Journal: Obstetrics & Gynecology Science

Article Title: The effect of salinomycin on ovarian cancer stem-like cells

doi: 10.5468/ogs.2016.59.4.261

Figure Lengend Snippet: (A) Increased expression of CD44 in OVCAR3 sphere forming cells. The expression of ovarian cancer stem cell marker CD44 was increased in OVCAR3 sphere forming cells as observed under fluorescence microscopy. Nuclei were stained with Hoechst (×100). (B) Analysis of surface marker expressions by flow cytometry. CD44 and CD117 were increased approximately two folds in OVCAR3 sphere cells. FITC, fluorescein isothiocyanate; APC, allophysocyanin.

Article Snippet: Second, resulting cells were then depleted of CD117 - subsets by using mouse antihuman CD117 antibody coupled to magnetic microbeads (Miltenyi Biotec), and CD44 + CD117 + cells were named as cancer stem-like cells (OVCAR3 CD44 + CD117 + ).

Techniques: Expressing, Marker, Fluorescence, Microscopy, Staining, Flow Cytometry

Journal: Obstetrics & Gynecology Science

Article Title: The effect of salinomycin on ovarian cancer stem-like cells

doi: 10.5468/ogs.2016.59.4.261

Figure Lengend Snippet: The expressions of stemness genes in OVCAR3 CD44 + CD117 + cells by Western blot (A) and by semiquantitative reverse transcription polymerase chain reaction (B). The amount of cDNA input was adjusted to equalize the expression level of GAPDH. Octamer-binding trascription factor 3/4 (OCT3/4), nanog homeobox (NANOG) and sex determining region Y-box 2 (SOX2) are known to be as stemness genes. The expressions of stemness genes were increased in OVCAR3 CD44 + CD117 + cells than those in OVCAR3. Each band was quantified by densitometric analysis and presented in a bar graph. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Second, resulting cells were then depleted of CD117 - subsets by using mouse antihuman CD117 antibody coupled to magnetic microbeads (Miltenyi Biotec), and CD44 + CD117 + cells were named as cancer stem-like cells (OVCAR3 CD44 + CD117 + ).

Techniques: Western Blot, Reverse Transcription, Polymerase Chain Reaction, Expressing, Binding Assay

Journal: Obstetrics & Gynecology Science

Article Title: The effect of salinomycin on ovarian cancer stem-like cells

doi: 10.5468/ogs.2016.59.4.261

Figure Lengend Snippet: Effect of salinomycin in growth inhibition of ovarian cancer stem-like cells. The cells were exposed to various concentrations of (A) paclitaxel (1, 10, 100 and 200 nM) and (B) salinomycin (0.1, 0.5, 1 and 5 µM) in OVCAR3 and OVCAR3 CD44 + CD117 + cells for 48 hours to evaluate effect in growth inhibition of ovarian cancer stem-like cells. (C) OVCAR3 CD44 + CD117 + cells were treated with paclitaxel (10 nM) and/or salinomycin (0.1 µM) for 48 hours. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Values are mean±standard deviation of three measurements. CTL, control; PTX, paclitaxel; Sal, salinomycin. * P <0.05.

Article Snippet: Second, resulting cells were then depleted of CD117 - subsets by using mouse antihuman CD117 antibody coupled to magnetic microbeads (Miltenyi Biotec), and CD44 + CD117 + cells were named as cancer stem-like cells (OVCAR3 CD44 + CD117 + ).

Techniques: Inhibition, MTT Assay, Standard Deviation, Control

Journal: Journal of Research of the National Institute of Standards and Technology

Article Title: Improved Near-Infrared Spectral Responsivity Scale

doi: 10.6028/jres.105.055

Figure Lengend Snippet: The internal quantum efficiency of a trap detector measured by the absolute cryogenic radiometer (ACR)-based setup (filled diamonds) and by the Spectral Comparator Facility (SCF) (squares).

