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At least 811 records · Page 45
All sourcesNASA NTRSDOE OSTI
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 OSTI2015

Structural, Functional, and Immunogenic Insights on Cu,Zn Superoxide Dismutase Pathogenic Virulence Factors from Neisseria meningitidis and Brucella abortus

ABSTRACT Bacterial pathogens Neisseria meningitidis and Brucella abortus pose threats to human and animal health worldwide, causing meningococcal disease and brucellosis, respectively. Mortality from acute N. meningitidis infections remains high despite antibiotics, and brucellosis presents alimentary and health consequences. Superoxide dismutases are master regulators of reactive oxygen and general pathogenicity factors and are therefore therapeutic targets. Cu,Zn superoxide dismutases (SODs) localized to the periplasm promote survival by detoxifying superoxide radicals generated by major host antimicrobial immune responses. We discovered that passive immunization with an antibody directed at N. meningitidis SOD (NmSOD) was protective in a mouse infection model. To define the relevant atomic details and solution assembly states of this important virulence factor, we report high-resolution and X-ray scattering analyses of NmSOD and of SOD from B. abortus (BaSOD). The NmSOD structures revealed an auxiliary tetrahedral Cu-binding site bridging the dimer interface; mutational analyses suggested that this metal site contributes to protein stability, with implications for bacterial defense mechanisms. Biochemical and structural analyses informed us about electrostatic substrate guidance, dimer assembly, and an exposed C-terminal epitope in the NmSOD dimer. In contrast, the monomeric BaSOD structure provided insights for extending immunogenic peptide epitopes derived from the protein. These collective results reveal unique contributions of SOD to pathogenic virulence, refine predictive motifs for distinguishing SOD classes, and suggest general targets for antibacterial immune responses. The identified functional contributions, motifs, and targets distinguishing bacterial and eukaryotic SOD assemblies presented here provide a foundation for efforts to develop SOD-specific inhibitors of or vaccines against these harmful pathogens. IMPORTANCE By protecting microbes against reactive oxygen insults, SODs aid survival of many bacteria within their hosts. Despite the ubiquity and conservation of these key enzymes, notable species-specific differences relevant to pathogenesis remain undefined. To probe mechanisms that govern the functioning of Neisseria meningitidis and Brucella abortus SODs, we used X-ray structures, enzymology, modeling, and murine infection experiments. We identified virulence determinants common to the two homologs, assembly differences, and a unique metal reservoir within meningococcal SOD that stabilizes the enzyme and may provide a safeguard against copper toxicity. The insights reported here provide a rationale and a basis for SOD-specific drug design and an extension of immunogen design to target two important pathogens that continue to pose global health threats.

