, Nestin, and Sox2 and preadipocytes (CD24, CD34, S100, Pref-1, GATA2, and C/EBP-delta. NCDASCs were able to differentiate into adipocytes with high efficiency but their osteogenic and chondrogenic potential was markedly attenuated, indicating their commitment to adipogenesis. In vivo, a very small proportion of adipocytes were originated from the neural crest. In addition, p75NTR-positive neural crest-derived cells were identified along the vessels within the subcutaneous adipose tissue, but they were negative for mural and endothelial markers. These results demonstrate that ASCs contain neural crest-derived adipocyte-restricted progenitors whose phenotype is distinct from that of non-neural crest derivatives.

  1. HPV-E7 Delivered by Engineered Exosomes Elicits a Protective CD8+ T Cell-Mediated Immune Response

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    Paola Di Bonito


    Full Text Available We developed an innovative strategy to induce a cytotoxic T cell (CTL immune response against protein antigens of choice. It relies on the production of exosomes, i.e., nanovesicles spontaneously released by all cell types. We engineered the upload of huge amounts of protein antigens upon fusion with an anchoring protein (i.e., HIV-1 Nefmut, which is an inactive protein incorporating in exosomes at high levels also when fused with foreign proteins. We compared the immunogenicity of engineered exosomes uploading human papillomavirus (HPV-E7 with that of lentiviral virus-like particles (VLPs incorporating equivalent amounts of the same antigen. These exosomes, whose limiting membrane was decorated with VSV-G, i.e., an envelope protein inducing pH-dependent endosomal fusion, proved to be as immunogenic as the cognate VLPs. It is noteworthy that the immunogenicity of the engineered exosomes remained unaltered in the absence of VSV-G. Most important, we provide evidence that the inoculation in mouse of exosomes uploading HPV-E7 induces production of anti-HPV E7 CTLs, blocks the growth of syngeneic tumor cells inoculated after immunization, and controls the development of tumor cells inoculated before the exosome challenge. These results represent the proof-of-concept about both feasibility and efficacy of the Nefmut-based exosome platform for the induction of CD8+ T cell immunity.

  2. Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions.

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    Sandra Schwarz

    Full Text Available Bacteria that live in the environment have evolved pathways specialized to defend against eukaryotic organisms or other bacteria. In this manuscript, we systematically examined the role of the five type VI secretion systems (T6SSs of Burkholderia thailandensis (B. thai in eukaryotic and bacterial cell interactions. Consistent with phylogenetic analyses comparing the distribution of the B. thai T6SSs with well-characterized bacterial and eukaryotic cell-targeting T6SSs, we found that T6SS-5 plays a critical role in the virulence of the organism in a murine melioidosis model, while a strain lacking the other four T6SSs remained as virulent as the wild-type. The function of T6SS-5 appeared to be specialized to the host and not related to an in vivo growth defect, as ΔT6SS-5 was fully virulent in mice lacking MyD88. Next we probed the role of the five systems in interbacterial interactions. From a group of 31 diverse bacteria, we identified several organisms that competed less effectively against wild-type B. thai than a strain lacking T6SS-1 function. Inactivation of T6SS-1 renders B. thai greatly more susceptible to cell contact-induced stasis by Pseudomonas putida, Pseudomonas fluorescens and Serratia proteamaculans-leaving it 100- to 1000-fold less fit than the wild-type in competition experiments with these organisms. Flow cell biofilm assays showed that T6S-dependent interbacterial interactions are likely relevant in the environment. B. thai cells lacking T6SS-1 were rapidly displaced in mixed biofilms with P. putida, whereas wild-type cells persisted and overran the competitor. Our data show that T6SSs within a single organism can have distinct functions in eukaryotic versus bacterial cell interactions. These systems are likely to be a decisive factor in the survival of bacterial cells of one species in intimate association with those of another, such as in polymicrobial communities present both in the environment and in many infections.

  3. CD34(-) cells at the apex of the human hematopoietic stem cell hierarchy have distinctive cellular and molecular signatures. (United States)

    Anjos-Afonso, Fernando; Currie, Erin; Palmer, Hector G; Foster, Katie E; Taussig, David C; Bonnet, Dominique


    In addition to well-characterized CD34(+) hematopoietic stem and progenitor cells (HSPCs), the human hematopoietic stem cell (HSC) hierarchy contains a rare CD34(-) population with severe combined immunodeficiency-repopulating capacity. However, little is known about the molecular characteristics of these CD34(-) cells or their relationship to the CD34(+) populations. Here, we show that the self-renewing Lin(-)CD34(-)CD38(-)CD93(hi) population contains cells that not only function as HSCs, but can also be placed above the CD34(+) populations in the hematopoietic hierarchy. These cells have an active Notch pathway, in which signaling through Delta4 is crucial for maintenance of the primitive state, and combined signals from Jagged1 and TGF-β are important in controlling its quiescence. They are also refractory to proliferative signals and show a repressed canonical Wnt pathway, in part regulated by Notch. Overall, therefore, CD34(-) cells represent an immature and quiescent human HSC population maintained through a distinctive network of cellular signaling interactions.

  4. Lsr2 of Mycobacterium leprae and Its Synthetic Peptides Elicit Restitution of T Cell Responses in Erythema Nodosum Leprosum and Reversal Reactions in Patients with Lepromatous Leprosy (United States)

    Saini, Chaman; Prasad, H. K.; Rani, Rajni; Murtaza, A.; Misra, Namita; Shanker Narayan, N. P.


    The Lsr2 protein of Mycobacterium leprae and its synthetic peptides have been shown to elicit lymphoproliferation and gamma interferon (IFN-γ) release by peripheral blood mononuclear cells (PBMCs) of patients with lepromatous leprosy (M. Chaduvula, A. Murtaza, N. Misra, N. P. Narayan, V. Ramesh, H. K. Prasad, R. Rani, R. K. Chinnadurai, I. Nath, Infect. Immun. 80:742–752, 2012). PBMCs from 16 patients with lepromatous leprosy who were undergoing erythema nodosum leprosum (ENL) (type 2) and 5 patients with reversal reactions (RR) (type 1) were stimulated with M. leprae, recombinant Lsr2, and six end-to-end synthetic peptides (A through F) spanning the Lsr2 sequence. During the reaction all patients with ENL showed lymphoproliferation (stimulation index, >2) in response to peptides A and F, with other peptides eliciting responses in 75 to 88% of the subjects. In PBMC cultures, both lymphoproliferation and IFN-γ release for peptide E were significantly higher than for peptides B and C and recombinant Lsr2 (P < 0.05, Wilcoxon signed-rank test). Five patients with RR also showed enhanced lymphoproliferative responses and IFN-γ release in response to Lsr2, M. leprae, and peptide E. Six months postreaction, 14 patients with ENL continued to exhibit responses to Lsr2 and its peptides, with the highest responses being elicited by peptide E. However, 5 subjects showed no lymphoproliferation and had reduced IFN-γ release in response to Lsr2 peptides (P < 0.001, Kruskal-Wallis test) but responded to recombinant Lsr2. Six patients with ENL had HLA-A*68.01, which the STFPEITHI program showed to have high peptide-binding scores of 20 to 21 for peptides E, B, and C. Eleven patients had HLA-DRB1*1501 and HLA-DRB1*1502, which had high binding scores for peptides C and E. Thus, Lsr2 and its peptides are recognized in leprosy reactions during and well after the subsidence of clinical signs. PMID:23446220

  5. Enhanced accumulation of phytosterols and phenolic compounds in cyclodextrin-elicited cell suspension culture of Daucus carota. (United States)

    Miras-Moreno, Begoña; Almagro, Lorena; Pedreño, M A; Sabater-Jara, Ana Belén


    In this work, suspension-cultured cells of Daucus carota were used to evaluate the effect of β-cyclodextrins on the production of isoprenoid and phenolic compounds. The results showed that the phytosterols and phenolic compounds were accumulated in the extracellular medium (15100μgL(-1) and 477.46μgL(-1), respectively) in the presence of cyclodextrins. Unlike the phytosterol and phenolic compound content, β-carotene (1138.03μgL(-1)), lutein (25949.54μgL(-1)) and α-tocopherol (8063.82μgL(-1)) chlorophyll a (1625.13μgL(-1)) and b (9.958 (9958.33μgL(-1)) were mainly accumulated inside the cells. Therefore, cyclodextrins were able to induce the cytosolic mevalonate pathway, increasing the biosynthesis of phytosterols and phenolic compounds, and accumulate them outside the cells. However, in the absence of these cyclic oligosaccharidic elicitors, carrot cells mainly accumulated carotenoids through the methylerythritol 4-phosphate pathway. Therefore, the use of cyclodextrins would allow the extracellular accumulation of both phytosterols and phenolic compounds by diverting the carbon flux towards the cytosolic mevalonate/phenylpropanoid pathway.

  6. The K1 Serotype Capsular Polysaccharide of Porphyromonas gingivalis Elicits Chemokine Production from Murine Macrophages That Facilitates Cell Migration ▿


    d'Empaire, Gabriela; Baer, Michael T.; Gibson, Frank C.


    Porphyromonas gingivalis is the principal organism associated with aggressive forms of generalized periodontal disease. Previous reports have suggested that encapsulated P. gingivalis strains are more virulent than unencapsulated strains; however, the contribution of capsular polysaccharide (CPS) to the virulence of this organism is poorly understood. Since periodontal disease presents with a complex inflammatory cell lesion comprised of neutrophils and monocytes, we cultured murine peritonea...

  7. Vaccination with experimental feline immunodeficiency virus vaccines, based on autologous infected cells, elicits enhancement of homologous challenge infection.

    NARCIS (Netherlands)

    J.A. Karlas (Jos); C.H.J. Siebelink (Kees); M.A. van Peer (Maartje); W. Huisman (Willem); A.M. Cuisinier; G.F. Rimmelzwaan (Guus); A.D.M.E. Osterhaus (Albert)


    textabstractCats were vaccinated with fixed autologous feline immunodeficiency virus (FIV)-infected cells in order to present viral proteins to the immune system of individual cats in an MHC-matched fashion. Upon vaccination, a humoral response against Gag was induced. Furthermore, virus-neutralizin

  8. Vaccination with experimental feline immunodeficiency virus vaccines, based on autologous infected cells, elicits enhancement of homologous challenge infection

    NARCIS (Netherlands)

    J.A. Karlas (Jos); C.H.J. Siebelink (Kees); M.A. Peer; W. Huisman (Willem); A.M. Cuisinier; G.F. Rimmelzwaan (Guus); A.D.M.E. Osterhaus (Ab)


    textabstractCats were vaccinated with fixed autologous feline immunodeficiency virus (FIV)-infected cells in order to present viral proteins to the immune system of individual cats in an MHC-matched fashion. Upon vaccination, a humoral response against Gag was induced. Furthermore,

  9. lin-28 controls the succession of cell fate choices via two distinct activities.

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    Bhaskar Vadla

    Full Text Available lin-28 is a conserved regulator of cell fate succession in animals. In Caenorhabditis elegans, it is a component of the heterochronic gene pathway that governs larval developmental timing, while its vertebrate homologs promote pluripotency and control differentiation in diverse tissues. The RNA binding protein encoded by lin-28 can directly inhibit let-7 microRNA processing by a novel mechanism that is conserved from worms to humans. We found that C. elegans LIN-28 protein can interact with four distinct let-7 family pre-microRNAs, but in vivo inhibits the premature accumulation of only let-7. Surprisingly, however, lin-28 does not require let-7 or its relatives for its characteristic promotion of second larval stage cell fates. In other words, we find that the premature accumulation of mature let-7 does not account for lin-28's precocious phenotype. To explain let-7's role in lin-28 activity, we provide evidence that lin-28 acts in two steps: first, the let-7-independent positive regulation of hbl-1 through its 3'UTR to control L2 stage-specific cell fates; and second, a let-7-dependent step that controls subsequent fates via repression of lin-41. Our evidence also indicates that let-7 functions one stage earlier in C. elegans development than previously thought. Importantly, lin-28's two-step mechanism resembles that of the heterochronic gene lin-14, and the overlap of their activities suggests a clockwork mechanism for developmental timing. Furthermore, this model explains the previous observation that mammalian Lin28 has two genetically separable activities. Thus, lin-28's two-step mechanism may be an essential feature of its evolutionarily conserved role in cell fate succession.

  10. Mapping the distinctive populations of lymphatic endothelial cells in different zones of human lymph nodes.

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    Saem Mul Park

    Full Text Available The lymphatic sinuses in human lymph nodes (LNs are crucial to LN function yet their structure remains poorly defined. Much of our current knowledge of lymphatic sinuses derives from rodent models, however human LNs differ substantially in their sinus structure, most notably due to the presence of trabeculae and trabecular lymphatic sinuses that rodent LNs lack. Lymphatic sinuses are bounded and traversed by lymphatic endothelial cells (LECs. A better understanding of LECs in human LNs is likely to improve our understanding of the regulation of cell trafficking within LNs, now an important therapeutic target, as well as disease processes that involve lymphatic sinuses. We therefore sought to map all the LECs within human LNs using multicolor immunofluorescence microscopy to visualize the distribution of a range of putative markers. PROX1 was the only marker that uniquely identified the LECs lining and traversing all the sinuses in human LNs. In contrast, LYVE1 and STAB2 were only expressed by LECs in the paracortical and medullary sinuses in the vast majority of LNs studied, whilst the subcapsular and trabecular sinuses lacked these molecules. These data highlight the existence of at least two distinctive populations of LECs within human LNs. Of the other LEC markers, we confirmed VEGFR3 was not specific for LECs, and CD144 and CD31 stained both LECs and blood vascular endothelial cells (BECs; in contrast, CD59 and CD105 stained BECs but not LECs. We also showed that antigen-presenting cells (APCs in the sinuses could be clearly distinguished from LECs by their expression of CD169, and their lack of expression of PROX1 and STAB2, or endothelial markers such as CD144. However, both LECs and sinus APCs were stained with DCN46, an antibody commonly used to detect CD209.

  11. Distinct and site-specific phosphorylation of the retinoblastoma protein at serine 612 in differentiated cells.

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    Takayuki Hattori

    Full Text Available The retinoblastoma susceptibility protein (pRB is a phosphoprotein that regulates cell cycle progression at the G1/S transition. In quiescent and early G1 cells, pRB predominantly exists in the active hypophosphorylated form. The cyclin/cyclin-dependent protein kinase complexes phosphorylate pRB at the late G1 phase to inactivate pRB. This event leads to the dissociation and activation of E2F family transcriptional factors. At least 12 serine/threonine residues in pRB are phosphorylated in vivo. Although there have been many reports describing bulk phosphorylation of pRB, detail research describing the function of each phosphorylation site remains unknown. Besides its G1/S inhibitory function, pRB is involved in differentiation, prevention of cell death and control of tissue fate. To uncover the function of phosphorylation of pRB in various cellular conditions, we have been investigating phosphorylation of each serine/threonine residue in pRB with site-specific phospho-serine/threonine antibodies. Here we demonstrate that pRB is specifically phosphorylated at Ser612 in differentiated cells in a known kinase-independent manner. We also found that pRB phosphorylated at Ser612 still associates with E2F-1 and tightly binds to nuclear structures including chromatin. Moreover, expression of the Ser612Ala mutant pRB failed to induce differentiation. The findings suggest that phosphorylation of Ser612 provides a distinct function that differs from the function of phosphorylation of other serine/threonine residues in pRB.

  12. Belief Elicitation in Experiments

    DEFF Research Database (Denmark)

    Blanco, Mariana; Engelmann, Dirk; Koch, Alexander

    Belief elicitation in economics experiments usually relies on paying subjects according to the accuracy of stated beliefs in addition to payments for other decisions. Such incentives, however, allow risk-averse subjects to hedge with their stated beliefs against adverse outcomes of other decisions...... in the experiment. This raises two questions: (i) can we trust the existing belief elicitation results, (ii) can we avoid potential hedging confounds? Our results instill confidence regarding both issues. We propose an experimental design that eliminates hedging opportunities, and use this to test for the empirical...

  13. Holistic systems biology approaches to molecular mechanisms of human helper T cell differentiation to functionally distinct subsets. (United States)

    Chen, Z; Lönnberg, T; Lahesmaa, R


    Current knowledge of helper T cell differentiation largely relies on data generated from mouse studies. To develop therapeutical strategies combating human diseases, understanding the molecular mechanisms how human naïve T cells differentiate to functionally distinct T helper (Th) subsets as well as studies on human differentiated Th cell subsets is particularly valuable. Systems biology approaches provide a holistic view of the processes of T helper differentiation, enable discovery of new factors and pathways involved and generation of new hypotheses to be tested to improve our understanding of human Th cell differentiation and immune-mediated diseases. Here, we summarize studies where high-throughput systems biology approaches have been exploited to human primary T cells. These studies reveal new factors and signalling pathways influencing T cell differentiation towards distinct subsets, important for immune regulation. Such information provides new insights into T cell biology and into targeting immune system for therapeutic interventions.

  14. Dnmt3a and Dnmt3b have overlapping and distinct functions in hematopoietic stem cells. (United States)

    Challen, Grant A; Sun, Deqiang; Mayle, Allison; Jeong, Mira; Luo, Min; Rodriguez, Benjamin; Mallaney, Cates; Celik, Hamza; Yang, Liubin; Xia, Zheng; Cullen, Sean; Berg, Jonathan; Zheng, Yayun; Darlington, Gretchen J; Li, Wei; Goodell, Margaret A


    Epigenetic regulation of hematopoietic stem cells (HSCs) ensures lifelong production of blood and bone marrow. Recently, we reported that loss of de novo DNA methyltransferase Dnmt3a results in HSC expansion and impaired differentiation. Here, we report conditional inactivation of Dnmt3b in HSCs either alone or combined with Dnmt3a deletion. Combined loss of Dnmt3a and Dnmt3b was synergistic, resulting in enhanced HSC self-renewal and a more severe block in differentiation than in Dnmt3a-null cells, whereas loss of Dnmt3b resulted in a mild phenotype. Although the predominant Dnmt3b isoform in adult HSCs is catalytically inactive, its residual activity in Dnmt3a-null HSCs can drive some differentiation and generates paradoxical hypermethylation of CpG islands. Dnmt3a/Dnmt3b-null HSCs displayed activated β-catenin signaling, partly accounting for the differentiation block. These data demonstrate distinct roles for Dnmt3b in HSC differentiation and provide insights into complementary de novo methylation patterns governing regulation of HSC fate decisions.

  15. Two distinct secretory vesicle-priming steps in adrenal chromaffin cells. (United States)

    Liu, Yuanyuan; Schirra, Claudia; Edelmann, Ludwig; Matti, Ulf; Rhee, JeongSeop; Hof, Detlef; Bruns, Dieter; Brose, Nils; Rieger, Heiko; Stevens, David R; Rettig, Jens


    Priming of large dense-core vesicles (LDCVs) is a Ca(2+)-dependent step by which LDCVs enter a release-ready pool, involving the formation of the soluble N-ethyl-maleimide sensitive fusion protein attachment protein (SNAP) receptor complex consisting of syntaxin, SNAP-25, and synaptobrevin. Using mice lacking both isoforms of the calcium-dependent activator protein for secretion (CAPS), we show that LDCV priming in adrenal chromaffin cells entails two distinct steps. CAPS is required for priming of the readily releasable LDCV pool and sustained secretion in the continued presence of high Ca(2+) concentrations. Either CAPS1 or CAPS2 can rescue secretion in cells lacking both CAPS isoforms. Furthermore, the deficit in the readily releasable LDCV pool resulting from CAPS deletion is reversed by a constitutively open form of syntaxin but not by Munc13-1, a priming protein that facilitates the conversion of syntaxin to the open conformation. Our data indicate that CAPS functions downstream of Munc13s but also interacts functionally with Munc13s in the LDCV-priming process.

  16. Crude venom from nematocysts of Pelagia noctiluca (Cnidaria: Scyphozoa) elicits a sodium conductance in the plasma membrane of mammalian cells (United States)

    Morabito, Rossana; Costa, Roberta; Rizzo, Valentina; Remigante, Alessia; Nofziger, Charity; La Spada, Giuseppa; Marino, Angela; Paulmichl, Markus; Dossena, Silvia


    Cnidarians may negatively impact human activities and public health but concomitantly their venom represents a rich source of bioactive substances. Pelagia noctiluca is the most venomous and abundant jellyfish of the Mediterranean Sea and possesses a venom with hemolytic and cytolytic activity for which the mechanism is largely unknown. Here we show that exposure of mammalian cells to crude venom from the nematocysts of P. noctiluca profoundly alters the ion conductance of the plasma membrane, therefore affecting homeostatic functions such as the regulation and maintenance of cellular volume. Venom-treated cells exhibited a large, inwardly rectifying current mainly due to permeation of Na+ and Cl‑, sensitive to amiloride and completely abrogated following harsh thermal treatment of crude venom extract. Curiously, the plasma membrane conductance of Ca2+ and K+ was not affected. Current-inducing activity was also observed following delivery of venom to the cytosolic side of the plasma membrane, consistent with a pore-forming mechanism. Venom-induced NaCl influx followed by water and consequent cell swelling most likely underlie the hemolytic and cytolytic activity of P. noctiluca venom. The present study underscores unique properties of P. noctiluca venom and provides essential information for a possible use of its active compounds and treatment of envenomation.

  17. Distinct molecular signature of human skin Langerhans cells denotes critical differences in cutaneous dendritic cell immune regulation. (United States)

    Polak, Marta E; Thirdborough, Stephen M; Ung, Chuin Y; Elliott, Tim; Healy, Eugene; Freeman, Tom C; Ardern-Jones, Michael R


    Langerhans cells (LCs) are professional antigen-presenting cells (APCs) residing in the epidermis. Despite their high potential to activate T lymphocytes, current understanding of human LC biology is limited. Genome-wide comparison of the transcriptional profiles of human skin migratory CD1a+ LCs and CD11c+ dermal dendritic cells (DDCs) demonstrated significant differences between these "dendritic cell (DC)" types, including preferential expression of 625 genes (Pmolecular networks activated after stimulation with tumor necrosis factor-α (TNF-α) confirmed the unique molecular signature of LCs. Although LCs conformed to the phenotype of professional APC, inflammatory signaling activated primarily genes associated with cellular metabolism and mitochondrial activation (e.g., CYB561 and MRPS35), cell membrane re-organization, and antigen acquisition and degradation (CAV1 and PSMD14; P<0.05-P<0.0001). Conversely, TNF-α induced classical activation in DDCs with early downregulation of surface receptors (mannose receptor-1 (MRC1) and C-type lectins), and subsequent upregulation of cytokines, chemokines (IL1a, IL1b, and CCL18), and matrix metalloproteinases (MMP1, MMP3, and MMP9; P<0.05-P<0.0001). Functional interference of caveolin abrogated LCs superior ability to cross-present antigens to CD8+ T lymphocytes, highlighting the importance of these networks to biological function. Taken together, these observations support the idea of distinct biological roles of cutaneous DC types.

  18. The naturally processed CD95L elicits a c-yes/calcium/PI3K-driven cell migration pathway.

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    Sébastien Tauzin


    Full Text Available Patients affected by chronic inflammatory disorders display high amounts of soluble CD95L. This homotrimeric ligand arises from the cleavage by metalloproteases of its membrane-bound counterpart, a strong apoptotic inducer. In contrast, the naturally processed CD95L is viewed as an apoptotic antagonist competing with its membrane counterpart for binding to CD95. Recent reports pinpointed that activation of CD95 may attract myeloid and tumoral cells, which display resistance to the CD95-mediated apoptotic signal. However, all these studies were performed using chimeric CD95Ls (oligomerized forms, which behave as the membrane-bound ligand and not as the naturally processed CD95L. Herein, we examine the biological effects of the metalloprotease-cleaved CD95L on CD95-sensitive activated T-lymphocytes. We demonstrate that cleaved CD95L (cl-CD95L, found increased in sera of systemic lupus erythematosus (SLE patients as compared to that of healthy individuals, promotes the formation of migrating pseudopods at the leading edge of which the death receptor CD95 is capped (confocal microscopy. Using different migration assays (wound healing/Boyden Chamber/endothelial transmigration, we uncover that cl-CD95L promotes cell migration through a c-yes/Ca²⁺/PI3K-driven signaling pathway, which relies on the formation of a CD95-containing complex designated the MISC for Motility-Inducing Signaling Complex. These findings revisit the role of the metalloprotease-cleaved CD95L and emphasize that the increase in cl-CD95L observed in patients affected by chronic inflammatory disorders may fuel the local or systemic tissue damage by promoting tissue-filtration of immune cells.

  19. The Immunodominance Change and Protection of CD4+ T-Cell Responses Elicited by an Envelope Protein Domain III-Based Tetravalent Dengue Vaccine in Mice.

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    Hsin-Wei Chen

    Full Text Available Dengue is the leading cause of mosquito-borne viral infections and no vaccine is available now. Envelope protein domain III (ED3 is the major target for the binding of dengue virus neutralizing antibodies; however, the ED3-specifc T-cell response is less well understood. To investigate the T-cell responses to four serotypes of dengue virus (DENV-1 to 4, we immunized mice using either a tetravalent ED3-based DNA or protein vaccine, or combined both as a DNA prime-protein boost strategy (prime-boost. A significant serotype-dependent IFN-γ or IL-4 response was observed in mice immunized with either the DNA or protein vaccine. The IFN-γ response was dominant to DENV-1 to 3, whereas the IL-4 response was dominant to DENV-4. Although the similar IgG titers for the four serotypes were observed in mice immunized with the tetravalent vaccines, the neutralizing antibody titers varied and followed the order of 2 = 3>1>4. Interestingly, the lower IFN-γ response to DENV-4 is attributable to the immunodominance change between two CD4+ T-cell epitopes; one T-cell epitope located at E349-363 of DENV-1 to 3 was more immunogenic than the DENV-4 epitope E313-327. Despite DENV-4 specific IFN-γ responses were suppressed by immunodominance change, either DENV-4-specific IFN-γ or neutralizing antibody responses were still recalled after DENV-4 challenge and contributed to virus clearance. Immunization with the prime-boost elicited both IFN-γ and neutralizing antibody responses and provided better protection than either DNA or protein immunization. Our findings shed light on how ED3-based tetravalent dengue vaccines sharpen host CD4 T-cell responses and contribute to protection against dengue virus.

  20. Hydrogen Peroxide Is Involved in Salicylic Acid-Elicited Rosmarinic Acid Production in Salvia miltiorrhiza Cell Cultures

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    Wenfang Hao


    Full Text Available Salicylic acid (SA is an elicitor to induce the biosynthesis of secondary metabolites in plant cells. Hydrogen peroxide (H2O2 plays an important role as a key signaling molecule in response to various stimuli and is involved in the accumulation of secondary metabolites. However, the relationship between them is unclear and their synergetic functions on accumulation of secondary metabolites are unknown. In this paper, the roles of SA and H2O2 in rosmarinic acid (RA production in Salvia miltiorrhiza cell cultures were investigated. The results showed that SA significantly enhanced H2O2 production, phenylalanine ammonia-lyase (PAL activity, and RA accumulation. Exogenous H2O2 could also promote PAL activity and enhance RA production. If H2O2 production was inhibited by NADPH oxidase inhibitor (IMD or scavenged by quencher (DMTU, RA accumulation would be blocked. These results indicated that H2O2 is secondary messenger for signal transduction, which can be induced by SA, significantly and promotes RA accumulation.

  1. An African horse sickness virus serotype 4 recombinant canarypox virus vaccine elicits specific cell-mediated immune responses in horses. (United States)

    El Garch, H; Crafford, J E; Amouyal, P; Durand, P Y; Edlund Toulemonde, C; Lemaitre, L; Cozette, V; Guthrie, A; Minke, J M


    A recombinant canarypox virus vectored vaccine co-expressing synthetic genes encoding outer capsid proteins, VP2 and VP5, of African horse sickness virus (AHSV) serotype 4 (ALVAC(®)-AHSV4) has been demonstrated to fully protect horses against homologous challenge with virulent field virus. Guthrie et al. (2009) detected weak and variable titres of neutralizing antibody (ranging from horses received two vaccinations twenty-eight days apart and three horses remained unvaccinated. The detection of VP2/VP5 specific IFN-γ responses was assessed by enzyme linked immune spot (ELISpot) assay and clearly demonstrated that all ALVAC(®)-AHSV4 vaccinated horses developed significant IFN-γ production compared to unvaccinated horses. More detailed immune responses obtained by flow cytometry demonstrated that ALVAC(®)-AHSV4 vaccinations induced immune cells, mainly CD8(+) T cells, able to recognize multiple T-epitopes through all VP2 and only the N-terminus sequence of VP5. Neither VP2 nor VP5 specific IFN-γ responses were detected in unvaccinated horses. Overall, our data demonstrated that an experimental recombinant canarypox based vaccine induced significant CMI specific for both VP2 and VP5 proteins of AHSV4.

  2. B7h-expressing dendritic cells and plasma B cells mediate distinct outcomes of ICOS costimulation in T cell-dependent antibody responses

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    Larimore Kevin


    Full Text Available Abstract Background The ICOS-B7h costimulatory receptor-ligand pair is required for germinal center formation, the production of isotype-switched antibodies, and antibody affinity maturation in response to T cell-dependent antigens. However, the potentially distinct roles of regulated B7h expression on B cells and dendritic cells in T cell-dependent antibody responses have not been defined. Results We generated transgenic mice with lineage-restricted B7h expression to assess the cell-type specific roles of B7h expression on B cells and dendritic cells in regulating T cell-dependent antibody responses. Our results show that endogenous B7h expression is reduced on B cells after activation in vitro and is also reduced in vivo on antibody-secreting plasma B cells in comparison to both naïve and germinal center B cells from which they are derived. Increasing the level of B7h expression on activated and plasma B cells in B-B7hTg mice led to an increase in the number of antibody-secreting plasma cells generated after immunization and a corresponding increase in the concentration of antigen-specific high affinity serum IgG antibodies of all isotypes, without affecting the number of responding germinal center B cells. In contrast, ICOS costimulation mediated by dendritic cells in DC-B7hTg mice contributed to germinal center formation and selectively increased IgG2a production without affecting the overall magnitude of antibody responses. Conclusions Using transgenic mice with lineage-restricted B7h expression, we have revealed distinct roles of ICOS costimulation mediated by dendritic cells and B cells in the regulation of T cell-dependent antibody responses.

  3. Oral Delivery of a Novel Recombinant Streptococcus mitis Vector Elicits Robust Vaccine Antigen-Specific Oral Mucosal and Systemic Antibody Responses and T Cell Tolerance.

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    Emily Xie

    Full Text Available The pioneer human oral commensal bacterium Streptococcus mitis has unique biologic features that make it an attractive mucosal vaccine or therapeutic delivery vector. S. mitis is safe as a natural persistent colonizer of the mouth, throat and nasopharynx and the oral commensal bacterium is capable of inducing mucosal antibody responses. A recombinant S. mitis (rS. mitis that stably expresses HIV envelope protein was generated and tested in the germ-free mouse model to evaluate the potential usefulness of this vector as a mucosal vaccine against HIV. Oral vaccination led to the efficient and persistent bacterial colonization of the mouth and the induction of both salivary and systemic antibody responses. Interestingly, persistently colonized animals developed antigen-specific systemic T cell tolerance. Based on these findings we propose the use of rS. mitis vaccine vector for the induction of mucosal antibodies that will prevent the penetration of the mucosa by pathogens such as HIV. Moreover, the first demonstration of rS. mitis having the ability to elicit T cell tolerance suggest the potential use of rS. mitis as an immunotherapeutic vector to treat inflammatory, allergic and autoimmune diseases.

  4. Treg/Th17 polarization by distinct subsets of breast cancer cells is dictated by the interaction with mesenchymal stem cells


    Patel, Shyam A.; Dave, Meneka A.; Bliss, Sarah A.; Giec-Ujda, Agata B.; Bryan, Margarette; Pliner, Lillian F.; Rameshwar, Pranela


    Breast cancer (BC) cells (BCCs) exist within a hierarchy beginning with cancer stem cells (CSCs). Unsorted BCCs interact with mesenchymal stem cells (MSCs) to induce regulatory T cells (Tregs). This study investigated how distinct BCC subsets interacted with MSCs to polarize T-cell response, Tregs versus T helper 17 (Th17). This study tested BC initiating cells (CSCs) and the relatively more mature early and late BC progenitors. CSCs interacted with the highest avidity to MSCs. This interacti...

  5. iNKT Cells Are Responsible for the Apoptotic Reduction of Basophils That Mediate Th2 Immune Responses Elicited by Papain in Mice Following γPGA Stimulation. (United States)

    Park, Hyun Jung; Lee, Sung Won; Park, Se-Ho; Hong, Seokmann


    Recent studies have demonstrated that Bacillus subtilis-derived poly-gamma glutamic acid (γPGA) treatment suppresses the development of allergic diseases such as atopic dermatitis (AD). Although basophils, an innate immune cell, are known to play critical roles in allergic immune responses and repeated long-term administration of γPGA results in decreased splenic basophils in an AD murine model, the underlying mechanisms by which γPGA regulates basophil frequency remain unclear. To investigate how γPGA modulates basophils, we employed basophil-mediated Th2 induction in vivo model elicited by the allergen papain protease. Repeated injection of γPGA reduced the abundance of basophils and their production of IL4 in mice, consistent with our previous study using NC/Nga AD model mice. The depletion of basophils by a single injection of γPGA was dependent on the TLR4/DC/IL12 axis. CD1d-dependent Vα14 TCR invariant natural killer T (iNKT) cells are known to regulate a variety of immune responses, such as allergy. Because iNKT cell activation is highly sensitive to IL12 produced by DCs, we evaluated whether the effect of γPGA on basophils is mediated by iNKT cell activation. We found that in vivo γPGA treatment did not induce the reduction of basophils in iNKT cell-deficient CD1d KO mice, suggesting the critical role of iNKT cells in γPGA-mediated basophil depletion at the early time points. Furthermore, increased apoptotic basophil reduction triggered by iNKT cells upon γPGA stimulation was mainly attributed to Th1 cytokines such as IFNγ and TNFα, consequently resulting in inhibition of papain-induced Th2 differentiation via diminishing basophil-derived IL4. Taken together, our results clearly demonstrate that γPGA-induced iNKT cell polarization toward the Th1 phenotype induces apoptotic basophil depletion, leading to the suppression of Th2 immune responses. Thus, elucidation of the crosstalk between innate immune cells will contribute to the design and

  6. DNA methylation in small cell lung cancer defines distinct disease subtypes and correlates with high expression of EZH2 (United States)

    Poirier, John T.; Gardner, Eric E.; Connis, Nick; Moreira, Andre L.; de Stanchina, Elisa; Hann, Christine L.; Rudin, Charles M.


    Small cell lung cancer (SCLC) is an aggressive malignancy characterized by early metastasis, rapid development of resistance to chemotherapy, and genetic instability. This study profiles DNA methylation in SCLC, patient-derived xenografts (PDXs) and cell lines at single nucleotide resolution. DNA methylation patterns of primary samples are distinct from those of cell lines, while PDXs maintain a pattern closely consistent with primary samples. Clustering of DNA methylation and gene expression of primary SCLC revealed distinct disease subtypes among histologically indistinguishable primary patient samples with similar genetic alterations. SCLC is notable for dense clustering of high-level methylation in discrete promoter CpG islands, in a pattern clearly distinct from other lung cancers and strongly correlated with high expression of the E2F target and histone methyltransferase gene EZH2. Pharmacologic inhibition of EZH2 in a SCLC PDX markedly inhibited tumor growth. PMID:25746006

  7. DNA methylation in small cell lung cancer defines distinct disease subtypes and correlates with high expression of EZH2. (United States)

    Poirier, J T; Gardner, E E; Connis, N; Moreira, A L; de Stanchina, E; Hann, C L; Rudin, C M


    Small cell lung cancer (SCLC) is an aggressive malignancy characterized by early metastasis, rapid development of resistance to chemotherapy and genetic instability. This study profiles DNA methylation in SCLC, patient-derived xenografts (PDX) and cell lines at single-nucleotide resolution. DNA methylation patterns of primary samples are distinct from those of cell lines, whereas PDX maintain a pattern closely consistent with primary samples. Clustering of DNA methylation and gene expression of primary SCLC revealed distinct disease subtypes among histologically indistinguishable primary patient samples with similar genetic alterations. SCLC is notable for dense clustering of high-level methylation in discrete promoter CpG islands, in a pattern clearly distinct from other lung cancers and strongly correlated with high expression of the E2F target and histone methyltransferase gene EZH2. Pharmacologic inhibition of EZH2 in a SCLC PDX markedly inhibited tumor growth.

  8. Distinct patterns of somatic genome alterations in lung adenocarcinomas and squamous cell carcinomas. (United States)

    Campbell, Joshua D; Alexandrov, Anton; Kim, Jaegil; Wala, Jeremiah; Berger, Alice H; Pedamallu, Chandra Sekhar; Shukla, Sachet A; Guo, Guangwu; Brooks, Angela N; Murray, Bradley A; Imielinski, Marcin; Hu, Xin; Ling, Shiyun; Akbani, Rehan; Rosenberg, Mara; Cibulskis, Carrie; Ramachandran, Aruna; Collisson, Eric A; Kwiatkowski, David J; Lawrence, Michael S; Weinstein, John N; Verhaak, Roel G W; Wu, Catherine J; Hammerman, Peter S; Cherniack, Andrew D; Getz, Gad; Artyomov, Maxim N; Schreiber, Robert; Govindan, Ramaswamy; Meyerson, Matthew


    To compare lung adenocarcinoma (ADC) and lung squamous cell carcinoma (SqCC) and to identify new drivers of lung carcinogenesis, we examined the exome sequences and copy number profiles of 660 lung ADC and 484 lung SqCC tumor-normal pairs. Recurrent alterations in lung SqCCs were more similar to those of other squamous carcinomas than to alterations in lung ADCs. New significantly mutated genes included PPP3CA, DOT1L, and FTSJD1 in lung ADC, RASA1 in lung SqCC, and KLF5, EP300, and CREBBP in both tumor types. New amplification peaks encompassed MIR21 in lung ADC, MIR205 in lung SqCC, and MAPK1 in both. Lung ADCs lacking receptor tyrosine kinase-Ras-Raf pathway alterations had mutations in SOS1, VAV1, RASA1, and ARHGAP35. Regarding neoantigens, 47% of the lung ADC and 53% of the lung SqCC tumors had at least five predicted neoepitopes. Although targeted therapies for lung ADC and SqCC are largely distinct, immunotherapies may aid in treatment for both subtypes.

  9. Fungal spore concentrations in two haematopoietic stem cell transplantation (HSCT) units containing distinct air control systems. (United States)

    Brun, C P; Miron, D; Silla, L M R; Pasqualotto, A C


    Invasive fungal diseases have emerged as important causes of morbidity and mortality in haematological patients. In this study air samples were collected in two haematopoietic stem cell transplantation (HSCT) units, in which distinct air-control systems were in place. In hospital 1 no high-efficiency particulate air (HEPA) filter was available whereas in hospital 2 HSCT rooms were equipped with HEPA filters, with positive air pressure in relation to the corridor. A total of 117 samples from rooms, toilets and corridors were obtained during December 2009 to January 2011, using a six-stage Andersen sampler. In both hospitals, the concentration of potentially pathogenic fungi in the air was reduced in patients' rooms compared to corridors (P < 0·0001). Despite the presence of a HEPA filter in hospital 2, rooms in both hospitals showed similar concentrations of potentially pathogenic fungi (P = 0·714). These findings may be explained by the implementation of additional protective measures in hospital 1, emphasizing the importance of such measures in protected environments.

  10. Distinct axo-somato-dendritic distributions of three potassium channels in CA1 hippocampal pyramidal cells. (United States)

    Kirizs, Tekla; Kerti-Szigeti, Katalin; Lorincz, Andrea; Nusser, Zoltan


    Potassium channels comprise the most diverse family of ion channels and play critical roles in a large variety of physiological and pathological processes. In addition to their molecular diversity, variations in their distributions and densities on the axo-somato-dendritic surface of neurons are key parameters in determining their functional impact. Despite extensive electrophysiological and anatomical investigations, the exact location and densities of most K(+) channels in small subcellular compartments are still unknown. Here we aimed at providing a quantitative surface map of two delayed-rectifier (Kv1.1 and Kv2.1) and one G-protein-gated inwardly rectifying (Kir3.2) K(+) channel subunits on hippocampal CA1 pyramidal cells (PCs). Freeze-fracture replica immunogold labelling was employed to determine the relative densities of these K(+) channel subunits in 18 axo-somato-dendritic compartments. Significant densities of the Kv1.1 subunit were detected on axon initial segments (AISs) and axon terminals, with an approximately eight-fold lower density in the latter compartment. The Kv2.1 subunit was found in somatic, proximal dendritic and AIS plasma membranes at approximately the same densities. This subunit has a non-uniform plasma membrane distribution; Kv2.1 clusters are frequently adjacent to, but never overlap with, GABAergic synapses. A quasi-linear increase in the Kir3.2 subunit density along the dendrites of PCs was detected, showing no significant difference between apical dendritic shafts, oblique dendrites or dendritic spines at the same distance from the soma. Our results demonstrate that each subunit has a unique cell-surface distribution pattern, and predict their differential involvement in synaptic integration and output generation at distinct subcellular compartments.

  11. Distinct cell clusters touching islet cells induce islet cell replication in association with over-expression of Regenerating Gene (REG protein in fulminant type 1 diabetes.

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    Kaoru Aida

    Full Text Available BACKGROUND: Pancreatic islet endocrine cell-supporting architectures, including islet encapsulating basement membranes (BMs, extracellular matrix (ECM, and possible cell clusters, are unclear. PROCEDURES: The architectures around islet cell clusters, including BMs, ECM, and pancreatic acinar-like cell clusters, were studied in the non-diabetic state and in the inflamed milieu of fulminant type 1 diabetes in humans. RESULT: Immunohistochemical and electron microscopy analyses demonstrated that human islet cell clusters and acinar-like cell clusters adhere directly to each other with desmosomal structures and coated-pit-like structures between the two cell clusters. The two cell-clusters are encapsulated by a continuous capsule composed of common BMs/ECM. The acinar-like cell clusters have vesicles containing regenerating (REG Iα protein. The vesicles containing REG Iα protein are directly secreted to islet cells. In the inflamed milieu of fulminant type 1 diabetes, the acinar-like cell clusters over-expressed REG Iα protein. Islet endocrine cells, including beta-cells and non-beta cells, which were packed with the acinar-like cell clusters, show self-replication with a markedly increased number of Ki67-positive cells. CONCLUSION: The acinar-like cell clusters touching islet endocrine cells are distinct, because the cell clusters are packed with pancreatic islet clusters and surrounded by common BMs/ECM. Furthermore, the acinar-like cell clusters express REG Iα protein and secrete directly to neighboring islet endocrine cells in the non-diabetic state, and the cell clusters over-express REG Iα in the inflamed milieu of fulminant type 1 diabetes with marked self-replication of islet cells.