Article Snippet: Photodetectors are currently calibrated for spectral power responsivity in the visible and near-infrared (NIR) wavelength regions at the National Institute of Standards and Technology Spectral Comparator Facility (SCF) [ , ].

Techniques:

Journal: Journal of Research of the National Institute of Standards and Technology

Article Title: Improved Near-Infrared Spectral Responsivity Scale

doi: 10.6028/jres.105.055

Figure Lengend Snippet: The relative combined standard uncertainty for InGaAs photodiode calibration. The wavelength region from 850 nm to 1600 nm was measured with an 850 nm longpass filter (diamonds) and a 1300 nm longpass filter was used for measurements from 1400 nm to 1800 nm (circles). Also shown is the Spectral Comparator Facility (SCF) uncertainty prior to this work (crosses).

Article Snippet: Photodetectors are currently calibrated for spectral power responsivity in the visible and near-infrared (NIR) wavelength regions at the National Institute of Standards and Technology Spectral Comparator Facility (SCF) [ , ].

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Inhibiting antibiotic-resistant Enterobacteriaceae by microbiota-mediated intracellular acidification

doi: 10.1084/jem.20181639

Figure Lengend Snippet: SCFAs inhibit Enterobacteriaceae isolates . (A) Acetate, propionate, and butyrate concentrations in antibiotic-naive or ampicillin mice. Total SCFA represents the sum of these three most-abundant SCFA ( n = 8 mice from two independent experiments; t test). (B) Growth of the indicated isolates of Enterobacteriaceae in LB with or without a mixture of SCFA at the indicated pH was measured after 16 h of incubation ( n = 6–8 wells from three to four independent experiments; one-way ANOVA followed by Dunnett’s multiple comparison test to the control LB condition for each isolate). (C) Growth of Enterobacteriaceae isolates in LB at the indicated pH values with a mixture of acetate, propionate, and butyrate was measured after 16 h of incubation ( n = 6 wells from two independent experiments). pH values observed directly in cecal contents are indicated in green (antibiotic naive) or orange (antibiotic treated). Dotted line indicates strain growth in LB alone ( n = 6 wells from two independent experiments). (D) The K. pneumoniae , E. coli , or P. mirabilis burdens in the intestinal content (CFU/g) of colonized mice given PBS or FMT treatments or collected before treatment is plotted against SCFA concentration in cecal contents (acetate + propionate + butyrate; n = 3–12 mice per group; Spearman correlation. CFU/g values are also plotted in ).

Article Snippet: After thawing, samples were extracted with 80% methanol containing internal standards of deuterated SCFA ( d -3 acetate, d -5 propionate, and d -7 butyrate; Cambridge Isotope Laboratories).

Techniques: Incubation, Comparison, Control, Concentration Assay

Journal: The Journal of Experimental Medicine

Article Title: Inhibiting antibiotic-resistant Enterobacteriaceae by microbiota-mediated intracellular acidification

doi: 10.1084/jem.20181639

Figure Lengend Snippet: Loss of SCFA production correlates with E. coli expansion in an allo-HCT patient. (A) Daily changes in the composition of the intestinal microbiota were monitored in a patient undergoing allo-HCT from 9 d before transplant to 5 d after transplant when a BSI with E. coli was detected (dashed red line). Select antibiotics administered to the patient are depicted. (B) The composition of the microbiota in daily fecal samples is plotted. Values are plotted as the percent abundance of each species determined using MetaPhlAn analysis. (C) The inverse Simpson diversity index of the microbiota in daily fecal samples. (D) Levels of the indicated β-lactamase genes in the microbiota from daily fecal samples. (E) Acetate, propionate, and butyrate concentrations in daily fecal samples from the patient. (F) Total SCFA in daily fecal samples from the patient calculated as the sum of acetate, propionate, and butyrate. (G) The relative abundance of E. coli (MetaPhlAn) compared with the total SCFA concentration in the daily fecal sample. At the point highlighted in red, a BSI with E. coli was detected ( n = 15 d; Spearman correlation).

