Numerical Study of a Direct Injection Internal Combustion Engine Burning a Blend of Hydrogen and Dimethyl Ether

In the reported study, various aspects of dimethyl ether/hydrogen combustion in a Reactivity Controlled Compression Ignition (RCCI) engine are numerically evaluated using Reynolds Averaged Navier-Stokes (RANS) and Large Eddy Simulation (LES). Early direct injection and mixture propagation were also explored, along with peculiaritis of dimethyl ether combustion modeling. The numerical models are validated using available experimental results of a partially premixed dimethyl ether jet flames and an optically accessible internal combustion engine with direct hydrogen injection. LES showed more predictive results in modeling both combustion and mixture propagation. The same models were applied to a full engine cycle of an RCCI engine with stratified reactivity, to gain phenomenological insight into the physical processes involved in stratified reactivity combustion. We showed that 3D and turbulence considerations had a great impact on simulation results, and the LES was able to capture the pressure oscillations typical for this type of combustion

The HTLV-1 gp21 fusion peptide inhibits antigen specific T-cell activation in-vitro and in mice.

The ability of the Lentivirus HIV-1 to inhibit T-cell activation by its gp41 fusion protein is well documented, yet limited data exists regarding other viral fusion proteins. HIV-1 utilizes membrane binding region of gp41 to inhibit T-cell receptor (TCR) complex activation. Here we examined whether this T-cell suppression strategy is unique to the HIV-1 gp41. We focused on T-cell modulation by the gp21 fusion peptide (FP) of the Human T-lymphotropic Virus 1 (HTLV-1), a Deltaretrovirus that like HIV infects CD4+ T-cells. Using mouse and human in-vitro T-cell models together with in-vivo T-cell hyper activation mouse model, we reveal that HTLV-1's FP inhibits T-cell activation and unlike the HIV FP, bypasses the TCR complex. HTLV FP inhibition induces a decrease in Th1 and an elevation in Th2 responses observed in mRNA, cytokine and transcription factor profiles. Administration of the HTLV FP in a T-cell hyper activation mouse model of multiple sclerosis alleviated symptoms and delayed disease onset. We further pinpointed the modulatory region within HTLV-1's FP to the same region previously identified as the HIV-1 FP active region, suggesting that through convergent evolution both viruses have obtained the ability to modulate T-cells using the same region of their fusion protein. Overall, our findings suggest that fusion protein based T-cell modulation may be a common viral trait

CD spectra analysis of the HTLV FP derived peptides by the CDNN secondary structure analysis program.

<p>CD spectra analysis of the HTLV FP derived peptides by the CDNN secondary structure analysis program.</p

Primary human T-cell activation is inhibited upon HTLV FP treatment.

<p>Human peripheral T lymphocytes were isolated from whole blood and activated using CD3 and CD28 antibodies, in the presence of HTLV FP or HTLV Scr at 10μM. Media was collected 48 hours following activation and secretion of IL2, IFN-γ and IL10 was measured by ELISA assay. HTLV FP treatment inhibits IL2 and IFN-γ and elevates IL10 secretion from primary human peripheral T-cells. n = 12. One-way ANOVA was used for statistical analysis. *<i>P</i><0.05; ***<i>P</i><0.001; ns, not significant.</p

Detection of the immune modulatory region within the HTLV FP.

<p>(<i>A</i>) Sequences of shorter peptides derived from the full length HTLV FP. (<i>B</i>) MOG_35-55–antigen specific T-cells were activated by APCs in the presence of HTLV FP derived peptides at 10μM and proliferative responses were assessed as described above. The HTLV FP and HTLV FP_5-13 inhibit T-cell proliferation with higher potency than HTLV FP_9-22 and HTLV FP_14-22. The data is presented as mean inhibition of proliferation. n = 12. (<i>C</i>) MOG_35-55–antigen specific T cells were activated by either (<i>i</i>) irradiated MOG_35-55 presenting APCs (black), (<i>ii</i>) antibodies against CD3 and CD28 (white), or (<i>iii</i>) PMA and Ionomycin (gray) in the presence of HTLV FP_5-13 and HIV FP_5-13 at 10μM. Proliferative responses were assessed as described above. The HTLV FP inhibits T-cell proliferation induced either through the TCR or downstream from the TCR with equal potencies. The data is presented as mean inhibition of proliferation. n = 12. (<i>D</i>) Secondary structures of HTLV FP derived peptides as revealed by CD spectroscopy. CD spectra were measured at 25μM in 5mM HEPES buffer containing 1% lyso-phosphatidylcholine (LPC). The HTLV FP and HTLV FP_5-13 exhibit a typical α-helical curve while the HTLV FP_9-22 and HTLV FP_14-22 exhibit a typical random coil. (<i>E</i>) MOG_35-55–antigen specific T-cells were activated by APCs in the presence of HTLV FP_5-13 D/L enantiomers at 10μM and proliferative responses were assessed as described above. Both HTLV FP_5-13 enantiomers inhibit T-cells proliferation with equal potencies. The data is presented as mean inhibition of proliferation. n = 12. One-way ANOVA was used for statistical analysis. *<i>P</i><0.05;<i>**P</i><0.01;***<i>P</i><0.001.</p

Inhibition of T-cell activation by the HTLV FP.

