Immunological and hematological outcomes following protracted low dose/low dose rate ionizing radiation and simulated microgravity

Using a ground-based model to simulate spaceflight [21-days of single-housed, hindlimb unloading (HLU) combined with continuous low-dose gamma irradiation (LDR, total dose of 0.04 Gy)], an in-depth survey of the immune and hematological systems of mice at 7-days post-exposure was performed. Collected blood was profiled with a hematology analyzer and spleens were analyzed by whole transcriptome shotgun sequencing (RNA-sequencing). The results revealed negligible differences in immune differentials. However, hematological system analyses of whole blood indicated large disparities in red blood cell differentials and morphology, suggestive of anemia. Murine Reactome networks indicated majority of spleen cells displayed differentially expressed genes (DEG) involved in signal transduction, metabolism, cell cycle, chromatin organization, and DNA repair. Although immune differentials were not changed, DEG analysis of the spleen revealed expression profiles associated with inflammation and dysregulated immune function persist to 1-week post-simulated spaceflight. Additionally, specific regulation pathways associated with human blood disease gene orthologs, such as blood pressure regulation, transforming growth factor-β receptor signaling, and B cell differentiation were noted. Collectively, this study revealed differential immune and hematological outcomes 1-week post-simulated spaceflight conditions, suggesting recovery from spaceflight is an unremitting process

Primate-specific endogenous retrovirus-driven transcription defines naive-like stem cells

Naive embryonic stem cells hold great promise for research and therapeutics as they have broad and robust developmental potential. While such cells are readily derived from mouse blastocysts it has not been possible to isolate human equivalents easily, although human naive-like cells have been artificially generated (rather than extracted) by coercion of human primed embryonic stem cells by modifying culture conditions or through transgenic modification. Here we show that a sub-population within cultures of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) manifests key properties of naive state cells. These naive-like cells can be genetically tagged, and are associated with elevated transcription of HERVH, a primate-specific endogenous retrovirus. HERVH elements provide functional binding sites for a combination of naive pluripotency transcription factors, including LBP9, recently recognized as relevant to naivety in mice. LBP9-HERVH drives hESC-specific alternative and chimaeric transcripts, including pluripotency-modulating long non-coding RNAs. Disruption of LBP9, HERVH and HERVH-derived transcripts compromises self-renewal. These observations define HERVH expression as a hallmark of naive-like hESCs, and establish novel primate-specific transcriptional circuitry regulating pluripotency

The Effects of Simulated Spaceflight Conditions on the Myometrium of the Mouse Uterus

As scientific discovery and human presence push further into space, it is necessary to investigate the effects of spaceflight on physiological systems. Research into the effects of the space flight environment on the human body is still in its relative infancy. Although initial studies have indicated harmful effects of spaceflight environments on certain body systems, this phenomenon still needs illumination with regards to the female reproductive system. Better understanding of these consequences can change the way society views space travel and colonization of other planets. The spaceflight environment consists of at least two major factors that could confer negative effects on physiology, namely radiation and microgravity. In this experiment, uterine smooth muscle, or the myometrium, was analyzed in 6-month old female C57BL/6 mice that were exposed to 21 days of low dose/low dose rate whole-body radiation with γ-radiation using 57Co plates (0.04 Gy at 0.01 cGy/h) and/or simulated microgravity (via hind limb unloading). Tissue samples were harvested 4 months after the 21-day simulated spaceflight period. Following embedding, sectioning, and hematoxylin and eosin staining (H&E), the tissues were examined, and the average thicknesses of the myometrial layers were measured. Three types of measurements were made 1 – outer longitudinal layer, 2 – inner circular layer, and 3 –total muscle layer thickness (outer and inner combined). Two-way ANOVA statistical tests were used to compare the thicknesses of the myometrial muscle layers between the various treatment groups. A statistical difference was found between the thicknesses in the outer longitudinal layer of smooth muscle between the control animals and the unloaded animals (P: 0.051)

The Effects of Simulated Spaceflight Conditions on the Mucin Lining of the Mouse Uterine Tube

To determine the effects of spaceflight on the mucin layer of uterine tubes, female mice were subjected to simulated microgravity and/or low dose rate radiation (LDR). Astronaut age-appropriate (6 months old), female C57BL/6 mice were exposed to anti-orthostatic tail suspension (AOS) for up 21 days to model the unloading, fluid shift, and physiological stress aspects of the microgravity component. Subsets of mice were also exposed to whole-body, gamma-irradiation (0.04Gy at 0.01cGy/h) using 57Co plates to simulate the LDR radiation component. Mice were then euthanized at 1, 4 or 9 months after the 21 day simulation. Tissues were harvested and quantitatively analyzed for mucin production by measuring the mucin layer thickness of the isthmus, ampulla, and infundibulum regions of the uterine tubes. Analyses conducted indicate that there were no significant reductions in the isthmus and ampulla sections across all treatment groups at the 1, 4, and 9 month time samples. The infundibulum section showed significant reductions at 4 and 9 months post treatment, but there was not a significant change in thickness at 1 month post treatment. These data indicate that both simulated microgravity and radiation exposure cause a thinning of the mucin layer in the infundibulum region of the uterine tube, but do not cause significant morphological changes in the isthmus and ampulla sections of the tube

Effects of skeletal unloading on the antibody repertoire of tetanus toxoid and/or CpG treated C57BL/6J mice.