Article Snippet: After thawing, samples were extracted with 80% methanol containing internal standards of deuterated SCFA ( d -3 acetate, d -5 propionate, and d -7 butyrate; Cambridge Isotope Laboratories).

Techniques: Concentration Assay

Journal: Gene therapy

Article Title: In vitro immune modulation by antibodies coupled to tumour cells.

doi: 10.1038/sj.gt.3300534

Figure Lengend Snippet: Figure 4 Biotinylated cells can bind streptavidin. 2 × 107 TIB232 cells in two 400 ml aliquots on ice, were either incubated with 3 ml of DMSO or 3 ml of biotinamidocaproate N-hydroxysuccinimide ester as described in Materials and methods. After 30 min incubation, cells were washed twice and resuspended in 2 ml HBSS + 1% FCS and further divided into five 400 ml aliquots to which was added 1 mg/ml streptavidin to a final concentration of 0 (b and d), 7 (e and f), 40 (g and h) and 160 mg/ml (i–k). After 30 min on ice, cells were washed twice in 10 ml HBSS + 1% FCS and 5 × 105 cells stained either with biotin-FITC (a, b, e, g, i and j) or streptavidin-FITC (c, d, f, h, k and l) and analyzed by FACS as described.

Article Snippet: This complex wasUK), 10 ng/ml recombinant human GM-CSF (kind gift from Schering-Plough, Bury St Edmunds, UK), 20 ng/ml then added to cells and incubated on ice for a further 30 min after which they were washed twice with HBSS +recombinant human stem cell factor (SCF) (225-SC, R&D Systems, Oxford, UK) and 10 ng/ml recombinant human 1% FCS and binding of the complex was detected as described under Immunofluoresence.

Techniques: Incubation, Concentration Assay, Staining

Journal: Gene therapy

Article Title: In vitro immune modulation by antibodies coupled to tumour cells.

