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All sources NASA NTRS DOE OSTI

DOE OSTI2022

Vaccination before or after SARS-CoV-2 infection leads to robust humoral response and antibodies that effectively neutralize variants

Current coronavirus disease 2019 (COVID-19) vaccines effectively reduce overall morbidity and mortality and are vitally important to controlling the pandemic. Individuals who previously recovered from COVID-19 have enhanced immune responses after vaccination (hybrid immunity) compared with their naïve-vaccinated peers; however, the effects of post-vaccination breakthrough infections on humoral immune response remain to be determined. Here, we measure neutralizing antibody responses from 104 vaccinated individuals, including those with breakthrough infections, hybrid immunity, and no infection history. We find that human immune sera after breakthrough infection and vaccination after natural infection broadly neutralize SARS-CoV-2 (severe acute respiratory coronavirus 2) variants to a similar degree. Although age negatively correlates with antibody response after vaccination alone, no correlation with age was found in breakthrough or hybrid immune groups. Together, our data suggest that the additional antigen exposure from natural infection substantially boosts the quantity, quality, and breadth of humoral immune response regardless of whether it occurs before or after vaccination.

60 APPLIED LIFE SCIENCES↗

NASA NTRS2001

Suppression of antigen-specific lymphocyte activation in modeled microgravity

Various parameters of immune suppression are observed in lymphocytes from astronauts during and after a space flight. It is difficult to ascribe this suppression to microgravity effects on immune cells in crew specimens, due to the complex physiological response to space flight and the resultant effect on in vitro immune performance. Use of isolated immune cells in true and modeled microgravity in immune performance tests, suggests a direct effect of microgravity on in vitro cellular function. Specifically, polyclonal activation of T-cells is severely suppressed in true and modeled microgravity. These recent findings suggest a potential suppression of oligoclonal antigen-specific lymphocyte activation in microgravity. We utilized rotating wall vessel (RWV) bioreactors as an analog of microgravity for cell cultures to analyze three models of antigen-specific activation. A mixed-lymphocyte reaction, as a model for a primary immune response, a tetanus toxoid response and a Borrelia burgdorferi response, as models of a secondary immune response, were all suppressed in the RWV bioreactor. Our findings confirm that the suppression of activation observed with polyclonal models also encompasses oligoclonal antigen-specific activation.

NASA Discipline Cell Biology↗

NASA NTRS2017

Effects of Simulated Microgravity on a Host-Pathogen System

While it has been shown that decades of astronauts and cosmonauts can suffer from illnesses both during and after spaceflight, the underlying causes are still poorly understood, due in part to the fact that there are so many variables to consider when investigating the human immune system in a complex environment. Invertebrates have become popular models for studying human disease because they are cheap, highly amenable to experimental manipulation, and have innate immune systems with a high genetic similarity to humans. Fruit flies (Drosophila melanogaster) have been shown to experience a dramatic shift in immune gene expression following spaceflight, but are still able to fight off infections when exposed to bacteria. However, the common bacterial pathogen Serratia marcescens was shown to become more lethal to fruit flies after being cultured in space, suggesting that not only do we need to consider host changes in susceptibility, but also changes in the pathogen itself after spaceflight conditions. Being able to simulate spaceflight conditions in a controlled environment on the ground gives us the ability to not only evaluate the effects of microgravity on the host immune system, but also how the microorganisms that cause immune disorders are being affected by these drastic environmental shifts. In this study, I use a ground-based simulated microgravity environment to examine the genetic changes associated with increased S. marcescens virulence in order to understand how microgravity is affecting this pathogen, as well as how these genetic changes influence and interact with the host immune system. This study will provide us with more directed approaches to studying the effects of spaceflight on human beings, with the ultimate goal of being able to counteract immune dysfunction in future space exploration.

