and ultrasonically cleaned using acetone, isopropyl alcohol and trichloroethylene. Before transferring the samples to the deposition chambers, they were dried using hot air. The substrate stage had a two-fold rota- tion facility and rotated at 4 rev min 21 during deposi- tion. Bipolar pulsed DC power supplies (advanced energy pinnacle plus) were used. Cathodes were ener- gised in current mode and the substrate was energised in voltage mode. A base pressure of better than 2610 23 Pa was achieved before initiating deposition, which was very similar to the base pressure reported by Musil and Hruby 9 and Zhou et al. 13 The deposition cycle consisted of sputter cleaning of the targets with shutters in closed position, followed by ion etching. Ion etching was
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Pulsed plasma processing has found increasing acceptance over last decades, particularly for the surface treatment of materials used in data storage, flat panel, semiconductor and other industrial applications. Pulsed DC processing is a sputtering technique combining the advantages of DC and RF sputtering. RF sputtering may produce better quality insulating films, but deposition rates are very low. The RF systems are usually complex and difficult to be used for the scale up of commercial products. The main problem in the DC reactive sputter deposition of insulating materials from an elemental metal target in a mixture of argon and reactive gas is that an insulating film may be formed on the target surface. This insulating layer is charged by the positive ions that are accelerated in the plasma and collected on this surface. If the insulating layer can sustain an infinite electric field strength this layer is ultimately charged to the applied power supply voltage V a . As a consequence, there will be no potential difference
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The MMC used as grid interface to klystron modulators requires the control of the AC active and reactive powers and the DC voltage, to recharge the capacitor bank before the next pulse. The proposed concept of the controller is presented in Fig. 3. The AC power references are used to compute the d, q current references for the PI phase current controllers  based on (1). The MMC has distributed energy storage, and the overall energy stored in the converter must be actively maintained. In  the energy stored in each phase is controlled by varying the circulating current reference and this approach is typical when the MMC is used as an inverter, e.g. drives. In the application analysed in this paper, the DC power is imposed by the load and the energy control loop can only act on the AC active power reference (Fig. 3). Naming P ACcorr ,
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an increase in welding speed or, in other words, a decrease in transfer of heat input to the weld zone improves tensile strength, even if by a small amount. During the tensile tests, none of the samples was fractured in the weld metal and the breakages of each sample occurred in areas close to the HAZ. It was determined according to the percentage elongation results of the welded samples that the said percentages decreased by a small amount in relation to the main material. The fact that the percentage elongation values of the welded samples were revealed to be lower than that of the main material can be said to cause the most of the deformation to occur in the HAZ and the area surrounding it. It can also be said that the welding current and the shielding argon gas have an effect on percentage elongation as well. Additionally, formation of a fine-grained structure in the weld metal through the effect of a low heat input caused the ductility of the welded samples to be lower in comparison to that of the main material. The temperature differences due to the heating and cooling in the weld zone which arise from the fact that the thermal expansion coefficients of aluminum and its alloys are high lead to severe internal stresses and distortions. In consideration of this adverse circumstance and as stated before, the test samples were joined with the pulsed direct current arc welding method. This method enabled the application of a strong pinch effect on every area of work that is desired, which in turn provided a suitable work environment via administration of a small heat input to the work piece without causing a short circuit. It also made it possible for molten metal drops of the desired number and size to be transferred to the work piece via adjustment of the frequency, which yielded a positive effect in enhancing the mechanical properties of the weld metal. In addition, the welding wire chosen for the study (AlMg5) is thought to have had a positive effect on the enhancement of the mechanical properties of the weld metal as well.
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Abstract: A high power pulsed DC glow discharge plasma (HPPGDP) system was employed to perform fast nitriding of AISI 316 austenitic stainless steel in Ar and N2 atmosphere. In-situ optical emission spectroscopy and Infrared pyrometer measurements were used during the plasma nitriding to investigate the effect of dynamic plasma on the nitriding behaviour. SEM and EDX, XRD, Knoop indentation, and tribo-tests were used to characterize microstructures and properties of the nitrided austenitic stainless steel samples.HPPGDP produced high ionization of both Ar and N2in the plasma that corresponded to dense ion bombardment on the biases steel samples to induce effective plasma surface heating and to form high nitrogen concentration on the biased steel surfaces, and therefore fast nitriding (> 10µm/hour) was achieved. Various phases were identified on the nitrided stainless steel samples formed from a predominantly a single phase of nitrogen supersaturated austenite toa multi-phase structure comprising chromium nitride, iron nitride and ferrite dependent on the nitriding time. All the nitrided AISI 316 austenitic stainless steel samples were evaluated with high hardness (up to 17.3 GPa) and exceptional wear resistance sliding to against hardened steel balls and tungsten carbide balls.
