Duane L. Pierson

The Dynamics of Herpesvirus and Polyomavirus Reactivation and Shedding in Healthy Adults: A 14-Month Longitudinal Study

Humans are infected with viruses that establish long-term persistent infections. To address whether immunocompetent individuals control virus reactivation globally or independently and to identify patterns of sporadic reactivation, we monitored herpesviruses and polyomaviruses in 30 adults, over 14 months. Epstein-Barr virus (EBV) DNA was quantitated in saliva and peripheral blood mononuclear cells (PBMCs), cytomegalovirus (CMV) was assayed in urine, and JC virus (JCV) and BK virus (BKV) DNAs were assayed in urine and PBMCs. All individuals shed EBV in saliva, whereas 67% had >or=1 blood sample positive for EBV. Levels of EBV varied widely. CMV shedding occurred infrequently but occurred more commonly in younger individuals (P<.03). JCV and BKV virurias were 46.7% and 0%, respectively. JCV shedding was age dependent and occurred commonly in individuals >or=40 years old (P<.03). Seasonal variation was observed in shedding of EBV and JCV, but there was no correlation among shedding of EBV, CMV, and JCV (P>.50). Thus, adults independently control persistent viruses, which display discordant, sporadic reactivations.

Epstein-Barr virus shedding by astronauts during space flight

Patterns of Epstein-Barr virus (EBV) reactivation in 32 astronauts and 18 healthy age-matched control subjects were characterized by quantifying EBV shedding. Saliva samples were collected from astronauts before, during, and after 10 space shuttle missions of 5-14 days duration. At one time point or another, EBV was detected in saliva from each of the astronauts. Of 1398 saliva specimens from 32 astronauts, polymerase chain reaction analysis showed that 314 (23%) were positive for EBV DNA. Examination by flight phase showed that 29% of the saliva specimens collected from 28 astronauts before flight were positive for EBV DNA, as were 16% of those collected from 25 astronauts during flight and 16% of those collected after flight from 23 astronauts. The mean number of EBV copies from samples taken during the flights was 417 per mL, significantly greater (p<.05) than the number of viral copies from the preflight (40) and postflight (44) phases. In contrast, the control subjects shed EBV DNA with a frequency of 3.7% and mean number of EBV copies of 40 per mL of saliva. Ten days before flight and on landing day, titers of antibody to EBV viral capsid antigen were significantly (p<.05) greater than baseline levels. On landing day, urinary levels of cortisol and catecholamines were greater than their preflight values. In a limited study (n=5), plasma levels of substance P and other neuropeptides were also greater on landing day. Increases in the number of viral copies and in the amount of EBV-specific antibody were consistent with EBV reactivation before, during, and after space flight.

Multiple latent viruses reactivate in astronauts during Space Shuttle missions.

Latent virus reactivation and diurnal salivary cortisol and dehydroepiandrosterone were measured prospectively in 17 astronauts (16 male and 1 female) before, during, and after short-duration (12-16 days) Space Shuttle missions. Blood, urine, and saliva samples were collected during each of these phases. Antiviral antibodies and viral load (DNA) were measured for Epstein-Barr virus (EBV), varicella-zoster virus (VZV), and cytomegalovirus (CMV). Three astronauts did not shed any virus in any of their samples collected before, during, or after flight. EBV was shed in the saliva in all of the remaining 14 astronauts during all 3 phases of flight. Seven of the 14 EBV-shedding subjects also shed VZV during and after the flight in their saliva samples, and 8 of 14 EBV-shedders also shed CMV in their urine samples before, during, and after flight. In 6 of 14 crewmembers, all 3 target viruses were shed during one or more flight phases. Both EBV and VZV DNA copies were elevated during the flight phase relative to preflight or post-flight levels. EBV DNA in peripheral blood was increased preflight relative to post-flight. Eighteen healthy controls were also included in the study. Approximately 2-5% of controls shed EBV while none shed VZV or CMV. Salivary cortisol measured preflight and during flight were elevated relative to post-flight. In contrast DHEA decreased during the flight phase relative to both preflight and post-flight. As a consequence, the molar ratio of the area under the diurnal curve of cortisol to DHEA with respect to ground (AUCg) increased significantly during flight. This ratio was unrelated to viral shedding. In summary, three herpes viruses can reactivate individually or in combination during spaceflight.