Immunology↗

NASA NTRS2017

Studying the Effect of Radiation in the Context of Deep Space Travel

Bhattacharya, Sharmila↗

DOE OSTI2024

Systemic immunological responses are dependent on sex and ovarian hormone presence following acute inhaled woodsmoke exposure

Background: Rural regions of the western United States have experienced a noticeable surge in both the frequency and severity of acute wildfire events, which brings significant challenges to both public safety and environmental conservation efforts, with impacts felt globally. Identifying factors contributing to immune dysfunction, including endocrinological phenotypes, is essential to understanding how hormones may influence toxicological susceptibility. Methods: This exploratory study utilized male and female C57BL/6 mice as in vivo models to investigate distinct responses to acute woodsmoke (WS) exposure with a focus on sex-based differences. In a second set of investigations, two groups were established within the female mouse cohort. In one group, mice experienced ovariectomy (OVX) to simulate an ovarian hormone-deficient state similar to surgical menopause, while the other group received Sham surgery as controls, to investigate the mechanistic role of ovarian hormone presence in driving immune dysregulation following acute WS exposure. Each experimental cohort followed a consecutive 2-day protocol with daily 4-h exposure intervals under two conditions: control HEPA-filtered air (FA) and acute WS to simulate an acute wildfire episode. Results: Metals analysis of WS particulate matter (PM) revealed significantly increased levels of 63 Cu, 182 W, 208 Pb, and 238 U, compared to filtered air (FA) controls, providing insights into the specific metal components most impacted by the changing dynamics of wildfire occurrences in the region. Male and female mice exhibited diverse patterns in lung mRNA cytokine expression following WS exposure, with males showing downregulation and females displaying upregulation, notably for IL-1β, TNF-α, CXCL-1, CCL-5, TGF-β, and IL-6. After acute WS exposure, there were notable differences in the responses of macrophages, neutrophils, and bronchoalveolar lavage (BAL) cytokines IL-10, IL-6, IL-1β, and TNF-α. Significant diverse alterations were observed in BAL cytokines, specifically IL-1β, IL-10, IL-6, and TNF-α, as well as in the populations of immune cells, such as macrophages and polymorphonuclear leukocytes, in both Sham and OVX mice, following acute WS exposure. These findings elucidated the profound influence of hormonal changes on inflammatory outcomes, delineating substantial sex-related differences in immune activation and revealing altered immune responses in OVX mice due to ovarian hormone deficiency. In addition, the flow cytometry analysis highlighted the complex interaction between OVX surgery, acute WS exposure, and their collective impact on immune cell populations within the hematopoietic bone marrow niche. Conclusions: In summary, both male and female mice, alongside females subjected to OVX and those who had sham surgery, exhibit significant variations in the expression of proinflammatory cytokines, chemokines, lung mRNA gene expression, and related functional networks linked to signaling pathways. These differences potentially act as mediators of sex-specific and hormonal influences in the systemic inflammatory response to acute WS exposure during a wildfire event. Understanding the regulatory roles of genes expressed differentially under environmental stressors holds considerable implications, aiding in identifying sex-specific therapeutic targets for addressing acute lung inflammation and injury.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Integrated Metabolomics and Proteomics Analyses in the Local Milieu of Islet Allografts in Rejection versus Tolerance

An understanding of the immune mechanisms that lead to rejection versus tolerance of allogeneic pancreatic islet grafts is of paramount importance, as it facilitates the development of innovative methods to improve the transplant outcome. Here, we used our established intraocular islet transplant model to gain novel insight into changes in the local metabolome and proteome within the islet allograft’s immediate microenvironment in association with immune-mediated rejection or tolerance. We performed integrated metabolomics and proteomics analyses in aqueous humor samples representative of the graft’s microenvironment under each transplant outcome. The results showed that several free amino acids, small primary amines, and soluble proteins related to the Warburg effect were upregulated or downregulated in association with either outcome. In general, the observed shifts in the local metabolite and protein profiles in association with rejection were consistent with established pro-inflammatory metabolic pathways and those observed in association with tolerance were immune regulatory. Taken together, the current findings further support the potential of metabolic reprogramming of immune cells towards immune regulation through targeted pharmacological and dietary interventions against specific metabolic pathways that promote the Warburg effect to prevent the rejection of transplanted islets and promote their immune tolerance.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Vaccine potential of ESAT-6 protein fused with consensus CD4+ T-cell epitopes of PE/PPE proteins against highly pathogenic Mycobacterium tuberculosis strain HN878