38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEA↗
DOE OSTI2017

On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires

Cas1 integrase is the key enzyme of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas adaptation module that mediates acquisition of spacers derived from foreign DNA by CRISPR arrays. In diverse bacteria, the cas1 gene is fused (or adjacent) to a gene encoding a reverse transcriptase (RT) related to group II intron RTs. An RT-Cas1 fusion protein has been recently shown to enable acquisition of CRISPR spacers from RNA. Phylogenetic analysis of the CRISPR-associated RTs demonstrates monophyly of the RT-Cas1 fusion, and coevolution of the RT and Cas1 domains. Nearly all such RTs are present within type III CRISPR-Cas loci, but their phylogeny does not parallel the CRISPR-Cas type classification, indicating that RT-Cas1 is an autonomous functional module that is disseminated by horizontal gene transfer and can function with diverse type III systems. To compare the sequence pools sampled by RT-Cas1-associated and RT-lacking CRISPR-Cas systems, we obtained samples of a commercially grown cyanobacterium— Arthrospira platensis . Sequencing of the CRISPR arrays uncovered a highly diverse population of spacers. Spacer diversity was particularly striking for the RT-Cas1-containing type III-B system, where no saturation was evident even with millions of sequences analyzed. In contrast, analysis of the RT-lacking type III-D system yielded a highly diverse pool but reached a point where fewer novel spacers were recovered as sequencing depth was increased. Matches could be identified for a small fraction of the non-RT-Cas1-associated spacers, and for only a single RT-Cas1-associated spacer. Thus, the principal source(s) of the spacers, particularly the hypervariable spacer repertoire of the RT-associated arrays, remains unknown. IMPORTANCE While the majority of CRISPR-Cas immune systems adapt to foreign genetic elements by capturing segments of invasive DNA, some systems carry reverse transcriptases (RTs) that enable adaptation to RNA molecules. From analysis of available bacterial sequence data, we find evidence that RT-based RNA adaptation machinery has been able to join with CRISPR-Cas immune systems in many, diverse bacterial species. To investigate whether the abilities to adapt to DNA and RNA molecules are utilized for defense against distinct classes of invaders in nature, we sequenced CRISPR arrays from samples of commercial-scale open-air cultures of Arthrospira platensis , a cyanobacterium that contains both RT-lacking and RT-containing CRISPR-Cas systems. We uncovered a diverse pool of naturally occurring immune memories, with the RT-lacking locus acquiring a number of segments matching known viral or bacterial genes, while the RT-containing locus has acquired spacers from a distinct sequence pool for which the source remains enigmatic. While the majority of CRISPR-Cas immune systems adapt to foreign genetic elements by capturing segments of invasive DNA, some systems carry reverse transcriptases (RTs) that enable adaptation to RNA molecules. From analysis of available bacterial sequence data, we find evidence that RT-based RNA adaptation machinery has been able to join with CRISPR-Cas immune systems in many, diverse bacterial species. To investigate whether the abilities to adapt to DNA and RNA molecules are utilized for defense against distinct classes of invaders in nature, we sequenced CRISPR arrays from samples of commercial-scale open-air cultures of Arthrospira platensis , a cyanobacterium that contains both RT-lacking and RT-containing CRISPR-Cas systems. We uncovered a diverse pool of naturally occurring immune memories, with the RT-lacking locus acquiring a number of segments matching known viral or bacterial genes, while the RT-containing locus has acquired spacers from a distinct sequence pool for which the source remains enigmatic.

horizontal gene transfer↗
DOE OSTI2017

Precancer Atlas to Drive Precision Prevention Trials

Cancer development is a complex process driven by inherited and acquired molecular and cellular alterations. Prevention is the holy grail of cancer elimination, but making this a reality will take a fundamental rethinking and deep understanding of premalignant biology. Here, we propose a national concerted effort to create a Precancer Atlas (PCA), integrating multi-omics and immunity – basic tenets of the neoplastic process. The biology of neoplasia caused by germline mutations has led to paradigm-changing precision prevention efforts, including: tumor testing for mismatch repair (MMR) deficiency in Lynch syndrome establishing a new paradigm, combinatorial chemoprevention efficacy in familial adenomatous polyposis (FAP), signal of benefit from imaging-based early detection research in high-germline risk for pancreatic neoplasia, elucidating early ontogeny in BRCA1-mutation carriers leading to an international breast cancer prevention trial, and insights into the intricate germline-somatic-immunity interaction landscape. Emerging genetic and pharmacologic (metformin) disruption of mitochondrial (mt) respiration increased autophagy to prevent cancer in a Li-Fraumeni mouse model (biology reproduced in clinical pilot) and revealed profound influences of subtle changes in mt DNA background variation on obesity, aging, and cancer risk. The elaborate communication between the immune system and neoplasia includes an increasingly complex cellular microenvironment and dynamic interactions between host genetics, environmental factors, and microbes in shaping the immune response. Cancer vaccines are in early murine and clinical precancer studies, building on the recent successes of immunotherapy and HPV vaccine immune prevention. Molecular monitoring in Barrett's esophagus to avoid overdiagnosis/treatment highlights an important PCA theme. Next generation sequencing (NGS) discovered age-related clonal hematopoiesis of indeterminate potential (CHIP). Ultra-deep NGS reports over the past year have redefined the premalignant landscape remarkably identifying tiny clones in the blood of up to 95% of women in their 50s, suggesting that potentially premalignant clones are ubiquitous. Similar data from eyelid skin and peritoneal and uterine lavage fluid provide unprecedented opportunities to dissect the earliest phases of stem/progenitor clonal (and microenvironment) evolution/diversity with new single-cell and liquid biopsy technologies. Cancer mutational signatures reflect exogenous or endogenous processes imprinted over time in precursors. In conclusion, accelerating the prevention of cancer will require a large-scale, longitudinal effort, leveraging diverse disciplines (from genetics, biochemistry, and immunology to mathematics, computational biology, and engineering), initiatives, technologies, and models in developing an integrated multi-omics and immunity PCA – an immense national resource to interrogate, target, and intercept events that drive oncogenesis.

