Peak Time Point Research Articles

Non-HIV Vaccine-Induced Immune Responses as Potential Baseline Immunogenicity Predictors of ALVAC-HIV and AIDSVAX B/E-Induced Immune Responses.

Identifying correlations between immune responses elicited via HIV and non-HIV vaccines could aid the search for correlates of HIV protection and increase statistical power in HIV vaccine-efficacy trial designs. An exploratory objective of the HVTN 097 phase 1b trial was to assess whether immune responses [focusing on those supported as correlates of risk (CoR) of HIV acquisition] induced via the RV144 pox-prime HIV vaccine regimen correlated with those induced via tetanus toxoid (TT) and/or hepatitis B virus (HBV) vaccines. We measured TT-specific and HBV-specific IgG-binding antibody responses and TT-specific and HBV-specific CD4+ T-cell responses at multiple time points in HVTN 097 participants, and we assessed their correlations at peak time points with HIV vaccine (ALVAC-HIV and AIDSVAX B/E)-induced responses. Four correlations were significant [false discovery rate-adjusted p-value (FDR) ≤ 0.2]. Three of these four were with IgG-binding antibody responses to TT measured one month after TT receipt, with the strongest and most significant correlation [rho = 0.368 (95% CI: 0.096, 0.588; p = 0.008; FDR = 0.137)] being with IgG-binding antibody responses to MN gp120 gDneg (B protein boost) measured two weeks after the second ALVAC-HIV and AIDSVAX B/E boost. The fourth significant correlation [(rho = 0.361; 95% CI: 0.049, 0.609; p = 0.021; FDR = 0.137)] was between CD4+ T-cell responses to a hepatitis B surface antigen peptide pool, measured 2 weeks after the third HBV vaccination, and IgG-binding antibody responses to gp70BCaseAV1V2 (B V1V2 immune correlate), measured two weeks after the second ALVAC-HIV and AIDSVAX B/E boost. These moderate correlations imply that either vaccine, TT or HBV, could potentially provide a moderately useful immunogenicity predictor for the ALVAC-HIV and AIDSVAX B/E HIV vaccine regimen.

Adjuvant effects of β-defensin on DNA vaccine OmpC against edwardsiellosis in flounder (Paralichthys olivaceus)

β-defensin of flounder plays an important role in immunomodulation by recruiting immune cells and has a potential vaccine adjuvant effect in addition to its bactericidal activity. In this study, adjuvant effects of β-defensin on DNA vaccine OmpC against edwardsiellosis in flounder (Paralichthys olivaceus) were investigated. The bicistronic eukaryotic expression plasmid pBudCE4.1 plasmid vector with two independent coding regions was selected to construct DNA vaccine of p-OmpC which express only the gene for the outer membrane protein of Edwardsiella tarda and the vaccine of p-OmpC-βdefensin which express both the outer membrane protein of the bacterium and β-defensin of flounder. In vitro and in vivo studies have shown that the constructed plasmids can be expressed in flounder embryonic cell lines and injection sites of muscles. After vaccination by intramuscular injection, both p-OmpC and p-OmpC-βdefensin groups showed significant upregulation of immune-response. Compared to the pBbudCE4.1 and the p-OmpC vaccinated groups, the p-OmpC-βdefensin vaccinated group showed significantly more cell aggregation at the injection site and intense immune response. The proportion of sIgM+ cells, as well as the CD4-1+ and CD4-2+ cells in both spleen and kidney was significantly higher in the p-OmpC-βdefensin vaccinated group at peak time point than in the control groups. The relative survival rate of the p-OmpC-βdefensin vaccine was 74.17%, which was significantly higher than that of the p-OmpC vaccinated group 48.33%. The results in this study determined that β-defensin enhances the responses in cellular and humoral immunity and evokes a high degree of protection against E. tarda, which is a promising candidate for vaccine adjuvant.

Port carbon emission peaking simulation and emission reduction pathway in China

In order to achieve the goal of “carbon peak before 2030 and carbon neutrality before 2060”, all industries are fully engaged in energy conservation and carbon reduction work. Carbon emissions from the transportation sector account for nearly 10% of China's carbon emissions[1]. Port is an extremely important part of China's transportation industry, and it is also one of the key areas of energy conservation and emission reduction in the transportation industry. It is very important to carry out research on the status evaluation of port carbon emissions, predict the future trend of carbon emissions, and explore the strategy and path of port carbon peak. In this study, the carbon emission scenario prediction model based on the detailed list of port equipment was established to simulate the port carbon emissions. It is also applied to a northern port in China to study and judge the peak value and time point of carbon emission in the future, and propose the path of carbon peak in the port.

