Chemotaxis of Cell Populations through Confined Spaces at Single-Cell Resolution

Cell migration is crucial for both physiological and pathological processes. Current in vitro cell motility assays suffer from various drawbacks, including insufficient temporal and/or optical resolution, or the failure to include a controlled chemotactic stimulus. Here, we address these limitations with a migration chamber that utilizes a self-sustaining chemotactic gradient to induce locomotion through confined environments that emulate physiological settings. Dynamic real-time analysis of both population-scale and single-cell movement are achieved at high resolution. Interior surfaces can be functionalized through adsorption of extracellular matrix components, and pharmacological agents can be administered to cells directly, or indirectly through the chemotactic reservoir. Direct comparison of multiple cell types can be achieved in a single enclosed system to compare inherent migratory potentials. Our novel microfluidic design is therefore a powerful tool for the study of cellular chemotaxis, and is suitable for a wide range of biological and biomedical applications

Surface Properties of Helicobacter pylori Urease Complex Are Essential for Persistence

The enzymatic activity of Helicobacter pylori's urease neutralises stomach acidity, thereby promoting infection by this pathogen. Urease protein has also been found to interact with host cells in vitro, although this property's possible functional importance has not been studied in vivo. To test for a role of the urease surface in the host/pathogen interaction, surface exposed loops that display high thermal mobility were targeted for inframe insertion mutagenesis. H. pylori expressing urease with insertions at four of eight sites tested retained urease activity, which in three cases was at least as stable as was wild-type urease at pH 3. Bacteria expressing one of these four mutant ureases, however, failed to colonise mice for even two weeks, and a second had reduced bacterial titres after longer term (3 to 6 months) colonisation. These results indicate that a discrete surface of the urease complex is important for H. pylori persistence during gastric colonisation. We propose that this surface interacts directly with host components important for the host-pathogen interaction, immune modulation or other actions that underlie H. pylori persistence in its special gastric mucosal niche

Stoichiometry of HLA Class II-Invariant Chain Oligomers

BACKGROUND: The HLA gene complex encodes three class II isotypes, DR, DQ, and DP. HLA class II molecules are peptide receptors that present antigens for recognition by T lymphocytes. In antigen presenting cells, the assembly of matched α and β subunits to heterodimers is chaperoned by invariant chain (Ii). Ii forms a homotrimer with three binding sites for class II heterodimers. The current model of class II and Ii structure states that three αβ heterodimers bind to an Ii trimer. METHODOLOGY/PRINCIPAL FINDINGS: [corrected] We have now analyzed the composition and size of the complexes of class II and Ii using epitope tagged class II subunits and density gradient experiments. We show here that class II-Ii oligomers consist of one class II heterodimer associated with one Ii trimer, such that the DR, DQ and DP isotypes are contained within separate complexes with Ii. CONCLUSION/SIGNIFICANCE: We propose a structural model of the class II-Ii oligomer and speculate that the pentameric class II-Ii complex is bent towards the cell membrane, inhibiting the binding of additional class II heterodimers to Ii

Promiscuous Binding of Invariant Chain-Derived CLIP Peptide to Distinct HLA-I Molecules Revealed in Leukemic Cells

Antigen presentation by HLA class I (HLA-I) and HLA class II (HLA-II) complexes is achieved by proteins that are specific for their respective processing pathway. The invariant chain (Ii)-derived peptide CLIP is required for HLA-II-mediated antigen presentation by stabilizing HLA-II molecules before antigen loading through transient and promiscuous binding to different HLA-II peptide grooves. Here, we demonstrate alternative binding of CLIP to surface HLA-I molecules on leukemic cells. In HLA-II-negative AML cells, we found plasma membrane display of the CLIP peptide. Silencing Ii in AML cells resulted in reduced HLA-I cell surface display, which indicated a direct role of CLIP in the HLA-I antigen presentation pathway. In HLA-I-specific peptide eluates from B-LCLs, five Ii-derived peptides were identified, of which two were from the CLIP region. In vitro peptide binding assays strikingly revealed that the eluted CLIP peptide RMATPLLMQALPM efficiently bound to four distinct HLA-I supertypes (-A2, -B7, -A3, -B40). Furthermore, shorter length variants of this CLIP peptide also bound to these four supertypes, although in silico algorithms only predicted binding to HLA-A2 or -B7. Immunization of HLA-A2 transgenic mice with these peptides did not induce CTL responses. Together these data show a remarkable promiscuity of CLIP for binding to a wide variety of HLA-I molecules. The found participation of CLIP in the HLA-I antigen presentation pathway could reflect an aberrant mechanism in leukemic cells, but might also lead to elucidation of novel processing pathways or immune escape mechanisms