59 BASIC BIOLOGICAL 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 OSTI2017

Bacterial amyloid curli acts as a carrier for DNA to elicit an autoimmune response via TLR2 and TLR9

Bacterial biofilms are associated with numerous human infections. The predominant protein expressed in enteric biofilms is the amyloid curli, which forms highly immunogenic complexes with DNA. Infection with curli-expressing bacteria or systemic exposure to purified curli-DNA complexes triggers autoimmunity via the generation of type I interferons (IFNs) and anti-double-stranded DNA antibodies. Here, we show that DNA complexed with amyloid curli powerfully stimulates Toll-like receptor 9 (TLR9) through a two-step mechanism. First, the cross beta-sheet structure of curli is bound by cell-surface Toll-like receptor 2 (TLR2), enabling internalization of the complex into endosomes. After internalization, the curli-DNA immune complex binds strongly to endosomal TLR9, inducing production of type I IFNs. Analysis of wild-type and TLR2-deficient macrophages showed that TLR2 is the major receptor that drives the internalization of curli-DNA complexes. Suppression of TLR2 internalization via endocytosis inhibitors led to a significant decrease in Ifnβ expression. Confocal microscopy analysis confirmed that the TLR2-bound curli was required for shuttling of DNA to endosomal TLR9. Structural analysis using small-angle X-ray scattering revealed that incorporation of DNA into curli fibrils resulted in the formation of ordered curli-DNA immune complexes. Curli organizes parallel, double-stranded DNA rods at an inter-DNA spacing that matches up well with the steric size of TLR9. We also found that production of anti-double-stranded DNA autoantibodies in response to curli-DNA was attenuated in TLR2- and TLR9-deficient mice and in mice deficient in both TLR2 and TLR9 compared to wild-type mice, suggesting that both innate immune receptors are critical for shaping the autoimmune adaptive immune response. We also detected significantly lower levels of interferon-stimulated gene expression in response to purified curli-DNA in TLR2 and TLR9 deficient mice compared to wild-type mice, confirming that TLR2 and TLR9 are required for the induction of type I IFNs. Finally, we showed that curli-DNA complexes, but not cellulose, were responsible elicitation of the immune responses to bacterial biofilms. This study defines the series of events that lead to the severe pro-autoimmune effects of amyloid-expressing bacteria and suggest a mechanism by which amyloid curli acts as a carrier to break immune tolerance to DNA, leading to the activation of TLR9, production of type I IFNs, and subsequent production of autoantibodies.