Alterations in adaptive immunity persist during long-duration spaceflight

Background:It is currently unknown whether immune system alterations persist during long-duration spaceflight. In this study various adaptive immune parameters were assessed in astronauts at three intervals during 6-month spaceflight on board the International Space Station (ISS).AIMS:To assess phenotypic and functional immune system alterations in astronauts participating in 6-month orbital spaceflight.Methods:Blood was collected before, during, and after flight from 23 astronauts participating in 6-month ISS expeditions. In-flight samples were returned to Earth within 48u2009h of collection for immediate analysis. Assays included peripheral leukocyte distribution, T-cell function, virus-specific immunity, and mitogen-stimulated cytokine production profiles.Results:Redistribution of leukocyte subsets occurred during flight, including an elevated white blood cell (WBC) count and alterations in CD8+ T-cell maturation. A reduction in general T-cell function (both CD4+ and CD8+) persisted for the duration of the 6-month spaceflights, with differential responses between mitogens suggesting an activation threshold shift. The percentage of CD4+ T cells capable of producing IL-2 was depressed after landing. Significant reductions in mitogen-stimulated production of IFNγ, IL-10, IL-5, TNFα, and IL-6 persisted during spaceflight. Following lipopolysaccharide (LPS) stimulation, production of IL-10 was reduced, whereas IL-8 production was increased during flight.Conclusions:The data indicated that immune alterations persist during long-duration spaceflight. This phenomenon, in the absence of appropriate countermeasures, has the potential to increase specific clinical risks for crewmembers during exploration-class deep space missions.

Suppressed expression of non-DSB repair genes inhibits gamma-radiation-induced cytogenetic repair and cell cycle arrest

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in regulating DSB repair and cell cycle progression. In this study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequency of micronuclei (MN) formation and chromosome aberrations were measured to determine efficiency of cytogenetic repair, especially DSB repair. In response to IR, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR-induced biological consequences. Furthermore, eight non-DBS repair genes showed involvement in regulating DSB repair, indicating that successful DSB repair requires both DSB repair mechanisms and non-DSB repair systems. These results reveal that many genes play previously unrecognized roles in multiple DNA repair responses, all of which are required for successful repair of IR-induced damage.

Fitness level impacts salivary antimicrobial protein responses to a single bout of cycling exercise

AbstractPurposenSalivary antimicrobial proteins (sAMPs) protect the upper respiratory tract (URTI) from invading microorganisms and have been linked with URTI infection risk in athletes. While high training volume is associated with increased URTI risk, it is not known if fitness affects the sAMP response to acute exercise. This study compared the sAMP responses to various exercising workloads of highly fit experienced cyclists with those who were less fit.MethodsSeventeen experienced cyclists (nine highly fit; eight less fit) completed three 30-min exercise trials atxa0workloads corresponding to −5, +5 and +15xa0% of the individual blood lactate threshold. Saliva samples were collected pre- and post-exercise to determine the concentration and secretion of α-amylase, human neutrophil proteins 1–3 (HNP1–3) lactoferrin, LL-37, lysozyme, and salivary SIgA.ResultsThe concentration and/or secretion of all sAMPs increased post-exercise, but only α-amylase was sensitive to exercise workload. Highly fit cyclists had lower baseline concentrations of α-amylase, HNP1–3, and lactoferrin, although secretion rates did not differ between the groups. Highly fit cyclists did, however, exhibit greater post-exercise increases in the concentration and/or secretion of a majority of measured sAMPs (percentage difference between highly fit and less fit in parentheses), including α-amylase concentration (+107xa0%) and secretion (+148xa0%), HNP1–3 concentration (+97xa0%) and secretion (+158xa0%), salivary SIgA concentration (+181xa0%), lactoferrin secretion (+209xa0%) and LL-37 secretion (+138xa0%).ConclusionWe show for the first time that fitness level is a major determinant of exercise-induced changes in sAMPs. This might be due to training-induced alterations in parasympathetic and sympathetic nervous system activation.

CHAPTER 40 – Reactivation of Latent Herpes Viruses in Astronauts

INTRODUCTION Reactivation of latent viruses may pose an important health risk for people living and working in extreme environments such as space. Stress-induced changes in levels of specific hormones lead to alterations in the immune response through the hypothalamus-pituitary-adrenal (HPA) axis resulting in increased shedding of latent viruses. Astronauts experience various stresses (e.g., sleep deprivation) that may result in increased reactivation of latent viruses during space flight, potentially increasing the risk of disease among crewmembers. This study was undertaken to determine the effects of space flight upon the incidence and magnitude of latent virus reactivation and shedding.