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In the past, the metal coating in form of thin films to im- prove the quality of material was done by electroplating process which is often also called electro-deposition. The disadvantage of electroplating was harmful to the environ- ment. Subsequently, the vacuum depositions were devel- oped in chemical vapor deposition (CVD) and physical va- por deposition (PVD). CVD is a technique whereby gase- ous reactants can be deposited onto a substrate. However, often dangerous by-products are removed by gas flow. PVD is a clean coating technology that involves evaporation and deposition of a material. Material vaporizes are removed from a source by physical processes such as evaporation sputtering and it is transported in the form of a vapor atomic beam through a vacuum to the substrate. Magnetron sput- tering is one of PVD methods, which are widely used in thin film technology. The various types of magnetron sput- tering technique are direct current (DC), alternating current (AC), radio frequency (RF), and pulsed-dc . Pulsed-dc magnetron sputtering is one of the latest developments of sputtering technology for thin films deposition, which has
In total, 56 patients will be included in the study. The primary endpoint is recurrence-free survival, and the secondary endpoints are natural killer T-cell-specific immune response, the frequency of toxic effects and safety, and overall survival. Discussion: In order to determine the efficacy of α -GalCer-pulsed DC therapy, the present study compares patients with stage II – III NSCLC who underwent complete surgical resection followed by postoperative adjuvant therapy with cisplatin plus vinorelbine, to those who did not receive additional treatment (surgical resection plus postoperative adjuvant chemotherapy only).
paper, we compare the protective efficacy of different pro- tocols of cancer vaccination based on a classical, widely adopted model system constituted by gp100 vaccination in B16F10 melanoma . Intriguingly, the comparison of different vaccination protocols, such as peptide plus adjuvant, gene immunization and peptide-pulsed DC immunization, showed different levels of protective effi- cacy among the three protocols, being DC vaccination the most effective one. This is a remarkable observation since suggests that adequate stimulation of a single (or limited number of) T cell clone(s), specific for one specific TAA epitope, as induced by peptide-pulsed DC vaccination, may have more pronounced protective effects than multi- epitope mediated stimulation of a wide TAA-specific T cell repertoire, as induced by vaccination with a gene coding for the entire TAA molecule. Importantly, the protective effects of identical protocols performed in syngeneic or xenogeneic settings differed greatly. This is not surprising since the efficacy of xenogeneic immunization has been already reported in different models of experimental tumor and associated with the expression of a heteroclitic epitope by the immunizing agent [36,37]. The use of xeno- geneic or modified tumor antigens, as a system for in- creasing the immunogenicity of the vaccine and breaking
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T cells undergo a robust proliferative response to MV but not to LV. During the first stimulation, we were unable to detect any proliferation of CD8 ⫹ or CD4 ⫹ T cells (Fig. 4A and B). Proliferation, measured by an increase in Ki-67 expression, began on the second day of the second stimulation only in response to MV (Fig. 4A). Proliferation reached a maximum on the fifth day, with a higher percentage of BrdU- and Ki-67- positive cells in CD8 ⫹ T cells than in CD4 ⫹ T cells (Fig. 4A, B, and D). Culture with mDC did not result in a higher prolifer- ation rate during the same period. However, while T cells FIG. 3. Expression of activation markers on the T-cell surface. The expression of several molecules involved in T-cell activation was analyzed by flow cytometry during the three rounds of stimulation (S1 to S3). T cells were cultured with MV (shaded bars)- or LV (filled bars)-infected iDC or mDC for the first stimulation and were restimulated twice with inactivated-MV- or inactivated-LV-pulsed DC, respectively. The percentage of T cells cultured with infected DC and restimulated with inactivated-virus-pulsed DC that express the markers minus the percentage of T cells cultured and restimulated with mock-infected DC and restimulated with culture medium that express the markers was calculated. Results are means and SEM from five independent experiments using different donors. Significant differences between mock-infected and infected cells are indicated as for Fig. 1. The expression of the respective marker in control T cells that had first been stimulated with mock-infected iDC was quantified 2 and 5 days after restimulation with DC pulsed with inactivated MV (open circles on shaded bars) or with inactivated LV (open circles on filled bars) and is expressed as the mean from two independent experiments using different donors. (A) The expression of CD154 on CD3 ⫹ CD4 ⫹ (top) and CD3 ⫹ CD8 ⫹ (bottom) T cells cultured with iDC or mDC is shown for 5 days after each stimulation. (B) CD154 mRNA synthesis was evaluated by qRT-PCR 2 days after each round of stimulation in T cells cultured with mock (open bars)-, MV (shaded bars)-, or LV (filled bars)-infected iDC. The means and SEM from five independent experiments using different donors are shown. Results are expressed as the gene/ ␤ -actin ratio. (C and D) The expression of CD137 (C) and HLA-DR (D) by CD3 ⫹ CD8 ⫹ and CD3 ⫹ CD4 ⫹ T cells cultured with iDC or mDC is shown for day 2 (d2) and d5 of the three rounds of stimulation. Dot plots showing the expression of CD8 and CD137 or of CD4 and CD154 or HLA-DR among CD3-gated cells 5 days after the second stimulation with mock-, MV-, or LV-infected iDC are presented.