  12. Issues in Requirements Elicitation (United States)


    systems approach: characterized by Checkland’s Soft Systems Methodology (SSM), which is discussed briefly in Appendix A.6; emphasizes the subjectivity of...methodology for requirements elicitation. A.6 Notes on SSM Both the definition of methodology and the philosophy behind soft systems methodology (SSM...1986. [Checkland 89a] Checkland, Peter. Soft Systems Methodology . Rational Analysis for a Problematic World. New York: John Wiley & Sons, 71-100

  13. Elicitation of Unstated Needs (United States)


    in these interviews. The interviews absolutely do not touch on the solution space. This is a challenging approach to interviewing customers/users...hate it when I have to constantly adjust my radio volume! I find classical music quite relaxing. 95 Requirements Elicitation (RE) Training...2014 Carnegie Mellon University Driving in Your Car Exercise Theme of communicating or listening to music in the car without distraction! 96

  14. Application of an integrated LC-UV-MS-NMR platform to the identification of secondary metabolites from cell cultures: benzophenanthridine alkaloids from elicited Eschscholzia californica (california poppy) cell cultures(). (United States)

    Gathungu, Rose M; Oldham, John T; Bird, Susan S; Lee-Parsons, Carolyn W T; Vouros, Paul; Kautz, Roger


    Plant cell and tissue cultures are a scalable and controllable alternative to whole plants for obtaining natural products of medical relevance. Cultures can be optimized for high yields of desired metabolites using rapid profiling assays such as HPLC. We describe an approach to establishing a rapid assay for profiling cell culture expression systems using a novel microscale LC-UV-MS-NMR platform, designed to acquire both MS and NMR each at their optimal sensitivity, by using nanosplitter MS from 4 mm analytical HPLC columns, and offline microdroplet NMR. The approach is demonstrated in the analysis of elicited Eschscholzia californica cell cultures induced with purified yeast extract to produce benzophenanthridine alkaloids. Preliminary HPLC-UV provides an overview of the changes in the production of alkaloids with time after elicitation. At the time point corresponding to the production of the most alkaloids, the integrated LC-MS-microcoil NMR platform is used for structural identification of extracted alkaloids. Eight benzophenanthridine alkaloids were identified at the sub-microgram level. This paper demonstrates the utility of the nanosplitter LC-MS/microdroplet NMR platform when establishing cell culture expression systems.

  15. The Anti-Warburg Effect Elicited by the cAMP-PGC1α Pathway Drives Differentiation of Glioblastoma Cells into Astrocytes

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    Fan Xing


    Full Text Available Glioblastoma multiforme (GBM is among the most aggressive of human cancers. Although differentiation therapy has been proposed as a potential approach to treat GBM, the mechanisms of induced differentiation remain poorly defined. Here, we established an induced differentiation model of GBM using cAMP activators that specifically directed GBM differentiation into astroglia. Transcriptomic and proteomic analyses revealed that oxidative phosphorylation and mitochondrial biogenesis are involved in induced differentiation of GBM. Dibutyryl cyclic AMP (dbcAMP reverses the Warburg effect, as evidenced by increased oxygen consumption and reduced lactate production. Mitochondrial biogenesis induced by activation of the CREB-PGC1α pathway triggers metabolic shift and differentiation. Blocking mitochondrial biogenesis using mdivi1 or by silencing PGC1α abrogates differentiation; conversely, overexpression of PGC1α elicits differentiation. In GBM xenograft models and patient-derived GBM samples, cAMP activators also induce tumor growth inhibition and differentiation. Our data show that mitochondrial biogenesis and metabolic switch to oxidative phosphorylation drive the differentiation of tumor cells.

  16. The Anti-Warburg Effect Elicited by the cAMP-PGC1α Pathway Drives Differentiation of Glioblastoma Cells into Astrocytes. (United States)

    Xing, Fan; Luan, Yizhao; Cai, Jing; Wu, Sihan; Mai, Jialuo; Gu, Jiayu; Zhang, Haipeng; Li, Kai; Lin, Yuan; Xiao, Xiao; Liang, Jiankai; Li, Yuan; Chen, Wenli; Tan, Yaqian; Sheng, Longxiang; Lu, Bingzheng; Lu, Wanjun; Gao, Mingshi; Qiu, Pengxin; Su, Xingwen; Yin, Wei; Hu, Jun; Chen, Zhongping; Sai, Ke; Wang, Jing; Chen, Furong; Chen, Yinsheng; Zhu, Shida; Liu, Dongbing; Cheng, Shiyuan; Xie, Zhi; Zhu, Wenbo; Yan, Guangmei


    Glioblastoma multiforme (GBM) is among the most aggressive of human cancers. Although differentiation therapy has been proposed as a potential approach to treat GBM, the mechanisms of induced differentiation remain poorly defined. Here, we established an induced differentiation model of GBM using cAMP activators that specifically directed GBM differentiation into astroglia. Transcriptomic and proteomic analyses revealed that oxidative phosphorylation and mitochondrial biogenesis are involved in induced differentiation of GBM. Dibutyryl cyclic AMP (dbcAMP) reverses the Warburg effect, as evidenced by increased oxygen consumption and reduced lactate production. Mitochondrial biogenesis induced by activation of the CREB-PGC1α pathway triggers metabolic shift and differentiation. Blocking mitochondrial biogenesis using mdivi1 or by silencing PGC1α abrogates differentiation; conversely, overexpression of PGC1α elicits differentiation. In GBM xenograft models and patient-derived GBM samples, cAMP activators also induce tumor growth inhibition and differentiation. Our data show that mitochondrial biogenesis and metabolic switch to oxidative phosphorylation drive the differentiation of tumor cells.

  17. Enhanced biosynthesis of withanolides by elicitation and precursor feeding in cell suspension culture of Withania somnifera (L.) Dunal in shake-flask culture and bioreactor. (United States)

    Sivanandhan, Ganeshan; Selvaraj, Natesan; Ganapathi, Andy; Manickavasagam, Markandan


    The present study investigated the biosynthesis of major and minor withanolides of Withania somnifera in cell suspension culture using shake-flask culture and bioreactor by exploiting elicitation and precursor feeding strategies. Elicitors like cadmium chloride, aluminium chloride and chitosan, precursors such as cholesterol, mevalonic acid and squalene were examined. Maximum total withanolides detected [withanolide A (7606.75 mg), withanolide B (4826.05 mg), withaferin A (3732.81 mg), withanone (6538.65 mg), 12 deoxy withanstramonolide (3176.63 mg), withanoside IV (2623.21 mg) and withanoside V (2861.18 mg)] were achieved in the combined treatment of chitosan (100 mg/l) and squalene (6 mM) along with 1 mg/l picloram, 0.5 mg/l KN, 200 mg/l L-glutamine and 5% sucrose in culture at 4 h and 48 h exposure times respectively on 28th day of culture in bioreactor. We obtained higher concentrations of total withanolides in shake-flask culture (2.13-fold) as well as bioreactor (1.66-fold) when compared to control treatments. This optimized protocol can be utilized for commercial level production of withanolides from suspension culture using industrial bioreactors in a short culture period.

  18. Distinct population of highly malignant cells in a head and neck squamous cell carcinoma cell line established by xenograft model

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    Jan Chia-Ing


    Full Text Available Abstract The progression and metastasis of solid tumors, including head and neck squamous cell carcinoma (HNSCC, have been related to the behavior of a small subpopulation of cancer stem cells. Here, we have established a highly malignant HNSCC cell line, SASVO3, from primary tumors using three sequential rounds of xenotransplantation. SASVO3 possesses enhanced tumorigenic ability both in vitro and in vivo. Moreover, SASVO3 exhibits properties of cancer stem cells, including that increased the abilities of sphere-forming, the number of side population cells, the potential of transplanted tumor growth and elevated expression of the stem cell marker Bmi1. Injection of SASVO3 into the tail vein of nude mice resulted in lung metastases. These results are consistent with the postulate that the malignant and/or metastasis potential of HNSCC cells may reside in a stem-like subpopulation.

  19. Distinct Redox Profiles of Selected Human Prostate Carcinoma Cell Lines: Implications for Rational Design of Redox Therapy

    Energy Technology Data Exchange (ETDEWEB)

    Chaiswing, Luksana [Department of Pathology and Laboratory Medicine, School of Medicine and Public Health, University of Wisconsin, 1111 Highland Ave., WIMR 7168, Madison, WI 53705 (United States); Zhong, Weixiong; Oberley, Terry D., E-mail: [Department of Pathology and Laboratory Medicine, School of Medicine and Public Health, University of Wisconsin, 1111 Highland Ave., WIMR 7168, Madison, WI 53705 (United States); Pathology and Laboratory Medicine Service, William S. Middleton Memorial Veterans Hospital, Rm A-35, 2500 Overlook Terrace, Madison, WI 53705 (United States)


    The effects of several cancer chemotherapeutic drugs and radiation are mediated, at least in part, by oxidative stress. To better understand this process, we analyzed certain biochemical properties affecting reduction-oxidation (redox) balance in normal prostate epithelial cells and several prostate cancer cell lines. Highly aggressive androgen-independent prostate cancer PC3 cells demonstrated significantly higher levels of total antioxidant capacity (AC) and intra- and extracellular glutathione (GSH)/glutathione disulfide (GSSG) ratios when compared with normal prostate epithelial PrEC cells. WPE1-NB26 cells, a prostate cancer cell line derived from immortalized RWPE1 human prostate epithelial cells, demonstrated significantly higher levels of total AC and intra- and extracellular GSH/GSSG ratios, but lower levels of intracellular reactive oxygen/nitrogen species and lipid peroxidation compared with RWPE1 cells. LNCaP-C4-2 cells, a more aggressive prostate cancer derived from less aggressive androgen-responsive LNCaP cells, exhibited higher levels of AC and extracellular GSH/GSSG ratio when compared to LNCaP cells. Specific cell types showed distinct cytotoxic responses to redox-modulating compounds. WPE1-NB26 cells were more sensitive to phenethyl isothiocyanate and tumor necrosis factor (TNF) than RWPE1 cells, while PC3 cells were more sensitive to TNF than PrEC cells. These results are consistent with the hypothesis that cancer cell redox state may modulate responses to redox-modulating therapeutic regimens.

  20. Distinct Redox Profiles of Selected Human Prostate Carcinoma Cell Lines: Implications for Rational Design of Redox Therapy

    Directory of Open Access Journals (Sweden)

    Luksana Chaiswing


    Full Text Available The effects of several cancer chemotherapeutic drugs and radiation are mediated, at least in part, by oxidative stress. To better understand this process, we analyzed certain biochemical properties affecting reduction-oxidation (redox balance in normal prostate epithelial cells and several prostate cancer cell lines. Highly aggressive androgen-independent prostate cancer PC3 cells demonstrated significantly higher levels of total antioxidant capacity (AC and intra- and extracellular glutathione (GSH/glutathione disulfide (GSSG ratios when compared with normal prostate epithelial PrEC cells. WPE1-NB26 cells, a prostate cancer cell line derived from immortalized RWPE1 human prostate epithelial cells, demonstrated significantly higher levels of total AC and intra- and extracellular GSH/GSSG ratios, but lower levels of intracellular reactive oxygen/nitrogen species and lipid peroxidation compared with RWPE1 cells. LNCaP-C4-2 cells, a more aggressive prostate cancer derived from less aggressive androgen-responsive LNCaP cells, exhibited higher levels of AC and extracellular GSH/GSSG ratio when compared to LNCaP cells. Specific cell types showed distinct cytotoxic responses to redox-modulating compounds. WPE1-NB26 cells were more sensitive to phenethyl isothiocyanate and tumor necrosis factor (TNF than RWPE1 cells, while PC3 cells were more sensitive to TNF than PrEC cells. These results are consistent with the hypothesis that cancer cell redox state may modulate responses to redox-modulating therapeutic regimens.


    NARCIS (Netherlands)



    Several human renal cell carcinomas with X;autosome translocations have been reported in recent years. The t(X; I)(p11.2;q21) appears to be a specific primary anomaly, suggesting that tumors with this translocation form a distinct subgroup of RCC. Here we report two new cases, one with a t(X;10)(p11

  2. HIV-1 Subtype C Mosaic Gag Expressed by BCG and MVA Elicits Persistent Effector T Cell Responses in a Prime-Boost Regimen in Mice.

    Directory of Open Access Journals (Sweden)

    Tsungai Ivai Jongwe

    Full Text Available Over 90% of HIV/AIDS positive individuals in sub-Saharan Africa are infected with highly heterogeneous HIV-1 subtype C (HIV-1C viruses. One of the best ways to reduce the burden of this disease is the development of an affordable and effective prophylactic vaccine. Mosaic immunogens are computationally designed to overcome the hurdle of HIV diversity by maximizing the expression of potential T cell epitopes. Mycobacterium bovis BCG ΔpanCD auxotroph and modified vaccinia Ankara (MVA vaccines expressing HIV-1C mosaic Gag (GagM were tested in a prime-boost regimen to demonstrate immunogenicity in a mouse study. The BCG-GagM vaccine was stable and persisted 11.5 weeks post vaccination in BALB/c mice. Priming with BCG-GagM and boosting with MVA-GagM elicited higher Gag-specific IFN-γ ELISPOT responses than the BCG-GagM only and MVA-GagM only homologous vaccination regimens. The heterologous vaccination also generated a more balanced and persistent CD4+ and CD8+ T cell Gag-specific IFN-γ ELISPOT response with a predominant effector memory phenotype. A Th1 bias was induced by the vaccines as determined by the predominant secretion of IFN-γ, TNF-α, and IL-2. This study shows that a low dose of MVA (104 pfu can effectively boost a BCG prime expressing the same mosaic immunogen, generating strong, cellular immune responses against Gag in mice. Our data warrants further evaluation in non-human primates. A low dose vaccine would be an advantage in the resource limited countries of sub-Saharan Africa and India (where the predominating virus is HIV-1 subtype C.

  3. Intranasal immunization with recombinant HA and mast cell activator C48/80 elicits protective immunity against 2009 pandemic H1N1 influenza in mice.

    Directory of Open Access Journals (Sweden)

    Shu Meng

    Full Text Available BACKGROUND: Pandemic influenza represents a major threat to global health. Vaccination is the most economic and effective strategy to control influenza pandemic. Conventional vaccine approach, despite being effective, has a number of major deficiencies including limited range of protection, total dependence on embryonated eggs for production, and time consuming for vaccine production. There is an urgent need to develop novel vaccine strategies to overcome these deficiencies. METHODOLOGY/PRINCIPAL FINDINGS: The major objective of this work was to develop a novel vaccine strategy combining recombinant haemagglutinin (HA protein and a master cell (MC activator C48/80 for intranasal immunization. We demonstrated in BALB/c mice that MC activator C48/80 had strong adjuvant activity when co-administered with recombinant HA protein intranasally. Vaccination with C48/80 significantly increased the serum IgG and mucosal surface IgA antibody responses against HA protein. Such increases correlated with stronger and durable neutralizing antibody activities, offering protection to vaccinated animals from disease progression after challenge with lethal dose of A/California/04/2009 live virus. Furthermore, protected animals demonstrated significant reduction in lung virus titers, minimal structural alteration in lung tissues as well as higher and balanced production of Th1 and Th2 cytokines in the stimulated splenocytes when compared to those without C48/80. CONCLUSIONS/SIGNIFICANCE: The present study demonstrates that the novel vaccine approach of combining recombinant HA and mucosal adjuvant C48/80 is safe and effective in eliciting protective immunity in mice. Future studies on the mechanism of action of C48/80 and potential combination with other vaccine strategies such as prime and boost approach may help to induce even more potent and broad immune responses against viruses from various clades.

  4. Immunization against Clostridium perfringens cells elicits protection against Clostridium tetani in mouse model: identification of cross-reactive proteins using proteomic methodologies

    Directory of Open Access Journals (Sweden)

    Singh Lokendra


    Full Text Available Abstract Background Clostridium tetani and Clostridium perfringens are among the medically important clostridial pathogens causing diseases in man and animals. Several homologous open reading frames (ORFs have been identified in the genomes of the two pathogens by comparative genomic analysis. We tested a likelihood of extensive sharing of common epitopes between homologous proteins of these two medically important pathogens and the possibility of cross-protection using active immunization. Results Eight predominant cross-reactive spots were identified by mass spectrometry and had hits in the C. tetani E88 proteome with significant MOWSE scores. Most of the cross-reactive proteins of C. tetani shared 65–78% sequence similarity with their closest homologues in C. perfringens ATCC13124. Electron transfer flavoprotein beta-subunit (CT3 was the most abundant protein (43.3%, followed by methylaspartate ammonia-lyase (36.8% and 2-phosphoglycerate dehydratase (35.6%. All the proteins were predicted to be cytoplasmic by PSORT protein localization algorithm. Active immunization with C. perfringens whole cells elicited cross-protective immunity against C. tetani infection in a mouse model. Conclusion Most of the dominant cross-reactive proteins of C. tetani belonged to the cluster of orthologous group (COG functional category, either of posttranslational modification, protein turnover, and chaperones (O or energy production and conversion (C. The homologs of the identified proteins have been shown to play role in pathogenesis in other Gram-positive pathogenic bacteria. Our findings provide basis for the search of potential vaccine candidates with broader coverage, encompassing more than one pathogenic clostridial species.

  5. Antibody-targeted NY-ESO-1 to mannose receptor or DEC-205 in vitro elicits dual human CD8+ and CD4+ T cell responses with broad antigen specificity. (United States)

    Tsuji, Takemasa; Matsuzaki, Junko; Kelly, Marcus P; Ramakrishna, Venky; Vitale, Laura; He, Li-Zhen; Keler, Tibor; Odunsi, Kunle; Old, Lloyd J; Ritter, Gerd; Gnjatic, Sacha


    Immunization of cancer patients with vaccines containing full-length tumor Ags aims to elicit specific Abs and both CD4(+) and CD8(+) T cells. Vaccination with protein Ags, however, often elicits only CD4(+) T cell responses without inducing Ag-specific CD8(+) T cells, as exogenous protein is primarily presented to CD4(+) T cells. Recent data revealed that Ab-mediated targeting of protein Ags to cell surface receptors on dendritic cells could enhance the induction of both CD4(+) and CD8(+) T cells. We investigated in this study if these observations were applicable to NY-ESO-1, a cancer-testis Ag widely used in clinical cancer vaccine trials. We generated two novel targeting proteins consisting of the full-length NY-ESO-1 fused to the C terminus of two human mAbs against the human mannose receptor and DEC-205, both internalizing molecules expressed on APC. These targeting proteins were evaluated for their ability to activate NY-ESO-1-specific human CD4(+) and CD8(+) T cells in vitro. Both targeted NY-ESO-1 proteins rapidly bound to their respective targets on APC. Whereas nontargeted and Ab-targeted NY-ESO-1 proteins similarly activated CD4(+) T cells, cross-presentation to CD8(+) T cells was only efficiently induced by targeted NY-ESO-1. In addition, both mannose receptor and DEC-205 targeting elicited specific CD4(+) and CD8(+) T cells from PBLs of cancer patients. Receptor-specific delivery of NY-ESO-1 to APC appears to be a promising vaccination strategy to efficiently generate integrated and broad Ag-specific immune responses against NY-ESO-1 in cancer patients.

  6. Syndecans promote integrin-mediated adhesion of mesenchymal cells in two distinct pathways

    DEFF Research Database (Denmark)

    Whiteford, James; Behrends, Volker; Kirby, Hishani;


    to form focal adhesions in response to fibronectin. Consistent with actin cytoskeleton organization, the process required Rho-GTP and Rho kinase. While syndecan-2 and -4 ectodomains could both promote integrin-mediated adhesion, their pathways were distinct, as shown by competition assays. Evidence...

  7. Interaction proteomics analysis of polycomb proteins defines distinct PRC1 complexes in mammalian cells

    DEFF Research Database (Denmark)

    Vandamme, Julien; Völkel, Pamela; Rosnoblet, Claire;


    1/RNF2, PHC, and BMI1/PCGF families. Mammalian genomes encode five CBX family members (CBX2, CBX4, CBX6, CBX7, and CBX8) that are believed to have distinct biological functions. Here, we applied a tandem affinity purification (TAP) approach coupled with tandem mass spectrometry (MS/MS) methodologies...

  8. Stable antigen is most effective for eliciting CD8+ T-cell responses after DNA vaccination and infection with recombinant vaccinia virus in vivo. (United States)

    Schliehe, Christopher; Bitzer, Annegret; van den Broek, Maries; Groettrup, Marcus


    The induction of strong CD8(+) T-cell responses against infectious diseases and cancer has remained a major challenge. Depending on the source of antigen and the infectious agent, priming of CD8(+) T cells requires direct and/or cross-presentation of antigenic peptides on major histocompatibility complex (MHC) class I molecules by professional antigen-presenting cells (APCs). However, both pathways show distinct preferences concerning antigen stability. Whereas direct presentation was shown to efficiently present peptides derived from rapidly degraded proteins, cross-presentation is dependent on long-lived antigen species. In this report, we analyzed the role of antigen stability on DNA vaccination and recombinant vaccinia virus (VV) infection using altered versions of the same antigen. The long-lived nucleoprotein (NP) of lymphocytic choriomeningitis virus (LCMV) can be targeted for degradation by N-terminal fusion to ubiquitin or, as we show here, to the ubiquitin-like modifier FAT10. Direct presentation by cells either transfected with NP-encoding plasmids or infected with recombinant VV in vitro was enhanced in the presence of short-lived antigens. In vivo, however, the highest induction of NP-specific CD8(+) T-cell responses was achieved in the presence of long-lived NP. Our experiments provide evidence that targeting antigens for proteasomal degradation does not improve the immunogenicity of DNA vaccines and recombinant VVs. Rather, it is the long-lived antigen that is superior for the efficient activation of MHC class I-restricted immune responses in vivo. Hence, our results suggest a dominant role for antigen cross-priming in DNA vaccination and recombinant VV infection.

  9. Phenotypic and Functional Analysis of LCMV gp33-41-Specific CD8 T Cells Elicited by Multiple Peptide Immunization in Mice Revealed the Up-regulation of PD-1 Expression on Antigen Specific CD8 T Cells

    Institute of Scientific and Technical Information of China (English)

    Yi Liu; Lihui Xu; Yiqun Jiang; Jianfang Sun; Xianhui He


    The phenotype and function of antigen-specific CD8 T cells are closely associated with the efficacy of a therapeutic vaccination. Here we showed that multiple immunizations with LCMV gp33-41 peptide (KAV) in Freund's adjuvant could induce KAV-specific CD8 T cells with low expression of CD127 and CD62L molecules. The inhibitory receptor PD-1 was also expressed on a substantial part of KAV-specific CD8 T cells, and its expression level on KAV-specific CD8 T cells in spleen and lymph nodes was much higher when compared to those in peripheral blood. Furthermore, KAV-specific CD8 T cells could specifically kill KAV-pulsed target cells in vivo but the efficiency was low. These data suggest that prime-boost vaccination schedule with peptide in Freund's adjuvant can elicit antigen-specific CD8 T cells of effector-like phenotype with partial functional exhaustion, which may only provide short-term protection against the pathogen.

  10. Inhibition of oxidative stress-elicited AKT activation facilitates PPARγ agonist-mediated inhibition of stem cell character and tumor growth of liver cancer cells.

    Directory of Open Access Journals (Sweden)

    Lanlan Liu

    Full Text Available Emerging evidence suggests that tumor-initiating cells (TICs are the most malignant cell subpopulation in tumors because of their resistance to chemotherapy or radiation treatment. Targeting TICs may be a key innovation for cancer treatment. In this study, we found that PPARγ agonists inhibited the cancer stem cell-like phenotype and attenuated tumor growth of human hepatocellular carcinoma (HCC cells. Reactive oxygen species (ROS initiated by NOX2 upregulation were partially responsible for the inhibitory effects mediated by PPARγ agonists. However, PPARγ agonist-mediated ROS production significantly activated AKT, which in turn promoted TIC survival by limiting ROS generation. Inhibition of AKT, by either pharmacological inhibitors or AKT siRNA, significantly enhanced PPARγ agonist-mediated inhibition of cell proliferation and stem cell-like properties in HCC cells. Importantly, in nude mice inoculated with HCC Huh7 cells, we demonstrated a synergistic inhibitory effect of the PPARγ agonist rosiglitazone and the AKT inhibitor triciribine on tumor growth. In conclusion, we observed a negative feedback loop between oxidative stress and AKT hyperactivation in PPARγ agonist-mediated suppressive effects on HCCs. Combinatory application of an AKT inhibitor and a PPARγ agonist may provide a new strategy for inhibition of stem cell-like properties in HCCs and treatment of liver cancer.

  11. HIV-1-infected monocyte-derived dendritic cells do not undergo maturation but can elicit IL-10 production and T cell regulation (United States)

    Granelli-Piperno, Angela; Golebiowska, Angelika; Trumpfheller, Christine; Siegal, Frederick P.; Steinman, Ralph M.


    Dendritic cells (DCs) undergo maturation during virus infection and thereby become potent stimulators of cell-mediated immunity. HIV-1 replicates in immature DCs, but we now find that infection is not accompanied by many components of maturation in either infected cells or uninfected bystanders. The infected cultures do not develop potent stimulating activity for the mixed leukocyte reaction (MLR), and the DCs producing HIV-1 gag p24 do not express CD83 and DC-lysosome-associated membrane protein maturation markers. If different maturation stimuli are applied to DCs infected with HIV-1, the infected cells selectively fail to mature. When DCs from HIV-1-infected patients are infected and cultured with autologous T cells, IL-10 was produced in 6 of 10 patients. These DC-T cell cocultures could suppress another immune response, the MLR. The regulation was partially IL-10-dependent and correlated in extent with the level of IL-10 produced. Suppressor cells only developed from infected patients, rather than healthy controls, and the DCs had to be exposed to live virus rather than HIV-1 gag peptides or protein. These results indicate that HIV-1-infected DCs have two previously unrecognized means to evade immune responses: maturation can be blocked reducing the efficacy of antigen presentation from infected cells, and T cell-dependent suppression can be induced.

  12. Mycobacterial secretion systems ESX-1 and ESX-5 play distinct roles in host cell death and inflammasome activation

    KAUST Repository

    Abdallah, Abdallah


    During infection of humans and animals, pathogenic mycobacteria manipulate the host cell causing severe diseases such as tuberculosis and leprosy. To understand the basis of mycobacterial pathogenicity, it is crucial to identify the molecular virulence mechanisms. In this study, we address the contribution of ESX-1 and ESX-5 - two homologous type VII secretion systems of mycobacteria that secrete distinct sets of immune modulators - during the macrophage infection cycle. Using wild-type, ESX-1- and ESX-5-deficient mycobacterial strains, we demonstrate that these secretion systems differentially affect subcellular localization and macrophage cell responses. We show that in contrast to ESX-1, the effector proteins secreted by ESX-5 are not required for the translocation of Mycobacterium tuberculosis or Mycobacterium marinum to the cytosol of host cells. However, the M. marinum ESX-5 mutant does not induce inflammasome activation and IL-1b activation. The ESX-5 system also induces a caspase-independent cell death after translocation has taken place. Importantly, by means of inhibitory agents and small interfering RNA experiments, we reveal that cathepsin B is involved in both the induction of cell death and inflammasome activation upon infection with wild-type mycobacteria. These results reveal distinct roles for two different type VII secretion systems during infection and shed light on how virulent mycobacteria manipulate the host cell in various ways to replicate and spread. Copyright © 2011 by The American Association of Immunologists, Inc.

  13. Two distinct functional patterns of hepatitis C Virus (HCV-specific T cell responses in seronegative, aviremic patients.

    Directory of Open Access Journals (Sweden)

    Yoon Seok Choi

    Full Text Available In hepatitis C Virus (HCV high-risk groups, HCV-specific T cell responses have been detected in seronegative, aviremic persons who have no evidence of HCV infection. Herein, we investigated functional profiles of HCV-specific T-cell responses in seronegative, aviremic patients of a HCV high-risk group. Seventy seven hemodialysis patients with chronic renal disease were analyzed by IFN-γ ELISpot assays, and eight of 71 (11.3% seronegative, aviremic patients displayed HCV-specific T-cell responses. Their HCV-specific memory T cells were characterized by assessing cytokine polyfunctionality, known to provide antiviral protection. By intracellular staining of IFN-γ, TNF-α, IL-2 and MIP-1β, we identified two distinct populations in the seronegative, aviremic patients: polyfunctional responders and TNF-α-predominant responders. In further analysis, occult HCV infection was excluded as a cause of the HCV-specific T cell response via secondary nested RT-PCR of HCV RNA in peripheral blood mononuclear cell samples. HCV-specific T cells targeted multiple epitopes including non-structural proteins in a single patient, implying that their T cells might have been primed by HCV proteins synthesized within the host. We conclude that HCV-specific memory T cells of seronegative, aviremic patients arise from authentic HCV replication in the host, but not from current occult HCV infection. By functional pattern of HCV-specific T cells, there are two distinct populations in these patients: polyfunctional responders and TNF-α-predominant responders.

  14. Multiplex and genome-wide analyses reveal distinctive properties of KIR+ and CD56+ T cells in human blood. (United States)

    Chan, Wing Keung; Rujkijyanont, Piya; Neale, Geoffrey; Yang, Jie; Bari, Rafijul; Das Gupta, Neha; Holladay, Martha; Rooney, Barbara; Leung, Wing


    Killer cell Ig-like receptors (KIRs) on NK cells have been linked to a wide spectrum of health conditions such as chronic infections, autoimmune diseases, pregnancy complications, cancers, and transplant failures. A small subset of effector memory T cells also expresses KIRs. In this study, we use modern analytic tools including genome-wide and multiplex molecular, phenotypic, and functional assays to characterize the KIR(+) T cells in human blood. We find that KIR(+) T cells primarily reside in the CD56(+) T population that is distinctively DNAM-1(high) with a genome-wide quiescent transcriptome, short telomere, and limited TCR excision circles. During CMV reactivation in bone marrow transplant recipients, KIR(+)CD56(+) T cells rapidly expanded in real-time but not KIR(+)CD56(-) T cells or KIR(+) NK cells. In CMV(+) asymptomatic donors, as much as 50% of CD56(+) T cells are KIR(+), and most are distinguishably KIR2DL2/3(+)NKG2C(+)CD57(+). Functionally, the KIR(+)CD56(+) T cell subset lyses cancer cells and CMVpp65-pulsed target cells in a dual KIR-dependent and TCR-dependent manner. Analysis of metabolic transcriptome confirms the immunological memory status of KIR(+)CD56(+) T cells in contrast to KIR(-)CD56(+) T cells that are more active in energy metabolism and effector differentiation. KIR(-)CD56(+) T cells have >25-fold higher level of expression of RORC than the KIR(+) counterpart and are a previously unknown producer of IL-13 rather than IL-17 in multiplex cytokine arrays. Our data provide fundamental insights into KIR(+) T cells biologically and clinically.

  15. Distinct roles for dystroglycan, beta1 integrin and perlecan in cell surface laminin organization

    DEFF Research Database (Denmark)

    Henry, M D; Satz, J S; Brakebusch, C;


    Dystroglycan (DG) is a cell surface receptor for several extracellular matrix (ECM) molecules including laminins, agrin and perlecan. Recent data indicate that DG function is required for the formation of basement membranes in early development and the organization of laminin on the cell surface....... Here we show that DG-mediated laminin clustering on mouse embryonic stem (ES) cells is a dynamic process in which clusters are consolidated over time into increasingly more complex structures. Utilizing various null-mutant ES cell lines, we define roles for other molecules in this process. In beta1...... integrin-deficient ES cells, laminin-1 binds to the cell surface, but fails to organize into more morphologically complex structures. This result indicates that beta1 integrin function is required after DG function in the cell surface-mediated laminin assembly process. In perlecan-deficient ES cells...

  16. Distinct T cell dynamics in lymph nodes during the induction of tolerance and immunity. (United States)

    Hugues, Stéphanie; Fetler, Luc; Bonifaz, Laura; Helft, Julie; Amblard, François; Amigorena, Sebastian


    Induction of immunity and peripheral tolerance requires contacts between antigen-bearing dendritic cells (DCs) and cognate T cells. Using real-time two-photon microscopy, we have analyzed the dynamics of CD8(+) T cells in lymph nodes during the induction of antigen-specific immunity or tolerance. At 15-20 h after the induction of immunity, T cells stopped moving and established prolonged interactions with DCs. In tolerogenic conditions, despite effective initial T cell activation and proliferation, naive T cells remained motile and established serial brief contacts with multiple DCs. Thus, stable DC-T cell interactions occur during the induction of priming, whereas brief contacts may contribute to the induction of T cell tolerance.

  17. Different meningitis-causing bacteria induce distinct inflammatory responses on interaction with cells of the human meninges. (United States)

    Fowler, Mark I; Weller, Roy O; Heckels, John E; Christodoulides, Myron


    The interactions of bacterial pathogens with cells of the human leptomeninges are critical events in the progression of meningitis. An in vitro model based on the culture of human meningioma cells was used to investigate the interactions of the meningeal pathogens Escherichia coli K1, Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae. A rank order of association with meningioma cells was observed, with N. meningitidis showing the highest levels of adherence, followed by E. coli, S. pneumoniae and H. influenzae. Neisseria meningitidis and H. influenzae did not invade meningioma cells or induce cell death, but induced a concentration-dependent secretion of inflammatory mediators. Neisseria meningitidis induced higher levels of IL-6, MCP-1, RANTES and GM-CSF than H. influenzae, but there was no significant difference in the levels of IL-8 induced by both pathogens. Streptococcus pneumoniae was also unable to invade meningioma cells, but low concentrations of bacteria failed to stimulate cytokine secretion. However, higher concentrations of pneumococci led to cell death. By contrast, only E. coli K1 invaded meningioma cells directly and induced rapid cell death before an inflammatory response could be induced. These data demonstrate that the interactions of different bacterial pathogens with human meningeal cells are distinct, and suggest that different intervention strategies may be needed in order to prevent the morbidity and mortality associated with bacterial meningitis.

  18. Distinct roles of Cdc42 in thymopoiesis and effector and memory T cell differentiation.

    Directory of Open Access Journals (Sweden)

    Fukun Guo

    Full Text Available Cdc42 of the Rho GTPase family has been implicated in cell actin organization, proliferation, survival, and migration but its physiological role is likely cell-type specific. By a T cell-specific deletion of Cdc42 in mouse, we have recently shown that Cdc42 maintains naïve T cell homeostasis through promoting cell survival and suppressing T cell activation. Here we have further investigated the involvement of Cdc42 in multiple stages of T cell differentiation. We found that in Cdc42(-/- thymus, positive selection of CD4(+CD8(+ double-positive thymocytes was defective, CD4(+ and CD8(+ single-positive thymocytes were impaired in migration and showed an increase in cell apoptosis triggered by anti-CD3/-CD28 antibodies, and thymocytes were hyporesponsive to anti-CD3/-CD28-induced cell proliferation and hyperresponsive to anti-CD3/-CD28-stimulated MAP kinase activation. At the periphery, Cdc42-deficient naive T cells displayed an impaired actin polymerization and TCR clustering during the formation of mature immunological synapse, and showed an enhanced differentiation to Th1 and CD8(+ effector and memory cells in vitro and in vivo. Finally, Cdc42(-/- mice exhibited exacerbated liver damage in an induced autoimmune disease model. Collectively, these data establish that Cdc42 is critically involved in thymopoiesis and plays a restrictive role in effector and memory T cell differentiation and autoimmunity.

  19. Common variable immunodeficiency and inclusion body myositis: a distinct myopathy mediated by natural killer cells. (United States)

    Dalakas, M C; Illa, I


    Inclusion body myositis developed in two men, 36 and 48 years old with long-standing common variable immunodeficiency. Immunophenotypic analysis of the endomysial cells showed an increased number of natural killer (NK) cells (defined as CD57+, CD56+, CD3-, CD8-, CD68-) accounting for 8.5 to 9.5% of the total cells, compared with a mean of 1% in sporadic inclusion body myositis. The remaining cells were CD8+, macrophages, and CD4+ T cells. NK cells were positive for intercellular cell adhesion molecule-1 and invaded muscle fibers negative for major histocompatibility complex (MHC) class I. In contrast to ubiquitous endomysial expression of MHC class I antigen in sporadic inclusion body myositis, the MHC class I in common variable immunodeficiency and inclusion body myositis was absent or weakly expressed in only some of the muscle fibers surrounded by CD8+ cells. Enteroviral or retroviral RNA sequences were not amplified. Treatment with intravenous immunoglobulin improved strength in 1 patient whose repeated muscle biopsy specimen showed normal NK cells. We conclude that inclusion body myositis can develop in patients with common variable immunodeficiency. Common variable immunodeficiency with inclusion body myositis is an immune myopathy mediated by NK cells in a non-MHC class I-restricted cytotoxicity, and by CD8+ cells in an MHC class I-restricted process. This is the first description of an inflammatory myopathy in which NK cells participate in the myocytotoxic process.

  20. Multiparameter Analysis of Human Bone Marrow Stromal Cells Identifies Distinct Immunomodulatory and Differentiation-Competent Subtypes

    NARCIS (Netherlands)

    S. James (Sally); J. Fox (James); F. Afsari (Farinaz); J. Lee (Jennifer); S. Clough (Sally); C. Knight (Charlotte); J. Ashmore (James); P. Ashton (Peter); O. Preham (Olivier); M.J. Hoogduijn (Martin); R.D.A.R. Ponzoni (Raquel De Almeida Rocha); Y. Hancock; M. Coles (Mark); P.G. Genever (Paul)


    textabstractBone marrow stromal cells (BMSCs, also called bone-marrow-derived mesenchymal stromal cells) provide hematopoietic support and immunoregulation and contain a stem cell fraction capable of skeletogenic differentiation. We used immortalized human BMSC clonal lines for multi-level analysis

  1. Cells release subpopulations of exosomes with distinct molecular and biological properties

    NARCIS (Netherlands)

    Willms, Eduard; Johansson, Henrik J; Mäger, Imre; Lee, Yi; Blomberg, K Emelie M; Sadik, Mariam; Alaarg, Amr; Smith, C I Edvard; Lehtiö, Janne; El Andaloussi, Samir; Wood, Matthew J A; Vader, Pieter


    Cells release nano-sized membrane vesicles that are involved in intercellular communication by transferring biological information between cells. It is generally accepted that cells release at least three types of extracellular vesicles (EVs): apoptotic bodies, microvesicles and exosomes. While a wi

  2. Peripheral and site-specific CD4(+) CD28(null) T cells from rheumatoid arthritis patients show distinct characteristics. (United States)

    Pieper, J; Johansson, S; Snir, O; Linton, L; Rieck, M; Buckner, J H; Winqvist, O; van Vollenhoven, R; Malmström, V


    Proinflammatory CD4(+) CD28(null) T cells are frequently found in the circulation of patients with rheumatoid arthritis (RA), but are less common in the rheumatic joint. In the present study, we sought to identify functional differences between CD4(+) CD28(null) T cells from blood and synovial fluid in comparison with conventional CD28-expressing CD4(+) T cells. Forty-four patients with RA, displaying a distinct CD4(+) CD28(null) T cell population in blood, were recruited for this study; the methylation status of the IFNG locus was examined in isolated T cell subsets, and intracellular cytokine production (IFN-γ, TNF, IL-17) and chemokine receptor expression (CXCR3, CCR6 and CCR7) were assessed by flow cytometry on T cells from the two compartments. Circulating CD4(+) CD28(null) T cells were significantly more hypomethylated in the CNS-1 region of the IFNG locus than conventional CD4(+) CD28(+) T cells and produced higher levels of both IFN-γ and TNF after TCR cross-linking. CD4(+) CD28(null) T cells from the site of inflammation expressed significantly more CXCR3 and CCR6 compared to their counterparts in blood. While IL-17A production could hardly be detected in CD4(+) CD28(null) cells from the blood, a significant production was observed in CD4(+) CD28(null) T cells from synovial fluid. CD4(+) CD28(null) T cells were not only found to differ from conventional CD4(+) CD28(+) T cells in the circulation, but we could also demonstrate that synovial CD4(+) CD28(null) T cells showed additional effector functions (IL-17 coproduction) as compared to the same subset in peripheral blood, suggesting an active role for these cells in the perpetuation of inflammation in the subset of patients having a CD28(null) population.

  3. Hidden in the crowd: primordial germ cells and somatic stem cells in the mesodermal posterior growth zone of the polychaete Platynereis dumerillii are two distinct cell populations

    Directory of Open Access Journals (Sweden)

    Rebscher Nicole


    Full Text Available Abstract Background In the polychaete Platynereis, the primordial germ cells (PGCs emerge from the vasa, piwi, and PL10 expressing mesodermal posterior growth zone (MPGZ at the end of larval development, suggesting a post-embryonic formation from stem cells. Methods In order to verify this hypothesis, embryos and larvae were pulse labeled with the proliferation marker 5-ethynyl-2'-deoxyuridine (EdU at different stages of development. Subsequently, the PGCs were visualized in 7-day-old young worms using antibodies against the Vasa protein. Results Surprisingly, the primordial germ cells of Platynereis incorporate EdU only shortly before gastrulation (6-8 hours post fertilization (hpf, which coincides with the emergence of four small blastomeres from the mesoblast lineage. We conclude that these so-called 'secondary mesoblast cells' constitute the definitive PGCs in Platynereis. In contrast, the cells of the MPGZ incorporate EdU only from the pre-trochophore stage onward (14 hpf. Conclusion While PGCs and the cells of the MPGZ in Platynereis are indistinguishable in morphology and both express the germline markers vasa, nanos, and piwi, a distinct cluster of PGCs is detectable anterior of the MPGZ following EdU pulse-labeling. Indeed the PGCs form independently from the stem cells of the MPGZ prior to gastrulation. Our data suggest an early PGC formation in the polychaete by preformation rather than by epigenesis.

  4. Differential expression of surface markers in mouse bone marrow mesenchymal stromal cell subpopulations with distinct lineage commitment.

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    Maria Rostovskaya

    Full Text Available Bone marrow mesenchymal stromal cells (BM MSCs represent a heterogeneous population of progenitors with potential for generation of skeletal tissues. However the identity of BM MSC subpopulations is poorly defined mainly due to the absence of specific markers allowing in situ localization of those cells and isolation of pure cell types. Here, we aimed at characterization of surface markers in mouse BM MSCs and in their subsets with distinct differentiation potential. Using conditionally immortalized BM MSCs we performed a screening with 176 antibodies and high-throughput flow cytometry, and found 33 markers expressed in MSCs, and among them 3 were novel for MSCs and 13 have not been reported for MSCs from mice. Furthermore, we obtained clonally derived MSC subpopulations and identified bipotential progenitors capable for osteo- and adipogenic differentiation, as well as monopotential osteogenic and adipogenic clones, and thus confirmed heterogeneity of MSCs. We found that expression of CD200 was characteristic for the clones with osteogenic potential, whereas SSEA4 marked adipogenic progenitors lacking osteogenic capacity, and CD140a was expressed in adipogenic cells independently of their efficiency for osteogenesis. We confirmed our observations in cell sorting experiments and further investigated the expression of those markers during the course of differentiation. Thus, our findings provide to our knowledge the most comprehensive characterization of surface antigens expression in mouse BM MSCs to date, and suggest CD200, SSEA4 and CD140a as markers differentially expressed in distinct types of MSC progenitors.