Pro-Glu/Pro-Pro-Glu (PE/PPE) family proteins in Mycobacterium tuberculosis (Mtb) are contributors to pathogenesis and immune evasion. These proteins have a unique structure in which the sequence is conserved. We investigated the vaccine potential of ESAT-6 fused with consensus CD4{sup +} T-cell epitopes of PE/PPE proteins against highly pathogenic Mtb strain HN878 in a murine model. We selected consensus CD4{sup +} T-cell epitopes of PE/PPE proteins by multiple alignments, investigated their IFN-γ response during Mtb infection, and produced their fused ESAT-6 vaccine antigens. Our results showed an increased immune response in PE/PPE peptide -ESAT-6 fusion protein immunization group compared to ESAT-6 only immunization group. After challenge with Mtb strain HN878, we observed that induced CD4{sup +} T-cells secreted double-positive cytokine IL-2{sup +}/IFN-γ{sup +}, which is considered to be associated with protective T-cell immunity. Additionally, lower numbers of colony-forming units were observed in the spleen of fusion protein immunization groups than in those of single ESAT-6 group. Therefore, conjugation of consensus CD4{sup +} T-cell epitopes in N terminus of PE/PPE to vaccine antigens could potentially increase the protective efficacy of subunit vaccine.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2018

Inhibition of PD1:PD-L1 interaction by an E. coli-derived optimized PD1 variant

Highlights: • Cancer cells interact with immune checkpoints to shut down T cell response. • An optimization system for the selection of modified immune checkpoint proteins. • Using the described system improved PD1 variants were identified. • The improved variants are active in a cellular assay that mimics T cell:cancer interaction. Immune-checkpoint receptors are a set of signal transduction proteins that can stimulate or inhibit specific anti-tumor responses. It is well established that cancer cells interact with different immune checkpoints to shut down T-cell response, thereby enabling cancer proliferation. Given the importance of immune checkpoint receptors, a structure-function analysis of these systems is imperative. However, recombinant expression and purification of these membrane originated proteins is still a challenge. Therefore, many attempts are being made to improve their expression and solubility while preserving their biological relevance. For this purpose, we designed an E. coli-based optimization system that enables the acquisition of mutations that increases protein solubility and affinity towards its native ligand, while maintaining biological activity. Here we focused on the well-characterized extracellular domain of the 'programmed cell death protein 1' (PD1), an immune checkpoint receptor known to inhibit T-cell proliferation by interacting with its ligands PD-L1 and PD-L2. The simple ELISA-based screening system shown here enabled the identification of high-affinity, highly soluble, functional variants derived from the extracellular domain of human PD1. The system was based on the expression of a GST-tagged variants library in E. coli, which enabled the selection of improved PD1 variants after a single optimization round. Within only two screening rounds, the most active variant showed a 5-fold higher affinity and 2.4-fold enhanced cellular activity as compared to the wild type protein. This scheme can be translated toward other types of challenging receptors toward development of research tools or alternative therapeutics.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2025

scRNA seq of an F1 cross of Marek’s disease resistant and susceptible chickens identifies allele specific expression signatures enriched in transcription modulators

Abstract Marek’s disease (MD), a T cell lymphoma disease in chickens, is caused by the Marek’s disease virus (MDV) found ubiquitously in the poultry industry. Genetically resistant Line 6 3 (L6) and susceptible Line 7 2 (L7) chickens have been instrumental to research on avian immune system response to MDV infection. In this study we characterized molecular signatures unique to splenic immune cell types across different genetic backgrounds 6 days after infection. Using three populations, L6, L7, and an F1 cross between L6xL7, we evaluated the immune cell transcriptome of responding cell types using single cell RNA sequencing. Several MDV genes were found expressed mainly in cytotoxic T cells while ICP4 and MEQ MDV genes were expressed across infected cell types. Using the F1 we quantified allele specific expression (ASE) of biallelic SNPs and found biased expression of parental alleles specific to immune cell subtypes. We identified 22 SNPs with ASE in response to MDV infection mapped to gene rich regions surrounding 59 genes of critical importance for chromatin remodeling and transcriptional regulation. Histone deacetylase genes (HDAC1 and HDAC8) had increased expression of L6 alleles, while small nuclear RNA genes (SNORA68 and SNORA72) expressed higher levels of L7 alleles with infection in T cell subsets. SNPs with ASE also mapped genes important for an adequate immune response including GNLY (cytotoxic activity) and PDIA3 (component of MHC class I peptide loading complex), and genes known to promote viral replication (MCM5 and EIF3M). These results show that functional variants associated with susceptibility to MD may have a bigger impact in subsets of immune cell types, and by characterizing the transcriptomes of these subtypes we can unravel molecular signatures specific to MD genomic resistance.