Head-to-Head Comparison of Novel Vaccine Technologies Comes with a Minefield of Challenges.

Modern vaccine development is having a golden period, with a variety of novel subunit technologies being introduced into clinical development in recent years. This opens the opportunity to find the best platform to use for novel vaccine antigen candidates through head-to-head comparative studies. Seldom appreciated is, however, the fact that these different technologies often do not have the same optimal antigen dose ratio, prime-boost regime and peak timepoint for measuring immunity. Instead, the preclinical studies that make the basis for platform selection use standard protocols not optimized for individual vaccines and fail to make selection on an informed basis. Here, I discuss the opportunities we have to optimize vaccine platform technologies through a better understanding of vaccine priming kinetics, the optimal antigen dose and sampling time and location.

Relevant Studies on Cytokines Level and CAR-T Expansion Associated to CD7 CAR-T Therapy

Background In the past few years, CD7 became a highly reliable marker of CAR-T therapy treating T cell acute lymphocytic leukemia(T-ALL). In this case, better understanding of CD7 CAR-T therapy on cellular level became meaningful. Aim To figure out the correlation of cytokines level and CAR-T expansion to CRS and outcomes of CD7 CAR-T therapy. Methods Peripheral blood samples were collected from 70 patients treated with CD7 CAR-T from Dec. 2020 to Nov. 2022 in Hebei Yanda Ludaopei Hospital. There were 56 males and 14 females, from 2-year old to 60 with median age 19. Based on criteria of CRS and assessment results, 70 patients were split in different groups. According to CRS level, two groups were set as low (CRS0-1, 56 of 70) group and high group (CRS2-4, 14 of 70). Due to assessment results, 55 of 70 were classified as CR/CRi group, 15 of 70 were Non-CR group. 24 Cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-17F, IL-22,TNFα, TNFβ, IFN-γ, IL-2RA/sCD25, MIP-1α, MCP-1, GM-CSF, IL-15, REG3a, Elafin, ST-2, Granzyme B, TNFRI) were measure with microbeads with 2 lasers 4 colour Calibur flow cytometer. CAR-T cells, target cells(CD7+ cells), T cells subsets(CD3+, CD4+, CD8+, CD8+TCM, CD8+TEM, Treg) were tested with 3 lasers 8-10 colour Canto flow cytometer. All the cellular variations were calculated by comparing value of peak-time to value of D0 which was used as baseline. Time points day0, 4, 7, 11, 14, 21, 30 were monitored after CAR-T infusion. FCM data was analyzed with Diva software. Prism 8 was used for statistic analyse. Results According to patient characteristics (shown in table 1a), no matter under which criteria, patients in all 4 groups had similar median age and transfection efficiency. First of all, based on criteria of CRS level, patients were split into low group and high group(upper table 1a). CAR-T peak time was appeared significantly later(p=0.0274) in high group. Along with that, in high group, CD8+TEM(effect memory T) cells was amplified significantly more than that in low group at peak time point(p=0.0258). Clearance rates of target cells(CD7+) in low group were significantly higher(p=0.003). According to cytokine secretion levels, the level of IL-4(p=0.0183), IL-6(p<0.0001), IL-8(p=0.043), IL-10(p<0.0001), IL-22(p=0.0463), IFN-γ(p<0.0001), sCD25(<0.0001), MCP-1(p=0.0092), IL-15(p=0.0029), Granzyme B(p=0.0027) were significant higher in high group(as shown in figure 1). All median peak time of those 10 cytokines in high group were calculated, from day 10 up to day 15 after CAR-T infusion. According to the time gap between each of 10 cytokines with CAR-T expansion, MCP-1 had the largest time gap, which means MCP-1 reached peak time 9 days earlier than that of CAR-T cells. The median peak-time gap between cytokines level with CAR-T cells expansion was 6 days(showed in table 1b). Secondly, due to criteria of assessment results of CD7 CAR-T therapies, 70 patients were classified as CR/CRi group and Non-CR group (lower table 1a). CAR-T expansion, T cell subsets variation showed no statistical difference between two groups, and only target cells (CD7+) clearance rate was significantly higher in CR group. For all 24 cytokines measured in this study showed no significant differences between both groups. Conclusions Base on our data, IL-4, IL-6, IL-8, IL-10, IL-15, IL-22, IFN-γ, sCD25, MCP-1, and Granzyme B were correlated to severity of CRS after CD7 CAR-T therapy. Those 10 cytokines showed positive correlation to the level of CRS, and reached peak time earlier than that of CAR-T expansion, which means all these 10 cytokines could be used as indicators to predict occurrence and severity of CRS. Concurrence time of peak expansion of CD7 CAR-T cells, and amplification of CD8+TEM cells were significantly related to CRS. For all the patients who reached complete remission or CRi, CD7+ clearance rates were higher with statistical significance. Our results proved potential indicators for CRS prediction and prognosis.