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Our previous findings in other systems suggest that manipulation of DC co-stimulatory molecules, either with chemicals  or siRNA [14,17] provides a simple and reproducible method of in vivo inducing antigen-spe- cific immune deviation. We sought to test the feasibility of immunomodulation with CD40 knockdown in the CIA model. However, unlike our previous experiments in which CD40 knockdown was performed ex vivo on anti- gen-pulsed DC to induce immune deviation , here siRNA to CD40 was systemically administered two days prior to immunization with CII. Using the hydrodynamic injection method, 50 μg of siRNA was administered via the tail vein two days before immunization. Proliferative recall response to CII was performed to assess modula- tion of T cell activation under the cover of CD40 silenc- ing. T cells isolated from lymph nodes (Figures 2A) and spleens (Figures 2B) exhibited reduced proliferation from animals pretreated with CD40 siRNA but not controls. In contrast, no modulation of response to T cell activation with anti-CD3/CD28 in vitro was observed, indicating that immune modulation was not associated with unspe- cific T cell suppression (data not shown).
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Plus pulsed DC. power supply operated in constant current mode was used to supply power to the cathode. The system pressure was monitored using a capacitance manometer pressure gauge. The system was pumped to obtain an acceptable base pressure of 5.5 x10 -4 Pa. The operating pressure of 7.5 x 10 -1 Pa was set and controlled using a 100 sccm range MKS argon mass flow controller controlled by an MKS multi gas controller model 147. Films were deposited on glass microscope slides that had been cleaned using isopropyl alcohol and acetone in an ultrasonic bath.
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We investigated the use of cytotoxic T-lymphocyte (CTL) epitopes in peptide immunotherapy for glioblastoma. Three peptides (ERBB2, BIRC5 and CD99) were selected based on their peptide-T2 cell binding affinities and combined in a multipeptide cocktail or a branched multipeptide synthesized with mini-polyethylene glycol spacers. Dendritic cells (DCs) pulsed with the multipeptide cocktail or branched multipeptide were compared based on their immunophenotype and cytokine secretion. FACS analysis of alpha-type 1 polarized dendritic cells (αDC1s) revealed that both groups highly expressed CD80, CD83 and CD86, indicating that both treatments efficiently generated mature αDC1s with the expected phenotype. Production of IL- 12p70, IL-12p40 and IL-10 also increased upon αDC1 maturation in both groups. CTLs stimulated by either αDC1 group (“DC-CTLs”) included numerous IFN-γ-secreting cells against T2 cells loaded with the corresponding multipeptides. Large numbers of IFN- γ-secreting cells were observed when human glioblastoma cell lines and primary cells were treated with multipeptide-pulsed DC-CTLs. Both multipeptide-pulsed DC-CTL groups exhibited cytotoxic activity of 40-60% against the U251 cell line and 60-80% against primary cells. Branched multipeptide from ERBB2, BIRC5 and CD99 stably bound with T2 cells, and its cytotoxicity toward target cells was similar to that of the multipeptide cocktail. Thus, branched multipeptides could be promising candidates for immunotherapeutic glioblastoma treatment.