Spaceflight affects the immune system, but the effects on the antibody repertoire, responsible for humoral immunity, has not been well explored. In particular, the complex gene assembly and expression process; including mutations, might make this process vulnerable. Complementarity determining region 3 (CDR3), composed of parts of the V-(D-)J-gene segments, is very important for antigen binding and can be used as an important measure of variability. Skeletal unloading, and the physiological effects of it, parallel many impacts of space flight. Therefore, we explored the impact of skeletal unloading using the antiorthostatic suspension (AOS) model. Animals were experimentally challenged with tetanus toxoid (TT) and/or the adjuvant CpG. Blood was analyzed for anti-TT antibody and corticosterone concentrations. Whole spleen tissue was prepared for repertoire characterization. AOS animals showed higher levels of corticosterone levels, but AOS alone did not affect anti-TT serum antibody levels. Administration of CpG significantly increased the circulating anti-TT antibody concentrations. AOS did alter constant gene usage resulting in higher levels of IgM and lower levels of IgG. CpG also altered constant gene region usage increasing usage of IgA. Significant changes could be detected in multiple V-, D-, and J-gene segments in both the heavy and light chains in response to AOS, TT, and CpG treatments. Analysis of class-switched only transcripts revealed a different pattern of V-gene segment usage than detected in the whole repertoire and also showed significant alterations in gene segment usage after challenge. Alterations in V/J pairing were also detected in response to challenge. CDR3 amino acid sequence overlaps were similar among treatment groups, though the addition of CpG lowered overlap in the heavy chain. We isolated 3,045 whole repertoire and 98 potentially TT-specific CDR3 sequences for the heavy chain and 569 for the light chain. Our results demonstrate that AOS alters the repertoire response to challenge with TT and/or CpG

Immunological and hematological outcomes following protracted low dose/low dose rate ionizing radiation and simulated microgravity

Using a ground-based model to simulate spaceflight [21-days of single-housed, hindlimb unloading (HLU) combined with continuous low-dose gamma irradiation (LDR, total dose of 0.04 Gy)], an in-depth survey of the immune and hematological systems of mice at 7-days post-exposure was performed. Collected blood was profiled with a hematology analyzer and spleens were analyzed by whole transcriptome shotgun sequencing (RNA-sequencing). The results revealed negligible differences in immune differentials. However, hematological system analyses of whole blood indicated large disparities in red blood cell differentials and morphology, suggestive of anemia. Murine Reactome networks indicated majority of spleen cells displayed differentially expressed genes (DEG) involved in signal transduction, metabolism, cell cycle, chromatin organization, and DNA repair. Although immune differentials were not changed, DEG analysis of the spleen revealed expression profiles associated with inflammation and dysregulated immune function persist to 1-week post-simulated spaceflight. Additionally, specific regulation pathways associated with human blood disease gene orthologs, such as blood pressure regulation, transforming growth factor-β receptor signaling, and B cell differentiation were noted. Collectively, this study revealed differential immune and hematological outcomes 1-week post-simulated spaceflight conditions, suggesting recovery from spaceflight is an unremitting process

Spaceflight influences gene expression, photoreceptor integrity, and oxidative stress-related damage in the murine retina

Extended spaceflight has been shown to adversely affect astronaut visual acuity. The purpose of this study was to determine whether spaceflight alters gene expression profiles and induces oxidative damage in the retina. Ten week old adult C57BL/6 male mice were flown aboard the ISS for 35 days and returned to Earth alive. Ground control mice were maintained on Earth under identical environmental conditions. Within 38 (+/-4) hours after splashdown, mice ocular tissues were collected for analysis. RNA sequencing detected 600 differentially expressed genes (DEGs) in murine spaceflight retinas, which were enriched for genes related to visual perception, the phototransduction pathway, and numerous retina and photoreceptor phenotype categories. Twelve DEGs were associated with retinitis pigmentosa, characterized by dystrophy of the photoreceptor layer rods and cones. Differentially expressed transcription factors indicated changes in chromatin structure, offering clues to the observed phenotypic changes. Immunofluorescence assays showed degradation of cone photoreceptors and increased retinal oxidative stress. Total retinal, retinal pigment epithelium, and choroid layer thickness were significantly lower after spaceflight. These results indicate that retinal performance may decrease over extended periods of spaceflight and cause visual impairment

Linking gene expression to clinical outcomes in pediatric Crohn’s disease using machine learning

Abstract Pediatric Crohn’s disease (CD) is characterized by a severe disease course with frequent complications. We sought to apply machine learning-based models to predict risk of developing future complications in pediatric CD using ileal and colonic gene expression. Gene expression data was generated from 101 formalin-fixed, paraffin-embedded (FFPE) ileal and colonic biopsies obtained from treatment-naïve CD patients and controls. Clinical outcomes including development of strictures or fistulas and progression to surgery were analyzed using differential expression and modeled using machine learning. Differential expression analysis revealed downregulation of pathways related to inflammation and extra-cellular matrix production in patients with strictures. Machine learning-based models were able to incorporate colonic gene expression and clinical characteristics to predict outcomes with high accuracy. Models showed an area under the receiver operating characteristic curve (AUROC) of 0.84 for strictures, 0.83 for remission, and 0.75 for surgery. Genes with potential prognostic importance for strictures (REG1A, MMP3, and DUOX2) were not identified in single gene differential analysis but were found to have strong contributions to predictive models. Our findings in FFPE tissue support the importance of colonic gene expression and the potential for machine learning-based models in predicting outcomes for pediatric CD