Oxytocin prevents dysregulation of the acute stress response and glucocorticoid-induced oxidative stress in chronically isolated prairie voles

Chronic social isolation can lead to dysregulation of many physiological and psychological processes, including the ability to respond to acute stressors. Previous work in our lab reported that six weeks of social isolation in prairie voles (Microtus ochrogaster) caused increased glucocorticoid levels, oxidative damage, telomere degradation and anhedonia, and that oxytocin treatment prevented all of these changes. Following these results, we investigated how chronic social isolation with and without oxytocin treatment affected glucocorticoid (CORT) and oxidative stress responses to an acute stressor, a 5-min resident-intruder (R-I) test at the end of the social isolation period. To investigate the effect of a brief acute stressor on CORT and oxidative stress, baseline blood samples were collected following six weeks of social isolation, 24-hrs before the R-I test. Two more blood samples were collected 15-min after the end of the R-I test, and again 25-min later to measure peak and recovery responses, respectively. Isolated animals had higher baseline, peak, recovery, and integrated levels of CORT and reactive oxygen metabolites (ROMs, a measure of oxidative stress), compared to animals that did not experience isolation. Importantly, oxytocin treatment throughout the isolation period prevented these elevations in CORT and ROMs. No significant changes were observed in total antioxidant capacity (TAC). Levels of CORT and ROMs at the peak and recovery time points were positively correlated. These data show that acute stress in chronically isolated prairie voles, then, is associated with increased glucocorticoid-induced oxidative stress (GiOS), and that oxytocin mitigates isolation-induced dysregulation of glucocorticoid and oxidative stress acute stress responses.

Intrinsic growth rate and cellobiohydrolase activity underlie the phylogenetic signal to fungal decomposer succession

During litter decay, different fungal decomposer genera reach their highest relative abundance at different times. We tested the long-standinghypothesis that this “peak decay stage” of fungi is related to the activity of their fungal extracellular enzymes that break down various plant biopolymers and related as well to the growth rate of fungi. Using 50 decomposer fungal species, spanning a range of peak decay stages, we measured (1) the activity of four polysaccharidases and two oxidases generated by each species, and (2) fungal species’ growth rates. We found that the activity of cellobiohydrolase and growth rate were negatively correlated with peak time point for filamentous fungi; fungi peaking early had greatest cellobiohydrolase activity and fastest growth. No relationships were found between peak decay stage and enzymes or growth for yeasts. These data suggest growth and resource use are important factors shaping succession during decay by the main fungal decomposers, but as-yetuninvestigated traits may explain the remainder of the variation in succession.

The rectal mucosal immune environment and HIV susceptibility among young men who have sex with men.

Young men who have sex with men (YMSM) represent a particularly high-risk group for HIV acquisition in the US, despite similarly reported rates of sexual activity as older, adult MSM (AMSM). Increased rates of HIV infection among YMSM compared to AMSM could be partially attributable to differences within the rectal mucosal (RM) immune environment associated with earlier sexual debut and less lifetime exposure to receptive anal intercourse. Using an ex vivo explant HIV challenge model, we found that rectal tissues from YMSM supported higher levels of p24 at peak viral replication timepoints compared to AMSM. Among YMSM, the RM was characterized by increased CD4+ T cell proliferation, as well as lower frequencies of tissue resident CD8+ T cells and pro-inflammatory cytokine producing CD4+ and CD8+ T cells. In addition, the microbiome composition of YMSM was enriched for anaerobic taxa that have previously been associated with HIV acquisition risk, including Prevotella, Peptostreptococcus, and Peptoniphilus. These distinct immunologic and microbiome characteristics were found to be associated with higher HIV replication following ex vivo challenge of rectal explants, suggesting the RM microenvironment of YMSM may be uniquely conducive to HIV infection.