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The patient was treated with adjuvant chemotherapy consisting of paclitaxel plus S-1 for 1 year and immuno- therapy with tumor lysate-pulsed DC-activated killer (T- DAK) cell immunotherapy for 5 years. Paclitaxel (120 mg/ body) was administered weekly, twice every 4 weeks for four courses, after which it was replaced by S-1 because of difficulty in securing vascular access. S-1 (80 mg/day) was administered orally for 2 weeks, followed by a 2-week rest period. The methods used to prepare the T-DAK cells have been described previously . Firstly, immature DCs were prepared from plastic-adherent PBMCs using recombinant human granulocyte/monocyte colony-stimulating factor (100 ng/ml; Primmune KK, Kobe, Japan) and recombinant human IL-4 (500 U/ml; Primmune KK) for 7 days. Tumor cells obtained from the tumor masses were lysed by five
The choices of antigen and adjuvant are of critical importance in all vaccination strategies. Using DC as an adjuvant for vac- cination was largely developed in cancer trials, where the use of synthetic peptides as antigens has several advantages: they are relatively easy to produce, they enable monitoring of the peptide-specific cellular responses, they exclude the possibility of infection from the vaccine product, and they minimize the risk of generating immunity to self antigens. For these reasons, HIV-1-specific synthetic peptides were chosen for this phase I safety and feasibility study of DC-based immunization in this ART-treated cohort. It remains unknown and is a subject of much study as to what will emerge as the optimal antigen for HIV-1 vaccination, whether therapeutic or prophylactic. Here we have shown that administration of only two doses of synthetic peptide-pulsed DC, albeit with relatively narrow specificity, represents a feasible and potentially immunogenic strategy.
FIG. 1. Mature DCs enhance HIV transmission to different types of target cells independently of C-type lectins. (A) Surface markers of iDCs and mDCs. Monocyte-derived iDCs were cultured with LPS to generate mDCs. Cell surface markers were stained with specific MAbs or isotype-matched IgG controls and analyzed by flow cytometry. The histogram peaks of CD11c staining on iDCs and mDCs were overlapped. (B) Enhanced HIV transmission by mDCs is independent of C-type lectins. DCs and Raji/DC-SIGN cells were preincubated separately with medium, anti-DC-SIGN cocktails, and mannan prior to HIV incubation, as described previously (57). Raji/DC-SIGN cells, iDCs, and mDCs were pulsed separately with single-cycle, luciferase reporter R5-tropic HIV-Luc/JRFL (multiplicity of infection [MOI], 0.2), washed, and cocultured separately with autologous PBLs, the CD4 ⫹ T-cell line Hut/CCR5, and the HIV indicator cells GHOST/R5. HIV infection was determined after 2 days by measuring the luciferase activity. (C) No detectable HIV cis-infection in DCs and Raji/DC-SIGN cells. Cells were infected with HIV-Luc/JRFL (MOI, 0.2), and viral infection was determined at 2 dpi. (D) mDCs enhance transmission of HIV pseudotyped with X4-tropic Env (HXB2). Transmission of HIV-Luc/HXB2 with DCs as donors and Hut/CCR5 cells as targets was performed as described for panel B. The asterisk indicates a significant difference (P ⬍ 0.05) compared with iDCs. The data show the means ⫾ standard deviations of triplicate samples. One representative experiment out of four is shown. cps, counts per second.
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The DNA negative ions are adsorbed onto the anode aluminum surface after irradiating the electrode, and the adsorption is maintained semipermanently . There- fore, the amount of DNA negative ions irradiated to the anode can be estimated by measuring the absorbance of the DNA solution after applying the DC and/or RF elec- tric fields . In this experiment, several types of single- stranded DNA are used. The notation of DNA is as fol- lows. The bases of adenine, thymine, guanine, and cyto- sine are represented by A, T, G, and C, respectively. In addition, the number of bases is denoted by subscripts; for example, a DNA molecule consisting of 15 adenines is rep- resented by A 15 .
500°C during 1-2 hours of pulsed plasma post-oxidation treatment. Yang et. al.  showed that the highest amount of magnetite occurs when oxidation is carried out at 400 and 450°C in an oxygen atmosphere while M. Ebrahimi et. al.  reported that nitriding process at 570 and 630°C enhances the formation of magnetite phase in oxidized layers after post-oxidation process. Although these recent researches on duplex nitriding plus post-oxidation treatment have been carried out, process parameters to get layers with the highest magnetite content has not been clearly defined.