Science & Technology - Other Topics↗

DOE OSTI2022

Lifelong cytomegalovirus and early‐LIFE irradiation synergistically potentiate age‐related defects in response to vaccination and infection

Abstract While whole‐body irradiation (WBI) can induce some hallmarks of immune aging, (re)activation of persistent microbial infection also occurs following WBI and may contribute to immune effects of WBI over the lifespan. To test this hypothesis in a model relevant to human immune aging, we examined separate and joint effects of lifelong latent murine cytomegalovirus (MCMV) and of early‐life WBI over the course of the lifespan. In late life, we then measured the response to a West Nile virus (WNV) live attenuated vaccine, and lethal WNV challenge subsequent to vaccination. We recently published that a single dose of non‐lethal WBI in youth, on its own, was not sufficient to accelerate aging of the murine immune system, despite widespread DNA damage and repopulation stress in hematopoietic cells. However, 4Gy sub‐lethal WBI caused manifest reactivation of MCMV. Following vaccination and challenge with WNV in the old age, MCMV‐infected animals experiencing 4Gy, but not lower, dose of sub‐lethal WBI in youth had reduced survival. By contrast, old irradiated mice lacking MCMV and MCMV‐infected, but not irradiated, mice were both protected to the same high level as the old non‐irradiated, uninfected controls. Analysis of the quality and quantity of anti‐WNV immunity showed that higher mortality in MCMV‐positive WBI mice correlated with increased levels of MCMV‐specific immune activation during WNV challenge. Moreover, we demonstrate that infection, including that by WNV, led to MCMV reactivation. Our data suggest that MCMV reactivation may be an important determinant of increased late‐life mortality following early‐life irradiation and late‐life acute infection.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2018

Dynamic balance of pro- and anti-inflammatory signals controls disease and limits pathology

Immune responses to pathogens are complex and not well understood in many diseases, and this is especially true for infections by persistent pathogens. One mechanism that allows for long-term control of infection while also preventing an over-zealous inflammatory response from causing extensive tissue damage is for the immune system to balance pro- and anti-inflammatory cells and signals. This balance is dynamic and the immune system responds to cues from both host and pathogen, maintaining a steady state across multiple scales through continuous feedback. Identifying the signals, cells, cytokines, and other immune response factors that mediate this balance over time has been difficult using traditional research strategies. Computational modeling studies based on data from traditional systems can identify how this balance contributes to immunity. Here we provide evidence from both experimental and mathematical/computational studies to support the concept of a dynamic balance operating during persistent and other infection scenarios. We focus mainly on tuberculosis, currently the leading cause of death due to infectious disease in the world, and also provide evidence for other infections. A better understanding of the dynamically balanced immune response can help shape treatment strategies that utilize both drugs and host-directed therapies.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2019

Burn and thoracic trauma alters fracture healing, systemic inflammation, and leukocyte kinetics in a rat model of polytrauma

Singular traumatic insults, such as bone fracture, typically initiate an appropriate immune response necessary to restore the host to pre-insult homeostasis with limited damage to self. However, multiple concurrent insults, such as a combination of fracture, blunt force trauma, and burns (polytrauma), are clinically perceived to result in abnormal immune response leading to inadequate healing and resolution. To investigate this phenomenon, we created a model rat model of polytrauma. To investigate relationship between polytrauma and delayed healing, we created a novel model of polytrauma in a rat which encompassed a 3-mm osteotomy, blunt chest trauma, and full-thickness scald burn. Healing outcomes were determined at 5 weeks where the degree of bone formation at the osteotomy site of polytrauma animals was compared to osteotomy only animals (OST). We observed significant differences in the bone volume fraction between polytrauma and OST animals indicating that polytrauma negatively effects wound healing. Polytrauma animals also displayed a significant decrease in their ability to return to pre-injury weight compared to osteotomy animals. Polytrauma animals also exhibited significantly altered gene expression in osteogenic pathways as well as the innate and adaptive immune response. Perturbed inflammation was observed in the polytrauma group compared to the osteotomy group as evidenced by significantly altered white blood cell (WBC) profiles and significantly elevated plasma high-mobility group box 1 protein (HMGB1) at 6 and 24 h post-trauma. Conversely, polytrauma animals exhibited significantly lower concentrations of plasma TNF-alpha (TNF-α) and interleukin 6 (IL-6) at 72 h post-injury compared to OST. Following polytrauma with burn injury, the local and systemic immune response is divergent from the immune response following a less severe singular injury (osteotomy). This altered immune response that follows was associated with a reduced capacity for wound healing.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2026

