Advanced Sheet Metal Manufacturing using Rapid Tooling 522

A closed loop process is proposed for making sheet metal prototyping parts by using advanced computer aided techniques and computer controlled machines. The key aspect of this process is the method used to fabricate and modify the sheet metal forming tools, which are not necessarily for mass production but should be suitable for short run production or design evaluation of sheet metal products where the prototyping cost and lead-time are greatly reduced. Various approaches are investigated in the preparation of the tooling for onward embossing on a sheet metal. The three indirect approaches use Selective Laser Sintering (SLS), Stereolithography(SLA), and high speed Computer Numerical Controlled (CNC) milling to build the masters from computer data models. And the masters are then served in the vacuum casting process to generate the non-ferrous tooling. The direct approach uses DTM’s RapidSteel to produce the metal tooling without going through any secondary process. Comparisons on quality, leading time and cost are presented.Mechanical Engineerin

Persistent Helicobacter pylori Specific Th17 Responses in Patients with Past H. pylori Infection Are Associated with Elevated Gastric Mucosal IL-1β

10.1371/journal.pone.0039199PLoS ONE76

The IL-17A response of CD4⁺ PBMCs to HP-pulsed APCs.

<p>The mean ± SEM (standard error of mean) has been depicted for measurements of IL-17A concentration in the supernatant of co-culture experiments performed as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039199#pone-0039199-g003" target="_blank">figure 3B</a>. CD4⁺ T cells were co-cultured with autologous HP-pulsed APCs either in the absence or presence of MHC Class II blocking antibody. The fold-decrease in response was calculated by dividing the supernatant concentration of IL-17A from the culture without MHC Class II blockade, by the IL-17A concentration in the culture with MHC Class II blockade. The mean fold-decrease was obtained for the indicated number of biological replicates in each group, and mean ± SEM has been reported in the table.</p

Elevated frequencies of cells that express IL-17A persist in individuals with past HP infection.

<p>(A) The percentage of CD3⁺CD8⁻CCR6⁺IL-17A⁺ cells as a function of CD3⁺CD8⁻ PBMCs was assessed by flow cytometry. PBMCs were activated with PMA and ionomycin for 5 hours in the presence of GolgiStop. Cells were stained for cell surface CD3, CD8, and CCR6, fixed, permeabilised, and stained for intracellular IL-17A. Th17 cells were defined as CCR6⁺IL-17A⁺ events within the CD3⁺CD8⁻ compartment. Representative flow cytometry plots of individuals from group A, P, and N have been depicted. (B) Scatter plot of CD3⁺CD8⁻CCR6⁺IL-17A⁺ cells as a percentage of CD3⁺CD8⁻ cells among PBMCs that had been stimulated with PMA and ionomycin. Group A (n = 44), group P (n = 47), and group N (n = 48). The median and interquartile ranges have been represented on the scatter plot as horizontal bars. (C) The frequency of CD3⁺CD8⁻CCR6⁺IL-17A⁺ events within the CD3⁺CD8⁻ compartment for individuals from group P divided according to years since HP treatment. 1 year (n = 13), 2–3 years (n = 9), 4–9 years (n = 7), and ≥10 years (n = 3). (D) Number of CD4⁺IL-17A⁺ cells per high powered field (HPF) in gastric biopsy samples. Immunofluorescence microscopy was performed on gastric biopsies obtained from 8 patients in group A, 17 patients in group P, 12 patients in group N. For each patient sample, ten HPFs were evaluated and the average number of CD4⁺IL-17A⁺ cells per HPF was represented on the scatter plot. (E) Number of CD4⁺IL-17A⁺ cells per HPF in samples from group P stratified according to years since HP treatment. 1 year (n = 3), 2–3 years (n = 5), 4–9 years (n = 1), and ≥10 years (n = 3). (F − I) Cytokine concentrations in clarified homogenate obtained from mechanically disrupted gastric biopsy samples were measured using the MILLIPLEX® xMAP® bead-based cytokine quantification assay. (F) IL-17A concentration in gastric biopsy samples obtained from patients in group A (n = 7), group P (n = 15), and group N (n = 9). (G) IL-17A concentration in gastric biopsy samples from group P individuals depicted in (F) who were further sub-grouped based on the presence (PC+) or absence (PC<b>−</b>) of histological evidence of pre-cancerous lesions (chronic atrophic gastritis or intestinal metaplasia) in the gastric mucosa. PC+ (n = 12), PC<b>−</b> (n = 3). (H) IFNγ concentration in gastric biopsy samples obtained from patients in group A (n = 7), group P (n = 15), and group N (n = 9). (I) IL-8 concentration in gastric biopsy samples obtained from patients in group A (n = 7), group P (n = 15), and group N (n = 9). NS: not significant, *p<0.05, **p<0.001, ***p<0.0001.</p