Association between microRNAs 10b/21/34a and acute toxicity in glioblastoma patients treated with radiotherapy and temozolomide

A personalized approach to chemoradiation is important in reducing its potential side effects and identifying a group of patients prone to toxicity. MicroRNAs have been shown to have a predictive potential for radiotoxicity. The goal of the study was to test if levels of miRNA in peripheral blood mononuclear cells of glioblastoma patients are associated with toxicity and to identify the peak time point for toxicity. MicroRNA-10b/21/34a levels were measured in 43 patients with and without toxicity, at baseline, at the 15th, and at the 30th fraction by Real-Time quantitative Polymerase Chain Reaction. MicroRNA-10b/21 levels increased with toxicity grade (p = 0.014; p = 0.013); miR-21/34a levels were significantly different between patients with and without toxicity at the 15th fraction (p = 0.030; p = 0.045), while miR-34a levels significantly changed during treatment (p < 0.001). All three miRNAs showed a significantly high positive correlation with one another. MiR-34a might be considered as a predictive factor for toxicity due to its changes during treatment, and differences between the groups with and without toxicity; miR-10b might be used to predict toxicity; miR-10b/21 might be used for predicting the grade of toxicity in GB patients.

Maternal immune activation and dietary soy isoflavone supplementation influence pig immune function but not muscle fiber formation.

The goals of this study were to determine the impact of maternal PRRSV infection on offspring muscle and immune development and the potential of dietary soy isoflavones to mitigate those effects. Thirteen first-parity gilts ("gilts") were randomly allotted into one of three treatments: not infected and fed a diet devoid of isoflavones (CON), infected with porcine reproductive and respiratory syndrome virus (PRRSV) and fed the control diet (POS) or that supplemented with 1,500 mg/kg soy-derived isoflavones (ISF). Gilts were inoculated with PRRSV intranasally on gestational day (GD) 70. After farrowing (GD 114 ± 2), 1-2 offspring ("pigs") closest to the average litter weight were selected either at birth (3 ± 2 d of age) or weaning (21 ± 2 d of age) to determine body, muscle, and organ weights as well as muscle cell number and size. Four weaned pigs of average body weight within each litter were selected for postnatal immune challenge. At PND 52, pigs were injected with 5 µg/kg BW lipopolysaccharide (LPS) intraperitoneally. Serum was collected at 0, 4, and 8 h following LPS administration to analyze tumor necrosis factor alpha (TNF-α). At PND 59, pigs were administered a novel vaccine to elicit an adaptive immune response. At PND 59, 66, and 73, peripheral blood mononuclear cells were isolated and T-cell populations determined by flow cytometry. Both POS and ISF pigs exhibited persistent PRRSV infections throughout the study (PND 1-73). At PND 3, whole body, muscle, and organ weights were not different (P > 0.22) between groups, with the exception of relative liver weight, which was increased (P < 0.05) in POS compared with CON pigs. At PND 21, ISF pigs had reduced (P ≤ 0.05) whole body and muscle weights, but greater (P < 0.05) kidney weight compared with CON, and greater (P < 0.05) relative liver weight compared with CON and POS. Muscle fiber number and size were not different (P > 0.39) between groups at birth or weaning. After LPS administration, TNF-α was greatest in ISF pigs (P < 0.05) at both 0 and 8 h post-challenge. At the peak time-point of 4 h post-challenge, ISF pigs had the greatest concentration of TNF-α and CON pigs had the lowest, with POS pigs being intermediate (P = 0.01). After vaccination, ISF offspring had shifts in T-cell populations indicating an impaired immune response. These data indicate that maternal PRRSV infection may impact offspring organ growth and immune function, particularly when the dam is supplemented with isoflavones.

A model combining pretreatment MRI radiomic features and tumor-infiltrating lymphocytes to predict response to neoadjuvant systemic therapy in triple-negative breast cancer

PurposeWe aimed to develop a predictive model based on pretreatment MRI radiomic features (MRIRF) and tumor-infiltrating lymphocyte (TIL) levels, an established prognostic marker, to improve the accuracy of predicting pathologic complete response (pCR) to neoadjuvant systemic therapy (NAST) in triple-negative breast cancer (TNBC) patients. MethodsThis Institutional Review Board (IRB) approved retrospective study included a preliminary set of 80 women with biopsy-proven TNBC who underwent NAST, pretreatment dynamic contrast enhanced MRI, and biopsy-based pathologic assessment of TIL. A threshold of ≥ 20% was used to define high TIL. Patients were classified into pCR and non-pCR based on pathologic evaluation of post-NAST surgical specimens. pCR was defined as the absence of invasive carcinoma in the surgical specimen. Segmentation and MRIRF extraction were done using a Food and Drug Administration (FDA) approved software QuantX. The top five features were combined into a single MRIRF signature value. ResultsOf 145 extracted MRIRF, 38 were significantly correlated with pCR. Five nonredundant imaging features were identified: volume, uniformity, peak timepoint variance, homogeneity, and variance. The accuracy of the MRIRF model, P = .001, 72.7% positive predictive value (PPV), 72.0% negative predictive value (NPV), was similar to the TIL model (P = .038, 65.5% PPV, 72.6% NPV). When MRIRF and TIL models were combined, we observed improved prognostic accuracy (P < .001, 90.9% PPV, 81.4% NPV). The models area under the receiver operating characteristic curve (AUC) was 0.632 (TIL), 0.712 (MRIRF) and 0.752 (TIL + MRIRF). ConclusionA predictive model combining pretreatment MRI radiomic features with TIL level on pretreatment core biopsy improved accuracy in predicting pCR to NAST in TNBC patients.