Helicobacter pylori Impairs Murine Dendritic Cell Responses to Infection

International audienceBACKGROUND: Helicobacter pylori, a human pathogen associated with chronic gastritis, peptic ulcer and gastric malignancies, is generally viewed as an extracellular microorganism. Here, we show that H. pylori replicates in murine bone marrow derived-dendritic cells (BMDCs) within autophagosomes. METHODOLOGY/PRINCIPAL FINDINGS: A 10-fold increase of CFU is found between 2 h and 6 h p.i. in H. pylori-infected BMDCs. Autophagy is induced around the bacterium and participates at late time points of infection for the clearance of intracellular H. pylori. As a consequence of infection, LC3, LAMP1 and MHC class II molecules are retained within the H. pylori-containing vacuoles and export of MHC class II molecules to cell surface is blocked. However, formalin-fixed H. pylori still maintain this inhibitory activity in BMDC derived from wild type mice, but not in from either TLR4 or TLR2-deficient mice, suggesting the involvement of H. pylori-LPS in this process. TNF-alpha, IL-6 and IL-10 expression was also modulated upon infection showing a TLR2-specific dependent IL-10 secretion. No IL-12 was detected favoring the hypothesis of a down modulation of DC functions during H. pylori infection. Furthermore, antigen-specific T cells proliferation was also impaired upon infection. CONCLUSIONS/SIGNIFICANCE: H. pylori can infect and replicate in BMDCs and thereby affects DC-mediated immune responses. The implication of this new finding is discussed for the biological life cycle of H. pylori in the host

WNT activation by lithium abrogates TP53 mutation associated radiation resistance in medulloblastoma

TP53 mutations confer subgroup specific poor survival for children with medulloblastoma. We hypothesized that WNT activation which is associated with improved survival for such children abrogates TP53 related radioresistance and can be used to sensitize TP53 mutant tumors for radiation. We examined the subgroup-specific role of TP53 mutations in a cohort of 314 patients treated with radiation. TP53 wild-type or mutant human medulloblastoma cell-lines and normal neural stem cells were used to test radioresistance of TP53 mutations and the radiosensitizing effect of WNT activation on tumors and the developing brain. Children with WNT/TP53 mutant medulloblastoma had higher 5-year survival than those with SHH/TP53 mutant tumours (100% and 36.6% +/- 8.7%, respectively (p < 0.001)). Introduction of TP53 mutation into medulloblastoma cells induced radioresistance (survival fractions at 2Gy (SF2) of 89% +/- 2% vs. 57.4% +/- 1.8% (p < 0.01)). In contrast, beta-catenin mutation sensitized TP53 mutant cells to radiation (p < 0.05). Lithium, an activator of the WNT pathway, sensitized TP53 mutant medulloblastoma to radiation (SF2 of 43.5% +/- 1.5% in lithium treated cells vs. 56.6 +/- 3% (p < 0.01)) accompanied by increased number of.H2AX foci. Normal neural stem cells were protected from lithium induced radiation damage (SF2 of 33% +/- 8% for lithium treated cells vs. 27% +/- 3% for untreated controls (p = 0.05). Poor survival of patients with TP53 mutant medulloblastoma may be related to radiation resistance. Since constitutive activation of the WNT pathway by lithium sensitizes TP53 mutant medulloblastoma cells and protect normal neural stem cells from radiation, this oral drug may represent an attractive novel therapy for high-risk medulloblastomas.B.R.A.I.N Child Canada; Cancer Research UK; Brain Tumour Charity; Hungarian Brain Research Program [KTIA_13_NAP-A-V/3]; Janos Bolyai Scholarship of the Hungarian Academy of Sciences [TAMOP-4.2.2. A-11/1/KONV-2012-0025]; German Cancer Aid/Dr. Mildred Scheel Foundation for Cancer Research; Cure Childhood Cancer Foundation; St. Baldrick's Foundation; Southeastern Brain Tumor Foundation; Action Medical Research; [CZ.1.05/2.1.00/03.0101]; [CZ.1.07/2.3.00/20.0183