doi: 10.1038/sj.gt.3300534

Figure Lengend Snippet: Figure 5 Cells labelled with avidin can bind biotinylated antibody. 2 × 107 TIB232 cells 400 ml on ice were incubated with 3 ml of biotinamidocapro- Discussion ate N-hydroxysuccinimide ester as described in Materials and methods. After 30 min, cells were washed twice in RPMI + 10% FCS, adjusted to The technique described in this study offers tremendous 8 × 105/ml and cultured overnight. After 18 h, 2 × 107 biotinylated and potential for introducing antibody/protein on to the sur- untreated cells were avidin- (or mock-) treated, washed as described in face of target cells making them able to provide immune Materials and methods and resuspended in HBSS + 1% FCS to a concen- costimulation to T cells. Our strategy has been designed tration of 2 × 107/ml. One hundred microlitres was taken and biotinylated specifically to use only readily available reagents and antibody bound as described in Materials and methods. Cells were stained thus, in theory, no costly or complicated reagents need to with either biotin-FITC (c and d) or FITC-conjugated F(ab′)2 fragment of rabbit anti-mouse immunoglobulins (a, b, e–h) and analyzed by FACS be manufactured for this strategy to be used in therapy. as described. Although similar approaches have been reported,12,13 we believe that this is the first report of the use of an activating antibody attached to a tumour cell in such a way that the active site of the antibody is not involved protein G′-biotin + anti-CD28 (B/AV/PG/CD28). Jurkat cells incubated with PMA alone show little IL-2 secretion, in the initial binding. The orientation of the bound anti- body is such that its antigen-binding variable region is and other conditions show no significant change until, once again, the PMA-treated cells are incubated with available for further interactions. The presented data show that cells can be readily biotinylated to a high level B/AV/PG/CD28. Under these conditions the stimuli appear synergistic. A surprising result is seen when the under conditions that are not toxic to the cells (data not shown). The biotin on the cell surface is progressively cells are stimulated with PHA. Here we see that both B/AV/PG/CD28 and control + avidin + protein G′-biotin lost, but this can be slowed down with the use of meta- bolic inhibitors. Since the initial efficiency of biotinylation + anti-CD28 (C/AV/PG/CD28) both synergise with PHA treatment of Jurkat cells. This is a reproducible phenom- is high, the rate of reduction even in the absence of meta- bolic inhibitors can be tolerated as even after 72 h in cul- enon, and odd since neither avidin or protein G′-biotin bind nonspecifically to a sufficient degree to anchor anti- ture the presence of biotin on the cell surface can be read- ily detected. CD28 to these cells as determined by FACS analysis. We can only conclude that firstly the threshold of synergistic We have demonstrated that potentially important sur- face proteins are targets for biotinylation, but not univer- interaction with CD28 and PMA is higher (ie less sensitive) than for PHA, and that the bioassay of the Jur- sally, so whilst the immunological profile of B7.1 on cells is altered this is not the case with CD54 (ICAM-1) or the kat cells detects lower levels of presented anti-CD28 than the FACS analysis. Finally the addition of anti-CD28 MHC class 1 on TIB232 cells. Additionally, after 24 h in culture the profile of detectable B7.1 returns to near nor- alone has little effect on the Jurkat cells, and the addition of more, with B/AV/PG/CD28, is not effective. Incu- mal levels. Putative tumour antigens may be targets for biotinylation and this may then destroy their immuno- bation of cells with anti-CD28 alone (control/anti-CD28, C/CD28) therefore does not allow this antibody to persist genicity, but we feel that it would be unlikely that biotin- ylation would interfere with all of them, and that fresh through the washing steps before the assay, neither does the presence of avidin and protein G′-biotin (B/AV/PG), product would not be re-expressed with additional cul- ture. This has been shown to be the case with the only without the anti-CD28 to stimulate IL-2 production in these cells. Therefore these results strengthen the sugges- surface marker found to be interfered with in our studies (ie B7.1). tion that the stimulation of IL-2 production by the T cell

Article Snippet: This complex wasUK), 10 ng/ml recombinant human GM-CSF (kind gift from Schering-Plough, Bury St Edmunds, UK), 20 ng/ml then added to cells and incubated on ice for a further 30 min after which they were washed twice with HBSS +recombinant human stem cell factor (SCF) (225-SC, R&D Systems, Oxford, UK) and 10 ng/ml recombinant human 1% FCS and binding of the complex was detected as described under Immunofluoresence.

Techniques: Avidin-Biotin Assay, Incubation, Cell Culture, Staining, Binding Assay, Control, Bioassay, Marker

Journal: Gene therapy

Article Title: In vitro immune modulation by antibodies coupled to tumour cells.

doi: 10.1038/sj.gt.3300534

Figure Lengend Snippet: Figure 6 Cells with bridged avidin can be used to assemble a complex with protein G′-biotin and antibody. 8 × 107 TIB232 cells in 1.6 ml on ice were incubated with 6 ml of biotinamidocaproate N-hydroxysuccinimide ester as described, washed twice in RPMI + 10% FCS and cultured at 37°C overnight. After 18 h, 2.4 × 107 biotinylated cells (b, d, e, g, h, i, k, m, n, p, q and r) and untreated equivalents (a, c, f, j, l and o) were avidin treated as described, after which cells were washed twice in HBSS + 1% FCS, resuspended in 300 ml of HBSS + 1% FCS, 100 ml aliquots (8 × 106 each) were then added either to carrier alone (a–i) or a preformed complex of 42 mg mouse anti-human CD28 + 8.5 mg protein G′-biotin (j–r). After a further 30 min, cells were washed twice in DMEM + 10% FCS (DMEM is deficient in biotin), and a sample taken immediately for staining and FACS analysis as described (T = 0 time-point, a, b, j and k), the remaining cells were divided into two and cultured either at 37°C for 3.5 h (c, e, l and n) and 6 h (f, h, o and q) or 4°C for 3.5 h (d and m) or 6 h (g and p) harvested and stained for FACS analysis. (i and r) Duplicates of b and k that were returned to culture after staining and incubated for a further 6 h at 37°C. Before processing the biotinylation efficiency was checked and found to have worked as normal (data not shown). All samples stained as indicated on the Figure and analyzed by FACS as described.