A Phase I Study of CD19 Chimeric Antigen Receptor-T Cells Generated By the PiggyBac Transposon Vector for Acute Lymphoblastic Leukemia

Introduction: Chimeric antigen receptor-modified T cells targeting CD19 (CD19.CAR-T cells) have shown clinical success in patients with hematological malignancies. Despite the encouraging results obtained with this novel therapy, a major concern to its global spread, particularly in developing countries, is its high cost. We developed a method of non-viral gene transfer using piggyBac transposon to reduce the cost of CAR-T therapy. In preclinical study, the median number and transduction efficiency of CAR-T cells obtained from 2x10 7 PBMC in 9 donors were 1.0x10 8 (range, 0.58-1.8x10 8) and 51% (range, 29-73%), respectively. The major subset of CAR-T cells was phenotypically CD8+CD45RA+CCR7+, closely related T-memory stem cells. Ex vivo, CD19.CAR-T cells showed cytotoxic effect on CD19 positive tumor cell lines. In NSG mice model, CD19.CAR-T cells successfully inhibit tumor growth. CAR gene integration sites were determined by inverse polymerase chain reaction and subsequent next-generation sequencing using MiSeq and equally distributed throughout the genome without preference for specific sites. The pre-clinical testing in mouse demonstrated safe toxicity profile at the 50 times dose of CD19.CAR-T cells. We started a human clinical trial to define feasibility, toxicity, maximum tolerated dose and clinical response of CD19.CAR-T cells (jRCTa040190099).Methods: We report the results of cohort 1 of the study in which the safety and efficacy of autologous CD19.CAR-T in patients with relapsed or refractory B-precursor acute lymphoblastic leukemia were evaluated. We engineered autologous T cells via the piggyBac transposon system with CD19.CAR-expression transposon vector and piggyBac transposase-expression vector to express CD19.CAR incorporating CD28 costimulatory domain. We designed this phase I trial using a modified 3 + 3 design to enroll 3-12 patients with relapsed or refractory acute lymphoblastic leukemia in both children and adults. In this study, patients in cohorts 1 (16-60 years old) and 2 (1-15 years old) receive 1 × 10 5 CAR-transduced T cells per kg. Patients in cohorts 3 and 4 (both 1-60 years old) receive 3 × 10 5 and 1 × 10 6 CAR-transduced T cells per kg, respectively. All patients receive 25mg/m 2/d of fludarabine and 250mg/m 2/d of cyclophosphamide for 3 days followed by a single infusion of CAR-T cells.Results: Three patients were enrolled in cohort 1 and infused with 1 × 10 5 CAR-transduced T cells per kg. All patients had previously undergone allogeneic hematopoietic stem cell transplantation. All patients had achieved a hematological complete response with salvage treatment before CAR-T therapy. None of the patients had dose-limiting toxicities (DLT) defined as nonhematological toxicities above grade 4 or cytokine release syndrome (CRS) above grade 4 or graft versus host disease (GVHD) above 4, or grade 3 nonhematological toxicities and GVHD not improved to grade 2 within 4 weeks after CAR-T infusion. There was no occurrence of non-hematological adverse events above grade 3. CRS was observed in one patient (grade 1) who also developed headache due to infiltration of CAR-T cells into the spinal fluid. In two patients, B cell aplasia lasted 2 and 9 months, respectively. Elevation of serum cytokine levels was observed in all patients and the peak time point was 7-21 days after CAR-T cell infusion.Conclusions: CD19.CAR-T cell infusion produced by the piggyBac transposon gene engineering system was safe in cohort 1 of our study. As no patients had DLT in cohort 1, we are enrolling the patients in further cohorts. DisclosuresMurata: MSD: Honoraria; Kyowa Kirin: Honoraria; Sumitomo Dainippon Pharma: Honoraria; FUJIFILM: Honoraria; Toyama Chemical: Honoraria; Novartis: Honoraria; JCR Pharmaceutical: Honoraria; Astellas: Honoraria; Miyarisan Pharmaceutical: Honoraria; Asahi Kasei: Honoraria; GlaxoSmithKline: Honoraria; Celgene: Honoraria; Otsuka Pharmaceutical: Honoraria.