Host-Directed, Bioelectronic Immunomodulation for Protection Against Emerging Pathogens

Acute care of patients with severe infections often relies on systemic administration of pharmaceuticals and monitoring of complex physiological symptoms to identify immune system dysfunction, which can lead to increased mortality. Furthermore, determining disease-specific treatment plans often leads to a delay in patient care. To address this, we proposed an immune modulation system that electrically detects and responds to a patient’s immune system status, creating an agnostic means of treating illness and infection. Two pieces of hardware were developed for this task: a minimally-invasive sensor and a vagus nerve stimulator. Stimulation of the vagus nerve is known to modulate the immune system. The sensor is a microfabricated, silicon-based microneedle array capable of interfacing with interstitial fluid to detect small molecules such as inflammatory proteins (cytokines) and pharmaceuticals (vancomycin). Process optimization to manufacture the needles refined the silicon etch process, creating needle patches long enough to penetrate skin and reach interstitial fluid. The needles were tested for mechanical strength and stability, and did not shatter when inserted into skin models. The needles are coated with a thin film metal, turning them into electrodes for electrochemical sensing of our target molecules. We hybridized aptamers to the surface of the electrode to act as the sensing layer and were able to detect changes in the conformation of the aptamer electrochemically in the presence of the target molecule. The stimulator was a cuff electrode that encircled the vagus nerve. Rodent studies were conducted in which rodents were exposed to an inflammatory event and vagus nerve stimulation (VNS) was applied. It was demonstrated that optimized electrical stimulation of the vagus nerve created measurably different levels of cytokines in blood samples, and certain cytokines released during the inflammatory event were either upregulated or downregulated. In sum, this project successfully developed new platforms and technologies that can, with further development, enable better temporal insight into biomarker changes in the body, letting healthcare providers know of possible immune system dysfunction before they are detected physiologically. We also demonstrated the value of VNS and its possible use in treating immune system response to inflammation and illness.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2008

Incidence of Latent Virus Shedding during Space Flight

Measurements of immune parameters of both cellular and innate immunity indicate alterations in immune function in astronauts. Immune changes are due to stress and perhaps other factors associated with launch, flight, and landing phases. Medical relevance of observed changes is not known. The reactivation of latent viruses has been identified as an important in vivo indicator of clinically relevant immune changes. The polymerase chain reaction (PCR) was used to detect the presence of specific viral DNA in body fluids. Initial studies demonstrated Epstein-Barr virus (EBV) reactivation during all 3 mission phases. EBV is shed in saliva following reactivation from B-cells. Incidence of EBV in saliva was higher than control subjects during all 3 mission phases. However, quantitative PCR revealed 10-fold higher levels of EBV DNA present in saliva collected during flight than found in pre- and post flight specimens. To determine if other latent viruses showed similar effects, cytomegalovirus (CMV), another herpes virus, shed in urine following reactivation was studied. A very low incidence (less than 2%) of CMV in urine is found in healthy, lowstressed individuals. However, 25-50% of astronauts shed CMV in their urine before, during, or after flight. Our studies are now focused on varicella-zoster virus (VZV), the etiological agent of chicken-pox during childhood and shingles later in life. We demonstrated reactivation of VZV and shedding of the virus during and after spaceflight in saliva of astronauts with no sign of active infection or symptoms. The maximum shedding of VZV occurred during the flight phase and diminishes rapidly during the first five days after landing. We have utilized the same PCR assay for VZV in a clinical study of shingles patients. Generally, shingles patients shed much more VZV in saliva than astronauts. However, the VZV levels in astronauts overlap with the lower range of VZV numbers in shingles patients. Saliva from shingles patients and astronauts were cultured and infectious VZV was recovered from both groups. We have concluded that multiple latent viruses do reactivate before, during, and after spaceflight and serve as very sensitive indicators for diminished cellular immunity. Future plans will be focused on the clinical risks posed by the reactivation of these viruses. Initial efforts will determine the effect of longer missions on the International Space Station on the reactivation patterns of these viruses.