Article Snippet: This complex wasUK), 10 ng/ml recombinant human GM-CSF (kind gift from Schering-Plough, Bury St Edmunds, UK), 20 ng/ml then added to cells and incubated on ice for a further 30 min after which they were washed twice with HBSS +recombinant human stem cell factor (SCF) (225-SC, R&D Systems, Oxford, UK) and 10 ng/ml recombinant human 1% FCS and binding of the complex was detected as described under Immunofluoresence.

Techniques: Avidin-Biotin Assay, Incubation, Cell Culture, Staining

Journal: Gene therapy

Article Title: In vitro immune modulation by antibodies coupled to tumour cells.

doi: 10.1038/sj.gt.3300534

Figure Lengend Snippet: Figure 8 Mouse anti-human antibody complexed transiently with cells costimulates as a function of the antigenic target. 7 × 107 TIB232 cells Figure 7 Mouse anti-human CD28 antibody complexed transiently with were biotinylated in 1.6 ml PBS pH 8.0 essentially as previously described cells is functional as a costimulator. 8 × 107 TIB232 cells were biotinylated and cultured overnight. Biotinylated cells were then taken and either pro- in 1.6 ml PBS pH 8.0 essentially as previously described and cultured cessed as previously described with 2 mg/ml avidin for every 3.6 × 107 overnight. Biotinylated and untreated cells were then taken and processed cells in 360 ml HBSS + 1% FCS, or mock avidin-treated, cells were washed as previously described with 1 mg/ml avidin for every 7 × 107 cells in 900 twice and resuspended in 100 ml HBSS + 1% FCS for every 1.2 × 107 ml HBSS + 1% FCS and then 20 mg protein G′-biotin in 200 ml for every cells. To this was added a premix (30 min) of 6.5 mg of protein G′-biotin 3.5 × 107 cells and 100 mg anti-CD28 in 200 ml for every 3.5 × 107 cells, and 34 mg of either anti-CD28, CD3 or CD14. After complex assembly in the combinations described below. After complex assembly cells were cells were washed four times and cell samples analyzed by FACS to con- washed four times and cell samples analyzed by FACS to confirm firm efficiency of the complex formation. The remaining cells were kept in efficiency of the complex formation. The remaining cells were kept in RPMI + 10% FCS on ice. Cells were then added to the Jurkat assay as RPMI + 10% FCS on ice. Cells were then added to the Jurkat assay as described. Conditions were either Jurkat cells alone (no addition), biotinyl- described. Conditions were either Jurkat cells alone, untreated (control) ated cells, no avidin, with protein G′-biotin + anti-CD28 (B + PG/CD28), cells incubated with mouse anti-CD28 (C/CD28), untreated (control) cells biotinylated cells incubated with avidin + protein G′-biotin + anti-CD28 incubated with avidin + protein G′-biotin + anti-CD28 (C/AV/PG/CD28), (B/AV + PG/CD28), biotinylated cells incubated with avidin + protein G′- biotinylated cells incubated with avidin + protein G′-biotin + anti-CD28 biotin + anti-CD3 (B/AV + PG/CD3), and finally biotinylated cells incu- (B/AV/PG/CD28) or biotinylated cells incubated with avidin and protein bated with avidin + protein G′-biotin + anti-CD14 (B/AV + PG/CD14). G′-biotin alone (B/AV/PG). The human IL-2 ELISA detects human IL-2 The human IL-2 ELISA detects human IL-2 alone, and does not cross- alone, and does not cross-react with recombinant murine IL-2 in our hands react with recombinant murine IL-2 in our hands (data not shown). Data (data not shown). Data points show the pg/ml of quadruplicate determi- points show the pg/ml of triplicate determinations of IL-2 (±1 standard nations of IL-2 (±1 standard deviation). deviation). Crossed bars represent those samples whose IL-2 concentration exceeded the accurate range of the ELISA and were plotted as simply more try to avoid interfering with antigen recognition14 