  5. Naïve and memory B cells exhibit distinct biochemical responses following BCR engagement. (United States)

    Moens, Leen; Kane, Alisa; Tangye, Stuart G


    Immunological memory is characterized by the rapid reactivation of memory B cells that produce large quantities of high-affinity antigen-specific antibodies. This contrasts the response of naïve B cells, and the primary immune response, which is much slower and of lower affinity. Memory responses are critical for protection against infectious diseases and form the basis of most currently available vaccines. Although we have known about the phenomenon of long-lived memory for centuries, the biochemical differences underlying these diverse responses of naïve and memory B cells is incompletely resolved. Here we investigated the nature of B-cell receptor (BCR) signaling in human splenic naïve, IgM(+) memory and isotype-switched memory B cells following multivalent BCR crosslinking. We observed comparable rapid and transient phosphorylation kinetics for proximal (phosphotyrosine and spleen tyrosine kinase) and propagation (B-cell linker, phospholipase Cγ2) signaling components in these different B-cell subsets. However, the magnitude of activation of downstream components of the BCR signaling pathway were greater in memory compared with naïve cells. Although no differences were observed in the magnitude of Ca(2+) mobilization between subsets, IgM(+) memory B cells exhibited a more rapid Ca(2+) mobilization and a greater depletion of the Ca(2+) endoplasmic reticulum stores, while IgG(+) memory B cells had a prolonged Ca(2+) uptake. Collectively, our findings show that intrinsic signaling features of B-cell subsets contribute to the robust response of human memory B cells over naïve B cells. This has implications for our understanding of memory B-cell responses and provides a framework to modulate these responses in the setting of vaccination and immunopathologies, such as immunodeficiency and autoimmunity.

  6. Memory CD8(+) T cells elicited by HIV-1 lipopeptide vaccines display similar phenotypic profiles but differences in term of magnitude and multifunctionality compared with FLU- or EBV-specific memory T cells in humans. (United States)

    Figueiredo, Suzanne; Charmeteau, Benedicte; Surenaud, Mathieu; Salmon, Dominique; Launay, Odile; Guillet, Jean-Gérard; Hosmalin, Anne; Gahery, Hanne


    Differentiation marker, multifunctionality and magnitude analyses of specific-CD8(+) memory T cells are crucial to improve development of HIV vaccines designed to generate cell-mediated immunity. Therefore, we fully characterized the HIV-specific CD8(+) T cell responses induced in volunteers vaccinated with HIV lipopeptide vaccines for phenotypic markers, tetramer staining, cytokine secretion, and cytotoxic activities. The frequency of ex vivo CD8(+) T cells elicited by lipopeptide vaccines is very rare and central-memory phenotype and functions of these cells were been shown to be important in AIDS immunity. So, we expanded them using specific peptides to compare the memory T cell responses induced in volunteers by HIV vaccines with responses to influenza (FLU) or Epstein Barr virus (EBV). By analyzing the differentiation state of IFN-γ-secreting CD8(+) T cells, we found a CCR7(-)CD45RA(-)CD28(+int)/CD28(-) profile (>85%) belonging to a subset of intermediate-differentiated effector T cells for HIV, FLU, and EBV. We then assessed the quality of the response by measuring various T cell functions. The percentage of single IFN-γ T cell producers in response to HIV was 62% of the total of secreting T cells compared with 35% for FLU and EBV, dual and triple (IFN-γ/IL-2/CD107a) T cell producers could also be detected but at lower levels (8% compared with 37%). Finally, HIV-specific T cells secreted IFN-γ and TNF-α, but not the dual combination like FLU- and EBV-specific T cells. Thus, we found that the functional profile and magnitude of expanded HIV-specific CD8(+) T precursors were more limited than those of to FLU- and EBV-specific CD8(+) T cells. These data show that CD8(+) T cells induced by these HIV vaccines have a similar differentiation profile to FLU and EBV CD8(+) T cells, but that the vaccine potency to induce multifunctional T cells needs to be increased in order to improve vaccination strategies.

  7. hESC Differentiation toward an Autonomic Neuronal Cell Fate Depends on Distinct Cues from the Co-Patterning Vasculature

    Directory of Open Access Journals (Sweden)

    Lisette M. Acevedo


    Full Text Available To gain insight into the cellular and molecular cues that promote neurovascular co-patterning at the earliest stages of human embryogenesis, we developed a human embryonic stem cell model to mimic the developing epiblast. Contact of ectoderm-derived neural cells with mesoderm-derived vasculature is initiated via the neural crest (NC, not the neural tube (NT. Neurovascular co-patterning then ensues with specification of NC toward an autonomic fate requiring vascular endothelial cell (EC-secreted nitric oxide (NO and direct contact with vascular smooth muscle cells (VSMCs via T-cadherin-mediated homotypic interactions. Once a neurovascular template has been established, NT-derived central neurons then align themselves with the vasculature. Our findings reveal that, in early human development, the autonomic nervous system forms in response to distinct molecular cues from VSMCs and ECs, providing a model for how other developing lineages might coordinate their co-patterning.

  8. Human Glioma–Initiating Cells Show a Distinct Immature Phenotype Resembling but Not Identical to NG2 Glia (United States)

    Barrantes-Freer, Alonso; Kim, Ella; Bielanska, Joanna; Giese, Alf; Mortensen, Lena Sünke; Schulz-Schaeffer, Walter J.; Stadelmann, Christine; Brück, Wolfgang


    Abstract Glioma-initiating cells (GICs) represent a potential important therapeutic target because they are likely to account for the frequent recurrence of malignant gliomas; however, their identity remains unsolved. Here, we characterized the cellular lineage fingerprint of GICs through a combination of electrophysiology, lineage marker expression, and differentiation assays of 5 human patient-derived primary GIC lines. Most GICs coexpressed nestin, NG2 proteoglycan, platelet-derived growth factor receptor-α, and glial fibrillary acidic protein. Glioma-initiating cells could be partially differentiated into astrocytic but not oligodendroglial or neural lineages. We also demonstrate that GICs have a characteristic electrophysiologic profile distinct from that of well-characterized tumor bulk cells. Together, our results suggest that GICs represent a unique type of cells reminiscent of an immature phenotype that closely resembles but is not identical to NG2 glia with respect to marker expression and functional membrane properties. PMID:23481707

  9. The cytokines interleukin 27 and interferon-γ promote distinct Treg cell populations required to limit infection-induced pathology. (United States)

    Hall, Aisling O'Hara; Beiting, Daniel P; Tato, Cristina; John, Beena; Oldenhove, Guillaume; Lombana, Claudia Gonzalez; Pritchard, Gretchen Harms; Silver, Jonathan S; Bouladoux, Nicolas; Stumhofer, Jason S; Harris, Tajie H; Grainger, John; Wojno, Elia D Tait; Wagage, Sagie; Roos, David S; Scott, Philip; Turka, Laurence A; Cherry, Sara; Reiner, Steven L; Cua, Daniel; Belkaid, Yasmine; Elloso, M Merle; Hunter, Christopher A


    Interferon-γ (IFN-γ) promotes a population of T-bet(+) CXCR3(+) regulatory T (Treg) cells that limit T helper 1 (Th1) cell-mediated pathology. Our studies demonstrate that interleukin-27 (IL-27) also promoted expression of T-bet and CXCR3 in Treg cells. During infection with Toxoplasma gondii, a similar population emerged that limited T cell responses and was dependent on IFN-γ in the periphery but on IL-27 at mucosal sites. Transfer of Treg cells ameliorated the infection-induced pathology observed in Il27(-/-) mice, and this was dependent on their ability to produce IL-10. Microarray analysis revealed that Treg cells exposed to either IFN-γ or IL-27 have distinct transcriptional profiles. Thus, IFN-γ and IL-27 have different roles in Treg cell biology and IL-27 is a key cytokine that promotes the development of Treg cells specialized to control Th1 cell-mediated immunity at local sites of inflammation.

  10. Distinct Responses of Cytotoxic Ganoderma lucidum Triterpenoids in Human Carcinoma Cells. (United States)

    Ruan, Weimei; Wei, Ying; Popovich, David G


    The medicinal mushroom Ganoderma lucidum is well recognized for its effective cancer-preventative and therapeutic properties, while specific components responsible for these anticancer effects are not well studied. Six triterpenoids that are ganolucidic acid E, lucidumol A, ganodermanontriol, 7-oxo-ganoderic acid Z, 15-hydroxy-ganoderic acid S, and ganoderic acid DM were isolated and identified from an extract of the mushroom. All compounds reduced cell growth in three human carcinoma cells (Caco-2, HepG2, and HeLa cells) dose dependently with LC50s from 20.87 to 84.36 μM. Moreover, the six compounds induced apoptosis in HeLa cells with a maximum increase (22%) of sub-G1 accumulations and 43.03% apoptotic cells in terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay (15-hydroxy-ganoderic acid S treatment). Apoptosis was further confirmed by annexin-V staining. Four of the compounds also caused apoptosis in Caco-2 cells with maximum 9.5% increase of sub-G1 accumulations (7-oxo-ganoderic acid Z treatment) and maximum 29.84% apoptotic cells in TUNEL assay (ganoderic acid DM treatment). Contrarily, none of the compounds induced apoptosis in HepG2 cells. The different responses of the three cell lines following these treatments indicated that the bioactive properties of these compounds may vary from cells of different sites of origin and are likely acting under diverse regulatory mechanisms.

  11. B-cell lymphomas with features intermediate between distinct pathologic entities. From pathogenesis to pathology. (United States)

    Carbone, Antonino; Gloghini, Annunziata; Aiello, Antonella; Testi, Adele; Cabras, Antonello


    Published in September 2008, the updated World Health Organization Classification of Tumors of Hematopoietic and Lymphoid Tissues introduces provisional borderline categories for lymphoma cases that demonstrate overlapping clinical, morphological, and/or immunophenotypic features between well-established entities. These overlapping features pose real diagnostic challenges especially in identifying atypical cases of diffuse large B-cell lymphoma, Hodgkin lymphoma, and Burkitt lymphoma. Lymphoma cases showing borderline features between T-cell/histiocyte-rich large B-cell lymphoma and nodular lymphocyte predominant Hodgkin lymphoma are not included within the borderline categories provisionally recognized by the updated classification. Within the borderline categories, there are cases combining features of primary mediastinal large B-cell lymphoma and classical Hodgkin lymphoma. Many of these cases resemble classical Hodgkin lymphoma but have a large number of tumor cells expressing CD20, CD45, and B-cell transcription factors. Alternatively, these cases may resemble primary mediastinal large B-cell lymphoma but contain tumor cells resembling Reed-Sternberg cells and displaying an aberrant phenotype such as CD20(-), CD15(-/+) CD45(+), CD30(+), Pax5(+), OCT2(+/-), and BOB1(+/-). Another new borderline category defining B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, represents a biologically heterogeneous group. Cases with morphologic features intermediate and with CD10/BCL6 coexpression should be placed in diffuse large B-cell lymphoma/Burkitt lymphoma category if tumor cells also show strong BCL2 staining and/or a Ki67 proliferation index of less than 90%. When MYC rearrangements are present in these cases, the lymphomas often have atypical features, including concurrent rearrangements of BCL2 and/or BCL6 genes (so-called double/triple-hit lymphomas) and more aggressive behavior. For the

  12. Cutting Edge: Murine Mast Cells Rapidly Modulate Metabolic Pathways Essential for Distinct Effector Functions. (United States)

    Phong, Binh; Avery, Lyndsay; Menk, Ashley V; Delgoffe, Greg M; Kane, Lawrence P


    There is growing appreciation that cellular metabolic and bioenergetic pathways do not play merely passive roles in activated leukocytes. Rather, metabolism has important roles in controlling cellular activation, differentiation, survival, and effector function. Much of this work has been performed in T cells; however, there is still very little information regarding mast cell metabolic reprogramming and its effect on cellular function. Mast cells perform important barrier functions and help control type 2 immune responses. In this study we show that murine bone marrow-derived mast cells rapidly alter their metabolism in response to stimulation through the FcεRI. We also demonstrate that specific metabolic pathways appear to be differentially required for the control of mast cell function. Manipulation of metabolic pathways may represent a novel point for the manipulation of mast cell activation.

  13. A distinct gene expression signature characterizes human neuroblastoma cancer stem cells. (United States)

    Ross, Robert A; Walton, Jeanette D; Han, Dan; Guo, Hong-Fen; Cheung, Nai-Kong V


    Neuroblastoma, a malignancy of multipotent embryonic neural crest cells, is the most common extracranial solid cancer in childhood and most common cancer in infancy. Cellular phenotype has been shown to be an important determinant of the malignant potential in human neuroblastoma cells and tumors. Whereas neuroblastic (N-type) are moderately malignant and nonneuronal (S-type) cells are nonmalignant, I-type stem cells are highly tumorigenic, irrespective of N-myc amplification status. In the present study, we sought to determine which genes were overexpressed in the I-type cells which might characterize and maintain the stem cell state and/or malignancy of human neuroblastoma cancer stem cells. We used a microarray platform to compare the steady-state expression levels of mRNAs from 13 human neuroblastoma cell lines representing the three cellular phenotypes. Using qRT-PCR and Western blot analyses, we identified seven genes whose expression is consistently elevated exclusively in neuroblastoma cancer stem cells: CD133, KIT, NOTCH1, GPRC5C, PIGF2, TRKB, and LNGFR. Moreover, we show that the genes are phenotype specific, as differentiation of I-type BE(2)-C cells to either an N- or S-type morphology results in significantly reduced mRNA expression. Finally, we show that NOTCH1 plays an important role in maintaining the stem cell phenotype. The identification and characterization of these genes, elevated in highly malignant neuroblastoma stem cells, could provide the basis for developing novel therapies for treatment of this lethal childhood cancer.

  14. Distinctions in sensitivity and repair of cells of children with some hereditary diseases

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    Zasukhina, G.D.; Barashnev, Yu.I.; Vasil' eva, I.M.; Sdirkova, N.I.; Semyachkina, A.N. (AN SSSR, Moscow. Inst. Obshchej Genetiki)

    A study was made of blood cell sensitivity of children with some hereditary diseases, to ..gamma..-radiation and 4-nitro-quinoline-1-oxide. Using the host cell reactivation and chromatographic methods we revealed the increase in the sensitivity to the above mentioned agents and inhibition of the repair function in cells of patients with the following diseases: Marfan's disease, histidinemia, osteogenesis imperfecta, Sylvere-Russelle, Laurence, Franchescetti, and Losch-Nychane syndromes.

  15. Vaccination with peptides derived from cancer-testis antigens in combination with CpG-7909 elicits strong specific CD8+ T cell response in patients with metastatic esophageal squamous cell carcinoma. (United States)

    Iwahashi, Makoto; Katsuda, Masahiro; Nakamori, Mikihito; Nakamura, Masaki; Naka, Teiji; Ojima, Toshiyasu; Iida, Takeshi; Yamaue, Hiroki


    Potent helper action is necessary for peptide-based vaccines to efficiently induce antitumor immune responses against advanced cancer. A phase I trial for advanced esophageal squamous cell carcinoma was carried out for patients with HLA-A*2402 using epitope peptides derived from novel cancer-testis antigens, LY6K and TTK, in combination with CpG-7909 (NCT00669292). This study investigated the feasibility and the toxicity as well as induction of tumor antigen-specific immune responses. Nine patients were vaccinated on days 1, 8, 15, and 22 of each 28-day treatment cycle with peptide LY6K-177, peptide TTK-567, and CpG-7909 (level-1; 0, level-2; 0.02, level-3; 0.1 mg/kg) and all were tolerated by this treatment. LY6K-specific T cell responses in PBMCs were detected in two of the three patients in each level. In particular, two patients in level-2/3 showed potent LY6K-specific T cell responses. In contrast, only two patients in level-2/3 showed TTK-567-specific T cell responses. The frequency of LY6K-177 or TTK-567-specific CD8+ T cells increased in patients in level-2/3 (with CpG). The vaccination with peptides and CpG-7909 increased and activated both plasmacytoid dendritic cells and natural killer cells, and increased the serum level of α-interferon. There were no complete response (CR) and partial response (PR), however, one of three patients in level-1, and four of six patients in level-2/3 showed stable disease (SD). In conclusion, vaccination with LY6K-177 and TTK-567 in combination with CpG-7909 successfully elicited antigen-specific CD8+ T cell responses and enhanced the innate immunity of patients with advanced esophageal squamous cell carcinoma. This vaccine protocol is therefore recommended to undergo further phase II trials.

  16. Brown adipose tissue harbors a distinct sub-population of regulatory T cells.

    Directory of Open Access Journals (Sweden)

    Dasa Medrikova

    Full Text Available Regulatory T (Treg cells are critical determinants of both immune responses and metabolic control. Here we show that systemic ablation of Treg cells compromised the adaptation of whole-body energy expenditure to cold exposure, correlating with impairment in thermogenic marker gene expression and massive invasion of pro-inflammatory macrophages in brown adipose tissue (BAT. Indeed, BAT harbored a unique sub-set of Treg cells characterized by a unique gene signature. As these Treg cells respond to BAT activation upon cold exposure, this study defines a BAT-specific Treg sub-set with direct implications for the regulation of energy homeostasis in response to environmental stress.

  17. Eomesodermin and T-bet mark developmentally distinct human natural killer cells (United States)

    Liu, Kang; Reiner, Steven L.


    Immaturity of the immune system of human fetuses and neonates is often invoked to explain their increased susceptibility to infection; however, the development of the fetal innate immune system in early life remains incompletely explored. We now show that the most mature NK cells found in adult (or postnatal) human circulation (CD94–CD16+) are absent during ontogeny. Human fetal NK cells were found to express the 2 signature T-box transcription factors essential for the development of all murine NK and NK-like cells, eomesodermin (Eomes) and T-bet. The single-cell pattern of Eomes and T-bet expression during ontogeny, however, revealed a stereotyped pattern of reciprocal dominance, with immature NK cells expressing higher amounts of Eomes and more mature NK cells marked by greater abundance of T-bet. We also observed a stereotyped pattern of tissue-specific NK cell maturation during human ontogeny, with fetal liver being more restrictive to NK cell maturity than fetal bone barrow, spleen, or lung. These results support the hypothesis that maturation of human NK cells has a discrete restriction until postnatal life, and provide a framework to better understand the increased susceptibility of fetuses and newborns to infection. PMID:28289707

  18. CD161 Defines a Transcriptional and Functional Phenotype across Distinct Human T Cell Lineages

    Directory of Open Access Journals (Sweden)

    Joannah R. Fergusson


    Full Text Available The C-type lectin CD161 is expressed by a large proportion of human T lymphocytes of all lineages, including a population known as mucosal-associated invariant T (MAIT cells. To understand whether different T cell subsets expressing CD161 have similar properties, we examined these populations in parallel using mass cytometry and mRNA microarray approaches. The analysis identified a conserved CD161++/MAIT cell transcriptional signature enriched in CD161+CD8+ T cells, which can be extended to CD161+ CD4+ and CD161+TCRγδ+ T cells. Furthermore, this led to the identification of a shared innate-like, TCR-independent response to interleukin (IL-12 plus IL-18 by different CD161-expressing T cell populations. This response was independent of regulation by CD161, which acted as a costimulatory molecule in the context of T cell receptor stimulation. Expression of CD161 hence identifies a transcriptional and functional phenotype, shared across human T lymphocytes and independent of both T cell receptor (TCR expression and cell lineage.

  19. Multiparametric flow cytometry for identification and fluorescence activated cell sorting of five distinct B-cell subpopulations in normal tonsil tissue. (United States)

    Kjeldsen, Malene Krag; Perez-Andres, Martin; Schmitz, Alexander; Johansen, Preben; Boegsted, Martin; Nyegaard, Mette; Gaihede, Michael; Bukh, Anne; Johnsen, Hans E; Orfao, Alberto; Dybkaer, Karen


    The purpose of this study was to establish a procedure capable of isolating distinct B-cell subpopulations from human tonsils as a basis for subsequent molecular analyses. Overall, 5 distinct B-cell subpopulations were purified from fresh tonsils based on their fluorescence surface marker expression: naive B cells, centroblasts, centrocytes, memory B cells, and plasmablasts. The immunophenotypic identity of the subpopulations was verified by quantitative real-time reverse transcriptase-polymerase chain reaction using the proliferation marker MKI-67 and 6 B-cell-associated differentiation markers (BACH2, BCL6, PAX5, IRF4, PRDM1, and XBP1). Furthermore, within the centroblast compartment, large and small centroblasts could be distinguished and large centroblasts were shown to proliferate with a morphologic appearance of a "centroblast"-like cell but with lower gene expression of the germinal center markers BCL6 and BACH2 vs small centroblasts. This study has established a detailed and fast procedure for simultaneous sorting of up to 5 distinct maturation-associated B-cell subpopulations from human tonsils.

  20. Distinct patterns of stomatitis with concurrent cetuximab and radiotherapy for head and neck squamous cell carcinoma. (United States)

    Pryor, D I; Burmeister, E; Burmeister, B H; Poulsen, M G; Porceddu, S V


    To describe the distinct patterns of severe anterior stomatitis seen with concurrent cetuximab and radiotherapy (RT) compared to chemoRT or altered fractionation RT (AFRT) and explore potential associations with clinical and dosimetric parameters. We reviewed acute toxicity data from 42 patients receiving cetuximab-RT and a matched cohort of 36 patients receiving chemoRT or AFRT. The occurrence of grade ≥3 oral toxicities was analysed with respect to clinical (age, gender, smoking/alcohol history, tumour subsite, grade of acneiform rash) and dosimetric parameters. Cetuximab-RT patients experienced higher rates of grade ≥3 cheilitis (26% vs 6%, p=0.01) and anterior stomatitis (38% vs 6%, p=0.002), despite these structures receiving low RT doses (median maximum dose to lips 9.3 Gy, anterior oral cavity 20 Gy). Multivariable analyses identified increasing severity of acneiform rash as the strongest predictor of grade ≥3 cheilitis whilst increasing RT dose was weakly correlated. A trend was observed for increasing pack years of smoking on univariate analysis only. The combination of cetuximab and low doses of RT to the anterior oral cavity has resulted in a distinctive pattern of cheilitis and anterior stomatitis in our patients. Further exploration of this phenomenon may yield additional insights into the interaction of cetuximab with RT in non-target tissues.

  1. pRB and E2F4 play distinct cell-intrinsic roles in fetal erythropoiesis. (United States)

    Zhang, Jing; Lee, Eunice Y; Liu, Yangang; Berman, Seth D; Lodish, Harvey F; Lees, Jacqueline A


    The retinoblastoma tumor suppressor protein pRB functions, at least in part, by directly binding to and modulating the activity of the E2F transcription factors. Previous studies have shown that both E2F4 and pRB play important roles in fetal erythropoiesis. Given that these two proteins interact directly we investigated the overlap of E2F4 and pRB function in this process by analyzing E2f4(-/-), conditional Rb knockout (Rb(1lox/1lox)), and compound E2f4(-/-);Rb(1lox/1lox) embryos. At E15.5 E2f4(-/-) and Rb(1lox/1lox) fetal erythroid cells display distinct abnormalities in their differentiation profiles. When cultured in vitro, both E2f4(-/-) and Rb(1lox/1lox) erythroid cells show defects in cell cycle progression. Surprisingly, analysis of cell cycle profiling suggests that E2F4 and pRB control cell cycle exit through different mechanisms. Moreover, only pRB, but not E2F4, promotes cell survival in erythroid cells. We observed an additive rather than a synergistic impact upon the erythroid defects in the compound E2f4(-/-);Rb(1lox/1lox) embryos. We further found that fetal liver macrophage development is largely normal regardless of genotype. Taken together, our results show that E2F4 and pRB play independent cell-intrinsic roles in fetal erythropoiesis.

  2. Surface Ig on rabbit lymphocytes. Rabbit B and T cells are distinct populations

    NARCIS (Netherlands)

    Bast, B J; Catty, D; Manten-Slingerland, R; Jansen, J T; Veldhuis, Dick H.; Roholl, P; Ballieux, R E


    Rabbit peripheral blood lymphocytes (PBL) were analyzed by immunofluorescence using anti-T cell conjugates and anti-Fab, anti-a1 allotype, anti-IgM and anti-IgA conjugates. In addition, T cells were demonstrated by rosetting with papain-treated homologous erythrocytes. Control experiments, using aci

  3. PLGA-encapsulated perfluorocarbon nanoparticles for simultaneous visualization of distinct cell populations by (19)F MRI

    NARCIS (Netherlands)

    Srinivas, M.; Tel, J.; Schreibelt, G.; Bonetto, F.J.; Cruz, L.J.; Amiri, H.; Heerschap, A.; Figdor, C.G.; Vries, I.J.M. de


    AIM: In vivo imaging using (19)F MRI is advantageous, due to its ability to quantify cell numbers, but is limited for a lack of suitable labels. Here, we formulate two stable and clinically applicable labels for tracking two populations of primary human dendritic cells (DCs) simultaneously. MATERIAL

  4. The epidermis comprises autonomous compartments maintained by distinct stem cell populations

    DEFF Research Database (Denmark)

    Page, Mahalia E; Lombard, Patrick; Ng, Felicia


    populations. In contrast, upon wounding, stem cell progeny from multiple compartments acquire lineage plasticity and make permanent contributions to regenerating tissue. We further show that oncogene activation in Lrig1(+ve) cells drives hyperplasia but requires auxiliary stimuli for tumor formation...

  5. P2X7R activation drives distinct IL-1 responses in dendritic cells compared to macrophages. (United States)

    Englezou, Pavlos C; Rothwell, Simon W; Ainscough, Joseph S; Brough, David; Landsiedel, Robert; Verkhratsky, Alexei; Kimber, Ian; Dearman, Rebecca J


    The P2X(7)R is a functionally distinct member of the P2X family of non-selective cation channels associated with rapid activation of the inflammasome complex and signalling interleukin (IL)-1β release in macrophages. The main focus of this investigation was to compare P2X(7)R-driven IL-1 production by primary murine bone marrow derived dendritic cells (BMDC) and macrophages (BMM). P2X(7)R expression in murine BMDC and BMM at both transcriptional (P2X(7)A variant) and protein levels was demonstrated. Priming with lipopolysaccharide (LPS) and receptor activation with adenosine triphosphate (ATP) resulted in markedly enhanced IL-1 (α and β) secretion in BMDC compared with BMM. In both cell types IL-1 production was profoundly inhibited with a P2X(7)R-specific inhibitor (A-740003) demonstrating that this release is predominantly a P2X(7)R-dependent process. These data also suggest that P2X(7)R and caspase-1 activation drive IL-1α release from BMDC. Both cell types expressed constitutively the gain-of-function P2X(7)K as well as the full P2X(7)A variant at equivalent levels. LPS priming reduced significantly levels of P2X(7)A but not P2X(7)K transcripts in both BMDC and BMM. P2X(7)R-induced pore formation, assessed by YO-PRO-1 dye uptake, was greater in BMDC, and these cells were protected from cell death. These data demonstrate that DC and macrophages display distinct patterns of cytokine regulation, particularly with respect to IL-1, as a consequence of cell-type specific differences in the physicochemical properties of the P2X(7)R. Understanding the cell-specific regulation of these cytokines is essential for manipulating such responses in health and disease.

  6. Coilin phosphomutants disrupt Cajal body formation, reduce cell proliferation and produce a distinct coilin degradation product.

    Directory of Open Access Journals (Sweden)

    Zunamys I Carrero

    Full Text Available Coilin is a nuclear phosphoprotein that accumulates in Cajal bodies (CBs. CBs participate in ribonucleoprotein and telomerase biogenesis, and are often found in cells with high transcriptional demands such as neuronal and cancer cells, but can also be observed less frequently in other cell types such as fibroblasts. Many proteins enriched within the CB are phosphorylated, but it is not clear what role this modification has on the activity of these proteins in the CB. Coilin is considered to be the CB marker protein and is essential for proper CB formation and composition in mammalian cells. In order to characterize the role of coilin phosphorylation on CB formation, we evaluated various coilin phosphomutants using transient expression. Additionally, we generated inducible coilin phosphomutant cell lines that, when used in combination with endogenous coilin knockdown, allow for the expression of the phosphomutants at physiological levels. Transient expression of all coilin phosphomutants except the phosphonull mutant (OFF significantly reduces proliferation. Interestingly, a stable cell line induced to express the coilin S489D phosphomutant displays nucleolar accumulation of the mutant and generates a N-terminal degradation product; neither of which is observed upon transient expression. A N-terminal degradation product and nucleolar localization are also observed in a stable cell line induced to express a coilin phosphonull mutant (OFF. The nucleolar localization of the S489D and OFF coilin mutants observed in the stable cell lines is decreased when endogenous coilin is reduced. Furthermore, all the phosphomutant cells lines show a significant reduction in CB formation when compared to wild-type after endogenous coilin knockdown. Cell proliferation studies on these lines reveal that only wild-type coilin and the OFF mutant are sufficient to rescue the reduction in proliferation associated with endogenous coilin depletion. These results emphasize

  7. Endocrine disrupting chemicals affect the adipogenic differentiation of mesenchymal stem cells in distinct ontogenetic windows

    Energy Technology Data Exchange (ETDEWEB)

    Biemann, Ronald, E-mail: [Department of Anatomy and Cell Biology, Martin Luther University, Faculty of Medicine, Halle (Germany); Navarrete Santos, Anne [Department of Anatomy and Cell Biology, Martin Luther University, Faculty of Medicine, Halle (Germany); Navarrete Santos, Alexander [Department of Cardiothoracic Surgery, Martin Luther University, Faculty of Medicine, Halle (Germany); Riemann, Dagmar [Department of Immunology, Martin Luther University, Faculty of Medicine, Halle (Germany); Knelangen, Julia [Department of Anatomy and Cell Biology, Martin Luther University, Faculty of Medicine, Halle (Germany); Blueher, Matthias [Department of Medicine, University of Leipzig, Leipzig (Germany); Koch, Holger [Institute for Prevention and Occupational Medicine of the German Social Accident Insurance, Institute of the Ruhr-University Bochum (IPA), Ruhr-University Bochum, Bochum (Germany); Fischer, Bernd [Department of Anatomy and Cell Biology, Martin Luther University, Faculty of Medicine, Halle (Germany)


    Highlights: Black-Right-Pointing-Pointer Endocrine disrupting chemicals affect adipogenesis in mesenchymal stem cells (MSC). Black-Right-Pointing-Pointer The adipogenic impact depends strongly on the window of exposure. Black-Right-Pointing-Pointer Bisphenol A reduces the potential of MSC to differentiate into adipocytes. Black-Right-Pointing-Pointer DEHP and TBT trigger the adipogenic differentiation of mesenchymal stem cells. Black-Right-Pointing-Pointer BPA, DEHP and TBT did not affect adipogenesis in embryonic stem cells. -- Abstract: Endocrine disrupting chemicals (EDC) like bisphenol A (BPA), bis(2-ethylhexyl)phthalate (DEHP) and tributyltin (TBT) are ubiquitously present in the environment and in human tissues. They bind to nuclear hormone receptors and affect cellular and developmental processes. In this study, we show that BPA, DEHP and TBT affect the adipogenic differentiation of murine mesenchymal stem cells (MSC, C3H/10T1/2) in a concentration-, stage- and compound-specific manner. C3H/10T1/2 cells and embryonic stem cells (CGR8) were exposed to BPA, DEHP or TBT at different stages of cell determination and differentiation (undifferentiated growth, adipogenic induction and terminal adipogenic differentiation). The final amount of differentiated adipocytes, cellular triglyceride content and mRNA expression of adipogenic marker genes (adiponectin, FABP4, PPAR{gamma}2, LPL) were quantified and compared with corresponding unexposed cells. BPA (10 {mu}M) decreased subsequent adipogenic differentiation of MSC, when cells were exposed during undifferentiated growth. In contrast, DEHP (100 {mu}M) during the hormonal induction period, and TBT (100 nM) in all investigated stages, enhanced adipogenesis. Importantly, exposure of undifferentiated murine embryonic stem cells did not show any effect of the investigated EDC on subsequent adipogenic differentiation.

  8. Evidence of distinct tumour-propagating cell populations with different properties in primary human hepatocellular carcinoma.

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    Federico Colombo

    Full Text Available BACKGROUND AND AIMS: Increasing evidence that a number of malignancies are characterised by tumour cell heterogeneity has recently been published, but there is still a lack of data concerning liver cancers. The aim of this study was to investigate and characterise tumour-propagating cell (TPC compartments within human hepatocellular carcinoma (HCC. METHODS: After long-term culture, we identified three morphologically different tumour cell populations in a single HCC specimen, and extensively characterised them by means of flow cytometry, fluorescence microscopy, karyotyping and microarray analyses, single cell cloning, and xenotransplantation in NOD/SCID/IL2Rγ/⁻ mice. RESULTS: The primary cell populations (hcc-1, -2 and -3 and two clones generated by means of limiting dilutions from hcc-1 (clone-1/7 and -1/8 differently expressed a number of tumour-associated stem cell markers, including EpCAM, CD49f, CD44, CD133, CD56, Thy-1, ALDH and CK19, and also showed different doubling times, drug resistance and tumorigenic potential. Moreover, we found that ALDH expression, in combination with CD44 or Thy-1 negativity or CD56 positivity identified subpopulations with a higher clonogenic potential within hcc-1, hcc-2 and hcc-3 primary cell populations, respectively. Karyotyping revealed the clonal evolution of the cell populations and clones within the primary tumour. Importantly, the primary tumour cell population with the greatest tumorigenic potential and drug resistance showed more chromosomal alterations than the others and contained clones with epithelial and mesenchymal features. CONCLUSIONS: Individual HCCs can harbor different self-renewing tumorigenic cell types expressing a variety of morphological and phenotypical markers, karyotypic evolution and different gene expression profiles. This suggests that the models of hepatic carcinogenesis should take into account TPC heterogeneity due to intratumour clonal evolution.

  9. KIF7 Controls the Proliferation of Cells of the Respiratory Airway through Distinct Microtubule Dependent Mechanisms.

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    Garry L Coles


    Full Text Available The cell cycle must be tightly coordinated for proper control of embryonic development and for the long-term maintenance of organs such as the lung. There is emerging evidence that Kinesin family member 7 (Kif7 promotes Hedgehog (Hh signaling during embryonic development, and its misregulation contributes to diseases such as ciliopathies and cancer. Kif7 encodes a microtubule interacting protein that controls Hh signaling through regulation of microtubule dynamics within the primary cilium. However, whether Kif7 has a function in nonciliated cells remains largely unknown. The role Kif7 plays in basic cell biological processes like cell proliferation or cell cycle progression also remains to be elucidated. Here, we show that Kif7 is required for coordination of the cell cycle, and inactivation of this gene leads to increased cell proliferation in vivo and in vitro. Immunostaining and transmission electron microscopy experiments show that Kif7dda/dda mutant lungs are hyperproliferative and exhibit reduced alveolar epithelial cell differentiation. KIF7 depleted C3H10T1/2 fibroblasts and Kif7dda/dda mutant mouse embryonic fibroblasts have increased growth rates at high cellular densities, suggesting that Kif7 may function as a general regulator of cellular proliferation. We ascertained that in G1, Kif7 and microtubule dynamics regulate the expression and activity of several components of the cell cycle machinery known to control entry into S phase. Our data suggest that Kif7 may function to regulate the maintenance of the respiratory airway architecture by controlling cellular density, cell proliferation, and cycle exit through its role as a microtubule associated protein.

  10. Distinct effects of SIRT1 in cancer and stromal cells on tumor promotion. (United States)

    Shin, Dong Hoon; Choi, Yong-Joon; Jin, Peng; Yoon, Haejin; Chun, Yang-Sook; Shin, Hyun-Woo; Kim, Ja-Eun; Park, Jong-Wan


    The lysyl deacetylase SIRT1 acts as a metabolic sensor in adjusting metabolic imbalance. To explore the role of SIRT1 in tumor-stroma interplay, we designed an in vivo tumor model using SIRT1-transgenic mice. B16F10 mouse melanoma grew more quickly in SIRT1-transgenic mice than in wild-type mice, whereas SIRT1-overexpressing one grew slowly in both mice. Of human tumors, SIRT1 expression in stromal fibroblasts was found to correlate with poor prognosis in ovarian cancer. B16F10 and human ovarian cancer (SKOV3 and SNU840) cells were more proliferative in co-culture with SIRT1-overexpressiong fibroblasts. In contrast, SIRT1 within cancer cells has a negative effect on cell proliferation. In conditioned media from SIRT1-overexpressing fibroblasts, matrix metalloproteinase-3 (MMP3) was identified in cytokine arrays to be secreted from fibroblasts SIRT1-dependently. Fibroblast-derived MMP3 stimulated cancer cell proliferation, and such a role of MMP3 was also demonstrated in cancer/fibroblast co-grafts. In conclusion, SIRT1 plays differential roles in cancer and stromal cells. SIRT1 in stromal cells promotes cancer growth by producing MMP3, whereas SIRT1 in cancer cells inhibits growth via an intracellular event. The present study provides a basis for setting new anticancer strategies targeting SIRT1.

  11. The distinct distributions of immunocompetent cells in rat dentin pulp after pulpotomy. (United States)

    Zhang, Min; Kokabu, Shoichiro; Nakatomi, Chihiro; Sugiyama, Goro; Matsuo, Kou; Jimi, Eijiro


    Pulpotomy involves the removal of the coronal portion of pulp, including the diseased tissue, with the intent of maintaining the vitality of the remaining pulpal tissue via a therapeutic dressing. Once odontoblasts suffer injuries, the differentiation of mesenchymal cells is induced from the precursor cell population in the dental pulp, and these cells are recruited to the injured site to differentiate into odontoblasts. However, the involvement of immunocompetent cells during pulpal regeneration remains unclear. Thus, the purpose of this study was to investigate the properties of macrophages that infiltrated wound healing sites in rats between 1 and 28 days after pulpotomy (dap). During the inflammatory phase, ED1(+) (CD68(+) ) macrophages significantly increased throughout root pulp, especially apical to the demarcation zone, and this population persisted until 3 dap before decreasing gradually until 28 dap. OX6(+) macrophages expressing class II MHC also increased in the apical pulp at 1 dap and declined thereafter. However, OX6(+) cells appeared prior to dentin bridge formation at 3 dap and appeared again apical to the dentin bridge during the healing stage at 14 dap. The shift from ED1(+) cells in the inflammation phase to OX6(+) cells during dentin bridge formation might contribute to wound healing.

  12. Members of the heat-shock protein 70 family promote cancer cell growth by distinct mechanisms

    DEFF Research Database (Denmark)

    Rohde, Mikkel; Daugaard, Mads; Jensen, Mette Hartvig;


    Whereas the stress-inducible heat-shock protein 70 (Hsp70) has gained plenty of attention as a putative target for tumor therapy, little is known about the role of other Hsp70 proteins in cancer. Here we present the first thorough analysis of the expression and function of the cytosolic Hsp70...... the survival of tumorigenic as well as nontumorigenic cells depended on Hsc70. Cancer cells depleted for Hsp70 and Hsp70-2 displayed strikingly different morphologies (detached and round vs. flat senescent-like), cell cycle distributions (G2/M vs. G1 arrest) and gene expression profiles. Only Hsp70-2 depletion...

  13. Amino acids and mTOR mediate distinct metabolic checkpoints in mammalian G1 cell cycle.

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    Mahesh Saqcena

    Full Text Available OBJECTIVE: In multicellular organisms, cell division is regulated by growth factors (GFs. In the absence of GFs, cells exit the cell cycle at a site in G1 referred to as the restriction point (R and enter a state of quiescence known as G0. Additionally, nutrient availability impacts on G1 cell cycle progression. While there is a vast literature on G1 cell cycle progression, confusion remains - especially with regard to the temporal location of R relative to nutrient-mediated checkpoints. In this report, we have investigated the relationship between R and a series of metabolic cell cycle checkpoints that regulate passage into S-phase. METHODS: We used double-block experiments to order G1 checkpoints that monitor the presence of GFs, essential amino acids (EEAs, the conditionally essential amino acid glutamine, and inhibition of mTOR. Cell cycle progression was monitored by uptake of [(3H]-thymidine and flow cytometry, and analysis of cell cycle regulatory proteins was by Western-blot. RESULTS: We report here that the GF-mediated R can be temporally distinguished from a series of late G1 metabolic checkpoints mediated by EAAs, glutamine, and mTOR - the mammalian/mechanistic target of rapamycin. R is clearly upstream from an EAA checkpoint, which is upstream from a glutamine checkpoint. mTOR is downstream from both the amino acid checkpoints, close to S-phase. Significantly, in addition to GF autonomy, we find human cancer cells also have dysregulated metabolic checkpoints. CONCLUSION: The data provided here are consistent with a GF-dependent mid-G1 R where cells determine whether it is appropriate to divide, followed by a series of late-G1 metabolic checkpoints mediated by amino acids and mTOR where cells determine whether they have sufficient nutrients to accomplish the task. Since mTOR inhibition arrests cells the latest in G1, it is likely the final arbiter for nutrient sufficiency prior to committing to replicating the genome.

  14. Burkholderia type VI secretion systems have distinct roles in eukaryotic and bacterial cell interactions

    DEFF Research Database (Denmark)

    Schwarz, Sandra; West, T Eoin; Boyer, Frédéric


    . From a group of 31 diverse bacteria, we identified several organisms that competed less effectively against wild-type B. thai than a strain lacking T6SS-1 function. Inactivation of T6SS-1 renders B. thai greatly more susceptible to cell contact-induced stasis by Pseudomonas putida, Pseudomonas...... fluorescens and Serratia proteamaculans-leaving it 100- to 1000-fold less fit than the wild-type in competition experiments with these organisms. Flow cell biofilm assays showed that T6S-dependent interbacterial interactions are likely relevant in the environment. B. thai cells lacking T6SS-1 were rapidly...

  15. Identification and Analysis of Distinct Features in Imaging Thin-Film Solar Cells: Preprint

    Energy Technology Data Exchange (ETDEWEB)

    Zaunbrecher, K. N.; Johnston, S. W.; Sites, J. R.


    Electroluminescence and photoluminescence (EL and PL) are two imaging techniques employed at NREL that are used to qualitatively evaluate solar cells. In this work, imaging lab-scale CdTe and CIGS devices provides information about small-area PV response, which will aid in determining the effects of non-uniformities on cell performance. EL, PL, and dark lock-in thermography signatures are first catalogued. Their responses to varying conditions are then studied. Further analysis includes acquiring spectral data, making microscopy measurements, and correlating luminescence to device performance. The goal of this work is to quantitatively determine non-uniformity effects on cell performance using rapid imaging techniques.

  16. Association of Neisseria gonorrhoeae Opa(CEA with dendritic cells suppresses their ability to elicit an HIV-1-specific T cell memory response.