Comprehensive Secretome Profiling Elucidates Novel Disease Biology and Identifies Pre-Infusion Candidate Biomarkers to Predict the Development of Severe Cytokine Release Syndrome in Pediatric Patients Receiving CART19

Comprehensive Secretome Profiling Elucidates Novel Disease Biology and Identifies Pre-Infusion Candidate Biomarkers to Predict the Development of Severe Cytokine Release Syndrome in Pediatric Patients Receiving CART19

Age-dependent immune response in pigs against foot-and-mouth disease virus in vitro.

Foot-and-mouth disease, one of the most contagious diseases in cloven-hoofed animals, causes significant economic losses. The pathogenesis of foot-and-mouth disease virus (FMDV) infection is known to differ with age of the animals. In this study, we aimed to reveal the difference in immunological response in the initial stage of FMDV infection between piglets and adult pigs. Peripheral blood mononuclear cells (PBMCs) were isolated from 3 piglets (8 weeks old) and 3 pigs (35 weeks old) that were not vaccinated against FMDV. O-type FMDV (2 × 102 median tissue culture infectious dose) was inoculated into porcine PBMCs and the cells were incubated at 37.0°C under 5% CO2 for various time periods (0, 1, 3, 6, 12, 24, and 48 h). The total RNA was obtained from the FMDV-inoculated PBMCs after each time point, and the virus titer was investigated in these RNA samples. Furthermore, dynamics of mRNA expression of the six tested cytokines (interferon [IFN]-α, IFN-γ, ‎interleukin [IL]-6, IL-8, IL-10, and tumor necrosis factor [TNF]-α) in FMDV-inoculated porcine PBMCs were evaluated by time-series analysis to determine the differences, if any, based on the age of the pigs. The PBMCs of piglets contained the highest quantity of FMDV mRNA at 6 hours post-inoculation (hpi), and the PBMCs of pigs had the highest quantity of FMDV mRNA at 3 hpi. The mean cycle threshold-value in the PBMCs steadily decreased after the peak time point in the piglets and pigs (6 and 3 hpi, respectively). The dynamics of mRNA expression of all cytokines except TNF-α showed age-dependent differences in FMDV-inoculated PBMCs. The mRNA expression of most cytokines was more pronounced in the piglets than in the pigs, implying that the immune response against FMDV showed an age-dependent difference in pigs. In conclusion, within 48 hpi, the 8-week-old piglets responded more rapidly and were more sensitive to FMDV infection than the 35-week-old pigs, which could be associated with the difference in the pathogenesis of FMDV infection among the pigs. These results provide valuable insights into the mechanisms underlying the age-dependent differences in immune response in pigs against FMDV infection.

One hundred kilojoules of energy storage in water wire electric explosion deposition energy study

In this experiment, the electro-explosive deposition energy in water of aluminum-magnesium welding wire model ER5356 at 100 kJ capacitive storage energy was investigated. The loop current and the load discharge voltage during the wire electrical explosion were measured using a self-integrating Roche coil and a capacitive voltage divider, respectively. The physical process of electrical explosion and the energy deposition process were delineated by the measured loop currents and load voltages. The current waveform and load voltage of the electric explosion in water of 1.2 mm-3.0 mm diameter Al-Mg wire at 100 kJ stored energy were measured; the changes of load resistance value, load power and deposition energy of the wire loaded with electric explosion were calculated. The results show that the peak circuit current and peak time point decrease and then increase with increasing diameter, and the minimum value is achieved at 1.6 mm wire diameter; the load voltage and load resistance values gradually decrease with increasing diameter; the load power and total deposited energy of discharge achieve the maximum value at 2.0 mm diameter. At 100 kJ energy storage, there is an optimal range between 1.6 mm and 2.4 mm wire diameter.