<p>(<i>A</i>) MOG_35-55–antigen specific T-cells were activated by irradiated MOG_35-55 presenting APCs, in the presence of several viral derived FPs at 10μM. The proliferative responses were assessed by H³-thymidine incorporation assay, and normalized to the proliferation of non-activated T-cells. The HTLV FP inhibits T-cell proliferation with the same potency as the HIV FP. The data is presented as mean inhibition of proliferation. n = 12. (<i>B</i>) MOG_35-55–antigen specific T cells were activated by either (<i>i</i>) irradiated MOG_35-55 presenting APCs (black), (<i>ii</i>) antibodies against CD3 and CD28 (white), or (<i>iii</i>) PMA and Ionomycin (gray) in the presence of the HTLV and HIV FPs at 10μM. Proliferation was assessed as described above. The HTLV FP inhibits T-cell proliferation induced either through the TCR or downstream from the TCR with equal potencies. The data is presented as mean inhibition of proliferation. n = 12. One-way ANOVA was used for statistical analysis. **<i>P</i><0.01;***<i>P</i><0.001.</p

Reduced T-bet expression and elevated Gata3 expression induced by the HTLV FP.

<p>(<i>A-B</i>) MOG_35-55–antigen specific T-cells were activated by irradiated MOG_35-55 presenting APCs, in the presence of the HTLV FP at 10μM. Total RNA was isolated 24 hours following activation and mRNA expression levels were determined by qRT-PCR. The values of each gene were normalized to Rpl13a as a housekeeping control. The HTLV FP reduces <i>TBX21</i> (T-bet) mRNA expression and elevates <i>GATA3</i> mRNA expression. The data is presented as arbitrary units. n≥3. (<i>C-H</i>) MOG_35-55–antigen specific T-cells were activated by irradiated MOG_35-55 presenting APCs, in the presence of either HTLV or HIV FP at 10μM. Samples were fixed in 4% PFA and stained with anti T-bet-APC and anti Gata3-FITC antibodies (<i>C-D</i>) 24, (<i>E-F</i>) 48 and (<i>G-H</i>) 72 hours following activation. Each time point is represented as cell count vs. APC or FITC fluorescence histogram. Analysis was performed using LSR-II flow cytometer (BD) and FlowJo cell analysis software (FlowJo, LLC). The HTLV FP reduces T-bet expression and elevates Gata3 expression. n = 3. One-way ANOVA was used for statistical analysis. ns, not significant; *<i>P</i><0.05<i>;** P<0</i>.<i>01</i>.</p

Modulation of Th1/Th2 gene expression and cytokine secretion from activated T-cells by the HTLV FP.

<p>MOG_35-55–antigen specific T-cells were activated by irradiated MOG_35-55 presenting APCs, in the presence of the HTLV FP at 10μM. (<i>A</i>) Total RNA was isolated 24 hours following activation and mRNA expression levels were determined by qRT-PCR. The values of each gene were normalized to Rpl13a as a housekeeping control. The HTLV FP reduces mRNA expression of Th1 specific genes (<i>IFNG</i>, <i>LTA</i>, and <i>STAT4</i>) and elevates mRNA expression of Th2 specific genes (<i>IL4</i> and <i>IL10</i>). The data is presented as arbitrary units. n≥3. (<i>B</i>) Media was collected 24 hours following activation and secretion of cytokines was measured by ELISA assay. IFN-γ secretion is inhibited, IL4 secretion is elevated and TNFα is not affected by the HTLV FP. n = 12. One-way ANOVA was used for statistical analysis. *<i>P</i><0.05; **<i>P</i><0.01<i>;***P</i><0.001.</p

Administration of HTLV FP alleviates MOG35-55-induced EAE.

<p>(<i>A-B</i>) MOG_35-55–antigen specific T-cells were activated by irradiated MOG_35-55 presenting APCs, in the presence of HTLV FP or HTLV Scr at 10μM. (<i>A</i>) The proliferative responses were assessed by H³-thymidine incorporation assay, and normalized to the proliferation of non-activated T-cells. The data is presented as mean inhibition of proliferation. n = 12. (<i>B</i>) IFN-γ was measured by ELISA assay. n = 12. (<i>C-D</i>) EAE was induced in C57BL/6 female mice that were treated with a single dose of HTLV FP, HTLV Scr or vehicle. Two indexes to measure clinical disease severity are displayed. (<i>C</i>) Direct clinical measurement of EAE phenotype in a 5-point scale with increased disease symptoms correlating with higher score value. The data is presented as mean EAE clinical score. (<i>D</i>) Mice were weighed the day before EAE induction, and the change (as a percentage) in weight was recorded. The data is presented as mean change from the initial weight. One-way ANOVA was used for statistical analysis. ^<b>a</b> HTLV FP ≠ Vehicle; ^<b>b</b> HTLV FP ≠ Vehicle & HTLV Scr; ^<b>c</b> HTLV FP ≠ HTLV Scr. ^<b>a,b,c</b><i>P</i><0.05. (<i>E</i>) Spleenocytes were harvested at 26 DPI and stimulated using MOG35-55. The proliferative responses were assessed by H³-thymidine incorporation assay and normalized to the proliferation of non-activated T-cells. The data is presented as counts per minute (CPM). n = 7. (<i>F-G</i>) Spleenocytes were harvested at 26 DPI and stimulated using MOG35-55. Media was collected 48 hours following activation and secretion of IFN-γ and IL4 was measured by ELISA assay. HTLV FP treatment inhibits IFN-γ and elevates IL4 secretion from MOG35-55-reactive T-cells. n>8. One-way ANOVA was used for statistical analysis. *<i>P</i><0.05;**<i>P</i><0.01;***<i>P</i><0.001; ns, not significant.</p