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    Qigui Yu

    Full Text Available Infection with Neisseria gonorrhoeae (N. gonorrhoeae can trigger an intense local inflammatory response at the site of infection, yet there is little specific immune response or development of immune memory. Gonococcal surface epitopes are known to undergo antigenic variation; however, this is unlikely to explain the weak immune response to infection since individuals can be re-infected by the same serotype. Previous studies have demonstrated that the colony opacity-associated (Opa proteins on the N. gonorrhoeae surface can bind human carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1 on CD4⁺ T cells to suppress T cell activation and proliferation. Interesting in this regard, N. gonorrhoeae infection is associated with impaired HIV-1 (human immunodeficiency virus type 1-specific cytotoxic T-lymphocyte (CTL responses and with transient increases in plasma viremia in HIV-1-infected patients, suggesting that N. gonorrhoeae may also subvert immune responses to co-pathogens. Since dendritic cells (DCs are professional antigen presenting cells (APCs that play a key role in the induction of an adaptive immune response, we investigated the effects of N. gonorrhoeae Opa proteins on human DC activation and function. While morphological changes reminiscent of DC maturation were evident upon N. gonorrhoeae infection, we observed a marked downregulation of DC maturation marker CD83 when the gonococci expressing CEACAM1-specific Opa(CEA, but not other Opa variants. Consistent with a gonococcal-induced defect in maturation, Opa(CEA binding to CEACAM1 reduced the DCs' capacity to stimulate an allogeneic T cell proliferative response. Moreover, Opa(CEA-expressing N. gonorrhoeae showed the potential to impair DC-dependent development of specific adaptive immunity, since infection with Opa(CEA-positive gonococci suppressed the ability of DCs to stimulate HIV-1-specific memory CTL responses. These results reveal a novel mechanism to explain

  17. Elicitation threshold of cobalt chloride

    DEFF Research Database (Denmark)

    Fischer, Louise A; Johansen, Jeanne D; Voelund, Aage


    BACKGROUND: Cobalt is a strong skin sensitizer (grade 5 of 5 in the guinea-pig maximization test) that is used in various industrial and consumer applications. To prevent sensitization to cobalt and elicitation of allergic cobalt dermatitis, information about the elicitation threshold level...... of cobalt is important. OBJECTIVE: To identify the dermatitis elicitation threshold levels in cobalt-allergic individuals. MATERIALS AND METHODS: Published patch test dose-response studies were reviewed to determine the elicitation dose (ED) levels in dermatitis patients with a previous positive patch test...... reaction to cobalt. A logistic dose-response model was applied to data collected from the published literature to estimate ED values. The 95% confidence interval (CI) for the ratio of mean doses that can elicit a reaction in 10% (ED(10)) of a population was calculated with Fieller's method. RESULTS...

  18. Immunization with Ty21a live oral typhoid vaccine elicits crossreactive multifunctional CD8+ T-cell responses against Salmonella enterica serovar Typhi, S. Paratyphi A, and S. Paratyphi B in humans. (United States)

    Wahid, R; Fresnay, S; Levine, M M; Sztein, M B


    Previously we have extensively characterized Salmonella enterica serovar Typhi (S. Typhi)-specific cell-mediated immune (CMI) responses in volunteers orally immunized with the licensed Ty21a typhoid vaccine. In this study we measured Salmonella-specific multifunctional (MF) CD8+ T-cell responses to further investigate whether Ty21a elicits crossreactive CMI against S. Paratyphi A and S. Paratyphi B that also cause enteric fever. Ty21a-elicited crossreactive CMI responses against all three Salmonella serotypes were predominantly observed in CD8+ T effector/memory (T(EM)) and, to a lesser extent, in CD8+CD45RA+ T(EM) (T(EMRA)) subsets. These CD8+ T-cell responses were largely mediated by MF cells coproducing interferon-γ and macrophage inflammatory protein-1β and expressing CD107a with or without tumor necrosis factor-α. Significant proportions of Salmonella-specific MF cells expressed the gut-homing molecule integrin α4β7. In most subjects, similar MF responses were observed to S. Typhi and S. Paratyphi B, but not to S. Paratyphi A. These results suggest that Ty21a elicits MF CMI responses against Salmonella that could be critical in clearing the infection. Moreover, because S. Paratyphi A is a major public concern and Ty21a was shown in field studies not to afford cross-protection to S. Paratyphi A, these results will be important in developing a S. Typhi/S. Paratyphi A bivalent vaccine against enteric fevers.

  19. Transcriptional Reprogramming of Mature CD4+ T helper Cells generates distinct MHC class II-restricted Cytotoxic T Lymphocytes (United States)

    Mucida, Daniel; Husain, Mohammad Mushtaq; Muroi, Sawako; van Wijk, Femke; Shinnakasu, Ryo; Naoe, Yoshinori; Reis, Bernardo Sgarbi; Huang, Yujun; Lambolez, Florence; Docherty, Michael; Attinger, Antoine; Shui, Jr-Wen; Kim, Gisen; Lena, Christopher J.; Sakaguchi, Shinya; Miyamoto, Chizuko; Wang, Peng; Atarashi, Koji; Park, Yunji; Nakayama, Toshinori; Honda, Kenya; Ellmeier, Wilfried; Kronenberg, Mitchell; Taniuchi, Ichiro; Cheroutre, Hilde


    TCRαβ thymocytes differentiate to either CD8αβ cytotoxic T lymphocytes or CD4+ T helper cells. This functional dichotomy is controlled by key transcription factors, including the T helper master regulator, ThPOK, which suppresses the cytolytic program in MHC class II-restricted CD4+ thymocytes. ThPOK continues to repress CD8-lineage genes in mature CD4+ T cells, even as they differentiate to T helper effector subsets. Here we show that the T helper-fate was not fixed and that mature antigen-stimulated CD4+ T cells could terminate Thpok expression and reactivate CD8-lineage genes. This unexpected plasticity resulted in the post-thymic termination of the T helper-program and the functional differentiation of distinct MHC class II-restricted CD4+ cytotoxic T lymphocytes. PMID:23334788

  20. Several distinct properties of the IgE repertoire determine effector cell degranulation in response to allergen challenge

    DEFF Research Database (Denmark)

    Christensen, Lars Harder; Holm, Jens-Christian; Lund, Gitte;


    BACKGROUND: On cross-linking of receptor-bound IgE antibodies by allergens, effector cells (basophils and mast cells) involved in type I allergic reactions degranulate and release the potent chemical mediators stored inside their granules. Total and allergen-specific IgE concentrations, Ig......E affinity for allergen, and IgE clonality are all distinct properties of allergic patients' IgE repertoires. However, the inability to isolate individual IgE antibodies from allergic patients' sera presents a major barrier to understanding the importance of patient-specific IgE repertoires...... for the manifestation and severity of allergic symptoms. OBJECTIVE: We sought to investigate how individual properties of an IgE repertoire affect effector cell degranulation. METHODS: A panel of recombinant IgE (rIgE) antibodies specific for the major house dust mite allergen Der p 2 was developed and characterized...

  1. Novel Model of Tendon Regeneration Reveals Distinct Cell Mechanisms Underlying Regenerative and Fibrotic Tendon Healing (United States)

    Howell, Kristen; Chien, Chun; Bell, Rebecca; Laudier, Damien; Tufa, Sara F.; Keene, Douglas R.; Andarawis-Puri, Nelly; Huang, Alice H.


    To date, the cell and molecular mechanisms regulating tendon healing are poorly understood. Here, we establish a novel model of tendon regeneration using neonatal mice and show that neonates heal via formation of a ‘neo-tendon’ that differentiates along the tendon specific lineage with functional restoration of gait and mechanical properties. In contrast, adults heal via fibrovascular scar, aberrant differentiation toward cartilage and bone, with persistently impaired function. Lineage tracing identified intrinsic recruitment of Scx-lineage cells as a key cellular mechanism of neonatal healing that is absent in adults. Instead, adult Scx-lineage tenocytes are not recruited into the defect but transdifferentiate into ectopic cartilage; in the absence of tenogenic cells, extrinsic αSMA-expressing cells persist to form a permanent scar. Collectively, these results establish an exciting model of tendon regeneration and uncover a novel cellular mechanism underlying regenerative vs non-regenerative tendon healing. PMID:28332620

  2. Cytologic diagnosis of acinic cell carcinoma of minor salivary gland: a distinct rarely described entity

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    Rana Sherwani


    Full Text Available A rare case of acinic cell carcinoma of minor salivary gland with cervical lymph node metastasis in a 50-year-old man is reported and the literature regarding this type of tumor is reviewed. These tumors arise from either an intercalated duct stem cell or the reserve cell of the salivary gland terminal tubule but not from both simultaneously. Rarely these neoplasms arise from more mature acinar cells. It is clear that these tumors behave ominously. The 25 year determinate survival rate is 50%, with a 20% incidence of metastasis. Surgical excision is the treatment of choice. Radiotherapy, especially neutron therapy, has a place in the treatment of this tumor but the role of chemotherapy is not exactly known at this time.

  3. Influence of electrospun scaffolds prepared from distinct polymers on proliferation and viability of endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Matveeva, V. G., E-mail:; Antonova, L. V., E-mail:; Velikanova, E. A.; Sergeeva, E. A.; Krivkina, E. O.; Glushkova, T. V.; Kudryavtseva, Yu. A.; Barbarash, O. L.; Barbarash, L. S. [Research Institute for Complex Issues of Cardiovascular Diseases, Kemerovo, 650002 (Russian Federation)


    We compared electrospun nonwoven scaffolds from polylactic acid (PLA), polycaprolactone (PCL), and polyhydroxybutyrate/valerate (PHBV)/polycaprolactone (PHBV/PCL). The surface of PHBV/PCL and PCL scaffolds was highly porous and consisted of randomly distributed fibers, whilst the surface of PLA scaffolds consisted of thin straight fibers, which located more sparsely, forming large pores. Culture of EA.hy 926 endothelial cells on these scaffolds during 7 days and further fluorescent microscopy demonstrated that the surface of PHBV/PCL scaffolds was most favorable for efficient adhesion, proliferation, and viability of endothelial cells. The lowest proliferation rate and cell viability were detected on PLA scaffolds. Therefore, PHBV/PCL electrospun nonwoven scaffolds demonstrated the best results regarding endothelial cell proliferation and viability as compared to PCL and PLA scaffolds.

  4. Distinct effects of rotenone, 1-methyl-4-phenylpyridinium and 6-hydroxydopamine on cellular bioenergetics and cell death.

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    Samantha Giordano

    Full Text Available Parkinson's disease is characterized by dopaminergic neurodegeneration and is associated with mitochondrial dysfunction. The bioenergetic susceptibility of dopaminergic neurons to toxins which induce Parkinson's like syndromes in animal models is then of particular interest. For example, rotenone, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP and its active metabolite 1-methyl-4-phenylpyridinium (MPP(+, and 6-hydroxydopamine (6-OHDA, have been shown to induce dopaminergic cell death in vivo and in vitro. Exposure of animals to these compounds induce a range of responses characteristics of Parkinson's disease, including dopaminergic cell death, and Reactive Oxygen Species (ROS production. Here we test the hypothesis that cellular bioenergetic dysfunction caused by these compounds correlates with induction of cell death in differentiated dopaminergic neuroblastoma SH-SY5Y cells. At increasing doses, rotenone induced significant cell death accompanied with caspase 3 activation. At these concentrations, rotenone had an immediate inhibition of mitochondrial basal oxygen consumption rate (OCR concomitant with a decrease of ATP-linked OCR and reserve capacity, as well as a stimulation of glycolysis. MPP(+ exhibited a different behavior with less pronounced cell death at doses that nearly eliminated basal and ATP-linked OCR. Interestingly, MPP(+, unlike rotenone, stimulated bioenergetic reserve capacity. The effects of 6-OHDA on bioenergetic function was markedly less than the effects of rotenone or MPP(+ at cytotoxic doses, suggesting a mechanism largely independent of bioenergetic dysfunction. These studies suggest that these dopaminergic neurotoxins induce cell death through distinct mechanisms and differential effects on cellular bioenergetics.

  5. A Pathway Switch Directs BAFF Signaling to Distinct NFκB Transcription Factors in Maturing and Proliferating B Cells

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    Jonathan V. Almaden


    Full Text Available BAFF, an activator of the noncanonical NFκB pathway, provides critical survival signals during B cell maturation and contributes to B cell proliferation. We found that the NFκB family member RelB is required ex vivo for B cell maturation, but cRel is required for proliferation. Combined molecular network modeling and experimentation revealed Nfkb2 p100 as a pathway switch; at moderate p100 synthesis rates in maturing B cells, BAFF fully utilizes p100 to generate the RelB:p52 dimer, whereas at high synthesis rates, p100 assembles into multimeric IκBsome complexes, which BAFF neutralizes in order to potentiate cRel activity and B cell expansion. Indeed, moderation of p100 expression or disruption of IκBsome assembly circumvented the BAFF requirement for full B cell expansion. Our studies emphasize the importance of p100 in determining distinct NFκB network states during B cell biology, which causes BAFF to have context-dependent functional consequences.

  6. Different concentrations of kaempferol distinctly modulate murine embryonic stem cell function. (United States)

    Correia, Marcelo; Rodrigues, Ana S; Perestrelo, Tânia; Pereira, Sandro L; Ribeiro, Marcelo F; Sousa, Maria I; Ramalho-Santos, João


    Kaempferol (3,4',5,7-tetrahydroxyflavone) is a natural flavonoid with several beneficial and protective effects. It has been demonstrated that kaempferol has anticancer properties, particularly due to its effects on proliferation, apoptosis and the cell cycle. However, possible effects on pluripotent embryonic stem cell function have not yet been addressed. Embryonic stem cells have the ability to self-renew and to differentiate into all three germ layers with potential applications in regenerative medicine and in vitro toxicology. We show that exposure of murine embryonic stem cells (mESC) to high concentrations of kaempferol (200 μM) leads to decreased cell numbers, although the resulting smaller cell colonies remain pluripotent. However, lower concentrations of this compound (20 μM) increase the expression of pluripotency markers in mESCs. Mitochondrial membrane potential and mitochondrial mass are not affected, but a dose-dependent increase in apoptosis takes place. Moreover, mESC differentiation is impaired by kaempferol, which was not related to apoptosis induction. Our results show that low concentrations of kaempferol can be beneficial for pluripotency, but inhibit proper differentiation of mESCs. Additionally, high concentrations induce apoptosis and increase mitochondrial reactive oxygen species (ROS).

  7. Distinct cell stress responses induced by ATP restriction in quiescent human fibroblasts

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    Nirupama Yalamanchili


    Full Text Available Quiescence is the prevailing state of many cell types under homeostatic conditions. Yet, surprisingly little is known about how quiescent cells respond to energetic and metabolic challenges. To better understand compensatory responses of quiescent cells to metabolic stress, we established, in human primary dermal fibroblasts, an experimental ‘energy restriction’ model. Quiescence was achieved by short-term culture in serum-deprived media and ATP supply restricted using a combination of glucose transport inhibitors and mitochondrial uncouplers. In aggregate, these measures led to markedly reduced intracellular ATP levels while not compromising cell viability over the observation period of 48 h. Analysis of the transcription factor landscape induced by this treatment revealed alterations in several signal transduction nodes beyond the expected biosynthetic adaptations. These included increased abundance of NF-κB regulated transcription factors and altered transcription factor subsets regulated by Akt and p53. The observed changes in gene regulation and corresponding alterations in key signaling nodes are likely to contribute to cell survival at intracellular ATP concentrations substantially below those achieved by growth factor deprivation alone. This experimental model provides a benchmark for the investigation of cell survival pathways and related molecular targets that are associated with restricted energy supply associated with biological aging and metabolic diseases.

  8. SOX2 co-occupies distal enhancer elements with distinct POU factors in ESCs and NPCs to specify cell state.

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    Michael A Lodato

    Full Text Available SOX2 is a master regulator of both pluripotent embryonic stem cells (ESCs and multipotent neural progenitor cells (NPCs; however, we currently lack a detailed understanding of how SOX2 controls these distinct stem cell populations. Here we show by genome-wide analysis that, while SOX2 bound to a distinct set of gene promoters in ESCs and NPCs, the majority of regions coincided with unique distal enhancer elements, important cis-acting regulators of tissue-specific gene expression programs. Notably, SOX2 bound the same consensus DNA motif in both cell types, suggesting that additional factors contribute to target specificity. We found that, similar to its association with OCT4 (Pou5f1 in ESCs, the related POU family member BRN2 (Pou3f2 co-occupied a large set of putative distal enhancers with SOX2 in NPCs. Forced expression of BRN2 in ESCs led to functional recruitment of SOX2 to a subset of NPC-specific targets and to precocious differentiation toward a neural-like state. Further analysis of the bound sequences revealed differences in the distances of SOX and POU peaks in the two cell types and identified motifs for additional transcription factors. Together, these data suggest that SOX2 controls a larger network of genes than previously anticipated through binding of distal enhancers and that transitions in POU partner factors may control tissue-specific transcriptional programs. Our findings have important implications for understanding lineage specification and somatic cell reprogramming, where SOX2, OCT4, and BRN2 have been shown to be key factors.

  9. Pilot Study on the Use of DNA Priming Immunization to Enhance Y. pestis LcrV-Specific B Cell Responses Elicited by a Recombinant LcrV Protein Vaccine

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    Wei Li


    Full Text Available Recent studies indicate that DNA immunization is powerful in eliciting antigen-specific antibody responses in both animal and human studies. However, there is limited information on the mechanism of this effect. In particular, it is not known whether DNA immunization can also enhance the development of antigen-specific B cell development. In this report, a pilot study was conducted using plague LcrV immunogen as a model system to determine whether DNA immunization is able to enhance LcrV-specific B cell development in mice. Plague is an acute and often fatal infectious disease caused by Yersinia pestis (Y. pestis. Humoral immune responses provide critical protective immunity against plague. Previously, we demonstrated that a DNA vaccine expressing LcrV antigen can protect mice from lethal mucosal challenge. In the current study, we further evaluated whether the use of a DNA priming immunization is able to enhance the immunogenicity of a recombinant LcrV protein vaccine, and in particular, the development of LcrV-specific B cells. Our data indicate that DNA immunization was able to elicit high-level LcrV antibody responses when used alone or as part of a prime-boost immunization approach. Most significantly, DNA immunization was also able to increase the levels of LcrV-specific B cell development. The finding that DNA immunization can enhance antigen-specific B cell responses is highly significant and will help guide similar studies in other model antigen systems.

  10. Distinct and atypical intrinsic and extrinsic cell death pathways between photoreceptor cell types upon specific ablation of Ranbp2 in cone photoreceptors.

    Directory of Open Access Journals (Sweden)

    Kyoung-In Cho


    Full Text Available Non-autonomous cell-death is a cardinal feature of the disintegration of neural networks in neurodegenerative diseases, but the molecular bases of this process are poorly understood. The neural retina comprises a mosaic of rod and cone photoreceptors. Cone and rod photoreceptors degenerate upon rod-specific expression of heterogeneous mutations in functionally distinct genes, whereas cone-specific mutations are thought to cause only cone demise. Here we show that conditional ablation in cone photoreceptors of Ran-binding protein-2 (Ranbp2, a cell context-dependent pleiotropic protein linked to neuroprotection, familial necrotic encephalopathies, acute transverse myelitis and tumor-suppression, promotes early electrophysiological deficits, subcellular erosive destruction and non-apoptotic death of cones, whereas rod photoreceptors undergo cone-dependent non-autonomous apoptosis. Cone-specific Ranbp2 ablation causes the temporal activation of a cone-intrinsic molecular cascade highlighted by the early activation of metalloproteinase 11/stromelysin-3 and up-regulation of Crx and CoREST, followed by the down-modulation of cone-specific phototransduction genes, transient up-regulation of regulatory/survival genes and activation of caspase-7 without apoptosis. Conversely, PARP1+ -apoptotic rods develop upon sequential activation of caspase-9 and caspase-3 and loss of membrane permeability. Rod photoreceptor demise ceases upon cone degeneration. These findings reveal novel roles of Ranbp2 in the modulation of intrinsic and extrinsic cell death mechanisms and pathways. They also unveil a novel spatiotemporal paradigm of progression of neurodegeneration upon cell-specific genetic damage whereby a cone to rod non-autonomous death pathway with intrinsically distinct cell-type death manifestations is triggered by cell-specific loss of Ranbp2. Finally, this study casts new light onto cell-death mechanisms that may be shared by human dystrophies with distinct

  11. Distinct Gut-Derived Bacteria Differentially Affect Three Types of Antigen-Presenting Cells and Impact on NK- and T-Cell Responses

    DEFF Research Database (Denmark)

    Fink, Lisbeth Nielsen; Hansen, Anne Marie Valentin; Frøkiær, Hanne

    Objectives Gut bacteria are assumed essential for development and maintenance of a balanced immune system. Specifically, stimulation of antigen-presenting cells (APCs) by gut bacteria is important for polarisation of the immune response. This experiment was designed to reveal similarities...... and differences between the reaction patterns of three types of human APCs when stimulated with intestinal bacteria. Furthermore, the effect of these APCs on NK-cells and T-cells was examined. Methodology The APCs used in this study were blood monocytes, blood dendritic cells, and dendritic cells differentiated...... previously been examined, but this study revealed that their effect on other kinds of APCs is markedly different. When APCs matured by different bacteria were added to either NK-cells or T-cells, different APCs combined with distinct strains of bacteria caused the production of varying amounts of cytokines...

  12. Effects of Corroded and Non-Corroded Biodegradable Mg and Mg Alloys on Viability, Morphology and Differentiation of MC3T3-E1 Cells Elicited by Direct Cell/Material Interaction.

    Directory of Open Access Journals (Sweden)

    Sepideh Mostofi

    Full Text Available This study investigated the effect of biodegradable Mg and Mg alloys on selected properties of MC3T3-E1 cells elicited by direct cell/material interaction. The chemical composition and morphology of the surface of Mg and Mg based alloys (Mg2Ag and Mg10Gd were analysed by scanning electron microscopy (SEM and EDX, following corrosion in cell culture medium for 1, 2, 3 and 8 days. The most pronounced difference in surface morphology, namely crystal formation, was observed when Pure Mg and Mg2Ag were immersed in cell medium for 8 days, and was associated with an increase in atomic % of oxygen and a decrease of surface calcium and phosphorous. Crystal formation on the surface of Mg10Gd was, in contrast, negligible at all time points. Time-dependent changes in oxygen, calcium and phosphorous surface content were furthermore not observed for Mg10Gd. MC3T3-E1 cell viability was reduced by culture on the surfaces of corroded Mg, Mg2Ag and Mg10Gd in a corrosion time-independent manner. Cells did not survive when cultured on 3 day pre-corroded Pure Mg and Mg2Ag, indicating crystal formation to be particular detrimental in this regard. Cell viability was not affected when cells were cultured on non-corroded Mg and Mg alloys for up to 12 days. These results suggest that corrosion associated changes in surface morphology and chemical composition significantly hamper cell viability and, thus, that non-corroded surfaces are more conducive to cell survival. An analysis of the differentiation potential of MC3T3-E1 cells cultured on non-corroded samples based on measurement of Collagen I and Runx2 expression, revealed a down-regulation of these markers within the first 6 days following cell seeding on all samples, despite persistent survival and proliferation. Cells cultured on Mg10Gd, however, exhibited a pronounced upregulation of collagen I and Runx2 between days 8 and 12, indicating an enhancement of osteointegration by this alloy that could be valuable for

  13. Distinct Functions of Different scl Isoforms in Zebrafish Definitive Hematopoietic Stem Cell Initiation and Maintenance (United States)

    Lan, Yahui


    The establishment of entire blood system relies on the multi-potent hematopoietic stem cells (HSCs), thus identifying the molecular mechanism in HSC generation is of importance for not only complementing the fundamental knowledge in stem cell biology, but also providing insights to the regenerative therapies. Recent researches have documented the formation of nascent HSCs through a direct transition from ventral aortic endothelium, named as endothelial hematopoietic transition (EHT) process. However, the precise genetic program engaged in this process remains largely elusive. The transcription factor scl plays pivotal and conserved roles in embryonic and adult hematopoiesis from teleosts to mammals. Our lab have previously identified a new truncated scl isoform, scl-beta, which is indispensible for the specification of HSCs in the ventral wall of dorsal aorta (VDA), the zebrafish equivalent of mammalian fetal hematopoietic organ. Here we observe that, by combining time-lapse confocal imaging of transgenic zebrafish and genetic epistasis analysis, scl-beta is expressed in a subset of ventral aortic endothelial cells and critical for their forthcoming transformation to hemogenic endothelium; in contrast, runx1 is required downstream to govern the successful egress of the hemogenic endothelial cells to become naive HSCs. In addition, the traditional known full-length scl-alpha isoform is firstly evidenced to be required for the maintenance or survival of newly formed HSCs in VDA. Collectively our data has established the genetic hierarchy controlling discrete steps in the consecutive process of HSC formation from endothelial cells and further development in VDA.

  14. The requirements for natural Th17 cell development are distinct from those of conventional Th17 cells


    Kim, Jiyeon S.; Smith-Garvin, Jennifer E; Koretzky, Gary A.; Jordan, Martha S.


    CD4+ T helper 17 (Th17) cells play a critical role in the adaptive immune response against extracellular pathogens. Most studies to date have focused on understanding the differentiation of Th17 cells from naive CD4+ T cells in peripheral effector sites. However, Th17 cells are present in the thymus. In this study, we demonstrate that a population of Th17 cells, natural Th17 cells (nTh17 cells), which acquire effector function during development in the thymus before peripheral antigen exposur...

  15. Distinct Dasatinib-Induced Mechanisms of Apoptotic Response and Exosome Release in Imatinib-Resistant Human Chronic Myeloid Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Juan Liu


    Full Text Available Although dasatinib is effective in most imatinib mesylate (IMT-resistant chronic myeloid leukemia (CML patients, the underlying mechanism of its effectiveness in eliminating imatinib-resistant cells is only partially understood. This study investigated the effects of dasatinib on signaling mechanisms driving-resistance in imatinib-resistant CML cell line K562 (K562RIMT. Compared with K562 control cells, exsomal release, the phosphoinositide 3-kinase (PI3K/protein kinase B (Akt/ mammalian target of rapamycin (mTOR signaling and autophagic activity were increased significantly in K562RIMT cells and mTOR-independent beclin-1/Vps34 signaling was shown to be involved in exosomal release in these cells. We found that Notch1 activation-mediated reduction of phosphatase and tensin homolog (PTEN was responsible for the increased Akt/mTOR activities in K562RIMT cells and treatment with Notch1 γ-secretase inhibitor prevented activation of Akt/mTOR. In addition, suppression of mTOR activity by rapamycin decreased the level of activity of p70S6K, induced upregulation of p53 and caspase 3, and led to increase of apoptosis in K562RIMT cells. Inhibition of autophagy by spautin-1 or beclin-1 knockdown decreased exosomal release, but did not affect apoptosis in K562RIMT cells. In summary, in K562RIMT cells dasatinib promoted apoptosis through downregulation of Akt/mTOR activities, while preventing exosomal release and inhibiting autophagy by downregulating expression of beclin-1 and Vps34. Our findings reveal distinct dasatinib-induced mechanisms of apoptotic response and exosomal release in imatinib-resistant CML cells.

  16. P2X7R activation drives distinct IL-1 responses in dendritic cells compared to macrophages


    Englezou, Pavlos C.; Rothwell, Simon W.; Ainscough, Joseph S.; Brough, David; Landsiedel, Robert; Verkhratsky, Alexei; Kimber, Ian; Dearman, Rebecca J


    The P2X7R is a functionally distinct member of the P2X family of non-selective cation channels associated with rapid activation of the inflammasome complex and signalling interleukin (IL)-1β release in macrophages. The main focus of this investigation was to compare P2X7R-driven IL-1 production by primary murine bone marrow derived dendritic cells (BMDC) and macrophages (BMM). P2X7R expression in murine BMDC and BMM at both transcriptional (P2X7A variant) and protein levels was demonstrated....

  17. Chronic mast cell leukemia: a novel leukemia-variant with distinct morphological and clinical features. (United States)

    Valent, Peter; Sotlar, Karl; Sperr, Wolfgang R; Reiter, Andreas; Arock, Michel; Horny, Hans-Peter


    Mast cell leukemia (MCL) is a rare form of systemic mastocytosis characterized by leukemic expansion of mostly immature mast cells, organ damage, drug-resistance, and a poor prognosis. Even when treated with chemotherapy, most patients have a life-expectancy of less than one year. However, there are rare patients with MCL in whom the condition is less aggressive and does not cause organ damage within a short time. In these patients, mast cells exhibit a more mature morphology when compared to acute MCL. A recently proposed classification suggests that these cases are referred to as chronic MCL. In the present article, we discuss clinical, histopathological and morphological aspects of acute and chronic MCL.

  18. Distinct host cell fates for human malignant melanoma targeted by oncolytic rodent parvoviruses. (United States)

    Vollmers, Ellen M; Tattersall, Peter


    The rodent parvoviruses are known to be oncoselective, and lytically infect many transformed human cells. Because current therapeutic regimens for metastatic melanoma have low response rates and have little effect on improving survival, this disease is a prime candidate for novel approaches to therapy, including oncolytic parvoviruses. Screening of low-passage, patient-derived melanoma cell lines for multiplicity-dependent killing by a panel of five rodent parvoviruses identified LuIII as the most melanoma-lytic. This property was mapped to the LuIII capsid gene, and an efficiently melanoma tropic chimeric virus shown to undergo three types of interaction with primary human melanoma cells: (1) complete lysis of cultures infected at very low multiplicities; (2) acute killing resulting from viral protein synthesis and DNA replication, without concomitant expansion of the infection, due to failure to export progeny virions efficiently; or (3) complete resistance that operates at an intracellular step following virion uptake, but preceding viral transcription.

  19. Variability in the recognition of distinctive immunofluorescence patterns in different brands of HEp-2 cell slides

    Directory of Open Access Journals (Sweden)

    Alessandra Dellavance


    Full Text Available INTRODUCTION: Indirect immunofluorescence on HEp-2 cells is considered the gold standard for the detection of autoantibodies against cellular antigens. However, the culture conditions, cell fixation and permeabilization processes interfere directly in the preservation and spatial distribution of antigens. Therefore, one can assume that certain peculiarities in the processing of cellular substrate may affect the recognition of indirect immunofluorescence patterns associated with several autoantibodies. OBJECTIVE: To evaluate a panel of serum samples representing nuclear, nucleolar, cytoplasmic, mitotic apparatus, and chromosome plate patterns on HEp-2 cell substrates from different suppliers. MATERIALS AND METHODS: Seven blinded observers, independent from the three selected reference centers, evaluated 17 samples yielding different nuclear, nucleolar, cytoplasmic and mitotic apparatus patterns on HEp-2 cell slides from eight different brands. The slides were coded to maintain confidentiality of both brands and participating centers. RESULTS: The 17 HEp-2 cell patterns were identified on most substrates. Nonetheless, some slides showed deficit in the expression of several patterns: nuclear coarse speckled/U1-ribonucleoprotein associated with antibodies against RNP (U1RNP, centromeric protein F (CENP-F, proliferating cell nuclear antigen (PCNA, cytoplasmic fine speckled associated with anti-Jo-1 antibodies (histidyl synthetase, nuclear mitotic apparatus protein 1 (NuMA-1 and nuclear mitotic apparatus protein 2 (NuMA-2. CONCLUSION: Despite the overall good quality of the assessed HEp-2 substrates, there was considerable inconsistency in results among different commercial substrates. The variations may be due to the evaluated batches, hence generalizations cannot be made as to the respective brands. It is recommended that each new batch or new brand be tested with a panel of reference sera representing the various patterns.

  20. Multiple steady states with distinct cellular metabolism in continuous culture of mammalian cells. (United States)

    Europa, A F; Gambhir, A; Fu, P C; Hu, W S


    Mammalian cells have the ability to proliferate under different nutrient environments by utilizing different combinations of the nutrients, especially glucose and the amino acids. Under the conditions often used in in vitro cultivation, the cells consume glucose and amino acids in great excess of what is needed for making up biomass and products. They also produce large amounts of metabolites with lactate, ammonia, and some non-essential amino acids such as alanine as the most dominant ones. By controlling glucose and glutamine at low levels, cellular metabolism can be altered and can result in reduced glucose and glutamine consumption as well as in reduced metabolite formation. Using a fed-batch reactor to manipulate glucose at a low level (as compared to a typical batch culture), cell metabolism was altered to a state with substantially reduced lactate production. The culture was then switched to a continuous mode and allowed to reach a steady-state. At this steady-state, the concentrations of cells and antibody were substantially higher than a control culture that was initiated from a batch culture without first altering cellular metabolism. The lactate and other metabolite concentrations were also substantially reduced as compared to the control culture. This newly observed steady-state was achieved at the same dilution rate and feed medium as the control culture. The paths leading to the two steady-states, however, were different. These results demonstrate steady-state multiplicity. At this new steady-state, not only was glucose metabolism altered, but the metabolism of amino acids was altered as well. The amino acid metabolism in the new steady-state was more balanced, and the excretion of non-essential amino acids and ammonia was substantially lower. This approach of reaching a more desirable steady-state with higher concentrations of cells and product opens a new avenue for high-density- and high-productivity-cell culture.

  1. Multiparametric profiling of non–small-cell lung cancers reveals distinct immunophenotypes (United States)

    Lizotte, Patrick H.; Ivanova, Elena V.; Awad, Mark M.; Jones, Robert E.; Keogh, Lauren; Liu, Hongye; Dries, Ruben; Herter-Sprie, Grit S.; Santos, Abigail; Feeney, Nora B.; Paweletz, Cloud P.; Kulkarni, Meghana M.; Bass, Adam J.; Rustgi, Anil K.; Yuan, Guo-Cheng; Kufe, Donald W.; Jänne, Pasi A.; Hammerman, Peter S.; Sholl, Lynette M.; Hodi, F. Stephen; Richards, William G.; Bueno, Raphael; English, Jessie M.; Bittinger, Mark A.


    BACKGROUND. Immune checkpoint blockade improves survival in a subset of patients with non–small-cell lung cancer (NSCLC), but robust biomarkers that predict response to PD-1 pathway inhibitors are lacking. Furthermore, our understanding of the diversity of the NSCLC tumor immune microenvironment remains limited. METHODS. We performed comprehensive flow cytometric immunoprofiling on both tumor and immune cells from 51 NSCLCs and integrated this analysis with clinical and histopathologic characteristics, next-generation sequencing, mRNA expression, and PD-L1 immunohistochemistry (IHC). RESULTS. Cytometric profiling identified an immunologically “hot” cluster with abundant CD8+ T cells expressing high levels of PD-1 and TIM-3 and an immunologically “cold” cluster with lower relative abundance of CD8+ T cells and expression of inhibitory markers. The “hot” cluster was highly enriched for expression of genes associated with T cell trafficking and cytotoxic function and high PD-L1 expression by IHC. There was no correlation between immunophenotype and KRAS or EGFR mutation, or patient smoking history, but we did observe an enrichment of squamous subtype and tumors with higher mutation burden in the “hot” cluster. Additionally, approximately 20% of cases had high B cell infiltrates with a subset producing IL-10. CONCLUSIONS. Our results support the use of immune-based metrics to study response and resistance to immunotherapy in lung cancer. FUNDING. The Robert A. and Renée E. Belfer Family Foundation, Expect Miracles Foundation, Starr Cancer Consortium, Stand Up to Cancer Foundation, Conquer Cancer Foundation, International Association for the Study of Lung Cancer, National Cancer Institute (R01 CA205150), and the Damon Runyon Cancer Research Foundation. PMID:27699239

  2. Lipopolysaccharide Could Be Internalized into Human Peripheral Blood Mononuclear Cells and Elicits TNF-α Release,but not via the Pathway of Toll-Like Receptor 4 on the Cell Surface

    Institute of Scientific and Technical Information of China (English)

    HongZhou; GuofuDing; WeiLiu; LiangxiWang; YonglingLu; HongweiCao; JiangZheng


    Lipopolysaccharide (LPS [endotoxin]), the principal component of the outer membrane of gram-negative bacteria, stimulate various cell types to release numerous proinflammatory mediators such as TNF-α, IL-6 and IL-12, which may damage cells and lead to organ injury, even sepsis and septic shock. Toll-like receptor 4(TLR4) has been identified as the receptor involved in the recognition of LPS, but the role of LPS uptake inactivating signal transduction remains controversial. In the present study, TNF-α was used as a marker of macrophages/monocytes activated by LPS, and CQ was used as an inhibitor of endosome mature in order to definitude what stage the signal transduction elicited by LPS was interrupted. We found that there indeed existed internalization of LPS and internalization partially participated in LPS signaling since CQ inhibitedcytokine release, and decreased accumulation of FITC-LPS in hPBMC. In contrast, anti-hTLR4 antibodycould decrease cytokines' release, but no inhibition on accumulation of FITC-LPS. This result revealed thatinhibition of cytokine release was related to reduction of FITC-LPS accumulation in the cells. But TLR4 on thecell surface didn't possibly participated in internalization of LPS. Thus, LPS signaling and internalizationcannot be viewed as mutually independent processes. Cellular & Molecular Immunology. 2004;1(5):373-377.

  3. Brachyury and SMAD signalling collaboratively orchestrate distinct mesoderm and endoderm gene regulatory networks in differentiating human embryonic stem cells. (United States)

    Faial, Tiago; Bernardo, Andreia S; Mendjan, Sasha; Diamanti, Evangelia; Ortmann, Daniel; Gentsch, George E; Mascetti, Victoria L; Trotter, Matthew W B; Smith, James C; Pedersen, Roger A


    The transcription factor brachyury (T, BRA) is one of the first markers of gastrulation and lineage specification in vertebrates. Despite its wide use and importance in stem cell and developmental biology, its functional genomic targets in human cells are largely unknown. Here, we use differentiating human embryonic stem cells to study the role of BRA in activin A-induced endoderm and BMP4-induced mesoderm progenitors. We show that BRA has distinct genome-wide binding landscapes in these two cell populations, and that BRA interacts and collaborates with SMAD1 or SMAD2/3 signalling to regulate the expression of its target genes in a cell-specific manner. Importantly, by manipulating the levels of BRA in cells exposed to different signalling environments, we demonstrate that BRA is essential for mesoderm but not for endoderm formation. Together, our data illuminate the function of BRA in the context of human embryonic development and show that the regulatory role of BRA is context dependent. Our study reinforces the importance of analysing the functions of a transcription factor in different cellular and signalling environments.

  4. Morphological and biochemical analysis of the secretory pathway in melanoma cells with distinct metastatic potential

    NARCIS (Netherlands)

    Ayala, [No Value; Babia, T; Baldassarre, M; Pompeo, A; Fabra, A; Kok, JW; Luini, A; Buccione, R; Egea, G


    In this report, we have investigated whether alterations of the morphological and functional aspects of the biosecretory membrane system are associated with the metastatic potential of tumor cells. To this end, we have analyzed the morphology of the Golgi complex, the cytoskeleton organization and m

  5. Two distinct modes for propagation of histone PTMs across the cell cycle

    DEFF Research Database (Denmark)

    Alabert, Constance; Barth, Teresa K; Reverón-Gómez, Nazaret;


    of new histone deposition. Importantly, within one cell cycle, all PTMs are restored. In general, new histones are modified to mirror the parental histones. However, H3K9 trimethylation (H3K9me3) and H3K27me3 are propagated by continuous modification of parental and new histones because the establishment...

  6. FGFR3 isoforms have distinct functions in the regulation of growth and cell morphology. (United States)

    Shimizu, Akio; Takashima, Yuji; Kurokawa-Seo, Misuzu


    We have previously cloned the alternatively spliced isoform of fibroblast growth factor receptor 3 (FGFR3DeltaAB) that lacks the acid box in the extracellular region. To understand the biological functions and signal transduction of these FGFR3 isoforms, we analyzed the effect of FGF1 in ATDC5 cells, chondroprogenitor cell lines overexpressing these isoforms. In response to FGF1, FGFR3 induced a marked cell-morphology change to a round shape, while FGFR3DeltaAB did not. Furthermore, FGFR3 induced complete growth arrest, whereas FGFR3DeltaAB induced only moderate growth inhibition. Both receptors induced the expression of the CDK inhibitor p21(CIP1). However, only FGFR3 induced STAT1 phosphorylation that mediates the transcriptional induction of p21(CIP1), although both FGFR3 isoforms could induce a strong activation of mitogen-activated protein (MAP) kinases. Taken together, the different biological responses mediated by FGFR3 and FGFR3DeltaAB appear to be due to a difference in their ability to utilize STAT1 pathway and signals involved in cell rounding.

  7. Distinct behaviour of the homeodomain derived cell penetrating peptide penetratin in interaction with different phospholipids.

    Directory of Open Access Journals (Sweden)

    Ofelia Maniti

    Full Text Available BACKGROUND: Penetratin is a protein transduction domain derived from the homeoprotein Antennapedia. Thereby it is currently used as a cell penetrating peptide to introduce diverse molecules into eukaryotic cells, and it could also be involved in the cellular export of transcription factors. Moreover, it has been shown that it is able to act as an antimicrobial agent. The mechanisms involved in all these processes are quite controversial. METHODOLOGY/PRINCIPAL FINDINGS: In this article, we report spectroscopic, calorimetric and biochemical data on the penetratin interaction with three different phospholipids: phosphatidylcholine (PC and phosphatidylethanolamine (PE to mimic respectively the outer and the inner leaflets of the eukaryotic plasma membrane and phosphatidylglycerol (PG to mimic the bacterial membrane. We demonstrate that with PC, penetratin is able to form vesicle aggregates with no major change in membrane fluidity and presents no well defined secondary structure organization. With PE, penetratin aggregates vesicles, increases membrane rigidity and acquires an α-helical structure. With PG membranes, penetratin does not aggregate vesicles but decreases membrane fluidity and acquires a structure with both α-helical and β-sheet contributions. CONCLUSIONS/SIGNIFICANCE: These data from membrane models suggest that the different penetratin actions in eukaryotic cells (membrane translocation during export and import and on prokaryotes may result from different peptide and lipid structural arrangements. The data suggest that, for eukaryotic cell penetration, penetratin does not acquire classical secondary structure but requires a different conformation compared to that in solution.

  8. Digitoxin-induced cytotoxicity in cancer cells is mediated through distinct kinase and interferon signaling networks. (United States)

    Prassas, Ioannis; Karagiannis, George S; Batruch, Ihor; Dimitromanolakis, Apostolos; Datti, Alessandro; Diamandis, Eleftherios P


    Cardiac glycosides (e.g., digoxin, digitoxin) constitute a diverse family of plant-derived sodium pump inhibitors that have been in clinical use for the treatment of heart-related diseases (congestive heart failure, atrial arrhythmia) for many years. Recently though, accumulating in vitro and in vivo evidence highlight potential anticancer properties of these compounds. Despite the fact that members of this family have advanced to clinical trial testing in cancer therapeutics, their cytotoxic mechanism is not yet elucidated. In this study, we investigated the cytotoxic properties of cardiac glycosides against a panel of pancreatic cancer cell lines, explored their apoptotic mechanism, and characterized the kinetics of cell death induced by these drugs. Furthermore, we deployed a high-throughput kinome screening approach and identified several kinases of the Na-K-ATPase-mediated signal transduction circuitry (epidermal growth factor receptor, Src, pkC, and mitogen-activated protein kinases) as important mediators downstream of cardiac glycoside cytotoxic action. To further extend our knowledge on their mode of action, we used mass-spectrometry-based quantitative proteomics (stable isotope labeling of amino acids in cell culture) coupled with bioinformatics to capture large-scale protein perturbations induced by a physiological dose of digitoxin in BxPC-3 pancreatic cancer cells and identified members of the interferon family as key regulators of the main protein/protein interactions downstream of digitoxin action. Hence, our findings provide more in-depth information regarding the molecular mechanisms underlying cardiac glycoside-induced cytotoxicity.

  9. Immunization with vaccinia virus induces polyfunctional and phenotypically distinctive CD8+ T cell responses (United States)

    Precopio, Melissa L.; Betts, Michael R.; Parrino, Janie; Price, David A.; Gostick, Emma; Ambrozak, David R.; Asher, Tedi E.; Douek, Daniel C.; Harari, Alexandre; Pantaleo, Giuseppe; Bailer, Robert; Graham, Barney S.; Roederer, Mario; Koup, Richard A.