59 BASIC BIOLOGICAL 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↗
DOE OSTI2013

Therapeutic targeting of regulatory T cells enhances tumor-specific CD8+ T cell responses in Epstein–Barr virus associated nasopharyngeal carcinoma

Epstein–Barr virus (EBV) is associated with multiple malignancies including nasopharyngeal carcinoma (NPC). In nasopharynx cancer, CD8+ T cells specific for EBV Nuclear Antigen-1 (EBNA-1) and Latent Membrane Protein 2 (LMP2) are important components of anti-tumor immunity since both are consistently expressed in NPC. We have previously shown that EBNA-1-specific CD8+ T cell responses were suppressed in NPC patients compared to healthy controls. We now find that CD8+ T cell responses specific for LMP2 are also abnormal in NPC patients, and both EBNA-1- and LMP2-specific responses are suppressed by regulatory T cells (Treg). EBNA-1 and LMP2-specific CD8+ T cell responses, as well as immune control of EBV-infected cells in vitro, could be restored by the depletion of Tregs and by use of a clinically approved drug targeting Tregs. Thus, in vivo modulation of Tregs may be an effective means of enhancing these anti-tumor immune responses in NPC patients. - Highlights: • Viral proteins are tumor antigens in Epstein–Barr virus associated Nasopharyngeal Carcinoma. • CD8+ T cell responses against EBV proteins EBNA-1 and LMP2 are suppressed in NPC patients. • T regulatory cells are responsible for suppressing EBV immunity in NPC patients. • Depletion of Tregs with Ontak can rescue EBV-specific CD8+ T cell responses in NPC patients. • This clinically approved drug may be effective for enhancing anti-tumor immunity in NPC patients.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Tissue distribution of aryl hydrocarbon receptor in the intestine: Implication of putative roles in tumor suppression

Intestinal homeostasis is maintained by complex interactions between intestinal microorganisms and the gut immune system. Dysregulation of gut immunity may lead to inflammatory disorders and tumorigenesis. We previously have shown the tumor suppressive effects of aryl hydrocarbon receptor (AhR) in intestinal carcinogenesis. In the present study, we investigated AhR distribution in the mouse and human intestine by histochemical analysis. In the normal intestine, AhR was mainly localized in the stroma containing immune cells in the lamina propria and lymphoid follicles. On the other hand, in the tumor tissue from human colon cancer and that developed in Apc{sup Min/+}mice, AhR expression was elevated. AhR immunostaining was found in both stromal and tumor cells. Although AhR was localized in the cytoplasm of tumor cells in most cases, nuclear AhR was also observed in some. AhR knockdown using siRNA resulted in significant promotion of cell growth in colon cancer cell lines. Furthermore, AhR activation by AhR ligands supplemented in culture medium suppressed cell growth. Our study results suggest that tumor suppressive roles of AhR are estimated in two distinct ways: in normal tissue, AhR is associated with tumor prevention by regulating gut immunity, whereas in tumor cells, it is involved in growth suppression. - Highlights: • In the normal intestine, AhR was mainly localized in stroma containing immune cells. • In the tumor tissue, AhR expression was found in both stromal and tumor cells. • AhR knockdown promoted cell growth in colon cancer cell lines.

3-METHYLCHOLANTHRENE↗
DOE OSTI2015

Recombination enhances HIV-1 envelope diversity by facilitating the survival of latent genomic fragments in the plasma virus population

HIV-1 is subject to immune pressure exerted by the host, giving variants that escape the immune response an advantage. Virus released from activated latent cells competes against variants that have continually evolved and adapted to host immune pressure. Nevertheless, there is increasing evidence that virus displaying a signal of latency survives in patient plasma despite having reduced fitness due to long-term immune memory. We investigated the survival of virus with latent envelope genomic fragments by simulating within-host HIV-1 sequence evolution and the cycling of viral lineages in and out of the latent reservoir. Our model incorporates a detailed mutation process including nucleotide substitution, recombination, latent reservoir dynamics, diversifying selection pressure driven by the immune response, and purifying selection pressure asserted by deleterious mutations. We evaluated the ability of our model to capture sequence evolution in vivo by comparing our simulated sequences to HIV-1 envelope sequence data from 16 HIV-infected untreated patients. Empirical sequence divergence and diversity measures were qualitatively and quantitatively similar to those of our simulated HIV-1 populations, suggesting that our model invokes realistic trends of HIV-1 genetic evolution. Moreover, reconstructed phylogenies of simulated and patient HIV-1 populations showed similar topological structures. Our simulation results suggest that recombination is a key mechanism facilitating the persistence of virus with latent envelope genomic fragments in the productively infected cell population. Recombination increased the survival probability of latent virus forms approximately 13-fold. Prevalence of virus with latent fragments in productively infected cells was observed in only 2% of simulations when we ignored recombination, while the proportion increased to 27% of simulations when we allowed recombination. We also found that the selection pressures exerted by different fitness landscapes influenced the shape of phylogenies, diversity trends, and survival of virus with latent genomic fragments. Furthermore, our model predicts that the persistence of latent genomic fragments from multiple different ancestral origins increases sequence diversity in plasma for reasonable fitness landscapes.

