Faculty Bibliography

Unconventional T lymphocyte populations are emerging as important regulators of tumor immunity. Despite this, the role of TCRαβ+CD4-CD8-NK1.1- innate αβ T-cells (iαβTs) in pancreatic ductal adenocarcinoma (PDA) has not been explored. We found that iαβTs represent ~10% of T-lymphocytes infiltrating PDA in mice and humans. Intra-tumoral iαβTs express a distinct TCR-repertoire and profoundly immunogenic phenotype compared to their peripheral counterparts and conventional lymphocytes. iαβTs comprised ~75% of the total intra-tumoral IL-17+ cells. Moreover, iαβT cell adoptive transfer is protective in both murine models of PDA and human organotypic systems. We show iαβT cells induce a CCR5-dependent immunogenic macrophage reprogramming, thereby enabling marked CD4+ and CD8+ T cell expansion/activation and tumor protection. Collectively, iαβTs govern fundamental intra-tumoral crosstalk between innate and adaptive immune populations and are attractive therapeutic targets.

Animals have evolved specialized neural circuits to defend themselves from pain- and injury-causing stimuli. Using a combination of optical, behavioral and genetic approaches in the larval zebrafish, we describe a novel role for hypothalamic oxytocin (OXT) neurons in the processing of noxious stimuli. In vivo imaging revealed that a large and distributed fraction of zebrafish OXT neurons respond strongly to noxious inputs, including the activation of damage-sensing TRPA1 receptors. OXT population activity reflects the sensorimotor transformation of the noxious stimulus, with some neurons encoding sensory information and others correlating more strongly with large-angle swims. Notably, OXT neuron activation is sufficient to generate this defensive behavior via the recruitment of brainstem premotor targets, whereas ablation of OXT neurons or loss of the peptide attenuates behavioral responses to TRPA1 activation. These data highlight a crucial role for OXT neurons in the generation of appropriate defensive responses to noxious input.

Atherosclerosis is a progressive vascular disease triggered by interplay between abnormal shear stress and endothelial lipid retention. A combination of these and, potentially, other factors leads to a chronic inflammatory response in the vessel wall, which is thought to be responsible for disease progression characterized by a buildup of atherosclerotic plaques. Yet molecular events responsible for maintenance of plaque inflammation and plaque growth have not been fully defined. Here we show that endothelial TGFβ signaling is one of the primary drivers of atherosclerosis-associated vascular inflammation. Inhibition of endothelial TGFβ signaling in hyperlipidemic mice reduces vessel wall inflammation and vascular permeability and leads to arrest of disease progression and regression of established lesions. These pro-inflammatory effects of endothelial TGFβ signaling are in stark contrast with its effects in other cell types and identify it as an important driver of atherosclerotic plaque growth and show the potential of cell-type specific therapeutic intervention aimed at control of this disease.

PURPOSE/OBJECTIVE:To identify and characterize amyloid-like substance (ALS) in human and mouse oocytes and preimplantation embryos. METHODS:An experimental prospective pilot study. A total of 252 mouse oocytes and preimplantation embryos and 50 immature and in vitro matured human oocytes and parthenogenetic human embryos, from 11 consenting fertility patients, ages 18-45. Fluorescence intensity from immunofluorescent staining and data from confocal microscopy were quantified. Data were compared by one-way analysis of variance, with the least square-MEANS post-test, Pearson correlation coefficients (r), and bivariate analyses (t tests). ALS morphology was verified using transmission electron microscopy. RESULTS:Immunostaining for ALS appears throughout the zona pellucida, as well as in the cytoplasm and nucleus of mouse and human oocytes, polar bodies, and parthenogenetic embryos, and mouse preimplantation embryos. In mouse, 2-cell embryos exhibited the highest level of ALS (69000187.4 ± 6733098.07). Electron microscopy confirmed the presence of ALS. In humans, fresh germinal vesicle stage oocytes exhibited the highest level of ALS (4164.74088 ± 1573.46) followed by metaphase I and II stages (p = 0.008). There was a significant negative association between levels of ALS and patient body mass index, number of days of ovarian stimulation, dose of gonadotropin used, time between retrieval and fixation, and time after the hCG trigger. Significantly higher levels of ALS were found in patients with AMH between 1 and 3 ng/ml compared to < 1 ng/ml. CONCLUSION/CONCLUSIONS:We demonstrate for the first time the presence, distribution, and change in ALS throughout some stages of mouse and human oocyte maturation and embryonic development. We also determine associations between ALS in human oocytes with clinical characteristics.