    Vaccinia virus immunization provides lifelong protection against smallpox, but the mechanisms of this exquisite protection are unknown. We used polychromatic flow cytometry to characterize the functional and phenotypic profile of CD8+ T cells induced by vaccinia virus immunization in a comparative vaccine trial of modified vaccinia virus Ankara (MVA) versus Dryvax immunization in which protection was assessed against subsequent Dryvax challenge. Vaccinia virus–specific CD8+ T cells induced by both MVA and Dryvax were highly polyfunctional; they degranulated and produced interferon γ, interleukin 2, macrophage inflammatory protein 1β, and tumor necrosis factor α after antigenic stimulation. Responding CD8+ T cells exhibited an unusual phenotype (CD45RO−CD27intermediate). The unique phenotype and high degree of polyfunctionality induced by vaccinia virus also extended to inserted HIV gene products of recombinant NYVAC. This quality of the CD8+ T cell response may be at least partially responsible for the profound efficacy of these vaccines in protection against smallpox and serves as a benchmark against which other vaccines can be evaluated. PMID:17535971

  10. Distinct roles for ROCK1 and ROCK2 in the regulation of cell detachment. (United States)

    Shi, Jianjian; Wu, Xiangbing; Surma, Michelle; Vemula, Sasidhar; Zhang, Lumin; Yang, Yu; Kapur, Reuben; Wei, Lei


    This study, using mouse embryonic fibroblast (MEF) cells derived from ROCK1(-/-) and ROCK2(-/-) mice, is designed to dissect roles for ROCK1 and ROCK2 in regulating actin cytoskeleton reorganization induced by doxorubicin, a chemotherapeutic drug. ROCK1(-/-) MEFs exhibited improved actin cytoskeleton stability characterized by attenuated periphery actomyosin ring formation and preserved central stress fibers, associated with decreased myosin light chain 2 (MLC2) phosphorylation but preserved cofilin phosphorylation. These effects resulted in a significant reduction in cell shrinkage, detachment, and predetachment apoptosis. In contrast, ROCK2(-/-) MEFs showed increased periphery membrane folding and impaired cell adhesion, associated with reduced phosphorylation of both MLC2 and cofilin. Treatment with inhibitor of myosin (blebbistatin), inhibitor of actin polymerization (cytochalasin D), and ROCK pan-inhibitor (Y27632) confirmed the contributions of actomyosin contraction and stress fiber instability to stress-induced actin cytoskeleton reorganization. These results support a novel concept that ROCK1 is involved in destabilizing actin cytoskeleton through regulating MLC2 phosphorylation and peripheral actomyosin contraction, whereas ROCK2 is required for stabilizing actin cytoskeleton through regulating cofilin phosphorylation. Consequently, ROCK1 and ROCK2 can be functional different in regulating stress-induced stress fiber disassembly and cell detachment.

  11. Mesenchymal stem cells from different organs are characterized by distinct topographic Hox codes. (United States)

    Ackema, Karin B; Charité, Jeroen


    Mesenchymal stem cells (MSC) are multipotent cells found as part of the stromal compartment of the bone marrow and in many other organs. They can be identified in vitro as CFU-F (colony forming unit-fibroblast) based on their ability to form adherent colonies of fibroblast-like cells in culture. MSC expanded in vitro retain characteristics appropriate to their tissue of origin. This is reflected in their propensity for differentiating towards specific lineages, and their capacity to generate, upon retransplantation in vivo, a stroma supporting typical lineages of hematopoietic cells. Hox genes encode master regulators of regional specification and organ development in the embryo and are widely expressed in the adult. We investigated whether they could be involved in determining tissue-specific properties of MSC. Hox gene expression profiles of individual CFU-F colonies derived from various organs and anatomical locations were generated, and the relatedness between these profiles was determined using hierarchical cluster analysis. This revealed that CFU-F have characteristic Hox expression signatures that are heterogeneous but highly specific for their anatomical origin. The topographic specificity of these Hox codes is maintained during differentiation, suggesting that they are an intrinsic property of MSC. Analysis of Hox codes of CFU-F from vertebral bone marrow suggests that MSC originate over a large part of the anterioposterior axis, but may not originate from prevertebral mesenchyme. These data are consistent with a role for Hox proteins in specifying cellular identity of MSC.

  12. Single gene-based distinction of individual microbial genomes from a mixed population of microbial cells

    Directory of Open Access Journals (Sweden)

    Manu Valtteri Tamminen


    Full Text Available Recent progress in environmental microbiology has revealed vast populations of microbes in any given habitat that cannot be detected by conventional culturing strategies. The use of sensitive genetic detection methods such as CARD-FISH and in situ PCR have been limited by the cell wall permeabilization requirement that cannot be performed similarly on all cell types without lysing some and leaving some unpermeabilized. Furthermore, the detection of low copy targets such as genes present in single copies in the microbial genomes, has remained problematic. We describe an emulsion-based procedure to trap individual microbial cells into picoliter-volume polyacrylamide droplets that provide a rigid support for genetic material and therefore allow complete degradation of cellular material to expose the individual genomes. The polyacrylamide droplets are subsequently converted into picoliter-scale reactors for genome amplification. The amplified genomes are labelled based on the presence of a target gene and differentiated from those that do not contain the gene by flow cytometry. Using the Escherichia coli strains XL1 and MC1061, which differ with respect to the presence (XL1 or absence (MC1061 of a single copy of a tetracycline resistance gene per genome, we demonstrate that XL1 genomes present at 0.1% of MC1061 genomes can be differentiated using this method. Using a spiked sediment microbial sample, we demonstrate that the method is applicable to highly complex environmental microbial communities as a target gene-based screen for individual microbes. The method provides a novel tool for enumerating functional cell populations in complex microbial communities. We envision that the method could be optimized for fluorescence-activated cell sorting to enrich genetic material of interest from complex environmental samples.

  13. Distinction of neurons, glia and endothelial cells in the cerebral cortex: an algorithm based on cytological features

    Directory of Open Access Journals (Sweden)

    Miguel Ángel García-Cabezas


    Full Text Available The estimation of the number or density of neurons and types of glial cells and their relative proportions in different brain areas are at the core of rigorous quantitative neuroanatomical studies. Unfortunately, the lack of detailed, updated, systematic, and well-illustrated descriptions of the cytology of neurons and glial cell types, especially in the primate brain, makes such studies especially demanding, often limiting their scope and broad use. Here, following extensive analysis of histological materials and the review of current and classical literature, we compile a list of precise morphological criteria that can facilitate and standardize identification of cells in stained sections examined under the microscope. We describe systematically and in detail the cytological features of neurons and glial cell types in the cerebral cortex of the macaque monkey and the human using semithin and thick sections stained for Nissl. We used this classical staining technique because it labels all cells in the brain in distinct ways. In addition, we corroborate key distinguishing characteristics of different cell types in sections immunolabeled for specific markers counterstained for Nissl and in ultrathin sections processed for electron microscopy. Finally, we summarize the core features that distinguish each cell type in easy-to-use tables and sketches, and structure these key features in an algorithm that can be used to systematically distinguish cellular types in the cerebral cortex. Moreover, we report high inter-observer algorithm reliability, which is a crucial test for obtaining consistent and reproducible cell counts in unbiased stereological studies. This protocol establishes a consistent framework that can be used to reliably identify and quantify cells in the cerebral cortex of primates as well as other mammalian species in health and disease.

  14. Human decidual macrophages and NK cells differentially express Toll-like receptors and display distinct cytokine profiles upon TLR stimulation.

    Directory of Open Access Journals (Sweden)

    Marion eDuriez


    Full Text Available Maternofetal pathogen transmission is partially controlled at the level of the maternal uterine mucosa at the fetal implantation site (the decidua basalis, where maternal and fetal cells are in close contact. Toll-like receptors (TLRs may play an important role in initiating rapid immune responses against pathogens in the decidua basalis, however the tolerant microenvironment should be preserved in order to allow fetal development. Here we investigated the expression and functionality of TLRs expressed by decidual macrophages (dMs and NK cells (dNKs, the major decidual immune cell populations.We report for the first time that both human dMs and dNK cells express mRNAs encoding TLRs 1-9, albeit with a higher expression level in dMs. TLR2, TLR3 and TLR4 protein expression checked by flow cytometry was positive for both dMs and dNK cells. In vitro treatment of primary dMs and dNK cells with specific TLR2, TLR3, TLR4, TLR7/8 and TLR9 agonists enhanced their secretion of pro- and anti-inflammatory cytokines, as well as cytokines and chemokines involved in immune cell crosstalk. Only dNK cells released IFN-γ, whereas only dMs released IL-1β, IL-10 and IL-12. TLR9 activation of dMs resulted in a distinct pattern of cytokine expression compared to the other TLRs. The cytokine profiles expressed by dMs and dNK cells upon TLR activation are compatible with maintenance of the fetotolerant immune environment during initiation of immune responses to pathogens at the maternofetal interface.

  15. Bacillus cereus enterotoxins act as major virulence factors and exhibit distinct cytotoxicity to different human cell lines. (United States)

    Jeßberger, Nadja; Dietrich, Richard; Bock, Stefanie; Didier, Andrea; Märtlbauer, Erwin


    A comparative analysis on the relevance of the Bacillus cereus enterotoxins Nhe (nonhemolytic enterotoxin), HBL (haemolysin BL) and CytK (cytotoxin K) was accomplished, concerning their toxic activity towards different target cell lines. Overall, among the components secreted by the reference strains for Nhe and HBL, the enterotoxin complexes accounted for over 90% of the total toxicity. Vero and primary endothelial cells (HUVEC) were highly susceptible to Nhe, whereas Hep-G2, Vero and A549 reacted most sensitive to Nhe plus HBL. For CytK the highest toxicity was observed on CaCo-2 cells. As HBL positive strains always produce Nhe in parallel, the specific contribution of both enterotoxin complexes to the overall observed cytotoxic effects was determined by consecutively removing their single components. While in most cell lines Nhe and HBL contributed more or less equally (40-60%) to cytotoxicity, the relative activity of Nhe was approximately 90% in HUVEC, and that of HBL 75% in A549 cells. With U937, a nearly Nhe resistant cell line was identified for the first time. This distinct susceptibility of cell lines was confirmed by investigating a set of 37 B. cereus strains. Interestingly, whereas Nhe is the enterotoxin mainly responsible for cell death as determined by WST-1 bioassays, more rapid pore formation was observed when HBL was present, pointing to a different mode of action of the two enterotoxin complexes. Furthermore, correlation was observed between cytotoxicity of solely Nhe producing strains and NheB. Cytotoxicity of Nhe/HBL producing isolates correlated with the expression of HBL L1, NheB and HBL B. In conclusion, the observed susceptibilities of target cell lines of different histological origin underline that B. cereus enterotoxins represent major virulence factors and that toxicity is not restricted to gastrointestinal infections. The varying contribution of Nhe and HBL to total cytotoxicity strongly indicates that Nhe as well as HBL specific B

  16. EV71 virus-like particles produced by co-expression of capsid proteins in yeast cells elicit humoral protective response against EV71 lethal challenge


    Wang, Xiaowen; Xiao, Xiangqian; Zhao, Miao; Wei LIU; Pang, Lin; Sun, Xin; Cen, Shan; Burton B Yang; Huang, Yuming; Sheng, Wang; Zeng, Yi


    Background Enterovirus 71 (EV71) is the most common causative pathogens of hand, foot and mouth disease (HFMD) associated with severe neurological complications. There is a great need to develop prophylactic vaccine against EV71 infection. Results EV71 virus-like particle (VLP) was produced in yeast expression system by the co-expression of four EV71 structural proteins VP1–VP4. Immunization with the recombinant VLPs elicited potent anti-EV71 antibody responses in adult mice and anti-VLP sera...

  17. Distinct gene expression signatures in human embryonic stem cells differentiated towards definitive endoderm at single-cell level

    DEFF Research Database (Denmark)

    Norrman, Karin; Strömbeck, Anna; Semb, Henrik;


    of anterior definitive endoderm (DE). Here, we differentiated human embryonic stem cells towards DE using three different activin A based treatments. Differentiation efficiencies were evaluated by gene expression profiling over time at cell population level. A panel of key markers was used to study DE...... for the three activin A based protocols applied. Our data provide novel insights in DE gene expression at the cellular level of in vitro differentiated human embryonic stem cells, and illustrate the power of using single-cell gene expression profiling to study differentiation heterogeneity and to characterize......Characterization of directed differentiation of pluripotent stem cells towards therapeutically relevant cell types, including pancreatic beta-cells and hepatocytes, depends on molecular markers and assays that resolve the signature of individual cells. Pancreas and liver both have a common origin...

  18. Distinct populations of dendritic cells are present in the subepithelial dome and T cell regions of the murine Peyer's patch (United States)


    Despite the fact that the Peyer's patch (PP) is the primary site for antigen uptake in the intestine, the cellular basis of antigen handling after transport into the PP is poorly understood. We performed immunohistology of murine PPs using the dendritic cell (DC)-reactive monoclonal antibodies N418, NLDC-145, M342, and 2A1, as well as antibodies to other T cell, B cell, and macrophage markers. N418+, 2A1+, NLDC-145-, M342- cells form a dense layer of cells in the subepithelial dome (SED), just beneath the follicle epithelium, and are scattered throughout the follicle, sparing the germinal center. In contrast, N418+, 2A1+, NLDC-145+, and M342+ DCs are present in the interfollicular T cell regions (IFR). CD3+ and CD4+, but no CD8+ T cells were present in the SED and the follicle, including the germinal center, while CD3+, CD4+, and CD8+ T cells were present in the IFR. B cells and macrophages were poorly represented in the SED as no B220+ cells, only few Mac-1lo cells, and no F4/80+ cells were present at this site. In contrast, Mac-1hi cells were found in the IFR and lamina propria of intestinal villi, while F4/80+ cells were found only in the latter. In further phenotypic studies, we analyzed surface molecules of PP and spleen DCs by flow cytometry and found that these cells had similar fluorescence profiles when stained with N418, NLDC-145, and 33D1 DC-reactive antibodies, and antibodies to the costimulatory molecules B7-1 (1G10) and B7-2 (GL1). In contrast, PP DCs expressed 5- 10-fold higher levels of major histocompatibility complex class II antigens (IEk) than spleen DCs. Finally, in functional studies, we demonstrated that both PP and spleen DCs process soluble protein antigens during overnight culture and induce similar levels of proliferation in CD3+ T cells, and CD4+/Mel 14hi T cells from T cell receptor transgenic mice. The in vivo relevance of such presentation was shown by the fact that PP DCs isolated from Balb/c mice after being fed ovalbumin stimulated

  19. A novel supervised trajectory segmentation algorithm identifies distinct types of human adenovirus motion in host cells. (United States)

    Helmuth, Jo A; Burckhardt, Christoph J; Koumoutsakos, Petros; Greber, Urs F; Sbalzarini, Ivo F


    Biological trajectories can be characterized by transient patterns that may provide insight into the interactions of the moving object with its immediate environment. The accurate and automated identification of trajectory motifs is important for the understanding of the underlying mechanisms. In this work, we develop a novel trajectory segmentation algorithm based on supervised support vector classification. The algorithm is validated on synthetic data and applied to the identification of trajectory fingerprints of fluorescently tagged human adenovirus particles in live cells. In virus trajectories on the cell surface, periods of confined motion, slow drift, and fast drift are efficiently detected. Additionally, directed motion is found for viruses in the cytoplasm. The algorithm enables the linking of microscopic observations to molecular phenomena that are critical in many biological processes, including infectious pathogen entry and signal transduction.

  20. Human Cells Cultured under Physiological Oxygen Utilize Two Cap-binding Proteins to recruit Distinct mRNAs for Translation. (United States)

    Timpano, Sara; Uniacke, James


    Translation initiation is a focal point of translational control and requires the binding of eIF4E to the 5' cap of mRNA. Under conditions of extreme oxygen depletion (hypoxia), human cells repress eIF4E and switch to an alternative cap-dependent translation mediated by a homolog of eIF4E, eIF4E2. This homolog forms a complex with the oxygen-regulated hypoxia-inducible factor 2α and can escape translation repression. This complex mediates cap-dependent translation under cell culture conditions of 1% oxygen (to mimic tumor microenvironments), whereas eIF4E mediates cap-dependent translation at 21% oxygen (ambient air). However, emerging evidence suggests that culturing cells in ambient air, or "normoxia," is far from physiological or "normal." In fact, oxygen in human tissues ranges from 1-11% or "physioxia." Here we show that two distinct modes of cap-dependent translation initiation are active during physioxia and act on separate pools of mRNAs. The oxygen-dependent activities of eIF4E and eIF4E2 are elucidated by observing their polysome association and the status of mammalian target of rapamycin complex 1 (eIF4E-dependent) or hypoxia-inducible factor 2α expression (eIF4E2-dependent). We have identified oxygen conditions where eIF4E is the dominant cap-binding protein (21% normoxia or standard cell culture conditions), where eIF4E2 is the dominant cap-binding protein (1% hypoxia or ischemic diseases and cancerous tumors), and where both cap-binding proteins act simultaneously to initiate the translation of distinct mRNAs (1-11% physioxia or during development and stem cell differentiation). These data suggest that the physioxic proteome is generated by initiating translation of mRNAs via two distinct but complementary cap-binding proteins.

  1. Two distinct signaling pathways regulate peroxynitrite-induced apoptosis in PC12 cells. (United States)

    Shacka, J J; Sahawneh, M A; Gonzalez, J D; Ye, Y-Z; D'Alessandro, T L; Estévez, A G


    The mechanisms of peroxynitrite-induced apoptosis are not fully understood. We report here that peroxynitrite-induced apoptosis of PC12 cells requires the simultaneous activation of p38 and JNK MAP kinase, which in turn activates the intrinsic apoptotic pathway, as evidenced by Bax translocation to the mitochondria, cytochrome c release to the cytoplasm and activation of caspases, leading to cell death. Peroxynitrite induces inactivation of the Akt pathway. Furthermore, overexpression of constitutively active Akt inhibits both peroxynitrite-induced Bax translocation and cell death. Peroxynitrite-induced death was prevented by overexpression of Bcl-2 and by cyclosporin A, implicating the involvement of the intrinsic apoptotic pathway. Selective inhibition of mixed lineage kinase (MLK), p38 or JNK does not attenuate the decrease in Akt phosphorylation showing that inactivation of the Akt pathway occurs independently of the MLK/MAPK pathway. Together, these results reveal that peroxynitrite-induced activation of the intrinsic apoptotic pathway involves interactions with the MLK/MAPK and Akt signaling pathways.

  2. Innate control of actin nucleation determines two distinct migration behaviours in dendritic cells. (United States)

    Vargas, Pablo; Maiuri, Paolo; Bretou, Marine; Sáez, Pablo J; Pierobon, Paolo; Maurin, Mathieu; Chabaud, Mélanie; Lankar, Danielle; Obino, Dorian; Terriac, Emmanuel; Raab, Matthew; Thiam, Hawa-Racine; Brocker, Thomas; Kitchen-Goosen, Susan M; Alberts, Arthur S; Sunareni, Praveen; Xia, Sheng; Li, Rong; Voituriez, Raphael; Piel, Matthieu; Lennon-Duménil, Ana-Maria


    Dendritic cell (DC) migration in peripheral tissues serves two main functions: antigen sampling by immature DCs, and chemokine-guided migration towards lymphatic vessels (LVs) on maturation. These migratory events determine the efficiency of the adaptive immune response. Their regulation by the core cell locomotion machinery has not been determined. Here, we show that the migration of immature DCs depends on two main actin pools: a RhoA-mDia1-dependent actin pool located at their rear, which facilitates forward locomotion; and a Cdc42-Arp2/3-dependent actin pool present at their front, which limits migration but promotes antigen capture. Following TLR4-MyD88-induced maturation, Arp2/3-dependent actin enrichment at the cell front is markedly reduced. Consequently, mature DCs switch to a faster and more persistent mDia1-dependent locomotion mode that facilitates chemotactic migration to LVs and lymph nodes. Thus, the differential use of actin-nucleating machineries optimizes the migration of immature and mature DCs according to their specific function.

  3. PRIMA-1met (APR-246) inhibits growth of colorectal cancer cells with different p53 status through distinct mechanisms. (United States)

    Li, Xiao-Lan; Zhou, Jianbiao; Chan, Zit-Liang; Chooi, Jing-Yuan; Chen, Zhi-Rong; Chng, Wee-Joo


    PRIMA-1met (APR-246) is a methylated derivative and structural analog of PRIMA-1 (p53 re-activation and induction of massive apoptosis). PRIMA-1met has been reported to restore both the wild type (wt) structure and function of mutant p53. Here, we show that PRIMA-1met is highly effective at limiting the growth of CRC cells regardless of p53 status. However, PRIMA-1met induces robust apoptosis only in CRC cells with mutant p53. Upregulation of Noxa, a proapoptotic molecule, is crucial for PRIMA-1met mediated activity. In human xenograft model of disease, PRIMA-1met effectively suppresses CRC tumor growth. Our results uncover distinct mechanisms of PRIMA-1met in CRC with different p53 status, thus providing a mechanistic rationale to evaluate the clinical efficacy of PRIMA-1met in CRC patients with different p53 status.

  4. Segregated Cell Populations Enable Distinct Parallel Encoding within the Radial Axis of the CA1 Pyramidal Layer (United States)

    Geiller, Tristan; Royer, Sebastien


    Numerous studies have implicated the hippocampus in the encoding and storage of declarative and spatial memories. Several models have considered the hippocampus and its distinct subfields to contain homogeneous pyramidal cell populations. Yet, recent studies have led to a consensus that the dorso-ventral and proximo-distal axes have different connectivities and physiologies. The remaining deep-superficial axis of the pyramidal layer, however, remains relatively unexplored due to a lack of techniques that can record from neurons simultaneously at different depths. Recent advances in transgenic mice, two-photon imaging and dense multisite recording have revealed extensive disparities between the pyramidal cells located in the deep and the superficial layers. Here, we summarize differences between the two populations in terms of gene expression and connectivity with other intra-hippocampal subregions and local interneurons that underlie distinct learning processes and spatial representations. A unified picture will emerge to describe how such local segregations can increase the capacity of the hippocampus to compute and process numerous tasks in parallel. PMID:28243162

  5. Distinct Neurodegenerative Changes in an Induced Pluripotent Stem Cell Model of Frontotemporal Dementia Linked to Mutant TAU Protein

    Directory of Open Access Journals (Sweden)

    Marc Ehrlich


    Full Text Available Frontotemporal dementia (FTD is a frequent form of early-onset dementia and can be caused by mutations in MAPT encoding the microtubule-associated protein TAU. Because of limited availability of neural cells from patients’ brains, the underlying mechanisms of neurodegeneration in FTD are poorly understood. Here, we derived induced pluripotent stem cells (iPSCs from individuals with FTD-associated MAPT mutations and differentiated them into mature neurons. Patient iPSC-derived neurons demonstrated pronounced TAU pathology with increased fragmentation and phospho-TAU immunoreactivity, decreased neurite extension, and increased but reversible oxidative stress response to inhibition of mitochondrial respiration. Furthermore, FTD neurons showed an activation of the unfolded protein response, and a transcriptome analysis demonstrated distinct, disease-associated gene expression profiles. These findings indicate distinct neurodegenerative changes in FTD caused by mutant TAU and highlight the unique opportunity to use neurons differentiated from patient-specific iPSCs to identify potential targets for drug screening purposes and therapeutic intervention.

  6. Opposite modulation of cell migration by distinct subregions of urokinase connecting peptide. (United States)

    Franco, Paola; Carotenuto, Alfonso; Marcozzi, Cristina; Votta, Giuseppina; Sarno, Ciro; Iaccarino, Ingram; Brancaccio, Diego; De Vincenzo, Anna; Novellino, Ettore; Grieco, Paolo; Stoppelli, Maria Patrizia


    Functional analysis of isolated protein domains may uncover cryptic activities otherwise missed. The serine protease urokinase (uPA) has a clear-cut motogen activity that is catalytically independent and resides in its amino-terminal growth factor domain (GFD, residues 1-49) and connecting peptide region (CP, residues 132-158). To functionally dissect the CP region, we analysed the biological activity of two synthetic peptides corresponding to the N-terminal [uPA-(135-143), residues 135-143] and C-terminal [uPA-(144-158), residues 144-158] CP subregions. Most of the chemotactic activity of connecting peptide-derived peptide (CPp, [uPA-(135-158)]) for embryonic kidney HEK293/uPAR-25 cells is retained by uPA-(144-158) at nanomolar concentrations. In contrast, uPA-(135-143) inhibits basal, CPp -, vitronectin- and fibronectin-induced cell migration. Radioreceptor binding assays on intact HEK293 cells revealed that uPA-(135-143) and uPA-(144-158) are both able to compete with [(125)I]-CPp, albeit with different binding affinities. The consequences of phospho-mimicking, S138E substitution, were studied using [138E]uPA-(135-158) and [138E]uPA-(135-143) peptides. Unlike CPp, [138E]uPA-(135-158) and [138E]uPA-(135-143) exhibit remarkable inhibitory properties. Finally, analysis of the conformational preferences of the peptides allowed to identify secondary structure elements exclusively characterising the stimulatory CPp and uPA-(144-158) versus the inhibitory uPA-(135-143), [138E]uPA-(135-158) and [138E]uPA-(135-143) peptides. In conclusion, these data shed light on the cryptic activities of uPA connecting peptide, revealing the occurrence of two adjacent regions, both competing for binding to cell surface but conveying opposite signalling on cell migration.

  7. T cell clones from Schistosoma haematobium infected and exposed individuals lacking distinct cytokine profiles for Th1/Th2 polarisation

    Directory of Open Access Journals (Sweden)

    Mduluza T


    Full Text Available T cell clones were derived from peripheral blood mononuclear cells of Schistosoma haematobium infected and uninfected individuals living in an endemic area. The clones were stimulated with S. haematobium worm and egg antigens and purified protein derivative. Attempts were made to classify the T cell clones according to production of the cytokines IL-4, IL-5 and IFN-gamma. All the T cell clones derived were observed to produce cytokines used as markers for the classification of Th1/Th2 subsets. However, the 'signature' cytokines marking each subset were produced at different levels. The classification depended on the dominating cytokine type, which was having either Th0/1 or Th0/2 subsets. The results indicated that no distinct cytokine profiles for polarisation of Th1/Th2 subsets were detected in these S. haematobium infected humans. The balance in the profiles of cytokines marking each subset were related to infection and re-infection status after treatment with praziquantel. In the present study, as judged by the changes in infection status with time, the T cell responses appeared to be less stable and more dynamic, suggesting that small quantitative changes in the balance of the cytokines response could result in either susceptibility or resistant to S. haematobium infection.

  8. Cell fate decisions in malignant hematopoiesis: leukemia phenotype is determined by distinct functional domains of the MN1 oncogene.

    Directory of Open Access Journals (Sweden)

    Courteney K Lai

    Full Text Available Extensive molecular profiling of leukemias and preleukemic diseases has revealed that distinct clinical entities, like acute myeloid (AML and T-lymphoblastic leukemia (T-ALL, share similar pathogenetic mutations. It is not well understood how the cell of origin, accompanying mutations, extracellular signals or structural differences in a mutated gene determine the phenotypic identity of leukemias. We dissected the functional aspects of different protein regions of the MN1 oncogene and their effect on the leukemic phenotype, building on the ability of MN1 to induce leukemia without accompanying mutations. We found that the most C-terminal region of MN1 was required to block myeloid differentiation at an early stage, and deletion of an extended C-terminal region resulted in loss of myeloid identity and cell differentiation along the T-cell lineage in vivo. Megakaryocytic/erythroid lineage differentiation was blocked by the N-terminal region. In addition, the N-terminus was required for proliferation and leukemogenesis in vitro and in vivo through upregulation of HoxA9, HoxA10 and Meis2. Our results provide evidence that a single oncogene can modulate cellular identity of leukemic cells based on its active gene regions. It is therefore likely that different mutations in the same oncogene may impact cell fate decisions and phenotypic appearance of malignant diseases.

  9. Immune Reactions against Gene Gun Vaccines Are Differentially Modulated by Distinct Dendritic Cell Subsets in the Skin.

    Directory of Open Access Journals (Sweden)

    Corinna Stefanie Weber

    Full Text Available The skin accommodates multiple dendritic cell (DC subsets with remarkable functional diversity. Immune reactions are initiated and modulated by the triggering of DC by pathogen-associated or endogenous danger signals. In contrast to these processes, the influence of intrinsic features of protein antigens on the strength and type of immune responses is much less understood. Therefore, we investigated the involvement of distinct DC subsets in immune reactions against two structurally different model antigens, E. coli beta-galactosidase (betaGal and chicken ovalbumin (OVA under otherwise identical conditions. After epicutaneous administration of the respective DNA vaccines with a gene gun, wild type mice induced robust immune responses against both antigens. However, ablation of langerin+ DC almost abolished IgG1 and cytotoxic T lymphocytes against betaGal but enhanced T cell and antibody responses against OVA. We identified epidermal Langerhans cells (LC as the subset responsible for the suppression of anti-OVA reactions and found regulatory T cells critically involved in this process. In contrast, reactions against betaGal were not affected by the selective elimination of LC, indicating that this antigen required a different langerin+ DC subset. The opposing findings obtained with OVA and betaGal vaccines were not due to immune-modulating activities of either the plasmid DNA or the antigen gene products, nor did the differential cellular localization, size or dose of the two proteins account for the opposite effects. Thus, skin-borne protein antigens may be differentially handled by distinct DC subsets, and, in this way, intrinsic features of the antigen can participate in immune modulation.

  10. Distinct abscisic acid signaling pathways for modulation of guard cell versus mesophyll cell potassium channels revealed by expression studies in Xenopus laevis oocytes (United States)

    Sutton, F.; Paul, S. S.; Wang, X. Q.; Assmann, S. M.; Evans, M. L. (Principal Investigator)


    Regulation of guard cell ion transport by abscisic acid (ABA) and in particular ABA inhibition of a guard cell inward K(+) current (I(Kin)) is well documented. However, little is known concerning ABA effects on ion transport in other plant cell types. Here we applied patch clamp techniques to mesophyll cell protoplasts of fava bean (Vicia faba cv Long Pod) plants and demonstrated ABA inhibition of an outward K(+) current (I(Kout)). When mesophyll cell protoplast mRNA (mesophyll mRNA) was expressed in Xenopus laevis oocytes, I(Kout) was generated that displayed similar properties to I(Kout) observed from direct analysis of mesophyll cell protoplasts. I(Kout) expressed by mesophyll mRNA-injected oocytes was inhibited by ABA, indicating that the ABA signal transduction pathway observed in mesophyll cells was preserved in the frog oocytes. Co-injection of oocytes with guard cell protoplast mRNA and cRNA for KAT1, an inward K(+) channel expressed in guard cells, resulted in I(Kin) that was similarly inhibited by ABA. However, oocytes co-injected with mesophyll mRNA and KAT1 cRNA produced I(Kin) that was not inhibited by ABA. These results demonstrate that the mesophyll-encoded signaling mechanism could not substitute for the guard cell pathway. These findings indicate that mesophyll cells and guard cells use distinct and different receptor types and/or signal transduction pathways in ABA regulation of K(+) channels.

  11. Lhx2 expression promotes self-renewal of a distinct multipotential hematopoietic progenitor cell in embryonic stem cell-derived embryoid bodies.

    Directory of Open Access Journals (Sweden)

    Lina Dahl

    Full Text Available The molecular mechanisms regulating the expansion of the hematopoietic system including hematopoietic stem cells (HSCs in the fetal liver during embryonic development are largely unknown. The LIM-homeobox gene Lhx2 is a candidate regulator of fetal hematopoiesis since it is expressed in the fetal liver and Lhx2(-/- mice die in utero due to severe anemia. Moreover, expression of Lhx2 in embryonic stem (ES cell-derived embryoid bodies (EBs can lead to the generation of HSC-like cell lines. To further define the role of this transcription factor in hematopoietic regulation, we generated ES cell lines that enabled tet-inducible expression of Lhx2. Using this approach we observed that Lhx2 expression synergises with specific signalling pathways, resulting in increased frequency of colony forming cells in developing EB cells. The increase in growth factor-responsive progenitor cells directly correlates to the efficiency in generating HSC-like cell lines, suggesting that Lhx2 expression induce self-renewal of a distinct multipotential hematopoietic progenitor cell in EBs. Signalling via the c-kit tyrosine kinase receptor and the gp130 signal transducer by IL-6 is necessary and sufficient for the Lhx2 induced self-renewal. While inducing self-renewal of multipotential progenitor cells, expression of Lhx2 inhibited proliferation of primitive erythroid precursor cells and interfered with early ES cell commitment, indicating striking lineage specificity of this effect.

  12. Immunogold electron microscopy and confocal analyses reveal distinctive patterns of histone H3 phosphorylation during mitosis in MCF-7 cells. (United States)

    Yan, Yitang; Cummings, Connie A; Sutton, Deloris; Yu, Linda; Castro, Lysandra; Moore, Alicia B; Gao, Xiaohua; Dixon, Darlene


    Histone phosphorylation has a profound impact on epigenetic regulation of gene expression, chromosome condensation and segregation, and maintenance of genome integrity. Histone H3 Serine 10 is evolutionally conserved and heavily phosphorylated during mitosis. To examine Histone H3 Serine 10 phosphorylation (H3S10ph) dynamics in mitosis, we applied immunogold labeling and confocal microscopy to visualize H3S10ph expression in MCF-7 cells. Confocal observations showed that MCF-7 cells had abundant H3S10ph expression in prophase and metaphase. In anaphase, the H3S10ph expression was significantly decreased and displayed only sparsely localized staining that mainly associated with the chromatid tips. We showed that immunogold bead density distribution followed the H3S10ph expression patterns observed in confocal analysis. At a higher magnification in metaphase, the immunogold beads were readily visible and the bead distribution along the condensed chromosomes was distinctive, indicating the specificity and reliability of the immunogold staining procedure. In anaphase, the beads were found to distribute focally in specific regions of chromatids, reinforcing the confocal observations of differential H3 phosphorylation. To our knowledge, this is the first report to show the specific H3S10ph expression with an immunogold technique and transmission electron microscopy. Additionally, with confocal microscopy, we analyzed H3S10ph expression in an immortalized cell line derived from benign uterine smooth muscle tumor cells. H3S10ph epitope was expressed more abundantly during anaphase in the benign tumor cells, and there was no dramatic differential expression within the condensed chromatid clusters as observed in MCF-7 cells. The differences in H3S10ph expression pattern and dynamics may contribute to the differential proliferative potential between benign tumor cells and MCF-7 cells.

  13. Ultrastructural and molecular distinctions between the porcine inner cell mass and epiblast reveal unique pluripotent cell states

    DEFF Research Database (Denmark)

    Hall, V. J.; Jacobsen, Janus Valentin; Rasmussen, M. A.


    Characterization of the pluripotent cell populations within the porcine embryo is essential for understanding pluripotency and self-renewal regulation in the inner cell mass (ICM) and epiblast. In this study, we perform detailed ultrastructural and molecular characterization of the developing...... pluripotent cell population as it develops from the ICM to the late epiblast. The ultrastructural observations revealed that the outer cells of the ICM have a high nuclear:cytoplasmic ratio but are transcriptionally inactive and contain mitochondria with few cristae. In contrast, the epiblast cells have...

  14. Pregnane X receptor activation and silencing promote steatosis of human hepatic cells by distinct lipogenic mechanisms. (United States)

    Bitter, Andreas; Rümmele, Petra; Klein, Kathrin; Kandel, Benjamin A; Rieger, Jessica K; Nüssler, Andreas K; Zanger, Ulrich M; Trauner, Michael; Schwab, Matthias; Burk, Oliver


    In addition to its well-characterized role in the regulation of drug metabolism and transport by xenobiotics, pregnane X receptor (PXR) critically impacts on lipid homeostasis. In mice, both ligand-dependent activation and knockout of PXR were previously shown to promote hepatic steatosis. To elucidate the respective pathways in human liver, we generated clones of human hepatoma HepG2 cells exhibiting different PXR protein levels, and analyzed effects of PXR activation and knockdown on steatosis and expression of lipogenic genes. Ligand-dependent activation as well as knockdown of PXR resulted in increased steatosis in HepG2 cells. Activation of PXR induced the sterol regulatory element-binding protein (SREBP) 1-dependent lipogenic pathway via PXR-dependent induction of SREBP1a, which was confirmed in primary human hepatocytes. Inhibiting SREBP1 activity by blocking the cleavage-dependent maturation of SREBP1 protein impaired the induction of lipogenic SREBP1 target genes and triglyceride accumulation by PXR activation. On the other hand, PXR knockdown resulted in up-regulation of aldo-keto reductase (AKR) 1B10, which enhanced the acetyl-CoA carboxylase (ACC)-catalyzed reaction step of de novo lipogenesis. In a cohort of human liver samples histologically classified for non-alcoholic fatty liver disease, AKR1B10, SREBP1a and SREBP1 lipogenic target genes proved to be up-regulated in steatohepatitis, while PXR protein was reduced. In summary, our data suggest that activation and knockdown of PXR in human hepatic cells promote de novo lipogenesis and steatosis by induction of the SREBP1 pathway and AKR1B10-mediated increase of ACC activity, respectively, thus providing mechanistic explanations for a putative dual role of PXR in the pathogenesis of steatohepatitis.

  15. Trypanosoma cruzi-elicited CD8+ T cell-mediated myocarditis: chemokine receptors and adhesion molecules as potential therapeutic targets to control chronic inflammation?

    Directory of Open Access Journals (Sweden)

    Joseli Lannes-Vieira


    Full Text Available In Chagas disease, during the acute phase, the establishment of inflammatory processes is crucial for Trypanosoma cruzi control in target tissues and for the establishment of host/parasite equilibrium. However, in about 30% of the patients, inflammation becomes progressive, resulting in chronic disease, mainly characterized by myocarditis. Although several hypothesis have been raised to explain the pathogenesis of chagasic myocardiopathy, including the persistence of the parasite and/or participation of autoimmune processes, the molecular mechanisms underlying the establishment of the inflammatory process leading to parasitism control but also contributing to the maintenance of T. cruzi-elicited chronic myocarditis remain unsolved. Trying to shed light on these questions, we have for several years been working with murine models for Chagas disease that reproduce the acute self-resolving meningoencephalitis, the encephalitis resulting of reactivation described in immunodeficient individuals, and several aspects of the acute and chronic myocarditis. In the present review, our results are summarized and discussed under the light of the current literature. Furthermore, rational therapeutic intervention strategies based on integrin-mediated adhesion and chemokine receptor-driven recruitment of leukocytes are proposed to control T. cruzi-elicited unbalanced inflammation.

  16. Two distinct sites in sonic Hedgehog combine for heparan sulfate interactions and cell signaling functions

    DEFF Research Database (Denmark)

    Chang, Shu-Chun; Mulloy, Barbara; Magee, Anthony I


    Hedgehog (Hh) proteins are morphogens that mediate many developmental processes. Hh signaling is significant for many aspects of embryonic development, whereas dysregulation of this pathway is associated with several types of cancer. Hh proteins require heparan sulfate proteoglycans (HSPGs......) for their normal distribution and signaling activity. Here, we have used molecular modeling to examine the heparin-binding domain of sonic hedgehog (Shh). In biochemical and cell biological assays, the importance of specific residues of the putative heparin-binding domain for signaling was assessed...

  17. HLA class II haplotypes distinctly associated with vaso-occlusion in children with sickle cell disease. (United States)

    Mahdi, Najat; Al-Ola, Khadija; Al-Subaie, Abeer M; Ali, Muhallab E; Al-Irhayim, Zaid; Al-Irhayim, A Qader; Almawi, Wassim Y


    We investigated the association of HLA class II alleles and haplotypes with sickle cell anemia vaso-occlusive crisis (VOC). DRB1*100101 was positively associated, while DRB1*140101, DRB1*150101, and DQB1*060101 were negatively associated, with VOC. Both susceptible (DRB1*100101-DQB1*050101) and protective (DRB1*110101-DQB1*030101 and DRB1*150101-DQB1*060101) haplotypes were identified, indicating that HLA class II haplotypes influence VOC risk.

  18. Distinct short-lived and long-lived antibody-producing cell populations. (United States)

    Ho, F; Lortan, J E; MacLennan, I C; Khan, M


    This report analyzes the life span of Ig-containing cells (IgCC) in different sites of antibody production. The experimental approach was based upon the observations that most IgCC are derived from proliferating precursors while IgCC themselves are mainly nondividing end cells. Rats were given a continuous infusion of [3H] thymidine via an osmotic pump inserted in the peritoneal cavity. At intervals of 1, 3, 5 or 10 days after starting infusions, tissues were taken and analyzed by a combination of immunohistology and autoradiography to identify the proportions of IgCC which had gone through S phase of the cell cycle during the period of infusion. After 3 days infusion the median and (range) percent-labeled IgCC in the medullary cords of mesenteric and cervical lymph nodes and the red pulp of the spleen were, respectively, 88 (81-90), 75 (66-77) and 88 (82-93). Conversely that for IgCC in bone marrow was only 13 (11-17) and that in the lamina propria of the jejunum 47 (33-68). The rate of increase in labeling of bone marrow IgCC with length of infusion was approximately linear. Extrapolation of this slope suggests that bone marrow IgCC have a life span in excess of 3 weeks. The slopes of increase in IgCC labeled with time for lymph nodes and spleen were clearly biphasic suggesting that while most IgCC in these tissues have a life span of less than 3 days, there is also a minor population of long-lived IgCC. The lamina propria appears to have approximately equal proportions of long and short-lived IgCC. The life span of IgCC, with the exception of IgMCC, appears to be a feature of the site of antibody production rather than the Ig class produced. Almost all IgM-containing cells were found to be short lived.

  19. Langerhans cells are generated by two distinct PU.1-dependent transcriptional networks. (United States)

    Chopin, Michaël; Seillet, Cyril; Chevrier, Stéphane; Wu, Li; Wang, Hongsheng; Morse, Herbert C; Belz, Gabrielle T; Nutt, Stephen L


    Langerhans cells (LCs) are the unique dendritic cells found in the epidermis. While a great deal of attention has focused on defining the developmental origins of LCs, reports addressing the transcriptional network ruling their differentiation remain sparse. We addressed the function of a group of key DC transcription factors-PU.1, ID2, IRF4, and IRF8-in the establishment of the LC network. We show that although steady-state LC homeostasis depends on PU.1 and ID2, the latter is dispensable for bone marrow-derived LCs. PU.1 controls LC differentiation by regulating the expression of the critical TGF-β responsive transcription factor RUNX3. PU.1 directly binds to the Runx3 regulatory elements in a TGF-β-dependent manner, whereas ectopic expression of RUNX3 rescued LC differentiation in the absence of PU.1 and promoted LC differentiation from PU.1-sufficient progenitors. These findings highlight the dual molecular network underlying LC differentiation, and show the central role of PU.1 in these processes.