Gastric mucosal expression of cytokines that modulate Th17 responses.

<p>(A) Semi-quantitative real-time PCR was used to determine gene expression of CCL20 in gastric biopsy samples, normalised to expression of β-actin. Group A (n = 17), group P (n = 27), group N (n = 20). (B) Semi-quantitative real-time PCR was used to determine gene expression of hBD-2 in gastric biopsy samples, normalised to expression of β-actin. Group A (n = 16), group P (n = 11), and group N (n = 12). (C) Semi-quantitative real-time PCR was used to determine gene expression of IL-23p19 in gastric biopsy samples, normalised to expression of β-actin. Group A (n = 15), group P (n = 31), and group N (n = 30). (D – G) <i>Ex vivo</i> protein concentrations of IL-6, TNF-α, IL-1β, and IL-1Ra in homogenised gastric biopsy samples were measured by MILLIPLEX® xMAP® bead-based cytokine quantification assay. Group A (n = 6), group P (n = 14), and group N (n = 7). (H) IL-1 receptor antagonist blocks IL-17A production by CD4⁺ T cells co-cultured with HP-pulsed APCs. PBMCs obtained from a group P individual were pulsed with HP and prepared as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039199#pone-0039199-g003" target="_blank">figure 3B</a> for use as APCs. These APCs were co-cultured for 48 hours with purified autologous CD4⁺ T cells at a ratio of 5 APCs: 1 CD4⁺ T cell, either in the absence or presence of IL-1Ra (final concentration 200 ng/ml). IL-17A levels in culture supernatant were measured using ELISA. Representative data from 1 of 2 independent experiments has been depicted. NS: not significant, *p<0.05, **p<0.001, ***p<0.0001.</p

IL-22 expression in the gastric mucosa.

<p>(A) Number of IL-22⁺ cells per high powered field (HPF) in gastric biopsy samples. Immunofluorescence microscopy was performed on gastric biopsies obtained from 13 patients in group A, 20 patients in group P, and 9 patients in group N. Ten HPFs were evaluated per sample, and the average number of IL-22⁺ cells per HPF was represented on the scatter plot. (B) <i>Ex vivo</i> concentration of IL-22 in gastric biopsies. Cytokine concentrations in clarified homogenate obtained from mechanically disrupted gastric biopsy samples were measured by ELISA. Group A (n = 8), group P (n = 17), and group N (n = 9). (C) The ratio of gastric mucosal IL-17A to IL-22 was determined by dividing the concentration of IL-17A in gastric mucosal homogenate with the concentration of IL-22 found in the same biopsy sample obtained from a given individual. Group A (n = 7), group P (n = 14), and group N (n = 8). NS: not significant, *p<0.05, ***p<0.0001.</p

The IL-17A response of expanded LPMCs to HP-pulsed APCs.

<p>The mean ± SEM (standard error of mean) has been depicted for measurements of IL-17A concentration in the supernatant of co-culture experiments performed as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039199#pone-0039199-g003" target="_blank">figure 3C</a>. LPMCs were co-cultured with autologous HP-pulsed APCs, either in the absence or presence of MHC Class II blocking antibody. The fold-decrease in response was calculated by dividing the supernatant concentration of IL-17A from the culture without MHC Class II blockade, by the IL-17A concentration in the culture with MHC Class II blockade. The mean fold-decrease was obtained for the indicated number of biological replicates in each group, and mean ± SEM has been reported in the table.</p

Primers used to perform real time PCR.

<p>Primers used to perform real time PCR.</p