Objective: During preimplantation embryo development, the telomere aging clock resets and 2-cell genes establish totipotency. Like most long -lived cells, oocytes have short telomeres, but telomeres elongate markedly during early embryo development, even in the absence of telomerase (1). Recent studies report that the retrotransposon, LINE-1, is activated after fertilization, when telomeres elongate. Retrotransposons protect chromosome ends in many species, so we hypothesized that inhibiting retrotransposition of LINE-1 during mouse early embryos would disrupt telomere reprogramming in mouse preimplantation embryos. We also evaluated the effect of LINE-1 on the 2-cell, totipotency genes.
Design(s): Laboratory experiment
Material(s) and Method(s): Sixty thawed mouse zygotes (B6C3F1 X B6D2F1) (Embryotech Laboratories) were grouped (15 zygotes/group) and cultured in Global medium containing 0.4% BSA with or without 1muM of the reverse transcriptase inhibitor, AZT, for up to 15 (mid 2-cell stage) or 24 hours (late 2-cell stage). DNA and mRNA were isolated from embryos. Gene expression was measured by RT-qPCR, and LINE-1 copy number and telomere length were measured by qPCR. Data (Mean+/-SEM) was analized with GraphPad Prism 8 software by one-way ANOVA Tukey's multiple comparisons test.
Result(s): As expected, AZT inhibited LINE-1 copy number in late 2-cell embryos compared to controls (1.000+/-0.113 vs 1.942+/-0.089, P<0.0001). Telomeres were longer in late 2-cell (1.563+/-0.107) than mid 2-cell embryos (1.128+/-0.059) (P=0.001), consistent with prior observations of progressive telomere elongation during early development(1). AZT blocked telomere elongation between mid- and late-2 cell stages stage (1.152+/-0.039 vs 1.000+/-0.107, P=0.1994). Telomere length of late 2-cell embryos exposed to AZT was shorter than untreated controls (1.000+/-0.107 vs 1.563+/-0.107, P=0.0002). Transcripts of LINE-1, as well as the 2-cell genes, DUX and Zscan4, were highly expressed in mid- 2-cell compared to late 2-cell embryos (P <0.0001), regardless of inhibition of LINE-1. This suggests that 2-cell genes are activated at synthesis phase of the 2-cell stage. Moreover, AZT decreased expression of DUX, Zscan4d and LINE-1 in mid- 2-cell embryos (0.312+/-0.021, 0.727+/-0.055 and 1.266+/-0.066 respectively) compared to control embryos (1.913+/-0.197, 1.912+/-0.202 and 1.913+0.216 respectively, P<0.001).
Conclusion(s): The retrotransposon, LINE-1, regulates telomere lengthening during preimplantation embryo development- inhibition of LINE-1 synthesis by AZT blocks telomere elongation. AZT also inhibits the 2-cell genes, DUX and Zscan4d, suggesting that LINE-1 is essential not only for telomere reprogramming but also for the establishment of totipotency during early development. References: 1. Liu L, Bailey SM, Okuka M, Munoz P, Li C, Zhou L, Wu C, Czerwiec E, Sandler L, Seyfang A, Blasco MA, Keefe DL. Telomere lengthening early in development. Nat Cell Biol. 2007 Dec; 9(12):1436-41.
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Objective: Clinical guidelines on the optimal duration of controlled ovarian stimulation and ideal follicle size were developed for fresh embryo transfer cycles. Whether these apply to freeze all cycles remain unclear. We evaluated the impact of lead follicle size and duration of stimulation on the probability of euploid embryos in women undergoing IVF/PGT-A Design: Cross-sectional study
Material(s) and Method(s): Data from 721 patients undergoing at least two cycles of COS for IVF with preimplantation genetic testing for aneuploidy (PGT-A) via Next Generation Sequencing (NGS) (1859 cycles). Mixed-effect logistic regression, which can account for correlations among repeated outcomes within sample patients, was used to evaluate the association between independent variables and probability of achieving euploid embryos. We first conducted a mixed-effect logistic regression in a univariate manner. All variables then were evaluated in a multivariate model to control for confounding effects. Significant variables to p<0.05 were retained in the final model. p-values <0.05 were considered significant. Statistical analyses were performed using "nlme" and "lme4" package from R project. Results are reported as odds ratios (OR) with 95% confidence intervals (CI).
Result(s): Increasing sum (1.034 [1.022 1.046]/p<0.001) and mean diameter (1.129 [1.046 1.219] p=0.002) of the 5 largest follicles increased the probability of forming euploid embryos. Increasing days of stimulation showed a non-significant trend toward lower chance of forming euploid embryos (0.976 [0.923 1.031]/p=0.382) (Table 1).
Conclusion(s): Allowing the lead follicles to exceed 18mm increases the total number of euploid embryos formed per cycle, presumably by enabling retrieval of additional mature oocytes. Evidence of a detrimental effect of excessive follicle size was not evident in our study, though the number of cycles with follicles exceeding 24 mm was limited. The non-significant trend toward decreased euploid embryos following prolonged stimulation may reflect the effects of poor responders. [Figure presented]
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Germ granules are hallmarks of all germ cells. Early ultrastructural studies in Drosophila first described these membraneless granules in the oocyte and early embryo as filled with amorphous to fibrillar material mixed with RNA. Genetic studies identified key protein components and specific mRNAs that regulate germ cell specific functions. More recently these ultrastructural studies have been complemented by biophysical analysis describing germ granules as phase transitioned condensates. In this review, we provide an overview that connects the composition of germ granules with their function in controlling germ cell specification, formation and migration, and illuminate these mysterious condensates as the gatekeepers of the next generation. This article is protected by copyright. All rights reserved.

OBJECTIVE:mice revealed an increase in the stress granule-specific markers Ras-G3BP (GTPase-activating protein SH3 domain-binding protein) and PABP (poly-A-binding protein) in intimal macrophages and smooth muscle cells that correlated with disease progression. In vitro, PABP+ and G3BP+ SGs were rapidly induced in VSMC and bone marrow-derived macrophages in response to atherosclerotic stimuli, including oxidized low-density lipoprotein and mediators of mitochondrial or oxidative stress. We observed an increase in eIF2α phosphorylation, a requisite for stress granule formation, in cells exposed to these stimuli. Interestingly, SG formation, PABP expression, and eIF2α phosphorylation in VSMCs is reversed by treatment with the anti-inflammatory cytokine interleukin-19. Microtubule inhibitors reduced stress granule accumulation in VSMC, suggesting cytoskeletal regulation of stress granule formation. SG formation in VSMCs was also observed in other vascular disease pathologies, including vascular restenosis. Reduction of SG component G3BP1 by siRNA significantly altered expression profiles of inflammatory, apoptotic, and proliferative genes. CONCLUSIONS:These results indicate that SG formation is a common feature of the vascular response to injury and disease, and that modification of inflammation reduces stress granule formation in VSMC.