L-cell Arntl is required for rhythmic glucagon-like peptide-1 secretion and maintenance of intestinal homeostasis

ObjectiveRecent studies using whole-body clock-disrupted animals identified a disruption in the circadian rhythm of the intestinal L-cell incretin hormone, glucagon-like peptide-1 (GLP-1). Although GLP-1 plays an essential role in metabolism through enhancement of both glucose-stimulated insulin secretion and satiety, recent evidence has also demonstrated its importance in regulating intestinal and microbial homeostasis. Therefore, using in vivo and in vitro models, this study assessed the role of the core circadian clock gene Arntl in the regulation of time-dependent GLP-1 secretion and its impact on the intestinal environment. MethodsOral glucose tolerance tests were conducted at zeitgeber time 2 and 14 in control and inducible Gcg-Arntl knockout (KO) mice. Colonic intraepithelial lymphocytes were isolated, mucosal gene expression analysis was conducted, and 16S rRNA gene sequencing of colonic feces as well as analysis of microbial metabolites were performed. Time-dependent GLP-1 secretion and transcriptomic analysis were conducted in murine (m) GLUTag L-cells following siRNA-mediated knockdown of Arntl. ResultsGcg-Arntl KO mice displayed disrupted rhythmic release of GLP-1 associated with reduced secretion at the established peak time point. Analysis of the intestinal environment in KO mice revealed a decreased proportion of CD4+ intraepithelial lymphocytes in association with increased proinflammatory cytokine gene expression and increased colonic weight. Moreover, increased Actinobacteria within the colonic microbiome was found following L-cell Arntl disruption, as well as reductions in the microbial products, short chain fatty acids, and bile acids. Finally, siRNA-mediated knockdown of Arntl in mGLUTag L-cells resulted in both impaired time-dependent GLP-1 secretion and the disruption of pathways related to key cellular processes. ConclusionsThese data establish, for the first time, the essential role of Arntl in the intestinal L-cell in regulating time-dependent GLP-1 secretion. Furthermore, this study revealed the integral role of L-cell Arntl in mediating the intestinal environment, which ultimately may provide novel insight into the development of therapeutics for the treatment of intestinal and metabolic disorders.

PRMT5 Promotes Cyclin E1 and Cell Cycle Progression in CD4 Th1 Cells and Correlates With EAE Severity.

Multiple Sclerosis (MS) is a debilitating central nervous system disorder associated with inflammatory T cells. Activation and expansion of inflammatory T cells is thought to be behind MS relapses and influence disease severity. Protein arginine N-methyltransferase 5 (PRMT5) is a T cell activation-induced enzyme that symmetrically dimethylates proteins and promotes T cell proliferation. However, the mechanism behind PRMT5-mediated control of T cell proliferation and whether PRMT5 contributes to diseases severity is unclear. Here, we evaluated the role of PRMT5 on cyclin/cdk pairs and cell cycle progression, as well as PRMT5’s link to disease severity in an animal model of relapsing-remitting MS. Treatment of T helper 1 (mTh1) cells with the selective PRMT5 inhibitor, HLCL65, arrested activation-induced T cell proliferation at the G1 stage of the cell cycle, suggesting PRMT5 promotes cell cycle progression in CD4+ T cells. The Cyclin E1/Cdk2 pair promoting G1/S progression was also decreased after PRMT5 inhibition, as was the phosphorylation of retinoblastoma. In the SJL mouse relapsing-remitting model of MS, the highest PRMT5 expression in central nervous system-infiltrating cells corresponded to peak and relapse timepoints. PRMT5 expression also positively correlated with increasing CD4 Th cell composition, disease severity and Cyclin E1 expression. These data indicate that PRMT5 promotes G1/S cell cycle progression and suggest that this effect influences disease severity and/or progression in the animal model of MS. Modulating PRMT5 levels may be useful for controlling T cell expansion in T cell-mediated diseases including MS.

260-OR: The L-Cell Clock Is Required for Circadian GLP-1 Secretion and Maintenance of Intestinal Homeostasis