As we previously hypothesized  NLRP3-inflammasome could play a role in response against HIV and could par- ticipate to full dendritic cell activation and to increased proliferation of specific HIV-induced T cells. However our new findings point out that the protocol implied to differ- entiate monocytes to DC could affect inflammasome biol- ogy. Our results agreed with previously reported data by Guarda et al.  about the ability of type I IFNs to sup- press pro-IL-1 levels and decrease the activity of NLRP1 and NLRP3-inflammasomes in mice. Moreover it is well known that the activation of NLRP3-inflammasome in DC is related with the ability to induce a Th17 polarization in naïve T cells , while IFN-DC are able to induce Th1 but fail to polarize naïve T cells into Th17 , letting us hypothesize that in IFN-DC the inflammasomes are not induced.
There is no strictly defined standard duration of cul- ture to generate human PBMC-derived DCs. To date, most clinical studies have used a 7-day culture with GM-CSF and IL-4. However, data from Czerniecki and colleagues have shown that fully functional antigen- presenting cells (APC) can be rapidly developed from CD14 + PBMC cells in as little as 40 hours [135,136]. Al- though, these “rapid DC” are efficient in presenting class I and II peptides, data from our laboratory revealed that at least four days of differentiation with GM-CSF and IL-4 were required for elutriated peripheral blood mono- cytes to acquire the phenotype and functional properties of cross-presenting APCs capable of processing lysate antigen . These 4-day DCs pulsed with lysate under- went proper maturation into DC1 cells when exposed to bacterial lipopolysaccharide (LPS) combined with IFN-γ, producing high levels of IL-12, but required a concomi- tant – not sequential – exposure to the two maturing agents . We selected these cells as the proposed vac- cine platform, which will be administered intranodally, since intranodal administration of DCs allows adminis- tration of a defined quantity of DCs directly to the site of T-cell sensitization. This approach also allows the peak IL-12 secretion to be synchronized with their prox- imity to T-cells, where IL-12 can exert its full effects during antigen presentation . IL-12 is paramount as dendritic cells that are able to produce high levels of IL- 12 can induce long-lived type 1 T-cell responses against tumor-associated antigens more efficiently than standard mature DCs. The benefit of high IL-12 producing DCs was highlighted in several recent papers [138-140] dem- onstrating the importance of IL-12 production with re- gard to the induction of tumor-specific CTLs in vitro and its ability to predict prolongation of progression-free survival of patients with advanced cancer . Okada et al. showed in a Phase I/II cancer vaccine trial of ma- lignant glioma patients that IL-12 production levels by αDC1 positively correlated with time to progression.
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T lymphocytes orchestrate adaptive immunity by directly killing pathogen-infected and tumor cells and by releasing soluble factors (cytokines) that selectively activate and regulate other cells of the immune system. They can detect speci ﬁ c antigens using clonogenic antigen receptors (TCR). Most TCRs display speci ﬁ city for peptide antigens presented on major histocompatibility complex (MHC) molecules on antigen-presenting cells (APC). However, some T cells respond to lipid-based antigens presented by the CD1 family of MHC-like molecules, which are typically expressed by APC (Bendelac et al. 2007; Salio et al. 2014). The most extensively stud- ied lipid-reactive T cell is the invariant natural killer T (iNKT) cell, which expresses a semi-invariant TCR α -chain (V α 24J α 18 in humans and V α 14J α 18 in mice) paired with one of a limited number of β -chains, which recognizes glycolipid antigens bound to CD1d. A number of self (Brennan et al. 2011; Facciotti et al. 2012) and microbial (Kinjo et al. 2005; Mattner et al. 2005) glycosphingolipids have been shown to bind to CD1d and stimulate iNKT cells, but most of our understanding of iNKT cells comes from studies using the xenogeneic glycolipid, α -galactosylceramide ( α -GalCer). Upon activation with α -GalCer, iNKT cells kill target cells and secrete a diverse range of growth factors and cytokines, allowing them to contribute to the activation of T cells (Gumperz et al. 2002; O ’ Reilly et al. 2011), natural killer cells (Carnaud et al. 1999) and macrophages (Lynch et al. 2015). Activated iNKT cells can also interact directly with other cells of the immune system and can induce the maturation of dendritic cells (DC) into APC (Kitamura et al. 1999; Vincent et al. 2002) and of B cells into antibody- secreting plasma cells (Galli et al. 2003; Zeng et al. 2013). Thus, immune recognition of glycolipids plays a central role in the activa- tion and regulation of innate and adaptive immune responses.
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