Mehta, Satish K.↗

NASA NTRS2019

Effects of Spaceflight and Simulated Microgravity on a Host-Pathogen System

While evidence suggests that astronauts and cosmonauts suffer from immune disorders both during and after spaceflight, the underlying causes are still poorly understood, due in part to the fact that there are so many variables to consider when investigating the human immune system in a complex environment. Furthermore, research has shown that common human pathogens also become more virulent after experiencing spaceflight, which can be especially concerning in the context of potentially immunocompromised astronauts. Invertebrates have become popular models for studying human disease because they have immune systems with a high genetic similarity to humans. Recently, the common bacterial pathogen Serratia marcescens was shown to become more lethal to the fruit fly, Drosophila melanogaster, after being cultured in space, suggesting that not only do we need to consider host changes in susceptibility, but also changes in the pathogen itself after exposure to spaceflight conditions. Being able to simulate spaceflight conditions in a controlled environment on the ground gives us the ability to understand how the microorganisms that cause immune disorders are being affected by these drastic environmental shifts. In this study, I use both spaceflight and Low-shear modeled microgravity (LSMMG) environments to examine the genetic changes associated with increased S. marcescens virulence in order to understand how microgravity is affecting this pathogen, as well as how these genetic changes influence and interact with the host immune system. I also examined the effects of nutrient composition and altered growth conditions on the LSMMG-induced increase in virulence, as well as changes in gene expression mediated by both nutrient composition and exposure to LSMMG. This study will provide us with more directed approaches to studying the effects of spaceflight on human beings, with the ultimate goal of being able to prevent human immune dysfunction in future space exploration.

Gilbert, Rachel↗

NASA NTRSDate not supplied

Validation of Multisystem Countermeasures Protocol for Spaceflight during Antarctica Winter-over at Palmer Station (Palmer Countermeasures)

Exploration-class missions beyond the Van Allen belt to the Moon and then Mars will begin soon. Low-Earth orbital spaceflight results in the persistent perturbation of the human immune system, characterized by reductions in T and NK cell function, altered cytokine profiles, and the reactivation of latent herpesviruses. While these alterations have not caused widespread clinical issues, some crewmembers experience immune-related adverse events, including manifestations of symptomatic herpes viral reactivation, allergy, and respiratory distress. Because future deep-space exploration missions will be of unprecedented duration, it is reasonable to hypothesize that the immune perturbations observed aboard International Space Station (ISS) will intensify during longer missions in deep space, thereby placing crewmembers at elevated clinical risk. Thus, it is imperative to preserve the immune vigilance of astronauts by developing a countermeasure strategy. Of all the Earth analogs studied to date, an Antarctica winter-over (AWO) mission most closely reproduces the spaceflight experience: prolonged deployment, extreme environment, circadian misalignment, isolation, station lifestyle, and personal risk. The US maintains three primary stations in Antarctica: South Pole Station, McMurdo, and Palmer. Previous studies suggest that stations located near the interior of Antarctica (South Pole, McMurdo) have confounding effects on the immune system due to persistent hypobaric hypoxia. Thus, it was hypothesized that winter-over at a coastal station (Palmer) would be more akin to spaceflight due to its normoxic but still extreme environment. Therefore, AWO at Palmer Station was chosen as the platform for testing and validating the effectiveness of a NASA multi-system countermeasures protocol designed for deep space missions. The array of countermeasure protocols and monitoring methods deployed for each AWO will consist of diet modifications, nutritional supplementation, prescribed aerobic and resistive exercise, and a protocol of stress relieving virtual reality exercises. A multitude of biological sample types, including blood, saliva, and hair will be collected in tandem with the countermeasures in order to examine the combined effectiveness of the countermeasures. Samples and logs from subjects will be transported from Palmer Station to Johnson Space Center for further processing and distribution to co-investigators at the end of each winter-over. Extracted samples will be analyzed by appropriate testing platforms (Multiplex, qPCR, ELISA, etc.) to monitor alterations in leukocyte distribution, T cell and NK function, cytokine profiles, reactivation of latent herpesviruses, and nutritional factors. The data collected will be compared to a control year in which no countermeasures were deployed to evaluate the overall effectiveness of the analog and to validate the candidate immune countermeasure strategy. With the completion of the Antarctica Winter-Over (WO) 2022 control year, samples for 13 subjects have been successfully returned from Antarctica to NASA/JSC for further processing and distribution to Co-Investigators. WO 2023, the first countermeasure year, has also commenced with 11 subject consenting and performing their base line data collections (BDC) held in Chile. Another 5 subjects, who were already stationed at Palmer Station, Antarctica, joined as participates in the investigation. These 5 subjects were consented, but no BDC was able to be collected due to their joining in-mission. Therefore, there will be a total of 16 subjects participating in Antarctica's 2023 Winter-Over.