Microbiological Lessons Learned From the Space Shuttle

After 30 years of being the centerpiece of NASA’s human spacecraft, the Space Shuttle will retire. This highly successful program provided many valuable lessons for the International Space Station (ISS) and future spacecraft. Major microbiological risks to crewmembers include food, water, air, surfaces, payloads, animals, and other crewmembers, and ground support personnel. Adverse effects of microorganisms are varied and can jeopardize crew health and safety, spacecraft systems, and mission objectives. Engineering practices and operational procedures can minimize the negative effects of microorganisms. To minimize problems associated with microorganisms, appropriate steps must begin in the design phase of new spacecraft or space habitats. Spacecraft design must include requirements to control accumulation of water including humidity, leaks, and condensate on surfaces. Materials used in habitable volumes must not contribute to microbial growth. Use of appropriate materials and the implementation of robust housekeeping that utilizes periodic cleaning and disinfection will prevent high levels of microbial growth on surfaces. Air filtration can ensure low levels of bioaerosols and particulates in the breathing air. The use of physical and chemical steps to disinfect drinking water coupled with filtration can provide safe drinking water. Thorough preflight examination of flight crews, consumables, and the environment can greatly reduce pathogens in spacecraft. The advances in knowledge of living and working onboard the Space Shuttle formed the foundation for environmental microbiology requirements and operations for the International Space Station (ISS) and future spacecraft. Research conducted during the Space Shuttle Program resulted in an improved understanding of the effects of spaceflight on human physiology, microbial properties, and specifically the host-microbe interactions. Host-microbe interactions are substantially affected by spaceflight. Astronaut immune functions were found to be altered. Selected microorganisms were found to become more virulent during spaceflight. The increased knowledge gained on the Space Shuttle resulted in further studies of the host-microbe interactions on the ISS to determine if countermeasures were necessary. Lessons learned from the Space Shuttle Program were integrated into the ISS resulting in the safest space habitat to date.

Preventing Infectious Diseases in Spacecraft and Space Habitats

Spacecraft crewmembers live and work in a closed environment that is monitored to ensure health and safety. Lessons learned from previous spaceflight missions have been incorporated into the design and development of the International Space Station (ISS). The microbial control actions on the ISS include engineering designs, such as high efficiency particulate air filtering of the air, microbial monitoring of the air, surfaces, and water, as well as remediation procedures when needed. This chapter will describe an overview of microbial risks of spaceflight focusing on measures to prevent infectious disease. The information discussed in this chapter is focused on the microbial monitoring activities in United States Operating Segment (USOS) of the ISS and experimental data obtained on USOS crewmembers.

Simulated microgravity ‘disarms’ human Natural Killer cells and suppresses cytotoxic activity against tumor target cells

Maintaining immune system integrity is of paramount importance for astronauts embarking on long-duration spaceflight missions. We have shown that Natural killer (NK) cell function is impaired during spaceflight; however, the mechanisms responsible for this effect are unknown. We exposed primary human blood NK cells to 12xa0h of simulated microgravity (SMG) using a rotating wall vessel zero gravity cell culture analog. Compared to static and vertical axis rotational controls, SMG (horizontal axis rotation) was found to decrease subsequent NK-cell cytotoxic function in 1G against allogeneic target cell lines of leukemia (K562), multiple myeloma (U266), B-lymphoma (721.221) and HLA-E transfected lymphoma (221.AEH) origin. Flow cytometric analysis revealed that SMG had little effect on the surface expression of a wide-range of NK-cell activating and inhibitory receptors, but did decrease intracellular perforin expression. Using cytometry by time of flight (CyTOF), SMG was found to lower NK-cell degranulation as determined by CD107a expression following co-incubation with K562 target cells in 1G. Moreover, SMG reduced NK-cell expression of the pro-inflammatory cytokines TNF-alpha and IFN-gamma in response to K562 co-culture at 1G. These results indicate that microgravity may be involved in spaceflight-associated reductions in NK cell function. Specifically, pre-exposure to SMG appears to disrupt NK-cell killing by ‘disarming’ them of cytolytic granules and impairing their ability to secrete effector cytokines when confronted with tumor target cells in the 1G environment.