  20. Coexistence of a colon carcinoma with two distinct renal cell carcinomas: a case report

    Directory of Open Access Journals (Sweden)

    Giannopoulos Lambros A


    Full Text Available Abstract Introduction We present the case of a patient with two tumors in his left kidney and a synchronous colon cancer. While coexisting tumors have been previously described in the same kidney or the kidney and other organs, or the colon and other organs, to the best of our knowledge no such concurrency of three primary tumors has been reported in the literature to date. Case presentation A 72-year-old man of Greek nationality presenting with pain in the right hypochondrium underwent a series of examinations that revealed gallstones, a tumor in the hepatic flexure of the colon and an additional tumor in the upper pole of the left kidney. He was subjected to a right hemicolectomy, left nephrectomy and cholecystectomy, and his postoperative course was uneventful. Histopathology examinations showed a mucinous colon adenocarcinoma, plus two tumors in the left kidney, a papillary renal cell carcinoma and a chromophobe renal cell carcinoma. Conclusion This case underlines the need to routinely scan patients pre-operatively in order to exclude coexisting tumors, especially asymptomatic renal tumors in patients with colorectal cancer, and additionally to screen concurrent tumors genetically in order to detect putative common genetic alterations.

  1. P. falciparum isolate-specific distinct patterns of induced apoptosis in pulmonary and brain endothelial cells.

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    Nadine N'Dilimabaka

    Full Text Available The factors implicated in the transition from uncomplicated to severe clinical malaria such as pulmonary oedema and cerebral malaria remain unclear. It is known that alterations in vascular integrity due to endothelial cell (EC activation and death occur during severe malaria. In this study, we assessed the ability of different P. falciparum clinical isolates to induce apoptosis in ECs derived from human lung and brain. We observed that induction of EC apoptosis was sensitive to the environmental pH and required direct contact between the parasite and the cell, though it was not correlated to the ability of the parasite to cytoadhere. Moreover, the extent of induced apoptosis in the two EC types varied with the isolate. Analysis of parasite genes transcript led us to propose that the activation of different pathways, such as Plasmodium apoptosis-linked pathogenicity factors (PALPF, PALPF-2, PALPF-5 and PF11_0521, could be implied in EC death. These observations provide an experimental framework to decipher the molecular mechanism implicated in the genesis of severe malaria.

  2. Different BAG-1 isoforms have distinct functions in modulating chemotherapeutic-induced apoptosis in breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    Hongyu LIU; Zhuomin WANG; Yun BAI; Min WANG; Ying LI; Sen WEI; Qinghua ZHOU; Jun CHEN


    Aim:BAG-1 is a multifunctional anti-apoptotic gene with four isoforms,and different BAG-1 isoforms have different anti-apoptotic functions.In this study,we transfected BAG-1 isoforms into the human breast cancer cell lines Hs578T (ER nega-tive) and MCF-7 (ER positive) to study their effect on apoptosis with or without estrogens.Methods: The constructed recombinant expression vectors carrying individual BAG-1 isoforms was used to transfect human breast cancer cell lines Hs578T (ER negative) and MCF-7 (ER positive).After stable cell lines were made,a variety of apoptosis-inducing agents,including doxorubicin,docetaxel,and 5-FU,was used to treat these cell lines with or without estrogen to test the role of BAG-1.The mechanism by which BAG-1 affected the function of Bcl-2 was exploredby using the cycloheximide chase assay.Results: The BAG-1 p50 and p46 isoforms significantly enhanced the resistance to apoptosis in both cell lines according to flow cytometry analysis.BAG-1 p33 and p29 failed to protect the transfected cells from apoptosis.The cell viability assay showed that only BAG-1 p50,but not p46,p33,or p29,increased estrogen-dependent function in ER-positive cell line MCF-7.Only BAG-1 p50 dramatically increased its anti-apoptotic ability in the presence of estrogen,while estrogen has very little effect on the anti-apoptotic ability of other BAG-1 isoforms.In the detection of the expression of K-ras,Hsp70,cytochrome c,Raf-1,ER-α,and Bcl-2 in MCF-7 cells by Western blot,only Bcl-2 protein expression was significantly increased in MCF-7 cells transfected with BAG-1 p50 and p46,respectively.Furthermore,the cycloheximide chase assay indicated that the degradation of Bcl-2 protein was extended in the BAG-1 p50 and p46 transfected MCF-7 cells.Conclusion: Distinct isoforrns of BAG-1 have different anti-apoptotic functions in breast cancer cells,and that the BAG-1 p50 isoform can potentiate the role of estrogen in ER-positive breast cancer.

  3. Identification of Distinct Breast Cancer Stem Cell Populations Based on Single-Cell Analyses of Functionally Enriched Stem and Progenitor Pools

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    Nina Akrap


    Full Text Available The identification of breast cancer cell subpopulations featuring truly malignant stem cell qualities is a challenge due to the complexity of the disease and lack of general markers. By combining extensive single-cell gene expression profiling with three functional strategies for cancer stem cell enrichment including anchorage-independent culture, hypoxia, and analyses of low-proliferative, label-retaining cells derived from mammospheres, we identified distinct stem cell clusters in breast cancer. Estrogen receptor (ERα+ tumors featured a clear hierarchical organization with switch-like and gradual transitions between different clusters, illustrating how breast cancer cells transfer between discrete differentiation states in a sequential manner. ERα− breast cancer showed less prominent clustering but shared a quiescent cancer stem cell pool with ERα+ cancer. The cellular organization model was supported by single-cell data from primary tumors. The findings allow us to understand the organization of breast cancers at the single-cell level, thereby permitting better identification and targeting of cancer stem cells.

  4. Targeting of apoptotic pathways by SMAC or BH3 mimetics distinctly sensitizes paclitaxel-resistant triple negative breast cancer cells. (United States)

    Panayotopoulou, Effrosini G; Müller, Anna-Katharina; Börries, Melanie; Busch, Hauke; Hu, Guohong; Lev, Sima


    Standard chemotherapy is the only systemic treatment for triple-negative breast cancer (TNBC), and despite the good initial response, resistance remains a major therapeutic obstacle. Here, we employed a High-Throughput Screen to identify targeted therapies that overcome chemoresistance in TNBC. We applied short-term paclitaxel treatment and screened 320 small-molecule inhibitors of known targets to identify drugs that preferentially and efficiently target paclitaxel-treated TNBC cells. Among these compounds the SMAC mimetics (BV6, Birinapant) and BH3-mimetics (ABT-737/263) were recognized as potent targeted therapy for multiple paclitaxel-residual TNBC cell lines. However, acquired paclitaxel resistance through repeated paclitaxel pulses result in desensitization to BV6, but not to ABT-263, suggesting that short- and long-term paclitaxel resistance are mediated by distinct mechanisms. Gene expression profiling of paclitaxel-residual, -resistant and naïve MDA-MB-231 cells demonstrated that paclitaxel-residual, as opposed to -resistant cells, were characterized by an apoptotic signature, with downregulation of anti-apoptotic genes (BCL2, BIRC5), induction of apoptosis inducers (IL24, PDCD4), and enrichment of TNFα/NF-κB pathway, including upregulation of TNFSF15, coupled with cell-cycle arrest. BIRC5 and FOXM1 downregulation and IL24 induction was also evident in breast cancer patient datasets following taxane treatment. Exposure of naïve or paclitaxel-resistant cells to supernatants of paclitaxel-residual cells sensitized them to BV6, and treatment with TNFα enhanced BV6 potency, suggesting that sensitization to BV6 is mediated, at least partially, by secreted factor(s). Our results suggest that administration of SMAC or BH3 mimetics following short-term paclitaxel treatment could be an effective therapeutic strategy for TNBC, while only BH3-mimetics could effectively overcome long-term paclitaxel resistance.

  5. A Distinct Subpopulation of Bone Marrow Mesenchymal Stem Cells, Muse Cells, Directly Commit to the Replacement of Liver Components. (United States)

    Katagiri, H; Kushida, Y; Nojima, M; Kuroda, Y; Wakao, S; Ishida, K; Endo, F; Kume, K; Takahara, T; Nitta, H; Tsuda, H; Dezawa, M; Nishizuka, S S


    Genotyping graft livers by short tandem repeats after human living-donor liver transplantation (n = 20) revealed the presence of recipient or chimeric genotype cases in hepatocytes (6 of 17, 35.3%), sinusoidal cells (18 of 18, 100%), cholangiocytes (15 of 17, 88.2%) and cells in the periportal areas (7 of 8, 87.5%), suggesting extrahepatic cell involvement in liver regeneration. Regarding extrahepatic origin, bone marrow mesenchymal stem cells (BM-MSCs) have been suggested to contribute to liver regeneration but compose a heterogeneous population. We focused on a more specific subpopulation (1-2% of BM-MSCs), called multilineage-differentiating stress-enduring (Muse) cells, for their ability to differentiate into liver-lineage cells and repair tissue. We generated a physical partial hepatectomy model in immunodeficient mice and injected green fluorescent protein (GFP)-labeled human BM-MSC Muse cells intravenously (n = 20). Immunohistochemistry, fluorescence in situ hybridization and species-specific polymerase chain reaction revealed that they integrated into regenerating areas and expressed liver progenitor markers during the early phase and then differentiated spontaneously into major liver components, including hepatocytes (≈74.3% of GFP-positive integrated Muse cells), cholangiocytes (≈17.7%), sinusoidal endothelial cells (≈2.0%), and Kupffer cells (≈6.0%). In contrast, the remaining cells in the BM-MSCs were not detected in the liver for up to 4 weeks. These results suggest that Muse cells are the predominant population of BM-MSCs that are capable of replacing major liver components during liver regeneration.

  6. Improved flow cytometric assessment reveals distinct microvesicle (cell-derived microparticle signatures in joint diseases.

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    Bence György

    Full Text Available INTRODUCTION: Microvesicles (MVs, earlier referred to as microparticles, represent a major type of extracellular vesicles currently considered as novel biomarkers in various clinical settings such as autoimmune disorders. However, the analysis of MVs in body fluids has not been fully standardized yet, and there are numerous pitfalls that hinder the correct assessment of these structures. METHODS: In this study, we analyzed synovial fluid (SF samples of patients with osteoarthritis (OA, rheumatoid arthritis (RA and juvenile idiopathic arthritis (JIA. To assess factors that may confound MV detection in joint diseases, we used electron microscopy (EM, Nanoparticle Tracking Analysis (NTA and mass spectrometry (MS. For flow cytometry, a method commonly used for phenotyping and enumeration of MVs, we combined recent advances in the field, and used a novel approach of differential detergent lysis for the exclusion of MV-mimicking non-vesicular signals. RESULTS: EM and NTA showed that substantial amounts of particles other than MVs were present in SF samples. Beyond known MV-associated proteins, MS analysis also revealed abundant plasma- and immune complex-related proteins in MV preparations. Applying improved flow cytometric analysis, we demonstrate for the first time that CD3(+ and CD8(+ T-cell derived SF MVs are highly elevated in patients with RA compared to OA patients (p=0.027 and p=0.009, respectively, after Bonferroni corrections. In JIA, we identified reduced numbers of B cell-derived MVs (p=0.009, after Bonferroni correction. CONCLUSIONS: Our results suggest that improved flow cytometric assessment of MVs facilitates the detection of previously unrecognized disease-associated vesicular signatures.

  7. The neural cell adhesion molecule L1 is distinct from the N-CAM related group of surface antigens BSP-2 and D2

    DEFF Research Database (Denmark)

    Faissner, A; Kruse, J; Goridis, C


    The neural cell adhesion molecule L1 and the group of N-CAM related molecules, BSP-2 and D2 antigen, are immunochemically distinct molecular species. The two groups of surface molecules are also functionally distinct entities, since inhibition of Ca2+-independent adhesion among early post-natal m......-natal mouse cerebellar cells by Fab fragments of both antibodies are at least additive, when compared with equal concentrations of the individual antibodies....

  8. Quinuclidine compounds differently act as agonists of Kenyon cell nicotinic acetylcholine receptors and induced distinct effect on insect ganglionic depolarizations. (United States)

    Mathé-Allainmat, Monique; Swale, Daniel; Leray, Xavier; Benzidane, Yassine; Lebreton, Jacques; Bloomquist, Jeffrey R; Thany, Steeve H


    We have recently demonstrated that a new quinuclidine benzamide compound named LMA10203 acted as an agonist of insect nicotinic acetylcholine receptors. Its specific pharmacological profile on cockroach dorsal unpaired median neurons (DUM) helped to identify alpha-bungarotoxin-insensitive nAChR2 receptors. In the present study, we tested its effect on cockroach Kenyon cells. We found that it induced an inward current demonstrating that it bounds to nicotinic acetylcholine receptors expressed on Kenyon cells. Interestingly, LMA10203-induced currents were completely blocked by the nicotinic antagonist α-bungarotoxin. We suggested that LMA10203 effect occurred through the activation of α-bungarotoxin-sensitive receptors and did not involve α-bungarotoxin-insensitive nAChR2, previously identified in DUM neurons. In addition, we have synthesized two new compounds, LMA10210 and LMA10211, and compared their effects on Kenyon cells. These compounds were members of the 3-quinuclidinyl benzamide or benzoate families. Interestingly, 1 mM LMA10210 was not able to induce an inward current on Kenyon cells compared to LMA10211. Similarly, we did not find any significant effect of LMA10210 on cockroach ganglionic depolarization, whereas these three compounds were able to induce an effect on the central nervous system of the third instar M. domestica larvae. Our data suggested that these three compounds could bind to distinct cockroach nicotinic acetylcholine receptors.

  9. A Distinct Slow-Cycling Cancer Stem-like Subpopulation of Pancreatic Adenocarcinoma Cells is maintained in Vivo

    Energy Technology Data Exchange (ETDEWEB)

    Dembinski, Jennifer L., E-mail:; Krauss, Stefan [Cellular and Genetic Therapy, Department of Microbiology, Cancer Stem Cell Innovation Center (CAST), Oslo University Hospital, Rikshospitalet, Oslo (Norway)


    Pancreatic adenocarcinoma has the worst prognosis of any major malignancy, with <5% of patients surviving five years. This can be contributed to the often late diagnosis, lack of sufficient treatment and metastatic spread. Heterogeneity within tumors is increasingly becoming a focus in cancer research, as novel therapies are required to target the most aggressive subpopulations of cells that are frequently termed cancer stem cells (CSCs). In the current study, we describe the identification of a slow-cycling cancer stem-like population of cells in vivo in BxPC-3 and Panc03.27 xenografts. A distinct slow-cycling label-retaining population of cells (DiI+/SCC) was found both at the edge of tumors, and in small circumscribed areas within the tumors. DiI+/SCC in these areas display an epithelial-to-mesenchymal transition (EMT) fingerprint, including an upregulation of the mesenchymal markers vimentin and N-cadherin and a loss of the epithelial marker E-cadherin. DiI+/SCC also displayed a critical re-localization of beta-catenin from the membrane to the nucleus. Additionally, the DiI+/SCC population was found to express the developmental signaling molecule sonic hedgehog. This study represents a novel step in defining the biological activities of a tumorigenic subpopulation within the heterogeneous tumor microenvironment in vivo. Understanding the interactions and functions of a CSC population within the context of the tumor microenvironment is critical to design targeted therapeutics.

  10. Distinct Sources of Hematopoietic Progenitors Emerge before HSCs and Provide Functional Blood Cells in the Mammalian Embryo

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    Kathleen E. McGrath


    Full Text Available Hematopoietic potential arises in mammalian embryos before adult-repopulating hematopoietic stem cells (HSCs. At embryonic day 9.5 (E9.5, we show the first murine definitive erythro-myeloid progenitors (EMPs have an immunophenotype distinct from primitive hematopoietic progenitors, maturing megakaryocytes and macrophages, and rare B cell potential. EMPs emerge in the yolk sac with erythroid and broad myeloid, but not lymphoid, potential. EMPs migrate to the fetal liver and rapidly differentiate, including production of circulating neutrophils by E11.5. Although the surface markers, transcription factors, and lineage potential associated with EMPs overlap with those found in adult definitive hematopoiesis, they are present in unique combinations or proportions that result in a specialized definitive embryonic progenitor. Furthermore, we find that embryonic stem cell (ESC-derived hematopoiesis recapitulates early yolk sac hematopoiesis, including primitive, EMP, and rare B cell potential. EMPs do not have long-term potential when transplanted in immunocompromised adults, but they can provide transient adult-like RBC reconstitution.

  11. Hyperosmolarity invokes distinct anti-inflammatory mechanisms in pulmonary epithelial cells: evidence from signaling and transcription layers.

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    Franklin L Wright

    Full Text Available Hypertonic saline (HTS has been used intravenously to reduce organ dysfunction following injury and as an inhaled therapy for cystic fibrosis lung disease. The role and mechanism of HTS inhibition was explored in the TNFα and IL-1β stimulation of pulmonary epithelial cells. Hyperosmolar (HOsm media (400 mOsm inhibited the production of select cytokines stimulated by TNFα and IL-1β at the level of mRNA translation, synthesis and release. In TNFα stimulated A549 cells, HOsm media inhibited I-κBα phosphorylation, NF-κB translocation into the nucleus and NF-κB nuclear binding. In IL-1β stimulated cells HOsm inhibited I-κBα phosphorylation without affecting NF-κB translocation or nuclear binding. Incubation in HOsm conditions inhibited both TNFα and IL-1β stimulated nuclear localization of interferon response factor 1 (IRF-1. Additional transcription factors such as AP-1, Erk-1/2, JNK and STAT-1 were unaffected by HOsm. HTS and sorbitol supplemented media produced comparable outcomes in all experiments, indicating that the effects of HTS were mediated by osmolarity, not by sodium. While not affecting MAPK modules discernibly in A549 cells, both HOsm conditions inhibit IRF-1 against TNFα or IL-1β, but inhibit p65 NF-kB translocation only against TNFα but not IL-1β. Thus, anti-inflammatory mechanisms of HTS/HOsm appear to disrupt cytokine signals at distinct intracellular steps.

  12. Antigen-Specific Th17 Cells Are Primed by Distinct and Complementary Dendritic Cell Subsets in Oropharyngeal Candidiasis.

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    Kerstin Trautwein-Weidner


    Full Text Available Candida spp. can cause severe and chronic mucocutaneous and systemic infections in immunocompromised individuals. Protection from mucocutaneous candidiasis depends on T helper cells, in particular those secreting IL-17. The events regulating T cell activation and differentiation toward effector fates in response to fungal invasion in different tissues are poorly understood. Here we generated a Candida-specific TCR transgenic mouse reactive to a novel endogenous antigen that is conserved in multiple distant species of Candida, including the clinically highly relevant C. albicans and C. glabrata. Using TCR transgenic T cells in combination with an experimental model of oropharyngeal candidiasis (OPC we investigated antigen presentation and Th17 priming by different subsets of dendritic cells (DCs present in the infected oral mucosa. Candida-derived endogenous antigen accesses the draining lymph nodes and is directly presented by migratory DCs. Tissue-resident Flt3L-dependent DCs and CCR2-dependent monocyte-derived DCs collaborate in antigen presentation and T cell priming during OPC. In contrast, Langerhans cells, which are also present in the oral mucosa and have been shown to prime Th17 cells in the skin, are not required for induction of the Candida-specific T cell response upon oral challenge. This highlights the functional compartmentalization of specific DC subsets in different tissues. These data provide important new insights to our understanding of tissue-specific antifungal immunity.

  13. Distinct roles of Shh and Fgf signaling in regulating cell proliferation during zebrafish pectoral fin development

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    Neumann Carl J


    Full Text Available Abstract Background Cell proliferation in multicellular organisms must be coordinated with pattern formation. The major signaling pathways directing pattern formation in the vertebrate limb are well characterized, and we have therefore chosen this organ to examine the interaction between proliferation and patterning. Two important signals for limb development are members of the Hedgehog (Hh and Fibroblast Growth Factor (Fgf families of secreted signaling proteins. Sonic hedgehog (Shh directs pattern formation along the anterior/posterior axis of the limb, whereas several Fgfs in combination direct pattern formation along the proximal/distal axis of the limb. Results We used the genetic and pharmacological amenability of the zebrafish model system to dissect the relative importance of Shh and Fgf signaling in regulating proliferation during development of the pectoral fin buds. In zebrafish mutants disrupting the shh gene, proliferation in the pectoral fin buds is initially normal, but later is strongly reduced. Correlating with this reduction, Fgf signaling is normal at early stages, but is later lost in shh mutants. Furthermore, pharmacological inhibition of Hh signaling for short periods has little effect on either Fgf signaling, or on expression of G1- and S-phase cell-cycle genes, whereas long periods of inhibition lead to the downregulation of both. In contrast, even short periods of pharmacological inhibition of Fgf signaling lead to strong disruption of proliferation in the fin buds, without affecting Shh signaling. To directly test the ability of Fgf signaling to regulate proliferation in the absence of Shh signaling, we implanted beads soaked with Fgf protein into shh mutant fin buds. We find that Fgf-soaked beads rescue proliferation in the pectoral find buds of shh mutants, indicating that Fgf signaling is sufficient to direct proliferation in zebrafish fin buds in the absence of Shh. Conclusion Previous studies have shown that both

  14. Lipopolysaccharide (LPS) inhibits steroid production in theca cells of bovine follicles in vitro: distinct effect of LPS on theca cell function in pre- and post-selection follicles. (United States)

    Magata, Fumie; Horiuchi, Maya; Miyamoto, Akio; Shimizu, Takashi


    In postpartum dairy cows, lipopolysaccharide (LPS) derived from gram-negative bacteria such as Escherichia coli causes uterine inflammation and leads to ovarian dysfunction. The aim of this study was to determine the effect of LPS on steroid production in bovine theca cells at different stages of follicular development. Theca cells isolated from pre- and post-selection follicles (PRFs, 8.5 mm in diameter, respectively) of bovine ovaries were exposed to LPS under luteinizing hormone (LH) conditions, estradiol (E2) conditions or both conditions in vitro. Bovine theca cells expressed the LPS receptor gene complex: Toll-like receptor 4 (TLR4), CD14 and MD2. LPS suppressed progesterone (P4) and androstenedione (A4) production with downregulation of steroidogenic enzyme transcripts when theca cells were stimulated with LH. By contrast, LPS did not affect P4 or A4 production when theca cells were stimulated with E2. P4 and A4 production in theca cells from PRFs was suppressed by LPS as early as at 48 h of culture, whereas the effect of LPS on theca cells from POFs was observed at 96 h of culture. The results demonstrate that LPS inhibits steroid production in theca cells under LH conditions. Moreover, theca cells from POFs showed a slower response to LPS compared with that of theca cells from PRFs, which might imply a distinct effect of LPS on follicles at different developmental stages. These findings suggest a possible mechanism of ovarian dysfunction and subsequent infertility in cows with endometritis.

  15. Distinct Rayleigh scattering from hot spot mutant p53 proteins reveals cancer cells. (United States)

    Jun, Ho Joon; Nguyen, Anh H; Kim, Yeul Hong; Park, Kyong Hwa; Kim, Doyoun; Kim, Kyeong Kyu; Sim, Sang Jun


    The scattering of light redirects and resonances when an electromagnetic wave interacts with electrons orbits in the hot spot core protein and oscillated electron of the gold nanoparticles (AuNP). This report demonstrates convincingly that resonant Rayleigh scattering generated from hot spot mutant p53 proteins is correspondence to cancer cells. Hot spot mutants have unique local electron density changes that affect specificity of DNA binding affinity compared with wild types. Rayleigh scattering changes introduced by hot-spot mutations were monitored by localized surface plasmon resonance (LSPR) shift changes. The LSPR λmax shift for hot-spot mutants ranged from 1.7 to 4.2 nm for mouse samples and from 0.64 nm to 2.66 nm for human samples, compared to 9.6 nm and 15 nm for wild type and mouse and human proteins, respectively with a detection sensitivity of p53 concentration at 17.9 nM. It is interesting that hot-spot mutants, which affect only interaction with DNA, launches affinitive changes as considerable as wild types. These changes propose that hot-spot mutants p53 proteins can be easily detected by local electron density alterations that disturbs the specificity of DNA binding of p53 core domain on the surface of the DNA probed-nanoplasmonic sensor.

  16. Turtle Dorsal Cortex Pyramidal Neurons Comprise Two Distinct Cell Types with Indistinguishable Visual Responses.

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    Thomas Crockett

    Full Text Available A detailed inventory of the constituent pieces in cerebral cortex is considered essential to understand the principles underlying cortical signal processing. Specifically, the search for pyramidal neuron subtypes is partly motivated by the hypothesis that a subtype-specific division of labor could create a rich substrate for computation. On the other hand, the extreme integration of individual neurons into the collective cortical circuit promotes the hypothesis that cellular individuality represents a smaller computational role within the context of the larger network. These competing hypotheses raise the important question to what extent the computational function of a neuron is determined by its individual type or by its circuit connections. We created electrophysiological profiles from pyramidal neurons within the sole cellular layer of turtle visual cortex by measuring responses to current injection using whole-cell recordings. A blind clustering algorithm applied to these data revealed the presence of two principle types of pyramidal neurons. Brief diffuse light flashes triggered membrane potential fluctuations in those same cortical neurons. The apparently network driven variability of the visual responses concealed the existence of subtypes. In conclusion, our results support the notion that the importance of diverse intrinsic physiological properties is minimized when neurons are embedded in a synaptic recurrent network.

  17. Tropomyosin variants describe distinct functional subcellular domains in differentiated vascular smooth muscle cells. (United States)

    Gallant, Cynthia; Appel, Sarah; Graceffa, Philip; Leavis, Paul; Lin, Jim Jung-Ching; Gunning, Peter W; Schevzov, Galina; Chaponnier, Christine; DeGnore, Jon; Lehman, William; Morgan, Kathleen G


    Tropomyosin (Tm) is known to be an important gatekeeper of actin function. Tm isoforms are encoded by four genes, and each gene produces several variants by alternative splicing, which have been proposed to play roles in motility, proliferation, and apoptosis. Smooth muscle studies have focused on gizzard smooth muscle, where a heterodimer of Tm from the α-gene (Tmsm-α) and from the β-gene (Tmsm-β) is associated with contractile filaments. In this study we examined Tm in differentiated mammalian vascular smooth muscle (dVSM). Liquid chromatography-tandem mass spectrometry (LC MS/MS) analysis and Western blot screening with variant-specific antibodies revealed that at least five different Tm proteins are expressed in this tissue: Tm6 (Tmsm-α) and Tm2 from the α-gene, Tm1 (Tmsm-β) from the β-gene, Tm5NM1 from the γ-gene, and Tm4 from the δ-gene. Tm6 is by far most abundant in dVSM followed by Tm1, Tm2, Tm5NM1, and Tm4. Coimmunoprecipitation and coimmunofluorescence studies demonstrate that Tm1 and Tm6 coassociate with different actin isoforms and display different intracellular localizations. Using an antibody specific for cytoplasmic γ-actin, we report here the presence of a γ-actin cortical cytoskeleton in dVSM cells. Tm1 colocalizes with cortical cytoplasmic γ-actin and coprecipitates with γ-actin. Tm6, on the other hand, is located on contractile bundles. These data indicate that Tm1 and Tm6 do not form a classical heterodimer in dVSM but rather describe different functional cellular compartments.

  18. Differentiated neuroprogenitor cells incubated with human or canine adenovirus, or lentiviral vectors have distinct transcriptome profiles.

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    Stefania Piersanti

    Full Text Available Several studies have demonstrated the potential for vector-mediated gene transfer to the brain. Helper-dependent (HD human (HAd and canine (CAV-2 adenovirus, and VSV-G-pseudotyped self-inactivating HIV-1 vectors (LV effectively transduce human brain cells and their toxicity has been partly analysed. However, their effect on the brain homeostasis is far from fully defined, especially because of the complexity of the central nervous system (CNS. With the goal of dissecting the toxicogenomic signatures of the three vectors for human neurons, we transduced a bona fide human neuronal system with HD-HAd, HD-CAV-2 and LV. We analysed the transcriptional response of more than 47,000 transcripts using gene chips. Chip data showed that HD-CAV-2 and LV vectors activated the innate arm of the immune response, including Toll-like receptors and hyaluronan circuits. LV vector also induced an IFN response. Moreover, HD-CAV-2 and LV vectors affected DNA damage pathways--but in opposite directions--suggesting a differential response of the p53 and ATM pathways to the vector genomes. As a general response to the vectors, human neurons activated pro-survival genes and neuron morphogenesis, presumably with the goal of re-establishing homeostasis. These data are complementary to in vivo studies on brain vector toxicity and allow a better understanding of the impact of viral vectors on human neurons, and mechanistic approaches to improve the therapeutic impact of brain-directed gene transfer.

  19. Nuclear morphometry identifies a distinct aggressive cellular phenotype in cutaneous squamous cell carcinoma. (United States)

    Glazer, Evan S; Bartels, Peter H; Prasad, Anil R; Yozwiak, Michael L; Bartels, Hubert G; Einspahr, Janine G; Alberts, David S; Krouse, Robert S


    By identifying aggressive cutaneous squamous cell carcinoma (cSCC) in patients who are at high risk for recurrences or second primaries after resection, intensive surveillance and therapy may decrease morbidity and mortality. We investigated the role of nuclear morphometry (karyometry) in differentiating between aggressive and nonaggressive cSCC. We retrospectively analyzed cSCC lesions from 40 male patients. Twenty-two patients had evidence of aggressive cSCC (local/regional recurrence or a second primary cSCC), and 18 patients were identified with similar ages and sites of disease as control patients with nonaggressive cSCC (no evidence of recurrence, metastasis, or second primary). We carried out karyometric analysis to identify nuclear features that discriminate between aggressive and nonaggressive cSCC nuclei. We used statistically significant differences (Kruskal-Wallis test, P < 0.0001) to compose a quantitative aggressive classification score (proportion of aggressive nuclei from 0% to 100%). For comparisons, we used Fisher's exact test or Student's t test. The mean age was 79 ± 7 years for aggressive cSCC and 80 ± 9 years for nonaggressive cSCC (P = 0.66). We analyzed a mean of 96 nuclei in each group. The mean classification score for aggressive cSCC was significantly higher (69% ± 6%) than for nonaggressive cSCC (28% ± 5%, P = 0.00002). Overall, the classification score accurately categorized 80% of our patients (P = 0.0004). In most patients, karyometry differentiated between aggressive and nonaggressive cSCC. We found that classification scores, which provide information on individual lesions, could be used for risk stratification.

  20. Epigenetic control of Ccr7 expression in distinct lineages of lung dendritic cells. (United States)

    Moran, Timothy P; Nakano, Hideki; Kondilis-Mangum, Hrisavgi D; Wade, Paul A; Cook, Donald N


    Adaptive immune responses to inhaled allergens are induced following CCR7-dependent migration of precursor of dendritic cell (pre-DC)-derived conventional DCs (cDCs) from the lung to regional lymph nodes. However, monocyte-derived (moDCs) in the lung express very low levels of Ccr7 and consequently do not migrate efficiently to LN. To investigate the molecular mechanisms that underlie this dichotomy, we studied epigenetic modifications at the Ccr7 locus of murine cDCs and moDCs. When expanded from bone marrow precursors, moDCs were enriched at the Ccr7 locus for trimethylation of histone 3 lysine 27 (H3K27me3), a modification associated with transcriptional repression. Similarly, moDCs prepared from the lung also displayed increased levels of H3K27me3 at the Ccr7 promoter compared with migratory cDCs from that organ. Analysis of DC progenitors revealed that epigenetic modification of Ccr7 does not occur early during DC lineage commitment because monocytes and pre-DCs both had low levels of Ccr7-associated H3K27me3. Rather, Ccr7 is gradually silenced during the differentiation of monocytes to moDCs. Thus, epigenetic modifications of the Ccr7 locus control the migration and therefore the function of DCs in vivo. These findings suggest that manipulating epigenetic mechanisms might be a novel approach to control DC migration and thereby improve DC-based vaccines and treat inflammatory diseases of the lung.

  1. A prospective study on contrast-enhanced magnetic resonance imaging of testicular lesions: distinctive features of Leydig cell tumours

    Energy Technology Data Exchange (ETDEWEB)

    Manganaro, Lucia; Vinci, Valeria; Saldari, Matteo; Bernardo, Silvia; Cantisani, Vito; Catalano, Carlo [Sapienza University of Rome, Department of Radiology, Rome (Italy); Pozza, Carlotta; Gianfrilli, Daniele; Pofi, Riccardo; Lenzi, Andrea; Isidori, Andrea M. [Sapienza University of Rome, Department of Experimental Medicine, Rome (Italy); Scialpi, Michele [Perugia University, S. Maria della Misericordia Hospital, Department of Surgical and Biomedical Sciences, Division of Radiology 2, Perugia (Italy)


    Up to 20 % of incidentally found testicular lesions are benign Leydig cell tumours (LCTs). This study evaluates the role of contrast-enhanced magnetic resonance imaging (MRI) in the identification of LCTs in a large prospective cohort study. We enrolled 44 consecutive patients with at least one solid non-palpable testicular lesion who underwent scrotal MRI. Margins of the lesions, signal intensity and pattern of wash-in and wash-out were analysed by two radiologists. The frequency distribution of malignant and benign MRI features in the different groups was compared by using the chi-squared or Fisher's exact test. Sensitivity, specificity, positive and negative predictive value, and diagnostic accuracy were calculated. The sensitivity of scrotal MRI to diagnose LCTs was 89.47 % with 95.65 % specificity; sensitivity for malignant lesions was 95.65 % with 80.95 % specificity. A markedly hypointense signal on T2-WI, rapid and marked wash-in followed by a prolonged washout were distinctive features significantly associated with LCTs. Malignant lesions were significantly associated with blurred margins, weak hypointense signal on T2-WI,and weak and progressive wash-in. The overall diagnostic accuracy was 93 %. LCTs have distinctive contrast-enhanced MRI features that allow the differential diagnosis of incidental testicular lesions. (orig.)

  2. T-cell-rich large B-cell lymphoma. A study of 30 cases, supporting its histologic heterogeneity and lack of clinical distinctiveness. (United States)

    Krishnan, J; Wallberg, K; Frizzera, G


    To determine whether correlations existed between morphologic and immunophenotypic findings and clinical characteristics, 30 cases of T-cell-rich large B-cell lymphomas (TBL) were evaluated by histopathology, immunostaining, and polymerase chain reaction on paraffin-embedded material. All were characterized by a polymorphic cell composition, including a variable mixture of small and large lymphoid cells and reactive cell. Most cases (87%) fitted into one of three main histologic types of non-Hodgkin's lymphoma (diffuse, mixed cell; diffuse, large cell; follicular and diffuse, mixed cell), and one group of eight cases had the prototypic features described by Ramsay et al. (17). All cases showed a component of large CD20(L26)+ MB2+ B cells in a predominant back-ground of reactive T cells (> 50% of the total lymphoid forms). Clonality was demonstrated by light chain restriction in 67% of cases and by rearrangement of the immunoglobulin heavy chain gene and bcl-2 gene in 64% and 28% of cases, respectively. The patients were predominantly men (70%), ages 18-83 years (median of 62.5), and were initially seen predominantly with nodal disease (and extranodal involvement in 20%) at advanced stages (III-IV: 77%). Treatment was mostly aggressive chemotherapy, and the outcomes were favorable (84% alive and well). These features are not distinctive as compared with those of typical large-cell lymphoma, nor did subgroups within the series (prototypic cases versus others; cases with less [ 70%] T-cell infiltration) significantly differ in clinical presentation or outcome. Thus, this study confirms that TBL, while useful as a diagnostic variant to be distinguished from both peripheral T-cell lymphoma and Hodgkin's disease, is a heterogeneous assortment of diverse histopathologic categories rather than a clinicopathologic entity. The term "T-cell rich" might, however, be usefully retained as a morphologic specification to be added to recognized histologic categories of lymphoma.

  3. Imaging Cellular Dynamics with Spectral Relaxation Imaging Microscopy: Distinct Spectral Dynamics in Golgi Membranes of Living Cells (United States)

    Lajevardipour, Alireza; Chon, James W. M.; Chattopadhyay, Amitabha; Clayton, Andrew H. A.


    Spectral relaxation from fluorescent probes is a useful technique for determining the dynamics of condensed phases. To this end, we have developed a method based on wide-field spectral fluorescence lifetime imaging microscopy to extract spectral relaxation correlation times of fluorescent probes in living cells. We show that measurement of the phase and modulation of fluorescence from two wavelengths permit the identification and determination of excited state lifetimes and spectral relaxation correlation times at a single modulation frequency. For NBD fluorescence in glycerol/water mixtures, the spectral relaxation correlation time determined by our approach exhibited good agreement with published dielectric relaxation measurements. We applied this method to determine the spectral relaxation dynamics in membranes of living cells. Measurements of the Golgi-specific C6-NBD-ceramide probe in living HeLa cells revealed sub-nanosecond spectral dynamics in the intracellular Golgi membrane and slower nanosecond spectral dynamics in the extracellular plasma membrane. We interpret the distinct spectral dynamics as a result of structural plasticity of the Golgi membrane relative to more rigid plasma membranes. To the best of our knowledge, these results constitute one of the first measurements of Golgi rotational dynamics.

  4. BMP Sustains Embryonic Stem Cell Self-Renewal through Distinct Functions of Different Krüppel-like Factors

    Directory of Open Access Journals (Sweden)

    Masato Morikawa


    Full Text Available Bone morphogenetic protein (BMP signaling exerts paradoxical roles in pluripotent stem cells (PSCs; it sustains self-renewal of mouse embryonic stem cells (ESCs, while it induces differentiation in other PSCs, including human ESCs. Here, we revisit the roles of BMP-4 using mouse ESCs (mESCs in naive and primed states. SMAD1 and SMAD5, which transduce BMP signals, recognize enhancer regions together with KLF4 and KLF5 in naive mESCs. KLF4 physically interacts with SMAD1 and suppresses its activity. Consistently, a subpopulation of cells with active BMP-SMAD can be ablated without disturbing the naive state of the culture. Moreover, Smad1/5 double-knockout mESCs stay in the naive state, indicating that the BMP-SMAD pathway is dispensable for it. In contrast, the MEK5-ERK5 pathway mediates BMP-4-induced self-renewal of mESCs by inducing Klf2, a critical factor for the ground state pluripotency. Our study illustrates that BMP exerts its self-renewing effect through distinct functions of different Krüppel-like factors.

  5. Distinct pathways for modification of the bacterial cell wall by non-canonical D-amino acids. (United States)

    Cava, Felipe; de Pedro, Miguel A; Lam, Hubert; Davis, Brigid M; Waldor, Matthew K


    Production of non-canonical D-amino acids (NCDAAs) in stationary phase promotes remodelling of peptidoglycan (PG), the polymer that comprises the bacterial cell wall. Impairment of NCDAAs production leads to excessive accumulation of PG and hypersensitivity to osmotic shock; however, the mechanistic bases for these phenotypes were not previously determined. Here, we show that incorporation of NCDAAs into PG is a critical means by which NCDAAs control PG abundance and strength. We identified and reconstituted in vitro two (of at least three) distinct processes that mediate NCDAA incorporation. Diverse bacterial phyla incorporate NCDAAs into their cell walls, either through periplasmic editing of the mature PG or via incorporation into PG precursor subunits in the cytosol. Production of NCDAAs in Vibrio cholerae requires the stress response sigma factor RpoS, suggesting that NCDAAs may aid bacteria in responding to varied environmental challenges. The widespread capacity of diverse bacteria, including non-producers, to incorporate NCDAAs suggests that these amino acids may serve as both autocrine- and paracrine-like regulators of chemical and physical properties of the cell wall in microbial communities.

  6. Distinct ErbB2 receptor populations differentially interact with beta1 integrin in breast cancer cell models (United States)

    Toscani, Andrés Martín; Sampayo, Rocío G.; Barabas, Federico Martín; Fuentes, Federico; Simian, Marina


    ErbB2 is a member of the ErbB family of tyrosine kinase receptors that plays a major role in breast cancer progression. Located at the plasma membrane, ErbB2 forms large clusters in spite of the presence of growth factors. Beta1 integrin, membrane receptor of extracellular matrix proteins, regulates adhesion, migration and invasiveness of breast cancer cells. Physical interaction between beta1 integrin and ErbB2 has been suggested although published data are contradictory. The aim of the present work was to study the interaction between ErbB2 and beta1 integrin in different scenarios of expression and activation. We determined that beta1 integrin and ErbB2 colocalization is dependent on the expression level of both receptors exclusively in adherent cells. In suspension cells, lack of focal adhesions leave integrins free to diffuse on the plasma membrane and interact with ErbB2 even at low expression levels of both receptors. In adherent cells, high expression of beta1 integrin leaves unbound receptors outside focal complexes that diffuse within the plasma membrane and interact with ErbB2 membrane domains. Superresolution imaging showed the existence of two distinct populations of ErbB2: a major population located in large clusters and a minor population outside these structures. Upon ErbB2 overexpression, receptors outside large clusters can freely diffuse at the membrane and interact with integrins. These results reveal how expression levels of beta1 integrin and ErbB2 determine their frequency of colocalization and show that extracellular matrix proteins shape membrane clusters distribution, regulating ErbB2 and beta1 integrin activity in breast cancer cells. PMID:28306722

  7. Recursive Distinctioning

    CERN Document Server

    Isaacson, Joel


    Recursive distinctioning (RD) is a name coined by Joel Isaacson in his original patent document describing how fundamental patterns of process arise from the systematic application of operations of distinction and description upon themselves. Recursive distinctioning means just what it says. A pattern of distinctions is given in a space based on a graphical structure (such as a line of print or a planar lattice or given graph). Each node of the graph is occupied by a letter from some arbitrary alphabet. A specialized alphabet is given that can indicate distinctions about neighbors of a given node. The neighbors of a node are all nodes that are connected to the given node by edges in the graph. The letters in the specialized alphabet (call it SA) are used to describe the states of the letters in the given graph and at each stage in the recursion, letters in SA are written at all nodes in the graph, describing its previous state. The recursive structure that results from the iteration of descriptions is called ...

  8. Correlation of cell surface proteins of distinct Beauveria bassiana cell types and adaption to varied environment and interaction with the host insect. (United States)

    Yang, Zhi; Jiang, Hongyan; Zhao, Xin; Lu, Zhuoyue; Luo, Zhibing; Li, Xuebing; Zhao, Jing; Zhang, Yongjun


    The insect fungal pathogen Beauveria bassiana produces a number of distinct cell types that include aerial conidia, blastospores and haemolymph-derived cells, termed hyphal bodies, to adapt varied environment niches and within the host insect. These cells display distinct biochemical properties and surface structures, and a highly ordered outermost brush-like structure uniquely present on hyphal bodies, but not on any in vitro cells. Here, we found that the outermost structure on the hyphal bodies mainly consisted of proteins associated to structural wall components in that most of it could be removed by dithiothreitol (DTT) or proteinase K. DTT-treatment also caused delayed germination, decreased tolerance to ultraviolet irradiation and virulence of conidia or blastospores, with decreased adherence and alternated carbohydrate epitopes, suggesting involvement in fungal development, stress responses and virulence. To characterize these cell surface molecules, proteins were released from the living cells using DTT, and identified and quantitated using label-free quantitative mass spectrometry. Thereafter, a series of bioinformatics programs were used to predict cell surface-associated proteins (CSAPs), and 96, 166 and 54 CSAPs were predicted from the identified protein pools of conidia, blastospores and hyphal bodies, respectively, which were involved in utilization of carbohydrate, nitrogen, and lipid, detoxification, pathogen-host interaction, and likely other cellular processes. Thirteen, sixty-nine and six CSAPs were exclusive in conidia, blastospores and hyphal bodies, respectively, which were verified by eGFP-tagged proteins at their N-terminus. Our data provide a crucial cue to understand mechanism of B. bassiana to adapt to varied environment and interaction with insect host.