Glucagon-like peptide-1 (GLP-1) is an incretin hormone released by the intestinal L cell in response to nutrient intake. Recent studies identified a circadian rhythm in GLP-1 secretion that is disrupted in whole-body Bmal1 KO mice. However, the extent to which the L cell clock regulates circadian GLP-1 secretion and overall diurnal metabolic and intestinal homeostasis is unknown. Gcg-creERT2/+;Bmal1flox/flox KO mice with a disrupted clock in L cells, alpha cells and some CNS cells were thus generated. M/F- and age-matched KO mice had normal body weight and 24hr food intake patterns vs. all genetic and chemical controls. Upon oral glucose gavage at the known peak and trough timepoints of GLP-1 release, KO animals lost the rhythmic GLP-1 secretory pattern in association with impaired secretion (p&amp;lt;0.001) at the peak timepoint. Interestingly, the reduced GLP-1 secretion did not translate to decreased insulin or hyperglycemia, perhaps due to decreased glucagon levels (p&amp;lt;0.05). However, L cell Bmal1 KO caused increases in colonic weight and interleukin-6 (p&amp;lt;0.05) and interferon gamma (p&amp;lt;0.05) expression, suggesting a pro-inflammatory state. In line with findings that altered intestinal epithelial cell function decreases the frequency of intraepithelial CD4+ T cells, KO animals had reduced proportions of these immune cells (p&amp;lt;0.05). Furthermore, 16S rRNA gene analysis revealed increased actinobacteria in the colon, consistent with data from GLP-1 receptor KO mice. KO mice also had decreased levels of cecal bile acids (p&amp;lt;0.05-0.001) and short-chain fatty acids (p&amp;lt;0.05) suggestive of a functional impairment in the KO microbiome. Finally, siRNA-mediated knockdown of Bmal1 in the murine GLUTag L cell line also resulted in impaired rhythmic GLP-1 secretion (p&amp;lt;0.001). Together, these data establish the L cell clock as an essential regulator of circadian GLP-1 secretion which is, then, critical to the maintenance of overall intestinal homeostasis. Disclosure S. E. Martchenko: None. A. Martchenko: None. A. Biancolin: None. P. L. Brubaker: None. Funding Canadian Institutes of Health Research (PJT-15308)

Vaccine-elicited T cells adopt an early memory-like transcriptional phenotype distinct from infection-elicited T cells

Abstract Protection from pathogens relies on both humoral (antibody-mediated) and cellular (T cell-mediated) responses. While infections robustly elicit both of these types of immunity, currently approved vaccine adjuvants largely fail to induce significant T cell responses. However, recent work by our lab and others suggests that the mechanisms governing vaccine-elicited T cells (Tvac) may be substantially different than those governing infection-elicited T cells (Tinf). We have recently demonstrated that optimal subunit vaccine-elicited T cell responses rely on different cytokine signals (IL-27 and 15) and metabolic function (oxidative phosphorylation vs. aerobic glycolysis) leading to phenotypically and functionally different outcomes (memory vs. effector). Our goal was to investigate the transcriptional programming that drives Tvac formation and function from immunization to memory timepoints. Using a single-cell RNA-seq approach along with functional assays, we compared Tvac and Tinf at various time points post-immunization or infectious challenge. Our data indicate that the gene expression of Tvac markedly diverges from Tinf at early and peak time points. Remarkably, while capable of being highly proliferative, Tvac adopt a distinct transcriptional phenotype with similarities to recently identified self-renewing stem cell memory T cells, such as TCF1 expression. This transcriptional program likely enables the persistence of Tvac in substantially greater abundance at memory time points conferring overall greater protection. Taken together, our data have identified a novel transcriptional program that supports a robust vaccine-elicited T cell response with implications for future therapeutic vaccine design.

CD8αα expression increases during demyelinating disease progression

Abstract Experimental autoimmune encephalomyelitis (EAE) is a well-described mouse model for multiple sclerosis (MS), a demyelinating autoimmune disorder of the central nervous system (CNS). While the role of CD4 T cells is well established, other immune cell types, particularly CD8 T cells, are key contributors to disease pathogenesis. Our study aims to categorize the functional contributions of CD8 T cell subsets to EAE kinetics and disease outcomes. The use of myelin oligodendrocyte glycoprotein (MOG) 35–55 peptide alongside an adjuvant to inoculate mice, provides a reproducible disease model. Demyelinating disease severity was analyzed at defined timepoints including, onset (d10–14), peak (d17–21), early chronic (d28–35) and late chronic (d80–120) stages. The total number of CD3+ T cells in the CNS remained consistent during EAE. However, it goes from a CD4 T cell dominated response at peak EAE to an equalized 1:1 ratio of CD4 and CD8 T cells at late chronic stage. Furthermore, differentiation of CD8 T cell subsets based on CD8α and CD8β protein expression revealed that CD8αα T cells numbers increase to comprise ~40% of the CD8+ population at late chronic EAE. In addition, CD8βLO cells become a prominent subset during late chronic stage, with a concomitant decrease in CD8αβHI T cells. Moreover, CD8αα T cells are activated and exhibit a central memory phenotype along with downregulation of exhaustion markers by FACS analysis, and yet have significantly reduced cytokine expression. These results are consistent with single cell RNA sequencing data obtained at the peak and late chronic time points. Thus, CD8αα T cells constitute a phenotypically distinct class of CD8 T cell subsets, with a blunted effector role in demyelinating disease outcome.