Cody L Gutierrez↗

DOE OSTI2020

Longitudinal proteomics analysis in the immediate microenvironment of islet allografts during progression of rejection

The applicability and benefits of pancreatic islet transplantation are limited due to various issues including the need to avoid immune-mediated rejection. In the present study, we used our experimental platform of allogeneic islet transplant in the anterior chamber of the eye (ACE-platform) to longitudinally monitor the progress of rejection in mice and obtain aqueous humor samples representative of the microenvironment of the graft for accurately-timed proteomic analyses. LC-MS/MS-based proteomics performed on such mass-limited samples (~5 μL) identified a total of 1296 proteins. Various analyses revealed distinct protein patterns associated with the mounting of the inflammatory and immune responses and their evolution with the progression of the rejection. Pathway analyses indicated predominant changes in cytotoxic functions, cell movement, and innate and adaptive immune responses. Furthermore, network prediction analyses revealed transition from humoral to cellular immune response and exacerbation of pro-inflammatory signaling. One of the proteins identified by this localized proteomics as a candidate biomarker of islet rejection, Cystatin 3, was further validated by ELISA in the aqueous humor. This study provides (1) experimental evidence demonstrating the feasibility of longitudinal localized proteomics using small aqueous humor samples and (2) proof-of-concept for the discovery of biomarkers of impending immune attack from the immediate local microenvironment of ACE-transplanted islets.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRSDate not supplied

Oxidative Stress and Neuroconsequences of the Spaceflight Environment –A 5-ion GCRsim Dose-response Study

The overall scope of this project is to test the hypothesis that Ionizing Radiation (IR), microgravity and social isolation combine synergistically to trigger an oxidative stress response that alters immune homeostasis, brain structure/function, and neurobehavioral/cognitive performance. Specific Aims for this project are to: (1) Determine dose-response curves for acute ‘Five-Ion GCR Simulation’ exposure for immune, brain and performance responses in crew age-matched adult male and female mice; (2) Determine effects of acute ‘Five-Ion GCR Simulation’ exposure singly and in combination with simulated microgravity and social isolation, on immune, brain and performance responses in crew age-matched male and female mice mimicking deep space missions; and (3) Determine efficacy of the dietary antioxidant, Nicotinamide Mononucleotide (NMN), a key intermediate in nicotinamide adenine dinucleotide (NAD+) biosynthesis. Here we exposed mature (23-24-week-old) male and female mice to simulated 5-Ion GCR (either 0, 5, 15, or 50cGy) at the NASA Space Radiation Laboratory (NSRL). Across ‘Acute’ (IR+72hrs and IR+14 days) and ‘Delayed’ (IR+28 to IR+124 days) phases of the 6-month-long study, we assessed immune, neural, and functional (sensorimotor/behavioral/cognitive) responses anticipated during a transit to Mars. This presentation will report on dose-response curves for 5-ion GCRsim related to potential biomarkers; structural/functional changes in the immune and nervous systems; and behavioral/cognitive performance deficits. This project addresses NASA’s efforts to characterize risks and identify appropriate countermeasures in anticipation of future deep space missions. Ensuring crew health and performance during extended transits necessitates that sensorimotor and cognitive abilities remain strong to avoid potentially catastrophic health and safety outcomes.

deep space↗