  9. Synthetic RNAs Mimicking Structural Domains in the Foot-and-Mouth Disease Virus Genome Elicit a Broad Innate Immune Response in Porcine Cells Triggered by RIG-I and TLR Activation. (United States)

    Borrego, Belén; Rodríguez-Pulido, Miguel; Revilla, Concepción; Álvarez, Belén; Sobrino, Francisco; Domínguez, Javier; Sáiz, Margarita


    The innate immune system is the first line of defense against viral infections. Exploiting innate responses for antiviral, therapeutic and vaccine adjuvation strategies is being extensively explored. We have previously described, the ability of small in vitro RNA transcripts, mimicking the sequence and structure of different domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome (ncRNAs), to trigger a potent and rapid innate immune response. These synthetic non-infectious molecules have proved to have a broad-range antiviral activity and to enhance the immunogenicity of an FMD inactivated vaccine in mice. Here, we have studied the involvement of pattern-recognition receptors (PRRs) in the ncRNA-induced innate response and analyzed the antiviral and cytokine profiles elicited in swine cultured cells, as well as peripheral blood mononuclear cells (PBMCs).

  10. Synthetic RNAs Mimicking Structural Domains in the Foot-and-Mouth Disease Virus Genome Elicit a Broad Innate Immune Response in Porcine Cells Triggered by RIG-I and TLR Activation

    Directory of Open Access Journals (Sweden)

    Belén Borrego


    Full Text Available The innate immune system is the first line of defense against viral infections. Exploiting innate responses for antiviral, therapeutic and vaccine adjuvation strategies is being extensively explored. We have previously described, the ability of small in vitro RNA transcripts, mimicking the sequence and structure of different domains in the non-coding regions of the foot-and-mouth disease virus (FMDV genome (ncRNAs, to trigger a potent and rapid innate immune response. These synthetic non-infectious molecules have proved to have a broad-range antiviral activity and to enhance the immunogenicity of an FMD inactivated vaccine in mice. Here, we have studied the involvement of pattern-recognition receptors (PRRs in the ncRNA-induced innate response and analyzed the antiviral and cytokine profiles elicited in swine cultured cells, as well as peripheral blood mononuclear cells (PBMCs.

  11. Synthetic RNAs Mimicking Structural Domains in the Foot-and-Mouth Disease Virus Genome Elicit a Broad Innate Immune Response in Porcine Cells Triggered by RIG-I and TLR Activation (United States)

    Borrego, Belén; Rodríguez-Pulido, Miguel; Revilla, Concepción; Álvarez, Belén; Sobrino, Francisco; Domínguez, Javier; Sáiz, Margarita


    The innate immune system is the first line of defense against viral infections. Exploiting innate responses for antiviral, therapeutic and vaccine adjuvation strategies is being extensively explored. We have previously described, the ability of small in vitro RNA transcripts, mimicking the sequence and structure of different domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome (ncRNAs), to trigger a potent and rapid innate immune response. These synthetic non-infectious molecules have proved to have a broad-range antiviral activity and to enhance the immunogenicity of an FMD inactivated vaccine in mice. Here, we have studied the involvement of pattern-recognition receptors (PRRs) in the ncRNA-induced innate response and analyzed the antiviral and cytokine profiles elicited in swine cultured cells, as well as peripheral blood mononuclear cells (PBMCs). PMID:26193305

  12. Nifedipine, a calcium channel blocker, inhibits advanced glycation end product (AGE)-elicited mesangial cell damage by suppressing AGE receptor (RAGE) expression via peroxisome proliferator-activated receptor-gamma activation

    Energy Technology Data Exchange (ETDEWEB)

    Matsui, Takanori [Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011 (Japan); Yamagishi, Sho-ichi, E-mail: [Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011 (Japan); Takeuchi, Masayoshi [Department of Pathophysiological Science, Faculty of Pharmaceutical Science, Hokuriku University, Kanazawa (Japan); Ueda, Seiji; Fukami, Kei; Okuda, Seiya [Department of Medicine, Kurume University School of Medicine, Kurume (Japan)


    The interaction between advanced glycation end products (AGE) and their receptor RAGE mediates the progressive alteration in renal architecture and loss of renal function in diabetic nephropathy. Oxidative stress generation and inflammation also play a central role in diabetic nephropathy. This study investigated whether and how nifedipine, a calcium channel blocker (CCB), blocked the AGE-elicited mesangial cell damage in vitro. Nifedipine, but not amlodipine, a control CCB, down-regulated RAGE mRNA levels and subsequently reduced reactive oxygen species (ROS) generation in AGE-exposed mesangial cells. AGE increased mRNA levels of vascular cell adhesion molecule-1 (VCAM-1) and induced monocyte chemoattractant protein-1 (MCP-1) production in mesangial cells, both of which were prevented by the treatment with nifedipine, but not amlodipine. The beneficial effects of nifedipine on AGE-exposed mesangial cells were blocked by the simultaneous treatment of GW9662, an inhibitor of peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}). Although nifedipine did not affect expression levels of PPAR-{gamma}, it increased the PPAR-{gamma} transcriptional activity in mesangial cells. Our present study provides a unique beneficial aspect of nifedipine on diabetic nephropathy; it could work as an anti-inflammatory agent against AGE by suppressing RAGE expression in cultured mesangial cells via PPAR-{gamma} activation.

  13. Primary cutaneous peripheral T-cell lymphoma, unspecified with an indolent clinical course: a distinct peripheral T-cell lymphoma?

    LENUS (Irish Health Repository)

    Ryan, A J A


    Primary cutaneous peripheral T-cell lymphomas (PTL), unspecified, are rare lymphomas, with a poor prognosis. They grow and disseminate rapidly, leading to widespread disease. We report a case of PTL, unspecified occurring on the nose. Despite its aggressive histology, this tumour behaved indolently. It is remarkably similar, clinically and histologically, to four recently described cases that occurred on the ear.

  14. Elicitation of ostomy pouch preferences

    DEFF Research Database (Denmark)

    Bonnichsen, Ole


    in ostomy pouch attributes. The theory, study design, elicitation procedure, and resulting preference structure of the sample is described. Methods: A discrete-choice experiment (DCE) was used to elicit preferences. Respondents were asked to choose between alternatives in choice sets, in which each...... pouches when cost is included as an attribute. A total of 254 patients responded to the survey and preferences were estimated using a random parameter logit econometric specification. Results: Respondents had significantly positive WTP for all potential attribute improvements presented in the survey...

  15. Distinctive response of CNS glial cells in oro-facial pain associated with injury, infection and inflammation

    Directory of Open Access Journals (Sweden)

    Ribeiro-da-Silva Alfredo


    Full Text Available Abstract Oro-facial pain following injury and infection is frequently observed in dental clinics. While neuropathic pain evoked by injury associated with nerve lesion has an involvement of glia/immune cells, inflammatory hyperalgesia has an exaggerated sensitization mediated by local and circulating immune mediators. To better understand the contribution of central nervous system (CNS glial cells in these different pathological conditions, in this study we sought to characterize functional phenotypes of glial cells in response to trigeminal nerve injury (loose ligation of the mental branch, infection (subcutaneous injection of lipopolysaccharide-LPS and to sterile inflammation (subcutaneous injection of complete Freund's adjuvant-CFA on the lower lip. Each of the three insults triggered a specific pattern of mechanical allodynia. In parallel with changes in sensory response, CNS glial cells reacted distinctively to the challenges. Following ligation of the mental nerve, both microglia and astrocytes in the trigeminal nuclear complex were highly activated, more prominent in the principal sensory nucleus (Pr5 and subnucleus caudalis (Sp5C area. Microglial response was initiated early (days 3-14, followed by delayed astrocytes activation (days 7-28. Although the temporal profile of microglial and astrocyte reaction corresponded respectively to the initiation and chronic stage of neuropathic pain, these activated glial cells exhibited a low profile of cytokine expression. Local injection of LPS in the lower lip skin also triggered a microglial reaction in the brain, which started in the circumventricular organs (CVOs at 5 hours post-injection and diffused progressively into the brain parenchyma at 48 hours. This LPS-induced microglial reaction was accompanied by a robust induction of IκB-α mRNA and pro-inflammatory cytokines within the CVOs. However, LPS induced microglial activation did not specifically occur along the pain signaling pathway. In

  16. Distinct effector memory CD4+ T cell signatures in latent Mycobacterium tuberculosis infection, BCG vaccination and clinically resolved tuberculosis. (United States)

    Adekambi, Toidi; Ibegbu, Chris C; Kalokhe, Ameeta S; Yu, Tianwei; Ray, Susan M; Rengarajan, Jyothi


    Two billion people worldwide are estimated to be latently infected with Mycobacterium tuberculosis (Mtb) and are at risk for developing active tuberculosis since Mtb can reactivate to cause TB disease in immune-compromised hosts. Individuals with latent Mtb infection (LTBI) and BCG-vaccinated individuals who are uninfected with Mtb, harbor antigen-specific memory CD4(+) T cells. However, the differences between long-lived memory CD4(+) T cells induced by latent Mtb infection (LTBI) versus BCG vaccination are unclear. In this study, we characterized the immune phenotype and functionality of antigen-specific memory CD4(+) T cells in healthy BCG-vaccinated individuals who were either infected (LTBI) or uninfected (BCG) with Mtb. Individuals were classified into LTBI and BCG groups based on IFN-γ ELISPOT using cell wall antigens and ESAT-6/CFP-10 peptides. We show that LTBI individuals harbored high frequencies of late-stage differentiated (CD45RA(-)CD27(-)) antigen-specific effector memory CD4(+) T cells that expressed PD-1. In contrast, BCG individuals had primarily early-stage (CD45RA(-)CD27(+)) cells with low PD-1 expression. CD27(+) and CD27(-) as well as PD-1(+) and PD-1(-) antigen-specific subsets were polyfunctional, suggesting that loss of CD27 expression and up-regulation of PD-1 did not compromise their capacity to produce IFN-γ, TNF-α and IL-2. PD-1 was preferentially expressed on CD27(-) antigen-specific CD4(+) T cells, indicating that PD-1 is associated with the stage of differentiation. Using statistical models, we determined that CD27 and PD-1 predicted LTBI versus BCG status in healthy individuals and distinguished LTBI individuals from those who had clinically resolved Mtb infection after anti-tuberculosis treatment. This study shows that CD4(+) memory responses induced by latent Mtb infection, BCG vaccination and clinically resolved Mtb infection are immunologically distinct. Our data suggest that differentiation into CD27(-)PD-1(+) subsets in

  17. Distinct effector memory CD4+ T cell signatures in latent Mycobacterium tuberculosis infection, BCG vaccination and clinically resolved tuberculosis.

    Directory of Open Access Journals (Sweden)

    Toidi Adekambi

    Full Text Available Two billion people worldwide are estimated to be latently infected with Mycobacterium tuberculosis (Mtb and are at risk for developing active tuberculosis since Mtb can reactivate to cause TB disease in immune-compromised hosts. Individuals with latent Mtb infection (LTBI and BCG-vaccinated individuals who are uninfected with Mtb, harbor antigen-specific memory CD4(+ T cells. However, the differences between long-lived memory CD4(+ T cells induced by latent Mtb infection (LTBI versus BCG vaccination are unclear. In this study, we characterized the immune phenotype and functionality of antigen-specific memory CD4(+ T cells in healthy BCG-vaccinated individuals who were either infected (LTBI or uninfected (BCG with Mtb. Individuals were classified into LTBI and BCG groups based on IFN-γ ELISPOT using cell wall antigens and ESAT-6/CFP-10 peptides. We show that LTBI individuals harbored high frequencies of late-stage differentiated (CD45RA(-CD27(- antigen-specific effector memory CD4(+ T cells that expressed PD-1. In contrast, BCG individuals had primarily early-stage (CD45RA(-CD27(+ cells with low PD-1 expression. CD27(+ and CD27(- as well as PD-1(+ and PD-1(- antigen-specific subsets were polyfunctional, suggesting that loss of CD27 expression and up-regulation of PD-1 did not compromise their capacity to produce IFN-γ, TNF-α and IL-2. PD-1 was preferentially expressed on CD27(- antigen-specific CD4(+ T cells, indicating that PD-1 is associated with the stage of differentiation. Using statistical models, we determined that CD27 and PD-1 predicted LTBI versus BCG status in healthy individuals and distinguished LTBI individuals from those who had clinically resolved Mtb infection after anti-tuberculosis treatment. This study shows that CD4(+ memory responses induced by latent Mtb infection, BCG vaccination and clinically resolved Mtb infection are immunologically distinct. Our data suggest that differentiation into CD27(-PD-1(+ subsets in LTBI is

  18. A synthetic lymph node containing inactivated Treponema pallidum cells elicits strong, antigen-specific humoral and cellular immune responses in mice. (United States)

    Stamm, Lola V; Drapp, Rebecca L


    The goal of this study was to investigate the use of a synthetic lymph node (SLN) for delivery of Treponema pallidum (Tp) antigens. Immune responses of C57BL/6 mice were analyzed at 4, 8, and 12 weeks after SLN implantation. Group 1 mice received SLN with no antigen; Group 2, SLN with formalin-inactivated Tp (f-Tp); and Group 3, SLN with f-Tp plus a CpG oligodeoxynucleotide. When tested by ELISA, sera from Group 2 and Group 3 mice showed stronger IgG antibody reactivity than sera from Group 1 mice to sonicates of f-Tp or untreated Tp, but not to sonicate of normal rabbit testicular extract at all times. The IgG1 level was higher than IgG2c level for Group 2 mice at all times and for Group 3 mice at 4 and 8 weeks. IgG1 and IgG2c levels were nearly equivalent for Group 3 mice at 12 weeks. Immunoblotting showed that IgG from Group 2 and Group 3 mice recognized several Tp proteins at all times. Supernatants of splenocytes from Group 2 and Group 3 mice contained significantly more IFNγ than those from Group 1 mice after stimulation with f-Tp at all times. A significant level of IL-4 was not detected in any supernatants. These data show that strong humoral and cellular immune responses to Tp can be elicited via a SLN.

  19. Alpha4beta1 integrin and erythropoietin mediate temporally distinct steps in erythropoiesis: integrins in red cell development. (United States)

    Eshghi, Shawdee; Vogelezang, Mariette G; Hynes, Richard O; Griffith, Linda G; Lodish, Harvey F


    Erythropoietin (Epo) is essential for the terminal proliferation and differentiation of erythroid progenitor cells. Fibronectin is an important part of the erythroid niche, but its precise role in erythropoiesis is unknown. By culturing fetal liver erythroid progenitors, we show that fibronectin and Epo regulate erythroid proliferation in temporally distinct steps: an early Epo-dependent phase is followed by a fibronectin-dependent phase. In each phase, Epo and fibronectin promote expansion by preventing apoptosis partly through bcl-xL. We show that alpha(4), alpha(5), and beta(1) are the principal integrins expressed on erythroid progenitors; their down-regulation during erythropoiesis parallels the loss of cell adhesion to fibronectin. Culturing erythroid progenitors on recombinant fibronectin fragments revealed that only substrates that engage alpha(4)beta(1)-integrin support normal proliferation. Collectively, these data suggest a two-phase model for growth factor and extracellular matrix regulation of erythropoiesis, with an early Epo-dependent, integrin-independent phase followed by an Epo-independent, alpha(4)beta(1)-integrin-dependent phase.

  20. Novel monofunctional platinum (II) complex Mono-Pt induces apoptosis-independent autophagic cell death in human ovarian carcinoma cells, distinct from cisplatin. (United States)

    Guo, Wen-Jie; Zhang, Yang-Miao; Zhang, Li; Huang, Bin; Tao, Fei-Fei; Chen, Wei; Guo, Zi-Jian; Xu, Qiang; Sun, Yang


    Failure to engage apoptosis appears to be a leading mechanism of resistance to traditional platinum drugs in patients with ovarian cancer. Therefore, an alternative strategy to induce cell death is needed for the chemotherapy of this apoptosis-resistant cancer. Here we report that autophagic cell death, distinct from cisplatin-induced apoptosis, is triggered by a novel monofunctional platinum (II) complex named Mono-Pt in human ovarian carcinoma cells. Mono-Pt-induced cell death has the following features: cytoplasmic vacuolation, caspase-independent, no nuclear fragmentation or chromatin condensation, and no apoptotic bodies. These characteristics integrally indicated that Mono-Pt, rather than cisplatin, initiated a nonapoptotic cell death in Caov-3 ovarian carcinoma cells. Furthermore, incubation of the cells with Mono-Pt but not with cisplatin produced an increasing punctate distribution of microtubule-associated protein 1 light chain 3 (LC3), and an increasing ratio of LC3-II to LC3-I. Mono-Pt also caused the formation of autophagic vacuoles as revealed by monodansylcadaverine staining and transmission electron microscopy. In addition, Mono-Pt-induced cell death was significantly inhibited by the knockdown of either BECN1 or ATG7 gene expression, or by autophagy inhibitors 3-methyladenine, chloroquine and bafilomycin A 1. Moreover, the effect of Mono-Pt involved the AKT1-MTOR-RPS6KB1 pathway and MAPK1 (ERK2)/MAPK3 (ERK1) signaling, since the MTOR inhibitor rapamycin increased, while the MAPK1/3 inhibitor U0126 decreased Mono-Pt-induced autophagic cell death. Taken together, our results suggest that Mono-Pt exerts anticancer effect via autophagic cell death in apoptosis-resistant ovarian cancer. These findings lead to increased options for anticancer platinum drugs to induce cell death in cancer.

  1. Grima: A Distinct Emotion Concept? (United States)

    Schweiger Gallo, Inge; Fernández-Dols, José-Miguel; Gollwitzer, Peter M.; Keil, Andreas


    People experience an unpleasant sensation when hearing a scratch on a board or plate. The present research focuses on this aversive experience known in Spanish as ‘grima’ with no equivalent term in English and German. We hypothesized that this aversive experience constitutes a distinctive, separate emotional concept. In Study 1, we found that the affective meaning of ‘grima’ was closer to disgust than to other emotion concepts. Thus, in Study 2 we explored the features of grima and compared them with disgust. As grima was reported to be predominantly elicited by certain auditory stimuli and associated with a distinctive physiological pattern, Study 3 used direct measures of physiological arousal to test the assumption of a distinctive pattern of physiological responses elicited by auditory stimuli of grima and disgust, and found different effects on heart rate but not on skin conductance. In Study 4, we hypothesized that only participants with an implementation intention geared toward down-regulating grima would be able to successfully weaken the grima- but not disgust- experience. Importantly, this effect was specific as it held true for the grima-eliciting sounds only, but did not affect disgust-related sounds. Finally, Study 5 found that English and German speakers lack a single accessible linguistic label for the pattern of aversive reactions termed by Spanish speaking individuals as ‘grima’, whereas the elicitors of other emotions were accessible and accurately identified by German, English, as well as Spanish speakers. PMID:28217102

  2. Distinct populations of hepatic stellate cells in the mouse liver have different capacities for retinoid and lipid storage.

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    Diana N D'Ambrosio

    Full Text Available Hepatic stellate cell (HSC lipid droplets are specialized organelles for the storage of retinoid, accounting for 50-60% of all retinoid present in the body. When HSCs activate, retinyl ester levels progressively decrease and the lipid droplets are lost. The objective of this study was to determine if the HSC population in a healthy, uninjured liver demonstrates heterogeneity in its capacity for retinoid and lipid storage in lipid droplets. To this end, we utilized two methods of HSC isolation, which leverage distinct properties of these cells, including their vitamin A content and collagen expression. HSCs were isolated either from wild type (WT mice in the C57BL/6 genetic background by flotation in a Nycodenz density gradient, followed by fluorescence activated cell sorting (FACS based on vitamin A autofluorescence, or from collagen-green fluorescent protein (GFP mice by FACS based on GFP expression from a GFP transgene driven by the collagen I promoter. We show that GFP-HSCs have: (i increased expression of typical markers of HSC activation; (ii decreased retinyl ester levels, accompanied by reduced expression of the enzyme needed for hepatic retinyl ester synthesis (LRAT; (iii decreased triglyceride levels; (iv increased expression of genes associated with lipid catabolism; and (v an increase in expression of the retinoid-catabolizing cytochrome, CYP2S1.Our observations suggest that the HSC population in a healthy, uninjured liver is heterogeneous. One subset of the total HSC population, which expresses early markers of HSC activation, may be "primed" and ready for rapid response to acute liver injury.

  3. Light, genotype, and abscisic acid affect chloroplast positioning in guard cells of Arabidopsis thaliana leaves in distinct ways. (United States)

    Königer, Martina; Jessen, Brita; Yang, Rui; Sittler, Dorothea; Harris, Gary C


    The goal of this study was to investigate the effects of light intensity, genotype, and various chemical treatments on chloroplast movement in guard cells of Arabidopsis thaliana leaves. After treatment at various light intensities (dark, low, and high light), leaf discs were fixed with glutaraldehyde, and imaged using confocal laser microscopy. Each chloroplast was assigned a horizontal (close to pore, center, or epidermal side) and vertical (outer, middle, inner) position. White light had a distinct effect on chloroplast positioning, most notably under high light (HL) when chloroplasts on the upper leaf surface of wild-type (WT) moved from epidermal and center positions toward the pore. This was not the case for phot1-5/phot2-1 or phot2-1 plants, thus phototropins are essential for chloroplast positioning in guard cells. In npq1-2 mutants, fewer chloroplasts moved to the pore position under HL than in WT plants, indicating that white light can affect chloroplast positioning also in a zeaxanthin-dependent way. Cytochalasin B inhibited the movement of chloroplasts to the pore under HL, while oryzalin did not, supporting the idea that actin plays a role in the movement. The movement along actin cables is dependent on CHUP1 since chloroplast positioning in chup1 was significantly altered. Abscisic acid (ABA) caused most chloroplasts in WT and phot1-5/phot2-1 to be localized in the center, middle part of the guard cells irrespective of light treatment. This indicates that not only light but also water stress influences chloroplast positioning.

  4. Ultrastructural cytochemical and ultrastructural morphological differences between human multinucleated giant cells elicited by wear particles from hip prostheses and artificial ligaments at the knee. (United States)

    Anazawa, Ukei; Hanaoka, Hideya; Morioka, Hideo; Morii, Takeshi; Toyama, Yoshiaki


    The authors investigated the ultrastructural cytochemical features of multinucleated and mononuclear cells in periprosthetic tissues associated with bone resorption (osteolysis) and those in tissues adjoining failed artificial ligaments having no relation to bone resorption. Clinical specimens of granulation tissue of each type, respectively numbering 4 and 3, were stained for tartrate-resistant acid phosphatase (TRAP) reactions and examined by light and electron microscopy. Both periprosthetic granulation tissues and those adjoining artificial ligaments contained TRAP-positive multinucleated and mononuclear cells. Near joint prostheses, multinucleated cells, including some giant cells, showed TRAP activity and cytoplasmic features resembling osteoclasts, while others had features consistent with foreign-body giant cells, and still others showed degenerative changes. Near artificial ligaments, TRAP-positive multinucleated cells lacked osteoclastic features. At both sites, TRAP-positive multinucleated cells had phagocytised wear particles. TRAP-positive mononuclear cells at both sites also showed phagocytic cytoplasmic features, but not osteoclastic cytoplasmic features. Human mononuclear phagocytes and multinucleated giant cells induced by wear particles possess TRAP activity. Those multinucleated giant cells at sites of osteolysis developed osteoclastic cytoplasmic features and have a phagocytic function.

  5. Persistence of autoreactive T cell drive is required to elicit anti-chromatin antibodies in a murine model of drug-induced lupus. (United States)

    Kretz-Rommel, A; Rubin, R L


    Long-term treatment with procainamide and numerous other medications is occasionally associated with the development of drug-induced lupus. We recently established a murine model for this syndrome by disrupting central T cell tolerance. Two intrathymic injections of procainamide-hydroxylamine (PAHA), a reactive metabolite of procainamide, into (C57BL/6 x DBA/2)F1 mice resulted in the appearance of chromatin-reactive T cells and anti-chromatin autoantibodies. The current study explores in this model the role of autoreactive T cells in autoantibody production and examines why autoantibodies after a single intrathymic drug injection were much more limited in isotype and specificity. Injection of as few as 5000 chromatin-reactive T cells into naive, syngeneic mice induced a rapid IgM anti-denatured DNA response, while injection of at least 100-fold greater number of activated T cells was required for induction of IgG anti-chromatin Abs, suggesting that small numbers of autoreactive T cells can be homeostatically controlled. Mice subjected to a single intrathymic PAHA injection after receiving splenic B cells from an intrathymic PAHA-injected syngeneic donor also developed anti-chromatin Abs, but adoptive transfer of similarly primed T cells or of B cells without intrathymic PAHA injection of the recipient failed to produce an anti-chromatin response. However, anti-chromatin Abs developed after a single intrathymic PAHA injection in Fas-deficient C57BL/6-lpr/lpr mice, suggesting that activation-induced cell death limited autoimmunity in normal mice. Taken together, these results imply that chromatin-reactive T cells produced by intrathymic PAHA created a B cell population primed to somatically mutate and Ig class switch when subjected to a heavy load or second wave of autoreactive T cells.

  6. Analysis of gene expression data from non-small cell lung carcinoma cell lines reveals distinct sub-classes from those identified at the phenotype level.

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    Andrew R Dalby

    Full Text Available Microarray data from cell lines of Non-Small Cell Lung Carcinoma (NSCLC can be used to look for differences in gene expression between the cell lines derived from different tumour samples, and to investigate if these differences can be used to cluster the cell lines into distinct groups. Dividing the cell lines into classes can help to improve diagnosis and the development of screens for new drug candidates. The micro-array data is first subjected to quality control analysis and then subsequently normalised using three alternate methods to reduce the chances of differences being artefacts resulting from the normalisation process. The final clustering into sub-classes was carried out in a conservative manner such that sub-classes were consistent across all three normalisation methods. If there is structure in the cell line population it was expected that this would agree with histological classifications, but this was not found to be the case. To check the biological consistency of the sub-classes the set of most strongly differentially expressed genes was be identified for each pair of clusters to check if the genes that most strongly define sub-classes have biological functions consistent with NSCLC.

  7. Deleted in malignant brain tumors 1 (DMBT1) elicits increased VEGF and decreased IL-6 production in type II lung epithelial cells

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    Müller, Hanna; Nagel, Christian; Weiss, Christel


    between VEGF and IL-6 levels to DMBT1 expression in the lungs of preterm and term infants and in lung epithelial cells in vitro. METHODS: We examined by ELISA VEGF levels in 120 tracheal aspirates of 57 preterm and term infants and tested for correlation with different perinatal factors as well...... as with DMBT1 levels. To examine the effect of DMBT1 on VEGF and IL-6 expression we compared type II lung epithelial A549 cells stably transfected with a DMBT1 expression plasmid (DMBT1+ cells) to A549 cells stably transfected with an empty expression plasmid (DMBT1- cells). The concentrations of VEGF and IL-6...... that DMBT1 promotes VEGF and suppresses IL-6 production in alveolar tissues, which could point to DMBT1 having a possible role in the transition from inflammation to regeneration and being a potentially useful clinical marker....

  8. Identification of ITGA4/ITGB7 and ITGAE/ITGB7 expressing subsets of decidual dendritic-like cells within distinct microdomains of the pregnant mouse uterus. (United States)

    Behrends, Jochen; Karsten, Christian M; Wilke, Sonja; Röbke, Astrid; Kruse, Andrea


    Several leukocyte populations have been described within the pregnant mouse uterus, some of which express the integrin beta 7 (ITGB7). Here we demonstrate that the majority of the ITGB7(+) decidual leukocytes belong to the dendritic cell (DC) lineage. By multiparameter flow cytometric analysis we demonstrated the existence of three distinct DC subsets, characterized by differential expression of ITGA4/ITGB7 (formerly alpha4beta7-integrin) and ITGAE/ITGB7 (formerly alphaEbeta7-integrin). Importantly, the predominant DC subsets reside in distinct microdomains of the Day 9 pregnant mouse uterus. ITGAX(+) ITGAM(med) ITGA4/ITGB7(+) ITGAE(-) (formerly CD11c(+) CD11b(med) alpha4beta7(+) alphaE(-)) cells represent the majority of DCs in the vascular zone (VZ), whereas ITGAX(+) ITGAM(-) ITGAE/ITGB7(+) (formerly CD11c(+) CD11b(-) alphaEbeta7(+)) DCs are mainly located in the lower central decidua basalis (cDB) and the underlying myometrium. A population of ITGAX(+) ITGAM(low) DCs lacking ITGB7 are restricted to the cDB. Confocal microscopy studies show direct contact of VZ DCs with uterine natural killer (uNK) cells, suggesting a functional relationship between both cell populations. Collectively, our data identify three phenotypically distinct DC subsets residing in distinct microdomains of the uterus. The differential expression of ITGA4/ITGB7 and ITGAE/ITGB7 suggests distinct functional roles of the different DC subsets during early pregnancy.

  9. Relevance of long-lived CD8+ T effector memory (TEM cells for protective immunity elicited by heterologous prime-boost vaccination.

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    José Ronnie Vasconcelos


    Full Text Available Owing to the importance of major histocompatibility complex class Ia–restricted CD8+ T cells for host survival following viral, bacterial, fungal, or parasitic infection, it has become largely accepted that these cells should be considered in the design of a new generation of vaccines. For the past 20 years, solid evidence has been provided that the heterologous prime-boost regimen achieves the best results in terms of induction of long-lived protective CD8+ T cells against a variety of experimental infections. Although this regimen has often been used experimentally, as is the case for many vaccines, the mechanism behind the efficacy of this vaccination regimen is still largely unknown. The main purpose of this review is to examine the characteristics of the protective CD8+ T cells generated by this vaccination regimen. Part of its efficacy certainly relies on the generation and maintenance of large numbers of specific lymphocytes. Other specific characteristics may also be important, and studies on this direction have only recently been initiated. So far, the characterization of these protective, long-lived T cell populations suggests that there is a high frequency of polyfunctional T cells; these cells cover a large breadth and display a T effector memory (TEM phenotype. These TEM cells are capable of proliferating after an infectious challenge and are highly refractory to apoptosis due to a control of the expression of pro-apoptotic receptors such as CD95. Also, they do not undergo significant long-term immunological erosion. Understanding the mechanisms that control the generation and maintenance of the protective activity of these long-lived TEM cells will certainly provide important insights into the physiology of CD8+ T cells and pave the way for the design of new or improved vaccines..

  10. Silencing of ribosomal protein S9 elicits a multitude of cellular responses inhibiting the growth of cancer cells subsequent to p53 activation.

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    Mikael S Lindström

    Full Text Available BACKGROUND: Disruption of the nucleolus often leads to activation of the p53 tumor suppressor pathway through inhibition of MDM2 that is mediated by a limited set of ribosomal proteins including RPL11 and RPL5. The effects of ribosomal protein loss in cultured mammalian cells have not been thoroughly investigated. Here we characterize the cellular stress response caused by depletion of ribosomal protein S9 (RPS9. METHODOLOGY/PRINCIPAL FINDINGS: Depletion of RPS9 impaired production of 18S ribosomal RNA and induced p53 activity. It promoted p53-dependent morphological differentiation of U343MGa Cl2:6 glioma cells as evidenced by intensified expression of glial fibrillary acidic protein and profound changes in cell shape. U2OS osteosarcoma cells displayed a limited senescence response with increased expression of DNA damage response markers, whereas HeLa cervical carcinoma cells underwent cell death by apoptosis. Knockdown of RPL11 impaired p53-dependent phenotypes in the different RPS9 depleted cell cultures. Importantly, knockdown of RPS9 or RPL11 also markedly inhibited cell proliferation through p53-independent mechanisms. RPL11 binding to MDM2 was retained despite decreased levels of RPL11 protein following nucleolar stress. In these settings, RPL11 was critical for maintaining p53 protein stability but was not strictly required for p53 protein synthesis. CONCLUSIONS: p53 plays an important role in the initial restriction of cell proliferation that occurs in response to decreased level of RPS9. Our results do not exclude the possibility that other nucleolar stress sensing molecules act upstream or in parallel to RPL11 to activate p53. Inhibiting the expression of certain ribosomal proteins, such as RPS9, could be one efficient way to reinitiate differentiation processes or to induce senescence or apoptosis in rapidly proliferating tumor cells.

  11. Genomic Copy Number Signatures Uncovered a Genetically Distinct Group from Adenocarcinoma and Squamous Cell Carcinoma in Non-Small Cell Lung Cancer. (United States)

    Lee, Eunjung; Moon, Ji Wook; Wang, Xianfu; Kim, Chungyeul; Li, Shibo; Shin, Bong Kyung; Jung, Wonkyung; Kim, Hyun Koo; Kim, Han Kyeom; Lee, Ji-Yun


    Adenocarcinoma (AC) and squamous cell carcinoma (SCC) of non-small cell lung carcinoma (NSCLC) have different clinical presentations, morphologies, treatments, and prognoses. Recent studies suggested that fundamental genetic alterations related to carcinogenesis of each tumor type may be different. In this study, we investigated the genomic alterations of 47 primary NSCLC samples (22 ACs and 25 SCCs) as well as the corresponding normal tissue using array comparative genomic hybridization. Frequent copy number alterations (CNAs), which were identified in more than 68% of all of the cases, were evaluated in each subtype (SCC and AC), and a CNA signature was established. Among these CNAs, 37 genes from the SCCs and 15 genes from the ACs were located in a region of gain, and 4 genes from the SCCs and 13 genes from the ACs were located in a region of loss. The most frequent gain was located on 3q26-29 including the gene TP63 in SCCs and 7q11.23 and 7q36.3 in ACs. Moreover, we identified 3 genetically distinct groups (group I [16 SCC] with CNA signature of SCC; group II [7 SCC + 8 AC], which has a genetically distinctive CNA signature from SCC and AC; and group III [2 SCC + 14 AC] with CNA signature of AC) by gene clustering extracted from CNAs, which are associated with a prognosis. The present study contributed to the molecular characterization of AC and SCC of NSCLC and showed a subtype of tumor that has a unique genetic CNA signature. However, further study about the significance of these 3 distinct groups and their usefulness as a diagnostic marker of identified CNAs is necessary.

  12. Distinct sets of PIWI proteins produce arbovirus and transposon-derived piRNAs in Aedes aegypti mosquito cells. (United States)

    Miesen, Pascal; Girardi, Erika; van Rij, Ronald P


    The PIWI-interacting RNA (piRNA) pathway is essential for transposon silencing in many model organisms. Its remarkable efficiency relies on a sophisticated amplification mechanism known as the ping-pong loop. In Alphavirus-infected Aedes mosquitoes, piRNAs with sequence features that suggest ping-pong-dependent biogenesis are produced from viral RNA. The PIWI family in Aedes mosquitoes is expanded when compared to other model organisms, raising the possibility that individual PIWI proteins have functionally diversified in these insects. Here, we show that Piwi5 and Ago3, but none of the other PIWI family members, are essential for piRNA biogenesis from Sindbis virus RNA in infected Aedes aegypti cells. In contrast, the production of piRNAs from transposons relies on a more versatile set of PIWI proteins, some of which do not contribute to viral piRNA biogenesis. These results indicate that functional specialization allows distinct mosquito PIWI proteins to process RNA from different endogenous and exogenous sources.

  13. Cell type-specific genes show striking and distinct patterns of spatial expression in the mouse brain. (United States)

    Ko, Younhee; Ament, Seth A; Eddy, James A; Caballero, Juan; Earls, John C; Hood, Leroy; Price, Nathan D


    To characterize gene expression patterns in the regional subdivisions of the mammalian brain, we integrated spatial gene expression patterns from the Allen Brain Atlas for the adult mouse with panels of cell type-specific genes for neurons, astrocytes, and oligodendrocytes from previously published transcriptome profiling experiments. We found that the combined spatial expression patterns of 170 neuron-specific transcripts revealed strikingly clear and symmetrical signatures for most of the brain's major subdivisions. Moreover, the brain expression spatial signatures correspond to anatomical structures and may even reflect developmental ontogeny. Spatial expression profiles of astrocyte- and oligodendrocyte-specific genes also revealed regional differences; these defined fewer regions and were less distinct but still symmetrical in the coronal plane. Follow-up analysis suggested that region-based clustering of neuron-specific genes was related to (i) a combination of individual genes with restricted expression patterns, (ii) region-specific differences in the relative expression of functional groups of genes, and (iii) regional differences in neuronal density. Products from some of these neuron-specific genes are present in peripheral blood, raising the possibility that they could reflect the activities of disease- or injury-perturbed networks and collectively function as biomarkers for clinical disease diagnostics.

  14. Distinct sets of PIWI proteins produce arbovirus and transposon-derived piRNAs in Aedes aegypti mosquito cells (United States)

    Miesen, Pascal; Girardi, Erika; van Rij, Ronald P.


    The PIWI-interacting RNA (piRNA) pathway is essential for transposon silencing in many model organisms. Its remarkable efficiency relies on a sophisticated amplification mechanism known as the ping-pong loop. In Alphavirus-infected Aedes mosquitoes, piRNAs with sequence features that suggest ping-pong-dependent biogenesis are produced from viral RNA. The PIWI family in Aedes mosquitoes is expanded when compared to other model organisms, raising the possibility that individual PIWI proteins have functionally diversified in these insects. Here, we show that Piwi5 and Ago3, but none of the other PIWI family members, are essential for piRNA biogenesis from Sindbis virus RNA in infected Aedes aegypti cells. In contrast, the production of piRNAs from transposons relies on a more versatile set of PIWI proteins, some of which do not contribute to viral piRNA biogenesis. These results indicate that functional specialization allows distinct mosquito PIWI proteins to process RNA from different endogenous and exogenous sources. PMID:26068474

  15. Distinct temporal changes in host cell lncRNA expression during the course of an adenovirus infection

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    Zhao, Hongxing, E-mail: [The Beijer Laboratory, Dept. of Immunology, Genetics and Pathology, Uppsala University, S-751 85 Uppsala (Sweden); Chen, Maoshan [Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086 (Australia); Lind, Sara Bergström [Department of Chemistry-BMC, Analytical Chemistry, Science for Life Laboratory, Uppsala University, Box 599, SE-751 24 Uppsala (Sweden); Pettersson, Ulf [The Beijer Laboratory, Dept. of Immunology, Genetics and Pathology, Uppsala University, S-751 85 Uppsala (Sweden)


    The deregulation of cellular long non-coding RNA (lncRNA) expression during a human adenovirus infection was studied by deep sequencing. Expression of lncRNAs increased substantially following the progression of the infection. Among 645 significantly expressed lncRNAs, the expression of 398 was changed more than 2-fold. More than 80% of them were up-regulated and 80% of them were detected during the late phase. Based on the genomic locations of the deregulated lncRNAs in relation to known mRNAs and miRNAs, they were predicted to be involved in growth, structure, apoptosis and wound healing in the early phase, cell proliferation in the intermediate phase and protein synthesis, modification and transport in the late phase. The most significant functions of cellular RNA-binding proteins, previously shown to interact with the deregulated lncRNAs identified here, are involved in RNA splicing, nuclear export and translation events. We hypothesize that adenoviruses exploit the lncRNA network to optimize their reproduction. - Highlights: • The expression of 398 lncRNAs showed a distinct temporal pattern during Ad2 infection. • 80% of the deregulated lncRNAs were up-regulated during the late phase of infection. • The deregulated lncRNAs potentiallyinteract with 33 cellular RNA binding proteins. • These RBPs are involved in RNA splicing, nuclear export and translation. • Adenovirus exploits the cellular lncRNA network to optimize its replication.

  16. Large particulate allergens can elicit mast cell-mediated anaphylaxis without exit from blood vessels as efficiently as do small soluble allergens. (United States)

    LiHua, Li; Yoshikawa, Soichiro; Ohta, Takuya; Horiguchi, Kayo; Kawano, Yohei; Ohtsu, Hiroshi; Yamanishi, Yoshinori; Karasuyama, Hajime


    Anaphylaxis is a rapid-onset, life-threatening allergic reaction in that IgE, mast cells and histamine are commonly involved. It can be experimentally induced in IgE-sensitized animals by intravenous injection of corresponding allergens, and the sign of anaphylactic reaction can be detected within minutes after allergen challenge. However, it remains puzzling why the anaphylactic reaction can be initiated in vivo so quickly, considering that allergens are delivered into the blood circulation while mast cells reside within peripheral tissues but not in the blood circulation. To address this issue, we compared two different forms of the same allergen, small soluble and large particulate ones, in their ability to induce anaphylaxis in IgE-sensitized mice. In contrast to our expectation, particulate allergens could induce anaphylaxis as quickly and efficiently as did soluble allergens, even though they remained inside of blood vessels. In vivo imaging analysis suggested the direct interaction of intravascular particulate allergens and perivascular mast cells across the capillary wall. Taken together with previous report that perivascular mast cells can capture IgE in the blood circulation by extending cell processes across the vessel wall, our findings imply that blood-circulating allergens, regardless of their size, can stimulate mast cells without exit from blood vessels, by means of cross-linking IgE on mast cell processes inserted into the vessel lumen, and hence initiate anaphylactic reaction so quickly.

  17. A SILAC-Based Approach Elicits the Proteomic Responses to Vancomycin-Associated Nephrotoxicity in Human Proximal Tubule Epithelial HK-2 Cells. (United States)

    Li, Zhi-Ling; Zhou, Shu-Feng


    Vancomycin, a widely used antibiotic, often induces nephrotoxicity, however, the molecular targets and underlying mechanisms of this side effect remain unclear. The present study aimed to examine molecular interactome and analyze the signaling pathways related to the vancomycin-induced nephrotoxicity in human proximal tubule epithelial HK-2 cells using the stable isotope labeling by amino acids in cell culture (SILAC) approach. The quantitative proteomic study revealed that there were at least 492 proteins interacting with vancomycin and there were 290 signaling pathways and cellular functions potentially regulated by vancomycin in HK-2 cells. These proteins and pathways played a critical role in the regulation of cell cycle, apoptosis, autophagy, EMT, and ROS generation. These findings suggest that vancomycin-induced proteomic responses in HK-2 cells involvefunctional proteins and pathways that regulate cell cycle, apoptosis, autophagy, and redox homeostasis. This is the first systemic study revealed the networks of signaling pathways and proteomic responses to vancomycin treatment in HK-2 cells, and the data may be used to discriminate the molecular and clinical subtypes and to identify new targets and biomarkers for vancomycin-induced nephrotoxic effect. Further studies are warranted to explore the potential of quantitative proteomic analysis in the identification of new targets and biomarkers for drug-induced renal toxicity.

  18. Butanol isomers exert distinct effects on voltage-gated calcium channel currents and thus catecholamine secretion in adrenal chromaffin cells.