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↗
DOE OSTI2025

Cohort-based pan-cancer analysis and experimental studies reveal ISG15 gene as a novel biomarker for prognosis and immunotherapy efficacy prediction

Abstract ISG15, an interferon-stimulated ubiquitin-like protein, plays a multifaceted role in tumorigenesis and immune regulation. This study comprehensively evaluates ISG15 as a prognostic biomarker and predictor of immunotherapy response through pan-cancer bioinformatics analysis and experimental validation. By integrating multiomics data from TCGA, GEO, and clinical cohorts, we found that ISG15 is significantly overexpressed in multiple cancers and generally correlates with poor prognosis. Elevated ISG15 expression is associated with increased immune checkpoint gene expression, particularly PD-L1, and immune infiltration, notably M2-like tumor-associated macrophages. Immunohistochemistry and multiplexed immunofluorescence confirmed a strong positive correlation between ISG15, PD-L1, and M2-TAM infiltration in lung and gastric cancer samples. Functional analysis at the single-cell level revealed significant associations between ISG15 and tumor proliferation, angiogenesis, and immune suppression. Immunotherapy cohort analysis demonstrated that tumors with high ISG15 expression responded favorably to PD-L1 inhibitors but exhibited resistance to CTLA-4 blockade, findings further validated in lung cancer patients receiving anti-PD-1 therapy. These results suggest that ISG15 is a promising biomarker for prognosis and immunotherapy response prediction across cancers. Its integration into clinical decision-making may enhance personalized treatment strategies, improve immunotherapy outcomes, and provide new insights into the tumor immune microenvironment, cancer progression, and potential therapeutic targets for future drug development.

Immunology↗
DOE OSTI2017

Antibiotics-induced gut microbiota dysbiosis promotes tumor initiation via affecting APC-Th1 development in mice

Gut microbiota is critical for maintaining body immune homeostasis and thus affects tumor growth and therapeutic efficiency. Here, we investigated the link between microbiota and tumorgenesis in a mice model of subcutaneous melanoma cell transplantation, and explored the underlying mechanism. We found disruption of gut microbiota by pretreating mice with antibiotics promote tumor growth and remodeling the immune compartment within the primary tumor. Indeed, gut microbial dysbiosis reduced the infiltrated mature antigen-presenting cells of tumor, together with lower levels of co-stimulators, such as CD80, CD86 and MHCII, as well as defective Th1 cytokines, including IFNγ, TNFα, IL12p40, and IL12p35. Meantime, splenic APCs displayed blunted ability in triggering T cell proliferation and IFNγ secretion. However, oral administration of LPS restored the immune surveillance effects and thus inhibited tumor growth in the antibiotics induced gut microbiota dysbiosis group. Taken together, these data highly supported that antibiotics induced gut microbiota dysbiosis promotes tumor initiation, while LPS supplementation would restore the effective immune surveillance and repress tumor initiation. - Highlights: • Antibiotics induced gut microbiota dysbiosis promotes tumor initiation in mice. • Gut microbiota disruption compromise APC-Th1 development in tumor. • Orally LPS administration rescues tumor immune surveillance in gut microbiota disrupted mice. • None of the authors have any conflict of interest.

60 APPLIED LIFE SCIENCES↗