at least than 2000 pg/ml. some bound antibody would be correctly orientated. A more directed strategy was utilized where the orien- tation of the bound antibody was more predictable. This length of time that the complex would need to remain for the strategy to be truly effective as a cell vaccine. A was done because the gift of the mouse anti-human CD28 was valuable and in relatively short supply (we wished number of antibody combinations and cytokines are cur- rently being examined in animal models in order to to avoid an antibody–biotin conjugation reaction) and the alternative strategy provides added flexibility to the determine this. Although low temperature and azide appear to be able to inhibit degradation to a certain potential user. Since we were not actually cross-linking in this study degree neither of them can be used in vivo, thus alterna- tive strategies are at present under investigation. we were somewhat surprised that the avidin protein G′- biotin complex once assembled on the cell surface disap- Other labelling strategies have been utilized to simplify the system as much as possible and reduce the size of the pears quite rapidly. At least one major component of this deterioration involved the internalization of the complex complex on the cell surface. One of the first, that proved ultimately fruitless, was to avoid the biotinylation com- into the cell, presumably by endocytosis. This process appears to be stimulated by the addition of more compo- pletely and avidinylate the cells directly with the com- mercially available maleimide and hydrazide derivatives nents to the cell membrane, given that the decrease in biotin level on the cells takes place over a period of days, of avidin. The problem with these was that the pretreat- ment required to optimize the presence of the correct tar- whilst avidin complexed to the cells deteriorates in hours. Even with the rapid deterioration however, a detectable gets (SH groups for the maleimide13 and modified cell surface glycoprotein for the hydrazide15,16), were in our amount remains after 6 h in culture, and sufficient remains attached, even after extensive washing of the hands quite cytotoxic and the labelling not very efficient. The high molecular weight of avidin compared with cells, for anti-CD28 to be used in the Jurkat costimu- lation assay. biotin also precluded the application of sufficiently high molar concentrations of derivatised avidin. Other We have not yet investigated whether an in vitro effect can be seen with tumour and effector cells from a reagents such as the N-(biotinoyl) dipalmitoyl-l-a-phos- phatidylethanolamine (biotin-DPPE12) conjugate was tumour-bearing patient. It is of course quite possible that the presence of anti-CD28 alone in this case would not capable of only minimal association with the membrane and was very unstable. Synthesis of biotinylated cationic be sufficient, because the tumour specific T cells may be in the anergic state. In addition little is known about the lipidophiles such as 1,1′-didodecyl-,3,3′,3′-tetramethyl-

Article Snippet: This complex wasUK), 10 ng/ml recombinant human GM-CSF (kind gift from Schering-Plough, Bury St Edmunds, UK), 20 ng/ml then added to cells and incubated on ice for a further 30 min after which they were washed twice with HBSS +recombinant human stem cell factor (SCF) (225-SC, R&D Systems, Oxford, UK) and 10 ng/ml recombinant human 1% FCS and binding of the complex was detected as described under Immunofluoresence.

Techniques: Functional Assay, Cell Culture, Avidin-Biotin Assay, Control, Incubation, Enzyme-linked Immunosorbent Assay, Recombinant, Standard Deviation, Concentration Assay, Conjugation Assay, In Vivo, Membrane, High Molecular Weight, In Vitro