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    Sarah McDavid

    Full Text Available Butanol (C4H10OH has been used both to dissect the molecular targets of alcohols/general anesthetics and to implicate phospholipase D (PLD signaling in a variety of cellular functions including neurotransmitter and hormone exocytosis. Like other primary alcohols, 1-butanol is a substrate for PLD and thereby disrupts formation of the intracellular signaling lipid phosphatidic acid. Because secondary and tertiary butanols do not undergo this transphosphatidylation, they have been used as controls for 1-butanol to implicate PLD signaling. Recently, selective pharmacological inhibitors of PLD have been developed and, in some cases, fail to block cellular functions previously ascribed to PLD using primary alcohols. For example, exocytosis of insulin and degranulation of mast cells are blocked by primary alcohols, but not by the PLD inhibitor FIPI. In this study we show that 1-butanol reduces catecholamine secretion from adrenal chromaffin cells to a much greater extent than tert-butanol, and that the PLD inhibitor VU0155056 has no effect. Using fluorescent imaging we show the effect of these drugs on depolarization-evoked calcium entry parallel those on secretion. Patch-clamp electrophysiology confirmed the peak amplitude of voltage-gated calcium channel currents (I(Ca is inhibited by 1-butanol, with little or no block by secondary or tert-butanol. Detailed comparison shows for the first time that the different butanol isomers exert distinct, and sometimes opposing, effects on the voltage-dependence and gating kinetics of I(Ca. We discuss these data with regard to PLD signaling in cellular physiology and the molecular targets of general anesthetics.

  19. Butanol isomers exert distinct effects on voltage-gated calcium channel currents and thus catecholamine secretion in adrenal chromaffin cells. (United States)

    McDavid, Sarah; Bauer, Mary Beth; Brindley, Rebecca L; Jewell, Mark L; Currie, Kevin P M


    Butanol (C4H10OH) has been used both to dissect the molecular targets of alcohols/general anesthetics and to implicate phospholipase D (PLD) signaling in a variety of cellular functions including neurotransmitter and hormone exocytosis. Like other primary alcohols, 1-butanol is a substrate for PLD and thereby disrupts formation of the intracellular signaling lipid phosphatidic acid. Because secondary and tertiary butanols do not undergo this transphosphatidylation, they have been used as controls for 1-butanol to implicate PLD signaling. Recently, selective pharmacological inhibitors of PLD have been developed and, in some cases, fail to block cellular functions previously ascribed to PLD using primary alcohols. For example, exocytosis of insulin and degranulation of mast cells are blocked by primary alcohols, but not by the PLD inhibitor FIPI. In this study we show that 1-butanol reduces catecholamine secretion from adrenal chromaffin cells to a much greater extent than tert-butanol, and that the PLD inhibitor VU0155056 has no effect. Using fluorescent imaging we show the effect of these drugs on depolarization-evoked calcium entry parallel those on secretion. Patch-clamp electrophysiology confirmed the peak amplitude of voltage-gated calcium channel currents (I(Ca)) is inhibited by 1-butanol, with little or no block by secondary or tert-butanol. Detailed comparison shows for the first time that the different butanol isomers exert distinct, and sometimes opposing, effects on the voltage-dependence and gating kinetics of I(Ca). We discuss these data with regard to PLD signaling in cellular physiology and the molecular targets of general anesthetics.

  20. HIF-1alpha and HIF-2alpha are differentially activated in distinct cell populations in retinal ischaemia.

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    Freya M Mowat

    Full Text Available BACKGROUND: Hypoxia plays a key role in ischaemic and neovascular disorders of the retina. Cellular responses to oxygen are mediated by hypoxia-inducible transcription factors (HIFs that are stabilised in hypoxia and induce the expression of a diverse range of genes. The purpose of this study was to define the cellular specificities of HIF-1alpha and HIF-2alpha in retinal ischaemia, and to determine their correlation with the pattern of retinal hypoxia and the expression profiles of induced molecular mediators. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the tissue distribution of retinal hypoxia during oxygen-induced retinopathy (OIR in mice using the bio-reductive drug pimonidazole. We measured the levels of HIF-1alpha and HIF-2alpha proteins by Western blotting and determined their cellular distribution by immunohistochemistry during the development of OIR. We measured the temporal expression profiles of two downstream mediators, vascular endothelial growth factor (VEGF and erythropoietin (Epo by ELISA. Pimonidazole labelling was evident specifically in the inner retina. Labelling peaked at 2 hours after the onset of hypoxia and gradually declined thereafter. Marked binding to Müller glia was evident during the early hypoxic stages of OIR. Both HIF-1alpha and HIF-2alpha protein levels were significantly increased during retinal hypoxia but were evident in distinct cellular distributions; HIF-1alpha stabilisation was evident in neuronal cells throughout the inner retinal layers whereas HIF-2alpha was restricted to Müller glia and astrocytes. Hypoxia and HIF-alpha stabilisation in the retina were closely followed by upregulated expression of the downstream mediators VEGF and EPO. CONCLUSIONS/SIGNIFICANCE: Both HIF-1alpha and HIF-2alpha are activated in close correlation with retinal hypoxia but have contrasting cell specificities, consistent with differential roles in retinal ischaemia. Our findings suggest that HIF-2alpha activation

  1. Oral Administration of T Cell Epitope Peptide Inhibits the Systemic IL-4 Response Elicited by an Egg-White Diet in a TCR Transgenic Mouse Model


    HIRAIDE, Erika; NAKAJIMA-ADACHI, Haruyo; Hachimura, Satoshi


    Oral immunotherapy with T cell epitope peptides is a promising treatment for food allergy. We examined the effect of oral administration of an ovalbumin T cell epitope peptide (OVA323-339) in a TCR transgenic mouse model (OVA23-3 mice). OVA23-3 mice were fed egg-white diet containing ovalbumin and subsequently orally administrated the OVA323-339 peptide. Cytokine measurements revealed that the IL-4 production of splenic CD4+ T cells was significantly decreased by feeding the OVA323-339 peptid...

  2. Quinacrine induces apoptosis in human leukemia K562 cells via p38 MAPK-elicited BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression

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    Changchien, Jung-Jung; Chen, Ying-Jung; Huang, Chia-Hui [Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan (China); Cheng, Tian-Lu [Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Lin, Shinne-Ren [Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Chang, Long-Sen, E-mail: [Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan (China); Department of Biotechnology, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China)


    Although previous studies have revealed the anti-cancer activity of quinacrine, its effect on leukemia is not clearly resolved. We sought to explore the cytotoxic effect and mechanism of quinacrine action in human leukemia K562 cells. Quinacrine induced K562 cell apoptosis accompanied with ROS generation, mitochondrial depolarization, and down-regulation of BCL2L1 and BCL2. Upon exposure to quinacrine, ROS-mediated p38 MAPK activation and ERK inactivation were observed in K562 cells. Quinacrine-induced cell death and mitochondrial depolarization were suppressed by the p38MAPK inhibitor SB202190 and constitutively active MEK1 over-expression. Activation of p38 MAPK was shown to promote BCL2 degradation. Further, ERK inactivation suppressed c-Jun-mediated transcriptional expression of BCL2L1. Over-expression of BCL2L1 and BCL2 attenuated quinacrine-evoked mitochondrial depolarization and rescued the viability of quinacrine-treated cells. Taken together, our data indicate that quinacrine-induced K562 cell apoptosis is mediated through mitochondrial alterations triggered by p38 MAPK-mediated BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression. - Highlights: • Quinacrine induces K562 cell apoptosis via down-regulation of BCL2 and BCL2L1. • Quinacrine induces p38 MAPK activation and ERK inactivation in K562 cells. • Quinacrine elicits p38 MAPK-mediated BCL2 down-regulation. • Quinacrine suppresses ERK/c-Jun-mediated BCL2L1 expression.

  3. Eliciting Epitope-Specific CD8+ T Cell Response by Immunization with Microbial Protein Antigens Formulated with α-Galactosylceramide: Theory, Practice, and Protocols. (United States)

    Gilchuk, Pavlo; Knight, Frances C; Wilson, John T; Joyce, Sebastian


    CD8+ cytotoxic T lymphocytes confer protection against infectious diseases caused by viruses, bacteria, and parasites. Hence, significant efforts have been invested into devising ways to generate CD8+ T cell-targeted vaccines. Generation of microbe-free protein subunit vaccines requires a thorough knowledge of protective target antigens. Such antigens are proteolytically processed peptides presented by MHC class I molecules. To induce a robust antigen-specific CD8+ T cell response through vaccination, it is essential to formulate the antigen with an effective adjuvant. Here, we describe a versatile method for generating high-frequency antigen-specific CD8+ T cells through immunization of mice using the invariant natural killer T cell agonist α-galactosylceramide as the adjuvant.

  4. Necrosis is the dominant cell death pathway in uropathogenic Escherichia coli elicited epididymo-orchitis and is responsible for damage of rat testis.

    Directory of Open Access Journals (Sweden)

    Yongning Lu

    Full Text Available Male infertility is a frequent medical condition, compromising approximately one in twenty men, with infections of the reproductive tract constituting a major etiological factor. Bacterial epididymo-orchitis results in acute inflammation most often caused by ascending canalicular infections from the urethra via the continuous male excurrent ductal system. Uropathogenic Escherichia coli (UPEC represent a relevant pathogen in urogenital tract infections. To explore how bacteria can cause damage and cell loss and thus impair fertility, an in vivo epididymo-orchitis model was employed in rats by injecting UPEC strain CFT073 into the vas deference in close proximity to the epididymis. Seven days post infection bacteria were found predominantly in the testicular interstitial space. UPEC infection resulted in severe impairment of spermatogenesis by germ cell loss, damage of testicular somatic cells, a decrease in sperm numbers and a significant increase in TUNEL (+ cells. Activation of caspase-8 (extrinsic apoptotic pathway, caspase-3/-6 (intrinsic apoptotic pathway, caspase-1 (pyroptosis pathway and the presence of 180 bp DNA fragments, all of which serve as indicators of the classical apoptotic pathway, were not observed in infected testis. Notably, electron microscopical examination revealed degenerative features of Sertoli cells (SC in UPEC infected testis. Furthermore, the passive release of high mobility group protein B1 (HMGB1, as an indication of necrosis, was observed in vivo in infected testis. Thus, necrosis appears to be the dominant cell death pathway in UPEC infected testis. Substantial necrotic changes seen in Sertoli cells will contribute to impaired spermatogenesis by loss of function in supporting the dependent germ cells.

  5. Necrosis is the dominant cell death pathway in uropathogenic Escherichia coli elicited epididymo-orchitis and is responsible for damage of rat testis. (United States)

    Lu, Yongning; Bhushan, Sudhanshu; Tchatalbachev, Svetlin; Marconi, Marcelo; Bergmann, Martin; Weidner, Wolfgang; Chakraborty, Trinad; Meinhardt, Andreas


    Male infertility is a frequent medical condition, compromising approximately one in twenty men, with infections of the reproductive tract constituting a major etiological factor. Bacterial epididymo-orchitis results in acute inflammation most often caused by ascending canalicular infections from the urethra via the continuous male excurrent ductal system. Uropathogenic Escherichia coli (UPEC) represent a relevant pathogen in urogenital tract infections. To explore how bacteria can cause damage and cell loss and thus impair fertility, an in vivo epididymo-orchitis model was employed in rats by injecting UPEC strain CFT073 into the vas deference in close proximity to the epididymis. Seven days post infection bacteria were found predominantly in the testicular interstitial space. UPEC infection resulted in severe impairment of spermatogenesis by germ cell loss, damage of testicular somatic cells, a decrease in sperm numbers and a significant increase in TUNEL (+) cells. Activation of caspase-8 (extrinsic apoptotic pathway), caspase-3/-6 (intrinsic apoptotic pathway), caspase-1 (pyroptosis pathway) and the presence of 180 bp DNA fragments, all of which serve as indicators of the classical apoptotic pathway, were not observed in infected testis. Notably, electron microscopical examination revealed degenerative features of Sertoli cells (SC) in UPEC infected testis. Furthermore, the passive release of high mobility group protein B1 (HMGB1), as an indication of necrosis, was observed in vivo in infected testis. Thus, necrosis appears to be the dominant cell death pathway in UPEC infected testis. Substantial necrotic changes seen in Sertoli cells will contribute to impaired spermatogenesis by loss of function in supporting the dependent germ cells.

  6. Centrifugal forces within usually-used magnitude elicited a transitory and reversible change in proliferation and gene expression of osteoblastic cells UMR-106. (United States)

    Li, Juan; Jiang, Lingyong; Liao, Ga; Chen, Guoping; Liu, Ying; Wang, Jun; Zheng, Yi; Luo, Songjiao; Zhao, Zhihe


    Centrifugation is an important step in biochemical and molecular biological researches. But the effects of centrifugal stress on cells are still unclear. In this study, osteoblastic cells UMR-106 were subjected to a moderate centrifugal stress at 209 x g for 10 min. Then the cell proliferation and gene transcription after centrifugation were analyzed with flow cytometry and Real-time RT-PCR techniques, respectively. The result showed that the cell proliferation and mRNA expression of Runx2/Cbfa1, Collagen I and osteocalcin changed shortly after centrifugal loading, but recovered to pre-load levels within 24 h. A dose-response study of exposure cells to centrifugal force at 209, 253 and 301 x g showed that the centrifugal forces within usually-used range can rapidly influenced the mRNA expression of the osteoblast-specific genes, but no statistical differences were found among the three centrifugal magnitudes. And the fast regulation in the investigated genes was proved to be related to increased c-fos mRNA levels and subsequent activation of RTK and integrity of cytoskeleton construction. The result showed that the osteoblastic cells displayed a fast auto-regulation to usually-used centrifugal stress through multiple signal pathways.

  7. IBU-octyl-cytisine, a novel bifunctional compound eliciting anti-inflammatory and cholinergic activity, ameliorates CNS inflammation by inhibition of T-cell activity. (United States)

    Nizri, Eran; Irony-Tur-Sinai, Michal; Lavon, Iris; Meshulam, Haim; Amitai, Gabi; Brenner, Talma


    Experimental autoimmune encephalomyelitis (EAE) is a central nervous system (CNS) inflammatory model in which MOG-specific T-cells initiate an autoimmune attack leading to demyelinization and consequently, neurological damage and morbidity. As EAE pathogenesis results from the involvement of immune cells, CNS resident-cells and inflammatory mediators, our treatment strategy was to use a bifunctional compound with dual anti-inflammatory properties: a non-steroidal anti-inflammatory moiety and a nicotinic agonist moiety, intended to interact with the alpha7 nicotinic receptor present on immune cells. We used IBU-Octyl-Cytisine, with an ibuprofen (IBU) moiety and Cytisine, as the nicotinic agonist. The two moieties are attached by an eight carbon (octyl) spacer. Treatment of EAE with IBU-Octyl-Cytisine (2.5 mg/kg/day, i.p.) reduced significantly (by 70%) disease severity and inflammatory infiltrates in the spinal cord. An equivalent dose of IBU was ineffective, whereas Cytisine was significantly toxic. Treatment with IBU-Octyl-Cytisine inhibited the T-cell response toward the encephalitogenic epitope of myelin oligodendrocyte glycoprotein (MOG). In addition, expression of CCR5 by CD4(+)T-cells was lower, indicating a reduced migratory capacity following treatment. IBU-Octyl-Cytisine reduced Th(1) but not Th(2) cytokine production. This reduction was accompanied by a drop in the level of T-bet mRNA, a transcription factor pivotal to Th(1) lineage differentiation. Thus, IBU-Octyl-Cytisine is an effective treatment for EAE, influencing T-cell responses in several stages of disease pathogenesis. This bifunctional compound was more efficient than IBU or Cytisine separately, as well as than both moieties unconjugated. Thus, it seems that this strategy may be applicable in wider context.

  8. Dengue type four viruses with E-Glu345Lys adaptive mutation from MRC-5 cells induce low viremia but elicit potent neutralizing antibodies in rhesus monkeys. (United States)

    Lin, Hsiao-Han; Lee, Hsiang-Chi; Li, Xiao-Feng; Tsai, Meng-Ju; Hsiao, Hung-Ju; Peng, Jia-Guan; Sue, Shih-Che; Qin, Cheng-Feng; Wu, Suh-Chin


    Knowledge of virulence and immunogenicity is important for development of live-attenuated dengue vaccines. We previously reported that an infectious clone-derived dengue type 4 virus (DENV-4) passaged in MRC-5 cells acquired a Glu345Lys (E-E345K) substitution in the E protein domain III (E-DIII). The same cloned DENV-4 was found to yield a single E-Glu327Gly (E-E327G) mutation after passage in FRhL cells and cause the loss of immunogenicity in rhesus monkeys. Here, we used site-directed mutagenesis to generate the E-E345K and E-E327G mutants from DENV-4 and DENV-4Δ30 infectious clones and propagated in Vero or MRC-5 cells. The E-E345K mutations were consistently presented in viruses recovered from MRC-5 cells, but not Vero cells. Recombinant E-DIII proteins of E345K and E327G increased heparin binding correlated with the reduced infectivity by heparin treatment in cell cultures. Different from the E-E327G mutant viruses to lose the immunogencity in rhesus monkeys, the E-E345K mutant viruses were able to induce neutralizing antibodies in rhesus monkeys with an almost a 10-fold lower level of viremia as compared to the wild type virus. Monkeys immunized with the E-E345K mutant virus were completely protected with no detectable viremia after live virus challenges with the wild type DENV-4. These results suggest that the E-E345K mutant virus propagated in MRC-5 cells may have potential for the use in live-attenuated DENV vaccine development.

  9. The transcriptional profile of mesenchymal stem cell populations in primary osteoporosis is distinct and shows overexpression of osteogenic inhibitors.

    Directory of Open Access Journals (Sweden)

    Peggy Benisch

    Full Text Available Primary osteoporosis is an age-related disease characterized by an imbalance in bone homeostasis. While the resorptive aspect of the disease has been studied intensely, less is known about the anabolic part of the syndrome or presumptive deficiencies in bone regeneration. Multipotent mesenchymal stem cells (MSC are the primary source of osteogenic regeneration. In the present study we aimed to unravel whether MSC biology is directly involved in the pathophysiology of the disease and therefore performed microarray analyses of hMSC of elderly patients (79-94 years old suffering from osteoporosis (hMSC-OP. In comparison to age-matched controls we detected profound changes in the transcriptome in hMSC-OP, e.g. enhanced mRNA expression of known osteoporosis-associated genes (LRP5, RUNX2, COL1A1 and of genes involved in osteoclastogenesis (CSF1, PTH1R, but most notably of genes coding for inhibitors of WNT and BMP signaling, such as Sclerostin and MAB21L2. These candidate genes indicate intrinsic deficiencies in self-renewal and differentiation potential in osteoporotic stem cells. We also compared both hMSC-OP and non-osteoporotic hMSC-old of elderly donors to hMSC of ∼30 years younger donors and found that the transcriptional changes acquired between the sixth and the ninth decade of life differed widely between osteoporotic and non-osteoporotic stem cells. In addition, we compared the osteoporotic transcriptome to long term-cultivated, senescent hMSC and detected some signs for pre-senescence in hMSC-OP.Our results suggest that in primary osteoporosis the transcriptomes of hMSC populations show distinct signatures and little overlap with non-osteoporotic aging, although we detected some hints for senescence-associated changes. While there are remarkable inter-individual variations as expected for polygenetic diseases, we could identify many susceptibility genes for osteoporosis known from genetic studies. We also found new candidates, e.g. MAB21L

  10. A Recombinant G Protein Plus Cyclosporine A-Based Respiratory Syncytial Virus Vaccine Elicits Humoral and Regulatory T Cell Responses against Infection without Vaccine-Enhanced Disease. (United States)

    Li, Chaofan; Zhou, Xian; Zhong, Yiwei; Li, Changgui; Dong, Aihua; He, Zhonghuai; Zhang, Shuren; Wang, Bin


    Respiratory syncytial virus (RSV) infection can cause severe disease in the lower respiratory tract of infants and older people. Vaccination with a formalin-inactivated RSV vaccine (FI-RSV) and subsequent RSV infection has led to mild to severe pneumonia with two deaths among vaccinees. The vaccine-enhanced disease (VED) was recently demonstrated to be due to an elevated level of Th2 cell responses following loss of regulatory T (Treg) cells from the lungs. To induce high levels of neutralizing Abs and minimize pathogenic T cell responses, we developed a novel strategy of immunizing animals with a recombinant RSV G protein together with cyclosporine A. This novel vaccine induced not only a higher level of neutralizing Abs against RSV infection, but, most importantly, also significantly higher levels of Treg cells that suppressed VED in the lung after RSV infection. The induced responses provided protection against RSV challenge with no sign of pneumonia or bronchitis. Treg cell production of IL-10 was one of the key factors to suppress VED. These finding indicate that G protein plus cyclosporine A could be a promising vaccine against RSV infection in children and older people.

  11. Coxiella burnetii and Leishmania mexicana residing within similar parasitophorous vacuoles elicit disparate host responses

    Directory of Open Access Journals (Sweden)

    Jess A Millar


    Full Text Available Coxiella burnetii is a bacterium that thrives in an acidic parasitophorous vacuole (PV derived from lysosomes. Leishmania mexicana, a eukaryote, has also independently evolved to live in a morphologically similar PV. As Coxiella and Leishmania are highly divergent organisms that cause different diseases, we reasoned that their respective infections would likely elicit distinct host responses despite producing phenotypically similar parasite-containing vacuoles. The objective of this study was to investigate, at the molecular level, the macrophage response to each pathogen. Infection of THP-1 (human monocyte/macrophage cells with Coxiella and Leishmania elicited disparate host responses. At 5 days post-infection, when compared to uninfected cells, 1057 genes were differentially expressed (746 genes up- and 311 genes down-regulated in C. burnetii infected cells, whereas 698 genes (534 genes up- and 164 genes down-regulated were differentially expressed in L. mexicana infected cells. Interestingly, of the 1755 differentially expressed genes identified in this study, only 126 genes (~7% are common to both infections. We also discovered that 1090 genes produced mRNA isoforms at significantly different levels under the two infection conditions, suggesting that alternate proteins encoded by the same gene might have important roles in host response to each infection. Additionally, we detected 257 micro RNAs (miRNAs that were expressed in THP-1 cells and identified miRNAs that were specifically expressed during Coxiella or Leishmania infections. Collectively, this study identified host mRNAs and miRNAs that were influenced by Coxiella and/or Leishmania infections. Intriguingly, our data indicate that although their PVs are morphologically similar, Coxiella and Leishmania have evolved different strategies that perturb distinct host processes to create and thrive within their respective intracellular niches.

  12. RSK is a principal effector of the RAS-ERK pathway for eliciting a coordinate promotile/invasive gene program and phenotype in epithelial cells

    DEFF Research Database (Denmark)

    Doehn, Ulrik; Hauge, Camilla; Frank, Scott R


    The RAS-stimulated RAF-MEK-ERK pathway confers epithelial cells with critical motile and invasive capacities during development, tissue regeneration, and carcinoma progression, often via promoting the epithelial-mesenchymal transition (EMT). Many mechanisms by which ERK exerts this control remain...... elusive. We demonstrate that the ERK-activated kinase RSK is necessary to induce mesenchymal motility and invasive capacities in nontransformed epithelial and carcinoma cells. RSK is sufficient to induce certain motile responses. Expression profiling analysis revealed that a primary role of RSK...... to stimulate motility and invasion. These findings uncover a mechanism whereby the RAS-ERK pathway controls epithelial cell motility by identifying RSK as a key effector, from which emanate multiple highly coordinate transcription-dependent mechanisms for stimulation of motility and invasive properties....

  13. CALHM1 ion channel elicits amyloid-β clearance by insulin-degrading enzyme in cell lines and in vivo in the mouse brain. (United States)

    Vingtdeux, Valérie; Chandakkar, Pallavi; Zhao, Haitian; Blanc, Lionel; Ruiz, Santiago; Marambaud, Philippe


    Alzheimer's disease is characterized by amyloid-β (Aβ) peptide accumulation in the brain. CALHM1, a cell-surface Ca(2+) channel expressed in brain neurons, has anti-amyloidogenic properties in cell cultures. Here, we show that CALHM1 controls Aβ levels in vivo in the mouse brain through a previously unrecognized mechanism of regulation of Aβ clearance. Using pharmacological and genetic approaches in cell lines, we found that CALHM1 ion permeability and extracellular Ca(2+) were required for the Aβ-lowering effect of CALHM1. Aβ level reduction by CALHM1 could be explained by an increase in extracellular Aβ degradation by insulin-degrading enzyme (IDE), extracellular secretion of which was strongly potentiated by CALHM1 activation. Importantly, Calhm1 knockout in mice reduced IDE enzymatic activity in the brain, and increased endogenous Aβ concentrations by up to ∼50% in both the whole brain and primary neurons. Thus, CALHM1 controls Aβ levels in cell lines and in vivo by facilitating neuronal and Ca(2+)-dependent degradation of extracellular Aβ by IDE. This work identifies CALHM1 ion channel as a potential target for promoting amyloid clearance in Alzheimer's disease.

  14. Induction of acetylation and bundling of cellular microtubules by 9-(4-vinylphenyl) noscapine elicits S-phase arrest in MDA-MB-231 cells. (United States)

    Cheriyamundath, Sanith; Mahaddalkar, Tejashree; Kantevari, Srinivas; Lopus, Manu


    Noscapine is an alkaloid present in the latex of Papaver somniferum. It has been known for its anticancer efficacy and lack of severe toxicities to normal tissues. Structural alterations in noscapine core architecture have produced a number of potent analogues of noscapine. Here, we report an unusual activity of a novel noscapine analogue, 9-(4-vinylphenyl)noscapine (VinPhe-Nos) on cancer cells. As we reported earlier, VinPhe-Nos inhibited MDA-MB-231 cell proliferation with an IC50 of 6μM. The present study elucidated a possible antiproliferative mechanism of action of VinPhe-Nos. The noscapinoid significantly inhibited clonogenic propagation of MDA-MB-231 cells. However, unlike the majority of tubulin-binding agents, it did not induce mitotic arrest; instead, it prolonged S-phase. Although prolonged presence of the drug show some disruption of cellular microtubule architecture, it did not affect microtubule recovery after cold-induced depolymerization. VinPhe-Nos, nevertheless, induced acetylation and bundling of microtubules. Our data suggest that rational modification of parent compound can alter its mechanism of action on cell cycle and that VinPhe-Nos can be investigated further as a less-toxic, S-phase-preferred, cytostatic anticancer agent.

  15. Induction of hematopoietic microchimerism by gene-modified BMT elicits antigen-specific B and T cell unresponsiveness toward gene therapy products

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    Jérémie Martinet


    Full Text Available Background: Gene therapy is a promising treatment option for hemophilia and other protein deficiencies. However, immune responses against the transgene product represent an obstacle to safe and effective gene therapy, urging for the implementation of tolerization strategies. Induction of a hematopoietic chimerism via bone marrow transplantation (BMT is a potent means for inducing immunological tolerance in solid organ transplantation. Objectives: We reasoned here that the same viral vector could be used firstly to transduce BM cells for inducing chimerism-associated transgene-specific immune tolerance and, secondly, for correcting protein deficiencies by vector-mediated systemic production of the deficient coagulation factor.Methods: Evaluation of strategies to induce B and T cell tolerance was performed using ex vivo gene transfer with lentiviral vectors encoding coagulation factor IX (FIX or the SIINFEKL epitope of ovalbumin. Following induction of microchimerism via BMT, animals were challenged with in vivo gene transfer with lentiviral vectors.Results: The experimental approach prevented humoral immune response against FIX, resulting in persistence of therapeutic levels of circulating FIX after lentiviral-mediated gene transfer in vivo. In an ovalbumin model, we also demonstrated that this approach effectively tolerized the CD8+ T cell compartment in an antigen-specific manner.Conclusions: These results provide the proof-of-concept that inducing a microchimerism by gene-modified BMT is a powerful tool to provide transgene-specific B and T cell tolerance in a gene therapy setting.

  16. Increased t cell breadth and antibody response elicited in prime-boost regimen by viral vector encoded homologous siv gag/env in outbred cd1 mice

    DEFF Research Database (Denmark)

    Andersson, Anne Marie Carola; Holst, Peter Johannes


    Background: A major obstacle for the development of HIV vaccines is the virus' worldwide sequence diversity. Nevertheless, the presence of T cell epitopes within conserved regions of the virus' structural Gag protein and conserved structures in the envelope (env) sequence raises the possibility...

  17. A synthetic peptide derived from the animo acid sequence of canine parvovirus structural proteins which defines a B cell epitope and elicits antiviral antibody in BALB c mice.

    NARCIS (Netherlands)

    G.F. Rimmelzwaan (Guus); J. Carlson; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)


    textabstractSynthetic peptides, recombinant fusion proteins and mouse monoclonal antibodies were used to delineate a B cell epitope of the VP'2 structural protein of canine parvovirus (CPV). Although this epitope is not preferentially recognized in the normal antibody response to CPV, virus-specific

  18. Clostridium perfringens enterotoxin elicits rapid and specific cytolysis of breast carcinoma cells mediated through tight junction proteins claudin 3 and 4. (United States)

    Kominsky, Scott L; Vali, Mustafa; Korz, Dorian; Gabig, Theodore G; Weitzman, Sigmund A; Argani, Pedram; Sukumar, Saraswati


    Clostridium perfringens enterotoxin (CPE) induces cytolysis very rapidly through binding to its receptors, the tight junction proteins CLDN 3 and 4. In this study, we investigated CLDN 3 and 4 expression in breast cancer and tested the potential of CPE-mediated therapy. CLDN 3 and 4 proteins were detected in all primary breast carcinomas tested (n = 21) and, compared to normal mammary epithelium, were overexpressed in approximately 62% and 26%, respectively. Treatment of breast cancer cell lines in culture with CPE resulted in rapid and dose-dependent cytolysis exclusively in cells that expressed CLDN 3 and 4. Intratumoral CPE treatment of xenografts of T47D breast cancer cells in immunodeficient mice resulted in a significant reduction in tumor volume (P = 0.007), with accompanying necrosis. Necrotic reactions were also seen in three freshly resected primary breast carcinoma samples treated with CPE for 12 hours, while isolated primary breast carcinoma cells underwent rapid and complete cytolysis within 1 hour. Thus, expression of CLDN 3 and 4 sensitizes primary breast carcinomas to CPE-mediated cytolysis and emphasizes the potential of CPE in breast cancer therapy.

  19. Inorganic mercury accumulation in brain following waterborne exposure elicits a deficit on the number of brain cells and impairs swimming behavior in fish (white seabream-Diplodus sargus). (United States)

    Pereira, Patrícia; Puga, Sónia; Cardoso, Vera; Pinto-Ribeiro, Filipa; Raimundo, Joana; Barata, Marisa; Pousão-Ferreira, Pedro; Pacheco, Mário; Almeida, Armando


    The current study contributes to fill the knowledge gap on the neurotoxicity of inorganic mercury (iHg) in fish through the implementation of a combined evaluation of brain morphometric alterations (volume and total number of neurons plus glial cells in specific regions of the brain) and swimming behavior (endpoints related with the motor activity and mood/anxiety-like status). White seabream (Diplodus sargus) was exposed to realistic levels of iHg in water (2μgL(-1)) during 7 (E7) and 14 days (E14). After that, fish were allowed to recover for 28 days (PE28) in order to evaluate brain regeneration and reversibility of behavioral syndromes. A significant reduction in the number of cells in hypothalamus, optic tectum and cerebellum was found at E7, accompanied by relevant changes on swimming behavior. Moreover, the decrease in the number of neurons and glia in the molecular layer of the cerebellum was followed by a contraction of its volume. This is the first time that a deficit on the number of cells is reported in fish brain after iHg exposure. Interestingly, a recovery of hypothalamus and cerebellum occurred at E14, as evidenced by the identical number of cells found in exposed and control fish, and volume of cerebellum, which might be associated with an adaptive phenomenon. After 28 days post-exposure, the optic tectum continued to show a decrease in the number of cells, pointing out a higher vulnerability of this region. These morphometric alterations coincided with numerous changes on swimming behavior, related both with fish motor function and mood/anxiety-like status. Overall, current data pointed out the iHg potential to induce brain morphometric alterations, emphasizing a long-lasting neurobehavioral hazard.

  20. Differential response to 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in non-small cell lung cancer cells with distinct oncogene mutations. (United States)

    Zhang, Qiuhong; Kanterewicz, Beatriz; Shoemaker, Suzanne; Hu, Qiang; Liu, Song; Atwood, Kristopher; Hershberger, Pamela


    We previously demonstrated that non-small cell lung cancer (NSCLC) cells and primary human lung tumors aberrantly express the vitamin D3-catabolizing enzyme, CYP24, and that CYP24 restricts transcriptional regulation and growth control by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) in NSCLC cells. To ascertain the basis for CYP24 dysregulation, we assembled a panel of cell lines that represent distinct molecular classes of lung cancer: cell lines were selected which harbored mutually exclusive mutations in either the K-ras or the Epidermal Growth Factor Receptor (EGFR) genes. We observed that K-ras mutant lines displayed a basal vitamin D receptor (VDR)(low)CYP24(high) phenotype, whereas EGFR mutant lines had a VDR(high)CYP24(low) phenotype. A mutation-associated difference in CYP24 expression was also observed in clinical specimens. Specifically, K-ras mutation was associated with a median 4.2-fold increase in CYP24 mRNA expression (p=4.8×10(-7)) compared to EGFR mutation in a series of 147 primary lung adenocarcinoma cases. Because of their differential basal expression of VDR and CYP24, we hypothesized that NSCLC cells with an EGFR mutation would be more responsive to 1,25(OH)2D3 treatment than those with a K-ras mutation. To test this, we measured the ability of 1,25(OH)2D3 to increase reporter gene activity, induce transcription of endogenous target genes, and suppress colony formation. In each assay, the extent of 1,25(OH)2D3 response was greater in EGFR mutation-positive HCC827 and H1975 cells than in K-ras mutation-positive A549 and 128.88T cells. We subsequently examined the effect of combining 1,25(OH)2D3 with erlotinib, which is used clinically in the treatment of EGFR mutation-positive NSCLC. 1,25(OH)2D3/erlotinib combination resulted in significantly greater growth inhibition than either single agent in both the erlotinib-sensitive HCC827 cell line and the erlotinib-resistant H1975 cell line. These data are the first to suggest that EGFR mutations may

  1. Localization of Brain-Derived Neurotrophic Factor to Distinct Terminals of Mossy Fiber Axons Implies Regulation of Both Excitation and Feedforward Inhibition of CA3 Pyramidal Cells


    Danzer, Steve C.; McNamara, James O.


    Hippocampal dentate granule cells directly excite and indirectly inhibit CA3 pyramidal cells via distinct presynaptic terminal specializations of their mossy fiber axons. This mossy fiber pathway contains the highest concentration of brain-derived neurotrophic factor (BDNF) in the CNS, yet whether BDNF is positioned to regulate the excitatory and/or inhibitory pathways is unknown. To localize BDNF, confocal microscopy of green fluorescent protein transgenic mice was combined with BDNF immunoh...

  2. Trichuris suis excretory secretory products (ESP) elicit interleukin-6 (IL-6) and IL-10 secretion from intestinal epithelial cells (IPEC-1). (United States)

    Parthasarathy, G; Mansfield, L S


    Immune responses to gastrointestinal helminth infections have received increasing attention due to similarities to allergen-induced responses. In fact, the whipworm parasite of swine, Trichuris suis, has been used in beginning clinical trials as an antidote to inflammatory bowel disease. This strategy was based on this similarity and the recognition that other worms have been documented to induce anti-inflammatory responses in the host. In an effort to understand the basis for this response, we hypothesized that the proteins and peptides secreted by T. suis stimulate local intestinal epithelial cells to produce anti-inflammatory cytokines. To test this hypothesis in a correlate system of the natural swine host, T. suis excretory secretory products (ESP) were used to treat both differentiated and undifferentiated intestinal pig epithelial cells (IPEC-1) in vitro as a model for the effect on villus tip and crypt epithelial cells in the vicinity of the worms. IPEC-1 were exposed to low-level doses (0.3mg/ml) of T. suis ESP, and IL-4, IL-6 and IL-10 cytokine responses were measured by an enzyme-linked immunosorbant assay (ELISA). IL-6 was the predominant cytokine produced, accompanied by moderate IL-10 secretion from both differentiated and undifferentiated cells. As expected, IL-4 was not produced by IPEC-1. Additionally, IL-6 and IL-10 cytokines were produced within 24h, suggesting that these two cytokines form part of the primary host response to T. suis infections. These data suggest that T. suis ESP could enhance host immune responses and modulation through the induction of enteric IL-6 and IL-10.

  3. Fusion-Expressed CTB Improves Both Systemic and Mucosal T-Cell Responses Elicited by an Intranasal DNA Priming/Intramuscular Recombinant Vaccinia Boosting Regimen

    Directory of Open Access Journals (Sweden)

    Sugan Qiu


    Full Text Available Previous study showed that CTB (Cholera toxin subunit B can be used as a genetic adjuvant to enhance the systemic immune responses. To further investigate whether it can also be used as a genetic adjuvant to improve mucosal immune responses, we constructed DNA and recombinant Tiantan vaccinia (rTTV vaccines expressing OVA-CTB fusion antigen. Female C57BL/6 mice were immunized with an intranasal DNA priming/intramuscular rTTV boosting regimen. OVA specific T-cell responses were measured by IFN-γ ELISPOT and specific antibody responses were determined by ELISA. Compared to the nonadjuvant group (pSV-OVA intranasal priming/rTTV-OVA intramuscular boosting, pSV-OVA-CTB intranasal priming/rTTV-OVA-CTB intramuscular boosting group significantly improved the magnitudes of T-cell responses at spleen (1562±567 SFCs/106 splenocytes versus 330±182 SFCs/106 splenocytes, P<0.01, mesenteric LN (96±83 SFCs/106 lymphocytes versus 1±2 SFCs/106 lymphocytes, P<0.05, draining LNs of respiratory tract (109±60 SFCs/106 lymphocytes versus 2±2 SFCs/106 lymphocytes, P<0.01 and female genital tract (89±48 SFCs/106 lymphocytes versus 23±21 SFCs/106 lymphocytes, P<0.01. These results collectively demonstrated that fusion-expressed CTB could act as a potent adjuvant to improve both systemic and mucosal T-cell responses.

  4. Glial Cell-Elicited Activation of Brain Microvasculature in Response to Brucella abortus Infection Requires ASC Inflammasome-Dependent IL-1β Production. (United States)

    Miraglia, M Cruz; Costa Franco, Miriam M; Rodriguez, Ana M; Bellozi, Paula M Q; Ferrari, Carina C; Farias, Maria I; Dennis, Vida A; Barrionuevo, Paula; de Oliveira, Antonio C P; Pitossi, Fernando; Kim, Kwang Sik; Delpino, M Victoria; Oliveira, Sergio Costa; Giambartolomei, Guillermo H


    Blood-brain barrier activation and/or dysfunction are a common feature of human neurobrucellosis, but the underlying pathogenic mechanisms are largely unknown. In this article, we describe an immune mechanism for inflammatory activation of human brain microvascular endothelial cells (HBMEC) in response to infection with Brucella abortus Infection of HBMEC with B. abortus induced the secretion of IL-6, IL-8, and MCP-1, and the upregulation of CD54 (ICAM-1), consistent with a state of activation. Culture supernatants (CS) from glial cells (astrocytes and microglia) infected with B. abortus also induced activation of HBMEC, but to a greater extent. Although B. abortus-infected glial cells secreted IL-1β and TNF-α, activation of HBMEC was dependent on IL-1β because CS from B. abortus-infected astrocytes and microglia deficient in caspase-1 and apoptosis-associated speck-like protein containing a CARD failed to induce HBMEC activation. Consistently, treatment of CS with neutralizing anti-IL-1β inhibited HBMEC activation. Both absent in melanoma 2 and Nod-like receptor containing a pyrin domain 3 are partially required for caspase-1 activation and IL-1β secretion, suggesting that multiple apoptosis-associated speck-like protein containing CARD-dependent inflammasomes contribute to IL-1β-induced activation of the brain microvasculature. Inflammasome-mediated IL-1β secretion in glial cells depends on TLR2 and MyD88 adapter-like/TIRAP. Finally, neutrophil and monocyte migration across HBMEC monolayers was increased by CS from Brucella-infected glial cells in an IL-1β-dependent fashion, and the infiltration of neutrophils into the brain parenchyma upon intracranial injection of B. abortus was diminished in the absence of Nod-like receptor containing a pyrin domain 3 and absent in melanoma 2. Our results indicate that innate immunity of the CNS set in motion by B. abortus contributes to the activation of the blood-brain barrier in neurobrucellosis and IL-1β mediates

  5. Lasting antibody responses are mediated by a combination of newly formed and established bone marrow plasma cells drawn from clonally distinct precursors. (United States)

    Chernova, Irene; Jones, Derek D; Wilmore, Joel R; Bortnick, Alexandra; Yucel, Mesut; Hershberg, Uri; Allman, David


    Current models hold that serum Ab titers are maintained chiefly by long-lived bone marrow (BM) plasma cells (PCs). In this study, we characterize the role of subpopulations of BM PCs in long-term humoral responses to T cell-dependent Ag. Surprisingly, our results indicate that 40-50% of BM PCs are recently formed cells, defined, in part, by rapid steady-state turnover kinetics and secretion of low-affinity IgM Abs. Further, for months after immunization with a hapten-protein conjugate, newly formed Ag-induced, IgM-secreting BM PCs were detected in parallel with longer-lived IgG-secreting cells, suggesting ongoing and parallel input to the BM PC pool from two distinct pools of activated B cells. Consistent with this interpretation, IgM and IgG Abs secreted by cells within distinct PC subsets exhibited distinct L chain usage. We conclude that long-term Ab responses are maintained by a dynamic BM PC pool composed of both recently formed and long-lived PCs drawn from clonally disparate precursors.

  6. Lasting antibody responses are mediated by a combination of newly formed and established bone marrow plasma cells drawn from clonally distinct precursors 1 (United States)

    Chernova, Irene; Jones, Derek D.; Wilmore, Joel R.; Bortnick, Alexandra; Yucel, Mesut; Hershberg, Uri; Allman, David


    Current models hold that serum antibody titers are maintained chiefly by long-lived bone marrow (BM) plasma cells (PCs). Here we characterize the role of subpopulations of BM PCs in long-term humoral responses to T-cell dependent antigen. Surprisingly, our results indicate that 40–50% of BM PCs are recently formed cells, defined in part by rapid steady state turnover kinetics and secretion of low affinity immunoglobulin-M (IgM) antibodies. Further, for months after immunization with a hapten-protein conjugate newly formed antigen-induced IgM-secreting BM PCs were detected in parallel with longer-lived IgG-secreting cells, suggesting ongoing and parallel input to the BM PC pool from two distinct pools of activated B cells. Consistent with this interpretation, IgM and IgG antibodies secreted by cells within distinct PC subsets exhibited distinct light chain usage. We conclude that long-term antibody responses are maintained by a dynamic BM PC pool comprised of both recently formed and long-lived PCs drawn from clonally disparate precursors. PMID:25326027

  7. Tricho- and atrichoblast cell files show distinct PIN2 auxin efflux carrier exploitations and are jointly required for defined auxin-dependent root organ growth. (United States)

    Löfke, Christian; Scheuring, David; Dünser, Kai; Schöller, Maria; Luschnig, Christian; Kleine-Vehn, Jürgen


    The phytohormone auxin is a vital growth regulator in plants. In the root epidermis auxin steers root organ growth. However, the mechanisms that allow adjacent tissues to integrate growth are largely unknown. Here, the focus is on neighbouring epidermal root tissues to assess the integration of auxin-related growth responses. The pharmacologic, genetic, and live-cell imaging approaches reve