Alignment of the CMS silicon tracker during commissioning with cosmic rays

This is the Pre-print version of the Article. The official published version of the Paper can be accessed from the link below - Copyright @ 2010 IOPThe CMS silicon tracker, consisting of 1440 silicon pixel and 15 148 silicon strip detector modules, has been aligned using more than three million cosmic ray charged particles, with additional information from optical surveys. The positions of the modules were determined with respect to cosmic ray trajectories to an average precision of 3–4 microns RMS in the barrel and 3–14 microns RMS in the endcap in the most sensitive coordinate. The results have been validated by several studies, including laser beam cross-checks, track fit self-consistency, track residuals in overlapping module regions, and track parameter resolution, and are compared with predictions obtained from simulation. Correlated systematic effects have been investigated. The track parameter resolutions obtained with this alignment are close to the design performance.This work is supported by FMSR (Austria); FNRS and FWO (Belgium); CNPq, CAPES, FAPERJ, and FAPESP (Brazil); MES (Bulgaria); CERN; CAS, MoST, and NSFC (China); COLCIENCIAS (Colombia); MSES (Croatia); RPF (Cyprus); Academy of Sciences and NICPB (Estonia); Academy of Finland, ME, and HIP (Finland); CEA and CNRS/IN2P3 (France); BMBF, DFG, and HGF (Germany); GSRT (Greece); OTKA and NKTH (Hungary); DAE and DST (India); IPM (Iran); SFI (Ireland); INFN (Italy); NRF (Korea); LAS (Lithuania); CINVESTAV, CONACYT, SEP, and UASLP-FAI (Mexico); PAEC (Pakistan); SCSR (Poland); FCT (Portugal); JINR (Armenia, Belarus, Georgia, Ukraine, Uzbekistan); MST and MAE (Russia); MSTDS (Serbia); MICINN and CPAN (Spain); Swiss Funding Agencies (Switzerland); NSC (Taipei); TUBITAK and TAEK (Turkey); STFC (United Kingdom); DOE and NSF (USA)

Commissioning and performance of the CMS pixel tracker with cosmic ray muons

This is the Pre-print version of the Article. The official published verion of the Paper can be accessed from the link below - Copyright @ 2010 IOPThe pixel detector of the Compact Muon Solenoid experiment consists of three barrel layers and two disks for each endcap. The detector was installed in summer 2008, commissioned with charge injections, and operated in the 3.8 T magnetic field during cosmic ray data taking. This paper reports on the first running experience and presents results on the pixel tracker performance, which are found to be in line with the design specifications of this detector. The transverse impact parameter resolution measured in a sample of high momentum muons is 18 microns.This work is supported by FMSR (Austria); FNRS and FWO (Belgium); CNPq, CAPES, FAPERJ, and FAPESP (Brazil); MES (Bulgaria); CERN; CAS, MoST, and NSFC (China); COLCIENCIAS (Colombia); MSES (Croatia); RPF (Cyprus); Academy of Sciences and NICPB (Estonia); Academy of Finland, ME, and HIP (Finland); CEA and CNRS/IN2P3 (France); BMBF, DFG, and HGF (Germany); GSRT (Greece); OTKA and NKTH (Hungary); DAE and DST (India); IPM (Iran); SFI (Ireland); INFN (Italy); NRF (Korea); LAS (Lithuania); CINVESTAV, CONACYT, SEP, and UASLP-FAI (Mexico); PAEC (Pakistan); SCSR (Poland); FCT (Portugal); JINR (Armenia, Belarus, Georgia, Ukraine, Uzbekistan); MST and MAE (Russia); MSTDS (Serbia); MICINN and CPAN (Spain); Swiss Funding Agencies (Switzerland); NSC (Taipei); TUBITAK and TAEK (Turkey); STFC (United Kingdom); DOE and NSF (USA)

Performance of the CMS drift-tube chamber local trigger with cosmic rays

The performance of the Local Trigger based on the drift-tube system of the CMS experiment has been studied using muons from cosmic ray events collected during the commissioning of the detector in 2008. The properties of the system are extensively tested and compared with the simulation. The effect of the random arrival time of the cosmic rays on the trigger performance is reported, and the results are compared with the design expectations for proton-proton collisions and with previous measurements obtained with muon beams